IDENTIFICATION OF INHIBITORS OF TCPC AND TIR NADase ACTIVITY

Abstract

The present invention provides compounds useful as inhibitors of TIR NADase activity, compositions thereof, and methods of using the same. The present invention is useful for inhibition of TIR-domain NADase activity and/or treating microbial infection and/or modulating microbial physiology.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application 62/563,447, filed Sep. 26, 2017, the disclosure of which is hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0003] This application relates to various compounds and compositions, and methods, useful for inhibition of TIR-domain NADase activity and/or treating microbial infection and/or modulating microbial physiology.

BACKGROUND

[0004] Antimicrobial resistance has emerged as one of the principal public health problems of the 21st century that threatens the effective prevention and treatment of an ever-increasing range of infections caused by bacteria, parasites, viruses and fungi no longer susceptible to the common medicines used to treat them. Without effective antimicrobials for prevention and treatment of infections, medical procedures such as organ transplantation, cancer chemotherapy, diabetes management and major surgery (e.g., caesarean sections or hip replacements) become very high risk. The problem of antimicrobial resistance is especially concerning regarding antibiotic resistance in bacteria. For instance, resistance in Escherichia coli to one of the most widely used medicines for the treatment of urinary tract infections (fluoroquinolone antibiotics) is very widespread. There are countries in many parts of the world where this treatment is now ineffective in more than half of patients. Moreover, resistance to first-line drugs to treat infections caused by Staphylococcus aureus, a common cause of severe infections in health facilities and the community, is widespread. People with methicillin-resistant Staphylococcus aureus (MRSA) are estimated to be 64% more likely to die than people with a non-resistant form of the infection. Hence, much effort and resources have been devoted to understanding resistance mechanisms and discovery of novel classes of antibiotics.

[0005] Accordingly, a need exists for compounds and compositions which are useful for treating microbial infections. Additionally, methods for screening potential compounds and compositions useful for treating microbial infections are critical to the discovery of novel antimicrobial agents.

SUMMARY

[0006] Among the various aspects of the present disclosure provide enzyme(s) as therapeutic target(s) for microbial infections and disease.

[0007] In some embodiments, the present disclosure provides assays for identifying and/or characterizing a TIR NADase inhibitor. In one aspect, the present disclosure provides certain vector constructs and polypeptides for use in these assays, including wild-type, mutant or fragments of microbial or mammalian TIR domain polypeptides, as well as tagged versions of the same. In another aspect, the disclosure provides compositions comprising a polypeptide and a solid support which is used for screening TIR NADase inhibitors.

[0008] The present disclosure further provides methods of using TIR NADase inhibitors to treat or prevent a microbial infection. In some embodiments, the disclosure provides administering an effective amount of a composition comprising an inhibitor of TcpC TIR NADase activity. In one aspect, the TcpC NADase inhibitor compositions treat infection and disease caused by uropathogenic Escherichia coli. In another aspect, the disclosure provides administering compositions comprising dequalinium, cetylpyridium, amiloride or combinations thereof, for use as inhibitors of TcpC NADase activity.

[0009] In some embodiments, the disclosure provides inhibitors of TirS NADase. In one aspect, TirS NADase inhibitors treat infection and disease caused by Staphylococcus aureus. In another aspect, the disclosure provides the recognition that acrinol, akrinol or combinations thereof are useful as inhibitors of TirS NADase activity.

[0010] In certain embodiments, the present invention relates to Tir NADase inhibitor compositions and methods for decreasing the infectivity, morbidity, and rate of mortality associated with a variety of pathogenic organisms. In some embodiments, inhibitors of TIR NADase activity are selected from dequalinium, cetylpyridium, amiloride, acrinol, akrinol, analogs or derivatives thereof, or a pharmaceutically acceptable salt thereof. Compounds of the present invention and pharmaceutically acceptable compositions thereof, are useful for treating a variety of infections, infectious diseases, or conditions.

[0011] Compounds provided by this invention are also useful for the study of TIR NADase activity in biological and pathological phenomena; the study of microbial physiology; and the comparative evaluation of new TIR NADase activity inhibitors in vitro or in vivo.

[0012] While multiple embodiments are disclosed, still other embodiments of the present invention will become apparent to those skilled in the art from the following detailed description, which shows and describes illustrative embodiments of the invention. Accordingly, the figures and detailed description are to be regarded as illustrative in nature and not restrictive.

BRIEF DESCRIPTION OF THE FIGURES

[0013] The application file contains at least one drawing executed in color. Copies of this patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0014] FIG. 1A, FIG. 1B and FIG. 1D shows multiple TIR domains from bacteria and archaea induce NAD.sup.+ loss in host E. coli FIG. 1A depicts the E. coli metabolite assay to screen candidate TIR domains for NAD+ cleavage activity. FIG. 1B shows the SARM1 domain structure and role in axon degeneration. MLS--Mitochondrial Localization Signal; ARM--Armadillo/HEAT Motifs; SAM--Sterile Alpha Motif; TIR--Toll/Interleukin 1 Receptor. FIG. 1C shows the domain structure of candidate prokaryotic TIR containing proteins. CC--Coiled Coil; Ax_dyn_L--Axonemal dynein light chain; TM--transmembrane; ATP_Syn_B--ATP Synthase B/B' CF(0); MPN--Mpr1p Pad1p N-terminal. FIG. 1D shows the endogenous levels of NAD.sup.+ in E. coli after IPTG induction. Data were generated from at least three independent biological experiments. Data are presented as mean.+-.SEM; error bars represent SEM. P<0.001 for SARM1-TIR, ApTir-TIR, AbTir-TIR, TcpA-TIR, TcpO-TIR for individual comparisons (+/- IPTG) using unpaired, two-tailed Student's t test. P<0.01 for TcpC-TIR, TcpF-TIR, BtpA-TIR, for individual comparisons (+/- IPTG) using unpaired, two-tailed Student's t test. P<0.05 for Pd-TIR for individual comparisons (+/- IPTG) using unpaired, two-tailed Student's t test. P<0.001 for TirS-TIR IPTG v.s. NC vector IPTG.

[0015] FIG. 2A, FIG. 2B, FIG. 2C, FIG. 2D, FIG. 2E FIG. 2F, FIG. 2G, FIG. 2H, FIG. 2I, FIG. 2J, FIG. 2K and FIG. 2L depicts the characterization of an intrinsic NAD.sup.+ glycohydrolase activity in a subset of prokaryotic TIR domains. FIG. 2A shows prokaryotic TIR proteins purified from cell-free translation system cleave NAD.sup.+ in NADase assay. FIG. 2B is a HPLC chromatogram showing the TIR domain from Staphylococcus aureus (TirS-TIR) cleaves NAD.sup.+ into Nam and ADPR. FIG. 2C is a HPLC chromatogram showing the TIR domain from uropathogenic Escherichia coli (TcpC-TIR) cleaves NAD.sup.+ into Nam and ADPR. FIG. 2D is a HPLC chromatogram showing the TIR domain from the archaea Thermoplasmatales archaeon (TcpA-TIR) cleaves NAD.sup.+ into Nam and ADPR. Retention time was as follows: Nam at t.about.2.40 min and ADPR at t.about.1.10 min. FIG. 2E is the quantification of metabolites generated by prokaryotic TIR proteins as displayed in 2A-2D (normalized to 0 min). Data were generated from at least three independent reaction experiments. Data are presented as mean.+-.SEM; error bars represent SEM. P<0.001 for all individual comparisons between 0 and 5 min or between 0 and 30 min for each TIR. FIG. 2F is the kinetic parameters for TirS-TIR and TcpC-TIR NAD.sup.+ cleavage reaction. V.sub.max and K.sub.m were determined by fitting the data to the Michaelis-Menten equation and are presented as mean.+-.SEM for two independent purified protein reactions. FIG. 2G depicts amino acid sequence alignment of TirS-TIR with MilB cytidine 5' monophosphate (CMP) hydrolase, and modeling of TirS-TIR domain on the crystal structure of CMP hydrolase bound to CMP. E216 of TirS aligns with the catalytic glutamate residue of CMP hydrolase (SEQ ID NO:1 and SEQ ID NO:11). FIG. 2H depicts amino acid sequence alignment of TcpC-TIR with CMP hydrolase, and modeling of TcpC-TIR domain on the crystal structure of CMP hydrolase bound to CMP. E244 of TcpC aligns with the catalytic glutamate residue of CMP hydrolase (SEQ ID NO:2 and SEQ ID NO:11). Tan--modeled bacterial TIR; cyan--MilB CMP hydrolase; yellow--cytidine-5'-monophosphate (ligand for MilB). FIG. 2I depicts purified TIR proteins with mutation of catalytic glutamate to alanine do not cleave NAD.sup.+ in NADase assay (normalized to control at 0 min). FIG. 2J-FIG. 2K show the quantification of metabolites from reactions using purified proteins (TirS-TIR and TcpC-TIR) and substrate analogs (NADP--Nicotinamide Adenine Dinucleotide Phosphate; 3apAD--3-acetylpyridine Adenine Dinucleotide; sNAD--thionicotinamide Adenine Dinucleotide; NHD--Nicotinamide Hypoxanthine Dinucleotide; NGD--Nicotinamide Guanine Dinucleotide; NaAD--Nicotinic Acid Adenine Dinucleotide; NADH--Nicotinamide Adenine Dinucleotide reduced; NMN--Nicotinamide Mononucleotide; ATP--Adenosine triphosphate; GTP--Guanine triphosphate). Metabolites were measured by HPLC and LC-MS analyses, and normalized to 0 min controls. FIG. 2L is a heat map summarizing relative levels of substrates after 30 minutes of reaction with the purified TIR proteins indicated. Values range from 0 (green), indicating complete cleavage of substrate within 30 minutes, and 1 (red) indicating relatively unchanged levels of substrates. Data from (I-L) were generated from at least three independent reaction experiments. Data are presented as mean.+-.SEM; error bars represent SEM.

[0016] FIG. 3A. FIG. 3B, FIG. 3C, FIG. 3D, FIG. 3E, FIG. 3F, FIG. 3G, FIG. 3H and FIG. 3I depicts a variant cyclic ADPR is generated by a subset of prokaryotic TIR domains. FIG. 3A-FIG. 3D are HPLC chromatograms of metabolites extracted from E. coli lysates expressing candidate prokaryotic TIR domains. Metabolites extracted from TcpO-TIR, and AbTir-TIR reveal a prominent previously unidentified peak (labeled "X") around retention time 1.7 minutes (FIG. 3C-FIG. 3D), which is not seen in metabolites extracted from SARM1-TIR or TirS-TIR (FIGS. 3A and 3B). FIG. 3E is a HPLC chromatogram showing purified TcpO-TIR cleaves NAD.sup.+ into Nicotinamide, ADPR, and a metabolite close to retention time of Metabolite X. FIG. 2F is a HPLC chromatogram of cADPR standard. FIG. 3G shows a merge of HPLC chromatograms of metabolites generated from purified TcpO-TIR recombinant protein reaction (FIG. 3E) cADPR standard (FIG. 3F). FIG. 3H shows MS/MS spectra of product ion scan of m/z 542 of cADPR standard and TcpO-TIR generated metabolite X. FIG. 3I depicts the proposed structure of variant cADPR generated by TcpO-TIR with the N7 adenine position involved in cyclization.

[0017] FIG. 4A, FIG. 4B, FIG. 4C, FIG. 4D, FIG. 4E, FIG. 4F and FIG. 4G show Full-length TirS from Staphylococcus aureus induces NAD.sup.+ loss in mammalian HEK293T cells. FIG. 4A Endogenous NAD.sup.+ levels in HEK293 cells expressing both wild type and mutant TirS-TIR and TcpC-TIR. FIG. 4B depicts a western blot of wild type and mutant TirS-TIR and TcpC-TIR proteins expressed in HEK293T cells. FIG. 4C shows a HPLC chromatogram showing purified full length TirS (TirS-FL) cleaves NAD+ into Nam and ADPR in NADase assay. FIG. 4D is the quantification of metabolites generated by TirS-FL NADase reaction as displayed in C (normalized to 0 min NAD+).

[0018] FIG. 4E shows SYPRO Ruby gel of purified TirS (wild type and mutant). FIG. 4F shows endogenous NAD+ levels in HEK293T cells expressing both wild type and mutant TirS-FL. FIG. 4G depicts a western blot of wild type and mutant TirS-FL proteins expressed in HEK293T cells. Data were generated from at least three independent experiments. Data are presented as mean.+-.SEM; error bars represent SEM. ***p<0.001, unpaired two-tailed Student's t test.

[0019] FIG. 5A and FIG. 5B shows multiple sequence alignments of prokaryotic TIR domain containing sequences used in E. coli IPTG experiments. Sequences were aligned using the ClustalO multiple sequence alignment tool in HH Pred (SEQ ID NOs:1-10).

[0020] FIG. 6A, FIG. 6B, FIG. 6C, FIG. 6D, FIG. 6E, FIG. 6F and FIG. 6G show mass spectra identifying products of prokaryotic TIR NAD.sup.+ cleavage reactions. (FIG. 6A-FIG. 6F) Nicotinamide and ADPR were detected by mass spec in reactions catalyzed by TirS-TIR, TcpC-TIR, and TcpA-TIR. FIG. 6G depicts trace amount of cADPR were detected in the reactions catalyzed by TcpA-TIR, but not TirS-TIR or TcpC-TIR.

[0021] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D and FIG. 7E show multiple sequence alignments and structure modeling of prokaryotic TIRs with MilB CMP hydrolase. FIG. 7A is a SYPRO gel showing purified proteins (wild type and mutant) used in NADase assay time course. FIG. 7B shows modeling of the TcpA-TIR domain on the crystal structure of CMP hydrolase bound to CMP. E267 aligns with the catalytic glutamate residue of CMP hydrolase. (FIG. 7C-7E) Amino acid sequence alignment of TirS-TIR, TcpC-TIR, and TcpA-TIR with MilB cytidine 5' monophosphate (CMP) hydrolase. CMP catalytic glutamic acid is highlighted by the red asterisk and aligns to a conserved glutamic acid in the prokaryotic TIR domains (SEQ ID NOs:1-3 and 11).

[0022] FIG. 8A. FIG. 8B and FIG. 8C show TIR domains cleave other substrates besides NAD.sup.+. FIG. 8A are chemical structures of NAD.sup.+ and nucleotide analogs tested in NADase assay. Functional groups highlighted in red depict modifications in analogs compared to NAD+. Nucleotide analogs include NADP--Nicotinamide Adenine Dinucleotide Phosphate; 3apAD--3-acetylpyridine Adenine Dinucleotide; sNAD--thionicotinamide Adenine Dinucleotide; NHD--Nicotinamide Hypoxanthine Dinucleotide; NGD--Nicotinamide Guanine Dinucleotide; NaAD--Nicotinic Acid Adenine Dinucleotide; NADH--Nicotinamide Adenine Dinucleotide reduced; NMN--Nicotinamide Mononucleotide; ATP--Adenosine triphosphate; GTP--Guanine triphosphate. (FIG. 8B and FIG. 8C) Quantification of metabolites from reactions using purified proteins (TcpA-TIR and SARM1-TIR) and nucleotide analogs from A. Metabolites were measured by HPLC and LC-MS analyses, and normalized to 0 min controls.

[0023] FIG. 9A, FIG. 9B, FIG. 9C, FIG. 9D and FIG. 9E show HPLC chromatograms identifying a variant cADPR molecule. FIG. 9A is a HPLC chromatogram of metabolites extracted from E. coli lysates expressing BtpA-TIR reveal a prominent, previously unidentified peak labeled X. FIG. 9B is a cADPR standard and Metabolite X from purified TcpO-TIR reaction have similar parent ion m/z of 542, but different retention times. The retention time for Metabolite X differ from than shown in A and FIG. 4 since these are two separate LC instruments. (FIG. 9C-FIG. 9E) The ion pair of 542/136 is confirmed in metabolites extracted from E. coli lysates expressing TcpO-TIR, AbTir-TIR, and BtpA-TIR.

[0024] FIG. 10A, FIG. 10B, FIG. 10C and FIG. 10D shows the identification of TcpC inhibitors from Pharmacon library. FIG. 10A depicts the primary screen of compounds from the Pharmacon library identified Dequalinium as a potent inhibitor of the NADase activity of TcpC TIR. FIG. 10B depicts a dose response is shown for Dequalinium tested as an NADase inhibitor of the TIR domain from TcpC, Staph aureus TirS (called N=MSSA above), and SARM1 (called SAM-TIR). Note that dequalinium is a very potent inhibitor of TcpC-Tir with an IC50 of approximately 300 nM. FIG. 10C shows the inhibition by cetylpyridium of the NADase activity of the TIR domain from TcpC, Staph aureus TirS (called N=MSSA above), and SARM1 (called SAM-TIR). FIG. 10D shows the inhibition by amiloride of the NADase activity of the TIR domain from TcpC, Staph aureus TirS (called N=MSSA above), and SARM1 (called SAM-TIR).

[0025] FIG. 11A, FIG. 11B, FIG. 11C and FIG. 11D depicts the screen for inhibitors of the NADase activity of TirS from S. aureus. FIG. 11A shows the primary screen of inhibitors from the Pharmakon library identified Acrinol and Akrinol as inhibitors of the NADase activity of the TirS Tir domain of MSSA inhibitors. FIG. 11B depicts components of acrinol and akrinol and additional acridine compounds (listed in figure) were tested as inhibitors of the NADase activity of TirS. The most potent inhibition is mediated by 9-Aminoacridine. FIG. 11C, structure of Acrinol; FIG. 11D, structure of Akrinol (9-aminoacridine and 4-hexylresorcinol).

[0026] Reference to various embodiments does not limit the scope of the invention. Figures represented herein are not limitations to the various embodiments according to the invention and are presented for exemplary illustration of the invention.

DETAILED DESCRIPTION

[0027] The present disclosure provides, generally, methods and compositions for inhibiting NADase activity of TIR domain containing polypeptide. In particular, provided herein are compositions comprising TIR NADase inhibitor compounds such as dequalinium, cetylpyridium, amiloride, acrinol, akrinol, derivatives thereof or pharmaceutically acceptable salts thereof. Applicants have surprisingly discovered TIR domains constitute a new family of NADase enzymes. Hence, this large class of proteins is a central and previously unappreciated regulator of NAD+ biology and represents a new therapeutic window for modulating the virulence and/or survival of microbes.

[0028] Traditionally, the TIR domain is known as the signature domain of components of Toll-Like Receptor (TLR) signaling, and is present in numerous receptors and adaptor proteins in innate immune pathways (Akira et al., 2006; Burch-Smith and Dinesh-Kumar, 2007; O'Neill et al., 2013). These TIR domains serve as scaffolds that promote the assembly of signaling complexes to trigger activation of pro-inflammatory cytokines and other defense-related products (O'Neill et al., 2013). Bioinformatics analysis shows that TIR domains are present in proteins across all domains of life. In bacteria, TIR domain proteins are associated with virulence, and are thought to do so, in part, via blockade of innate immunity mediated by TLR signaling. While, a number of individual TIR domains from the pathogenic bacteria have been analyzed, the TIR domains from these pathogenic bacteria seem to differ in their modes of action and their roles in virulence. As a result targeting these domains for therapeutic intervention has been challenging and largely unsuccessful.

[0029] Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular compound is disclosed and discussed and a number of modifications that can be made to a number of molecules of the compound are discussed, specifically contemplated is each and every combination and permutation of the compound and the modifications that are possible unless specifically indicated to the contrary. Thus, if a class of molecules A, B, and C are disclosed as well as a class of molecules D, E, and F and an example of a combination molecule, A-D is disclosed, then even if each is not individually recited each is individually and collectively contemplated meaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F are considered disclosed. Likewise, any subset or combination of these is also disclosed. Thus, for example, the sub-group of A-E, B-F, and C-E would be considered disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.

[0030] Various aspects of the invention are described in further detail in the following sections.

(I) Compositions

[0031] One aspect of the present disclosure provides an inhibitor of TIR NADase activity. As used herein, an inhibitor of TIR NADase activity, or "TIR NADase inhibitor" includes any compound capable of downregulating or inactivating the NADase activity of a TIR domain containing polypeptide. In some embodiments, the inhibitors for use with the invention may function to inhibit the NADase activity of a TIR domain containing polypeptide, thereby increasing the concentration of NAD+ in a biological sample compared to that of a control or decreasing the concentration of one or more of ADPR, cADPR or nicotinamide relative to a control. In some embodiments, compounds that decrease TIR NADase activity also decrease the associated downstream signaling pathways.

[0032] A composition of the invention may optionally comprise one or more additional drug or therapeutically active agent in addition to the TIR NADase inhibitor. A composition of the invention may further comprise a pharmaceutically acceptable excipient, carrier, or diluent. Further, a composition of the invention may contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifiers, sweeteners, colorants, odorants, salts (substances of the present invention may themselves be provided in the form of a pharmaceutically acceptable salt), buffers, coating agents, or antioxidants.

[0033] Other aspects of the invention are described in further detail below.

(a) Dequalinium, Cetylpyridium, Amiloride, Acrinol, Akrinol, and Derivatives Thereof

[0034] In general, the compounds detailed herein include compounds comprising dequalinium, structure as diagrammed below. Dequalinium is a bis-quinolinium compound. Its chemical elements are expressed as C.sub.30H.sub.40N.sub.4, with a molecular weight of 527.578 g/mol. Dequalinium and its salts are commercially available, for example from Sigma Aldrich. Methods of making suitable derivatives are described in U.S. application Ser. No. 14/657,872, WO 97/48705, Galanakis et al. (1995) J. Med. Chem. 38: 595-606, Galanakis et al (1995) J. Med. Chem. 38: 3536-3546 and Galanakis et al (1996) J. Med. Chem. 39: 3592-3595, Abeywickrama et al. (2006) Bioorganic Medicinal Chem. 14: 7796-7803, Qin et al. (2000) J. Med. Chem. 43: 1413-1417, Campos Rosa et al (1996) J. Med. Chem. 39: 4247-4254, the contents of which are incorporated herein by reference in their entirety.

##STR00001##

[0035] Suitable derivatives of dequalinium include but are not limited to: 1-decanyl-2-methyl-4-aminoquinolinium iodide; 1-butyl-2-methyl-4-aminoquinolinium iodide; 1,1,1-triethyl-1-(10-iododecan-1-yl)ammonium iodide, 1-[1-(N,N,N-triethylammonium-1-yl)-2-methyl-4-aminoquinolinium diiodide; 1,1'-(decane-1,10-diyl)bis(4-aminopyridinium)diiodide; 1-(4-pentyn-1-yl)-4-aminopyridinium chloride; 1,1'-(deca-4,6-diyne-1,10-diyl)bis(4-aminopyridinium)dichloride dehydrate; 2,2'-N,N'-(decane-1,10-diyl)bis(2,4-diaminopyridine); 2,2'-N,N'-(decane-1,10-diyl)bis(2-aminopyridine); 2'-N,N'(decane-1,10-diyl)bis(1-methyl-2-aminopyridinium)diiodide; 1-(4-pentyn-1-yl)-2-methyl-4-aminoquinolinium iodide; 1,1'-(deca-4,6-diyne-1,10-diyl)bis(4-amino-2-methylquinolinium)diiodide hydrate; 1,1'-(decane-1,10-diyl)bis(quinolinium)diiodide; 1,1'-(decane-1,10-diyl)bis(9-amino-1,2,3,4-tetra-hydroacridinium)dibromid- e hydrate; 2,2'-(decane-1,10-diyl)bis(quinoline); 2,2'-(decane-1,10-diyl)bis(1-methylquinolinium)diiodide hydrate; 2,2'-(decane-1,10-diyl)bis(4-methoxyquinoline); 2,2'-(decane-1,10-diyl)bis(1-methyl-4-methoxyquinolinium)diiodide; 2,2'-(dodecane-1,12-diyl)bis(1-methylquinolinium)diiodide; 2,2'-(decane-1,10-diyl)bis(isoquinolinium)diiodide; 1,1'-(decane-1,10-diyl)bis(4-bromoisoquinolinium)diiodide; 1,1'-(decane-1,10-diyl)bis(1H-benzimidazole); 1,1'-(decane-1,10-diyl)bis(3-methylbenzimidazolium)diiodide hemihydrate; 1,1'-(decane-1,10-diyl)bis(2-methylbenzimidazole); 1,1'-(decane-1,10-diyl)bis(2,3-dimethylbenzimidazolium)diiodide; 1,10-bis[N-(acridin-9-yl)amino]decane dihydrochloride dihydrate; 1,1'-(1,10-Decanediyl)bis[4-amino-2-methyl quinolinium]diiodide; 1,1'-(1,10-Decanediyl)bis[4-aminoquinolinium]diiodide; 1,1'-(1,10-Decanediyl)bis[4-N,N,dimethylaminoquinolinium]diiodide; 1,1'-(1,10-Decanediyl)bis[2-methylquinolinium]diiodide; 1,1'-(1,10-Decanediyl)bis[quinolinium]diiodide [0195] 1,6-Bis[N-(1-methylquinolinium-2-methyl)amino]hexane diiodide; 1,1'-(1,10-Decanediyl)bis[1-amino isoquinolinium]diiodide; 1,1'-(1,10-Decanediyl)bis[2-methylbenzoxazolium]diiodide; 1,1'-(1,10-Decanediyl)bis[2-methylbenzothiazolium]diiodide; 1,1'-(1,10-Decanediyl)bis[2-amino-1-methylbenzimidazolium]diiodide; 1,1'-[(E)-5-Decene-1,10-diyl]bis[4-amino-2-methylquinolinium], diiodide; 1,1'-[(Z)-5-Decene-1,10-diyl]bis[4-amino-2-methylquinolinium], diiodide; 1,1'-(1,12-Dodecanediyl)bis[4-amino-2-methylquinolinium], diiodide; 1,1'-(1,14-Tetradecanediyl)bis[4-amino-2-methylquinolinium], diiodide; 1,1'-(1,16-Hexadecanediyl)bis[4-amino-2-methylquinolinium], diiodide; N-Decyl-4-aminoquinaldinium Iodide; 1,1'-[Biphenyl-3,3'-diylbis(methylene)]-bis(4-aminoquinolinium); Dibromide Hydrate; 1,1'-[Biphenyl-4,4'-diylbis(methylene)]bis(4-aminoquinolinium)Ditrifluoro- -acetate; 1,1'-(Phenanthrene-3,6-diylbis(methylene)]bis(4-aminoquinolinium- )Dibromide Hydrate Ethanoate; 1,1'-[Fluorene-2,7-diylbis(methylene)]-bis(4-aminoquinolinium)Ditrifluoro- acetate; 1,1'-[Methylenebis(benzene-1,4-diylmethylene)]bis(4-aminoquinolin- ium)Dibromide Hydrate; 1,1'-[Ethylenebis-(benzene-1,4-diylmethylene)]bis(4-aminoquinolinium)Dibr- omide Hydrate; (Z)-1,1'-[Stilbene-4,4'-diylbis(methylene)]-bis(4-aminoquinolinium)Dibrom- ide Sesquihydrate; (E)-1,1'-[Stilbene-4,4'-diylbis(methylene)]bis-(4-aminoquinolinium)Dibrom- ide Dihydrate; 1,1'-[Ethyne-1,2-diylbis(benzene-1,4-diylmethylene)]bis(4-aminoquinoliniu- m)Dibromide Sesquihydrate; 1,1'-[Propane-1,3-diylbis(benzene-1,4-diylmethylene)]bis(4-aminoquinolini- um)Dibromide Hemihydrate Ethanoate; 1,1'-[Pyridine-2,6-diylbis(benzene-1,4-diylmethylene)]-bis(4-aminoquinoli- nium)Dibromide Hydrate; 1,1'-[Butane-1,4-diylbis(benzene-1,4-diylmethylene)]bis-(4-aminoquinolini- um)Dibromide Hydrate; 1,1'-[1,1:4',1''-Terphenyl-4,4''-diylbis(methylene)]bis(4-aminoquinoliniu- m)Dibromide Trihydrate; 1,1'-[Naphthalene-2,6-diyl(bis(methylene)]bis(4-aminoquinolinium)Dibromid- e Hydrate; 1,1'-[Benzene-1,4-diylbis(methylene)]-bis(4-aminoquinolinium)Di- bromide Dihydrate; 1,1'-[Benzene-1,3-diylbis(methylene)]bis(4-aminoquinolinium)Dibromide Hemihydrate; 1,1'-(Propane-1,3-diyl)bis(4-aminoquinolinium)diiodide; 1,1'-(Butane-1,4-diyl)bis(4-aminoquinolinium)diiodide; 1,1'-(Pentane-1,5-diyl)bis(4-aminoquinolinium)diiodide

[0036] 1,1'-(Hexane-1,6-diyl)bis(4-aminoquinolinium)diiodide; 1,1'-(Octane-1,8-diyl)bis(4-aminoquinolinium)diiodide; 1,1'-(Dodecane-1,12-diyl)bis(4-aminoquinolinium)dibromide hemihydrate; 1,10-Bis[N-(2-methylquinolin-4-yl)amino]decane; 1,12-Bis[N-(2-methylquinolin-4-yl)amino]dodecane; 1,10-Bis[(2-methylquinolin-4-yl)amino]decane; 1,12-Bis[(2-methylquinolin-4-yl)amino]dodecane; 1,10-Bis(N-quinolin-4-ylamino)decane; 4,4'-[Decane-1,10-diylbis(oxy)]bis[quinoline]; 4,4'-[Decane-1,10-diylbis(thio)]bis[quinoline]; 4,4'-Dodecane-1,12-diylbis[quinoline]; 1,8-Bis(N-quinolin-4-yldiamino)octane; 1,8-Bis[N-(1-methylquinolinium-4-yl)amino]octane Diiodide Hydrate; 1,10-Bis[N-(1-methylquinolinium-4-yl)amino]decane Diiodide; 4,4'-[Decane-1,10-diylbis(oxy)]bis[1-methylquinolinium]Diiodide; 4,4'-[Decane-1,10-diylbis(thio)]bis[1-methylquinolinium]Diiodide Hydrate; 1,1-Dimethyl-4,4'-dodecane-1,12-diylbis[quinolinium]Diiodide; 4,4'-Decane-1,10-diylbis[quinoline]; 1,1-Dimethyl-4,4'-decane-1,10-diylbis[quinolinium]Diiodide; 1,10-Bis[N-(1-benzylquinolinium-4-yl)amino]decane Dibromide; 1,10-Bis[N-(1-benzyl-2-methylquinolinium-4-yl)amino]-decane Bis(trifluoroacetate); 1,12-Bis[N-(1-benzyl-2-methylquinolinium-4-yl)amino]-dodecane Bis(trifluoroacetate); 1-[N-(1-Benzyl-2-methylquinolinium-4-yl)amino]-10-[N'-(2-methylquinoliniu- -m-4-yl)amino]decane Bis(trifluoroacetate); 1-[N-(1-Benzyl-2-methylquinolinium-4-yl)amino]-12-(N'-(2-methylquinoliniu- m-4-yl)amino]dodecane Bis(trifluoroacetate)-3,5-Dimethoxybenzyl iodide; 1,10-Bis[N-[1-(3,5-dimethoxybenzyl)-2-methylquinolinium-4-yl]amino-]decan- e Bis(trifluoroacetate); 1-[N-[1-(3,5-Dimethoxybenzyl)-2-methylquinolinium-4-yl]amino]-10-[N'-(2-m- -ethylquinolinium-4-yl)amino]decane Bis(trifluoroacetate); 1,1'(3-Iodopropylidene)bis[benzene]; 1,10-Bis[N-[1-(3,3-diphenylprop-1-yl)-2-methylquinolinium-4-yl]amino]deca- ne Bis(trifluoroacetate); 4,7-Dichloro-1-methylquinolinium Iodide; 1,10-Bis[N-(7-chloro-1-methylquinolinium-4-yl)amino]-decane Diiodide Dihydrate.

[0037] In some embodiments, the compounds detailed herein include compounds comprising cetylpyridinium, structure as diagrammed below. Cetylpyridinium is a cationic quaternary ammonium compound as described in The Merck Index (The Merck Index, 11th Ed.; Merck & Co., Rahway, N.J. (1989); p. 311). Its chemical elements are expressed as C.sub.21H.sub.38CN.sup.+, with a molecular weight of 304.542 g/mol. Cetylpyridinium and its salts are commercially available, for example from Sigma Aldrich. Cetylpyridinium may be obtained by quaternization of pyridine with cetyl chloride. Methods of making suitable derivatives are described in WO 2016092499 A1, the contents of which are incorporated herein by reference in its entirety.

##STR00002##

[0038] In some embodiments, the compounds detailed herein include compounds comprising amiloride, structure as diagrammed below. Amiloride, also known as Midamor or 3,5-diamino-6-chloro-N-(diaminomethylidene)pyrazine-2-carboxamide, is a pyrazine compound. Its chemical elements are expressed as C.sub.6H.sub.8ClN.sub.7O, with a molecular weight of 229.628 g/mol. Amiloride is commercially available, for example from Chemieliva Pharmaceutical Co., Ltd. Methods of making amiloride and suitable derivatives are described Medicinal Chemistry Research February 2013, Volume 22, Issue 2, pp 1004-1010, Biochimica et Biophysica Acta (BBA)--Biomembranes Volume 1146, Issue 1, 23 Feb. 1993, Pages 59-64, U.S. Pat. No. 6,214,824 B1, the contents of which are incorporated herein by reference in their entirety. Suitable amiloride derivatives include but are not limited to, 5-(N-Ethyl-N-isopropyl)amiloride; 5-(N,N-Dimethyl)amiloride hydrochloride; 5-(N-Methyl-N-isobutyl)-amiloride; and 5-(N,N-Hexamethylene)amiloride.

##STR00003##

[0039] In some embodiments, the compounds detailed herein include compounds comprising acrinol, structure as diagrammed below. Acrinol, also known as ethacridine monolactate monohydrate, 6,9-Diamino-2-ethoxyacridine-DL-lactate monohydrate, and Rivanol, is an aromatic organic compound based on acridine. Its chemical elements are expressed as C.sub.18H.sub.21N.sub.3O.sub.4, with a molecular weight of 343.383 g/mol. Acrinol is commercially available, for example from Chemieliva Pharmaceutical Co., Ltd. Methods of making acrinol and suitable derivatives are described Chinese Journal of Organic Chemistry 38(3):594 (2018), the contents of which are incorporated herein by reference in their entirety. Suitable acrinol derivatives include but are not limited to, 6,9-Dichloro-2-methoxyacridine; 9-Amino-6-chloro-2-methoxyacridine; (4-(9-Acridinylamino)phenoxy)acetic acid; 2-[4-(Acridin-9-ylamino)phenoxy]pentanoic acid 2-[4-(acridin-9-ylamino)phenoxy]butanoic Acid; 2-[4-[(4-methylacridin-9-yl)amino]phenoxy]propanoic acid; 2-[4-(Acridin-9-ylamino)phenoxy]propanoic acid; 2-[4-[(4-methylacridin-9-yl)amino]phenoxy]butanoic acid; Ethacridine lactate monohydrate; (6,9-diamino-2-ethoxyacridin-1-yl) 2-hydroxypropanoate hydrate; 2-ethoxyacridine-1,3-diamine with 2-hydroxypropanoic acid; 7-ethoxyacridine-1,3-diamine with 2-hydroxypropanoic acid; 6-ethoxyacridine-3,9-diamine with 2-hydroxypropanoic acid; 7-ethoxyacridine-2,3-diamine with 2-hydroxypropanoic acid; and 1,3-Dihydroxy-9-acridinecarboxylic acid.

##STR00004##

[0040] In some embodiments, the compounds detailed herein include compounds comprising akrinol, structure as diagrammed below. Akrinol, also known as acrisorcin, is comprised of two active ingredients, 9-aminoacridine and 4-hexylresorcinol. Its chemical elements are expressed as C.sub.25H.sub.28N.sub.2O.sub.2, with a molecular weight of 388.511 g/mol. Akrinol is commercially available, for example from Chemieliva Pharmaceutical Co., Ltd. Suitable akrinol derivatives include but are not limited to, 2-(acridin-9-ylamino)benzene-1,3-diol.

##STR00005##

[0041] Compounds of this invention include those described generally above, and are further illustrated by the classes, subclasses, and species disclosed herein. As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. Additionally, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M. B. and March, J., John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0042] As used herein, the term "pharmaceutically acceptable salt" refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66, 1-19, incorporated herein by reference. Pharmaceutically acceptable salts of the compounds of this invention include those derived from suitable inorganic and organic acids and bases. Examples of pharmaceutically acceptable, nontoxic acid addition salts are salts of an amino group formed with inorganic acids such as hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methods used in the art such as ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate, phosphate, pivalate, propionate, stearate, succinate, sulfate, tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts, and the like.

[0043] Salts derived from appropriate bases include alkali metal, alkaline earth metal, ammonium and N+(C.sub.1-4alkyl).sub.4 salts. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like. Further pharmaceutically acceptable salts include, when appropriate, nontoxic ammonium, quaternary ammonium, and amine cations formed using counterions such as halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, loweralkyl sulfonate and aryl sulfonate.

[0044] Unless otherwise stated, structures depicted herein are also meant to include all isomeric (e.g., enantiomeric, diastereomeric, and geometric (or conformational)) forms of the structure; for example, the R and S configurations for each asymmetric center, Z and E double bond isomers, and Z and E conformational isomers. Therefore, single stereochemical isomers as well as enantiomeric, diastereomeric, and geometric (or conformational) mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, all tautomeric forms of the compounds of the invention are within the scope of the invention. Additionally, unless otherwise stated, structures depicted herein are also meant to include compounds that differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures including the replacement of hydrogen by deuterium or tritium, or the replacement of a carbon by a .sup.13C- or .sup.14C-enriched carbon are within the scope of this invention. Such compounds are useful, for example, as analytical tools, as probes in biological assays, or as therapeutic agents in accordance with the present invention.

(b) Components of the Composition

[0045] The present disclosure also provides pharmaceutical compositions. The pharmaceutical composition comprises a TIR NADase inhibitor, as an active ingredient, and at least one pharmaceutically acceptable excipient.

[0046] The pharmaceutically acceptable excipient may be a diluent, a binder, a filler, a buffering agent, a pH modifying agent, a disintegrant, a dispersant, a preservative, a lubricant, taste-masking agent, a flavoring agent, or a coloring agent. The amount and types of excipients utilized to form pharmaceutical compositions may be selected according to known principles of pharmaceutical science.

[0047] In each of the embodiments described herein, a composition of the invention may optionally comprise one or more additional drug or therapeutically active agent in addition to a TIR NADase inhibitor. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for treatment of the disease, disorder, or condition. In some embodiments, the additional drug or therapeutic agent maybe a small molecule, a polypeptide, a nucleic acid, a cell or parts thereof, and antibody or the like. In some embodiments, the administration of a TIR NADase inhibitor maybe administered before concurrently or after administration of an additional drug or therapeutic agent. In some embodiments, the additional drug or therapeutically active agent induces anti-inflammatory effects. In some embodiments, the secondary agent is selected from a corticosteroid, a non-steroidal anti-inflammatory drug (NSAID), an intravenous immunoglobulin, a kinase inhibitor, a fusion protein, a monoclonal antibody directed against one or more pro-inflammatory cytokines, and a combination thereof. In some embodiments, anti-inflammatory drugs include, but are not limited to, alclofenac, alclometasone dipropionate, algestone acetonide, alpha amylase, amcinafal, amcinafide, amfenac sodium, amiprilose hydrochloride, anakinra, anirolac, anitrazafen, apazone, balsalazide disodium, bendazac, benoxaprofen, benzydamine hydrochloride, bromelains, broperamole, budesonide, carprofen, cicloprofen, cintazone, cliprofen, clobetasol propionate, clobetasone butyrate, clopirac, cloticasone propionate, cormethasone acetate, cortodoxone, curcumin, deflazacort, desonide, desoximetasone, dexamethasone dipropionate, diclofenac potassium, diclofenac sodium, diflorasone diacetate, diflumidone sodium, diflunisal, difluprednate, diftalone, dimethyl sulfoxide, drocinonide, endrysone, enlimomab, enolicam sodium, epirizole, etodolac, etofenamate, felbinac, fenamole, fenbufen, fenclofenac, fenclorac, fendosal, fenpipalone, fentiazac, flazalone, fluazacort, flufenamic acid, flumizole, flunisolide acetate, flunixin, flunixin meglumine, fluocortin butyl, fluorometholone acetate, fluquazone, flurbiprofen, fluretofen, fluticasone propionate, furaprofen, furobufen, halcinonide, halobetasol propionate, halopredone acetate, ibufenac, ibuprofen, ibuprofen aluminum, ibuprofen piconol, ilonidap, indomethacin, indomethacin sodium, indoprofen, indoxole, intrazole, isoflupredone acetate, isoxepac, isoxicam, ketoprofen, lofemizole hydrochloride, lomoxicam, loteprednol etabonate, lysofylline, meclofenamate sodium, meclofenamic acid, meclorisone dibutyrate, mefenamic acid, mesalamine, meseclazone, methylprednisolone suleptanate, momiflumate, nabumetone, naproxen, naproxen sodium, naproxol, nimazone, olsalazine sodium, orgotein, orpanoxin, oxaprozin, oxyphenbutazone, paranyline hydrochloride, pentosan polysulfate sodium, phenbutazone sodium glycerate, piroxicam, piroxicam cinnamate, piroxicam olamine, pirprofen, prednazate, prifelone, prodolic acid, proquazone, proxazole, proxazole citrate, rimexolone, romazarit, salcolex, salnacedin, salsalate, sanguinarium chloride, seclazone, sermetacin, sudoxicam, sulindac, suprofen, talmetacin, talniflumate, talosalate, tebufelone, tenidap, tenidap sodium, tenoxicam, tesicam, tesimide, tetrydamine, tiopinac, tixocortol pivalate, tolmetin, tolmetin sodium, triclonide, triflumidate, zidometacin, zomepirac sodium, aspirin (acetylsalicylic acid), salicylic acid, corticosteroids, glucocorticoids, tacrolimus, pimecorlimus, mepolizumab, prodrugs thereof, and a combination thereof.

[0048] (i) Diluent

[0049] In one embodiment, the excipient may be a diluent. The diluent may be compressible (i.e., plastically deformable) or abrasively brittle. Non-limiting examples of suitable compressible diluents include microcrystalline cellulose (MCC), cellulose derivatives, cellulose powder, cellulose esters (i.e., acetate and butyrate mixed esters), ethyl cellulose, methyl cellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose, corn starch, phosphated corn starch, pregelatinized corn starch, rice starch, potato starch, tapioca starch, starch-lactose, starch-calcium carbonate, sodium starch glycolate, glucose, fructose, lactose, lactose monohydrate, sucrose, xylose, lactitol, mannitol, malitol, sorbitol, xylitol, maltodextrin, and trehalose. Non-limiting examples of suitable abrasively brittle diluents include dibasic calcium phosphate (anhydrous or dihydrate), calcium phosphate tribasic, calcium carbonate, and magnesium carbonate.

[0050] (ii) Binder

[0051] In another embodiment, the excipient may be a binder. Suitable binders include, but are not limited to, starches, pregelatinized starches, gelatin, polyvinylpyrrolidone, cellulose, methylcellulose, sodium carboxymethylcellulose, ethylcellulose, polyacrylamides, polyvinyloxoazolidone, polyvinylalcohols, C.sub.12-C.sub.18 fatty acid alcohol, polyethylene glycol, polyols, saccharides, oligosaccharides, polypeptides, oligopeptides, and combinations thereof.

[0052] (iii) Filler

[0053] In another embodiment, the excipient may be a filler. Suitable fillers include, but are not limited to, carbohydrates, inorganic compounds, and polyvinylpyrrolidone. By way of non-limiting example, the filler may be calcium sulfate, both di- and tri-basic, starch, calcium carbonate, magnesium carbonate, microcrystalline cellulose, dibasic calcium phosphate, magnesium carbonate, magnesium oxide, calcium silicate, talc, modified starches, lactose, sucrose, mannitol, or sorbitol.

[0054] (iv) Buffering Agent

[0055] In still another embodiment, the excipient may be a buffering agent. Representative examples of suitable buffering agents include, but are not limited to, phosphates, carbonates, citrates, tris buffers, and buffered saline salts (e.g., Tris buffered saline or phosphate buffered saline).

[0056] (v) pH Modifier

[0057] In various embodiments, the excipient may be a pH modifier. By way of non-limiting example, the pH modifying agent may be sodium carbonate, sodium bicarbonate, sodium citrate, citric acid, or phosphoric acid.

[0058] (vi) Disintegrant

[0059] In a further embodiment, the excipient may be a disintegrant. The disintegrant may be non-effervescent or effervescent. Suitable examples of non-effervescent disintegrants include, but are not limited to, starches such as corn starch, potato starch, pregelatinized and modified starches thereof, sweeteners, clays, such as bentonite, micro-crystalline cellulose, alginates, sodium starch glycolate, gums such as agar, guar, locust bean, karaya, pecitin, and tragacanth. Non-limiting examples of suitable effervescent disintegrants include sodium bicarbonate in combination with citric acid and sodium bicarbonate in combination with tartaric acid.

[0060] (vii) Dispersant

[0061] In yet another embodiment, the excipient may be a dispersant or dispersing enhancing agent. Suitable dispersants may include, but are not limited to, starch, alginic acid, polyvinylpyrrolidones, guar gum, kaolin, bentonite, purified wood cellulose, sodium starch glycolate, isoamorphous silicate, and microcrystalline cellulose.

[0062] (viii) Excipient

[0063] In another alternate embodiment, the excipient may be a preservative. Non-limiting examples of suitable preservatives include antioxidants, such as BHA, BHT, vitamin A, vitamin C, vitamin E, or retinyl palmitate, citric acid, sodium citrate; chelators such as EDTA or EGTA; and antimicrobials, such as parabens, chlorobutanol, or phenol.

[0064] (ix) Lubricant

[0065] In a further embodiment, the excipient may be a lubricant. Non-limiting examples of suitable lubricants include minerals such as talc or silica; and fats such as vegetable stearin, magnesium stearate, or stearic acid.

[0066] (x) Taste-Masking Agent

[0067] In yet another embodiment, the excipient may be a taste-masking agent. Taste-masking materials include cellulose ethers; polyethylene glycols; polyvinyl alcohol; polyvinyl alcohol and polyethylene glycol copolymers; monoglycerides or triglycerides; acrylic polymers; mixtures of acrylic polymers with cellulose ethers; cellulose acetate phthalate; and combinations thereof.

[0068] (xi) Flavoring Agent

[0069] In an alternate embodiment, the excipient may be a flavoring agent. Flavoring agents may be chosen from synthetic flavor oils and flavoring aromatics and/or natural oils, extracts from plants, leaves, flowers, fruits, and combinations thereof.

[0070] (xii) Coloring Agent

[0071] In still a further embodiment, the excipient may be a coloring agent. Suitable color additives include, but are not limited to, food, drug and cosmetic colors (FD&C), drug and cosmetic colors (D&C), or external drug and cosmetic colors (Ext. D&C).

[0072] The weight fraction of the excipient or combination of excipients in the composition may be about 99% or less, about 97% or less, about 95% or less, about 90% or less, about 85% or less, about 80% or less, about 75% or less, about 70% or less, about 65% or less, about 60% or less, about 55% or less, about 50% or less, about 45% or less, about 40% or less, about 35% or less, about 30% or less, about 25% or less, about 20% or less, about 15% or less, about 10% or less, about 5% or less, about 2%, or about 1% or less of the total weight of the composition.

[0073] The agents and compositions described herein can be formulated by any conventional manner using one or more pharmaceutically acceptable carriers or excipients as described in, for example, Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005), incorporated herein by reference in its entirety. Such formulations will contain a therapeutically effective amount of a biologically active agent described herein, which can be in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the subject.

[0074] The term "formulation" refers to preparing a drug in a form suitable for administration to a subject, such as a human. Thus, a "formulation" can include pharmaceutically acceptable excipients, including diluents or carriers.

[0075] The term "pharmaceutically acceptable" as used herein can describe substances or components that do not cause unacceptable losses of pharmacological activity or unacceptable adverse side effects. Examples of pharmaceutically acceptable ingredients can be those having monographs in United States Pharmacopeia (USP 29) and National Formulary (NF 24), United States Pharmacopeial Convention, Inc, Rockville, Md., 2005 ("USP/NF"), or a more recent edition, and the components listed in the continuously updated Inactive Ingredient Search online database of the FDA. Other useful components that are not described in the USP/NF, etc. may also be used.

[0076] The term "pharmaceutically acceptable excipient," as used herein, can include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic, or absorption delaying agents. The use of such media and agents for pharmaceutical active substances is well known in the art (see generally Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 21st edition, ISBN: 0781746736 (2005)). Except insofar as any conventional media or agent is incompatible with an active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0077] A "stable" formulation or composition can refer to a composition having sufficient stability to allow storage at a convenient temperature, such as between about 0.degree. C. and about 60.degree. C., for a commercially reasonable period of time, such as at least about one day, at least about one week, at least about one month, at least about three months, at least about six months, at least about one year, or at least about two years.

[0078] The formulation should suit the mode of administration. The agents of use with the current disclosure can be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, pulmonary, oral, topical, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, ophthalmic, buccal, and rectal. The individual agents may also be administered in combination with one or more additional agents or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the agent(s) or attached to the agent(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces.

[0079] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the agent(s) and reduce dosage frequency. Controlled-release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the agent, and consequently affect the occurrence of side effects. Controlled-release preparations may be designed to initially release an amount of an agent(s) that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an agent in the body, the agent can be released from the dosage form at a rate that will replace the amount of agent being metabolized or excreted from the body. The controlled-release of an agent may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

(d) Administration

[0080] (i) Dosage Forms

[0081] The composition can be formulated into various dosage forms and administered by a number of different means that will deliver a therapeutically effective amount of the active ingredient. Such compositions can be administered orally (e.g. inhalation), parenterally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, or intrasternal injection, or infusion techniques. Formulation of drugs is discussed in, for example, Gennaro, A. R., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. (18th ed, 1995), and Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Dekker Inc., New York, N.Y. (1980). In a specific embodiment, a composition may be a food supplement or a composition may be a cosmetic.

[0082] Solid dosage forms for oral administration include capsules, tablets, caplets, pills, powders, pellets, and granules. In such solid dosage forms, the active ingredient is ordinarily combined with one or more pharmaceutically acceptable excipients, examples of which are detailed above. Oral preparations may also be administered as aqueous suspensions, elixirs, or syrups. For these, the active ingredient may be combined with various sweetening or flavoring agents, coloring agents, and, if so desired, emulsifying and/or suspending agents, as well as diluents such as water, ethanol, glycerin, and combinations thereof. For administration by inhalation, the compounds are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0083] For parenteral administration (including subcutaneous, intraocular, intradermal, intravenous, intramuscular, intra-articular and intraperitoneal), the preparation may be an aqueous or an oil-based solution. Aqueous solutions may include a sterile diluent such as water, saline solution, a pharmaceutically acceptable polyol such as glycerol, propylene glycol, or other synthetic solvents; an antibacterial and/or antifungal agent such as benzyl alcohol, methyl paraben, chlorobutanol, phenol, thimerosal, and the like; an antioxidant such as ascorbic acid or sodium bisulfite; a chelating agent such as etheylenediaminetetraacetic acid; a buffer such as acetate, citrate, or phosphate; and/or an agent for the adjustment of tonicity such as sodium chloride, dextrose, or a polyalcohol such as mannitol or sorbitol. The pH of the aqueous solution may be adjusted with acids or bases such as hydrochloric acid or sodium hydroxide. Oil-based solutions or suspensions may further comprise sesame, peanut, olive oil, or mineral oil. The compositions may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carried, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0084] For topical (e.g., transdermal or transmucosal) administration, penetrants appropriate to the barrier to be permeated are generally included in the preparation. Pharmaceutical compositions adapted for topical administration may be formulated as ointments, creams, suspensions, lotions, powders, solutions, pastes, gels, sprays, aerosols, or oils. In some embodiments, the pharmaceutical composition is applied as a topical ointment or cream. When formulated in an ointment, the active ingredient may be employed with either a paraffinic or a water-miscible ointment base. Alternatively, the active ingredient may be formulated in a cream with an oil-in-water cream base or a water-in-oil base. Pharmaceutical compositions adapted for topical administration to the eye include eye drops wherein the active ingredient is dissolved or suspended in a suitable carrier, especially an aqueous solvent. Pharmaceutical compositions adapted for topical administration in the mouth include lozenges, pastilles, and mouth washes. Transmucosal administration may be accomplished through the use of nasal sprays, aerosol sprays, tablets, or suppositories, and transdermal administration may be via ointments, salves, gels, patches, or creams as generally known in the art.

[0085] In certain embodiments, a composition comprising the TIR NADase inhibitor, is encapsulated in a suitable vehicle to either aid in the delivery of the compound to target cells, to increase the stability of the composition, or to minimize potential toxicity of the composition. As will be appreciated by a skilled artisan, a variety of vehicles are suitable for delivering a composition of the present invention. Non-limiting examples of suitable structured fluid delivery systems may include nanoparticles, liposomes, microemulsions, micelles, dendrimers, and other phospholipid-containing systems. Methods of incorporating compositions into delivery vehicles are known in the art.

[0086] In one alternative embodiment, a liposome delivery vehicle may be utilized. Liposomes, depending upon the embodiment, are suitable for delivery of the TIR NADase inhibitor, in view of their structural and chemical properties. Generally speaking, liposomes are spherical vesicles with a phospholipid bilayer membrane. The lipid bilayer of a liposome may fuse with other bilayers (e.g., the cell membrane), thus delivering the contents of the liposome to cells. In this manner, the TIR NADase inhibitor may be selectively delivered to a cell by encapsulation in a liposome that fuses with the targeted cell's membrane.

[0087] Liposomes may be comprised of a variety of different types of phosolipids having varying hydrocarbon chain lengths. Phospholipids generally comprise two fatty acids linked through glycerol phosphate to one of a variety of polar groups. Suitable phospholids include phosphatidic acid (PA), phosphatidylserine (PS), phosphatidylinositol (PI), phosphatidylglycerol (PG), diphosphatidylglycerol (DPG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). The fatty acid chains comprising the phospholipids may range from about 6 to about 26 carbon atoms in length, and the lipid chains may be saturated or unsaturated. Suitable fatty acid chains include (common name presented in parentheses) n-dodecanoate (laurate), n-tretradecanoate (myristate), n-hexadecanoate (palmitate), n-octadecanoate (stearate), n-eicosanoate (arachidate), n-docosanoate (behenate), n-tetracosanoate (lignocerate), cis-9-hexadecenoate (palmitoleate), cis-9-octadecanoate (oleate), cis,cis-9,12-octadecandienoate (linoleate), all cis-9, 12, 15-octadecatrienoate (linolenate), and all cis-5,8,11,14-eicosatetraenoate (arachidonate). The two fatty acid chains of a phospholipid may be identical or different. Acceptable phospholipids include dioleoyl PS, dioleoyl PC, distearoyl PS, distearoyl PC, dimyristoyl PS, dimyristoyl PC, dipalmitoyl PG, stearoyl, oleoyl PS, palmitoyl, linolenyl PS, and the like.

[0088] The phospholipids may come from any natural source, and, as such, may comprise a mixture of phospholipids. For example, egg yolk is rich in PC, PG, and PE, soy beans contains PC, PE, PI, and PA, and animal brain or spinal cord is enriched in PS. Phospholipids may come from synthetic sources too. Mixtures of phospholipids having a varied ratio of individual phospholipids may be used. Mixtures of different phospholipids may result in liposome compositions having advantageous activity or stability of activity properties. The above mentioned phospholipids may be mixed, in optimal ratios with cationic lipids, such as N-(1-(2,3-dioleolyoxy)propyl)-N,N,N-trimethyl ammonium chloride, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchloarate, 3,3'-deheptyloxacarbocyanine iodide, 1,1'-dedodecyl-3,3,3',3'-tetramethylindocarbocyanine perchloarate, 1,1'-dioleyl-3, 3, 3',3'-tetramethylindo carbocyanine methanesulfonate, N-4-(delinoleylaminostyryl)-N-methylpyridinium iodide, or 1,1,-dilinoleyl-3,3,3',3'-tetramethylindocarbocyanine perchloarate.

[0089] Liposomes may optionally comprise sphingolipids, in which spingosine is the structural counterpart of glycerol and one of the one fatty acids of a phosphoglyceride, or cholesterol, a major component of animal cell membranes. Liposomes may optionally contain pegylated lipids, which are lipids covalently linked to polymers of polyethylene glycol (PEG). PEGs may range in size from about 500 to about 10,000 daltons.

[0090] Liposomes may further comprise a suitable solvent. The solvent may be an organic solvent or an inorganic solvent. Suitable solvents include, but are not limited to, dimethylsulfoxide (DMSO), methylpyrrolidone, N-methylpyrrolidone, acetronitrile, alcohols, dimethylformamide, tetrahydrofuran, or combinations thereof.

[0091] Liposomes carrying a TIR NADase inhibitor, may be prepared by any known method of preparing liposomes for drug delivery, such as, for example, detailed in U.S. Pat. Nos. 4,241,046; 4,394,448; 4,529,561; 4,755,388; 4,828,837; 4,925,661; 4,954,345; 4,957,735; 5,043,164; 5,064,655; 5,077,211; and 5,264,618, the disclosures of which are hereby incorporated by reference in their entirety. For example, liposomes may be prepared by sonicating lipids in an aqueous solution, solvent injection, lipid hydration, reverse evaporation, or freeze drying by repeated freezing and thawing. In a preferred embodiment the liposomes are formed by sonication. The liposomes may be multilamellar, which have many layers like an onion, or unilamellar. The liposomes may be large or small. Continued high-shear sonication tends to form smaller unilamellar lipsomes.

[0092] As would be apparent to one of ordinary skill, all of the parameters that govern liposome formation may be varied. These parameters include, but are not limited to, temperature, pH, concentration of a TIR NADase inhibitor, concentration and composition of lipid, concentration of multivalent cations, rate of mixing, presence of and concentration of solvent.

[0093] In another embodiment, a composition of the invention may be delivered to a cell as a microemulsion. Microemulsions are generally clear, thermodynamically stable solutions comprising an aqueous solution, a surfactant, and "oil." The "oil" in this case, is the supercritical fluid phase. The surfactant rests at the oil-water interface. Any of a variety of surfactants are suitable for use in microemulsion formulations including those described herein or otherwise known in the art. The aqueous microdomains suitable for use in the invention generally will have characteristic structural dimensions from about 5 nm to about 100 nm. Aggregates of this size are poor scatterers of visible light and hence, these solutions are optically clear. As will be appreciated by a skilled artisan, microemulsions can and will have a multitude of different microscopic structures including sphere, rod, or disc shaped aggregates. In one embodiment, the structure may be micelles, which are the simplest microemulsion structures that are generally spherical or cylindrical objects. Micelles are like drops of oil in water, and reverse micelles are like drops of water in oil. In an alternative embodiment, the microemulsion structure is the lamellae. It comprises consecutive layers of water and oil separated by layers of surfactant. The "oil" of microemulsions optimally comprises phospholipids. Any of the phospholipids detailed above for liposomes are suitable for embodiments directed to microemulsions. The TIR NADase inhibitor may be encapsulated in a microemulsion by any method generally known in the art.

[0094] In yet another embodiment, a TIR NADase inhibitor, may be delivered in a dendritic macromolecule, or a dendrimer. Generally speaking, a dendrimer is a branched tree-like molecule, in which each branch is an interlinked chain of molecules that divides into two new branches (molecules) after a certain length. This branching continues until the branches (molecules) become so densely packed that the canopy forms a globe. Generally, the properties of dendrimers are determined by the functional groups at their surface. For example, hydrophilic end groups, such as carboxyl groups, would typically make a water-soluble dendrimer. Alternatively, phospholipids may be incorporated in the surface of a dendrimer to facilitate absorption across the skin. Any of the phospholipids detailed for use in liposome embodiments are suitable for use in dendrimer embodiments. Any method generally known in the art may be utilized to make dendrimers and to encapsulate compositions of the invention therein. For example, dendrimers may be produced by an iterative sequence of reaction steps, in which each additional iteration leads to a higher order dendrimer. Consequently, they have a regular, highly branched 3D structure, with nearly uniform size and shape. Furthermore, the final size of a dendrimer is typically controlled by the number of iterative steps used during synthesis. A variety of dendrimer sizes are suitable for use in the invention. Generally, the size of dendrimers may range from about 1 nm to about 100 nm.

[0095] Generally, a safe and effective amount of a TIR NADase inhibitor is, for example, that amount that would cause the desired therapeutic effect in a subject while minimizing undesired side effects. In various embodiments, an effective amount of a TIR NADase inhibitor described herein can substantially inhibit TIR NADase activity and treat associated diseases. In some embodiments, an effective amount is an amount capable of increasing the concentration of NAD+ in a biological sample compared to that of a control or decreasing the concentration of one or more of ADPR, cADPR or nicotinamide relative to a control and as such represent a therapeutic option for treatment of diseases and disorders associated with TIR NADase activity. In some embodiments, compositions provided herein contain and/or deliver an amount of a TIR NADase inhibitor that is effective to measurably inhibit such TIR NADase activity and/or treat, and/or decrease the infectivity, morbidity, and rate of mortality associated with an infection or infectious disease or disorder in or on a subject when administered to the subject in an appropriate dosing regimen.

[0096] When used in the treatments described herein, a therapeutically effective amount of a TIR NADase inhibitor can be employed in pure form or, where such forms exist, in pharmaceutically acceptable salt form and with or without a pharmaceutically acceptable excipient. For example, the compounds of the present disclosure can be administered, at a reasonable benefit/risk ratio applicable to any medical treatment, in a sufficient amount to modulate diseases and disorders, for example, microbial infections associated with TIR NADase activity.

[0097] The amount of a composition described herein that can be combined with a pharmaceutically acceptable carrier to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of agent contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses.

[0098] Toxicity and therapeutic efficacy of compositions described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50, where larger therapeutic indices are generally understood in the art to be optimal.

[0099] The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the subject; the time of administration; the route of administration; the rate of excretion of the composition employed; the duration of the treatment; drugs used in combination or coincidental with the specific compound employed; and like factors well known in the medical arts (see e.g., Koda-Kimble et al. (2004) Applied Therapeutics: The Clinical Use of Drugs, Lippincott Williams & Wilkins, ISBN 0781748453; Winter (2003) Basic Clinical Pharmacokinetics, 4th ed., Lippincott Williams & Wilkins, ISBN 0781741475; Shargel (2004) Applied Biopharmaceutics & Pharmacokinetics, McGraw-Hill/Appleton & Lange, ISBN 0071375503). For example, it is well within the skill of the art to start doses of the composition at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. If desired, the effective daily dose may be divided into multiple doses for purposes of administration. Consequently, single dose compositions may contain such amounts or submultiples thereof to make up the daily dose. It will be understood, however, that the total daily usage of the compounds and compositions of the present disclosure will be decided by an attending physician within the scope of sound medical judgment.

[0100] Again, each of the states, diseases, disorders, and conditions, described herein, as well as others, can benefit from compositions and methods described herein. Generally, treating a state, disease, disorder, or condition includes preventing or delaying the appearance of clinical symptoms in a mammal that may be afflicted with or predisposed to the state, disease, disorder, or condition but does not yet experience or display clinical or subclinical symptoms thereof. Treating can also include inhibiting the state, disease, disorder, or condition, e.g., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof. Furthermore, treating can include relieving the disease, e.g., causing regression of the state, disease, disorder, or condition or at least one of its clinical or subclinical symptoms. A benefit to a subject to be treated can be either statistically significant or at least perceptible to the subject or to a physician.

[0101] Administration of a TIR NADase inhibitor can occur as a single event or over a time course of treatment. For example, a TIR NADase inhibitor can be administered daily, weekly, bi-weekly, or monthly. For treatment of acute conditions, the time course of treatment will usually be at least several days. Certain conditions could extend treatment from several days to several weeks. For example, treatment could extend over one week, two weeks, or three weeks. For more chronic conditions, treatment could extend from several weeks to several months or even a year or more.

[0102] The present disclosure encompasses pharmaceutical compositions comprising a TIR NADase inhibitor as disclosed above, so as to facilitate administration and promote stability of the active agent. For example, a TIR NADase inhibitor of this disclosure may be admixed with at least one pharmaceutically acceptable carrier or excipient resulting in a pharmaceutical composition which is capably and effectively administered (given) to a living subject, such as to a suitable subject (i.e. "a subject in need of treatment" or "a subject in need thereof"). For the purposes of the aspects and embodiments of the invention, the subject may be a human or any other animal. In particular embodiments, the subject is selected from the group consisting of primate, equine, ovine, caprine, leporine, avian, feline, rodent, or canine. Methods of preparing and administering a TIR NADase inhibitor disclosed herein to a subject in need thereof are well known to or are readily determined by those skilled in the art. The route of administration of a TIR NADase inhibitor can be, for example, peripheral, oral, parenteral, by inhalation or topical.

[0103] In some embodiments, the compounds as presented herein are administered as part of a pharmaceutically acceptable composition. In some embodiments, the compounds as presented herein are administered orally. In some embodiments, the compounds as presented herein are administered in a range of 0.01-100 mg/kg body weight of the patient.

(II) Methods

[0104] The present disclosure encompasses a method of measurably inhibiting TIR NADase activity of a TIR domain containing polypeptide. The present disclosure provides methods of inhibiting TIR NADase activity use in vitro, in vivo, in situ or ex vivo. Generally, the method comprises administration of an effective amount of a TIR NADase inhibitor, so as to down-regulate the NADase activity of a TIR domain containing polypeptide. In some embodiments, the TIR NADase inhibitor is administered to a biological sample. The term "biological sample", as used herein, includes, without limitation, tissues, cells, cell cultures or extracts thereof; biopsied material obtained from a subject or extracts thereof; and blood, saliva, urine, feces, semen, tears, or other body fluids or extracts thereof. Inhibition of enzymes in a biological sample is useful for a variety of purposes that are known to one of skill in the art. Examples of such purposes include, but are not limited to biological assays, gene expression studies, and biological target identification. In some embodiments, the TIR NADase inhibitor is administered to a subject in need thereof. Suitable compositions comprising a TIR NADase inhibitor are disclosed herein, for instance those described in Section I.

[0105] As used herein, the term "measurably inhibit" refers to a measurable change in TIR NADase activity between a sample comprising a TIR domain containing polypeptide contacted with a TIR NADase inhibitor, and an equivalent sample contacted with a control composition (e.g. a carrier) or in the absence of a TIR NADase inhibitor. In some embodiments, a TIR NADase inhibitor "measurably inhibits" TIR NADase activity by at least 1-fold, 2-fold, 3-fold, 4-fold, or greater as compared to the control. In some embodiments, a TIR NADase inhibitor "measurably inhibits" TIR NADase activity by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 50%, 75% or more as compared to control.

[0106] As used herein, the term "TIR NADase" refers to a polypeptide comprising a TIR domain and has NADase enzymatic activity. Unless otherwise indicated, "polypeptide" shall include a protein, protein domain, or peptide, and any fragment thereof. The ToII/II-1 Receptor (TIR) domain was first characterized due to homology between the intracellular regions of the mammalian IL-1 receptor (IL-1R) and the Drosophila protein Toll. Subsequently, six Toll-Like Receptors (TLRs) have been identified in Drosophila and more than twenty TLRs and IL-1Rs have been recognized in humans. Several adaptor proteins containing TIR domains have also been described. In general, the TIR domain consists of three `boxes` of conserved residues set in a core sequence ranging from about 135 to about 160 amino acids. Intervening residues may vary, as sequence conservation between domains is only 20-30%. Two interfaces are responsible for mediating TIR domain interactions, which include receptor/adaptor oligomerization and association between receptors and adaptors. TLR and IL-1R signaling pathways are key mediators of the innate immune response to bacteria and fungi in both Drosophila and mammals. TIR domain interactions between receptors and adaptors play a key role in activating conserved cellular signal transduction pathways in response to bacterial LPS, microbial and viral pathogens, cytokines and growth factors. Homotypic and heterotypic interactions are thought to mediate receptor signaling. Activation involves liberation of NF-.kappa.B resulting in lymphocyte activation, immunoglobulin isotype switching and expression of cytokines and their receptors.

[0107] The crystal structure of TIR domains from human TLR1 and TLR2 reveal a central five-stranded parallel .beta.-sheet surrounded by a total of five helices on both sides. Conserved residues are located in the hydrophobic core and large insertions or deletions can be present in several loop regions of different TIR domains. The BB loop, containing three highly conserved residues, protrudes from a large conserved surface patch and is thought to mediate heterodimeric interactions with TIR domain-containing adaptor proteins.

[0108] TIR domain containing polypeptides can be identified using techniques standard in the art, for example, those described in the Examples below. Unless otherwise specified a "TIR-domain containing polypeptide" or "TIR NADase" refers to a full length wild-type polypeptide which optionally includes a tag. In some embodiments, a mutant or fragment of a TIR NADase is a TIR-domain containing mutant or fragment having NADase activity. In some embodiments, the full length, mutant or fragment of a TIR NADase can consist of or consist essentially of a full length, mutant or fragment TIR NADase from any species encoding a TIR domain containing polypeptide. In some embodiments, the TIR NADase is a full length, mutant or fragment is from a prokaryotic organism. In some embodiments, a full length, mutant or fragment is a viral TIR NADase. In some embodiments, the full length, mutant or fragment of a TIR NADase is from a mammal. In some embodiments, the full length, mutant or fragment mammalian TIR NADase is from a human, with the provision that the TIR NADase is not SARM1. In some embodiments a full length, mutant or fragment is an insect TIR NADase. In some embodiments, the TIR NADase may comprise the amino acid sequence as set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, or 44. In another aspect, the TIR NADase may comprise an amino acid sequence with 80% identity, 81% identity, 82% identity, 83% identity, 84% identity, 85% identity, 86% identity, 87% identity, 88% identity, 89% identity, 90% identity, 91 identity, 92% identity, 93% identity, 94% identity, 95% identity, 96% identity, 97% identity, 98% identity, 99% identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, or 43.

[0109] According to an aspect of the present invention, the TIR NADase may comprise the amino acid sequence set forth in SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43 or their functional homologues, including derivatives and fragments. Homologues of a TIR NADase can be obtained, for example, by mutation of a TIR NADase-encoding nucleotide sequence, respectively, and expression from the mutated sequence and/or by use or derivation from related gene sequences. Alternatively, homologues can be obtained, for example by identifying gene sequences homologous to TIR domain containing polypeptides by screening databases containing either protein sequences or nucleotide sequences encoding proteins, for example using homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. A local sequence alignment program, e.g., BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal query is used in the present invention to filter hit sequences with significant E-values for ortholog identification. The reciprocal query entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal query's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. An acceptable level of homology over the whole sequence is at least about 20%, for example 30% homology, 40% homology, 50% homology, 60% homology, 70% homology, 80% homology, 90% homology or greater. The homology of a functional fragment of a TIR NADase may be at least 10% homology. A "homologous" amino acid sequence, as used herein, refers to an amino acid sequence that differs from a reference amino acid sequence, only by one or more (e.g., 1, 2, 3, 4 or 5) conservative amino acid substitutions, or by one or more (e.g., 1, 2, 3, 4 or 5) non-conservative amino acid substitutions, deletions, or additions located at positions at which they do not adversely affect the activity of the polypeptide. For example, the NADase activity of a TIR domain containing polypeptide can be measured. In some embodiments, such a sequence is at least 75%, 80%, 85%, 90% or 95% or greater identical to a reference amino acid sequence.

[0110] Homologous amino acid sequences include peptide sequences that are identical or substantially identical to a reference amino acid sequence. By "amino acid sequence substantially identical" is meant a sequence that is at least 90%, preferably 95%, more preferably 97%, and most preferably 99% identical to an amino acid sequence of reference and that preferably differs from the sequence of reference, if at all, by a majority of conservative amino acid substitutions.

[0111] Conservative amino acid substitutions typically include substitutions among amino acids of the same class. These classes include, for example, (a) amino acids having uncharged polar side chains, such as asparagine, glutamine, serine, threonine, and tyrosine; (b) amino acids having basic side chains, such as lysine, arginine, and histidine; (c) amino acids having acidic side chains, such as aspartic acid and glutamic acid; and (d) amino acids having nonpolar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and cysteine.

[0112] Another aspect of the present disclosure provides a method of inhibiting microbial NADase activity in a biological sample or in a subject in need thereof. In general, the method comprises administering a therapeutically effective amount of a composition comprising an inhibitor of TIR NADase activity for treatment of infections or infectious disorders. The inhibitors of TIR NADase activity as described herein are useful to decreasing the infectivity, morbidity, and rate of mortality associated with a variety of pathogenic organisms. According to one embodiment, the invention relates to a method of inhibiting microbial TIR NADase activity in a biological sample comprising the step of contacting said biological sample with a TIR NADase inhibitor as described herein. As used herein, the terms "treatment," "treat," and "treating" refer to reversing, alleviating, delaying the onset of, or inhibiting the progress of a disease or disorder, or one or more symptoms thereof, as described herein. In some embodiments, treatment may be administered after one or more symptoms have developed. In other embodiments, treatment may be administered in the absence of symptoms. For example, treatment may be administered to a susceptible individual prior to the onset of symptoms (e.g., in light of a history of symptoms and/or in light of genetic or other susceptibility factors). Treatment may also be continued after symptoms have resolved, for example to prevent or delay their recurrence.

[0113] In an aspect, the TIR NADase inhibitors are administered to treat a microbial infection. As used herein, "infection" and, equivalently, "infectious disease" shall refer to a disease arising from the presence of a foreign microorganism in and/or on the body of a subject. A foreign microorganism may be a virus, a bacterium, a fungus, or a parasite. Examples of infectious viruses include: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III, LAV or HTLV-III/LAV, or HIV-III; and other isolates, such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus; enteroviruses, human coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses); Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses); Birnaviridae; Hepadnaviridae (Hepatitis B virus); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMV), herpes viruses); Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swine fever virus); and unclassified viruses (e.g., the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class 1=internally transmitted; class 2=parenterally transmitted (i.e., Hepatitis C); Norwalk and related viruses, and astroviruses).

[0114] Examples of infectious bacteria include: Actinomyces israelii, Bacillus anthracis, Bacteroides spp., Borrelia burgdorferi, Chlamydia trachomatis, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Corynebacterium spp., Enterobacter aerogenes, Enterococcus sp., Erysipelothrix rhusiopathiae, Escherichia coli, Fusobacterium nucleatum, Haemophilus influenzae, Helicobacter pyloris, Klebsiella pneumoniae, Legionella pneumophilia, Leptospira, Listeria monocytogenes, Mycobacteria spp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii, M. gordonae), Neisseria gonorrhoeae, Neisseria meningitidis, Pasteurella multocida, pathogenic Campylobacter sp., Staphylococcus aureus, Streptobacillus moniliformis, Streptococcus (anaerobic spp.), Streptococcus (viridans group), Streptococcus agalactiae (Group B Streptococcus), Streptococcus bovis, Streptococcus faecalis, Streptococcus pneumoniae, Streptococcus pyogenes (Group A Streptococcus), Treponema paffidium, and Treponema pertenue.

[0115] Examples of infectious fungi include: Candida albicans, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, and Blastomyces dermatitidis.

[0116] Other infectious organisms (i.e., protists) include Plasmodium spp. such as Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax, and Toxoplasma gondii. Blood-borne and/or tissue parasites include Plasmodium spp., Babesia microti, Babesia divergens, Leishmania tropica, Leishmania spp., Leishmania braziliensis, Leishmania donovani, Trypanosoma gambiense and Trypanosoma rhodesiense (African sleeping sickness), Trypanosoma cruzi (Chagas' disease), and Toxoplasma gondii.

[0117] The foregoing list of viruses, bacteria, fungi and other infectious microorganisms is understood to be representative and not limiting. Other medically relevant microorganisms have been described extensively in the literature, e.g., see C. G. A Thomas, Medical Microbiology, Bailliere Tindall, Great Britain 1983, the entire contents of which is hereby incorporated by reference.

[0118] In another aspect, the present teachings include a host cell, that harbors a nucleic acid or product thereof that encodes an exogenous full length, mutant or fragment TIR NADase. As used herein, when referring to nucleic acids or a product thereof, the term "exogenous" refers to any nucleic acid sequence or polypeptide that is not naturally expressed in the particular cell where expression is desired. As used herein, the term "host cell" refers to any eukaryotic or prokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells, mammalian cells, avian cells, amphibian cells, fish cells and insect cells). Methods of transforming cells are well known in the art.

[0119] The nucleic acid can be DNA or RNA. In one embodiment the DNA can be present in a vector. The nucleic acid sequences which encode the reporter molecule of the invention can be operatively linked to expression control sequences. "Operatively linked" refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. An expression control sequence operatively linked to a coding sequence is achieved under conditions compatible with the expression control sequences. As used herein, the phrase "expression control sequences" refers to nucleic acid sequences that regulate the expression of a nucleic acid sequence to which it is operatively linked. Expression control sequences are operatively linked to a nucleic acid sequence when the expression control sequences control and regulate the transcription and, as appropriate, translation of the nucleic acid sequence. Thus, expression control sequences can include appropriate promoters, enhancers, transcription terminators, a start codon (i.e., ATG) in front of a protein-encoding gene, splicing signals for introns, and maintenance of the correct reading frame of that gene to permit proper translation of the mRNA, and stop codons. The term "control sequences" is intended to include, at a minimum, components whose presence can influence expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences. Expression control sequences can include a promoter.

[0120] By "promoter" is meant minimal sequence sufficient to direct transcription. Also included in the invention are those promoter elements which are sufficient to render promoter-dependent gene expression controllable for cell-type specific expression, tissue-specific expression, or expression inducible by external signals or agents; such elements may be located in the 5' or 3' regions of the gene. Both constitutive and inducible promoters, are included in the invention (see e.g., Bitter et al., Methods in Enzymology 153:516-544, 1987). For example, when cloning in bacterial systems, inducible promoters such as pL of bacteriophage .gamma., plac, ptrp, ptac (ptrp-lac hybrid promoter) and the like may be used. When cloning in mammalian cell systems, promoters derived from the genome of mammalian cells (e.g., metallothionein promoter) or from mammalian viruses (e.g., the retrovirus long terminal repeat; the adenovirus late promoter; the vaccinia virus 7.5K promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide for transcription of the nucleic acid sequences of the invention.

[0121] In some embodiments, the nucleic acid sequences encoding a TIR domain containing polypeptide of the invention may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a plasmid, virus or other vehicle known in the art that has been manipulated by insertion or incorporation of the nucleic acid sequences encoding the fusion peptides of the invention. The expression vector typically contains an origin of replication, a promoter, as well as specific genes which allow phenotypic selection of the transformed cells. Vectors suitable for use in the present invention include, but are not limited to the T7-based expression vector for expression in bacteria (Rosenberg, et al., Gene 56:125, 1987), the pMSXND expression vector for expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263:3521, 1988), baculovirus-derived vectors for expression in insect cells, cauliflower mosaic virus, CaMV; or tobacco mosaic virus, TMV. The nucleic acid sequences encoding a TIR domain containing polypeptide as described herein can also include a localization sequence to direct the indicator to particular cellular sites by fusion to appropriate organellar targeting signals or localized host proteins. A polynucleotide encoding a localization sequence, or signal sequence, can be used as a repressor and thus can be ligated or fused at the 5' terminus of a polynucleotide encoding the reporter polypeptide such that the signal peptide is located at the amino terminal end of the resulting fusion polynucleotide/polypeptide. The construction of expression vectors and the expression of genes in transfected cells involve the use of molecular cloning techniques also well known in the art. Sambrook et al., Molecular Cloning--A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989, and Current Protocols in Molecular Biology, M. Ausubel et al., eds., (Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., most recent Supplement). These methods include in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination/genetic recombination. (See, for example, the techniques described in Maniatis, et al., Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory, N.Y., 1989).

[0122] Depending on the vector utilized, any of a number of suitable transcription and translation elements, including constitutive and inducible promoters, transcription enhancer elements, transcription terminators, etc. may be used in the expression vector (see, e.g., Bitter, et al., Methods in Enzymology 153:516-544, 1987). These elements are well known to one of skill in the art.

[0123] By "transformation" is meant a permanent genetic change induce in a cell following incorporation of new DNA (i.e., DNA exogenous to the cell). Where the cell is a mammalian cell, the permanent genetic change is generally achieved by introduction of the DNA into the genome of the cell.

[0124] Transformation of a host cell with recombinant DNA may be carried out by conventional techniques well known to those skilled in the art. Where the host is prokaryotic, such as E. coli, competent cells which are capable of DNA uptake can be prepared from cells harvested after exponential growth phase and subsequently treated by the CaCl.sub.2) method by procedures well known in the art. Alternatively, MgCl2 or RbCl can be used. Transformation can also be performed after forming a protoplast of the host cell or by electroporation.

[0125] When the host is a eukaryote, such methods of transfection of DNA as calcium phosphate co-precipitates, conventional mechanical procedures such as microinjection, electroporation, insertion of a plasmid encased in liposomes, or virus vectors may be used. Eukaryotic cells can also be co-transfected with DNA sequences encoding the reporter molecules of the invention, and a second foreign DNA molecule encoding a selectable phenotype, such as the herpes simplex thymidine kinase gene. Another method is to use a eukaryotic viral vector, such as simian virus 40 (SV40) or bovine papilloma virus, to transiently infect or transform eukaryotic cells and express the protein. (Eukaryotic Viral Vectors, Cold Spring Harbor Laboratory, Gluzman ed., 1982). Preferably, a eukaryotic host is utilized as the host cell as described herein. The eukaryotic cell may be a yeast cell (e.g., Saccharomyces cerevisiae), or may be a mammalian cell. In one embodiment, the mammalian cell is a human cell.

[0126] Eukaryotic systems, and preferably mammalian expression systems, allow for proper post-translational modifications of expressed mammalian proteins to occur. Eukaryotic cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, phosphorylation, and, advantageously secretion of the gene product should be used as host cells for the expression of fluorescent indicator. Such host cell lines may include but are not limited to NRK1-HEK293T, CHO, VERO, BHK, HeLa, COS, MDCK, Jurkat, HEK-293, and W138. In one embodiment, the eukaryotic cell is a human cell.

[0127] Mammalian cell systems which utilize recombinant viruses or viral elements to direct expression may be engineered. For example, when using adenovirus expression vectors, the nucleic acid sequences encoding a TIR domain containing polypeptide of the invention may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence. This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non-essential region of the viral genome (e.g., region E1 or E3) will result in a recombinant virus that is viable and capable of expressing the fluorescent indicator in infected hosts (e.g., see Logan & Shenk, Proc. Natl. Acad. Sci. USA, 81:3655-3659, 1984). Alternatively, the vaccinia virus 7.5K promoter may be used. (e.g., see, Mackett, et al., Proc. Natl. Acad. Sci. USA, 79:7415-7419, 1982; Mackett, et al., J. Virol. 49:857-864, 1984; Panicali, et al., Proc. Natl. Acad. Sci. USA 79:4927-4931, 1982). Also contemplated are vectors based on bovine papilloma virus which have the ability to replicate as extrachromosomal elements (Sarver, et al., Mol. Cell. Biol. 1:486, 1981). Shortly after entry of this DNA into mouse cells, the plasmid replicates to about 100 to 200 copies per cell. Transcription of the inserted cDNA does not require integration of the plasmid into the host's chromosome, thereby yielding a high level of expression. These vectors can be used for stable expression by including a selectable marker in the plasmid, such as the neo gene. Alternatively, the retroviral genome can be modified for use as a vector capable of introducing and directing the expression of the fluorescent indicator gene in host cells (Cone & Mulligan, Proc. Natl. Acad. Sci. USA, 81:6349-6353, 1984). High level expression may also be achieved using inducible promoters, including, but not limited to, the metallothionine IIA promoter and heat shock promoters.

[0128] For long-term, high-yield production of recombinant proteins, stable expression may be preferred. Rather than using expression vectors which contain viral origins of replication, host cells can be transformed with the cDNA encoding a TIR domain containing polypeptide of the invention controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker. The selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines. For example, following the introduction of foreign DNA, engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media. A number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler, et al., Cell, 11:223, 1977), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026, 1962), and adenine phosphoribosyltransferase (Lowy, et al., Cell, 22:817, 1980) genes can be employed in tk-, hgprt- or aprt- cells respectively. Also, antimetabolite resistance can be used as the basis of selection for dhfr, which confers resistance to methotrexate (Wigler, et al., Proc. Natl. Acad. Sci. USA, 77:3567, 1980; O'Hare, et al., Proc. Natl. Acad Sci. USA, 8:1527, 1981); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA, 78:2072, 1981; neo, which confers resistance to the aminoglycoside G-418 (Colberre-Garapin, et al, J. Mol. Biol 150:1, 1981); and hygro, which confers resistance to hygromycin (Santerre, et al., Gene 30: 147, 1984) genes. Recently, additional selectable genes have been described, namely trpB, which allows cells to utilize indole in place of tryptophan; hisD, which allows cells to utilize histinol in place of histidine (Hartman & Mulligan, Proc. Natl. Acad. Sci. USA 85:8047, 1988); and ODC (ornithine decarboxylase) which confers resistance to the omithine decarboxylase inhibitor, 2-(difluoromethyl)-DL-omithine, DFMO (McConlogue L., In: Current Communications in Molecular Biology, Cold Spring Harbor Laboratory, ed., 1987).

[0129] A TIR domain containing polypeptide as described herein can be produced by expression of nucleic acid encoding the TIR domain containing polypeptide in prokaryotes. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors encoding a TIR domain containing polypeptide. The constructs can also be expressed in E. coli in large scale. Purification from bacteria is simplified when the sequences include tags for one-step purification by nickel-chelate chromatography. The construct can also contain a tag to simplify isolation of the fluorescent indicator. For example, a polyhistidine tag of, e.g., six histidine residues, can be incorporated at the amino terminal end of the fluorescent protein. The polyhistidine tag allows convenient isolation of the protein in a single step by nickel-chelate chromatography. The TIR domain containing polypeptide of the invention can also be engineered to contain a cleavage site to aid in protein recovery.

[0130] Techniques for the isolation and purification of either microbially or eukaryotically expressed polypeptides of the invention may be by any conventional means such as, for example, preparative chromatographic separations and immunological separations such as those involving the use of monoclonal or polyclonal antibodies or antigen.

[0131] In yet another aspect, the present invention provides methods to determine if a TIR-domain containing polypeptide is a TIR NADase, Such methods comprise: a) expressing an exogenous TIR-domain containing polypeptide or TIR-domain containing fragment thereof in a host cell, and; b) quantifying one or more of NAD.sup.+, ADPR, cADPR or nicotinamide in said TIR-domain expressing host cell or lysate thereof. In some embodiments, provided methods can further comprise c) identifying the TIR-domain containing polypeptide as a TIR NADase when the amount of NAD.sup.+ is decreased in said host cell and/or when the amount of ADPR, cADPR, nicotinamide or combinations thereof are increased in said host cell. In alternative embodiments, provided methods can further comprise d) identifying the TIR-domain containing polypeptide as a TIR NADase when the amount of NAD.sup.+ is decreased and/or ADPR cADPR, nicotinamide or combinations thereof are increased compared to a control host cell which does not express the TIR-domain containing polypeptide. In some embodiments, the host cell lysate is a NRK1-HEK293T cell. In some embodiments, one or more of NAD.sup.+, ADPR, nicotinamide or any combination thereof is quantified by any available analytical method, such as, for example, performing an HPLC analysis, a chemiluminescence assay, a liquid chromatography-mass spectroscopy analysis or a combination thereof.

[0132] In some embodiments, the present invention provides methods to determine if a TIR-domain containing polypeptide is a TIR NADase, Such methods comprise: a) expressing an exogenous TIR-domain containing polypeptide or TIR-domain containing fragment thereof in a cell-free system, and; b) determining if the TIR-domain polypeptide cleaves NAD.sup.+ by measuring NAD.sup.+, nicotinamide, ADPR, and cADPR. In some embodiments, provided methods can further comprise d) identifying the TIR-domain containing polypeptide as a TIR NADase when the amount of NAD.sup.+ is decreased in said cell-free system and/or when the amount of ADPR, cADPR, nicotinamide or combinations thereof are increased in said cell-free system. In alternative embodiments, provided methods can further comprise d) identifying the TIR-domain containing polypeptide as a TIR NADase when the amount of NAD.sup.+ is decreased and/or ADPR cADPR, nicotinamide or combinations thereof are increased compared to a cell-free system which does not express the TIR-domain containing polypeptide. In some embodiments, one or more of NAD+, ADPR, nicotinamide or any combination thereof is quantified by any available analytical method, such as, for example, performing an HPLC analysis, a chemiluminescence assay, a liquid chromatography-mass spectroscopy analysis or a combination thereof.

[0133] In some embodiments, the present invention provides methods of identifying if a TIR-domain containing polypeptide is a TIR NADase, which comprises: a) providing a mixture comprising i) NAD.sup.+ and; ii) a solid support to which is bound a polypeptide consisting of a TIR-domain containing polypeptide or fragment thereof; b) quantifying the NAD.sup.+ in the mixture; and c) identifying the TIR-domain containing polypeptide as a TIR NADase if the concentration of NAD.sup.+ is less than that of a control. By way of non-limiting example, the solid support to which a TIR domain containing polypeptide is bound may be beads, plastic, nitrocellulose, indium, tin, cadmium, silica, poly(ethylene glycol)methacrylate graft polymer, or variations thereof.

[0134] In some embodiments, the present invention provides methods of identifying a TIR NADase inhibitor. Such methods comprise: a) providing a mixture comprising i) a full length, mutant or fragment of a TIR NADase, ii) NAD.sup.+, and iii) a candidate inhibitor, wherein the mutant or fragment optionally has constitutive NADase activity; b) incubating the mixture; and c) quantifying NAD.sup.+, ADPR, nicotinamide or any combination thereof in the mixture after the incubating. In some embodiments, provided methods can further comprise d) determining the molar ratio of NAD.sup.+/ADPR; and e) identifying a candidate inhibitor compound as an NADase inhibitor if the molar ratio of NAD.sup.+/ADPR is greater than that of a control mixture that does not contain the candidate inhibitor. In some embodiments, one or more of NAD.sup.+, ADPR, nicotinamide or any combination thereof is quantified by any available analytical method, such as, for example, performing an HPLC analysis, a chemiluminescence assay, a liquid chromatography-mass spectroscopy analysis or a combination thereof. In some embodiments, the mixture comprises a cell lysate comprising a full length, mutant or fragment of a TIR NADase. In some embodiments, the cell lysate is a lysate of mammalian cells comprising, consisting of, or consisting essentially of a full length, mutant or fragment of a TIR NADase polypeptide that has NADase activity. In some embodiments, the cell lysate is from a host cell comprising, consisting of, or consisting essentially of a full length, mutant or fragment of a TIR NADase that has NADase activity. In some embodiments, the cell lysate is a lysate of NRK1-HEK293T cells comprising, consisting of, or consisting essentially of a full length, mutant or fragment of a TIR NADase that has NADase activity. In some embodiments, the mixture can comprise a purified TIR NADase polypeptide. In some embodiments, the NRK1-HEK293T cells is treated with nicotinamide riboside (NR), which can be useful for maintaining high NAD.sup.+ levels and increasing cell viability in the presence of constitutively active TIR NADase molecules. In some embodiments, an inhibitor is identified as an NADase inhibitor if the molar ratio of NAD.sup.+ to ADPR is greater than 4:1. In some embodiments, the candidate inhibitor compound is identified as a NADase inhibitor if the molar ratio of NAD.sup.+ to ADPR is greater than 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0135] In some embodiments, a method of identifying a TIR NADase inhibitor comprises: a) providing a mixture comprising i) a full length mutant or fragment of a TIR NADase, ii) NAD.sup.+ and iii) a candidate inhibitor, wherein the mutant or fragment optionally has constitutive NADase activity; b) incubating the mixture; c) quantifying NAD.sup.+ in the mixture after the incubating; and d) identifying the candidate inhibitor compound as an NADase inhibitor if the amount of NAD.sup.+ is greater than that of a control mixture that does not contain the candidate inhibitor. In some embodiments, the quantifying NAD.sup.+ in the mixture comprises, consists of, or consists essentially of performing a chemiluminescence assay. In some embodiments, the quantifying NAD.sup.+ in the mixture comprises, consists of, or consists essentially of performing an HPLC analysis. In some embodiments, the mixture can comprise a purified TIR containing fragment. In some embodiments, the mixture comprises a cell lysate comprising the mutant or fragment of a TIR NADase. In some embodiments, the cell lysate is a lysate of NRK1-HEK293T cells comprising the mutant or fragment of a TIR NADase. In some embodiments, the NRK1-HEK293T cells comprising the mutant or fragment of a TIR NADase is treated with NR.

[0136] In some embodiments, a TIR-domain containing polypeptide or TIR NADase comprises, consists of, or consists essentially of a) a full length, mutant or fragment of a TIR-domain containing polypeptide or TIR NADase, wherein the mutant or fragment optionally has constitutive NADase activity; and b) at least one tag. In non-limiting examples the at least one tag is a StrepTag, a polyhistidine tag, an antibody epitope (e.g., derived from myc), and the like and a combination thereof. In some embodiments, the polypeptide further includes at least one tag, such as an N-terminal tag. In some embodiments, the N-terminal tag is a tandem tag. In some embodiments, the at least one tag is a C-terminal tag. In some embodiments, the C-terminal tag is a polyhistidine tag. In some embodiments, the bead is a histidine tag purification bead. In some embodiments, the at least one tag is at least two tags. In some embodiments, the at least two tags is an N-terminal tag and a C-terminal tag. In some embodiments, the N-terminal tag is a tandem StrepTag and the C-terminal tag is a polyhistidine tag. In some embodiments, the polypeptide is immobilized on a solid support. In some embodiments, vectors include a plasmid or virus comprising a sequence encoding a polypeptide described herein.

[0137] In some embodiments, the present invention provides methods of identifying a TIR NADase inhibitor, which comprises: a) providing a mixture comprising NAD.sup.+ and a solid support to which is bound a polypeptide consisting of a full length, mutant or fragment of a TIR NADase optionally having constitutive NADase activity; b) adding a candidate inhibitor to the mixture; c) incubating the mixture; d) quantifying the NAD.sup.+ in the mixture; and e) identifying the candidate inhibitor compound as a TIR NADase inhibitor if the concentration of NAD.sup.+ is greater than that of a control. In some embodiments, provided methods include stopping NADase activity (if any) in the mixture after the incubating. In some embodiments, the quantifying NAD.sup.+ comprises performing an HPLC-based analysis. In some embodiments, the quantifying NAD.sup.+ and ADPR comprises performing an LC/MS-based analysis. In some embodiments, a candidate inhibitor compound is identified as a TIR NADase inhibitor if the molar ratio of NAD to ADPR is greater than 4:1. In some embodiments, a candidate inhibitor compound is identified as a TIR NADase inhibitor if the molar ratio of NAD to ADPR is greater than 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0138] In some embodiments, the present invention provides a full length TIR NADase polypeptide mutant or fragment. In some embodiments, a TIR NADase polypeptide, or mutant or fragment thereof may be bound to a solid support such as a bead. In some embodiments, the TIR NADase polypeptide, or mutant or fragment thereof bound to a solid support comprises, consists of, or consists essentially of a TIR domain, or a TIR NADase polypeptide deleted for an autoinhibitory domain. In some embodiments, the TIR NADase polypeptide mutant or fragment further comprises, consists of, or consists essentially of a tag. In some embodiments, a TIR NADase polypeptide mutant or fragment having NADase activity comprises, consists of, or consists essentially of a TIR NADase mutant or fragment bound to a solid support via a protein tag.

[0139] In some embodiments, a method of identifying a TIR NADase inhibitor comprises: a) providing a mixture comprising at least one cultured cell; b) adding a candidate TIR NADase inhibitor to the mixture; c) adding a labeled NAM to the mixture; d) incubating the mixture; and e) quantifying the amount of labeled and unlabeled NAD.sup.+ in the mixture. In some embodiments, provided methods can further comprise f) calculating the net rate of NAD.sup.+ consumption, for example by calculating the % decrease of unlabeled over total NAD.sup.+ (e.g., light NAD over total (light plus heavy) NAD+) over time. In some embodiments, the calculation is expressed, for example, as %/hr. In some embodiments, the labeled NAM is deuterium labeled ("heavy") NAM. In some embodiments, the labeled NAM is d4-NAM. In some embodiments, the quantifying of labeled and unlabeled NAD.sup.+ is performed using analytical methods such as LC-MS/MS. In some embodiments, the at least one cultured cell is a mammalian cell.

[0140] In each of the above embodiments, the candidate TIR NADase inhibitor may be chosen from a library of compounds. Suitable compounds include small molecules, pharmaceutically active compounds (i.e., drugs), natural products, carbohydrates, lipid molecules, amino acid derivatives, peptides, peptide mimetics, nucleic acids, antisense oligonucleotides, microRNAs, and so forth. In exemplary embodiments, the compounds are from a library of small molecules. In general, a small molecule is defined as a molecule having a molecular weight of less than about 1000 daltons (Da). In other embodiments, the plurality of compounds may comprise larger molecules that are cell permeable.

[0141] Libraries of small molecules are available through repositories or commercial sources, and means for generating libraries of small molecules are well known in the art. In some embodiments, the compounds to be screened may comprise a tag. Suitable tags include biotin, fluorophores, dyes, fluorocarbon tags, click chemistry tags, affinity tags, and the like. The presence of the tag may permit isolation of a complex comprising the compound and a cellular target with which the compound interacts.

[0142] The compounds to be screened generally will be dissolved in a suitable solvent (such as, e.g., DMSO or ethanol) and contacted with a sample comprising a TIR domain containing polypeptide. The candidate compounds may be distributed to the wells of a multi-well system using multichannel pipette systems or robotic liquid handling systems. Initially, the samples comprising a TIR domain containing polypeptide will be contacted with a single concentration of each compound. Additionally, compounds that affect the NADase activity of the TIR domain containing polypeptide can be rescreened at several different concentrations to determine the half maximal effective concentration (EC50). For statistical purposes, contact with a compound of interest will be performed in at least duplicate or triplicate.

[0143] During each screening procedure, a small percentage of samples comprising the TIR domain containing polypeptide can serve as untreated controls. That is, the untreated samples will not be contacted with any compounds of interest but rather will be contacted only with the solvent used to dissolve the compounds of interest, as appropriate.

(III) Kits

[0144] Also provided are kits. Such kits can include an agent or composition described herein and, in certain embodiments, instructions for administration. Such kits can facilitate performance of the methods described herein. When supplied as a kit, the different components of the composition can be packaged in separate containers and admixed immediately before use. Components include, but are not limited to compositions and pharmaceutical formulations comprising a TIR NADase inhibitor, isolated nucleic acids encoding a TIR NADase or cells exogenously expressing a TIR NADase polypeptide, as described herein. Such packaging of the components separately can, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the composition. The pack may, for example, comprise metal or plastic foil such as a blister pack. Such packaging of the components separately can also, in certain instances, permit long-term storage without losing activity of the components.

[0145] Kits may also include reagents in separate containers such as, for example, sterile water or saline to be added to a lyophilized active component packaged separately. For example, sealed glass ampules may contain a lyophilized component and in a separate ampule, sterile water, sterile saline or sterile each of which has been packaged under a neutral non-reacting gas, such as nitrogen. Ampules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, ceramic, metal or any other material typically employed to hold reagents. Other examples of suitable containers include bottles that may be fabricated from similar substances as ampules, and envelopes that may consist of foil-lined interiors, such as aluminum or an alloy. Other containers include test tubes, vials, flasks, bottles, syringes, and the like. Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle. Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix. Removable membranes may be glass, plastic, rubber, and the like.

[0146] In certain embodiments, kits can be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, mini-CD-ROM, CD-ROM, DVD-ROM, Zip disc, videotape, audio tape, and the like. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an Internet web site specified by the manufacturer or distributor of the kit.

[0147] Compositions and methods described herein utilizing molecular biology protocols can be according to a variety of standard techniques known to the art (see, e.g., Sambrook and Russel (2006) Condensed Protocols from Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, ISBN-10: 0879697717; Ausubel et al. (2002) Short Protocols in Molecular Biology, 5th ed., Current Protocols, ISBN-10: 0471250929; Sambrook and Russel (2001) Molecular Cloning: A Laboratory Manual, 3d ed., Cold Spring Harbor Laboratory Press, ISBN-10: 0879695773; Elhai, J. and Wolk, C. P. 1988. Methods in Enzymology 167, 747-754; Studier (2005) Protein Expr Purif. 41(1), 207-234; Gellissen, ed. (2005) Production of Recombinant Proteins: Novel Microbial and Eukaryotic Expression Systems, Wiley-VCH, ISBN-10: 3527310363; Baneyx (2004) Protein Expression Technologies, Taylor & Francis, ISBN-10: 0954523253).

Definitions

[0148] When introducing elements of the present disclosure or the preferred aspects(s) thereof, the articles "a," "an," "the," and "said" are intended to mean that there are one or more of the elements. The terms "comprising," "including," and "having" are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0149] As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this invention, the chemical elements are identified in accordance with the Periodic Table of the Elements, CAS version, and the Handbook of Chemistry and Physics, 75th Ed. 1994. Additionally, general principles of organic chemistry are described in "Organic Chemistry," Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry," 5th Ed., Smith, M. B. and March, J., eds. John Wiley & Sons, New York: 2001, the entire contents of which are hereby incorporated by reference.

[0150] The term "mmol", as used herein, is intended to mean millimole. The term "equiv", as used herein, is intended to mean equivalent. The term "mL", as used herein, is intended to mean milliliter. The term "g", as used herein, is intended to mean gram. The term "kg", as used herein, is intended to mean kilogram. The term ".mu.g", as used herein, is intended to mean micrograms. The term "h", as used herein, is intended to mean hour. The term "min", as used herein, is intended to mean minute. The term "M", as used herein, is intended to mean molar. The term ".mu.L", as used herein, is intended to mean microliter. The term ".mu.M", as used herein, is intended to mean micromolar. The term "nM", as used herein, is intended to mean nanomolar. The term "N", as used herein, is intended to mean normal. The term "amu", as used herein, is intended to mean atomic mass unit. The term ".degree. C.", as used herein, is intended to mean degree Celsius. The term "wt/wt", as used herein, is intended to mean weight/weight. The term "v/v", as used herein, is intended to mean volume/volume. The term "MS", as used herein, is intended to mean mass spectroscopy. The term "HPLC", as used herein, is intended to mean high performance liquid chromatograph. The term "RT", as used herein, is intended to mean room temperature. The term "e.g.", as used herein, is intended to mean example. The term "N/A", as used herein, is intended to mean not tested.

[0151] Specific embodiments disclosed herein may be further limited in the claims using "consisting of" or "consisting essentially of" language, rather than "comprising". When used in the claims, whether as filed or added per amendment, the transition term "consisting of" excludes any element, step, or ingredient not specified in the claims. The transition term "consisting essentially of" limits the scope of a claim to the specified materials or steps and those that do not materially affect the basic and novel characteristic(s). Embodiments of the invention so claimed are inherently or expressly described and enabled herein.

[0152] As various changes could be made in the above-described reporter molecules and methods without departing from the scope of the invention, it is intended that all matter contained in the above description and in the examples given below, shall be interpreted as illustrative and not in a limiting sense.

EXAMPLES

[0153] The following examples are included to demonstrate various embodiments of the present disclosure. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventors to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

Materials and Methods for Examples 1

[0154] Recombinant DNA and Endogenous NAD.sup.+ measurements in host E. coli.

[0155] Double tagged (N-terminal tandem StrepTag and C-terminal 6.times.HisTag) bacterial or archaea TIRs (see sequence in FIG. 5) were cloned into a pET30a+ plasmid. These constructs as well as non-recombinant pET30a+ were transformed into Shuffle T7 Express Competent E-coli (New England BioLabs). Single colonies were grown overnight and the next day, cultures were diluted in LB media, grown at 30.degree. C. until they reached A600 of approximately 0.4-0.8, when IPTG (0.1 mM final concentration) was added to induce protein expression. The cultures were then harvested approximately 2 hours later. The cultures were normalized to A600 of approximately 0.5.+-.0.05 and the pellet from 500 .mu.l of culture suspension was lysed by adding 200 .mu.L 0.5M perchloric acid (HCIO4). Samples were then placed on ice for at least 10 minutes, centrifuged, and supernatant collected. 1804 of supernatant was then added to approximately 674 of 3M K2CO3. Samples were placed on ice for at least 10 minutes, and centrifuged. NAD+ metabolites were then measured by HPLC as described below.

[0156] Cell-Free Protein Transcription and Translation.

[0157] In vitro cell-free protein transcription and translation was performed using the PURExpress In Vitro Protein Synthesis Kit (New England BioLabs Catalog # E6800S). For a total reaction volume of about 25 .mu.L, the reaction was assembled in the following order: 10 .mu.L of Solution A, 7.5 .mu.L of Solution B, 3 .mu.L of RNase inhibitor (40 U/uL), water, and 0.5-1.0 .mu.g of pET30a+ non-recombinant/recombinant DNA. The reaction was incubated at 37.degree. C. for 2.5 hours and stopped by placing on ice. Typically, two 25 .mu.L reactions were pooled together for Tandem Affinity purification of proteins, which is described below.

Mammalian Cell Culture and NAD.sup.+ Quantification.

[0158] HEK293T cells were maintained in 10% FBS in DMEM, supplemented with penicillin/streptomycin and glutamine, and passaged by suspending in 0.05% trypsin. For NAD.sup.+ measurements in mammalian cells, HEK293T cells were seeded in 12-well dishes (approximately 2.5.times.105 cells per well) and transiently transfected the following day with 500 ng of the indicated plasmid using polyethylenimine (3:1 ratio to plasmid DNA). Twenty-four hours later, cells were washed in cold phosphate buffered saline and NAD.sup.+ extracted using perchloric acid method. NAD.sup.+ was measured after separation by HPLC.

Identification of Prokaryotic TIR Domains and Database Search.

[0159] Candidate pathogenic bacterial TIRs were selected from the published literature where at least seven bacterial TIR domain proteins have been reported to promote virulence (Waldhuber et al., 2016). A. baumannii was included since carbapenem-resistant A. baumannii is classified under `Priority 1` of the 2017 WHO Priority Pathogens List for R&D of New Antibiotics. P. dentrificans TIR domain is not known to promote virulence but it was one of the first bacterial TIR domains where the crystal structure was available (Waldhuber et al., 2016). The archaeal TIR domains were identified by searching the non-redundant protein database in protein-BLAST, and selecting Archaea (taxaid:2157) under Organism. The blastp (protein-protein blast) algorithm was utilized and the search was performed using the sequence of the bacterial TIR domains TirS-TIR and TcpC-TIR (see sequence in FIG. 5). Both M. olleyae and T. archaeon ranked as the top 2 hits with highest max score, and lowest e-value in the search using TirS-TIR, and T. archaeon ranked the highest in the max score and identity, and lowest e-value in the search using TcpC-TIR. The TIR domain from Actinoplanes was identified from bioinformatics analyses establishing a phylogenetic relationship between SARM1-TIR and other TIR domains.

HPLC Metabolite Measurement.

[0160] Supernatant (90 .mu.L) containing the extracted metabolites was mixed with 0.5M Potassium Phosphate buffer (10 .mu.L), and metabolites were analyzed by HPLC (Nexera X2) with Kinetex (100.times.3 mm, 2.6 .mu.m; Phenomenex) column. Internal standards for NAD.sup.+, Nicotinamide (Nam), ADP Ribose (ADPR), and cyclic ADPR were used to generate standard curves for quantification of the respective compounds. The levels for each compound in each experimental sample were normalized to the 0 min time point that was analyzed concurrently.

Native Protein Purification.

[0161] Cell-free synthesized proteins were first purified by Strep Tag affinity methods where proteins were incubated in binding buffer (50 mM Sodium Phosphate buffer pH 7.6, 300 mM Sodium Chloride, 0.01% Tween-20) and 20 .mu.L MagStrep (Strep-Tactin) type 3 XT beads suspension (IBA Lifesciences) for 30 min. Proteins were then eluted from MagStrep type 3 XT beads with approximately 25 mM biotin (Sigma, B4501) for 25 min. The purified proteins were normally purified further by incubating the eluted protein with 10 .mu.L Co.sup.2+ Dynabead suspension (ThermoFisher) for 30 min to bind TIR proteins via the His tag. The beads were then washed at least two times with binding buffer and resuspended in 100 .mu.L of binding buffer for downstream applications.

NADase Assay and Metabolite Extraction.

[0162] For purified TIR domains, typically, 10 .mu.L of cobalt beads laden with purified protein were incubated with 5 .mu.M NAD.sup.+ and reaction buffer (92.4 mM NaCl and 0.64.times.PBS), for a total reaction volume of 50 .mu.L. Reactions were carried out at room temperature (25.degree. C.) for the indicated amount of time. For TirS-FL, 20 .mu.L of biotin eluted protein was incubated with 5 .mu.M NAD.sup.+, under the same buffer and temperature conditions. Reaction was stopped by addition of 1M of perchloric acid (HCIO.sub.4) and placing the tube on ice for at least 10 min. Neutralization was performed with 3M K.sub.2CO.sub.3. Samples were placed on ice for 10 min, and then separated by centrifugation. NAD.sup.+ metabolites were quantified by HPLC (see HPLC metabolite measurement). For LC-MS analysis, the extraction was performed using 50% Methanol in distilled water, and chloroform extraction (see LC-MS metabolite measurement for further details).

LC-MS Metabolite Measurement.

[0163] Samples were prepared by mixing the reactions with 50% methanol in distilled water. The samples were placed on ice, centrifuged, soluble metabolites in the supernatant were extracted with chloroform, and the aqueous phase was lyophilized and stored at -20.degree. C. until LC-MS analysis. For LC-MS, the metabolite samples were reconstituted with 5 mM ammonium formate, centrifuged 12,000.times.g for 10 min, and the cleared supernatant was applied to the LC-MS for metabolite identification and quantification. Liquid chromatography was performed by HPLC system (1290; Agilent) with Atlantis T3 (2.1.times.150 mm, 3 .mu.m; Waters) column. Samples (10 .mu.l) were injected at a flow rate of 0.15 ml/min with 5 mM ammonium formate for mobile phase A and 100% methanol for mobile phase B and metabolites were eluted with gradients of 2-6 min, 0-20% B; 6-8 min, 20-50% B; 8-10 min 50% B; 10-15 min, 50-0% B. The metabolites were detected with Triple Quad mass spectrometer (6460; Agilent) under positive ESI multiple reaction monitoring (MRM). Metabolites were quantified by MassHunter quantitative analysis tool (Agilent) with standard curves. Standard curves for each compound were generated by analyzing NAD.sup.+, cADPR, ADPR, and Nam reconstituted in 5 mM ammonium formate.

SYPRO Ruby Gel Staining.

[0164] Purified proteins were boiled in Laemmli buffer for 5-10 min and separated on a 4-12% Bis-Tris Plus gel. After electrophoresis, the gel was fixed in 50% Methanol/7% acetic acid for 30 min.times.2, then incubated overnight in SYPRO Ruby Protein Gel stain (Thermo Fisher). The next day, the gel was washed with 10% methanol/7% acetic acid solution for 30 min, rinsed in distilled water for 5 minutes.times.2, and stained proteins were visualized with a UV transilluminator.

Enzyme Kinetics Studies.

[0165] V.sub.max, and K.sub.m were determined from the initial reaction velocity or reaction velocity in the first 60s of product (ADPR) formation, and fitting the data to the Michaelis-Menten equation using nonlinear curve fit in GraphPad Prism 7. Data are presented as Mean.+-.SEM from two independents biological samples and a total of four independent reaction measurements.

Structural Modeling of Bacterial TIR Domains.

[0166] SWISS-Model (Arnold et al., 2006) was used to generate structural models of bacterial TIR domains. The crystal structure of MilB CMP hydrolase (PDB 4JEM) was used as the template. The modeled bacterial TIR domain and MilB crystal structures were visualized and superimposed with Chimera (www.rbvi.ucsf.edu/chimera). tan--modeled bacterial TIR; cyan--MilB CMP hydrolase; yellow--cytidine-5'-monophosphate (ligand for MilB).

HH Pred Protein Identification.

[0167] TirS-TIR, TcpC-TIR, and TcpA-TIR were analyzed for structural homologs using HH Pred. (Alva et al., 2016). Only known nucleotide enzymes with an E-value lower than 0.1 and a probability above 95% are shown in Table 1.

TABLE-US-00001 TABLE 1 Nucleotide hydrolase/transferase enzymes identified via HHPred. TirS-TIR, TcpC-TIR, and TcpA-TIR were each used as a query sequence in HHPred to identify candidate enzymes. PDB Protein Identification Probability E-value TirS-TIR 4JEM_B CMP/hydroxymethyl CMP hydrolase; CMP N- 97.83 0.000013 glycosidase, Hydrolase; HET: C5P; 1.553A {Streptomyces rimofaciens} 4JEM_A CMP/hydroxymethyl CMP hydrolase; CMP N- 97.83 0.000013 glycosidase, Hydrolase; HET: C5P; 1.553A {Streptomyces rimofaciens} 2KLH_B c-Myc-responsive protein Rcl; Protein, GMP, N- 97.73 0.000012 Glycosidase, Nucleus, Phosphoprotein; HET: 5GP; NMR {Rattus norvegicus} 4JEN_B Putative uncharacterized protein; CMP N- 97.51 0.000084 glycosidase, bacimethrin biosynthesis, Hydrolase; HET: PO4; 3.0A {Clostridium botulinum A} 4JEN_C Putative uncharacterized protein; CMP N- 97.51 0.000084 glycosidase, bacimethrin biosynthesis, Hydrolase; HET: PO4; 3.0A {Clostridium botulinum A} 4HX9_C Nucleoside deoxyribosyltransferase (E.C.2.4.2.6); 96.73 0.0034 Non-natural and design enzyme; HET: SO4, PG4; 2.68A {Lactobacillus leichmannii} 1S2D_B purine trans deoxyribosylase (E.C.2.4.2.6); ribosylate 96.62 0.0029 intermediate, PTD, AraA, TRANSFERASE; HET: ADE; 2.1A {Lactobacillus helveticus} SCOP: c.23.14.1 4P5E_B 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 96.57 0.002 (E.C.3.2.2.-); RCL, DNPH1, Inhibitor, Rossmann Fold; HET: N6P; 1.35A {Homo sapiens} 1F8Y_B NUCLEOSIDE 2-DEOXYRIBOSYLTRANSFERASE; 96.55 0.0064 active site, alpha/beta protein, biocatalyst; HET: 5MD; 2.4A {Lactobacillus leichmannii} SCOP: c.23. 14. 1 2F62_B Nucleoside 2-deoxyribosyltransferase (E.C.2.4.2.6); 96.02 0.0096 SGPP, Structural GENOMICS, PSI, PROTEIN; HET: GOL, 12M, SO4; 1.5A {Trypanosoma brucei} SCOP: c.23.14.1 2F2T_B Nucleoside 2-deoxyribosyltransferase (E.C.2.4.2.6); 96.02 0.0096 SGPP, Structural GENOMICS, PSI, PROTEIN; HET: GOL, 5IQ, SO4; 1.7A {Trypanosoma brucei} SCOP: c.23.14.1 TcpC-TIR 4JEN_B Putative uncharacterized protein; CMP N- 97.38 0.0002 glycosidase, bacimethrin biosynthesis, Hydrolase; HET: PO4; 3.0A {Clostridium botulinum A} 4JEN_C Putative uncharacterized protein; CMP N- 97.38 0.0002 glycosidase, bacimethrin biosynthesis, Hydrolase; HET: PO4; 3.0A {Clostridium botulinum A} 4JEM_B CMP/hydroxymethyl CMP hydrolase; CMP N- 97.34 0.00024 glycosidase, Hydrolase; HET: C5P; 1.553A {Streptomyces rimofaciens} 4JEM_A CMP/hydroxymethyl CMP hydrolase; CMP N- 97.34 0.00024 glycosidase, Hydrolase; HET: C5P; 1.553A {Streptomyces rimofaciens} 2KLH_B c-Myc-responsive protein Rcl; Protein, GMP, N- 97.18 0.00039 Glycosidase, Nucleus, Phosphoprotein; HET: 5GP; NMR {Rattus norvegicus} 4HX9_C Nucleoside deoxyribosyltransferase (E.C.2.4.2.6); 96.29 0.012 Non-natural and design enzyme; HET: SO4, PG4; 2.68A {Lactobacillus leichmannii} 1S2D_B purine trans deoxyribosylase (E.C.2.4.2.6); ribosylate 96.21 0.014 intermediate, PTD, AraA, TRANSFERASE; HET: ADE; 2.1A {Lactobacillus helveticus} SCOP: c.23.14.1 2F2T_B Nucleoside 2-deoxyribosyltransferase (E.C.2.4.2.6); 96.05 0.024 SGPP, Structural GENOMICS, PSI, PROTEIN; HET: GOL, 5IQ, SO4; 1.7A {Trypanosoma brucei} SCOP: c.23.14.1 2F62_B Nucleoside 2-deoxyribosyltransferase (E.C.2.4.2.6); 96.05 0.024 SGPP, Structural GENOMICS, PSI, PROTEIN; HET: GOL, 12M, SO4; 1.5A {Trypanosoma brucei} SCOP: c.23.14.1 4P5E_B 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 96 0.017 (E.C.3.2.2.-); RCL, DNPH1, Inhibitor, Rossmann Fold; HET: N6P; 1.35A {Homo sapiens} 1F8Y_B NUCLEOSIDE 2-DEOXYRIBOSYLTRANSFERASE; 95.79 0.053 active site, alpha/beta protein, biocatalyst; HET: 5MD; 2.4A {Lactobacillus leichmannii} SCOP: c.23. 14. 1 TcpA-TIR 4JEM_A CMP/hydroxymethyl CMP hydrolase; CMP N- 97.97 0.000003 glycosidase, Hydrolase; HET: C5P; 1.553A {Streptomyces rimofaciens} 4JEM_B CMP/hydroxymethyl CMP hydrolase; CMP N- 97.97 0.000003 glycosidase, Hydrolase; HET: C5P; 1.553A {Streptomyces rimofaciens} 4JEN_B Putative uncharacterized protein; CMP N- 97.86 0.000025 glycosidase, bacimethrin biosynthesis, Hydrolase; HET: PO4; 3.0A {Clostridium botulinum A} 4JEN_C Putative uncharacterized protein; CMP N- 97.86 0.000025 glycosidase, bacimethrin biosynthesis, Hydrolase; HET: PO4; 3.0A {Clostridium botulinum A} 2KLH_B c-Myc-responsive protein Rcl; Protein, GMP, N- 97.6 0.00004 Glycosidase, Nucleus, Phosphoprotein; HET: 5GP; NMR {Rattus norvegicus} 4HX9_C Nucleoside deoxyribosyltransferase (E.C.2.4.2.6); 97.16 0.00056 Non-natural and design enzyme; HET: SO4, PG4; 2.68A {Lactobacillus leichmannii} 1S2D_B purine trans deoxyribosylase (E.C.2.4.2.6); ribosylate 97.02 0.0006 intermediate, PTD, AraA, TRANSFERASE; HET: ADE; 2.1A {Lactobacillus helveticus} SCOP: c.23.14.1 1F8Y_B NUCLEOSIDE 2-DEOXYRIBOSYLTRANSFERASE; 96.92 0.002 active site, alpha/beta protein, biocatalyst; HET: 5MD; 2.4A {Lactobacillus leichmannii} SCOP: c.23. 14. 1 2F2T_B Nucleoside 2-deoxyribosyltransferase (E.C.2.4.2.6); 96.35 0.0048 SGPP, Structural GENOMICS, PSI, PROTEIN; HET: GOL, 5IQ, SO4; 1.7A {Trypanosoma brucei} SCOP: c.23.14.1 2F62_B Nucleoside 2-deoxyribosyltransferase (E.C.2.4.2.6); 96.35 0.0048 SGPP, Structural GENOMICS, PSI, PROTEIN; HET: GOL, 12M, SO4; 1.5A {Trypanosoma brucei} SCOP: c.23.14.1 4P5E_B 2'-deoxynucleoside 5'-phosphate N-hydrolase 1 96.28 0.012 (E.C.3.2.2.-); RCL, DNPH1, Inhibitor, Rossmann Fold; HET: N6P; 1.35A {Homo sapiens}

Quantification and Statistical Analysis.

[0168] Number of n is indicated in each figure legend. Unpaired two-tailed t-tests were used for individual comparisons with an assumption of equal variance between groups. All error bars represent SEM. Other data analyses were done with Graph Pad Prism 7, Microsoft Excel, Adobe Illustrator and Photoshop.

Example 1

[0169] This example illustrates a diverse set of prokaryotic TIR domains are capable of regulating levels of the central metabolic regulator NAD.sup.+ and thereby elucidate a previously unappreciated function of TIR domains separate from the scaffolding functions in TLR signaling and opening a new therapeutic window. Furthermore, this example illustrates a TIR domain assay for NADase activity.

Identification of Prokaryotic TIR Domains that Induce NAD.sup.+ Loss in Host E. coli.

[0170] Phylogenetic studies suggest that the TIR domain of SARM1, the NADase serving as the central executioner of injury-induced axonal death, is closely related to prokaryotic TIR domains (Zhang et al., 2011). This raises the question of whether TIR NADase activity is an evolved function unique to SARM1, or whether such an enzymatic activity may be the primordial function of TIR domains. To distinguish between these two possibilities, we developed an assay to screen for NADase activity in host E. coli expressing candidate TIR domains (FIG. 1A). We tested TIR domains from pathogenic bacteria, including those noted for extreme antibiotic resistance, TIR domains from non-pathogenic bacteria, TIR domains from archaea, as well as the SARM1 TIR domain (FIG. 1B, FIG. 1C; FIG. 5). As previously observed, IPTG protein induction of wild type (enzymatically active) SARM1-TIR induces NAD+ loss in host E. coli (FIG. 1D; Essuman et al., 2017). In contrast, NAD.sup.+ was not depleted after IPTG induction in E. coli harboring either a control plasmid or a plasmid encoding enzymatically dead SARM1-TIR (SARM1-TIR(E642A) (FIG. 1D). We therefore reasoned that NAD.sup.+ levels would be lower in E. coli expressing enzymatically active prokaryotic TIR domains. Consistent with this hypothesis, we found that expression of TIR domains from many pathogenic bacteria like Staphylococcus aureus (methicillin sensitive and methicillin resistant), uropathogenic Escherichia coli, and Acinetobacter baumannii induced NAD.sup.+ depletion in the host E. coli. Further, TIR domains from bacteria considered non-virulent such as Paracoccus dentrificans and Actinoplanes species, or from the archaea Theionarchaea archaeon, also caused loss of NAD.sup.+ in host E. coli (FIG. 1D). These observations indicate that, like SARM1, TIR domains from diverse prokaryotes induce losses of NAD.sup.+ in E. coli host.

Characterization of an Intrinsic NADase Activity in Prokaryotic TIR Domains.

[0171] The NAD.sup.+ depletion observed in E. coli expressing TIR domains from multiple prokaryotic proteins stimulated us to test whether this was due to an intrinsic NAD.sup.+ cleavage activity, or was a secondary effect on E. coli metabolism. To test the intrinsic TIR enzymatic activity hypothesis, we adopted a cell-free protein transcription and translation system reconstituted from purified components from E. coli to synthesize recombinant TIR protein (Shimizu et al., 2001). This system does not contain known NADases, and we experimentally verified that it is devoid of NAD.sup.+ cleavage activity (Essuman et al., 2017). We programmed this cell-free system with recombinant DNA encoding double tagged (StrepTag and 6.times.His) TIR domains of TirS and TcpC, from the Gram-positive bacteria Staphylococcus aureus and Gram-negative bacteria Escherichia coli CFT073 respectively. We also tested a TIR domain from the archaea Theionarchaea archaeon, which we name TcpA-TIR. We purified the synthesized TIR proteins by tandem affinity purification and tested their ability to cleave NAD+. Cobalt beads laden with purified TIR proteins were incubated with NAD+ (5 .mu.M), and metabolites were extracted and analyzed using High Performance Liquid Chromatography (HPLC). We found that all three prokaryotic TIR domains cleave NAD.sup.+, with the TIR domain of Staphylococcus aureus showing the most rapid cleavage (FIG. 2A).

[0172] Since different NAD.sup.+ consuming enzymes display uniquely characteristic reaction product profiles, we asked what products were formed by these prokaryotic TIR domains. Using HPLC and LC-MS, we found that all three prokaryotic TIR domains cleave NAD.sup.+ into Nicotinamide (Nam) and ADP-Ribose (ADPR) (FIG. 2B-2E; FIG. 6). However, unlike SARM1, the Staphylococcus aureus TirS-TIR and Escherichia coli CFT073 TcpC-TIR did not produce cADPR as a product (FIG. 6G). A minor trace peak corresponding to cADPR was detected in LC-MS samples of the archaeal protein TcpA-TIR, but was not observed on HPLC chromatograms (FIG. 6G and FIG. 2D). Hence, we conclude that the prokaryotic TIR domains from S. aureus and E. coli CFT073 possess a pure NAD.sup.+ glycohydrolase activity, which is distinct from the dual NAD.sup.+ glycohydrolase and cyclase activity of SARM1-TIR.

[0173] In characterizing the activity of these prokaryotic TIR domains, we examined their Michaelis-Menten parameters using the purified TIR proteins. The E.coli TcpC-TIR exhibited an estimated K.sub.m of 196 .mu.M and a V.sub.max of approximately 1.8 .mu.M/min, while the S. aureus TirS-TIR displayed an estimated K.sub.m of 490 .mu.M and a V.sub.max of .about.10 .mu.M/min (FIG. 2F). The E. coli TcpC-TIR K.sub.m of 196 .mu.M is very close to the reported 188 .mu.M K.sub.m value of the bacterial virulence effector SPN (Streptococcus Pyogenes NAD.sup.+ glycohydrolase) that is implicated in its virulence (Ghosh et al., 2010). Further, the K.sub.m values of both TcpC-TIR and TirS-TIR fall within the range of total NAD.sup.+ content in E. coli and mammalian cells (Canto et al., 2015; Zhou et al., 2011), suggesting that these TIRs could influence NAD.sup.+ levels in both eukaryotes and prokaryotes.

[0174] We next investigated structural elements within these prokaryotic TIR domains that contribute to NAD.sup.+ cleavage. The SARM1 TIR domain has structural similarities to nucleotide hydrolases, which led us to the identification of a glutamate residue within the proposed active site of SARM1-TIR that is absolutely essential for its catalytic activity (Essuman et al., 2017). Structural motif homology searches revealed that these prokaryotic TIRs share structural similarity with other nucleotide enzymes including cytidine monophosphate (CMP) hydrolase, and nucleoside 2-deoxyribosyltransferase (Table 1). No crystal structures have been reported for SARM1 TIR or the three prokaryotic TIRs; so using SWISS modeling (Arnold et al., 2006) and the known CMP hydrolase structure (PDB:4JEM), we generated structural models of the prokaryotic TIR domains. This analysis demonstrated that the catalytic glutamate of CMP hydrolase aligned with a conserved glutamate in the prokaryotic TIR domains (FIG. 2G, FIG. 2H and FIG. 7B). This glutamate is also the residue in SARM1-TIR that was essential for its catalytic activity (FIG. 5). We mutated this glutamate to alanine in each of the prokaryotic TIRs and found, in each case, that this abolished NADase activity (FIG. 2I). These experiments show that these TIR domains share structural homology to a family of nucleotidases, and that a conserved catalytic glutamic acid in the TIR proteins is essential for catalytic cleavage of NAD.sup.+.

[0175] In some instances, NADases are capable of cleaving a number of related molecules, such as the NAD+ precursor Nicotinamide Mononucleotide (NMN) in the case of CD38 (Sauve et al., 1998). Further, we wanted to determine whether prokaryotic TIRs could cleave additional substrates, providing additional paths to modifying cell metabolism. To examine the substrate specificity of the prokaryotic TIR domains, we tested their activity using a wide variety of metabolites and analogs. These included biologically important NAD.sup.+ analogs such as NADH and Nicotinamide Adenine Dinucleotide Phosphate (NADP.sup.+) that help maintain cellular redox state (Ying, 2008), key cellular energetic nucleotides like ATP and GTP, as well as NAD.sup.+ analogs like 3-acetylpyridine adenine dinucleotide (3-apAD), Nicotinic Acid Adenine Dinucleotide (NaAD), Nicotinamide Guanine Dinucleotide (NGD), and Nicotinamide Hypoxanthine Dinucleotide (NHD), which possess minor modifications compared to NAD.sup.+ that could indicate functional groups important for catalysis (FIG. 2J-2L; FIG. 8).

[0176] These studies demonstrated that all three prokaryotic TIR domains cleave NADP.sup.+, but have little effect on NMN (FIG. 2J-2L; FIG. 8). The cleavage of NADP.sup.+ suggests that these prokaryotic TIRs could modulate cellular function by altering the cellular NADP.sup.+ pools necessary for maintaining levels of NADPH needed for reductive biosynthesis and protection against reactive oxygen species (Ying, 2008). Interestingly, we also found that the SARM1-TIR domain cleaves NADP.sup.+, which provides an additional mechanism by which SARM1 could promote axon degeneration.

[0177] Next, we found that while all TIRs substantially cleaved 3-acetylpyridine adenine dinucleotide (3-apAD), and thionicotinamide adenine dinucleotide (sNAD), none cleaved Nicotinic Acid Adenine Dinucleotide (NaAD). These three NAD.sup.+ analogs possess only a single functional group modification on the NAD.sup.+ nicotinamide ring. For NaAD and 3apAD in particular, the amide (--NH2) group of the nicotinamide ring is substituted for a hydroxyl (--OH) in NaAD or a methyl (--CH3) group in 3apAD (FIG. 8A). The differential cleavage pattern suggests that this position on the NAD.sup.+ molecule is important for substrate recognition or catalysis by prokaryotic TIR domains.

[0178] Additionally, we noted that while prokaryotic TIRs did not cleave NHD or NGD, SARM1-TIR cleaved both of these substrates (FIG. 2J-2L; FIG. 8). Interestingly, both NHD and NGD are also cleaved by ADP-Ribosyl cyclases like CD38 (Graeff et al., 1994; Graeff et al., 1996). In particular, NGD has been used to probe the activity of proteins in the ADP-Ribosyl cyclase family that generate cADPR (Graeff et al., 1994; Graeff et al., 1996). These results clearly show clear differences between these prokaryotic TIR domains and their mammalian homolog SARM1, and are consistent with the lack of observed cyclase activity in the prokaryotic TirS-TIR and TcpC-TIR proteins.

[0179] Finally, we were unable to detect substantial cleavage of either ATP or GTP by any TIR domain, suggesting the substrate specificity may be limited to nucleotide analogs that more closely resemble the NAD.sup.+ molecule. Altogether, our results indicate that the prokaryotic TIRs are positioned to influence various aspects of cellular metabolism through their cleavage of NAD.sup.+ or its close analogs, and that functional groups associated with the nicotinamide ring of the NAD.sup.+ molecule dictate specificity for this class of enzymes.

Identification of a Variant Cyclic ADPR Generated by a Subset of TIR Domains.

[0180] Our findings demonstrate that many prokaryotic TIR domains cleave NAD.sup.+ to produce nicotinamide and ADPR. However, our results with the archaea TcpA-TIR suggested that, like SARM1, some prokaryotic TIR NADases may also be able to generate cADPR (FIG. 6G). Moreover, in our initial HPLC metabolite screen using the E. coli lysate assay, we observed a distinct and prominent peak (Metabolite X) produced by three different prokaryotic TIRs including the archaea Methanobrevibacter olleyae (TcpO-TIR), and the pathogenic bacteria, Acinetobacter baumannii (AbTir-TIR) and Brucella sp. (BtpA-TIR)(FIG. 3A-3D; FIG. 9A). The HPLC retention time of this Metabolite X did not correspond to retention times of any known NAD.sup.+ cleavage product such as ADPR, cADPR, or nicotinamide (FIG. 2B-2D) suggesting Metabolite X could be a previously unidentified product.

[0181] To identify Metabolite X, we asked if it was a direct cleavage product of NAD.sup.+, or if it was an E. coli response secondary to TIR signaling. Using our cell-free protein translation NADase assay, we tested the TIR domain from the archaea Methanobrevibacter olleyae (TcpO-TIR). We found that it cleaved NAD.sup.+ into Nicotinamide, ADP-Ribose, and a metabolite that had a retention time very close to that of Metabolite X (FIG. 3E). These data indicate that Metabolite X is a product of NAD.sup.+ cleavage and raises the intriguing possibility that these TIR domains metabolize NAD.sup.+ to a novel product.

[0182] To further identify Metabolite X, we performed LC-MS analysis on the extracted metabolites from the TcpO-TIR reaction. Examination of the MS spectra (m/z=70-1200) revealed metabolites produced by the reaction (compare 0 vs. 30 min) with m/z of 664, 123, 560, and 542, which correspond to NAD.sup.+, Nicotinamide, ADP-Ribose, and cADPR respectively (FIG. 9B and data not shown). Metabolite X is a molecule with an m/z of 542, which corresponds to cADPR; however, the LC retention time of m/z 542 was dramatically different from that of the cADPR standard (FIG. 9B). Further, on a separate LC instrument, both commercially available cADPR preparations (FIG. 3F and FIG. 3G) and cADPR generated from a SARM1-TIR programmed reaction (Essuman et al., 2017), had retention times distinct from Metabolite X indicating it is not cADPR.

[0183] MS/MS analysis was then used to further analyze the ion with m/z 542. We found that the major product ion had an m/z of 136, which likely corresponds to the adenine moiety of NAD.sup.+ (FIG. 3H). MS/MS spectra of standard cADPR showed a very similar fragmentation pattern to that of Metabolite X (FIG. 3H). To extend this analysis, we collected HPLC fractions corresponding to the Metabolite X peak in lysates prepared from E. coli expressing TcpO-TIR, AbTir-TIR, and Btpa-TIR. MS/MS analysis of these fractions showed the signature m/z 542/136 ion pair was produced by each of these TIR domains (FIG. 9C-9E).

[0184] These analyses indicate that a subset of prokaryotic TIR domains produce a variant of cADPR (Metabolite X) from NAD.sup.+ cleavage. The generation of cADPR by ADP-Ribosyl cyclases including the cyclase from Aplysia californica, and mammalian CD38 occurs via cyclization at the N1 position of the adenine ring (Graeff et al., 2009; Lee et al., 1994). However, CD38 can cyclize nicotinamide guanine dinucleotide (NGD), an NAD.sup.+ analog, using the N7 position of the guanine ring to produce N7-cGDPR (Graeff et al., 1996). Taken together with all of our metabolite analysis, it is likely that the variant cADPR (Metabolite X) is N7-cADPR (FIG. 3I), a previously unreported molecule. Taken together, our results show that prokaryotic TIR domains are NAD.sup.+ consuming enzymes, and that remarkable diversity in the reaction products exists even within TIRs from the same domain of life. Future studies should provide more insights into the structure of the variant cADPR molecule and how prokaryotic TIRs produce this unique reaction product.

Full Length TirS from Staphylococcus aureus Induces NAD.sup.+ Loss in Mammalian Cells.

[0185] A number of bacterial TIR domain proteins have been identified as secreted virulence factors (Askarian et al., 2014; Cirl et al., 2008; Patot et al., 2017). With the discovery that prokaryotic TIR domains function enzymatically to cleave NAD.sup.+, we tested TIR domain proteins associated with microbial virulence for their ability to degrade NAD.sup.+ in mammalian cells. First, we transiently expressed S. aureus TirS-TIR and E. coli TcpC-TIR in HEK293T cells, extracted metabolites, and measured NAD.sup.+ levels by HPLC. Consistent with results using purified recombinant protein in in vitro assays, we found that both wild type TirS-TIR and wild type TcpC-TIR induced NAD.sup.+ loss these cells (FIG. 4A). Moreover, this decline in NAD.sup.+ was not observed when catalytically dead mutant TirS-TIR or TcpC-TIR were transfected (FIG. 4A), indicating that the drop in NAD.sup.+ is due to intrinsic enzymatic activity of these TIR domains. We examined expression of these wildtype and mutant TIR domains and found that the mutants were expressed at remarkably higher levels (FIG. 4B), supporting the idea that mammalian cells grow poorly when expressing these potent NADases.

[0186] With confirmation that these prokaryotic TIR domains are active in mammalian cells, we set out to examine the effects of expressing the full-length S. aureus TirS virulence factor (TirS-FL) in HEK293T cells. First, we asked if TirS-FL purified from our cell-free protein translation system was able to cleave NAD+ in vitro. We found that wild type TirS-FL cleaves NAD.sup.+, whereas the mutant TirS-FL(E216A) was enzymatically inactive (FIG. 4C-4E). Similar to the purified TIR-only protein, HPLC analysis showed that TirS-FL cleaves NAD.sup.+ into Nicotinamide and ADP-Ribose (FIG. 4C and FIG. 4D).

[0187] To test if TirS-FL could induce NAD.sup.+ loss in mammalian cells, we transiently expressed it in HEK293T cells, extracted metabolites, and measured NAD.sup.+ levels. Consistent with data generated in vitro and in bacteria, we found that wild type TirS-FL induced NAD.sup.+ loss in mammalian cells, whereas enzymatically dead TirS-FL E216A did not (FIG. 4F, FIG. 4G). Again, we observed that the wildtype protein was expressed at much lower levels in HEK293T cells than its catalytically inactive mutant (FIG. 4G), suggesting that TirS is toxic to its mammalian host. These findings suggest that TIR domain proteins identified as virulence factors could promote virulence via their NADase activity and that therapeutic interventions targeting TIR NADases in pathogenic bacteria could restrict infections caused by these organisms.

Example 2

Identification of Inhibitors of TcpC and TirS NADase Activity

[0188] NRK1-HEK293T cells represent a polyclonal cell line that has been stably transfected with an FCIV expression vector that expresses human Nicotinamide Riboside Kinase 1 (NRK1), an enzyme that converts the NAD.sup.+ biosynthetic precursor nicotinamide riboside (NR) to NMN, the immediate precursor of NAD.sup.+. When these NRK1-expressing cells are supplemented with NR, NAD.sup.+ levels are augmented and cell viability is enhanced to enable efficient production and purification of the constitutively active TcpC TIR protein fragment. NRK1-HEK293T cells were seeded onto 150 cm2 plates at 20.times.106 cells per plate. The next day, the cells were transfected with 15 .mu.g FCIV-TcpC TIR (TcpC TIR expression plasmid) using X-tremeGENE 9 DNA Transfection Reagent (Roche product #06365787001). The cultures were supplemented with 1 mM NR at time of transfection to minimize toxicity from TcpC-TIR overexpression. Forty-eight hours after transfection, cells were harvested, pelleted by centrifugation at 1,000 rpm (Sorvall ST 16R centrifuge, Thermo Fisher), and washed once with cold PBS (0.01 M phosphate buffered saline NaCl 0.138 M; KCl 0.0027 M; pH 7.4). The cells were resuspended in PBS with protease inhibitors (Complete protease inhibitor cocktail, Roche product #11873580001) and cell lysates were prepared by sonication (Branson Sonifer 450, output=3, 20 episodes of stroke). The lysates were centrifuged (12,000.times.g for 10 min at 4.degree. C.) to remove cell debris and the supernatants (containing TcpC-TIR protein) were stored at -80.degree. C. for later use in the in vitro NADase assay (see below). Protein concentration was determined by the Bicinchoninic (BCA) method and used to normalize lysate concentrations.

[0189] Reaction mixtures were prepared by mixing TcpC-TIR cell lysate (0.9 .mu.g total protein), test compound stock (25 .mu.M final concentration, from Pharmakon library), and PBS (pH 7.4) to a final volume of 12 .mu.l. NAD (10 .mu.M final concentration) was then added for a final reaction volume of 20 .mu.l. The reaction was incubated at 37.degree. C. for 60 min, and then stopped by addition of 180 .mu.l of 0.55 M perchloric acid (HCIO4) and placed on ice. After 10 min on ice, the reaction plates were centrifuged for 10 min at 4,000 rpm (Sorvall ST 16R centrifuge). The supernatant (120 .mu.l) was transferred to a new plate and 10 .mu.l of 3M K2CO3 was added to neutralize the solution. Precipitated salts were removed by centrifugation 10 min at 4,000 rpm (Sorvall ST 16R centrifuge). The supernatant was transferred and analyzed by HPLC (Shimadzu Nexera X2) to quantify the amounts of NAD and ADPR, a product of the cleavage reaction, in each sample. From these values, the NAD/ADPR ratio was calculated for each compound as a measure of NAD cleavage activity. This ratio was compared to the ratio generated in the absence of compound inhibitors. A reduction of NADase activity (NAD/ADPR ratio>2) was used to identify compounds that inhibited the TcpC-TIR catalyzed NAD cleavage. A total of 320 drugs from Pharmakon library (MicroSource Discovery Systems, Inc.) were screened as candidate inhibitors of TcpC-Tir.

[0190] Positive hits were individually purchased from Sigma and tested its dose dependent inhibition in conditions described above. SARM1 Sam-Tir lysate and MSSA Tir were also used to test if these compounds could block these Tir NADase activities as well. Three inhibitors (dequalinium, cetylpyridium, and amiloride) are shown in FIG. 10.

[0191] The same protocol was used to screen for S. aureus TirS inhibitors, except that the TirS protein was generated using an in-vitro translation system (New England Biolab E6800). For this, 25 .mu.l reactions containing 500 ng TirS DNA and 20 units RNase Inhibitor were incubated at 37.degree. C. for 2.5 hours with the in vitro translation system, and the protein that was produced was used for screen. The original solution was diluted 250 fold and 1 ul of diluted solution was used for each 20 ul reaction (10 uM NAD, 25 uM compound, 37.degree. C. for 60 min).

[0192] Two hits (acrinol and akrinol) were obtained following screening of 960 drugs from the Pharmakon library (FIG. 11). Both drugs possess an acridine moiety, so additional compounds containing this structure were tested as candidate inhibitors of TirS. 9-aminoacridine and 2-ethoxy-6,9-diaminoacridine monolactate monohydrate (Acrinol) both inhibit TirS with an IC50 of about 30 .mu.M. Neither of these drugs inhibited SARM1 (Sam-Tir) or TcpC TIR NADase activity (data not shown).

Example 3

Preliminary Screen of Microbial TIR NADases

[0193] Preliminary counts indicate at least, about 137 new microbial TIRs have been tested and run on HPLC, with 26 shown as positive hits for NADase activity. That represents a 19% positive hit rate. The positive hits are shown below in Table 2.

TABLE-US-00002 TABLE 2 Microbial TIR NADases Organism Gene/Protein Coprococcus_eutactus_ATCC_27759 gi|2991|ref|COPEUT_02740| hypothetical protein Proteus_penneri_ATCC_35198 gi|3821|ref|PROPEN_03896| hypothetical protein Bacteroides_thetaiotaomicron_3731 gi|5683|ref|AMN69_RS28245| hypothetical protein Eubacterium_hallii_DSM_3353 gi|446|ref|EUBHAL_RS02150| TIR domain-containing protein Bacteroides_thetaiotaomicron_3731 gi|1307|ref|AMN69_RS06490| hypothetical protein Bacteroides_xylanisolvens_XB1A gi|3630|ref|BXY_397001|MTH538 TIR-like domain (DUF1863). Clostridium scindens_ATCC_35704 gi|726|ref|CLOSCI_021301|SEFIR domain protein Dorea_formicigenerans_ATCC_27755 gi|1847|ref|DORFOR_RS09155|TIR domain-containing protein Providencia_rustigianii_DSM_4541 gi|533|ref|PROVRUST_05034| hypothetical protein Bacteroides_coprophilus_DSM_18228 gi|713|ref|BACCOPRO_00713| SEFIR domain protein Bacteroides_thetaiotaomicron_7330 gi|632|ref|Btheta7330_RS03065| TIR domain-containing protein Bacteroides_thetaiotaomicron_7330 gi|4887|ref|Btheta7330_RS23835| hypothetical protein Roseburia_intestinalis_L1-82 gi|2906|ref|ROSINTL182_07906| hypothetical protein Bacteroides_ovatus_ATCC_8483 gi|5057|ref|BACOVA_05060| hypothetical protein Clostridium_bolteae_ATCC_BAA- gi|1498|ref|CLOBOL_01188| 613 hypothetical protein Eubacterium_cylindroides_DSM_3983 gi|308|ref|HMPREF0367_01592| Sel1 repeat protein Enterococcus phage EFC-1 TIR protein [Enterococcus phage EFC-1] Cyanothece sp. (strain PCC 8801) Middle23_0 (Putative lipoprotein) Leptolyngbya sp. PCC 7376 Lepto7376_4317 (YD repeat protein) Modestobacter marinus MODMU_1704 (uncharacterized protein) Scytonema sp. NIES-4073 GUN4 domain-containing protein Comamonas aquatica DA1877 AX13_15130 (Uncharacterized protein) Flavobacterium micromati TIR domain containing protein (SAMN05444372_11431) Lacunisphaera limnophila Invasion protein regulator (Verru16b_00714) Aquimarina amphilecti Uncharacterized protein (SAM N04487910_4078) Thioflavicoccus mobilis 8321 Uncharacterized protein (Thimo_2887)

Sequence CWU 1

1

431135PRTStaphylococcus aureus 1Glu Tyr Asp Val Phe Leu Ser His Ser Leu Asp Lys Glu Asp Tyr Val1 5 10 15Ser Lys Ile Ser Glu Lys Leu Ile Glu Lys Gly Leu Lys Phe Glu Asp 20 25 30Val Lys Val Phe Glu Ile Gly Lys Ser Gln Thr Glu Thr Met Asn Met 35 40 45Gly Ile Asn Ser Arg Phe Val Val Val Phe Leu Ser Pro Asn Phe Ile 50 55 60Glu Ser Gly Trp Ser Arg Tyr Glu Phe Leu Ser Phe Leu Asn Arg Glu65 70 75 80Ile Asn Glu His Val Ile Ile Leu Pro Ile Trp His Lys Val Ser Val 85 90 95Glu Asp Val Arg Ala Tyr Asn Pro Tyr Leu Val Asp Lys Tyr Ala Leu 100 105 110Asn Thr Ser Asp Phe Ser Ile Glu Glu Ile Val Glu Lys Ile Tyr Gln 115 120 125Val Ile Val Asn Ser Lys Asn 130 1352134PRTEscherichia coli 2His Tyr Asp Phe Phe Ile Ser His Ala Lys Glu Asp Lys Asp Thr Phe1 5 10 15Val Arg Pro Leu Val Asp Glu Leu Asn Arg Leu Gly Val Ile Trp Tyr 20 25 30Asp Glu Gln Thr Leu Glu Val Gly Asp Ser Leu Arg Arg Asn Ile Asp 35 40 45Leu Gly Leu Arg Lys Ala Asn Tyr Gly Ile Val Ile Leu Ser His Asn 50 55 60Phe Leu Asn Lys Lys Thr Gln Tyr Glu Leu Asp Ser Leu Ile Asn Arg65 70 75 80Ala Val Tyr Asp Asn Lys Ile Leu Pro Ile Trp His Asn Ile Asn Ala 85 90 95Gln Glu Val Ser Lys Tyr Ser His Tyr Leu Ala Asp Lys Met Ala Leu 100 105 110Gln Thr Ser Leu Tyr Ser Val Lys Glu Ile Ala Arg Glu Leu Ala Glu 115 120 125Ile Ala Tyr Arg Arg Arg 1303135PRTThermoplasmatales archaeon 3Glu Tyr Asp Val Phe Ile Cys His Ala His Glu Asp Lys Glu Phe Phe1 5 10 15Val Arg Glu Leu Ala Glu Lys Leu His Ser Lys Gly Leu Arg Val Trp 20 25 30Tyr Asp Glu Phe Thr Leu Ser Leu Gly Asp Asn Leu Arg Arg Ser Ile 35 40 45Glu Asn Gly Leu Ala Lys Ser Arg Tyr Gly Ile Val Val Leu Ser Lys 50 55 60Arg Phe Phe Glu Lys Glu Trp Pro Gln Lys Glu Leu Asp Gly Leu Val65 70 75 80Ala Lys Glu Val Glu Gly Lys Val Ile Leu Pro Val Trp His Gly Ile 85 90 95Thr Arg Glu Glu Val Gln Ser Phe Ser Ser Ile Leu Ala Asn Arg Leu 100 105 110Ala Ala Ser Ser Glu Lys Gly Ile Asp Tyr Val Val Asn Glu Ile Leu 115 120 125Arg Val Leu Arg Lys Lys Ser 130 1354173PRTActinoplanes 4Arg Tyr Asp Ala Phe Ile Ser Tyr Ser His Ala Asp Gly Ala Leu Ala1 5 10 15Pro Ala Val Gln Arg Gly Leu Gln Arg Leu Ala Arg Trp His Arg Pro 20 25 30Arg Ala Leu Glu Arg Asp Gln Thr Gly Leu Ala Val Ser His Ala Leu 35 40 45Trp Ser Ser Ile Lys Val Ala Leu Asp Gln Ser Glu Phe Phe Val Leu 50 55 60Leu Ala Ser Pro Glu Ala Ala Ala Ser Pro Trp Val Asn Gln Glu Ile65 70 75 80Glu His Trp Leu Ser Arg His Ser Val Asp Arg Leu Pro Val Thr Ser 85 90 95Gly Glu Trp Val Trp Asp Ala Asp Ala Gly Asp Val Asp Leu Glu Arg 100 105 110Ser Thr Ala Val Pro Pro Ala Leu Arg Gly Val Phe Gly Glu Glu Pro 115 120 125Arg His Leu Asp Leu Arg Trp Ala Arg Ala Glu His Glu Leu Asp Leu 130 135 140Arg His Gly Arg Phe Arg Asp Ala Ile Ala Glu Leu Ala Ala Gly Met145 150 155 160His Gly Met Ser Lys Glu Asp Leu Asp Gly Glu Asp Val 165 1705135PRTAcinetobacter baumannii 5Glu Tyr Asp Leu Phe Ile Ser His Ala Ser Glu Asp Lys Glu Asp Phe1 5 10 15Val Arg Pro Leu Ala Glu Thr Leu Gln Gln Leu Gly Val Asn Trp Tyr 20 25 30Asp Glu Phe Thr Leu Lys Val Gly Asp Ser Leu Arg Gln Lys Ile Asp 35 40 45Ser Gly Leu Arg Asn Ser Lys Tyr Gly Thr Val Val Leu Ser Thr Asp 50 55 60Ile Ile Lys Lys Asp Trp Thr Asn Tyr Glu Leu Asp Gly Leu Val Ala65 70 75 80Arg Glu Met Asn Gly His Lys Met Ile Leu Pro Ile Trp His Lys Ile 85 90 95Thr Lys Asn Asp Val Leu Asp Tyr Ser Pro Asn Leu Ala Asp Lys Val 100 105 110Ala Leu Asn Thr Ser Val Asn Ser Ile Glu Glu Ile Ala His Gln Leu 115 120 125Ala Asp Val Ile Leu Asn Arg 130 1356171PRTBrucella 6Gln Ala Val Lys Val Ala Gln Glu Gln Lys Arg Leu Ser Asp Glu Arg1 5 10 15Thr Lys His Glu Ala Phe Ile Lys Gln Ser Leu Ser Met Arg Thr Thr 20 25 30Ala Ser Ala Thr Met Glu Ala Glu Glu Tyr Asp Phe Phe Ile Ser His 35 40 45Ala Ser Glu Asp Lys Glu Ala Phe Val Gln Asp Leu Val Ala Leu Arg 50 55 60Asp Leu Gly Ala Lys Phe Tyr Asp Ala Tyr Thr Leu Lys Val Gly Asp65 70 75 80Ser Leu Arg Arg Lys Ile Asp Gln Gly Leu Ala Asn Ser Lys Phe Gly 85 90 95Ile Val Val Leu Ser Glu Ile Ile Phe Phe Ser Lys Gln Trp Pro Ala 100 105 110Arg Glu Leu Asp Gly Leu Thr Ala Met Glu Ile Gly Gln Thr Arg Ile 115 120 125Leu Pro Ile Trp His Lys Val Ser Tyr Asp Glu Val Arg Arg Phe Ser 130 135 140Pro Ser Leu Ala Asp Lys Val Ala Leu Asn Thr Ser Leu Lys Ser Val145 150 155 160Glu Glu Ile Ala Lys Glu Leu His Ser Leu Ile 165 1707168PRTEnterococcus faecalis 7Met Ser Asn Gly Lys Ile Phe Ile Ser His Ser Lys Asp Gln Glu Tyr1 5 10 15Val Asp Ala Phe Ile Gln Leu Leu Lys Phe Gly Phe Arg Thr Gln Asp 20 25 30Ile Phe Tyr Ser Thr Ile Glu Thr Gly Val Gln Pro Gly Glu Leu Ile 35 40 45Phe Asp Thr Ile Lys Arg Glu Leu Thr Asn Gln Pro Val Met Leu Tyr 50 55 60Phe Leu Ser Asp His Tyr Gln Ser Ile Pro Cys Leu Asn Glu Met Gly65 70 75 80Ala Ser Trp Met Leu Ser Asp Lys His Tyr Pro Ile Ala Leu Asn Asn 85 90 95Phe Ser Met Lys Asp Met Lys Gly Val Ile Ser Ser Glu Arg Leu Ala 100 105 110Ile Ala Phe Asn Asp Lys Thr Ser Thr Asn Glu Ile Asn Cys Leu Leu 115 120 125Lys Lys Leu Ser His Asp Thr Asp Val Gln Ala Glu Pro Asp Phe Glu 130 135 140Leu Asn Val Glu Lys Asn Ile Gln Pro Phe Gln Asn Lys Leu Thr Gln145 150 155 160Leu Ile Arg Gln Ala Ser Tyr Leu 1658148PRTParacoccus denitrificans 8Ser Ala Met Lys Pro Thr Ala Gly Pro Thr Asn Ala Asp Leu Thr Ser1 5 10 15Ala Pro His Asp Ile Phe Ile Ser His Ala Trp Glu Asp Lys Ala Asp 20 25 30Phe Val Glu Ala Leu Ala His Thr Leu Arg Ala Ala Gly Ala Glu Trp 35 40 45Tyr Asp Asp Phe Ser Leu Arg Pro Gly Asp Ser Leu Arg Ser Ile Asp 50 55 60Lys Gly Leu Gly Ser Arg Phe Gly Ile Val Leu Ser Thr His Phe Phe65 70 75 80Lys Lys Glu Trp Pro Gln Lys Glu Leu Asp Gly Leu Phe Gln Leu Glu 85 90 95Ser Ser Cys Arg Ser Arg Ile Leu Pro Ile Trp His Lys Val Ser Lys 100 105 110Asp Glu Val Ala Ser Phe Ser Pro Ile Met Ala Asp Lys Leu Ala Phe 115 120 125Asn Ile Ser Thr Lys Ser Val Asp Glu Ile Val Ala Asp Leu Met Ala 130 135 140Ile Ile Arg Asp1459133PRTM_olleaye 9Glu Tyr Asp Ile Phe Val Ser His Ser Glu Asp Lys Glu Asp Phe Val1 5 10 15Lys Glu Phe Val Asn Leu Leu Lys Gln Lys Gly Leu Ser Val Trp Tyr 20 25 30Asp Asp Asp Ile Val Lys Ile Gly His Asn Leu Arg Lys Arg Ile Ser 35 40 45Lys Gly Ile Lys Ser Asn Tyr Ala Val Ile Phe Ser Glu Asp Phe Phe 50 55 60Lys Ser Lys Trp Thr Asn Tyr Glu Tyr Asp Asn Ile Phe Leu Asp Phe65 70 75 80Tyr Asp Glu Lys Val Leu Pro Ile Leu His Asp Leu Thr Ile Glu Asp 85 90 95Leu Glu Lys Ile Asp Gly Ser Ile Pro Leu Ile Arg Ala Leu Ser Ile 100 105 110Lys Lys Phe Thr Val Glu Glu Ile Ile His Glu Ile Leu Glu Arg Ile 115 120 125Asn Glu Glu Lys Ser 13010162PRTHomo sapiens 10Thr Pro Asp Val Phe Ile Ser Tyr Arg Asn Ser Gly Ser Gln Leu Ala1 5 10 15Ser Leu Leu Lys Val His Leu Gln Leu His Gly Phe Ser Val Phe Ile 20 25 30Asp Val Glu Lys Leu Glu Ala Gly Lys Phe Glu Asp Lys Leu Ile Gln 35 40 45Ser Val Met Gly Ala Arg Asn Phe Val Leu Val Leu Ser Pro Gly Ala 50 55 60Leu Asp Lys Cys Met Gln Asp His Asp Cys Lys Asp Trp Val His Lys65 70 75 80Glu Ile Val Thr Ala Leu Ser Cys Gly Lys Asn Ile Val Pro Ile Asp 85 90 95Gly Phe Glu Trp Pro Glu Pro Gln Val Leu Pro Glu Asp Met Gln Ala 100 105 110Val Leu Thr Phe Asn Gly Ile Lys Trp Ser His Glu Tyr Gln Glu Ala 115 120 125Thr Ile Glu Lys Ile Ile Arg Phe Leu Gln Gly Arg Ser Ser Arg Asp 130 135 140Ser Ser Ala Gly Ser Asp Thr Ser Leu Glu Gly Ala Ala Pro Met Gly145 150 155 160Pro Thr11170PRTStreptomyces rimofaciens 11Gly His Met Thr Thr Thr Pro Lys Pro Arg Thr Ala Pro Ala Val Gly1 5 10 15Ser Val Phe Leu Gly Gly Pro Phe Arg Gln Leu Val Asp Pro Arg Thr 20 25 30Gly Val Met Ser Ser Gly Asp Gln Asn Val Phe Ser Arg Leu Ile Glu 35 40 45His Phe Glu Ser Arg Gly Thr Thr Val Tyr Asn Ala His Arg Arg Glu 50 55 60Ala Trp Gly Ala Glu Phe Leu Ser Pro Ala Glu Ala Thr Arg Leu Asp65 70 75 80His Asp Glu Ile Lys Ala Ala Asp Val Phe Val Ala Phe Pro Gly Val 85 90 95Pro Ala Ser Pro Gly Thr His Val Glu Ile Gly Trp Ala Ser Gly Met 100 105 110Gly Lys Pro Met Val Leu Leu Leu Glu Arg Asp Glu Asp Tyr Ala Phe 115 120 125Leu Val Thr Gly Leu Glu Ser Gln Ala Asn Val Glu Ile Leu Arg Phe 130 135 140Ser Gly Thr Glu Glu Ile Val Glu Arg Leu Asp Gly Ala Val Ala Arg145 150 155 160Val Leu Gly Arg Ala Gly Glu Pro Thr Val 165 17012152PRTRattus norvegicus 12Met Arg Arg Ser Val Tyr Phe Cys Gly Ser Ile Arg Gly Gly Arg Glu1 5 10 15Asp Gln Ala Leu Tyr Ala Arg Ile Val Ser Arg Leu Arg Arg Tyr Gly 20 25 30Lys Val Leu Thr Glu His Val Ala Asp Ala Glu Leu Glu Pro Leu Gly 35 40 45Glu Glu Ala Ala Gly Gly Asp Gln Phe Ile His Glu Gln Ala Leu Asn 50 55 60Trp Leu Gln Gln Ala Asp Val Val Val Ala Glu Val Thr Gln Pro Ser65 70 75 80Leu Gly Val Gly Tyr Glu Leu Gly Arg Ala Val Ala Leu Gly Lys Pro 85 90 95Ile Leu Cys Leu Phe Arg Pro Gln Ser Gly Arg Val Leu Ser Ala Met 100 105 110Ile Arg Gly Ala Ala Asp Gly Ser Arg Phe Gln Val Trp Asp Tyr Ala 115 120 125Glu Gly Glu Val Glu Thr Met Leu Asp Arg Tyr Phe Glu Ala Tyr Leu 130 135 140Val Glu His His His His His His145 15013176PRTEscherichia coli 13Met Gly Ser Asp Lys Ile His His His His His His Ser Ser Gly Glu1 5 10 15Asn Leu Tyr Phe Gln Gly His Met Ser Lys Arg Val Phe Leu Ala Ala 20 25 30Pro Phe Lys Gly Ala Ile Lys Glu Lys Gln Ser Ile Met Lys Glu Gln 35 40 45Glu Lys Lys Arg Ile Glu Asp Leu Ile Leu Phe Leu Glu Glu Lys Gly 50 55 60Trp Glu Val Asp Asn Ala His Lys Arg Glu Glu Trp Gly Ala Asn Phe65 70 75 80Met Ser Pro Asp Gln Cys Thr Lys Leu Asp Tyr Asp Ala Ile Lys Glu 85 90 95Cys Asp Leu Phe Ile Ala Phe Pro Gly Val Pro Val Ser Pro Gly Thr 100 105 110His Ile Glu Ile Gly Trp Ala Ser Ala Met Gly Lys Lys Ile Ile Leu 115 120 125Leu Leu Ala Glu Lys Glu Glu Asn Tyr Ala Tyr Leu Ile Arg Gly Leu 130 135 140His Thr Val Ser Asn Val His Tyr Ile Ile Tyr Asn Lys Glu Lys Glu145 150 155 160Tyr Leu Gln Lys Leu Asp Leu Tyr Leu Asp Gly Glu Asn Asn Glu Val 165 170 17514167PRTLactobacillus helveticus 14Met Lys Ala Val Val Pro Thr Gly Lys Ile Tyr Leu Gly Ser Pro Phe1 5 10 15Tyr Ser Asp Ala Gln Arg Glu Arg Ala Ala Lys Ala Lys Glu Leu Leu 20 25 30Ala Lys Asn Pro Ser Ile Ala His Val Phe Phe Pro Phe Asp Asp Gly 35 40 45Phe Thr Asp Pro Asp Glu Lys Asn Pro Glu Ile Gly Gly Ile Arg Ser 50 55 60Met Val Trp Arg Asp Ala Thr Tyr Gln Asn Asp Leu Thr Gly Ile Ser65 70 75 80Asn Ala Thr Cys Gly Val Phe Leu Tyr Asp Met Asp Gln Leu Asp Asp 85 90 95Gly Ser Ala Phe Glu Ile Gly Phe Met Arg Ala Met His Lys Pro Val 100 105 110Ile Leu Val Pro Phe Thr Glu His Pro Glu Lys Glu Lys Lys Met Asn 115 120 125Leu Met Ile Ala Gln Gly Val Thr Thr Ile Ile Asp Gly Asn Thr Glu 130 135 140Phe Glu Lys Leu Ala Asp Tyr Asn Phe Asn Glu Cys Pro Ser Asn Pro145 150 155 160Val Arg Gly Tyr Gly Ile Tyr 16515152PRTEscherichia coli 15Met Arg Pro Ala Leu Tyr Phe Cys Gly Ser Ile Arg Gly Gly Arg Glu1 5 10 15Asp Arg Thr Leu Tyr Glu Arg Ile Val Ser Arg Leu Arg Arg Phe Gly 20 25 30Thr Val Leu Thr Glu His Val Ala Ala Ala Glu Leu Gly Ala Arg Gly 35 40 45Glu Glu Ala Ala Gly Gly Asp Arg Leu Ile His Glu Gln Asp Leu Glu 50 55 60Trp Leu Gln Gln Ala Asp Val Val Val Ala Glu Val Thr Gln Pro Ser65 70 75 80Leu Gly Val Gly Tyr Glu Leu Gly Arg Ala Val Ala Phe Asn Lys Arg 85 90 95Ile Leu Cys Leu Phe Arg Pro Gln Ser Gly Arg Val Leu Ser Ala Met 100 105 110Ile Arg Gly Ala Ala Asp Gly Ser Arg Phe Gln Val Trp Asp Tyr Glu 115 120 125Glu Gly Glu Val Glu Ala Leu Leu Asp Arg Tyr Phe Glu Ala Asp Pro 130 135 140Leu Glu His His His His His His145 15016157PRTLactobacillus leichmannii 16Met Pro Lys Lys Thr Ile Tyr Phe Gly Ala Gly Trp Phe Thr Asp Arg1 5 10 15Gln Asn Lys Ala Tyr Lys Glu Ala Met Glu Ala Leu Lys Glu Asn Pro 20 25 30Thr Ile Asp Leu Glu Asn Ser Tyr Val Pro Leu Asp Asn Gln Tyr Lys 35 40 45Gly Ile Arg Val Asp Glu His Pro Glu Tyr Leu His Asp Lys Val Trp 50 55 60Ala Thr Ala Thr Tyr Asn Asn Asp Leu Asn Gly Ile Lys Thr Asn Asp65 70 75 80Ile Met Leu Gly Val Tyr Ile Pro Asp Glu Glu Asp Val Gly Leu Gly 85 90 95Met Glu Leu Gly Tyr Ala Leu Ser Gln Gly Lys Tyr Val Leu Leu Val 100 105 110Ile Pro Asp Glu Asp Tyr Gly Lys Pro Ile Asn Leu Met Ser Trp Gly 115

120 125Val Ser Asp Asn Val Ile Lys Met Ser Gln Leu Lys Asp Phe Asn Phe 130 135 140Asn Lys Pro Arg Phe Asp Phe Tyr Glu Gly Ala Val Tyr145 150 15517161PRTEscherichia coli 17Met Ala His His His His His His Met Arg Lys Ile Tyr Ile Ala Gly1 5 10 15Pro Ala Val Phe Asn Pro Asp Met Gly Ala Ser Tyr Tyr Asn Lys Val 20 25 30Arg Glu Leu Leu Lys Lys Glu Asn Val Met Pro Leu Ile Pro Thr Asp 35 40 45Asn Glu Ala Thr Glu Ala Leu Asp Ile Arg Gln Lys Asn Ile Gln Met 50 55 60Ile Lys Asp Cys Asp Ala Val Ile Ala Asp Leu Ser Pro Phe Arg Gly65 70 75 80His Glu Pro Asp Cys Gly Thr Ala Phe Glu Val Gly Cys Ala Ala Ala 85 90 95Leu Asn Lys Met Val Leu Thr Phe Thr Ser Asp Arg Arg Asn Met Arg 100 105 110Glu Lys Tyr Gly Ser Gly Val Asp Lys Asp Asn Leu Arg Val Glu Gly 115 120 125Phe Gly Leu Pro Phe Asn Leu Met Leu Tyr Asp Gly Val Glu Val Phe 130 135 140Asp Ser Phe Glu Ser Ala Phe Lys Tyr Phe Leu Ala Asn Phe Pro Ser145 150 155 160Lys18161PRTEscherichia coli 18Met Ala His His His His His His Met Arg Lys Ile Tyr Ile Ala Gly1 5 10 15Pro Ala Val Phe Asn Pro Asp Met Gly Ala Ser Tyr Tyr Asn Lys Val 20 25 30Arg Glu Leu Leu Lys Lys Glu Asn Val Met Pro Leu Ile Pro Thr Asp 35 40 45Asn Glu Ala Thr Glu Ala Leu Asp Ile Arg Gln Lys Asn Ile Gln Met 50 55 60Ile Lys Asp Cys Asp Ala Val Ile Ala Asp Leu Ser Pro Phe Arg Gly65 70 75 80His Glu Pro Asp Cys Gly Thr Ala Phe Glu Val Gly Cys Ala Ala Ala 85 90 95Leu Asn Lys Met Val Leu Thr Phe Thr Ser Asp Arg Arg Asn Met Arg 100 105 110Glu Lys Tyr Gly Ser Gly Val Asp Lys Asp Asn Leu Arg Val Glu Gly 115 120 125Phe Gly Leu Pro Phe Asn Leu Met Leu Tyr Asp Gly Val Glu Val Phe 130 135 140Asp Ser Phe Glu Ser Ala Phe Lys Tyr Phe Leu Ala Asn Phe Pro Ser145 150 155 160Lys19437PRTCoprococcus eutactus 19Met Lys Lys Asp Val Phe Ile Ser Tyr Ser Ser Lys Asp Phe Asp Lys1 5 10 15Val Asn Ser Val Lys Asn Ile Leu Glu Ile Asn Gly Ile Ser Cys Trp 20 25 30Met Ala Pro Gln Cys Ile Pro Pro Gly Ser Ser Tyr Ala Lys Glu Ile 35 40 45Pro Met Ala Ile Lys Asn Cys Lys Val Phe Leu Leu Met Leu Thr Ser 50 55 60Lys Ser Gln Glu Ser Gln Trp Val Pro Lys Glu Val Ser Leu Ala Leu65 70 75 80Ser Glu Thr Lys Cys Val Ile Pro Phe Val Ile Glu Asp Cys Ile Leu 85 90 95Thr Asp Met Phe Asn Phe Phe Leu Thr Asp Val Gln Arg Tyr Tyr Ala 100 105 110Phe Glu Leu Gln Ser Asp Ser Leu Arg Glu Leu Ile Glu Arg Ile Lys 115 120 125Phe Glu Cys Asn Lys Glu Ser Glu Thr Val Ser Ser Phe Ser Ile Asp 130 135 140Lys Ile Ser Asp Ile Glu Thr Trp Leu Ile Ser Lys Glu Arg Asn Ala145 150 155 160Leu Asn Arg Glu Phe Ser Ile Phe Leu Thr Lys Leu Lys Lys Met Tyr 165 170 175Lys Asp Asn Pro Asn Ala Glu Met Ile Glu Thr Trp Val Asp Lys Tyr 180 185 190Ala Val Glu Gln Tyr Thr Ile Arg Thr Val Met Glu Ser Leu Asp Lys 195 200 205Cys Ser Ile Tyr Leu Thr Gly Val Ala Glu Thr Lys Ile Gln Leu Ala 210 215 220Val Ile Tyr Ile His Ser Gly Val Lys Lys Tyr Ile Lys Asp Ala Arg225 230 235 240Asn Leu Leu Asn Ser Ala Ile Lys Thr Tyr Leu Asn Gln Ser Val Tyr 245 250 255Asp Asp Val Ala Phe Lys Arg Ile Val Tyr Ser Lys Trp Leu Ile Ala 260 265 270Val Thr Tyr Lys Gln Glu Arg Asn Phe Gly Tyr Ala Asn Glu Leu Cys 275 280 285Glu Asp Leu Ile Asp Cys Ile Asn Asp Glu Asn Gln Ile Phe Glu Val 290 295 300Pro Tyr Thr Asp Ser Leu Leu Leu Pro Gln Arg Glu Leu Ile Val Ile305 310 315 320Asn Lys Glu Glu Ile Met Ser Asp Phe Leu Leu Ile His Met Asp Asp 325 330 335Ile Ser Met Asn Pro Lys Glu Leu Phe Tyr Thr Gln Arg Arg Leu Leu 340 345 350Glu Leu Tyr Ile Leu Asn Asn Asp Phe Glu Lys Ala Lys Gly Ile Leu 355 360 365Pro Glu Leu Leu Ser Ser Phe Asp Arg Cys Lys Ser Phe Val Asp Glu 370 375 380Ile Tyr His Val Gly Leu Tyr Gln Asn Leu Phe Glu Tyr Tyr Ser Tyr385 390 395 400Val Gly Asp Lys Gln Ser Ala Glu Lys Tyr Tyr Arg Met Ala Leu Glu 405 410 415Asn Ala Lys Arg Asn Tyr Trp Lys Gly Lys Glu Lys Lys Leu Glu Asn 420 425 430Leu Lys Gln Val Phe 43520223PRTProteus penneri 20Met Ala Arg Cys Thr Ala Pro Val Arg Gly His Ser Ser Ala Ala Ala1 5 10 15Ala Ala Ala Cys Pro Ala Cys Arg Gly Arg Ser Ser Tyr Arg Tyr Ser 20 25 30Ser Ser Tyr Phe Pro Ala Ser Asn Ser Gly Val Ser Tyr Gly Ser Ser 35 40 45Ser Ser Ser Ser Ile Pro Arg Trp Ser Lys Ser Gly Ser Ser Ile Pro 50 55 60Tyr Thr Thr Ala Gln Val Gln Ser Leu Thr Pro Ile Arg Glu Thr Val65 70 75 80Glu Glu Arg Ala Ile Lys Gln Pro Asp Leu Arg Asp Val Phe Leu Cys 85 90 95His Ala Trp Asp Asp Arg Gln Gly Val Ala Lys Glu Leu His Asp Leu 100 105 110Leu Glu Ala Ala Gly Val Lys Val Trp Phe Ser Glu Lys Asp Leu Asn 115 120 125Leu Gly Val Pro Met Met Arg Ala Ile Asp Lys Gly Leu Ala Asn Ser 130 135 140Arg Ile Gly Leu Val Leu Val Thr Pro Ala Leu Leu Glu Arg Ile Gln145 150 155 160Arg Glu Gly Val Ala Asp Lys Glu Leu Ser Val Leu Leu Ala Gly Asn 165 170 175Gln Leu Val Pro Ile Val His Asn Thr Thr Tyr Glu Ala Leu Arg Asn 180 185 190Ile Ser Pro Leu Leu Ala Ser Arg Ser Gly Leu Asp Thr Gly Glu Asp 195 200 205Thr Leu Glu Ile Val Ala Thr Lys Ile Ser Glu Leu Val Thr Ile 210 215 22021352PRTBacteroides thetaiotaomicron 21Met Glu Glu Asn Lys Tyr Phe Ala Phe Ile Ser Tyr Ser Arg Lys Asp1 5 10 15Leu Gly Val Ala Lys Gln Ile Lys Asn Asn Ile Glu Ser Ile Thr Gly 20 25 30Lys Lys Cys Trp Met Asp Met Glu Gly Ile Glu Ser Gly Glu Gln Phe 35 40 45Val Asp Val Ile Val Ser Ala Ile Asp Asn Ser Lys Tyr Val Ile Phe 50 55 60Leu Leu Ser Asp Asn Ser Met Ala Ser Lys Tyr Ala Gln Lys Glu Ile65 70 75 80Thr Tyr Ala Asp Lys Val Gly Lys Lys Leu Ile Pro Leu Asn Ile Asp 85 90 95Arg Cys Thr Pro Lys Gly Trp Phe Leu Phe Asn Phe Gly Glu Val Asp 100 105 110Val Ile Asp Val Asn Val His Glu Gln Leu Glu Lys Phe Tyr Ser Asn 115 120 125Ile Lys Ser Trp Ser Ser Asn Gln Asn Leu Glu Lys Ser Phe Pro Ser 130 135 140Leu Phe Leu Asp Glu Ser Gly Gln Lys Lys Val Leu Tyr Leu Met Phe145 150 155 160Leu Ile Asp Ser Ser Gly Ser Met Tyr Gly Glu Arg Met Glu Ala Leu 165 170 175Asn Ala Ser Leu Ala Ser Val Phe Glu Asn Phe Ser Ile Ile Asn Pro 180 185 190Asp Ile Asp Ile Lys Ile Asn Ile Leu Gln Phe Ser Ser Gly Cys Gln 195 200 205Trp Met Tyr Ala Glu Pro Ile Asn Ile Glu Phe Phe Arg Trp Lys Leu 210 215 220Ile Asp Ala Arg Gly Leu Thr Asp Leu Gly Tyr Ala Cys His Glu Leu225 230 235 240Asp Asn Val Met Ser Lys Lys Ile Leu Phe Ser Leu Asp Asn Cys Pro 245 250 255Ala Gly Ile Ile Pro Pro Val Leu Phe Leu Ile Thr Asp Gly Glu Pro 260 265 270Thr Asp Glu Tyr Leu Pro His Met Ala Lys Leu Trp Asn Asn His Phe 275 280 285Phe Lys Lys Ser Asn Lys Phe Ala Ile Gly Leu Gly Gly Asp Phe Ser 290 295 300Ile Asp Ala Leu Lys Leu Phe Thr Lys Asn Gln Lys Gln Val Phe Thr305 310 315 320Ile Ser Asp Glu Ala Leu Asn Glu Phe Lys Ala Leu Val Thr Arg Leu 325 330 335Leu Thr Met Ser Leu Tyr Ala Gly Ser Met Ala Ser Leu Asn Glu Glu 340 345 35022285PRTEubacterium hallii 22Met Gly Val Glu Gln Tyr Gln Arg Lys Val Asn Ser Leu Asp Lys Glu1 5 10 15Ile Ala Asp Leu Glu Lys Lys Lys Ala Glu Glu Asp Arg Lys Ala Ala 20 25 30Asp Asn His Lys Lys Ala Ser Arg Val Ser Val Ser Lys Asn Ala Ser 35 40 45Glu Ser Met Ile Lys Ser Lys Met Arg Gln Ile Glu Asn Tyr Glu Glu 50 55 60Lys Ala Arg Lys Ala Glu Ala Ala Ser Ala Asp Tyr Gly Lys Lys Ile65 70 75 80Ser Asp Lys Arg Thr Lys Arg Asn Asp Ala Tyr Leu Lys Leu Gln Lys 85 90 95Glu Glu Gln Asn Glu Arg Lys Lys Gln Glu Lys Ser Ile Glu Asn Met 100 105 110Lys Arg Val Tyr Glu Gln Arg Ile Ser Glu Leu Glu Ser Leu Arg Leu 115 120 125Ser Lys Val Lys Asn Glu Phe Leu Glu Thr Val Ser Gly Asp Glu Pro 130 135 140Glu Tyr Asp Val Phe Val Ser His Ala Trp Glu Asp Lys Ala Asp Phe145 150 155 160Val Glu Ala Phe Val Gln Ala Leu Lys Asp Arg Glu Ile Lys Val Trp 165 170 175Tyr Asp Lys Ser Lys Ile Lys Trp Gly Asp Ser Met Arg Ala Arg Ile 180 185 190Asp Asp Gly Leu Arg Lys Ser Lys Phe Gly Val Ala Ile Leu Ser Pro 195 200 205Asn Tyr Ile Ala Glu Gly Lys Tyr Trp Thr Lys Ala Glu Leu Asp Gly 210 215 220Leu Phe Gln Met Glu Ser Ile Asn Gly Lys Thr Leu Leu Pro Ile Trp225 230 235 240His Asn Leu Thr Lys Lys Gln Val Met Asp Tyr Ser Pro Ile Leu Ala 245 250 255Ser Lys Leu Ala Met Thr Thr Ala Thr Met Thr Ala Glu Glu Ile Ala 260 265 270Asp Glu Leu Leu Asp Met Leu Ser Ser Glu Glu Asp Lys 275 280 28523413PRTBacteroides thetaiotaomicron 23Met Asp His Glu Val Phe Ile Ser Tyr Ser Ser Ala Asn Ile Gln Thr1 5 10 15Ala Gln Ala Ile Cys His Ala Leu Glu Ser Asn Arg Ile Lys Cys Trp 20 25 30Met Ala Pro Arg Asp Ile Arg Pro Gly Ala Glu Tyr Gly Asp Ile Ile 35 40 45Glu Glu Ala Ile Val Thr Cys Lys Val Phe Leu Ile Val Phe Ser Glu 50 55 60Thr Ser Gln Ile Ser Arg Trp Val Arg Ser Glu Leu Asn Ile Gly Phe65 70 75 80Ser Ser Asn Lys Pro Ile Ile Pro Phe Arg Ile Asp Pro Thr Asp Leu 85 90 95Lys Gly Ser Met Lys Leu Met Leu Asn Asp Lys His Trp Leu Asn Ala 100 105 110Tyr Pro Asn Pro Glu Glu Lys Phe Ser Glu Leu Ala Ala Val Ile Leu 115 120 125Asp Leu Leu Gln His Pro Ala Val Asp Thr Ile Ser Pro Pro Gly Phe 130 135 140Gln Pro Ala Ser Asn Val Ala Ser Ala Asp Gly Ser Asn Ala Gly Ala145 150 155 160Gln Gly Leu Phe Gly Lys Leu Leu Asn Gln Leu Phe Val Lys Arg Arg 165 170 175Val Lys Val Thr Ser Asp His Ala Ala Glu Ile Phe Leu Asn Gly Glu 180 185 190Arg Lys Gly Lys Leu Asp Ala Tyr Glu Thr Gly Asn Tyr Ser Val Ser 195 200 205Glu Asp Phe Tyr Gln Leu Glu Val Tyr Ser Cys Glu Tyr Gly Lys Lys 210 215 220Val Lys Cys Glu Tyr Ser Gly Ser Phe Ser Asn Ser Ser Thr Val Ile225 230 235 240Ile Ser Val Asp Met Ala Thr Ala Glu Lys Gln Tyr Leu Met Lys Ala 245 250 255Lys Asn Ile Thr Ser Gly Gln Ile Asn Asp Leu Gly Ile Ile Phe Leu 260 265 270Asp Glu Gly Lys Phe Glu Asp Ala Gln Glu Cys Phe Leu Lys Ala Ala 275 280 285Ser Met Gly Glu Ala Ala Ala Ala Tyr Asn Leu Gly Met Met Tyr His 290 295 300Phe Gly Lys Gly Val Glu Ile Asp Tyr Asp Val Ala Arg Glu Tyr Tyr305 310 315 320Glu Glu Ala Val Lys Ser Asp Tyr Pro Leu Ala Leu Asn Asn Leu Gly 325 330 335Ser Ile Tyr Tyr Asn Gly His Gly Val Arg Lys Asp Ile Ala Lys Ser 340 345 350Phe Pro Tyr Phe Cys Arg Ala Ala Glu Arg Gly Val Glu Ser Ala Gln 355 360 365Phe Thr Val Ala Thr Met Leu Phe Tyr Gly Gln Gly Val Ala Val Asp 370 375 380Lys Ala Lys Ala Lys Lys Trp Phe Gln Lys Ala Ala Ala Gln Gly Cys385 390 395 400Lys Asp Ser Gln Asn Tyr Leu Asn Ser Trp Val Asp Asp 405 41024173PRTbacteroides xylanisolvens 24Met Ala Leu Asn Arg Phe Tyr Leu Thr Glu Asn Ser Ile Ala Gln Arg1 5 10 15Ser Gly Leu Cys Ile Phe Ile Ser His Gln Lys Arg Asp Ala Asp Thr 20 25 30Ala Lys Lys Ile Ala Asp Tyr Phe Ile Thr Ser Gly Ile Asp Val Tyr 35 40 45Phe Asp Glu Tyr Asp Thr Lys Ile Asp Arg Thr Arg Pro Tyr Ser Val 50 55 60Val Ser Ala Ile Lys Thr Gly Ile Arg Asn Ser Thr His Met Leu Cys65 70 75 80Leu Leu Ser Gln Asn Ala Leu Asp Ser Lys Trp Ile Pro Trp Glu Val 85 90 95Gly Tyr Gly Tyr Asp Arg Thr Thr Val Val Gly Leu Thr Leu Lys Glu 100 105 110Ile Ser Gln Ser Val Leu Pro Glu Tyr Leu Gln Ile Val Pro Ile Leu 115 120 125Arg Gly Thr Lys Ser Leu Asn Asn Phe Ile Ser Asn Val Leu Lys Arg 130 135 140Asp Glu Ser Thr Leu Ile Asn Glu Arg Lys Leu Phe Ala Ala Tyr Gln145 150 155 160Ser Gln His Pro Leu Asp Ser Val Leu Asn Trp Glu Leu 165 17025482PRTClostridium scindens 25Met Lys Glu Asp Arg Ile Pro Lys Ile Phe Ile Ser Tyr Ser Trp Ser1 5 10 15Ser Asp Ala Ile Val Leu Glu Leu Ala Gln Arg Leu Val Ser His Gly 20 25 30Val Asp Val Val Leu Asp Lys Trp Asp Leu Lys Glu Gly Gln Asp Lys 35 40 45Tyr Ala Phe Met Glu Arg Cys Val Asn Asp Thr Glu Ile Thr Lys Val 50 55 60Leu Ile Val Cys Asp Lys Val Tyr Ala Gln Lys Ala Asn Asp Arg Thr65 70 75 80Gly Gly Val Gly Asp Glu Thr Val Ile Ile Ser Ser Glu Ile Tyr Gly 85 90 95Asn Thr Lys Gln Glu Lys Phe Ile Pro Val Ile Ala Glu Lys Ser Glu 100 105 110Asp Gly Lys Ala Tyr Val Pro Thr Tyr Ile Lys Thr Arg Ile Tyr Ile 115 120 125Asp Leu Ser Asp Pro Glu Thr Tyr Glu Ile Glu Tyr Glu Lys Leu Leu 130 135 140Arg Asn Ile Tyr Glu Lys Pro Gln Phe Thr Lys Pro Lys Leu Gly Lys145 150 155 160Met Pro Glu Trp Leu Asp Glu Glu Lys Thr Asp Phe Phe Gln Leu Lys 165 170 175Asp Leu Ile Arg Gln Ile His Gly Ser Asn Thr Asp Asn Lys Arg Lys 180 185 190Ser Cys Ile Ser Arg Phe Gln Ile Thr His Ile Glu Val Leu Lys Gly 195

200 205Tyr Ile Glu Lys Asn Ala Ser Pro Glu Arg Val Tyr Glu Ile Phe Leu 210 215 220Asn Thr Lys Pro Val Arg Asp Phe Tyr Leu Lys Phe Val Glu Thr Ile225 230 235 240Ala Glu Thr Glu Thr Asp Tyr Ala Glu Ile Ile Ala Glu Gly Phe Glu 245 250 255Thr Met Tyr Asn Gln Leu Thr Cys Ile Lys Ala Phe Val Pro Asn Ala 260 265 270Asn Ser Val Ser Gln Asn Asp Leu Asp Ile Phe Lys Ile Tyr Ile Trp 275 280 285Glu Leu Phe Ile Cys Val Ile Ala Phe Met Arg His Ile Lys Asp Tyr 290 295 300Ala Ala Ile Asn Glu Met Leu Thr Tyr Thr Tyr Phe Leu Glu Thr Ser305 310 315 320Ile Phe Gly Gly Ser Ile Lys Pro Asn Asn Tyr Thr Ala Phe Arg His 325 330 335His Ser Cys Val Ile Glu Glu Tyr Tyr Lys Pro Lys Thr Glu Met Lys 340 345 350Asn Lys Tyr Thr Leu Met Gly Asp Val Ile Cys Asn Gln Arg Glu Lys 355 360 365Leu Pro Ile Tyr Thr Gly Glu Ala Ile Ala Glu Ala Asp Leu Phe Leu 370 375 380Tyr Gln Val Cys Asn Ala Tyr Glu Leu Ser Glu Lys Glu Gln Ser Trp385 390 395 400His Lys Val Tyr Trp Phe Pro Thr Cys Tyr Val Tyr Val Lys Asn Met 405 410 415Pro Ile Glu Trp Glu Lys Met Lys Ser Arg Arg Tyr Cys Asp Lys Met 420 425 430Met Val Leu Phe Gly Val Asp Ser Leu Glu Asp Leu Lys Thr Val Ile 435 440 445Ser Arg Cys Thr Phe Asp Ser Gly Met Lys Tyr Pro Gly Ser Trp Asp 450 455 460Ala Ala Pro Ala Ile Leu Asn Cys Ile Gln Ile Glu Asp Ile Gly Met465 470 475 480Leu Ser26200PRTDorea formicigenerans 26Met Lys Tyr Tyr Tyr Asp Val Val Leu Ser Phe Ala Gly Glu Asp Arg1 5 10 15Glu Tyr Val Glu Glu Cys Ala Asp Ile Leu Thr Ala Leu Gly Ile Lys 20 25 30Val Phe Tyr Asp Ser Tyr Glu Gln Asp Val Leu Trp Gly Lys Asp Leu 35 40 45Tyr Thr Phe Leu Ala Asp Ile Tyr Ser Asn Lys Ala Arg Tyr Ala Ile 50 55 60Val Phe Ile Ser Gln His Tyr Val Lys Lys Cys Trp Thr Lys His Glu65 70 75 80Phe Lys Phe Ile Asn Glu Arg Met Phe Asn Ser Glu Thr Glu Tyr Leu 85 90 95Leu Pro Val Phe Leu Asp Asp Thr Lys Leu Cys Gly Ile Pro Glu Thr 100 105 110Gln Gly Tyr Leu Thr Asn Lys Thr Pro Tyr Glu Val Ala Val Met Phe 115 120 125Ala Lys Lys Ile Asn Lys Asp Ile Asp Val Glu Leu Met Lys Ser Glu 130 135 140Leu Gln Gln Ser Leu Pro Thr Tyr Glu Ile Thr Val Arg Gly Arg Asn145 150 155 160Val Arg Phe Tyr Ser Ala Val Glu Glu Phe Asp Ala Glu Tyr Pro Leu 165 170 175Ser Phe Leu Met Glu Leu Tyr Lys Leu Asp Met Met Tyr Asp Leu Phe 180 185 190Ile Leu Pro Glu Leu Val Pro Asn 195 20027136PRTProvidencia rustigianii 27Met Asn Ile Phe Val Ser Tyr Thr Thr Arg Asp Asp Asn Val Asn Lys1 5 10 15Lys Leu Leu Glu Arg Val Ser Glu Ile Val Ser Leu Tyr Gly His Cys 20 25 30Tyr Ile Asp Leu Leu His Asn Thr Lys Lys Asp Lys Gln Gln His Val 35 40 45Glu Phe Met Leu Ser Gln Ser Asn Leu Leu Ile Leu Ile Ala Ser Ser 50 55 60Ser Val Phe Thr Ser Lys Trp Val Gln Trp Glu Leu Asp Glu Ala Lys65 70 75 80Arg Cys Tyr Ile Pro Ile Ile Met Val Glu Ala Lys Ser Asp Met Asn 85 90 95Asn Ile Leu Lys Asn Leu Lys Ser Ile Leu Ala Thr Asn Ala Tyr Leu 100 105 110Leu Ser Asn His Arg Ser Glu Lys Phe Lys Asn Ser Ser Glu Asn Thr 115 120 125Met Ser Leu Leu Gly Gly Asn Val 130 13528267PRTBacteroides coprophilus 28Met Ser Ser Ser Lys Asn Leu Phe Val Ser Ile Ser Tyr Ser Gln Asp1 5 10 15Asn Leu Asp His Lys Arg Trp Val Ser Lys Leu Ala Gln Thr Leu Lys 20 25 30Asn Glu Gly Phe Lys Val Leu Val Asp Ile Phe Asn Leu His Gly Gly 35 40 45Glu Asp Ile Asn Glu Phe Met Glu Asn Val Ile Ile His Ser Asp Lys 50 55 60Val Leu Ile Ile Met Thr Gln Gly Tyr Lys Glu Lys Ala Asp Lys Arg65 70 75 80Phe Ala Gly Ala Gly Tyr Glu Ala Lys Leu Ile Ala Asn Leu Ile Lys 85 90 95Glu Asn Gln His Thr Arg Lys Phe Ile Pro Ile Lys Arg Asp Lys Asp 100 105 110Asp Cys Ile Pro Asn Phe Leu Lys Pro Leu Val Phe Ile Asp Met Ser 115 120 125Asp Asp Asn Leu Phe Asn Glu Lys Ile Glu Asp Leu Leu Leu Ala Ile 130 135 140Ser Gly Ser Asp Ser Asp Tyr Asp Asp Leu Ile Arg Glu Ile Asn Tyr145 150 155 160Gln Ile Asp Ser Leu Gln Pro Tyr Glu Glu Tyr Tyr Gly Asp Thr Ile 165 170 175Pro Leu Ile Glu Gln Lys Lys Tyr Gln Leu Ser Leu Lys Val Gln Asn 180 185 190Ala Gln Ser Glu His Asn Phe Leu Glu Val Ile Lys Tyr Lys Ile Gln 195 200 205Ile Ala Gln Ile Cys Gln Glu Gln Asn Lys Ser Gln Asp Ala Tyr Lys 210 215 220Leu Glu Glu Gly Ile Arg Glu Ala Tyr Ala Met Leu Ser Asp Phe Glu225 230 235 240Lys Val Gln Ala Lys Ser Ile Ile Ala Asp Tyr Asn Ser Asp Met Ala 245 250 255Leu Tyr Ile Asp Gln Asp Ile Asn Arg Asn Lys 260 26529287PRTBacteroides thetaiotaomicron 29Met Asn Ser Ser Tyr Tyr Gln Asn Gln Ile Asn Arg Leu Glu Lys Asp1 5 10 15Ile Ala Asp Leu Gln Lys Lys Ile Ala Asp Glu Asn Lys Lys Glu Ile 20 25 30Asp Lys Asn Lys Gln Ile Asp Ser Val His Arg Thr Ile Asn Lys Asn 35 40 45Thr Ser Ile Ser Thr Leu Asn Ser Lys Gln Arg Gln Ile Asp Gly Tyr 50 55 60Gln Lys Asp Ile Leu Asn Cys Arg Thr Lys Ile Ala Ser Tyr Gln Lys65 70 75 80Ser Ile Ala Thr Lys Ser Ala Glu Leu Gly Lys Lys Arg Gln Glu Leu 85 90 95Leu Lys Ala Gln Gln Ser Glu Gln Lys Lys Leu Gln Asp Asp Gln Leu 100 105 110Lys Phe Gln Lys Lys Leu Gln Ser Glu Ile Glu Ile Gln Lys Arg His 115 120 125Leu Glu Thr Leu Ile Ala Gln Asn Tyr Ser Thr Gln Asn Asn Lys Leu 130 135 140Val Ser Thr Glu Asp Ile Pro Glu Pro Thr Lys Gln Tyr Asp Phe Phe145 150 155 160Ile Ser His Ala Ser Glu Asp Lys Asp Asp Ile Val Arg Asp Leu Ala 165 170 175Glu Ala Leu Arg Asn Asn Gly Phe Glu Val Trp Tyr Asp Glu Phe Glu 180 185 190Leu Lys Ile Gly Asp Ser Leu Arg Lys Lys Ile Asp Tyr Gly Leu Ser 195 200 205Asn Ala Asn Tyr Gly Ile Val Ile Ile Ser Pro Ser Phe Val Lys Lys 210 215 220Asn Trp Thr Glu Tyr Glu Leu Asn Gly Met Val Ala Arg Glu Met Asn225 230 235 240Gly His Lys Val Ile Leu Pro Ile Trp His Lys Ile Thr Lys Asp Glu 245 250 255Val Leu Arg Phe Ser Pro Ser Leu Ala Asp Lys Leu Ala Leu Asn Thr 260 265 270Ser Ile His Thr Ile Asp Asp Ile Val Glu Asn Leu Lys Asn Leu 275 280 28530413PRTBacteroides thetaiotaomicron 30Met Asp His Glu Val Phe Ile Ser Tyr Ser Ser Ala Asn Ile Gln Thr1 5 10 15Ala Gln Ala Ile Cys His Ala Leu Glu Ser Asn Arg Ile Lys Cys Trp 20 25 30Met Ala Pro Arg Asp Ile Arg Pro Gly Ala Glu Tyr Gly Asp Ile Ile 35 40 45Glu Glu Ala Ile Val Thr Cys Lys Val Phe Leu Ile Val Phe Ser Glu 50 55 60Thr Ser Gln Ile Ser Arg Trp Val Arg Ser Glu Leu Asn Ile Gly Phe65 70 75 80Ser Ser Asn Lys Pro Ile Ile Pro Phe Arg Ile Asp Pro Thr Asp Leu 85 90 95Lys Gly Ser Met Lys Leu Met Leu Asn Asp Lys His Trp Leu Asn Ala 100 105 110Tyr Pro Asn Pro Glu Glu Lys Phe Ser Glu Leu Ala Ala Val Ile Leu 115 120 125Asp Leu Leu Gln His Pro Ala Val Asp Thr Ile Ser Pro Pro Gly Phe 130 135 140Gln Pro Ala Ser Asn Val Ala Ser Ala Asp Gly Ser Asn Ala Gly Ala145 150 155 160Gln Gly Leu Phe Gly Lys Leu Leu Asn Gln Leu Phe Val Lys Arg Arg 165 170 175Val Lys Val Thr Ser Asp His Ala Ala Glu Ile Phe Leu Asn Gly Glu 180 185 190Arg Lys Gly Lys Leu Asp Ala Tyr Glu Thr Gly Asn Tyr Ser Val Ser 195 200 205Glu Asp Phe Tyr Gln Leu Glu Val Tyr Ser Cys Glu Tyr Gly Lys Lys 210 215 220Val Lys Cys Glu Tyr Ser Gly Ser Phe Ser Asn Ser Ser Thr Val Ile225 230 235 240Ile Ser Val Asp Met Ala Thr Ala Glu Lys Gln Tyr Leu Met Lys Ala 245 250 255Lys Asn Ile Thr Ser Gly Gln Ile Asn Asp Leu Gly Ile Ile Phe Leu 260 265 270Asp Glu Gly Lys Phe Glu Asp Ala Gln Glu Cys Phe Leu Lys Ala Ala 275 280 285Ser Met Gly Glu Ala Ala Ala Ala Tyr Asn Leu Gly Met Met Tyr His 290 295 300Phe Gly Lys Gly Val Glu Ile Asp Tyr Asp Val Ala Arg Glu Tyr Tyr305 310 315 320Glu Glu Ala Val Lys Ser Asp Tyr Pro Leu Ala Leu Asn Asn Leu Gly 325 330 335Ser Ile Tyr Tyr Asn Gly His Gly Val Arg Lys Asp Ile Ala Lys Ser 340 345 350Phe Pro Tyr Phe Cys Arg Ala Ala Glu Arg Gly Val Glu Ser Ala Gln 355 360 365Phe Thr Val Ala Thr Met Leu Phe Tyr Gly Gln Gly Val Ala Val Asp 370 375 380Lys Ala Lys Ala Lys Lys Trp Phe Gln Lys Ala Ala Ala Gln Gly Cys385 390 395 400Lys Asp Ser Gln Asn Tyr Leu Asn Ser Trp Val Asp Asp 405 41031492PRTRoseburia intestinalis 31Met Phe Asp Ala Phe Ile Ser His Ser Ser Lys Asp Lys Glu Lys Ile1 5 10 15Val Leu Glu Leu Val Gln Lys Leu Glu Lys Asn Asn Ile Asp Val Trp 20 25 30Leu Asp Ala Asn Glu Ile Leu Ala Gly Asp Thr Ile Leu Asp Ala Val 35 40 45Glu Lys Gly Val Ala Thr Ala Leu Cys Thr Ile Leu Ile Ile Thr Pro 50 55 60Ala Phe Phe Glu Ser Phe Trp Thr Pro Val Glu Ile Gly Leu Ala Ile65 70 75 80Gly Gln Asn Asn Asn His Lys Leu Ile Pro Val Leu Cys Asn Val Ser 85 90 95Val Glu Asp Ile Ala Gln Arg Phe Pro Met Leu Leu Ser Leu Lys Tyr 100 105 110Ile Lys Leu Asp Pro Asp Asn Ile Asp Leu Cys Val Asp Glu Leu Cys 115 120 125Ser Asn Ile Ile Gln Ile Lys Lys Lys Tyr Glu Lys Asp Phe Pro Glu 130 135 140Ile Ser Phe His Lys Ala Val Lys Lys Phe His Ser Cys Asp Thr Pro145 150 155 160Thr Ala Asn Thr Ile Ser Ile Leu Leu Ser Glu Tyr Glu Gln Ile Val 165 170 175Glu Ile Asn Val Arg Thr Ala Val Leu His Ala Ser Gln Ile Ala Ile 180 185 190Thr Ile Ile Asn Asp Leu Phe Met Arg Leu Pro Ile Lys Gly Asn Ser 195 200 205Val Asp Thr Thr Ile Gly Lys Leu Asp Leu Ile Lys Val Ser Ser Ile 210 215 220Gly Leu Thr Glu Asn Val Tyr Glu His Leu Lys Leu Leu Ser Thr Pro225 230 235 240Ala Leu Asp Thr Asn Met Ser Leu Leu Thr Asn Asp Leu Asp Arg Arg 245 250 255Lys Leu Ala Glu Met Ser Met Thr Ala Ile Leu Glu Trp Tyr Ser Lys 260 265 270Tyr Leu Leu His Asn Lys Ile Ile Pro Gln Asp Arg Phe Glu Ile Val 275 280 285Trp Pro Asp Asp Leu Ser Tyr Glu Asp Phe Ile Thr Met Tyr Glu Ile 290 295 300Asp Cys Leu Val Leu Arg Glu Asp Leu Ile Ala Pro Pro Ser Ile Thr305 310 315 320Tyr Ala Trp Tyr Gln Tyr Asn Asn Tyr Thr His Ile Ala Val Arg Ser 325 330 335Thr Glu Thr Asn Arg Ile Val Gly Tyr Phe Thr Val Leu Pro Val Thr 340 345 350Asp Gln Leu Tyr Glu Asp Ile Gln Ser Gly Tyr Phe Lys Asp Asn Asp 355 360 365Leu Asn Thr Asp Asn Leu Arg Lys Tyr Asp Val Pro Asp Phe Tyr Lys 370 375 380Leu Tyr Ile Ala Cys Val Cys Ile His Pro Asp Tyr Gln Asn Thr Ser385 390 395 400Ala Phe Arg Lys Leu Tyr Asn Ala Leu Leu Gln Met Met Met Asp Leu 405 410 415Ala Val Glu Arg Glu Ile Tyr Ile Thr Asn Ile Ile Thr Glu Ala Ser 420 425 430Thr Leu Gln Gly Glu Lys Phe Cys Lys Ile Leu Gly Leu Asn Cys Met 435 440 445Leu Asn Thr Glu Ile Glu Thr Lys Ile Tyr Gly Ala Thr Leu Leu Pro 450 455 460Pro Ser Trp Gln Leu Arg Ser Ser Phe Gly Ser Lys Leu Met Lys Tyr465 470 475 480Tyr Lys Glu Lys Tyr Glu Glu Leu Lys Asp Leu Phe 485 49032439PRTBacteroides ovatus 32Met Lys Tyr Lys Tyr Asp Val Phe Ile Ser Tyr Ser Arg Arg Asp Tyr1 5 10 15Val Asp Glu Ser Tyr Asn Val Ile Pro Gly Asn Ala Ile Ala Glu Ile 20 25 30Gln Asn Val Phe Asp Glu Asn Gly Ile Thr Tyr Trp Phe Asp Lys Asp 35 40 45Gly Ile Tyr Ser Gly Gln Glu Phe Ile Glu Ile Ile Thr Gly Ala Ile 50 55 60Ala Glu Ser Lys Ile Leu Ile Phe Ile Ser Ser Lys His Ser Asn Glu65 70 75 80Ser Met Trp Thr Ala Gly Glu Ile Phe Glu Ala Leu Asp Gly Glu Lys 85 90 95Ala Ile Ile Pro Val Lys Ile Asp Asn Ser Gln Tyr Asn Lys Lys Phe 100 105 110Lys Leu Leu Ile Arg Pro Leu Asp Tyr Ile Asp Tyr Leu Glu Asn Pro 115 120 125Lys Asn Ala Leu Lys Asp Leu Leu Arg Ala Ile Asn Lys Val Lys Glu 130 135 140Asp Ile Ala Gln Lys Gln Arg Glu Glu Glu Lys Leu Arg Glu Glu Arg145 150 155 160Glu Ala Glu Ala Lys Lys Glu Lys Ile Lys Lys Glu Ile Ser Val Leu 165 170 175Ala Lys Asp Cys Gln Arg Leu Thr Leu Gln Gln Ile Asp Val Val Asn 180 185 190Gln Ile Phe Glu Lys Gln Ile Tyr Ile Gly Asn Thr Thr Lys Ile Cys 195 200 205Pro Ile Cys Asp Lys Glu Val Ser Ile Ser Ser Asn Phe Cys Asn Arg 210 215 220Cys Gly Trp Thr Phe Pro Ile Leu Tyr Cys Ile Asp Glu Asn Asn Thr225 230 235 240Tyr Pro Leu Asp Glu Glu Gln Leu Ser Ile Ala Arg Thr Asn Trp Cys 245 250 255Ser Ile Ile Lys Asn Ser Leu Glu Pro Glu Lys Gln Pro Leu Lys Ser 260 265 270Ser Pro Leu Gln Ala Thr Glu Glu His Lys Ser Met Thr Asp Asn Lys 275 280 285Tyr Ala Leu Gln Asn Ala Met Asp Glu Ile Glu Thr Leu Arg Lys Tyr 290 295 300Leu Leu Asp Glu Arg Thr Lys Val Lys Glu Ile Gln Glu Glu Tyr Glu305 310 315 320Asn Leu Leu Lys Val Gln Lys Asn Ile Glu Leu Lys Gln Glu Val Pro 325 330 335Ser Ala Arg Glu Ile Gly Asn Ile Ile Leu Asp Lys Phe Ser Thr Thr 340 345 350Asn Ser Lys Lys Tyr Gln Ser Phe Lys Asn Lys Asn Asp Val Phe Asn 355 360 365Phe Val Arg Ser Phe Cys Lys Ala Arg Phe Leu Thr Leu Asn Pro Ser 370

375 380Cys Tyr Val Ala Asp Ile Gln Tyr Thr Asp Leu Lys Asp Ala Leu Tyr385 390 395 400Glu Lys Tyr Asn Ile His Ile Gly Glu His Thr Leu Lys Cys His Ser 405 410 415Thr Leu Tyr Lys Leu Val Asp Ser Ile Trp Asn Glu Tyr Thr Lys His 420 425 430Ser Asn Ser Asp Ala Ser Arg 43533291PRTClostridium bolteae 33Met Ser Ser Glu Gln Tyr Gln Arg Thr Val Asn Ser Leu Asp Lys Glu1 5 10 15Ile Ala Asp Leu Glu Lys Lys Lys Ala Ala Lys Asp Arg Glu Val Ala 20 25 30Asn Leu Gln Gly Lys Ile Asn Thr Leu Lys Lys Ser Ile Asn Ser His 35 40 45Thr Ser Ala Ser Thr Leu Asn Ser Lys Met Arg Gln Ile Ala Ile His 50 55 60Glu Ser Asp Gln Ala Lys Lys Ser Gln Asp Ser Ala Asp Leu Gly Lys65 70 75 80Lys Ile Ala Glu Lys Arg Lys Lys Arg Ala Glu Ala Tyr Leu Arg Leu 85 90 95Gln Lys Glu Gln Gln Asn Glu Gln Lys Lys Gln Asp Lys Val Asn Gln 100 105 110Gln Ile Gln Ala Ser Tyr Glu Ala Arg Ile Lys Glu Leu Gln Gln Gln 115 120 125Leu Leu Gln Pro Val Val Thr Thr Thr Thr Thr Pro Ile Ala Ala Asp 130 135 140Glu Glu Tyr Asp Val Phe Val Ser His Ala Tyr Glu Asp Lys Glu Ser145 150 155 160Phe Val Asp Glu Phe Val Glu Ala Leu Arg Asn Gln Glu Leu Lys Val 165 170 175Trp Tyr Asp Met Asp Lys Leu Lys Trp Gly Asp Ser Met Arg Glu Lys 180 185 190Ile Asp Arg Gly Leu Ala Lys Ser Arg Tyr Gly Val Val Ile Leu Ser 195 200 205Pro Asn Tyr Ile Ala Glu His Lys Tyr Trp Thr Lys Ala Glu Leu Asn 210 215 220Gly Leu Phe Gln Val Glu Thr Val Asn Gly Lys Thr Ile Leu Pro Ile225 230 235 240Trp His Asn Leu Thr Lys Lys Gln Val Val Glu Tyr Ser Pro Ile Ile 245 250 255Ala Asp Arg Lys Ala Met Thr Thr Ala Leu Met Thr Pro Glu Glu Ile 260 265 270Ala Ala Glu Leu Lys Glu Leu Phe Thr Ala Glu Asp Thr Glu Asp Glu 275 280 285Asn Asn Gly 29034706PRTEubacterium cylindroides 34Met Glu Gln Thr Glu Ala Lys Glu Ile Tyr Gln Met Gly Val Glu Ala1 5 10 15Leu Asn Asp Cys Asp Tyr Arg Met Ala Gly Ala Tyr Phe Lys Asn Ala 20 25 30Ala Lys Met Asn Tyr Lys Pro Ala Ile Ala Ala Leu Glu Lys Leu Ile 35 40 45Leu Glu Gly Lys Tyr Lys Asn Val Gln Ser Lys Gln Thr Leu Ser Ser 50 55 60Asn Thr Glu Ser Lys Glu Leu Phe Glu Lys Gly Val Asp Tyr Tyr Lys65 70 75 80Gly Arg Asn Asn Thr Lys Ile Asp Tyr Val Lys Ala Phe Glu Cys Phe 85 90 95Leu Ser Ser Ser Ile Asp Gly Tyr Ala Phe Ala Met Asn Trp Leu Gly 100 105 110Tyr Cys Tyr Glu Tyr Gly Lys Gly Thr Lys Lys Asn Leu Asp Ser Ala 115 120 125Phe Gly Trp Tyr Glu Lys Ala Ala Asn Ala Asn Val Ala Asn Ala Ile 130 135 140Tyr His Leu Gly Arg Leu Tyr Tyr Asp Gly Lys Gly Val Gln Gln Asp145 150 155 160Tyr Asn Lys Ala Phe Gly Leu Phe Lys Lys Ala Cys Asp Leu Arg Glu 165 170 175Ser Leu Ala Tyr Glu Tyr Leu Ala Asp Cys Tyr Phe Tyr Gly Gln Gly 180 185 190Thr Lys Ile Asn Tyr Glu Lys Ala Leu Gln Phe Tyr Leu Lys Val Lys 195 200 205Pro Arg Gly Val Ile Glu Tyr Gln Ile Gly Cys Ile Tyr Ala Arg Gly 210 215 220Leu Ser Thr Gly Ser Asp Tyr Pro Lys Ala Ala Glu His Phe Gln Lys225 230 235 240Gly Val Asp Phe Gly His Ala Pro Ser Met Asn Asn Leu Gly Ala Leu 245 250 255Tyr Tyr Asn Gly Glu Gly Val Gln Lys Asp Tyr Asn Lys Ala Phe Glu 260 265 270Leu Phe Lys Lys Ala Ser Asp Leu Gly Tyr Thr Thr Ala His Gly Trp 275 280 285Leu Ala Asp Cys Tyr Tyr Tyr Gly Asn Gly Ala Lys Lys Asp Tyr Gln 290 295 300Lys Ala Leu Gln Leu Tyr Leu Lys Ala Lys Ser Ser Arg His Asn Glu305 310 315 320Gly Gln Ile Gly Tyr Ile Tyr Glu His Gly Leu Gly Thr Pro Ile Asp 325 330 335Ile Asn Lys Ala Ile Glu His Tyr Gln Lys Gly Val Asp Leu Gly Cys 340 345 350Thr Gly Cys Met His Asn Leu Gly Thr Leu Tyr Tyr Lys Gly Glu Gly 355 360 365Cys Gln Lys Asp Tyr Asn Lys Ala Phe Glu Leu Phe Lys Lys Ala Ser 370 375 380Asp Leu Gly Glu Ile Ala Ala His Gly Trp Leu Ala Lys Cys Tyr Phe385 390 395 400Tyr Gly Tyr Gly Thr Gln Lys Asp Tyr Gln Lys Ala Ile Glu His Tyr 405 410 415Gln Lys Ser Ile Asp Phe Gly Asn Thr Asp Ser Met Ile Ser Leu Gly 420 425 430Ile Leu Tyr Tyr Glu Gly Lys Gly Ile Gln Gln Asp Tyr Gln Lys Ala 435 440 445Phe Glu Leu Phe Lys Lys Ser Ser Asp Leu Glu Asn Lys Thr Ala His 450 455 460Ala Trp Leu Ala Glu Cys Tyr Tyr Tyr Gly Asn Gly Thr Lys Lys Asp465 470 475 480Tyr Gln Lys Ala Leu Gln Leu Tyr Leu Lys Gly Asn Leu Tyr Gly Tyr 485 490 495Thr Gln Ser Gln Ile Gly Arg Ile Tyr Glu Ser Gly Gln Gly Val Lys 500 505 510Lys Asn Leu Thr Glu Ala Leu Asn Trp Tyr Lys Lys Ala Asn Glu Ser 515 520 525Gly Lys Asp Cys Lys Ala Asp Ile Glu Arg Ile Asn Asn Leu Leu Asn 530 535 540Lys Lys Pro Glu Asn Lys Ser Ile Asp Lys Thr Lys Val Glu Thr Lys545 550 555 560Ala Asn Ser Asn Lys Pro Ile Lys Ala Tyr Glu Gly Asp Lys Pro Tyr 565 570 575Ile Phe Val Ser Tyr Ser His Thr Asp Ser Lys Val Val Lys Glu Val 580 585 590Leu Gln Met Leu Ile Asn Gln Gly Tyr Arg Ile Trp Phe Asp Glu Gly 595 600 605Ile Arg Ala Gly Ser Ser Trp Thr Glu Asn Leu Met Asn His Ile Lys 610 615 620Asn Ala Ser His Phe Ile Phe Phe Leu Ser Arg His Ser Ile Asn Ser625 630 635 640Lys Tyr Cys Leu Lys Glu Leu Arg Phe Ala Asp Arg Arg Asn Lys Ile 645 650 655Ile Ile Pro Ile Cys Ile Glu Asn Val Asn Val Ser Asp Glu Ile Asp 660 665 670Phe Leu Leu Gly Glu Val Gln Met Leu Phe Lys Asn Asn Leu Glu Lys 675 680 685Glu Glu Phe Met Glu Lys Phe Asn Ser Ser Asn His Ile Glu Ile Cys 690 695 700Ser Arg70535280PRTEnterococcus phage 35Met Ser Ile Asn Ser Leu Gln Gln Lys Glu Lys Ser Leu Leu Ser Asp1 5 10 15Ile Thr Lys Leu Glu Gly Asp Tyr Ala Arg Glu Gln Lys Lys Ile Ala 20 25 30Asn Ser Glu Lys Lys Ile Ala Asp Ser Ser Lys Lys Ile Glu Ser Ser 35 40 45Lys Ser Ile Ser Thr Ile Arg Ser Gln Ser Arg Ile Lys Glu Ser Glu 50 55 60Thr Lys Lys Ser Leu Ala Ser Lys Glu Lys Ser Ala Thr Ile Ser Ser65 70 75 80Lys Leu Ala Lys Lys Arg Lys Glu Leu Gly Asp Ile Gln Val Lys Leu 85 90 95Ser Lys Gln Arg Thr Ile Glu Asn Thr Gln Phe Gln Lys Asn Leu Lys 100 105 110Lys Thr Tyr Asp Thr Gln Ile Met Lys Ile Lys Glu Thr Gln Thr Ala 115 120 125Ala Leu Gln Asn Val Gln Ser Glu Leu Pro Asn Asp Pro Ser Leu Val 130 135 140Asn Lys Thr Tyr Asp Leu Phe Ile Ser Tyr Ala Ser Asp Asp Ser Glu145 150 155 160Tyr Val Asp Lys Leu Thr Gln Ala Phe Thr Asn Glu Gly Phe Ser Ile 165 170 175Trp Arg Asp Lys Ser Asp Ile Ala Trp Gly Gln Ser Ile Arg Gln Ser 180 185 190Ile Asp Ala Gly Leu Ser Asn Ser Lys Phe Gly Leu Val Val Leu Ser 195 200 205Ser Lys Tyr Ile Glu Lys Phe Trp Thr Asn Tyr Glu Leu Asp Gly Ile 210 215 220Leu Asn Lys Glu Ser Ala Thr Gly Arg Gln Met Ile Leu Pro Leu Trp225 230 235 240His Asn Ile Thr Lys Asp Glu Ile Asp Lys Lys Ser Pro Ser Leu Ser 245 250 255Asn Arg Leu Ala Leu Asp Thr Arg Ile Asn Ser Thr Asn Asp Ile Ile 260 265 270Asn Ala Phe Lys Ser Leu Leu Glu 275 280361808PRTLeptolyngbya sp. 36Met Ser Asn Thr Val Ser Pro Ser Ala Ser Lys Ser Phe Lys Asn Ala1 5 10 15Phe Ile Ser Tyr Gly Arg Ala Asp Ser Leu Asp Phe Ala Lys Trp Leu 20 25 30Asn Asp Lys Leu Val Asp Gln Gly Tyr Asp Ile Trp Phe Asp Phe Glu 35 40 45Asn Ile Pro Gln Gly Val Asp Tyr Gln Lys Gln Ile Asp Ala Gly Ile 50 55 60Glu Asn Ala Asp Asn Phe Ile Phe Val Ile Ala Pro His Ala Thr Asn65 70 75 80Ser Pro Tyr Cys Arg Lys Glu Val Glu Leu Ala Ile Ala Leu Asn Lys 85 90 95Arg Leu Val Pro Ile Met His Val Glu Glu Ile Ser Arg Glu Thr Trp 100 105 110Gln Gly Arg Asn Pro Asn Gly Thr Asp Glu Gln Trp Glu Ala Tyr Lys 115 120 125Thr Glu Gly Leu His Ser Cys Phe Thr Asn Leu His Pro Glu Leu Gly 130 135 140Lys Ile Asn Trp Asn Gln Val Ser Phe Lys Glu Gly Val Asn Asp Tyr145 150 155 160Glu Gln Ser Phe Gln Ala Leu Val Asp Ile Phe Glu Arg Gln Ser Asn 165 170 175Tyr Val Arg Gln His Thr Glu Phe Leu Asp Gly Ala Leu Lys Trp Glu 180 185 190Arg Gly Gln Lys Gln Ser Gln Leu Leu Leu Asp Glu Glu Gln Thr Leu 195 200 205Arg Ala Gln His Trp Leu Gln Thr Lys Phe His Gly Glu Gln Pro Pro 210 215 220Cys Val Pro Thr Asp Leu His Cys Glu Phe Ile Thr Glu Ser Leu Lys225 230 235 240Arg Leu Pro Asn Gly Met Ser Gln Leu Phe Phe Ala His Ala Asn Ser 245 250 255Asp Thr Glu Thr Met Gln Ser Leu Arg Gln Gln Met Arg Arg Ala Gly 260 265 270Tyr Ser Val Trp Thr Pro Glu Ala Asp Ile Gln Ser Gly Gln Asp Val 275 280 285Gln Ala Glu Ile Leu Arg Gly Ile Glu Gly Ala Asp Ser Phe Ile Tyr 290 295 300Leu Met Ser Ser Gln Ser Leu Glu Ser Ser Asp Cys Gln Ser Gln Leu305 310 315 320Gln Tyr Ala Gln Thr Leu Lys Lys Arg Ile Ile Pro Ile Arg Leu Glu 325 330 335Ser Ile Gly Ser Asp Thr Leu Pro Ala Asn Gln Gln Lys Phe Ser Trp 340 345 350Ile Asp Cys Thr Ser Asp Glu Ala Ala Ser Lys Gln Leu Ala Ile Ala 355 360 365Gln Val Leu Lys Ala Ile Lys Glu Glu Ser Ser Phe Tyr Glu Gln His 370 375 380Arg Gln Ile Leu Val Lys Ala Leu Ser Trp Asp Arg Gln Lys Arg Pro385 390 395 400Lys Ser Leu Leu Leu Gln Gly Gln Asp Phe Thr Leu Ala Glu Gln Trp 405 410 415Leu Ala Leu Ser Lys Thr Glu Gln Ser Ser Arg Ala Thr Asp Leu Gln 420 425 430Gln Ile Tyr Ile Lys Ala Ser Gln Glu Met Asn Gln Phe Phe Asp Val 435 440 445Phe Ile Ser Tyr Gly Arg Ala Asp Ser Lys Asp Phe Ala Thr Ile Leu 450 455 460His Asp Arg Leu Val Glu Gln Gly Phe Asn Val Trp Phe Asp Gln Asn465 470 475 480Asp Ile Pro Leu Gly Val Asp Phe Gln Glu Gln Ile Asn Ala Gly Ile 485 490 495Glu Lys Ala His Asn Phe Leu Phe Ile Ile Ala Pro His Ser Val Asn 500 505 510Ser Pro Tyr Cys Gly Lys Glu Val Asp Leu Ala Leu Asp Leu Asn Lys 515 520 525Arg Ile Ile Pro Ile Leu His Val Glu Glu Ile Ser Lys Glu Thr Trp 530 535 540Gln Gln Arg Asn Pro Tyr Lys Thr Glu Glu Ser Asp Trp Glu Glu Ala545 550 555 560Gln Lys Gln Gly Ala His Ser Ser Phe Asn Asn Met Asn Pro Glu Ile 565 570 575Gly Lys Ile Asn Trp Val Tyr Cys Arg Glu Gln Asp Asp Phe Glu Ala 580 585 590Ser Phe Glu Gly Leu Met Thr Leu Cys Arg Gln His Glu Asp Tyr Val 595 600 605Lys Gln His Thr Glu Ile Leu Ala Lys Ala Leu Ser Trp Glu Ser His 610 615 620Gln Lys Gln Ser Gln Tyr Leu Leu Val Gly Glu Asn Arg Ile Glu Ala625 630 635 640Glu Lys Trp Leu Leu Thr Arg Phe Thr Glu Thr Gln Ala Pro Cys Phe 645 650 655Pro Thr Asp Leu His Cys Glu Tyr Ile Thr Glu Ser Ile Lys Asn Ala 660 665 670Asn Asn Leu Met Thr Gln Val Phe Leu Ser His Ser Asp Glu Asp Gln 675 680 685Glu Ile Glu Asp Lys Val Arg Arg Ser Leu Met Arg Glu Gly Tyr Thr 690 695 700Val Trp Ser Ser Gln Arg Asp Ile Glu Ala Gly Val Asp Phe Asn Ala705 710 715 720Ala Ile Asn Arg Gly Ile Glu Thr Thr Asp Asn Ile Val Tyr Leu Leu 725 730 735Ser Pro Asn Ser Leu Ala Ser Glu Tyr Cys Gln Lys Glu Ile Asp Tyr 740 745 750Ala Leu Ser Leu Asn Lys Arg Ile Ile Pro Leu Asn Ile Ala Ala Val 755 760 765Asp Glu Thr Asn Leu Pro Ala Ala Ile Gln Ser Leu Gln Tyr Ile Asp 770 775 780Leu Thr Asn Gly Ile Glu Asp Glu Thr Tyr Gln Ala Arg Leu Asn Glu785 790 795 800Leu Leu Lys Ile Leu Asn Gln Asp Lys Ala Tyr Phe Glu Glu His Lys 805 810 815Ala Ile Leu Val Arg Gly Leu Lys Trp Gln Ala Gln Gln Lys Asn Asn 820 825 830Ser Ile Leu Leu Arg Gly His Asn Leu Arg Glu Ala Glu Ala Trp Leu 835 840 845Lys Leu Ala Ala Lys Arg Ser Glu Tyr Ala Ala Thr Pro Leu His Gln 850 855 860Glu Phe Ile Gln Ala Ser Leu Asp Gln Leu Glu Asp Val Ala Leu Asp865 870 875 880Val Phe Val Ser Tyr Ser Arg Ser Asp Ser Asp Phe Val Arg Gln Leu 885 890 895Asn Asp Ala Leu Gln Val Gln Gly Lys Thr Thr Trp Phe Asp Gln Glu 900 905 910Asn Ile Ala Ser Gly Thr Asp Phe Gln Ala Glu Ile Phe Gln Gly Ile 915 920 925Glu Thr Cys Asp Asn Phe Leu Phe Val Ile Ser Pro Asn Ser Val Asn 930 935 940Ser Pro Tyr Cys Ala Gly Glu Val Glu His Ala Ala Lys Leu Gly Lys945 950 955 960Arg Phe Val Thr Val Leu His Gln Ser Val Ser Ser Ala Ser Leu His 965 970 975Pro Glu Leu Glu Lys Val Gln Trp Ile Asp Phe Asn Lys Asn Asp Gly 980 985 990Asp Phe Tyr Ala Asn Phe Ser Glu Leu Ile Arg Thr Leu Asp Thr Asp 995 1000 1005Arg Glu His Val Arg Ser His Thr Lys Trp Ser Gln Arg Ala Leu 1010 1015 1020Asp Trp Val Asn Lys Lys Arg Gly Lys Asp Leu Leu Leu Arg Gly 1025 1030 1035Asn Glu Phe Ala Ile Ala Glu Asn Trp Leu Lys Glu Ala Asp Asp 1040 1045 1050Gly Lys Lys Thr Pro Lys Pro Thr Thr Leu Gln Arg Asp Phe Leu 1055 1060 1065Ile Ala Ser Lys Glu Ala Ile Glu Ala Ala Ile Arg Arg Glu Lys 1070 1075 1080Arg Ile Ser Met Ile Ile Arg Thr Leu Leu Gly Val Val Ser Val 1085 1090 1095Ala Cys Val Ile Ala Phe Ile Gln Tyr Arg Gln Ala Asn Phe Gln 1100 1105 1110Arg Lys Arg Ala Glu Arg

Val Gln Glu Gly Gln Ile Asn Ala Leu 1115 1120 1125Ser Asp Tyr Ser Val Ser Leu Leu Glu Asn His Gln Asp Leu Gln 1130 1135 1140Ser Leu Ile Glu Ala Ile Arg Ala Gly Arg Gln Leu Gln Leu Gln 1145 1150 1155Leu Lys Thr Val Asp Gln Ser Thr Val Asp Ala Val Thr Thr Thr 1160 1165 1170Leu Arg Asp Ala Leu Leu Asn Ile Glu Glu Val Asn Arg Ile Thr 1175 1180 1185Gly His Pro Ser Ala Gln Gly Ile Asn Ala Val Ala Tyr Ser Pro 1190 1195 1200Asp Glu Ser Met Val Ala Thr Gly Gly Ala Asp Gly Asn Ile Arg 1205 1210 1215Leu Trp Ser Ala Glu Gly Glu Ser Ile Arg Thr Leu Glu Asp His 1220 1225 1230Glu Ala Pro Ile Tyr Glu Met Glu Phe Ser Pro Asn Gly Lys Phe 1235 1240 1245Leu Leu Ser Gly Ser Glu Asp Phe Thr Ala Arg Leu Trp Asp Pro 1250 1255 1260Glu Thr Gly Glu Leu Leu Arg Thr Phe Glu Asp His Asp Asn Ser 1265 1270 1275Ile Tyr Gly Val Ser Phe Ser Pro Asp Ser Gln Ile Ile Ala Thr 1280 1285 1290Ala Ser Val Asp Gly Thr Val Asn Ile Tyr Ser Val Glu Gly Gln 1295 1300 1305Leu Leu Gln Thr Leu Glu Ile Asp Leu Glu Asn Tyr Asp Val Ser 1310 1315 1320Phe Asn Ala Asp Gly Ser Ala Ile Ala Thr Ala Ser Glu Asp Gly 1325 1330 1335Ile Leu Arg Phe Trp Asp Leu Glu Gly Glu Leu Arg Asn Glu Val 1340 1345 1350Glu Ala His Glu Asn Gly Ile Ser Thr Val Ala Phe Ser Pro Lys 1355 1360 1365Gly Asp Leu Val Ala Thr Gly Ser Trp Asp Gln Thr Ala Lys Leu 1370 1375 1380Trp Thr Ile Asp Gly Glu Ser Val Val Thr Leu Gln Gly His Thr 1385 1390 1395Asp Glu Val Asn His Leu Phe Phe Ser Asp Asp Gly Glu Phe Leu 1400 1405 1410Val Thr Thr Ser Tyr Asp Asn Leu Ala Lys Val Trp Ser Arg Glu 1415 1420 1425Gly Glu Leu Leu His Thr Ile Arg Gly His Glu Asp Gly Val Leu 1430 1435 1440Gly Val Ala Ile Ser Lys Asp Ser Ser Thr Val Lys Thr Thr Ser 1445 1450 1455Leu Asp Gly Thr Ala Arg Val Trp Asp Ile Ser Ser Leu Pro Asn 1460 1465 1470Val Lys Thr Phe Glu Asp Gln Thr Ala Asp Ile Phe Glu Val Glu 1475 1480 1485Phe Ser Pro Asp Glu Lys Trp Ile Gly Ser Ala Gly Asp Ser Gly 1490 1495 1500Ala Arg Ile Trp Asp Leu Glu Gly Asn Leu Ile Ser Asp Leu Asp 1505 1510 1515Gly Glu Asn Asn Ala Met Arg Asp Leu Ala Phe Ser Gln Asp Gly 1520 1525 1530Arg Tyr Leu Ala Thr Gly Glu Glu Asn Gly Val Val Lys Ile Trp 1535 1540 1545Glu Trp Thr Gly Asp Asn Phe Lys Leu Val Gln Thr Val Gln Asn 1550 1555 1560Glu Asp Ala Gly Lys Met Arg Ala Ile Ala Phe His Pro Glu Gly 1565 1570 1575Lys Tyr Leu Ala Thr Ala Gly Asp Gly Val Thr Val Gln Leu Trp 1580 1585 1590Thr Leu Asp Gly Glu Ser Val Phe Val Ser Glu Val Ala Asp Trp 1595 1600 1605Thr Asn Ser Ile Ala Phe Ser Pro Asp Gly Glu Phe Leu Ile Ser 1610 1615 1620Gly Gly Trp Asp Gln Met Ile Ser Leu Trp Asp Leu Glu Gly Asn 1625 1630 1635Leu Leu Asn Ser Trp Glu Ala His Pro Asp Ser Ile Asn Gly Leu 1640 1645 1650Ala Phe Gly Asn Asp Ala Gln Thr Ile Ile Ser Ala Ser Asn Asp 1655 1660 1665Gln Thr Ala Gln Ile Trp Gln Leu Asp Gly Ala Ser Gly Ser Pro 1670 1675 1680Thr Leu Thr Val Asn His Gly Ala Glu Val Asn Lys Ala Thr Leu 1685 1690 1695Ser Pro Asp Gly Lys Asn Leu Ile Thr Val Gly Gly Gln Thr Val 1700 1705 1710Lys Phe Trp Asp Leu Glu Gly Arg Leu Leu Gln Thr Ile Ala Ala 1715 1720 1725His Asn Asp Ile Ile Tyr Gly Phe Ala Leu Ser Ser Asp Gly Lys 1730 1735 1740Gln Phe Val Thr Gly Ser Tyr Asp Ser Thr Ala Arg Leu Trp Ser 1745 1750 1755Tyr Gln Pro Lys Val Ser Glu Lys Ala Thr Glu Ile Trp Gln Leu 1760 1765 1770Asp Leu Asn Ala Leu Val Asp His Ala Cys Gly Val Ala Arg Asn 1775 1780 1785Tyr Leu Thr Phe Asn Pro Asp Val Ser Asp Ser Asp Arg Leu Leu 1790 1795 1800Cys Glu Pro Asp Leu 180537166PRTModestobacter marinus 37Met Val Ser Tyr Thr Pro Thr Glu Trp Arg Thr Val Glu Pro Tyr Ala1 5 10 15Glu Lys Ala Val Glu Gln Ala Arg Val Asn Pro Asp Arg Arg Asp Leu 20 25 30Phe Leu Cys His Ala Trp Asp Asp Arg Gln Gly Ser Ala Ala Glu Leu 35 40 45His Gly Leu Leu Lys Thr Asn Gly Ala Thr Val Trp Phe Ser Glu Glu 50 55 60Asp Leu Pro Leu Gly Ser Leu Met Ile Arg Glu Ile Asp Lys Gly Leu65 70 75 80Arg Asn Ser Arg Ile Gly Ile Val Leu Val Thr Pro Ala Leu Leu Lys 85 90 95Ser Ile Glu Lys Glu Gly Ile Ala Glu Lys Glu Leu Ala Val Leu Leu 100 105 110Asn Ser Lys Arg Val Ile Pro Val Thr His Gly Val Thr Tyr Glu Gln 115 120 125Leu Leu Asp Ile Ser Pro Met Leu Ala Ser His Ala Gly Leu Ser Thr 130 135 140Lys Glu Ser Ser Leu Asp Asp Val Ala Ala Lys Leu Ala Ala Ala Ala145 150 155 160Ala Ala Leu Pro Ala Leu 16538237PRTScytonema sp 38Met Thr Asp Pro Leu Ile Val Ser Gly Thr Ala Asn Asp Ile Asp Ser1 5 10 15Leu Arg Gln Pro Leu Val Ala Gly Ser Ile Gly Val Gln Gln Gln Val 20 25 30Ile Pro Gln Leu Ala Asn Leu Gly Asp Thr Gly Leu Asp Val Leu Met 35 40 45Glu Phe Leu Phe Glu Arg Arg Glu Asn Pro Ala Thr Ser Val Asp Gly 50 55 60Lys Ala Tyr Gln Val Leu Tyr Asn Ser Asp Ser Pro Lys Ala Lys Glu65 70 75 80Phe Leu Gln Thr Tyr Phe Pro Phe Gly Ile Val Pro Leu Thr Ser Asp 85 90 95Gly Gly Ile Asp Tyr Ser Pro Leu Gln Gln Leu Leu Ala Thr Gln Asp 100 105 110Phe Gln Ala Ala Asp Arg Met Thr Leu Gln Lys Met Cys Glu Leu Ala 115 120 125Gly Ser Ala Ala Val Gln Arg Lys Trp Leu Tyr Phe Thr Glu Val Glu 130 135 140Asn Phe Pro Ile Thr Asp Leu Lys Thr Ile Asn Thr Leu Trp Leu Val145 150 155 160His Ser Glu Gly Lys Phe Gly Phe Thr Val Gln Arg Glu Ile Trp Leu 165 170 175Gly Leu Gly Lys Asn Trp Glu Asn Leu Trp Thr Lys Ile Gly Trp Lys 180 185 190Lys Gly Asn Asn Trp Thr Arg Tyr Pro Asn Glu Phe Thr Trp Asn Leu 195 200 205Ser Ala Pro Arg Gly His Leu Pro Leu Ser Asn Gln Leu Arg Gly Val 210 215 220Arg Val Ile Ala Ser Leu Leu Ser His Pro Ala Trp Asn225 230 23539147PRTComamonas aquatica 39Met Arg Arg Ser Ile Glu Ser Arg Ala Ala Leu Pro Asp Leu Arg Asp1 5 10 15Ile Phe Leu Cys His Ala Trp Asp Asp Arg Gln Gly Ala Ala Lys Glu 20 25 30Leu His Asp Gln Leu Glu Ser Arg Gly Val Ser Val Trp Phe Ser Glu 35 40 45Lys Asp Val Ala Leu Gly Thr Ser Leu Leu Arg Glu Ile Asp Lys Gly 50 55 60Leu Ala Lys Ser Arg Val Gly Ile Val Leu Val Thr Pro Ser Leu Leu65 70 75 80Ser Arg Val Arg Gly Glu Gly Ile Ala Glu Lys Glu Leu Ser Ala Leu 85 90 95Leu Ala Arg Asp Leu Leu Val Pro Ile Val His Asp Thr Thr Tyr Glu 100 105 110Ala Leu Arg Asp Val Ser Pro Leu Leu Gly Ser Arg Ser Gly Leu Ser 115 120 125Thr Lys Asp Ala Ser Met Ala Ala Val Ala Ala Lys Leu Ala Glu Leu 130 135 140Val Ala Val14540245PRTLacunisphaera limnophila 40Met Ser Arg Cys Thr Ala Pro Ile Arg Gly His Asn Ser Ala Ser Ala1 5 10 15Ala Glu Asn Cys Pro Val Cys Arg Tyr Lys Ser Arg Gly Tyr Ser Ser 20 25 30Tyr Ser Ser Asn Tyr Asp Ser Tyr Asn Ser Ser Arg Phe Ser Ser Ser 35 40 45Ser Gly Ser Asn Gly Ser Ser Ser Thr Ser Arg Gly Asn Tyr Asn Val 50 55 60Ser Lys Gly Arg Thr Ile Lys Pro Ser Trp Ser Arg Ser Thr Ser Pro65 70 75 80Ile Tyr Tyr Thr Pro Ala Glu Val Lys Thr Leu Thr Pro Val Arg Glu 85 90 95Asn Val Glu Lys Arg Val Val Lys Glu Asp Leu Arg Asp Val Phe Leu 100 105 110Cys His Ala Trp Asp Asp Arg Lys Gly Ser Ala Lys Asp Leu His Asp 115 120 125Leu Leu Glu Leu Lys Gly Val Ser Val Trp Phe Ser Glu Lys Asp Val 130 135 140Ile Ile Gly Ser Ser Leu Leu Arg Glu Ile Asp Lys Gly Leu Ala Arg145 150 155 160Ser Lys Val Gly Ile Val Leu Val Thr Pro Lys Phe Leu Glu Arg Ile 165 170 175Lys Asn Glu Gly Ile Ala Asp Lys Glu Leu Ser Ala Leu Leu Ala Arg 180 185 190Asp Leu Leu Val Pro Ile Val His Glu Thr Thr Phe Glu Glu Leu Arg 195 200 205Asp Glu Ser Pro Leu Leu Gly Ser Arg Ser Gly Leu Ser Thr Leu Glu 210 215 220Asp Ser Met Glu Asn Ile Ala Ser Lys Leu Ala Glu Leu Val Ser Asp225 230 235 240Lys Leu Ile Val Ile 245411102PRTLacunisphaera limnophila 41Met Ser Asp Pro Gly Lys Ala Val Phe Leu Ser Tyr Ala Ser Gln Asp1 5 10 15Ala Glu Ala Ala Lys Arg Ile Cys Asp Ala Leu Arg Ala Ala Gly Val 20 25 30Glu Val Trp Phe Asp Gln Asn Glu Leu Val Gly Gly Asp Gln Trp Asp 35 40 45Gly Lys Ile Arg Gly Gln Ile Ser Ser Cys Ala Leu Phe Val Pro Leu 50 55 60Ile Ser Ala Asn Thr Gln Ala Arg Leu Glu Gly Tyr Phe Arg Leu Glu65 70 75 80Trp Lys Leu Ala Ala Gln Arg Thr His Thr Met Ala Asp Glu Met Thr 85 90 95Phe Leu Leu Pro Val Leu Ile Asp Gly Thr Arg Asp Ala Glu Ala Arg 100 105 110Val Pro Ala Glu Phe Arg Ala Val Gln Trp Thr Lys Leu Pro Ala Gly 115 120 125Glu Ala Thr Pro Ala Phe Cys Val Arg Val Arg Lys Leu Leu Asp Gly 130 135 140Thr Ala Ala Gly Val Val Asp Pro Gly Leu Pro Gly Ser Thr Ser Pro145 150 155 160Ala Thr Ser Arg Pro Val Pro Pro Arg Arg Gly Ala Ser Lys Trp Trp 165 170 175Trp Val Leu Pro Ile Phe Gly Val Thr Met Ala Met Val Leu Val Met 180 185 190Lys Glu Ala Arg Lys Glu Pro Ala Pro Ser Ser Ser Asn Gly Pro Ala 195 200 205Pro Thr Met Thr Gln Pro Val Ser Glu Ala Arg Lys Leu Ala Asn Gln 210 215 220Ala Met Gln Leu Leu Glu Asp Pro Asn Phe Thr Arg Glu Thr Ser Trp225 230 235 240Leu Ala Asp Glu Leu Cys Gln Arg Ala Leu Ala Leu Asp Ala Gly Asp 245 250 255Ala Glu Val Trp Ala Val Ala Ala Phe Ala Ser His Asn Leu Phe Ser 260 265 270Asn Thr Tyr Asp Thr Ser Ala Gly Arg Arg Glu Lys Ala Arg Ser Gln 275 280 285Ala Ala Arg Ala Ser Gln Val Asp Pro Gln Ser Val Arg Ala Ala Leu 290 295 300Ala Val Ala Arg Cys Leu Glu Gly Thr Gly Asn Asp Gly Glu Leu Leu305 310 315 320Arg Ile Leu Gln Glu Leu His Arg Arg Ala Pro Ala Asp Gln Gln Val 325 330 335Leu Phe Ser Leu Val Arg Ala Glu Gly Gly Met Gly Asn Glu Thr Ala 340 345 350Val Gln Asp Leu Ile Arg Lys Phe Arg Ala Leu Pro Arg Thr Gly Leu 355 360 365Phe Pro Leu Ala Leu Trp Phe Glu Glu Leu Arg Leu Arg Thr Leu Gly 370 375 380Arg Tyr Ala Glu Ala Glu Ala Met Leu Asp Glu Met Leu Ala Asp Pro385 390 395 400Gly Val Leu Arg Gly Ala Tyr Tyr Glu Lys Leu Asn Leu Leu Met Arg 405 410 415Ser Trp His Asp Leu Ala Ala Ala Ala Pro Phe Ile Glu Lys Ile Pro 420 425 430Ala Arg Phe Arg Gln Glu Pro Ala Phe Gly Ser Ala Ile Ala Tyr Tyr 435 440 445Trp Leu Trp Arg Ala Glu Pro Glu Lys Ala Leu Gln Ala Leu Ala Gln 450 455 460Val Pro Gln Asp Tyr Phe Glu Glu Tyr Ala Ala Arg Glu Pro Lys Gly465 470 475 480Tyr Leu Thr Gly Trp Ala His Ala Ile Ala Lys Arg Pro Ala Ala Ala 485 490 495Gln Ala Glu Trp Arg Ala Ala Leu Thr Leu Val Asp Glu Arg Leu Lys 500 505 510Thr Asp Ala Arg Asn Pro Ser Leu Leu Asn Gln Arg Ala Leu Leu Leu 515 520 525Ala Leu Thr Gly Gln Arg Glu Ala Ala Arg Glu Ala Trp Gln Leu Arg 530 535 540Val Glu Leu Gly Gly Glu Gln Asn Pro Val Gly Leu Glu Ala Glu Thr545 550 555 560Gln Val Leu Val Ala Leu Asn Glu Pro Glu Ala Ala Leu Ala Ala Ile 565 570 575Glu Arg Glu Trp Ser Arg Arg Lys Pro Met Gly Arg Val Arg Ala Leu 580 585 590Pro Val Ile Arg Tyr His Pro Ala Tyr Ala Val Ile Arg Gly Asp Pro 595 600 605Arg Leu Gln Arg Ile Leu Lys Glu Asp Glu Ala Glu Leu His Val Leu 610 615 620Lys Glu Gln Gln Thr Arg Ser Ala Pro Ala Lys Glu Pro Thr Thr Ala625 630 635 640Ser Ser Ala Ala Asp Ala Lys Ser Val Ala Val Leu Ala Phe Ala Asn 645 650 655Leu Ser Asp Asp Lys Ala Asn Glu Tyr Phe Ser Asp Gly Ile Ser Glu 660 665 670Glu Leu Leu Asn Val Leu Ala Lys Val Pro Gly Leu Lys Val Ala Ala 675 680 685Arg Thr Ser Ala Phe Tyr Phe Lys Gly Lys Glu Val Pro Ile Pro Glu 690 695 700Ile Ala Arg Gln Leu Gly Val Ala Tyr Val Leu Glu Gly Ser Val Arg705 710 715 720Lys Gln Gly Asp Lys Val Arg Ile Thr Ala Gln Leu Ile Lys Ala Ala 725 730 735Asp Gly Phe Arg Val Trp Ser Asp Thr Phe Thr Arg Asp Leu Lys Asp 740 745 750Ile Phe Ala Val Gln Asp Glu Ile Ala Gly Leu Ile Ala Lys Asn Leu 755 760 765Glu Leu Lys Met Ser Met Gly Glu Ser Val Pro Arg Gln Ala Val Arg 770 775 780Pro Asp Ala Tyr Gln Ala Phe Leu Leu Gly Arg Ser Ala Ala Ala Lys785 790 795 800Ala Ser Val Val Glu Leu Arg Glu Ala Val Gly His Phe Glu Arg Ala 805 810 815Val Ala Leu Glu Pro Lys Tyr Thr Ala Ala Trp Val Gln Leu Ala Ser 820 825 830Ile His Thr Arg Leu Gly Arg Trp Gly Gly Ala Pro Thr Leu Ala Ser 835 840 845Trp Ala Ala Ala Arg Ala Ala Ile Asp Gln Ala Leu Ala Leu Glu Pro 850 855 860Glu Ser Pro Asp Val Leu Leu Ala Gln Gly Trp Ile Leu Arg Thr Ala865 870 875 880Glu Trp Asp Trp Arg Gly Ala Glu Arg Ser Phe Arg Arg Ala Leu Ala 885 890 895Leu Gln Pro Asn Asn Pro Glu Ile Leu Ala Gly Thr Ala Val Leu Leu 900 905 910Phe Asn Ile Gly Gln Thr Glu Glu Ala Phe Arg Leu Ala Arg Leu Ala 915 920 925Val Gln Leu Asp Pro Leu Asn Pro Ser Thr Gln Ile Asp Leu Ser Ile 930 935 940Met Phe Phe Glu Ser Lys Asn Trp Val Glu Ser Glu Arg Ala Ala Arg945 950 955 960Arg Ala Leu Gln Leu Ala Pro

Gly Gly Thr Ser Tyr His Ser Val Leu 965 970 975Ala Trp Pro Leu Ile Asn Gln Gly Arg Tyr Ala Glu Ala Glu Ala Ser 980 985 990Leu Ala Leu Asp Leu Asp Val Val Gln Gln Ser Gly Thr Arg Gly Leu 995 1000 1005Leu Gly Leu Ala Arg Gly Asp Leu Ala Met Val Arg Glu Met Leu 1010 1015 1020Ala Arg Leu Glu Gln Leu Ala Arg Thr Glu Pro Asp Arg Ala Asp 1025 1030 1035Leu Gln Thr Cys Thr Ala Trp Leu Tyr Ala Gly Leu Gly Asp Lys 1040 1045 1050Asp Arg Ala Phe Ala Ala Leu Glu Lys Ala Arg Ile Ser Arg Asp 1055 1060 1065Pro Ser Ile Ala Trp Leu Arg Glu Asn Thr Glu Leu Thr Pro Leu 1070 1075 1080Phe Ser Asp Pro Arg Trp Gln Glu Leu Met Arg Lys Val Gly Leu 1085 1090 1095Ala Asp Gly Gln 110042327PRTAquimarina amphilecti 42Met Lys Asn Arg Ser Tyr Glu Tyr Asp Val Ala Leu Ser Phe Ala Gly1 5 10 15Glu Asn Arg Ala Tyr Val Glu Arg Val Ala Asn Ser Leu Lys Thr Lys 20 25 30Gly Val Lys Val Phe Tyr Asp Leu Phe Glu Glu Ala Asn Leu Trp Gly 35 40 45Lys Asn Leu Tyr Glu Tyr Leu Ser Glu Ile Tyr Gln Asn Lys Ala Arg 50 55 60Tyr Thr Val Leu Phe Val Ser Ser Phe Tyr Asn Lys Lys Leu Trp Thr65 70 75 80Asn His Glu Arg Val Ser Met Gln Ala Arg Ala Phe Gln Glu Ser Arg 85 90 95Glu Tyr Ile Leu Pro Ala Arg Phe Asp Asp Thr Glu Ile Pro Gly Ile 100 105 110Leu Lys Thr Ile Gly Tyr Ile Asn Leu Glu Asn Arg Thr Pro Glu Glu 115 120 125Leu Ala Val Leu Ile Glu Asn Lys Leu Lys Lys Asp Gln Thr Phe Phe 130 135 140Lys Asn Arg Trp Ser Lys Leu Ser Thr Met Ile Ser Pro Lys Pro Phe145 150 155 160Ile Phe Thr Ile Lys Val Val Asp Glu Lys Ser Gln Leu Val Lys His 165 170 175Ala Lys Val Val Leu Val Ala Asn Asn Ser Thr Tyr Leu Glu Gly Tyr 180 185 190Thr Asp Glu Asn Gly Leu Ala His Phe Val Ile Arg Thr Arg Lys Leu 195 200 205Tyr Thr Val Leu Ile Ala His Ser Glu Tyr Pro Ala Val Val Phe Lys 210 215 220Ser Met Asn Pro Lys Glu Asp Ile Glu Val Thr Ile Glu Lys Thr Asn225 230 235 240Asn Ser Gly Ser Val Ile Ile Asn Lys Ser Gly Gln Ile Pro Gly Ile 245 250 255Ser Gly Lys Ile Glu Pro Val Leu Lys Ser Asp Lys Asn Leu Ser Val 260 265 270Tyr Ala Asp Asn Ile Ala Ile Glu Gly Gly Lys Asp Gln Pro Tyr Asp 275 280 285Phe Glu Leu Asn Lys Ser Ile Val Leu Glu Asp Asn Lys Gly Asn Ile 290 295 300Val His Leu Thr Phe Arg Phe Tyr Gln Ala Arg Ile Ala Leu Ile Asp305 310 315 320Phe Tyr Arg Gly Arg Ser Met 32543891PRTThioflavicoccus mobilis 43Met Pro Gly Tyr Asp Leu Phe Leu Ser Tyr Asn Arg Arg Asp Arg Ala1 5 10 15Ile Val Asp Pro Leu Ala Gly Ala Leu Cys Glu Arg Gly Leu Lys Val 20 25 30Phe Lys Asp Asp Trp Phe Leu Arg Pro Gly Glu Pro Trp Pro Leu Ala 35 40 45Leu Glu Lys Ser Leu Met Ala Ser Arg Ala Val Ala Val Ala Val Gly 50 55 60Arg Asn Gly Leu Gly Pro Trp Gln Gln Arg Glu Ala Ala Ala Ala Leu65 70 75 80Asp Leu Gln Ala Arg Arg Pro Pro Asp Asp Gly Leu Pro Val Ile Pro 85 90 95Val Leu Leu Asp Gln Asp Ser Ala Arg Gln Ala Gly Leu Ala Phe Leu 100 105 110Leu Gln Asn Thr Trp Val Glu His Trp Asp Pro Arg Ala Ala Asp Leu 115 120 125Ile Val Gly Ala Val Gln Gly Lys Ala Pro Ala Glu Leu Tyr Asp Thr 130 135 140Arg Asp Ala Asp Pro Arg Thr Arg Val Cys Pro Tyr Arg Gly Leu Gly145 150 155 160Val Phe Arg Glu Glu Asp Ala Gly Phe Tyr Val Gly Arg Glu Ser Asp 165 170 175Leu Glu Arg Leu Gly Asp Ala Ile Glu Arg His Pro Val Val Ala Val 180 185 190Val Gly Ala Ser Gly Ser Gly Lys Ser Ser Leu Val Arg Ala Gly Leu 195 200 205Ile Pro Arg Leu Arg Arg Ala Ser Gly Thr Arg Val Tyr Gln Val Ala 210 215 220Asp Met Met Pro Gly Arg Gly Pro Phe Leu Ala Leu Ala Arg Ala Leu225 230 235 240Leu Pro Leu Arg Glu Pro Glu Arg Val Leu Ser Trp Ser Lys Gly Gly 245 250 255Ile Asp Asp Glu Cys Glu Arg Leu Ser Ala Lys Leu Asp Gln Asn Gly 260 265 270Ala Glu His Leu Thr His Val Val Gly Gln Ile Leu Ala Glu Glu Pro 275 280 285Gly Thr Thr Gln Leu Leu Leu Leu Val Asp Gln Trp Glu Glu Leu Tyr 290 295 300Thr Tyr Arg Pro Ser Glu Ala Ala Ala Leu Arg Arg His Gly Glu Arg305 310 315 320Val Arg Arg Phe Ile Gly Met Leu Leu Glu Ala Ala Arg Leu Cys Pro 325 330 335Leu Gln Val Val Leu Thr Leu Arg Ala Asp Tyr Trp Gly Glu Val Leu 340 345 350Asn Asp Glu Pro Leu Ala Ala Ala Leu Cys Asp Pro Ala Leu Val His 355 360 365Leu Arg Ala Leu Asp Arg Ala Ala Leu Glu Arg Val Ile Arg His Pro 370 375 380Ala Glu Arg Val Gly Leu Gln Val Pro Asp Ala Leu Ala Glu Val Leu385 390 395 400Leu Asp Ala Ala Ala Gly Gln Pro Gly Asp Leu Pro Leu Leu Glu Phe 405 410 415Thr Leu Gln Gln Leu Trp Ala Glu Arg Ala Asp His Gly Gly Thr Leu 420 425 430Thr Leu Asp Ala Tyr Arg Ala Met Gly Gly Leu Glu Lys Ala Ile Val 435 440 445Ser Arg Ala Glu Thr Thr Leu Asp Arg Leu Ala Pro Ala Glu Arg Glu 450 455 460Ala Val Pro Gly Leu Phe Ala Ala Leu Val Gln Val Gly Glu Ala Arg465 470 475 480Thr Asp Leu Arg Arg Arg Ala Arg Leu Ala Glu Leu Gly Glu Pro Ala 485 490 495Arg Ala Ala Ala Cys Gln Leu Ala Asn Glu Arg Leu Leu Val Thr Gly 500 505 510Arg Asp Trp Thr Ser Gly Glu Glu Trp Val Glu Val Ala His Glu Ala 515 520 525Leu Leu Arg His Trp Pro Lys Leu Glu Gly Trp Ile Asp Thr Arg Arg 530 535 540Gly Ala Leu Leu Thr Val Arg Gln Leu Gln Ala Asp Thr Arg Thr Trp545 550 555 560Leu Glu Ser Gly Lys Arg Ser Gly Phe Leu Trp Ser His Glu Arg Ala 565 570 575Arg Glu Ala Ala Lys Ala Leu Thr Gln Leu Gly Met Glu Val Ser Leu 580 585 590Ser Ser Glu Glu Ala Glu Phe Leu Gly Pro Ile Asp Pro Gln Ala Met 595 600 605Leu Ala Glu Leu Glu Gln Pro Gln Thr Asp His Gln Arg Arg Ala Leu 610 615 620Ile Gly Ala Arg Leu Asp Leu Leu Gly Asp Pro Arg Pro Gly Ile Gly625 630 635 640Cys Thr Pro Gly Gly Thr Pro Asp Ile Ala Trp Tyr Pro Val Pro Gly 645 650 655Gly Glu Val Thr Ile Glu Val Glu Arg Leu Ile Arg Gly Gly Thr Lys 660 665 670Pro Lys Val Lys Ala Ile Ala Pro Phe His Ile Ala Arg Tyr Pro Val 675 680 685Thr Val Thr Gln Tyr Arg Ala Phe Leu Gln Ala Glu Asp Gly Trp Arg 690 695 700Asp Pro Gln Trp Trp Ala Asn Asp Leu Asp Arg Asp Pro Glu Gly Asp705 710 715 720Ser Tyr Asp Leu Gly Arg Tyr Asp Asn His Pro Ala Leu Tyr Val Ser 725 730 735Trp Phe Asp Ala Met Ala Tyr Cys Arg Trp Leu Ser Asn Arg Leu Gly 740 745 750Leu Leu Ile Arg Leu Pro Asp Glu Trp Glu Trp Gln Gln Ala Ala Thr 755 760 765Gly Gly Asp Ala Ala Gln Ile Phe Pro Trp Gly Ala Glu Trp Asn Pro 770 775 780Lys Thr Glu Pro Cys Arg Ala Asn Thr Phe Glu Ala Arg Leu Gly Gly785 790 795 800Val Thr Ala Val Gly Met Tyr Pro Ala Gly Ala Ser Pro Gly Gly Val 805 810 815Val Asp Met Ala Gly Thr Val Trp Glu Trp Cys Leu Asn Lys His Asp 820 825 830Ser Pro Leu Ala Thr Glu Ser Arg Pro Asp Asn Phe Asp Arg Arg Val 835 840 845Val Arg Gly Gly Ser Trp Tyr Val Val Gln Ser Tyr Ala Arg Ser Ala 850 855 860Ala Arg Tyr Arg Ser Asn Pro Ser Ser Arg Asp Ser Gly Leu Gly Phe865 870 875 880Arg Val Leu Cys Ala Ser Pro Ile Pro Gly His 885 890

Claims

1. A method of inhibiting TIR NADase activity in a biological sample comprising a TIR NADase, the method comprising, contacting the biological sample with a composition comprising an effective amount of a TIR NADase inhibitor, wherein the TIR NADase inhibitor is one or more of: ##STR00006## and derivatives thereof.

2.-15. (canceled)

16. A method of identifying a TIR NADase inhibitor, comprising: a) providing a mixture comprising; i) a full length, mutant or fragment of a TIR NADase, ii) NAD+, and; iii) a candidate inhibitor, wherein optionally the mutant or fragment has constitutive NADase activity; b) incubating the mixture; c) quantifying one or more of NAD+ ADPR, cADPR or nicotinamide in the mixture.

17. The method of claim 16, further comprising: a) identifying the candidate inhibitor compound as an NADase inhibitor if one or more of the following is true, the amount of NAD+ is greater or the amount of ADPR, cADPR or nicotinamide is less than that of a control mixture that does not contain the candidate inhibitor.

18. The method of claim 16, further comprising: a) determining the molar ratio of NAD+: ADPR; and; b) identifying the candidate inhibitor compound as an NADase inhibitor if the molar ratio is greater than that of a control mixture that does not contain the candidate inhibitor.

19. The method of claim 16, wherein the quantifying NAD+ and ADPR in the mixture comprises performing an HPLC analysis, performing a chemiluminescence assay, performing liquid chromatography-mass spectroscopy analysis or a combination thereof.

20. The method of claim 16, wherein the mixture comprises a cell lysate from a host cell comprising the full length, mutant or fragment of a TIR NADase.

21.-22. (canceled)

23. The method of claim 16, wherein the full length, mutant or fragment TIR NADase is from TcpC.

24. The method of claim 16, wherein the full length, mutant or fragment TIR NADase is from TirS.

25.-27. (canceled)

28. A method of treating a microbial infection in a subject in need thereof, wherein the microbe encodes a TIR NADase, the method comprising: administering to said subject a therapeutically effective amount of pharmaceutical composition comprising a TIR NADase inhibitor.

29. The method of claim 28, wherein the TIR NADase inhibitor is Dequalinium ##STR00007## or a derivative thereof.

30. The method of claim 28, wherein the TIR NADase inhibitor is Cetylpyridinium ##STR00008## or a derivative thereof.

31. The method of claim 28, wherein the TIR NADase inhibitor is Amiloride ##STR00009## or a derivative thereof.

32. The method of claim 28, wherein the TIR NADase inhibitor is Acrinol ##STR00010## or a derivative thereof.

33. The method of claim 28, wherein the TIR NADase inhibitor is Akrinol ##STR00011## or a derivative thereof.

34. The method of claim 28, wherein the infection is caused by a pathogenic bacteria, virus, fungus or protozoa.

35. The method of claim 34, wherein the bacteria is uropathogenic E. coli.

36. The method of claim 35, wherein the TIR NADase is TcpC.

37. The method of claim 34, wherein the bacteria is S. aureus.

38. The method of claim 37, wherein the TIR NADase is TirS.

39. The method of claim 28, wherein the TIR NADase inhibitor is administered with one or more additional therapeutically active agents.

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