U.S. patent application number 10/337699 was filed with the patent office on 2004-01-15 for broad spectrum inhibitors.
Invention is credited to Eissenstat, Michael, Erickson, John W., Gulnik, Sergei, Silva, Abelardo.
Application Number | 20040009890 10/337699 |
Document ID | / |
Family ID | 26994100 |
Filed Date | 2004-01-15 |
United States Patent
Application |
20040009890 |
Kind Code |
A1 |
Erickson, John W. ; et
al. |
January 15, 2004 |
Broad spectrum inhibitors
Abstract
The invention features a method of designing broad spectrum
inhibitors using structural data, compositions having broad
spectrum activity, and methods for treating disease using those
compositions.
Inventors: |
Erickson, John W.;
(Frederick, MD) ; Eissenstat, Michael; (Frederick,
MD) ; Silva, Abelardo; (Columbia, MD) ;
Gulnik, Sergei; (Frederick, MD) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Family ID: |
26994100 |
Appl. No.: |
10/337699 |
Filed: |
January 7, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60344788 |
Jan 7, 2002 |
|
|
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60383575 |
May 29, 2002 |
|
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Current U.S.
Class: |
514/1 ;
703/11 |
Current CPC
Class: |
A61P 31/12 20180101;
C07K 2299/00 20130101; G16B 15/00 20190201; A61K 31/337 20130101;
G16B 20/20 20190201; Y02A 90/10 20180101; C07D 307/20 20130101;
C12N 9/88 20130101; C07D 275/03 20130101; G16C 20/50 20190201; C12N
9/506 20130101; Y02A 50/30 20180101; C07D 493/04 20130101; G16B
15/20 20190201; G16B 20/00 20190201; C07D 495/04 20130101; C07D
491/04 20130101; A61P 31/18 20180101 |
Class at
Publication: |
514/1 ;
703/11 |
International
Class: |
A61K 031/00; G06G
007/48; G06G 007/58 |
Claims
Other embodiments are within the claims. What we claim is:
1. A method for the structure-based design of a drug that can act
as an inhibitor of at least two different biological entities, said
method comprising the steps of: (a) providing at least one
structure of a wild type target protein or an inhibitor-wild type
target protein complex; (b) providing at least one structure of a
variant target protein or an inhibitor-variant target protein
complex; (c) comparing at least one structure from step (a) with at
least one structure from step (b) to determine whether there exists
a common three-dimensionally conserved substructure comprising the
atomic coordinates of the structurally conserved atoms of the
inhibitors and structurally conserved atoms of the target proteins;
and (d) if a conserved substructure exists, using said atomic
coordinates of said conserved substructure to select a compound
having atoms matching those of said structurally conserved atoms of
the inhibitors, wherein the selection of said compound is performed
using computer modeling.
2. A method for the structure-based drug design of a broad spectrum
inhibitor, said method comprising the steps of: (a) providing at
least one structure of a wild type target protein or an
inhibitor-wild type target protein complex; (b) providing at least
one structure of a variant target protein or an inhibitor-variant
target protein complex; (c) comparing at least one structure from
step (a) with at least one structure from step (b) to determine
whether there exists a common three-dimensionally conserved
substructure comprising the atomic coordinates of the structurally
conserved atoms the target proteins or a common three-dimensionally
conserved substructure comprising the atomic coordinates of the
structurally conserved atoms of the inhibitors and structurally
conserved atoms of the target proteins; and (d) if a conserved
substructure exists, using said atomic coordinates of said
conserved substructure to select a compound having atoms matching
those of said structurally conserved atoms of the inhibitors or to
design a compound that binds to said target protein, wherein the
selection of said compound is performed using computer
modeling.
3. The method of claim 1, further comprising the steps of: (e)
comparing at least one structure from step (a) with at least one
structure from step (b) to determine whether there exists a
three-dimensionally non-conserved substructure comprising the
atomic coordinates of the structurally non-conserved atoms of the
inhibitors and structurally non-conserved atoms of the target
proteins; and (f) if a non-conserved substructure exists, using
said atomic coordinates of said non-conserved substructure to
reject a compound having atoms matching those of said structurally
non-conserved atoms of the inhibitors, wherein the rejection of
said compound is performed in conjunction with computer
modeling.
4. The method of claim 1, wherein at least two structures from step
b are used in step c.
5. The method of claim 4, wherein at least four structures from
step b are used in step c.
6. The method of claim 4, wherein said target proteins comprise at
least two variant forms.
7. The method of claim 6, wherein said target proteins comprise at
least four variant forms
8. The method of claim 1, wherein the inhibitors in said
inhibitor-wild type target protein complex and said
inhibitor-variant target protein complex are the same.
9. The method of claim 1, wherein the inhibitors in said
inhibitor-wild type target protein complex and said
inhibitor-variant target protein complex are different.
10. The method of claim 1, wherein said inhibitors are competitive
inhibitors.
11. The method of claim 1, wherein said inhibitors are
noncompetitive inhibitors.
12. The method of claim 1, wherein said inhibitors are reversible
inhibitors.
13. The method of claim 1, wherein said inhibitors are irreversible
inhibitors.
14. The method of claim 1, wherein said variant target protein is a
homologous target protein.
15. The method of claim 1, wherein said variant target protein is a
mutant target protein.
16. The method of claim 1, wherein at least one of said structures
is a crystal structure.
17. The method of claim 1, wherein at least one of said structures
is an nmr structure.
18. The method of claim 1, wherein at least one of said structures
is derived using computational methods.
19. The method of claim 1, wherein said target protein is expressed
in a microbe and said microbe is selected from the group consisting
of viruses, bacteria, protozoa, or fungi.
20. The method of claim 1, wherein said target protein is expressed
in a neoplasm.
21. The method of claims 19 or 20, wherein said target protein is
selected from the group consisting of an enzyme, a receptor, a
structural protein, a component of a macromolecular complex, a
component of a metabolic pathway, or an assembly of biological
molecules.
22. The method of claim 21, wherein said enzyme is selected from
the group consisting of reverse transcriptases, proteases, DNA and
RNA polymerases, methylases, oxidases, hydratases, esterases, acyl
transferases, helicases, topoisomerases, and kinases.
23. The method of claim 22, wherein said enzyme is HIV
protease.
24. The method of claim 23, wherein said inhibitors are selected
from the group consisting of indinavir, nelfinavir, ritonavir,
saquinavir, amprenavir, lopinavir, and UIC-94003.
25. The method of claim 23, wherein said structurally conserved
atoms of the inhibitor and structurally conserved atoms of the
protease have the atomic structural coordinates as provided in
Table 8.
26. The method of claim 22, wherein said enzyme is 3-dehydroquinate
dehydratase.
27. The method of claim 26, wherein said structurally conserved
atoms of the 3-dehydroquinate dehydratase have the atomic
structural coordinates as provided in Table 12.
28. A compound having a chemical structure selected using the
method of claim 19, wherein said compound has broad spectrum
activity against wild type and variant microbes.
29. A compound having a chemical structure selected using the
method of claim 20, wherein said compound has broad spectrum
activity against wild type and variant neoplasms.
30. The compound of claims 28 or 29, wherein said compound has an
IC.sub.50, variant/IC.sub.50, wild type ratio of less than 20.
31. The compound of claim 30, wherein said IC.sub.50,
variant/IC.sub.50, wildtype ratio is less than 6.
32. The compound of claims 28 or 29, wherein said compound has
broad spectrum activity against at least 3 mutant biological
entities.
33. The compound of claim 28, wherein said compound has broad
spectrum activity against at least 3 different organisms expressing
homologous target proteins.
34. A pharmaceutical composition comprising a compound of claim 28
and a pharmaceutically acceptable carrier or diluent.
35. A pharmaceutical composition comprising a compound of claim 29
and a pharmaceutically acceptable carrier or diluent.
36. A compound having a chemical structure selected using the
method of any one of claims 23-25, wherein said compound has broad
spectrum activity against HIV protease.
37. The compound of claim 36, wherein said compound has an
IC.sub.50, variant/IC.sub.50, wild type ratio of less than 10.
38. The compound of claim 37, wherein said IC.sub.50,
variant/IC.sub.50, wild type ratio is less than 6.
39. The compound of claim 36, wherein said compound has broad
spectrum activity against at least 3 mutant biological
entities.
40. A pharmaceutical composition comprising a compound of claim 36
and a pharmaceutically acceptable carrier or diluent.
41. A compound having a chemical structure selected using the
method of claims 26 or 27, wherein said compound has broad spectrum
activity against 3-dehydroquinate dehydratase.
42. The compound of claim 41, wherein said compound has an
IC.sub.50, variant/IC.sub.50, wild type ratio of less than 20.
43. The compound of claim 42, wherein said IC.sub.50,
variant/IC.sub.50, wild type ratio is less than 10.
44. The compound of claim 43, wherein said compound has broad
spectrum activity against at least 3 mutant biological
entities.
45. The compound of claim 41, wherein said compound has broad
spectrum activity against at least 3 different organisms expressing
homologous target proteins.
46. A pharmaceutical composition comprising a compound of claim 41
and a pharmaceutically acceptable carrier or diluent.
47. A method of treating a microbial infection in a patient, said
method comprising the step of administering to said patient a
pharmaceutical composition of claim 34 in an amount effective to
prevent or treat said infection.
48. A method of treating a neoplasm in a patient in need thereof,
said method comprising the step of administering to said patient a
pharmaceutical composition of claim 35 in amounts effective to
treat said neoplasm.
49. A method of treating an HIV infection in a patient in need
thereof, said method comprising the step of administering to said
patient a pharmaceutical composition of claim 40 in amounts
effective to treat said infection.
50. A method of treating a bacterial infection in a patient in need
thereof, said method comprising the step of administering to said
patient a pharmaceutical composition of claim 46 in amounts
effective to treat said infection.
51. The method of claim 50, wherein said bacterial infection is
caused by a bacterium selected from the group consisting of C
jejuni, V. cholerae, Y pestis, B. anthracis, P. putidas, and M.
tuberculosis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The application claims benefit of U.S. Provisional
Application No. 0/344,788, filed Jan. 7, 2002, and No. 60/383,575,
filed May 29, 2002, each of which is hereby incorporated by
reference.
BACKGROUND OF THE INVENTION
[0002] The present invention relates to the field of inhibitors and
methods for identifying or designing broad spectrum therapeutics
for use in the treatment of infectious diseases and cancers,
particularly where drug resistance is, or could reasonably
predicted to be, an obstacle to successful long term therapy.
[0003] The development of drug resistance is one of the most common
causes of drug failure in the treatment of diseases involving
replicating biological entities (i.e., cancer and infectious
diseases). Drug resistance often results from a reduction in
drug-binding affinity and can be quantified by the ratio of drug
binding affinity (Kd) for variant and wild type target proteins.
Administration of a drug introduces a selective pressure upon the
replicating biological entity. The result is the emergence of drug
resistant strains.
[0004] Drug resistance is a major obstacle to the successful
treatment of many cancers and infections, both bacterial and viral.
For example, increased resistance of bacterial infections to
antibiotic treatment has been extensively documented and has now
become a generally recognized medical problem, particularly with
nosocomial infections. See, for example, Jones et al., Diagn.
Microbiol. Infect. Dis. 31:379 (1998); Murray, Adv. Intern. Med.
42:339 (1997); and Nakae, Microbiologia 13:273 (1997).
[0005] Drug resistance has complicated the treatment for HIV as new
mutant strains of HIV have emerged that are resistant to multiple,
structurally diverse, experimental and chemotherapeutic
antiretrovirals, including HIV protease inhibitors (PIs),
nucleoside and non-nucleoside HIV reverse transcriptase inhibitors
(NRTIs and NNRTIs), and HIV fusion inhibitors (FIs).
[0006] More than 60 million people have been infected by HIV in the
last two decades, and 20 million people have died from HIV/AIDS.
While the development of highly active antiretrovirals to treat
HIV/AIDS has led to significant reductions in the mortality and
morbidity of AIDS, the rapid emergence and spread of drug-resistant
mutant strains of HIV is rendering current drugs ineffective, and
is the major cause of treatment failure. Recent estimates are that
nearly 50% of drug-experienced patients in North America harbor HIV
that is resistant to one or more of the 16 FDA-approved
antiretroviral agents used in multi-drug `cocktails` (Ref. dont
have this ref). Moreover, it has been estimated that drug-resistant
HIV accounts for up to 12% of new infections (Little et al., N.
Engl. J. Med., 347:385 (2002)).
[0007] Accordingly, drug resistant HIV strains represent distinct
infectious entities from a therapeutic viewpoint, and pose new
challenges for drug design as well as drug treatment of existing
infections. Substitutions have been documented in over 45 of the 99
amino acids of the HIV protease monomer in response to protease
inhibitor treatment (Mellors, et al., International Antiviral News,
3:8 (1995); Eastman, et al., J. Virol., 72:5154 (1998); Kozal, et
al., Nat. Med., 2:753 (1996)). The particular sequence and pattern
of mutations selected by PIs is believed to be somewhat
drug-specific and often patient-specific, but high level resistance
is typified by multiple mutations in the protease gene which give
rise to cross-resistance to all of the PIs.
[0008] In view of the foregoing problems, there exists a need for
inhibitors against drug resistant and mdrHIV strains. Further,
there exists a need for inhibitors against drug resistant and
multi-drug resistant HIV proteases (mdrPR). Further still, there
exists a need for inhibitors of HIV that can prevent or slow the
emergence of drug resistant and mdrHIV strains in infected
individuals.
[0009] Inhibitors with the ability to inhibit mdrHIV strains, and
to slow the emergence of drug resistant strains in wild type HIV
infections, are defined as "resistance-repellent" inhibitors.
[0010] There also exists a need for robust methods that can be used
to design "resistance-repellent" inhibitors.
[0011] More generally, there is a need for therapeutic regimens
that minimize the likelihood that resistance will occur in a
disease involving a replicating biological entity. In one approach,
drugs may be designed which have similar activity against both the
wild type and mutant forms of their target. Such drugs minimize the
probability of a mutant population emerging by reducing the
selective pressure introduced by the drug when used to treat wild
type infections. Such drugs also can be used to treat mutant
infections and can be used for salvage therapy.
[0012] There is also an urgent need to develop potent,
broad-spectrum, and mechanistically-novel antimicrobials suitable
for tackling the growing problem of antibiotic-resistant bacteria
strains, and for treating and/or preventing outbreaks of infectious
diseases, including diseases caused by bioterrorism agents like
anthrax, plague, cholera, gastroenteritis, multidrug-resistant
tuberculosis (MDR TB). The recent anthrax attack of 2001
underscored the reality of large-scale aerosol bioweapons attack by
terrorist groups. It also revealed that there is an urgent and
pressing need to discover and develop novel and potent
antimicrobials that can be used therapeutically and
prophylactically for biodefense against new bioattacks. The NIH and
CDC have identified a number of High Priority pathogens based on
their likelihood of causing widespread contagious disease and/or
death to the general population. Research on methods of protection
against potential agents of bioterrorism has been a priority for
several years at the NIH. A recent analysis suggested the existence
of ongoing offensive biological weapons programs in at least 13
countries (Inglesby, T. V., et al., JAMA, 287:2236, (2002)).
[0013] The widespread use of antibiotics in human medical as well
as in agricultural applications has promoted the emergence and
spread of drug resistant bacteria that are no longer sensitive to
existing drugs. The ease with which drug resistant microorganisms
can be selected in a simple laboratory setting is a further concern
when contemplating pharmaceutical-based strategies for biodefense.
There is an urgent need to discover and develop novel therapeutic
agents to combat pathogens that are likely to be used in a
bioterrorist scenario.
[0014] A list of selected agents rated by likelihood to cause the
greatest harm in a bioterrorist attack has been compiled by the CDC
and NIAID (Lane, H. C., et al., Nat Med., 7:1271 (2001)). B.
anthracis; the bacterium that causes anthrax, is one of the most
serious of the group A pathogens. Dissemination of B. anthracis
spores via the US Postal Service in 2001 established the
feasibility of large-scale aerosol bioweapons attack. It has been
estimated that between 130,000 and 3 million deaths would follow
the release of 100 kg of B. anthracis, a lethality matching that of
a hydrogen bomb (Inglesby, T. V., et al., JAMA, 287:2236, (2002)).
Penicillin, doxycycline and ciprofloxacin are currently approved by
the FDA for the treatment of inhalation anthrax infections.
However, it was advised that antibiotic resistance to penicillin-
and tetracycline-class antibiotics should be assumed following a
terrorist attack (Inglesby, T. V., et al., JAMA, 281:1735-45
(1999)). Moreover, in vitro selection of a B. anthracis strain that
is resistant to ofloxacin (a fluoroquinilone closely related to
ciprofloxacin) has been reported (Choe, C. H., et al., Antimicrob.
Agents. Chemother., 44:1766 (2000)). Following the anthrax attacks
of 2001, the CDC advocated the use of a combination of 2-3
antibiotics. As a post-exposure prophylaxis, 60 days of treatment
with ciprofloxacin is currently recommended. Strict compliance to
this drug regimen is complicated by moderate to severe
gastrointestinal tract intolerance.
[0015] Another group A pathogen, Y. pestis, is the causative agent
of plague. If 50 kg of Y. pestis were released as an aerosol over a
city of 5 million, pneumonic plague would afflict an estimated
150,000 individuals and result in 36,000 deaths (Inglesby, T. V.,
et al., JAMA, 283: 2281, (2000)). Streptomycin, tetracycline and
doxycycline are the FDA-approved treatment for plague. Wide spread
use of these antibiotics in the US raises concerns about possible
resistance. A US-licensed, formaldehyde-killed whole bacilli
vaccine was discontinued by its manufacturers in 1999 and is no
longer available.
[0016] C. jejuni and V. cholerae are category B pathogens which can
present a significant threat to the safety of food and water
supplies. C. jejuni infections are one of the most commonly
identified causes of acute bacterial gastroenteritis worldwide and
area frequent cause of Traveler's diarrhea (Allos, B. M., Clin
Infect Dis, 32:1201 (2001)). Currently, the CDC estimates that 2.4
million cases of Campylobacter infection occur in the United States
each year, affecting almost 1% of the entire population. In the
past few years, a rapidly increasing proportion of Campylobacter
strains all over the world have been found to be
fluoroquinolone-resistant. High rates of resistance make
tetracycline, amoxicillin, ampicillin, metronidazole, and
cephalasporins poor choices for the treatment of C. jejuni
infections. All Campylobacter species are inherently resistant to
vancomycin, rifampin, and trimethoprim. V. cholerae, a causative
agent of cholera, is responsible for 120,000 deaths annually
(Faruque, S. M., et al., Microbiol Mol Biol Rev, 62:1301 (1998))
and is characterized by a rapidly changing pattern of antibiotic
resistance.
[0017] TB is one of the most common and devastating infectious
diseases known to man. An estimated one third of the global
population is infected with Mycobacteria tuberculosis. Eight
million people develop an active infection and 2 million victims
die yearly (Dye, C., et al., JAMA, 282:677 (1999.)). Currently, a
combination of four drugs is recommended for TB treatment:
isoniazid, rifampicin, pyrazinamide and ethambutole. The treatment
course lasts 6 months. Such a multidrug combination together with
the lengthy duration of treatment is prone to side-effects and
adherence problems, which in turn can often lead to the development
of drug resistance. The current drugs used to treat TB infections
were introduced into clinical practice more than 30 years ago, in
the absence of any knowledge of molecular mechanism. There is an
urgent need to identify novel, effective, non-toxic and specific
drugs that can shorten the duration of treatment, reduce
side-effects, combat latent infection and reduce the spread of MDR
TB strains. In addition, it is important to recognize the need for
mechanistically novel drugs, i.e., antimicrobial agents that target
biochemical pathways distinct from those of existing TB drugs, in
order to be effective against MDR TB strains.
[0018] In summary, there is a clear need for the discovery of
novel, non-toxic, broad spectrum antibiotics that can be used to
(1) treat drug-resistant bacterial infections, and (2) protect
civilians and military personnel in case of bioterrorist attacks.
In one approach, drugs may be designed which have similar activity
against both the wild type and variant forms of their target. Such
drugs should minimize the probability of the emergence of mutant
populations by reducing the selective pressure introduced by the
drug when used to treat wild type infections. Such drugs also can
be used to treat mutant infections and can be used for salvage
therapy. In another approach, drugs may be designed which have
similar activity against various isotypes of a homologous target.
Such drugs can be used to treat multiple species of pathogenic
microorganisms since they will be active against the target of each
species. In a third approach, drugs can be designed that combine
the properties and the uses of both of the above approaches.
[0019] There also exists a need for robust methods that can be used
to design such broad spectrum antibiotics.
SUMMARY OF THE INVENTION
[0020] In a first aspect the invention features a method for the
structure-based design of a drug that can act as an inhibitor of at
least two different biological entities, the method comprising the
steps of: (a) providing at least one structure of a wild type
target protein or an inhibitor-wild type target protein complex;
(b) providing at least one structure of a variant target protein or
an inhibitor-variant target protein complex; (c) comparing at least
one structure from step (a) with at least one structure from step
(b) to determine whether there exists a common three-dimensionally
conserved substructure comprising the atomic coordinates of the
structurally conserved atoms of the inhibitors and structurally
conserved atoms of the target proteins; and (d) if a conserved
substructure exists, using the atomic coordinates of the conserved
substructure to select a compound having atoms matching those of
the structurally conserved atoms of the inhibitors, wherein the
selection of the compound is performed using computer modeling.
[0021] The invention also features a method for the structure-based
drug design of a broad spectrum compound, the method comprising the
steps of: (a) providing at least one structure of a wild type
target protein or an inhibitor-wild type target protein complex;
(b) providing at least one structure of a variant target protein or
an inhibitor-variant target protein complex; (c) comparing at least
one structure from step (a) with at least one structure from step
(b) to determine whether there exists a common three-dimensionally
conserved substructure comprising the atomic coordinates of the
structurally conserved atoms of the target proteins or a common
three-dimensionally conserved substructure comprising the atomic
coordinates of the structurally conserved atoms of the inhibitors
and structurally conserved atoms of the target proteins; and (d) if
a conserved substructure exists, using the atomic coordinates of
the conserved substructure to select a compound having atoms
matching those of the structurally conserved atoms of the
inhibitors or to design a compound that binds to the target
protein, wherein the selection of the compound is performed using
computer modeling.
[0022] Desirably, the above method further comprises the steps of:
(e) comparing at least one structure from step (a) with at least
one structure from step (b) to determine whether there exists a
three-dimensionally non-conserved substructure comprising the
atomic coordinates of the structurally non-conserved atoms of the
inhibitors and structurally non-conserved atoms of the target
proteins; and (f) if a non-conserved substructure exists, using the
atomic coordinates of the non-conserved substructure to reject a
compound having atoms matching those of the structurally
non-conserved atoms of the inhibitors, wherein the rejection of the
compound is performed in conjunction with computer modeling.
[0023] In any of the above methods, at least two, four, six, or
eight structures from step b can be used in step c. The methods can
be applied using several structures, including at least two, four,
six, or eight variant forms of the target protein.
[0024] The inhibitors used in the inhibitor-wild type target
protein complex and the inhibitor-variant target protein complex
can be the same or different. The inhibitors can be selected from
competitive or noncompetitive inhibitors. Furthermore, the
inhibitors can be selected from reversible, or irreversible
inhibitors.
[0025] In any of the above methods, the variant target protein can
be a homologous protein or a mutant protein.
[0026] In any of the above methods, the structures can be selected
from crystal structures, NMR structures, computer models, any
acceptable experimental, theoretical or computational method of
deriving a three-dimensional representation of a structure, or a
combination thereof.
[0027] Target proteins for use in the present invention include any
therapeutically relevant protein. The target protein can be a
viral, bacterial, protozoan, or fungal protein. In some instances,
the target protein is one that is expressed in a neoplasm.
[0028] Preferably, the target protein can be an enzyme, a receptor,
a structural protein, a component of a macromolecular complex, a
component of a metabolic pathway, or an assembly of biological
molecules. Desirably, the target protein is necessary for the
survival of the replicating biological entity. For example, the
target protein can be an enzyme selected from the group consisting
of reverse transcriptases, proteases, DNA and RNA polymerases,
methylases, oxidases, esterases, acyl transferases, helicases,
topoisomerases, and kinases. The target protein can be a component
of a metabolic pathway, such as the shikimate pathway. Desirable
target proteins include HIV protease or 3-dehydroquinate
dehydratase, among others.
[0029] Where the target protein is HIV protease, suitable
inhibitors for use in the methods of the invention include those
selected from the group consisting of indinavir, nelfinavir,
ritonavir, saquinavir, amprenavir, lopinavir, and UIC-94003.
[0030] A broad spectrum protease inhibitor can be designed using
the susbstructure of structurally conserved atoms described by the
atomic coordinates in Table 8, which includes the structurally
conserved atoms of the inhibitor and structurally conserved atoms
of the protease. A broad spectrum protease inhibitor can also be
designed using the structurally conserved atoms of the inhibitor
alone. These are described by the atomic coordinates in Table
8.
[0031] A broad spectrum 3-dehydroquinate dehydratase inhibitor can
be designed using the susbstructure of structurally conserved atoms
described by the atomic coordinates in Table 12, which includes the
structurally conserved atoms of the 3-dehydroquinate dehydratase. A
broad spectrum 3-dehydroquinate dehydratase inhibitor can also be
designed using the structurally conserved atoms of the inhibitor
alone. These are described by the atomic coordinates in Table
12.
[0032] The invention also features compounds having a chemical
structure selected using any of the methods above. Such compounds
are broad spectrum inhibitors and have broad spectrum activity
against replicating biological entities expressing a particular
target protein. Thus, if the target protein is expressed by a
microbe or a neoplasm, the compound will have broad spectrum
activity against the microbe or neoplasm, respectively.
[0033] The invention features a compound having broad spectrum
activity against HIV protease wherein the compound has a chemical
structure selected using the methods above, including those methods
utilizing the atomic coordinates of Table 8.
[0034] The invention features a compound having broad spectrum
activity against 3-dehydroquinate dehydratase wherein the compound
has a chemical structure selected using the methods above,
including those methods utilizing the atomic coordinates of Table
12.
[0035] The compounds of the invention exclude bis-THF compounds
(e.g., analogs of compounds 1 and 3) as described in J. Med. Chem.
39:3278-3290 (1996) (compounds 49-52 and 58-60), Bioorg. Med. Chem.
Lett. 8:979-982 (1998), WO99/65870, U.S. Pat. No. 6,319,946,
WO02/08657, WO02/092595, WO99/67417, EP00/9917, and WO00/76961; and
also exclude fused ring THF structures as described in Bioorg. Med.
Chem. Lett. 8:687-690 (1998) and U.S. Pat. No. 5,990,155.
[0036] For any of the broad spectrum inhibitors of the invention,
broad spectrum activity can be measured by the ratio of the
inhibitory concentrations of the broad spectrum inhibitor for the
variant and wild type biological entities (IC.sub.50,
variant/IC.sub.50, wild type). Desirably, the IC.sub.50,
variant/IC.sub.50, wild type ratio for a broad spectrum inhibitor
is less than 100, 80, 60, 40, 30, 20, 10, 8, 6, or, most desirably,
less than 3.
[0037] A broad spectrum inhibitor can be active against several
different mutant biological entities. Desirably, the inhibitor will
have broad spectrum activity against at least 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, or 12 mutant biological entities.
[0038] A broad spectrum inhibitor can also be active against
different organisms or neoplastic cell types expressing homologous
target proteins that possess sufficient structural similarity.
Desirably, the inhibitor will have broad spectrum activity against
at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, or 20
different organisms or neoplastic cell types expressing homologous
target proteins.
[0039] The invention also features a pharmaceutical composition
that includes a broad spectrum inhibitor described herein in any
pharmaceutically acceptable form, including isomers such as
diastereomers and enantiomers, salts, solvates, and polymorphs
thereof. The composition can include an inhibitor of the invention
along with a pharmaceutically acceptable carrier or diluent.
[0040] The invention also features methods of treating disease in a
patient in need thereof, which includes the administration of a
pharmaceutical composition of the invention to the patient in an
amount sufficient to treat the disease. The pharmaceutical
composition includes any broad spectrum inhibitor described herein.
Such broad spectrum inhibitors have broad spectrum activity against
replicating biological entities expressing a particular target
protein. Thus, if the target protein is expressed by a microbe or a
neoplasm, the disease to be treated will be a microbial infection
or neoplasm, respectively.
[0041] The invention features a method of treating an HIV infection
in a patient in need thereof, the method including the step of
administering to the patient a pharmaceutical composition including
a broad spectrum protease inhibitor described herein in amounts
effective to treat the HIV infection.
[0042] The invention features a method of treating a bacterial
infection in a patient in need thereof, the method including the
step of administering to the patient a pharmaceutical composition
including a broad spectrum 3-dehydroquinate dehydratase inhibitor
described herein in amounts effective to treat the bacterial
infection. The bacterial infection to be treated using the above
method can be caused by a bacterium selected from the group
consisting of C. jejuni, V. cholerae, Y. pestis, B. anthracis, P.
putidas, and M. tuberculosis. Furthermore, this method can be used
to treat infections by any microbe the utilizes 3-dehydroquinate
dehydratase.
[0043] The invention also features the use of a pharmaceutical
composition described herein in the manufacture of a medicament for
the treatment of a disease. The pharmaceutical composition includes
any broad spectrum inhibitor described herein. Such broad spectrum
inhibitors and have broad spectrum activity against replicating
biological entities expressing a particular target protein. Thus,
if the target protein is HIV protease or 3-dehydroquinate
dehydratase, the disease to be treated will be an HIV infection or
bacterial infection, respectively.
[0044] The term "replicating biological entity" includes, for
example, bacteria, fungi, yeasts, viruses, protozoa, prions and
neoplasms
[0045] Neoplasms include, for example, carcinomas of the bladder,
breast, colon, kidney, liver, lung, head and neck, gall-bladder,
ovary, pancreas, stomach, cervix, thyroid, prostate, or skin; a
hematopoietic tumor of lymphoid lineage; a hematopoietic tumor of
myeloid lineage; a tumor of mesenchymal origin; a tumor of the
central or peripheral nervous system; melanoma; seminoma;
teratocarcinoma; osteosarcoma; thyroid follicular cancer; and
Kaposi's sarcoma. Hematopoietic tumors of lymphoid lineage can be
leukemia, acute lymphocytic leukemia, acute lymphoblastic leukemia,
B-cell lymphoma, T-cell-lymphoma, Hodgkin's lymphoma, non-Hodgkin's
lymphoma, hairy cell lymphoma and Burkett's lymphoma.
[0046] By "wild type target protein" is meant a protein obtained
from a replicating biological entity that has not been subjected to
drug selection pressure, and could include polymorphisms or
isoforms thereof. A replicating biological entity that expresses
wild type target protein is referred to herein as a wild type
biological entity.
[0047] By "variant target protein" is meant a mutant target protein
or a homologous target protein. A replicating biological entity
that expresses variant target protein is referred to herein as a
variant biological entity.
[0048] By "mutant target protein" is meant a target protein that
contains one or more amino acid substitutions with respect to the
wild type target protein, including proteins from the same organism
that have evolved under drug selection pressure. In general, mutant
target proteins will have one or more amino acid substitutions and
should be readily identified as related to the cognate wild type
protein using standard sequence comparison methods. A replicating
biological entity that expresses mutant target protein is referred
to herein as a mutant biological entity.
[0049] By "homologous target protein" is meant a variant target
protein that is expressed in a different species or neoplastic cell
type than the wild type target protein, but has the same, or
similar, function.
[0050] By "structurally conserved target substructure", and by
"structurally" or "three-dimensionally conserved substructure" as
applied to target proteins, is meant the regions of the target
protein structure which are not significantly affected by amino
acid mutations or substitutions. Such regions can be defined using
standard methods of comparative analysis of three-dimensional
structures of proteins, such as superposition analysis, for
example. In the case of HIV protease, these regions were identified
using a pair wise superposition analysis of wild type and mutant
protease structures complexed with inhibitors. The superposition of
structures can be performed using the iterative procedure described
herein. In the case of DHQase, these regions were identified using
a pair wise superposition analysis of wild type and homologous
DHQase structures from different bacterial species with and without
inhibitors. It is apparent that the overall compositions of
structurally conserved target substructures will likely differ for
different, non-homologous target proteins, especially when the
frequency of amino acid substitutions in high. However, a
quantitative definition can be derived from the superposition
analysis, which provides both the identities and the positions of
the atoms that comprise these substructures. The regions that
comprise structurally conserved target substructures contain atoms
whose superimposed pairs have three-dimensional atomic coordinates
that match to within a distance of 1 .ANG., 0.6 .ANG., 0.4 .ANG.,
or 0.2 .ANG..
[0051] By "broad spectrum inhibitor" is meant a compound having
broad spectrum activity, i.e., an inhibitor that is active against
two different-biological entities, e.g., both a wild type
biological entity and one or more variants of that biological
entity. Thus, broad spectrum activity can be described by the
inhibitor's action against a particular target protein (e.g., broad
spectrum activity against protease) or a particular target organism
(e.g., broad spectrum activity against HIV). Broad spectrum
inhibitors will have medically insignificant interactions with
non-conserved regions. Broad spectrum inhibitors can be useful for
the treatment and/or prevention of infectious diseases caused by
multiple infectious agents, as well as for decreasing the
development of drug-resistance by these organisms.
[0052] As used herein, the term "treating" refers to administering
a pharmaceutical composition for prophylactic and/or therapeutic
purposes. To "prevent disease" refers to prophylactic treatment of
a patient who is not yet ill, but who is susceptible to, or
otherwise at risk of, a particular disease. To "treat disease" or
use for "therapeutic treatment" refers to administering treatment
to a patient already suffering from a disease to ameliorate the
disease and improve the patient's condition. Thus, in the claims
and embodiments, treating is the administration to a patient either
for therapeutic or prophylactic purposes.
[0053] The term "microbial infection" refers to the invasion of the
host patient by pathogenic microbes (e.g., bacteria, fungi, yeasts,
viruses, protozoa). This includes the excessive growth of microbes
that are normally present in or on the body of a patient. More
generally, a microbial infection can be any situation in which the
presence of a microbial population(s) is damaging to a host
patient. Thus, a patient is "suffering" from a microbial infection
when excessive numbers of a microbial population are present in or
on a patient's body, or when-the presence of a microbial
population(s) is damaging the cells or other tissue of a
patient.
[0054] The term "microbes" includes, for example, bacteria, fungi,
yeasts, viruses and protozoa. The term "administration" or
"administering" refers to a method of giving a dosage of a
pharmaceutical composition to a patient, where the method is, e.g.,
topical, oral, intravenous, intraperitoneal, or intramuscular. The
preferred method of administration can vary depending on various
factors, e.g., the components of the pharmaceutical composition,
site of the potential or actual disease and severity of
disease.
[0055] The term "patient" includes humans, cattle, pigs, sheep,
horses, dogs, and cats, and also includes other vertebrate, most
preferably, mammalian species.
[0056] Where "atomic coordinates" are provided, or otherwise
referred to, these coordinates define a three dimensional
structure. That such a structure may be defined by more than one
different coordinate system, e.g., by translation or rotation of
the coordinates, does not change the relative positions of the
atoms in the structure. Accordingly, any reference to atomic
coordinates herein is intended to include any equivalent three
dimensional structure defined by the coordinates.
[0057] By "computer modeling" is meant the use of a computer to
visualize or compute a compound, a portion of a compound, a target
protein, a portion of a target protein, a complex between a
compound and a target protein, or a portion of a complex between a
compound and a target protein.
[0058] Other features and advantages of the invention will be
apparent from the following detailed description and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0059] FIG. 1 is a table depicting the structures of compounds 1-7,
gt33, and qxa.
[0060] FIG. 2 illustrates the amino acid alignment of type II
DHQases. Fully conserved residues are framed. Catalytically
important amino acids are marked by stars. Arrows denote amino
acids that make hydrogen bonds and ionic interactions in the
structure of M. tuberculosis DHQase complexed with the inhibitor,
3-dehydroquinic acid oxime.
[0061] FIG. 3 illustrates the key interactions of the
substrate-based inhibitor, DHQO, with the active site residues for
the Type II DHQase from M. tuberculosis.
DETAILED DESCRIPTION
[0062] We have discovered that the comparative analysis of the
structures of complexes of inhibitors bound to wild type and
variant forms of a target protein can be used to design compounds
that are broad spectrum inhibitors.
[0063] The methods of the invention entail the design of compounds
having a particular structure. The methods rely upon the use of
structural information to arrive at these compounds. The structural
data define a three dimensional array-of the important contact
atoms in an inhibitor that bind to the target protein in a fashion
that results in broad spectrum activity against biological entities
expressing variants of the target protein.
[0064] Inhibitor-Target Protein Structures
[0065] Atomic structural coordinates can be selected from crystal
structures, NMR structures, computer models, any acceptable
experimental, theoretical or computational method of deriving a
three-dimensional representation of a structure, or a combination
thereof. Atomic coordinates for use in the methods of the invention
can be obtained from publicly available sources, e.g. from the
Protein Data Bank, or obtained using known experimental or
computational methods.
[0066] Atomic structural coordinates for use in the methods of the
invention include crystal structures of HIV protease/inhibitor
complexes derived from wild type and drug-resistant mutant
proteases, and of DHQase and DHQase inhibitor complexes derived
from two or more bacterial species, among others. In examples 1-3,
the methods of the invention are applied using the coordinates of
wild type HIV protease complexed with amprenavir, wild type HIV
protease complexed with UIC-94003, and V82F/184V mutant HIV
protease complexed with UIC-94003. In example 4, the methods of the
invention are applied using the coordinates of wild type DHQase
from M. tuberculosis and from Pseudomonas putidas. a complex
between a compound and a target protein. The coordinates of other
representative structures of HIV protease and DHQase should be
useful for performing the methods of the present invention.
[0067] Conserved Substructures
[0068] Conserved substructures can be identified for target
proteins, for target protein-inhibitor complexes, and/or for
inhibitors, depending on the nature of the structures that are used
in the comparative superposition analysis. In one approach, at
least one structure of a wild type target protein is compared to at
least one structure of a mutant or homologous target protein to
determine whether a common three-dimensionally conserved
substructure is present among the wild type protein and the mutant
or homologous proteins, respectively. In another approach, at least
one structure of an inhibitor-wild type target protein complex and
at least one structure of an inhibitor-mutant target protein
complex are compared to determine whether a common
three-dimensionally conserved substructure is present among the
mutant and wild type complexes. In a third approach, at least one
structure of an inhibitor-wild type target protein complex and at
least one structure of a mutant or homologous target protein
without inhibitor are compared to determine whether a common
three-dimensionally conserved substructure is present among the
respective mutant or homologous protein and the wild type
complexes. Variations of the approached described above can also be
used. In each case, such a comparison can be made by means of (a)
an overall superposition of the atoms of the protein structures;
and, where feasible, (b) a study of the distances from atoms of the
inhibitors to atoms of the protein. This analysis requires
three-dimensional atomic coordinates of the protein structures and
of the bound inhibitor.
[0069] The superposition of the protein structures can be performed
in a two step process: 1) the distance between all pairs of
corresponding C atoms (C atom of residue number 1 in one protein to
C atom of residue number 1 in the second protein; C atom of residue
number 2 in one protein to C atom of residue number 2 in the second
protein; and so on) of the polypeptide chains is minimized by means
of a least-square algorithm; 2) the superposition is refined by
minimizing, in an iterative process, the distances between
corresponding C atoms that are closer than a given distance (0.25 A
for example), thus eliminating regions of the structures having
large conformational differences to compute the superposition
parameters. Furthermore, where a partial structure is provided
(e.g., from NMR data) the available coordinates are
superimposed.
[0070] The conserved substructure identifies the relevant portion
of the target protein that is the active site, or binding region,
defined by that part of the target protein interacting with
inhibitor. Important interactions between the target protein and
inhibitor are identified by mapping the contacts between the two.
Structurally conserved regions of the target protein not near the
binding site are generally not relevant to the design of the broad
spectrum inhibitor. Accordingly, the selection of the meaningful
substructure is identified using the above mentioned contacts.
[0071] Design of a Broad Spectrum Inhibitor
[0072] The coordinates of the conserved inhibitor substructure are
used to design an inhibitor having atoms matching those of the
three-dimensionally structurally conserved atoms of the inhibitors.
The result is an inhibitor for which IC.sub.50, variant and IC50,
wild type are similar, minimizing the selective pressure introduced
by the drug.
[0073] The methods of the invention can employ computer-based
methods for designing broad spectrum inhibitors. These
computer-based methods fall into two broad classes: database
methods and de novo design methods. In database methods the
compound of interest is compared to all compounds present in a
database of chemical structures and compounds whose structure is in
some way similar to the compound of interest are identified. The
structures in the database are based on either experimental data,
generated by NMR or x-ray crystallography, or modeled
three-dimensional structures based on two-dimensional (i.e.,
sequence) data. In de novo design methods, models of compounds
whose structure is in some way similar to the compound of interest
are generated by a computer program using information derived from
known structures, e.g., data generated by x-ray crystallography
and/or theoretical rules. Such design methods can build a compound
having a desired structure in either an atom-by-atom manner or by
assembling stored small molecular fragments.
[0074] The success of both database and de novo methods in
identifying compounds having the desired activity depends on the
identification of the functionally relevant portion of the compound
of interest. The functionally relevant portion of the compound, the
pharmacophore, is defined by the structurally conserved
substructure. A pharmacophore then is an arrangement of structural
features and functional groups important for obtaining an inhibitor
having broad spectrum activity.
[0075] Not all identified compounds having the desired
pharmacophore will act as broad spectrum inhibitors. The actual
activity can be finally determined only by measuring the activity
of the compound in relevant biological assays. However, the methods
of the invention are extremely valuable because they can be used to
greatly reduce the number of compounds which must be tested to
identify those likely to exhibit broad spectrum activity.
[0076] Programs suitable for generating predicted three-dimensional
structures from two-dimensional data include: Concord (Tripos
Associated, St. Louis, Mo.), 3-D Builder (Chemical Design Ltd.,
Oxford, U.K.), Catalyst (Bio-CAD Corp., Mountain View, Calif.), and
Daylight (Abbott Laboratories, Abbott Park, Ill.).
[0077] Programs suitable for searching three-dimensional databases
to identify molecules bearing a desired pharmacophore include:
MACCS-3D and ISIS/3D (Molecular Design Ltd., San Leandro, Calif.),
ChemDBS-3D (Chemical Design Ltd., Oxford, U.K.), and Sybyl/3DB
Unity (Tripos Associates, St. Louis, Mo.).
[0078] Programs suitable for pharmacophore selection and design
include: DISCO (Abbott Laboratories, Abbott Park, Ill.), Catalyst
(Bio-CAD Corp., Mountain View, Calif.), and ChemDBS-3D (Chemical
Design Ltd., Oxford, U.K.).
[0079] Databases of chemical structures are available from
Cambridge Crystallographic Data Centre (Cambridge, U.K.) and
Chemical Abstracts Service (Columbus, Ohio).
[0080] De novo design programs include Ludi (Biosym Technologies
Inc., San Diego, Calif.) and Aladdin (Daylight Chemical Information
Systems, Irvine Calif.).
[0081] One skilled in the art may use one of several methods to
screen chemical entities for their ability to match the conserved
substructure. This process may begin by visual inspection of, for
example, the active site on the computer screen based on the atomic
coordinates for the target protein. Docking may be accomplished
using software such as Quanta and Sybyl, followed by energy
minimization and molecular dynamics with standard molecular
mechanics forcefields, such as CHARMM and AMBER.
[0082] Specialized computer programs may also assist in the process
of selecting chemical entities. These include:
[0083] 1. GRID (Goodford, P. J., "A Computational Procedure for
Determining Energetically Favorable Binding Sites on Biologically
Important Macromolecules," J. Med. Chem., 28:849 (1985)). GRID is
available from Oxford University, Oxford, UK.
[0084] 2. MCSS (Miranker, A. and M. Karplus, "Functionality Maps of
Binding Sites: A Multiple Copy Simultaneous Search Method."
Proteins: Structure, Function, and Genetics, 11:29 (1991)). MCSS is
available from Molecular Simulations, Burlington, Mass.
[0085] 3. AUTODOCK (Goodsell, D. S. and A. J. Olsen, "Automated
Docking of Substrates to Proteins by Simulated Annealing,"
Proteins: Structure, Function, and Genetics, 8:195 (1990)).
AUTODOCK is available from Scripps Research Institute, La Jolla,
Calif.
[0086] 4. DOCK (Kuntz, L. D. et al., "A Geometric Approach to
Macromolecule-Ligand Interactions," J. Mol. Biol., 161:269 (1982)).
DOCK is available from University of California, San Francisco,
Calif.
[0087] Once the conserved substructure for the inhibitor has been
identified, the conserved atoms of the inhibitor can be selected
for assembly into a single inhibitor. Assembly may be proceed by
visual inspection of the relationship of the fragments to each
other on the three-dimensional image displayed on a computer screen
in relation to the structure coordinates of the target protein.
This may be followed by manual model building using software such
as Quanta or Sybyl.
[0088] Useful programs to aid one of skill in the art in assembly
of the individual chemical entities or fragments include:
[0089] 1. CAVEAT (Bartlett, P. A. et al, "CAVEAT: A Program to
Facilitate the Structure-Derived Design of Biologically Active
Molecules". In "Molecular Recognition in Chemical and Biological
Problems," Special Pub., Royal Chem. Soc., 78:182 (1989)). CAVEAT
is available from the University of Calif., Berkeley, Calif.
[0090] 2. 3D Database systems such as MACCS-3D (MDL Information
Systems, San Leandro, Calif.). This area is reviewed in Martin, Y.
C., "3D Database Searching in Drug Design," J. Med. Chem., 35:2145
(1992)).
[0091] 3. HOOK (available from Molecular Simulations, Burlington,
Mass.).
[0092] Other molecular modeling techniques may also be employed in
accordance with this invention. See, e.g., Cohen, N. C. et al.,
"Molecular Modeling Software and Methods for Medicinal Chemistry,"
J. Med. Chem., 33:883 (1990). See also, Navia, M. A. and M. A.
Murcko, "The Use of Structural Information in Drug Design," Current
Opinions in Structural Biology, 2:202 (1992).
[0093] Once a broad spectrum inhibitor has been optimally designed,
as described above, substitutions may then be made in some of its
atoms or side groups in order to improve or modify its binding
properties. Generally, initial substitutions are conservative,
i.e., the replacement group will have approximately the same size,
shape, hydrophobicity and charge as the original group. It should,
of course, be understood that components known in the art to alter
conformation should be avoided.
[0094] In general, inhibitors designed using the methods of the
invention can be tested for broad spectrum activity using any of
the to in vitro and/or in vivo methods described below, among
others.
[0095] Broad Spectrum Inhibitors
[0096] Broad spectrum inhibitors match the pharmacophore defined by
the structurally conserved substructure. The pharmacophore is the
arrangement of structural features and functional groups important
for obtaining an inhibitor having broad spectrum activity. This
pharmacophore is derived using structural data for known inhibitors
complexed to a target protein. Accordingly, broad spectrum
inhibitors will often be structurally related to known compounds
lacking broad spectrum activity, but useful in the design of broad
spectrum inhibitors using the methods disclosed herein. These known
inhibitors serve as lead compounds for both the design and
synthesis of a broad spectrum inhibitor. Using the synthetic
methods for making the lead compounds and standard synthetic
methods as described by, for example, J. March, Advanced Organic
Chemistry: Reactions, Mechanisms and Structure," John Wiley &
Sons, Inc., 1992; T. W. Green and P. G. M. Wuts, "Protective Groups
in Organic Synthesis" (2.sup.nd Ed.), John Wiley & Sons, 1991;
and P. J. Kocienski, "Protecting Groups," Georg Thieme Verlag,
1994, one can synthesize the broad spectrum inhibitors described
herein.
[0097] Typically the lead compounds bear varied functional groups
which are present in the pharmacophore, including hydrogen-bond
donors, hydrogen-bond acceptors, ionic moieties, polar moieties,
hydrophobic moieties, aromatic centers, and electron-donors and
acceptors. These are linked by a structural scaffold which imparts
the appropriate a three dimension arrangement of the functional
groups.
[0098] Numerous modifications of the lead compound can be made
using techniques known in the art. These include changing a
functional group by replacing it with another moiety of the same
group. For example, one hydrogen-bond donor may be substituted by
another. A good hydrogen bond donor has an H atom bonded to a very
electronegative atom (e.g., O--H or N--H). Examples of
hydrogen-bond donors include alcohols, carboxylic acids, oximes,
and amides, among others. Similarly, one hydrogen-bond acceptor may
be substituted by another. A good hydrogen bond acceptor has an
electronegative element with lone pairs (e.g., O, N, or F).
Examples of hydrogen bond acceptors include water, halogen atoms,
alcohols, amines, carbonyls, ethers, and amides, among others. It
may also be desirable to alter the distance between functional
groups in a lead compound. This is achieved by employing synthetic
methods analogous to those used to prepare the lead compound, but
replacing the scaffold with a structurally related scaffold that
provides the desired distance (e.g., a scaffold that incorporates
more or fewer atoms linking the relevant functional groups). In
some instances it may also be desirable to alter the
stereochemistry in a lead compound. This can be accomplished by
employing racemic starting materials, or by employing reaction
conditions that result in racemization of the relevant chiral
center, followed by separation of the enantiomeric or
diastereomeric mixture.
[0099] Assays
[0100] Inhibitors designed using the methods disclosed herein may
be further assayed, using standard in vitro models or animal
models, to evaluate therapeutic activity and toxicity. These assays
are described in the literature and are familiar to those skilled
in the art. These include but are not limited to assays for
monitoring or measuring efficacy against HIV, bacteria, and
neoplasms.
[0101] One skilled in the art will be familiar with methods of
measuring the IC.sub.50's of a broad spectrum inhibitor described
herein. The IC.sub.50 value is determined by plotting percent
activity versus inhibitor concentration in the assay and
identifying the concentration at which 50% of the activity (e.g.,
growth, enzymatic activity, protein production, etc.) remains.
Inhibitors can be tested for antimicrobial activity against a panel
of organisms according to standard procedures described by the
National Committee for Clinical Laboratory Standards (NCCLS
document 7-A3, Vol. 13, No. 25, 1993/NCCLS document M27-P, Vol. 12,
No. 25, 1992). Inhibitors can be dissolved (0.1 .mu.g/ml-500
.mu.g/ml) in microbial growth media, diluted, and added to wells of
a microtiter plate containing bacteria or fungal cells in a final
volume of an appropriate media (Mueller-Hinton Broth; Haemophilus
Test Media; Mueller-Hinton Broth+5% Sheep Blood; or RPMI 1690).
Typically, the plates are incubated overnight at an appropriate
temperature (30.degree. C. to 37.degree. C.) and optical densities
(measure of cell growth) are measured using a commercial plate
reader.
[0102] IC.sub.50 's for broad spectrum protease inhibitors can be
measured against wild type HIV and clinically isolated mutant HIV
isolates, utilizing the PHA-PBMC exposed to HIV-1 (50 TCID.sub.50
dose/X0.sup.6 PBMC) as target cells and using the inhibition of p24
Gag protein production as an endpoint. The amounts of p24 antigen
produced by the cells can be determined on day 7 in culture using a
commercially available radioimmunoassay kit. Drug concentrations
resulting in 50% inhibition (IC.sub.50's) of p24 antigen production
can be determined by comparison with the p24 production level in
drug-free control cell cultures.
[0103] Therapy
[0104] The invention features a method of identifying a compound
having broad spectrum activity. Broad spectrum inhibitors of the
present invention may be administered by any appropriate route for
treatment or prevention of a disease or condition associated with a
bacterial infection, viral infection, or neoplastic disorder, among
others. These may be administered to humans, domestic pets,
livestock, or other animals with a pharmaceutically acceptable
diluent, carrier, or excipient, in unit dosage form. Administration
may be topical, parenteral, intravenous, intra-arterial,
subcutaneous, intramuscular, intracranial, intraorbital,
ophthalmic, intraventricular, intracapsular, intraspinal,
intracistemal, intraperitoneal, intranasal, aerosol, by
suppositories, or oral administration.
[0105] Therapeutic formulations may be in the form of liquid
solutions or suspensions; for oral administration, formulations may
be in the form of tablets or capsules; and for intranasal
formulations, in the form of powders, nasal drops, or aerosols.
[0106] Methods well known in the art for making formulations are
found, for example, in "Remington: The Science and Practice of
Pharmacy" (20th ed., ed. A. R. Gennaro AR., 2000, Lippincott
Williams & Wilkins). Formulations for parenteral administration
may, for example, contain excipients, sterile water, or saline,
polyalkylene glycols such as polyethylene glycol, oils of vegetable
origin, or hydrogenated napthalenes. Biocompatible, biodegradable
lactide polymer, lactide/glycolide copolymer, or
polyoxyethylene-polyoxypropylene copolymers may be used to control
the release of the compounds. Nanoparticulate formulations (e.g.,
biodegradable nanoparticles, solid lipid nanoparticles, liposomes)
may be used to control the biodistribution of the compounds. Other
potentially useful parenteral delivery systems include
ethylene-vinyl acetate copolymer particles, osmotic pumps,
implantable infusion systems, and liposomes. Formulations for
inhalation may contain excipients, for example, lactose, or may be
aqueous solutions containing, for example, polyoxyethylene-9-lauryl
ether, glycholate and deoxycholate, or may be oily solutions for
administration in the form of nasal drops, or as a gel. The
concentration of the broad spectrum inhibitor in the formulation
will vary depending upon a number of factors, including the dosage
of the drug to be administered, and the route of
administration.
[0107] The broad spectrum inhibitor may be optionally administered
as a pharmaceutically acceptable salt, such as a non-toxic acid
addition salts or metal complexes that are commonly used in the
pharmaceutical industry. Examples of acid addition salts include
organic acids such as acetic, lactic, pamoic, maleic, citric,
malic, ascorbic, succinic, benzoic, palmitic, suberic, salicylic,
tartaric, methanesulfonic, toluenesulfonic, or trifluoroacetic
acids or the like; polymeric acids such as tannic acid,
carboxymethyl cellulose, or the like; and inorganic acid such as
hydrochloric acid, hydrobromic acid, sulfuric acid phosphoric acid,
or the like. Metal complexes include zinc, iron, and calcium, among
others.
[0108] Administration of compounds in controlled release
formulations is useful where the broad spectrum inhibitor has (i) a
narrow therapeutic index (e.g., the difference between the plasma
concentration leading to harmful side effects or toxic reactions
and the plasma concentration leading to a therapeutic effect is
small; generally, the therapeutic index, TI, is defined as the
ratio of median lethal dose (LD.sub.50) to median effective dose
(ED.sub.50)); (ii) a narrow absorption window in the
gastro-intestinal tract; or (iii) a short biological half-life, so
that frequent dosing during a day is required in order to sustain
the plasma level at a therapeutic level.
[0109] Many strategies can be pursued to obtain controlled release
in which the rate of release outweighs the rate of metabolism of
the broad spectrum inhibitor. For example, controlled release can
be obtained by the appropriate selection of formulation parameters
and ingredients, including, e.g., appropriate controlled release
compositions and coatings. Examples include single or multiple unit
tablet or capsule compositions, oil solutions, suspensions,
emulsions, microcapsules, microspheres, nanoparticles, patches, and
liposomes.
[0110] Formulations for oral use include tablets containing the
active ingredient(s) in a mixture with non-toxic pharmaceutically
acceptable excipients. These excipients may be, for example, inert
diluents or fillers (e.g., sucrose and sorbitol), lubricating
agents, glidants, and antiadhesives (e.g., magnesium stearate, zinc
stearate, stearic acid, silicas, hydrogenated vegetable oils, or
talc).
[0111] Formulations for oral use may also be provided as chewable
tablets, or as hard gelatin capsules wherein the active ingredient
is mixed with an inert solid diluent, or as soft gelatin capsules
wherein the active ingredient is mixed with water or an oil
medium.
[0112] Pharmaceutical formulations of broad spectrum inhibitor
described herein include isomers such as diastereomers and
enantiomers, mixtures of isomers, including racemic mixtures,
salts, solvates, and polymorphs thereof.
[0113] The formulations can be administered to human patients in
therapeutically effective amounts. For example, when the broad
spectrum inhibitor is an antimicrobial drug, an amount is
administered which prevents, stabilizes, eliminates, or reduces a
microbial infection. Typical dose ranges are from about 0.01
.mu.g/kg to about 2 mg/kg of body weight per day. The exemplary
dosage of drug to be administered is likely to depend on such
variables as the type and extent of the disorder, the overall
health status of the particular patient, the formulation of the
compound excipients, and its route of administration. Standard
clinical trials maybe used to optimize the dose and dosing
frequency for any particular broad spectrum inhibitor.
[0114] The following examples are meant to illustrate, but in no
way limit, the claimed invention.
EXAMPLE I
[0115] This example illustrates the method by which
experimentally-determined crystal structures of the same inhibitor
in complex with wild type and mutant species of HIV protease can be
compared and analyzed for the existence of a three-dimensionally
conserved substructure.
[0116] The structures of wild type HIV-1 protease and a mutant,
V82F/184V, HIV-1 protease, both in complexes with the inhibitor
shown in FIG. 1 were determined using conventional x-ray
crystallography techniques. The structures were analyzed by means
of (a) an overall superposition of the atoms of the protein
structures; and, (b) a study of the distances from atoms of the
inhibitors to atoms of the protein. This analysis requires three
dimensional atomic coordinates of the protein structures and of the
bound inhibitor.
[0117] The superposition of the protein structures was performed in
a two step process: 1) the distance between all pairs of
corresponding C atoms (C atom of residue number 1 in one protein to
C atom of residue number 1 in the second protein; C atom of residue
number 2 in one protein to C atom of residue number 2 in the second
protein; and so on) of the polypeptide chains is minimized by means
of a least-square algorithm; 2) the superposition is refined by
minimizing, in an iterative process, the distances between
corresponding C atoms that are closer than a given distance (0.25
.ANG. in this example), thus eliminating regions of the structures
having large conformational differences to compute the
superposition parameters. The distances between equivalenced C
atoms after the minimization procedure are shown in Table 4.
1TABLE 4 Distances between equivalent C atoms Molecule 1: HIV-1 PR
wt: 1 Molecule 2: HIV-1 PR V82F/I84V mutant: 1 Molecule 1 Molecule
2 distance [.ANG.] CA PRO 1 CA PRO 1 0.455 CA GLN 2 CA GLN 2 0.434
CA ILE 3 CA ILE 3 0.418 CA THR 4 CA THR 4 0.317 CA LEU 5 CA LEU 5
0.172 CA TRP 6 CA TRP 6 0.228 CA GLN 7 CA GLN 7 0.364 CA ARG 8 CA
ARG 8 0.166 CA PRO 9 CA PRO 9 0.057 CA LEU 10 CA LEU 10 0.183 CA
VAL 11 CA VAL 11 0.194 CA THR 12 CA THR 12 0.168 CA ILE 13 CA ILE
13 0.146 CA LYS 14 CA LYS 14 0.229 CA ILE 15 CA ILE 15 0.266 CA GLY
16 CA GLY 16 0.662 CA GLY 17 CA GLY 17 0.491 CA GLN 18 CA GLN 18
0.267 CA LEU 19 CA LEU 19 0.112 CA LYS 20 CA LYS 20 0.128 CA GLU 21
CA GLU 21 0.190 CA ALA 22 CA ALA 22 0.169 CA LEU 23 CA LEU 23 0.218
CA LEU 24 CA LEU 24 0.233 CA ASP 25 CA ASP 25 0.160 CA THR 26 CA
THR 26 0.200 CA GLY 27 CA GLY 27 0.303 CA ALA 28 CA ALA 28 0.169 CA
ASP 29 CA ASP 29 0.150 CA ASP 30 CA ASP 30 0.038 CA THR 31 CA THR
31 0.047 CA VAL 32 CA VAL 32 0.173 CA LEU 33 CA LEU 33 0.194 CA GLU
34 CA GLU 34 0.310 CA GLU 35 CA GLU 35 0.260 CA MET 36 CA MET 36
0.136 CA SER 37 CA SER 37 0.494 CA LEU 38 CA LEU 38 0.607 CA PRO 39
CA PRO 39 0.094 CA GLY 40 CA GLY 40 0.774 CA ARG 41 CA ARG 41 0.448
CA TRP 42 CA TRP 42 0.204 CA LYS 43 CA LYS 43 0.596 CA PRO 44 CA
PRO 44 0.625 CA LYS 45 CA LYS 45 0.541 CA MET 46 CA MET 46 0.643 CA
ILE 47 CA ILE 47 0.361 CA GLY 48 CA GLY 48 0.240 CA GLY 49 CA GLY
49 0.182 CA ILE 50 CA ILE 50 0.110 CA GLY 51 CA GLY 51 0.243 CA GLY
52 CA GLY 52 0.200 CA PHE 53 CA PHE 53 0.119 CA ILE 54 CA ILE 54
0.255 CA LYS 55 CA LYS 55 0.295 CA VAL 56 CA VAL 56 0.108 CA ARG 57
CA ARG 57 0.129 CA GLN 58 CA GLN 58 0.074 CA TYR 59 CA TYR 59 0.372
CA ASP 60 CA ASP 60 0.496 CA GLN 61 CA GLN 61 0.780 CA ILE 62 CA
ILE 62 0.406 CA LEU 63 CA LEU 63 0.211 CA ILE 64 CA ILE 64 0.260 CA
GLU 65 CA GLU 65 0.193 CA ILE 66 CA ILE 66 0.181 CA CYS 67 CA CYS
67 0.518 CA GLY 68 CA GLY 68 0.641 CA HIS 69 CA HIS 69 0.319 CA LYS
70 CA LYS 70 0.179 CA ALA 71 CA ALA 71 0.265 CA ILE 72 CA ILE 72
0.350 CA GLY 73 CA GLY 73 0.253 CA THR 74 CA THR 74 0.301 CA VAL 75
CA VAL 75 0.187 CA LEU 76 CA LEU 76 0.186 CA VAL 77 CA VAL 77 0.070
CA GLY 78 CA GLY 78 0.306 CA PRO 79 CA PRO 79 0.047 CA THR 80 CA
THR 80 0.470 CA PRO 81 CA PRO 81 0.404 CA VAL 82 CA PHE 82 0.556 CA
ASN 83 CA ASN 83 0.146 CA ILE 84 CA VAL 84 0.196 CA ILE 85 CA ILE
85 0.163 CA GLY 86 CA GLY 86 0.224 CA ARG 87 CA ARG 87 0.127 CA ASN
88 CA ASN 88 0.048 CA LEU 89 CA LEU 89 0.081 CA LEU 90 CA LEU 90
0.197 CA THR 91 CA THR 91 0.226 CA GLN 92 CA GLN 92 0.176 CA ILE 93
CA ILE 93 0.151 CA GLY 94 CA GLY 94 0.338 CA CYS 95 CA CYS 95 0.233
CA THR 96 CA THR 96 0.305 CA LEU 97 CA LEU 97 0.089 CA ASN 98 CA
ASN 98 0.260 CA PHE 99 CA PHE 99 0.250 CA PRO 101 CA PRO 101 0.227
CA GLN 102 CA GLN 102 0.108 CA ILE 103 CA ILE 103 0.206 CA THR 104
CA THR 104 0.169 CA LEU 105 CA LEU 105 0.125 CA TRP 106 CA TRP 106
0.363 CA GLN 107 CA GLN 107 0.296 CA ARG 108 CA ARG 108 0.400 CA
PRO 109 CA PRO 109 0.173 CA LEU 110 CA LEU 110 0.182 CA VAL 111 CA
VAL 111 0.085 CA THR 112 CA THR 112 0.123 CA ILE 113 CA ILE 113
0.107 CA LYS 114 CA LYS 114 0.368 CA ILE 115 CA ILE 115 0.226 CA
GLY 116 CA GLY 116 0.638 CA GLY 117 CA GLY 117 0.516 CA GLN 118 CA
GLN 118 0.414 CA LEU 119 CA LEU 119 0.102 CA LYS 120 CA LYS 120
0.191 CA GLU 121 CA GLU 121 0.206 CA ALA 122 CA ALA 122 0.197 CA
LEU 123 CA LEU 123 0.231 CA LEU 124 CA LEU 124 0.145 CA ASP 125 CA
ASP 125 0.235 CA THR 126 CA THR 126 0.311 CA GLY 127 CA GLY 127
0.200 CA ALA 128 CA ALA 128 0.102 CA ASP 129 CA ASP 129 0.143 CA
ASP 130 CA ASP 130 0.261 CA THR 131 CA THR 131 0.172 CA VAL 132 CA
VAL 132 0.232 CA LEU 133 CA LEU 133 0.103 CA GLU 134 CA GLU 134
0.175 CA GLU 135 CA GLU 135 0.190 CA MET 136 CA MET 136 0.220 CA
SER 137 CA SER 137 0.739 CA LEU 138 CA LEU 138 0.277 CA PRO 139 CA
PRO 139 0.325 CA GLY 140 CA GLY 140 0.390 CA ARG 141 CA ARG 141
0.174 CA TRP 142 CA TRP 142 0.168 CA LYS 143 CA LYS 143 0.304 CA
PRO 144 CA PRO 144 0.194 CA LYS 145 CA LYS 145 0.456 CA MET 146 CA
MET 146 0.362 CA ILE 147 CA ILE 147 0.178 CA GLY 148 CA GLY 148
0.390 CA GLY 149 CA GLY 149 0.434 CA ILE 150 CA ILE 150 0.050 CA
GLY 151 CA GLY 151 0.199 CA GLY 152 CA GLY 152 0.152 CA PHE 153 CA
PHE 153 0.455 CA ILE 154 CA ILE 154 0.198 CA LYS 155 CA LYS 155
0.470 CA VAL 156 CA VAL 156 0.590 CA ARG 157 CA ARG 157 0.607 CA
GLN 158 CA GLN 158 0.465 CA TYR 159 CA TYR 159 0.301 CA ASP 160 CA
ASP 160 0.294 CA GLN 161 CA GLN 161 0.308 CA ILE 162 CA ILE 162
0.274 CA LEU 163 CA LEU 163 0.235 CA ILE 164 CA ILE 164 0.367 CA
GLU 165 CA GLU 165 0.410 CA ILE 166 CA ILE 166 0.201 CA CYS 167 CA
CYS 167 0.409 CA GLY 168 CA GLY 168 0.406 CA HIS 169 CA HIS 169
0.410 CA LYS 170 CA LYS 170 0.282 CA ALA 171 CA ALA 171 0.273 CA
ILE 172 CA ILE 172 0.317 CA GLY 173 CA GLY 173 0.563 CA THR 174 CA
THR 174 0.129 CA VAL 175 CA VAL 175 0.237 CA LEU 176 CA LEU 176
0.155 CA VAL 177 CA VAL 177 0.240 CA GLY 178 CA GLY 178 0.386 CA
PRO 179 CA PRO 179 0.340 CA THR 180 CA THR 180 0.335 CA PRO 181 CA
PRO 181 0.446 CA VAL 182 CA PHE 182 0.343 CA ASN 183 CA ASN 183
0.205 CA ILE 184 CA VAL 184 0.262 CA ILE 185 CA ILE 185 0.096 CA
GLY 186 CA GLY 186 0.118 CA ARG 187 CA ARG 187 0.202 CA ASN 188 CA
ASN 188 0.073 CA LEU 189 CA LEU 189 0.108 CA LEU 190 CA LEU 190
0.127 CA THR 191 CA THR 191 0.177 CA GLN 192 CA GLN 192 0.175 CA
ILE 193 CA ILE 193 0.241 CA GLY 194 CA GLY 194 0.118 CA CYS 195 CA
CYS 195 0.375 CA THR 196 CA THR 196 0.437 CA LEU 197 CA LEU 197
0.167 CA ASN 198 CA ASN 198 0.178
[0118] Table 4 shows that the 184V, V82F mutations induce
structural changes relative to the wild type structure in some
parts of the enzyme, but that other regions are less affected. The
regions of the protein structure which are not significantly
affected by the amino acid mutations are defined as structurally
conserved regions. In the present example, the mutations result in
localized structural changes in the backbone of HIV protease over a
wide range, from 0.038-0.774 .ANG..
[0119] The distances between the strongly interacting atoms of the
inhibitor to atoms of the wild type and mutant protein, that is
hydrogen-bond donors and acceptors, were computed and they are
displayed in Table 5.
2TABLE 5 Distances between atoms of the inhibitor and atoms of the
protein HIV PR wt: 1 V82F/I84V: 1 O2-Wat301 2.92 2.89 N1-027 3.36
3.46 O6-N30 3.30 3.61 06-N29 3.19 3.55 O7-N29 2.84 2.87 O7-OD1 29
3.42 3.54 O7-O1 3.31 3.19 O3-OD 25 (out) 2.50 2.94 O3-OD 25 (in)
2.65 2.67 O3-OD125 (out) 3.27 3.21 O3-OD125 (in) 2.80 2.67
O5-Wat301 2.70 2.79 O8-N130 3.16 2.96
[0120] Table 5 shows that the atoms of the inhibitor interact with
the same atoms of the two different proteins, in this case the wild
type and V82F/184V mutant HIV proteases. From Table 5, it can be
seen that the atoms of the enzymes with which the inhibitor
interacts belong to the structurally conserved regions. The effects
of mutations on the protein-inhibitor interactions can be
quantified in terms of the distances between interacting pairs of
atoms from the inhibitor and from atoms of the three-dimensionally
conserved substructure of the protein. These distances are similar
in the wild type and in the mutant complexes; the average of their
differences is only 0.07 .ANG.. The range of the differences is
0.02-0.36 .ANG..
EXAMPLE 2
[0121] This example illustrates the method by which
experimentally-determined crystal structures of two different
inhibitors in complexes with wild type HIV protease can be compared
and analyzed for the existence of a three-dimensionally conserved
substructure. The structures of wild type HIV-1 protease in
complexes with inhibitor 1 and with Amprenavir (inhibitor 2) were
analyzed by means of (a) an overall superposition of the protein
structures; and (b) a study of the distances from atoms of the
inhibitors to atoms of the protein.
[0122] The superposition of the protein structures is performed in
a two step process: 1) the distance between all pairs of
corresponding C atoms (C atom of residue number 1 in one protein to
C atom of residue number 1 in the second protein; C atom of residue
number 2 in one protein to C atom of residue number 2 in the second
protein; and so on) of the polypeptide chains is minimized by means
of a least-square algorithm; 2) the superposition is refined by
minimizing, in an iterative process, the distances between
corresponding C atoms that are closer than a given distance (0.25
.ANG. in this example), thus eliminating regions of the structures
having large conformational differences to compute the
superposition parameters. The distances between equivalenced C
atoms after the minimization procedure are shown in Table 6.
3TABLE 6 Distances between equivalent C atoms Molecule 1: HIV-1 PR
wt: 1 Molecule 2: HIV-1 PR wt: 2 Molecule 1 Molecule 2 distance
[.ANG.] CA PRO 1 CA PRO 1 0.200 CA GLN 2 CA GLN 2 0.320 CA ILE 3 CA
ILE 3 0.147 CA THR 4 CA THR 4 0.405 CA LEU 5 CA LEU 5 0.225 CA TRP
6 CA TRP 6 0.296 CA GLN 7 CA GLN 7 0.317 CA ARG 8 CA ARG 8 0.154 CA
PRO 9 CA PRO 9 0.143 CA LEU 10 CA LEU 10 0.259 CA VAL 11 CA VAL 11
0.275 CA THR 12 CA THR 12 0.307 CA ILE 13 CA ILE 13 0.207 CA LYS 14
CA LYS 14 0.273 CA ILE 15 CA ILE 15 0.434 CA GLY 16 CA GLY 16 0.469
CA GLY 17 CA GLY 17 0.414 CA GLN 18 CA GLN 18 0.319 CA LEU 19 CA
LEU 19 0.161 CA LYS 20 CA LYS 20 0.155 CA GLU 21 CA GLU 21 0.196 CA
ALA 22 CA ALA 22 0.338 CA LEU 23 CA LEU 23 0.246 CA LEU 24 CA LEU
24 0.292 CA ASP 25 CA ASP 25 0.142 CA THR 26 CA THR 26 0.109 CA GLY
27 CA GLY 27 0.176 CA ALA 28 CA ALA 28 0.193 CA ASP 29 CA ASP 29
0.087 CA ASP 30 CA ASP 30 0.118 CA THR 31 CA THR 31 0.111 CA VAL 32
CA VAL 32 0.087 CA LEU 33 CA LEU 33 0.306 CA GLU 34 CA GLU 34 0.333
CA GLU 35 CA GLU 35 0.399 CA MET 36 CA MET 36 0.296 CA SER 37 CA
SER 37 0.454 CA LEU 38 CA LEU 38 0.451 CA PRO 39 CA PRO 39 0.397 CA
GLY 40 CA GLY 40 0.444 CA ARG 41 CA ARG 41 0.535 CA TRP 42 CA TRP
42 0.346 CA LYS 43 CA LYS 43 0.442 CA PRO 44 CA PRO 44 0.548 CA LYS
45 CA LYS 45 0.307 CA MET 46 CA MET 46 0.320 CA ILE 47 CA ILE 47
0.403 CA GLY 48 CA GLY 48 0.237 CA GLY 49 CA GLY 49 0.280 CA ILE 50
CA ILE 50 0.206 CA GLY 51 CA GLY 51 0.368 CA GLY 52 CA GLY 52 0.315
CA PHE 53 CA PHE 53 0.378 CA ILE 54 CA ILE 54 0.180 CA LYS 55 CA
LYS 55 0.149 CA VAL 56 CA VAL 56 0.302 CA ARG 57 CA ARG 57 0.098 CA
GLN 58 CA GLN 58 0.219 CA TYR 59 CA TYR 59 0.279 CA ASP 60 CA ASP
60 0.385 CA GLN 61 CA GLN 61 0.431 CA ILE 62 CA ILE 62 0.343 CA LEU
63 CA LEU 63 0.473 CA ILE 64 CA ILE 64 0.344 CA GLU 65 CA GLU 65
0.456 CA ILE 66 CA ILE 66 0.481 CA CYS 67 CA CYS 67 0.920 CA GLY 68
CA CLY 68 0.999 CA HIS 69 CA HIS 69 0.295 CA LYS 70 CA LYS 70 0.406
CA ALA 71 CA ALA 71 0.446 CA ILE 72 CA ILE 72 0.374 CA GLY 73 CA
GLY 73 0.259 CA THR 74 CA THR 74 0.276 CA VAL 75 CA VAL 75 0.165 CA
LEU 76 CA LEU 76 0.220 CA VAL 77 CA VAL 77 0.202 CA GLY 78 CA GLY
78 0.231 CA PRO 79 CA PRO 79 0.131 CA THR 80 CA THR 80 0.374 CA PRO
81 CA PRO 81 0.472 CA VAL 82 CA VAL 82 0.554 CA ASN 83 CA ASN 83
0.149 CA ILE 84 CA ILE 84 0.261 CA ILE 85 CA ILE 85 0.223 CA GLY 86
CA GLY 86 0.130 CA ARG 87 CA ARG 87 0.165 CA ASN 88 CA ASN 88 0.103
CA LEU 89 CA LEU 89 0.072 CA LEU 90 CA LEU 90 0.076 CA THR 91 CA
THR 91 0.114 CA GLN 92 CA GLN 92 0.115 CA ILE 93 CA ILE 93 0.204 CA
GLY 94 CA GLY 94 0.220 CA CYS 95 CA CYS 95 0.068 CA THR 96 CA THR
96 0.185 CA LEU 97 CA LEU 97 0.095 CA ASN 98 CA ASN 98 0.311 CA PHE
99 CA PHE 99 0.216 CA PRO 101 CA PRO 101 0.455 CA GLN 102 CA GLN
102 0.121 CA ILE 103 CA ILE 103 0.120 CA THR 104 CA THR 104 0.109
CA LEU 105 CA LEU 105 0.128 CA TRP 106 CA TRP 106 0.205 CA GLN 107
CA GLN 107 0.229 CA ARG 108 CA ARG 108 0.211 CA PRO 109 CA PRO 109
0.195 CA LEU 110 CA LEU 110 0.135 CA VAL 111 CA VAL 111 0.086 CA
THR 112 CA THR 112 0.166 CA ILE 113 CA ILE 113 0.199 CA LYS 114 CA
LYS 114 0.333 CA ILE 115 CA ILE 115 0.356 CA GLY 116 CA GLY 116
0.671 CA GLY 117 CA GLY 117 0.709 CA GLN 118 CA GLN 118 0.370 CA
LEU 119 CA LEU 119 0.258 CA LYS 120 CA LYS 120 0.156 CA GLU 121 CA
GLU 121 0.250 CA ALA 122 CA ALA 122 0.276 CA LEU 123 CA LEU 123
0.103 CA LEU 124 CA LEU 124 0.112 CA ASP 125 CA ASP 125 0.078 CA
THR 126 CA THR 126 0.057 CA GLY 127 CA GLY 127 0.121 CA ALA 128 CA
ALA 128 0.098 CA ASP 129 CA ASP 129 0.190 CA ASP 130 CA ASP 130
0.302 CA THR 131 CA THR 131 0.073 CA VAL 132 CA VAL 132 0.178 CA
LEU 133 CA LEU 133 0.147 CA GLU 134 CA GLU 134 0.239 CA GLU 135 CA
GLU 135 0.101 CA MET 136 CA MET 136 0.235 CA SER 137 CA SER 137
0.391 CA LEU 138 CA LEU 138 0.364 CA PRO 139 CA PRO 139 0.532 CA
GLY 140 CA GLY 140 0.213 CA ARG 141 CA ARG 141 0.448 CA TRP 142 CA
TRP 142 0.133 CA LYS 143 CA LYS 143 0.195 CA PRO 144 CA PRO 144
0.082 CA LYS 145 CA LYS 145 0.359 CA MET 146 CA MET 146 0.306 CA
ILE 147 CA ILE 147 0.076 CA GLY 148 CA GLY 148 0.214 CA GLY 149 CA
GLY 149 0.205 CA ILE 150 CA ILE 150 0.163 CA GLY 151 CA GLY 151
0.287 CA GLY 152 CA GLY 152 0.318 CA PHE 153 CA PHE 153 0.125 CA
ILE 154 CA ILE 154 0.189 CA LYS 155 CA LYS 155 0.384 CA VAL 156 CA
VAL 156 0.510 CA ARG 157 CA ARG 157 0.405 CA GLN 158 CA GLN 158
0.139 CA TYR 159 CA TYR 159 0.361 CA ASP 160 CA ASP 160 0.252 CA
GLN 161 CA GLN 161 0.414 CA ILE 162 CA ILE 162 0.337 CA LEU 163 CA
LEU 163 0.202 CA ILE 164 CA ILE 164 0.359 CA GLU 165 CA GLU 165
0.463 CA ILE 166 CA ILE 166 0.347 CA CYS 167 CA CYS 167 0.256 CA
GLY 168 CA GLY 168 0.471 CA HIS 169 CA HIS 169 0.658 CA LYS 170 CA
LYS 170 0.489 CA ALA 171 CA ALA 171 0.445 CA ILE 172 CA ILE 172
0.396 CA GLY 173 CA GLY 173 0.523 CA THR 174 CA THR 174 0.130 CA
VAL 175 CA VAL 175 0.156 CA LEU 176 CA LEU 176 0.077 CA VAL 177 CA
VAL 177 0.129 CA GLY 178 CA GLY 178 0.276 CA PRO 179 CA PRO 179
0.272 CA THR 180 CA THR 180 0.580 CA PRO 181 CA PRO 181 0.436 CA
VAL 182 CA VAL 182 0.328 CA ASN 183 CA ASN 183 0.180 CA ILE 184 CA
ILE 184 0.151 CA ILE 185 CA ILE 185 0.104 CA GLY 186 CA GLY 186
0.059 CA ARG 187 CA ARG 187 0.058 CA ASN 188 CA ASN 188 0.183 CA
LEU 189 CA LEU 189 0.164 CA LEU 190 CA LEU 190 0.051 CA THR 191 CA
THR 191 0.216 CA GLW 192 CA GLN 192 0.162 CA ILE 193 CA ILE 193
0.158 CA GLY 194 CA GLY 194 0.047 CA CYS 195 CA CYS 195 0.050 CA
THR 196 CA THR 196 0.200 CA LEU 197 CA LEU 197 0.165 CA ASN 198 CA
ASN 198 0.074
[0123] The distances between the atoms of the inhibitors 1 and 2 to
atoms of the protein, that is, hydrogen-bond donors and acceptors,
were computed and are shown in Table 7.
4TABLE 7 Distances between atoms of inhibitors and atoms of the
proteins Wt: 1 complex Wt: 2 complex O2-Wat301 2.92 3.02 N1-027
3.36 3.58 O6-N30 3.30 3.50 06-N29 3.19 3.51 O7-N29 2.84 -- O7-OD1
29 3.42 -- O7-O1 3.31 -- O3-OD 25 (out) A 2.50 2.80 O3-OD 25 (in) A
2.65 2.66 O3-OD 25 (out) B 3.27 3.07 O3-OD 25 (in) B 2.80 2.68
O5-Wat301 2.70 2.77 O8-N 30 3.16 -- N3-N 30 3.17 N3-OD2 30 3.15
[0124] Inhibitors 1 (FIG. 1) and 2 (Amprenavir) have similar
structural elements, in particular their core, i.e. groups at the
P1-P1'60 positions. However, 2 has a THF group while 1 has a
bis-THF group at the P2' position. The P2 groups are identical
except for the substitution of an ether oxygen atom in 1 as
compared to an amine nitrogen atom at the same position in 2. Table
7 shows that 1 forms more interactions with the atoms of the
protein that were previously identified as belonging to the
structurally conserved substructure than does compound 2. For
example, the O7 oxygen atom in compound 1, that forms an
interaction with N29 nitrogen of the protease, has no counterpart
in compound 2. Instead, the O6 oxygen atom of 2 forms longer (and
presumably weaker) hydrogen bonds with both N30 (3.50 .ANG.) and
N29 (3.51 .ANG.). In contrast, the O6 oxygen of compound 1 forms a
shorter (and presumably stronger) hydrogen bond with N29 (3.19
.ANG.). Additionally, as can be seen in Table 7, where both
compounds 1 and 2 form interactions with atoms in the structurally
conserved substructure of HIV protease, the distances between
interacting atoms are consistently shorter for compound 1,
indicative of presumably stronger binding interactions.
[0125] Examples 1 and 2 were used to identify a three
dimensionally-conserved substructure of HIV protease that is
involved in the binding of HIV protease inhibitors and, in
particular, to identify atoms of these substructural elements that
are involved in forming interactions with atoms of HIV protease
inhibitors. This substructure is defined by the set of atomic
coordinates (in orthogonal coordinates) provided in Table 8 and any
equivalent set derived by applying arbitrary rotations and
translations to the set of atomic coordinates in Table 8. The
values of the coordinates (X,Y,Z) of the atoms defining the
substructure are affected by a standard error .sigma.. Therefore
(X,Y,Z) values for each atom are those defined in the intervals
(X-.sigma., X+.sigma.) for coordinate X, (Y-.sigma., Y+.sigma.) for
coordinate Y, and (Z-.sigma., Z+.sigma.) for coordinate Z.
5TABLE 8 Three dimensionally-conserved substructure of HIV protease
Atom X [.ANG.] Y [.ANG.] Z [.ANG.] .sigma. [.ANG.] Description
Substructure of the protein atoms Oxygen -7.9 13.6 27.4 0.5 Oxygen
atom of water molecule coordinated to main chain amide nitrogen
atoms of amino acid Gly 49 and Gly 149 O27 -13.8 17.7 30.4 0.5 Main
Chain carbonyl oxygen atom of amino acid Gly 27 N29 -13.4 18.2 34.5
0.5 Main chain amide nitrogen atom of amino acid Asp 29 N30 -11.9
18.6 36.7 0.5 Main chain amide nitrogen atom of amino acid Asp 30
OD1 25 -11.3 21.2 28.7 0.5 Carboxylate oxygen atom of aminoacid Asp
25 OD2 25 -9.4 20.4 29.3 0.5 Carboxylate oxygen atom of aminoacid
Asp 25 OD1 125 -12.7 20.3 26.4 0.5 Carboxylate oxygen atom of
aminoacid Asp 125 OD2 125 -12.7 20.3 26.4 0.5 Carboxylate oxygen
atom of aminoacid Asp 125 N129 -8.9 20.5 20.7 0.5 Main chain amide
nitrogen atom of amino acid Asp 129 N130 -10.1 19.5 18.6 0.5 Main
chain amide nitrogen atom of amino acid Asp 130 Substructure of the
inhibitor atoms Hydrogen -8.8 17.5 25.7 0.5 Interacting with main
chain Bond carbonyl oxygen atom of amino donor acid Gly 27 Atom
Hydrogen -8.5 15.3 25.1 0.5 Interacting with Oxygen atom of Bond
water molecule coordinated to acceptor main chain amide nitrogen
Atom atoms of amino acid Gly 49 and Gly 149 Hydrogen -10.4 19.1
27.4 0.5 Interacting with carboxylate Bond oxygen atoms of
aminoacids donor- Asp 25 and Asp 125 acceptor Atom Hydrogen -8.9
14.0 29.8 0.5 Interacting with Oxygen atom of Bond water molecule
coordinated to acceptor main chain amide nitrogen Atom atoms of
amino acid Gly 49 and Gly 149 Hydrogen -8.6 17.3 20.7 0.5 Main
chain amide nitrogen atom Bond of amino acid Asp 30 acceptor Atom
Hydrogen -6.9 18.7 21.4 0.5 Interacting with main chain Bond amide
nitrogen atom of amino acceptor acid Asp 29 Atom O8 -10.7 15.8 35.8
0.5 Interacting with main chain amide nitrogen atom of amino acid
Asp 130
EXAMPLE 3
[0126] The following example demonstrates that a protease inhibitor
that contains atoms that can make favorable interactions with the
atoms of the substructure may exhibit broad spectrum activity.
[0127] Compounds 1 and 3 contain a Bis-THF group at the P2 position
that contains two atoms, in particular, hydrogen bond acceptor
oxygen atoms, that can form hydrogen bonds with the two hydrogen
atoms attached to the backbone amide nitrogen atoms on the protein
at residues 29 and 30. Compound 2 is similar to 1 except that 2
contains a THF group at P2 with only a single hydrogen bond
acceptor oxygen atom. All three compounds differ in the P2'
substituent. Compounds 1 and 3 both are unaffected by the two
active site mutations, V82F and 184V, and Ki values for wild type
and mutant enzymes are similar for both compounds. In contrast,
compound 2, which contains only a single hydrogen bond acceptor
atom in the P2 substitutent, is dramatically affected by the active
site mutations, which demonstrate high level resistance to 2.
[0128] The antiviral activity of compounds 1 and 3 against HIV
derived from patient isolates that contain multiple mutations are
equivalent to their activity against wild type HIV strains. In
contrast, compound 2 is much less effective against the same mutant
viruses. None of the patients from whom virus was isolated had ever
been exposed to any of the compounds tested herein. Nonetheless,
compound 2 exhibited cross resistance to these virus strains that
is typically seen with all clinically useful HIV protease
inhibitors -4 (Saquinavir), 5 (Ritonavir), 6 (Indinavir) and 7
(Nelfinavir). Compounds 2, 4, 5, 6, and 7 have very different
chemical structures, but nonetheless behave as a single class with
respect to their antiviral behavior against wild type and multidrug
resistant HIV strains. All compounds are dramatically less potent
against the multidrug resistant strains of HIV.
[0129] In sharp contrast, compounds 1 and 3, which closely resemble
each other as well as compound 2, exhibit broad spectrum activity
in that they are equally effective against wild type and mutant HIV
strains that exhibit high level multidrug resistance towards
compounds 2, 4, 5, 6, and 7. The broad spectrum activity of
compound 1 was completely unexpected and contrasts with the common
and typical loss of antiviral potency experienced with compounds
like 2, 4, 5, 6, 7, and indeed most other HIV protease inhibitors
represented as similar or different structures that have been
reported.
[0130] The development and application of the 3D motif method
described above successfully revealed the presence of a unique,
three dimensionally-conserved substructure of HIV protease that is
useful in the design of broad spectrum inhibitors. Based on this
method, compound 3 was predicted, on the basis of comparative
molecular modeling using the coordinates of the complexes of
compound 1 with wild type and V82F/184V mutant HIV proteases, to be
able to make the same key interaction as compound 1 and thereby to
exhibit broad spectrum activity. Based on these data, it is
feasible to design protease inhibitors that are predicted to have
broad spectrum activity, and are predicted to be useful for the
treatment of both wild type (first line therapy) and drug resistant
(salvage therapy) HIV infections.
EXAMPLE 4
[0131] This example illustrates the method by which
experimentally-determined crystal structures of two different
target proteins, DHQases, from two different bacterial species can
be compared and analyzed for the existence of a three-dimensionally
conserved substructure even in the absence of readily discernible
or statistically significant sequence similarity. DHQases from
different bacterial species typically exhibit less than 30%
sequence identity (FIG. 2). A schematic map showing the key
interactions of the substrate-based inhibitor, DHQO, with the
active site residues for the Type II DHQase from M. tuberculosis is
provided in FIG. 3.
[0132] The structures of wild type DHQase from M. tuberculosis and
a homologous DHQase from Pseudomonas putidas were determined using
conventional x-ray crystallography techniques. The structures were
analyzed by means of (a) an overall superposition of the atoms of
the protein structures. This analysis requires three dimensional
atomic coordinates of the protein structures.
[0133] The superposition of the protein structures was performed in
a two step process: 1) the distance between all pairs of
corresponding C atoms (C atom of residue number 1 in one protein to
C atom of residue number 1 in the second protein; C atom of residue
number 2 in one protein to C atom of residue number 2 in the second
protein; and so on) of the polypeptide chains is minimized by means
of a least-square algorithm; 2) the superposition is refined by
minimizing, in an iterative process, the distances between
corresponding C atoms that are closer than a given distance (0.4
.ANG. in this example), thus eliminating regions of the structures
having large conformational differences to compute the
superposition parameters. The distances between equivalenced C
atoms after the minimization procedure are shown in Table 9.
6TABLE 9 Distances between equivalent C atoms Molecule 1: DHQase P.
putida wt: qxa Molecule 2: DHQase M. tuberculosis wt: gt33 Molecule
1 Molecule 2 distance [.ANG.] CA MET 2 CA GLU 2 1.078 CA ALA 3 CA
LEU 3 1.504 CA THR 4 CA ILE 4 1.800 CA LEU 5 CA VAL 5 1.283 CA LEU
6 CA ASN 6 0.911 CA VAL 7 CA VAL 7 0.715 CA LEU 8 CA ILE 8 0.298 CA
HIS 9 CA ASN 9 0.211 CA GLY 10 CA GLY 10 0.591 CA PRO 11 CA PRO 11
0.599 CA ASN 12 CA ASN 12 0.487 CA LEU 13 CA LEU 13 0.428 CA ASN 14
CA GLY 14 0.229 CA LEU 15 CA ARG 15 0.685 CA LEU 16 CA LEU 16 0.541
CA GLY 17 CA GLY 17 1.693 CA THR 18 CA ARG 18 2.287 CA ARG 19 CA
ARG 19 2.956 CA GLN 20 CA GLN 20 3.475 CA PRO 21 CA PRO 21 3.390 CA
GLY 22 CA ALA 22 4.037 CA THR 23 CA VAL 23 3.770 CA TYR 24 CA TYR
24 2.521 CA GLY 25 CA GLY 25 1.170 CA SER 26 CA GLY 26 1.642 CA THR
27 CA THR 27 1.454 CA THR 28 CA THR 28 1.532 CA LEU 29 CA HIS 29
1.471 CA GLY 30 CA ASP 30 1.632 CA GLN 31 CA GLU 31 1.966 CA ILE 32
CA LEU 32 1.586 CA ASN 33 CA VAL 33 1.875 CA GLN 34 CA ALA 34 2.230
CA ASP 35 CA LEU 35 2.343 CA LEU 36 CA ILE 36 1.927 CA GLU 37 CA
GLU 37 2.284 CA ARG 38 CA ARG 38 2.980 CA ARG 39 CA GLU 39 2.917 CA
ALA 40 CA ALA 40 2.719 CA ARG 41 CA ALA 41 3.367 CA GLU 42 CA GLU
42 3.534 CA ALA 43 CA LEU 43 3.281 CA GLY 44 CA GLY 44 3.161 CA HIS
45 CA LEU 45 2.899 CA HIS 46 CA LYS 46 1.844 CA LEU 47 CA ALA 47
1.599 CA LEU 48 CA VAL 48 1.201 CA HIS 49 CA VAL 49 2.053 CA LEU 50
CA ARG 50 1.045 CA GLN 51 CA GLN 51 0.266 CA SER 52 CA SER 52 0.300
CA ASN 53 CA ASP 53 0.282 CA ALA 54 CA SER 54 0.348 CA GLU 55 CA
GLU 55 0.326 CA TYR 56 CA ALA 56 0.238 CA GLU 57 CA GLN 57 0.380 CA
LEU 58 CA LEU 58 0.455 CA ILE 59 CA LEU 59 0.413 CA ASP 60 CA ASP
60 0.984 CA ARG 61 CA TRP 61 1.452 CA ILE 62 CA ILE 62 1.338 CA HIS
63 CA HIS 63 1.310 CA ALA 64 CA GLN 64 2.327 CA ALA 65 CA ALA 65
2.526 CA ARG 66 CA ALA 66 3.063 CA ASP 67 CA ASP 67 3.449 CA GLU 68
CA CA GLY 69 CA ALA 68 2.318 CA VAL 70 CA ALA 69 1.691 CA ASP 71 CA
GLU 70 0.812 CA PHE 72 CA PRO 71 0.515 CA ILE 73 CA VAL 72 0.561 CA
ILE 74 CA ILE 73 0.547 CA LEU 75 CA LEU 74 0.380 CA ASN 76 CA ASN
75 0.277 CA PRO 77 CA ALA 76 0.369 CA ALA 78 CA GLY 77 0.952 CA ALA
79 CA GLY 78 0.421 CA PHE 80 CA LEU 79 0.714 CA THR 81 CA THR 80
0.575 CA HIS 82 CA HIS 81 0.142 CA THR 83 CA THR 82 0.222 CA SER 84
CA SER 83 0.741 CA VAL 85 CA VAL 84 0.719 CA ALA 86 CA ALA 85 0.415
CA LEU 87 CA LEU 86 0.667 CA ARG 88 CA ARG 87 0.660 CA ASP 89 CA
ASP 88 0.426 CA ALA 90 CA ALA 89 0.697 CA LEU 91 CA CYS 90 1.233 CA
LEU 92 CA ALA 91 1.319 CA ALA 93 CA GLU 92 2.852 CA VAL 94 CA LEU
93 4.165 CA SER 95 CA SER 94 3.605 CA ILE 96 CA ALA 95 3.840 CA PRO
97 CA PRO 96 2.414 CA PHE 98 CA LEU 97 0.314 CA ILE 99 CA ILE 98
0.251 CA GLU 100 CA GLU 99 0.095 CA VAL 101 CA VAL 100 0.131 CA HIS
102 CA HIS 101 0.318 CA ILE 103 CA ILE 102 0.117 CA SER 104 CA SER
103 0.229 CA ASN 105 CA ASN 104 0.203 CA VAL 106 CA VAL 105 0.193
CA HIS 107 CA HIS 106 0.499 CA LYS 108 CA ALA 107 0.498 CA ARG 109
CA ARG 108 0.292 CA GLU 110 CA GLU 109 0.333 CA PRO 111 CA GLU 110
0.377 CA PHE 112 CA PHE 111 0.651 CA ARG 113 CA ARG 112 0.611 CA
ARG 114 CA ARG 113 0.469 CA HIS 115 CA HIS 114 0.467 CA SER 116 CA
SER 115 0.293 CA TYR 117 CA TYR 116 0.483 CA PHE 118 CA LEU 117
0.468 CA SER 119 CA SER 118 0.367 CA ASP 120 CA PRO 119 0.676 CA
VAL 121 CA ILE 120 0.445 CA ALA 122 CA ALA 121 0.334 CA VAL 123 CA
THR 122 0.405 CA GLY 124 CA GLY 123 0.372 CA VAL 125 CA VAL 124
0.375 CA ILE 126 CA ILE 125 0.250 CA CYS 127 CA VAL 126 0.328 CA
GLY 128 CA GLY 127 0.332 CA LEU 129 CA LEU 128 0.473 CA GLY 130 CA
GLY 129 0.272 CA ALA 131 CA ILE 130 0.551 CA THR 132 CA GLN 131
0.564 CA GLY 133 CA GLY 132 0.289 CA TYR 134 CA TYR 133 0.276 CA
ARG 135 CA LEU 134 0.476 CA LEU 136 CA LEU 135 0.556 CA ALA 137 CA
ALA 136 0.677 CA LEU 138 CA LEU 137 0.703 CA GLU 139 CA ARG 138
0.861 CA SER 140 CA TYR 139 0.876 CA ALA 141 CA LEU 140 1.330 CA
LEU 142 CA ALA 141 1.529 CA GLU 143 CA GLU 142 1.492 CA GLN 144 CA
HIS 143 1.738 CA LEU 145 CA VAL 144 3.487
[0134] Table 9 shows that the two structures are remarkably similar
overall despite their low level sequence identity. However, the
structures exhibit very large deviations in some regions, and are
highly conserved in others. In particular, this analysis reveals
that regions of the enzyme are minimally affected by the large
number of amino acid sequence substitutions. The regions of the
protein structure which are not significantly affected by the amino
acid substitutions are defined as structurally conserved regions.
In the present example, the substitutions result in localized
structural changes in the backbone of DHQase over a wide range,
from 0.095-4.165 .ANG..
[0135] The distances between the strongly interacting atoms of the
inhibitor to atoms of the homologous DHQase proteins, that is P.
putida wt: qxa and M. tuberculosis wt: gt33 complexes, were
computed and they are displayed in Tables 10 and 11,
respectively.
7TABLE 10 Distances between atoms of the inhibitor and atoms of the
protein DHQase P. putida wt: gxa distance [.ANG.] C6-PRO11 (O) 3.31
C6-ASN12 (CB) 3.10 N7-TYR24 (OH) 2.45 O12-ASN76 (HD2) 2.96
O13-HIS102 (CB) 3.38 O12-SER104 (N) 3.35 C6-ASN12 (CB) 3.10
[0136]
8TABLE 11 Distances between atoms of the inhibitor and atoms of the
protein DHQase M. tuberculosis wt: gt33 distance [.ANG.] N14-PRO11
(O) 3.35 O15-PRO11 (O) 3.01 O15-LEU 13 (CG) 3.36 O15-ARG 19 (NH1)
3.26 O15-ARG 19 (NH2) 3.32 O7-ASN 75 (OD1) 2.50 O13-ASN 75 (ND2)
2.98 N14-GLY 77 (CA) 3.01 O9-HIS 81 (NE2) 2.82 O7-HIS 101 (ND1)
3.25 O11-ILE 102 (N) 3.33 O13-ILE 102 (N) 2.77 O11-SER 103 (N) 2.96
O11-SER 103 (OG) 2.68 O9-ARG 112 (NH2) 3.09 O10-ARG 112 (NH2)
3.02
[0137] The methods of Examples 1-3 were applied to the DHQase data
to identify a three dimensionally-conserved substructure of DHQase
that is involved in the binding of DHQase inhibitors, in
particular, to identify the relevant target substructure for
developing broad spectrum inhibitors. This substructure is defined
by the set of atomic coordinates (in orthogonal coordinates)
provided in Table 12 and any equivalent set derived by applying
arbitrary rotations and translations to the set of atomic
coordinates in Table 12. The values of the coordinates (X,Y,Z) of
the atoms defining the substructure are affected by a standard
error .sigma.. Therefore (X,Y,Z) values for each atom are those
defined in the intervals (X-.sigma., X+.sigma.) for coordinate X,
(Y-.sigma., Y+.sigma.) for coordinate Y, and (Z-.sigma., Z+.sigma.)
for coordinate Z.
9TABLE 12 Three dimensionally-conserved substructure of DHQase, M.
tuberculosis Atom X [.ANG.] Y [.ANG.] Z [.ANG.] .sigma. [.ANG.]
Description Substructure of the protein atoms OD1 26.265 68.912
21.219 0.5 Side chain carbonyl ASN75 oxygen atom of amino acid ASN
75 ND2 27.336 66.960 20.951 0.5 Side chain nitrogen ASN 75 atom of
amino acid ASN 75 NE2 28.343 76.425 22.111 0.5 Side chain nitrogen
HIS 81 atom of amino acid HIS 81 ND1 28.079 70.604 23.662 0.5 Side
chain nitrogen HIS 101 atom of amino acid HIS 101 N ILE 31.227
67.167 22.168 0.5 Main chain amide 102 nitrogen atom of atom acid
ILE 102 N SER 33.754 68.315 21.558 0.5 Main chain amide 103
nitrogen atom of amino acid Ser 103 OG SER 33.946 71.059 20.735 0.5
Side chain hydroxyl 103 oxygen atom of amino acid SER 103
Substructure of the inhibitor Hydrogen 29.600 68.554 20.298 0.5
Interacting with main bond chain nitrogen atom of acceptor ILE 102
and side chain atom nitrogen atom of ASN 75 Hydrogen 28.031 70.739
20.422 0.5 Interacting with side bond chain oxygen atom of donor-
ASN75 and side chain acceptor nitrogen atom of atom HIS 101
Hydrogen 29.664 74.658 20.493 0.5 Interacting side chain bond
nitrogen atom of HIS donor- 81 acceptor atom Hydrogen 31.451 69.835
20.531 0.5 Interacting with main bond chain nitrogen atom acceptor
and side chain oxygen atom atom of SER 103
Other Embodiments
[0138] All publications and patent applications, and patents
mentioned in this specification are herein incorporated by
reference.
[0139] While the invention has been described in connection with
specific embodiments, it will be understood that it is capable of
further modifications. Therefore, this application is intended to
cover any variations, uses, or adaptations of the invention that
follow, in general, the principles of the invention, including
departures from the present disclosure that come within known or
customary practice within the art.
* * * * *