U.S. patent application number 11/505196 was filed with the patent office on 2007-05-03 for crystal structure of a deacetylase and inhibitors thereof.
Invention is credited to Ronald Breslow, Jill Donigian, Michael Finnin, Paul A. Marks, Nikola Pavletich, Victoria M. Richon, Richard A. Rifkind.
Application Number | 20070100559 11/505196 |
Document ID | / |
Family ID | 22544290 |
Filed Date | 2007-05-03 |
United States Patent
Application |
20070100559 |
Kind Code |
A1 |
Pavletich; Nikola ; et
al. |
May 3, 2007 |
Crystal structure of a deacetylase and inhibitors thereof
Abstract
The present invention provides three-dimensional structural
information from the hyperthermophilic bacterium Aquifex aeolicus
which is a histone deacetylase-like protein (HDLP). HDLP shares
35.2% amino acid sequence identity with human histone deacetylase
(HDAC1). The present invention further provides three-dimensional
structural information of HDLP bound by inhibitor molecules. The
three-dimensional structural information of the present invention
is useful to design, isolate and screen deacetylase inhibitor
compounds capable of inhibiting HDLP, HDAC family members and
HDLP-related molecules. The invention also relates to nucleic acids
encoding a mutant HDLP which facilitates the determination of the
three-dimensional structure of HDLP in the presence of a zinc
atom.
Inventors: |
Pavletich; Nikola; (New
York, NY) ; Finnin; Michael; (Alexandria, VA)
; Donigian; Jill; (North Arlington, NJ) ; Richon;
Victoria M.; (Rye, NY) ; Rifkind; Richard A.;
(New York, NY) ; Marks; Paul A.; (Washington,
CT) ; Breslow; Ronald; (Englewood, NJ) |
Correspondence
Address: |
MINTZ LEVIN, COHEN, FERRIS, et al.
24th Floor
666 Third Avenue
New York
NY
10017
US
|
Family ID: |
22544290 |
Appl. No.: |
11/505196 |
Filed: |
August 16, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10095109 |
Mar 8, 2002 |
7124068 |
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11505196 |
Aug 16, 2006 |
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PCT/US00/24700 |
Sep 8, 2000 |
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10095109 |
Mar 8, 2002 |
|
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60152753 |
Sep 8, 1999 |
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Current U.S.
Class: |
702/19 ;
435/197 |
Current CPC
Class: |
C12N 9/16 20130101 |
Class at
Publication: |
702/019 ;
435/197 |
International
Class: |
G06F 19/00 20060101
G06F019/00; C12N 9/18 20060101 C12N009/18 |
Goverment Interests
GOVERNMENT SUPPORT
[0002] The invention was supported, in whole or in part, by a grant
RO1 CA-65698 from the National Institutes of Health. The Government
has certain rights in the invention.
Claims
1-19. (canceled)
20. A crystal of an enzyme comprising deacetylase activity wherein
said crystal effectively diffracts X-rays for the determination of
the atomic coordinates of said enzyme to a resolution of greater
than 4 .ANG. and wherein the structure of said enzyme comprises a
conserved core .alpha./.beta. structure characteristic fold wherein
said conserved .alpha./.beta. fold comprises an eight-stranded
parallel .beta. sheet and eight .alpha. helices and wherein four of
the helices pack on either face of said parallel .beta. sheet and
wherein said structure of said enzyme comprises an rmsd of less
than or equal to 1.5 .ANG.in the positions of C.alpha. atoms for at
least 2/3 or more of the amino acids of HDLP as defined by the
atomic coordinates of HDLP.
21. The crystal of claim 20, wherein said protein structure further
comprises: (a) eight .alpha. helices positioned near one side of
the .beta. sheet; and (b) at least seven large, well defined loops
originating from the C-terminal ends of the .beta.-strands of said
eight-stranded parallel .beta. sheet wherein the eight extra
helices and the seven large loops are associated with a significant
extension of the structure beyond the core .alpha./.beta. motif and
wherein said extension of the structure gives rise to a deep,
narrow pocket and an internal cavity adjacent to the pocket.
22. The crystal of claim 20, wherein said enzyme comprising
deacetylase activity is selected from the group consisting of HDLP,
HDLP-related proteins, HDAC1, HDAC2, HDAC3, HDAC4, HDAC5, HDAC6,
HDAC-related proteins, APAH, AcuC, and functional derivatives
thereof.
23. The crystal of claim 21 further comprising a specifically bound
zinc atom in the active site of said enzyme.
24. The crystal of claim 21 further comprising a specifically bound
deacetylase inhibitor compound in the active site of said
enzyme.
25. The crystal of claim 21 defined by the atomic coordinates
according to FIG. 16.
26. A method for solving the structure of an HDAC family member
crystal comprising the steps of: (a) collecting X-ray diffraction
data of said crystal wherein said data diffracts to a high
resolution limit of greater than 4 .ANG.; (b) using the atomic
coordinates of HDLP according to FIG. 16 to perform molecular
replacement or refinement and difference fourier with said X-ray
diffraction data of said HDAC family member crystal to determine
the structure of said HDAC family member; and (c) refining said
structure of said HDAC family member.
27. The method of claim 26, wherein said HDAC family member is
HDAC1.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of International
Application No. PCT/US00/24700, which designated the United States
and was filed on Sep. 8, 2000, published in English, which claims
the benefit of U.S. Provisional Application No. 60/152,753, filed
on Sep. 8, 1999. The entire teachings of the above application(s)
are incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0003] The present invention relates to a histone deacetylase
homologue from the hyperthermophilic bacterium Aquifex aeolicus,
HDLP (histone deacetylase-like protein; also known as AcuC1), which
shares 35.2% sequence identity with human histone deacetylase
(HDAC1), that can be co-crystallized with an inhibitory ligand, and
more particularly, to the detailed crystallographic data obtained
from said co-crystallization which is disclosed herein. The
invention also relates to methods of using the crystal structure
and x-ray crystallographic coordinates of the apo-HDLP
inhibitor-bound HDLP to design, isolate and screen compounds which
bind to and inhibit the active site of HDLP and HDLP-related
proteins, such as those proteins belonging to the HDAC family,
including HDAC1.
[0004] The reversible modification of histones by acetylation is
associated with changes in nucleosome conformation and chromatin
structure, and plays an important role in the regulation of gene
expression (reviewed in Davie and Chadee, 1998, J. Cell Biochem.
Suppl. 30-31:203-213). The histone acetylase and deacetylase
enzymes that carry out these modifications are involved in many
cellular processes such as cell cycle progression and
differentiation, and their is deregulation is associated with
several types of human cancer (reviewed in Kouzarides, 1999, Curr.
Opin. Genet. Dev. 9:40-48; Hassig et al., 1997, Chem. Biol.
4:783-789; Fenrick and Heibert, 1998, J. Cell. Biochem. Suppl.
30-31:194-202).
[0005] Recently, several experimental antitumor compounds, such as
trichostatin A (TSA), trapoxin, suberoylanilide hydroxamic acid
(SAHA), and phenylbutyrate have been shown to act, at least in
part, by inhibiting histone deacetylases. Richon et al., 1998,
Proc. Natl. Acad. Sci., USA 95:3003-3007; Yoshida et al., 1990, J.
Biol. Chem. 265:17174-17179; Kijima et al., 1993, J. Biol. Chem.
268:22429-22435. Additionally, diallyl sulfide and related
molecules (Lea et al., 1999, Int. J. Oncol. 2:347-352), oxamflatin
(Kim et al., 1999, Oncogene 15:2461-2470), MS-27-275, a synthetic
benzamide derivative (Saito et al., 1999, Proc. Natl. Acad. Sci.
96:4592-4597), butarate derivatives (Lea and Tulsyan, 1995,
Anticancer Res. 15:879-883), FR901228 (Nokajima et al., 1998, Exp.
Cell Res. 241:126-133), depudecin (Kwon et al., 1998, Proc. Natl.
Acad. Sci. USA 95:3356-3361) and m-carboxysinnamic acid
bishydroxamide (CBHA; Richon et al., Proc. Natl. Acad. Sci. USA
95:3003-3007) have been shown to inhibit histone deacetylases. In
vitro, these compounds can inhibit the growth of fibroblast cells
by causing cell cycle arrest in the G1 and G2 phases (Richon et
al., 1996, Proc. Natl. Acad. Sci. USA 93:5705-5708; Kim et al.,
1999, Oncogene 18:2461-2470; Yoshida et al., 1995, Bioessays
17:423-430; Yoshida & Beppu, 1988, Exp. Cell. Res.
177:122-131), and can lead to the terminal differentiation and loss
of transforming potential of a variety of transformed cell lines.
Richon et al., 1996, Proc. Natl. Acad. Sci. USA 93:5705-5708; Kim
et al., 1999, Oncogene 18:2461-2470; Yoshida et al., 1987, Cancer
Res. 47:3688-3691. In vivo, phenylbutyrate is effective in the
treatment of acute promyelocytic leukemia in conjunction with
retinoic acid. Warrell et al., 1998, J. Natl. Cancer Inst.
90:1621-1625. SAHA is effective in preventing the formation of
mammary tumors in rats, and lung tumors in mice. Desai et al.,
1999, Proc. AACR 40: abstract #2396; Cohen et al., Cancer Res.,
submitted.
[0006] Histone deacetylases catalyze the removal of acetyl groups
from the .epsilon.-amino groups of lysine residues clustered near
the N-terminus of nucleosomal histones, and this process is
associated with transcriptional repression (reviewed in Struhl,
1998, Genes Dev. 12:599-606). Deletion of the yeast histone
deacetylase gene, rpd3, or its pharmacological inactivation with
trichostatin A reduces the transcriptional repression in a subset
of promoters, such as those of Ume6-regulated genes. Kadosh &
Struhl, 1998, Mol. Cell. Biol. 18:5121-5127. This is accompanied by
the increased acetylation of H4 histones in the repressed promoter
and its vicinity but has no effect on histones at promoter distal
regions. Kadosh & Struhl, 1998, Mol. Cell. Biol. 18:5121-5127;
Rundlett et al., 1998, Nature 392:831-835.
[0007] Histone deacetylases are recruited to specific promoters by
associating with DNA-binding transcriptional repressors, either
directly or through co-repressors that bridge the deacetylase to
the transcriptional repressors. For example, the Mad and Ume6
repressors bind to the co-repressor Sin3A (Laherty et al., 1997,
Cell 89:349-356; Hassig et al., 1997, Cell 89:341-347; Kadosh &
Struhl, 1997, Cell 89:365-371), and the nuclear receptors bind
N-CoR and the related SMRT co-repressors. Nagy et al., 1997, Cell
89:373-380; Alland et al, 1997, Nature 387:49-55; Heinzel et al,
1997, Nature 387:43-48.
[0008] The deregulation of histone deacetylase recruitment appears
to be one of the mechanisms through which these enzymes contribute
to tumorigenesis. In acute promyelocytic leukemia (APL),
chromosomal translocations fuse the retinoic acid receptor-.alpha.
(RAR.alpha.) to either PLZF or to PML. These fusion oncoproteins
have aberrant transcriptional repression activity resulting, in
part, through the recruitment of a co-repressor and, in turn,
HDACs. Grignani et al, 1998, Nature 391:815-818; Lin et al., 1998,
Nature 391:811-814. Treatment of PLZF-RAR.alpha. APL cells with TSA
enhances their responsiveness to retinoic acid-induced
differentiation. Grignani et al, 1998, Nature 391:815-818; Lin et
al., 1998, Nature 39-1:811-814.
[0009] The histone deacetylases comprise a large family of
proteins, conserved from yeast to man, and are divided into two
related classes. Class I is characterized by human HDAC1, 2, 3
(Taunton et al., 1996, Science 272:408-411; Yang et al., 1996,
Proc. Natl. Acad. Sci. USA 93:12845-12850; Emiliani et al., 1998,
Proc. Natl. Acad. Sci. USA 95:2795-2800), and yeast RPD3 (Videl
& Gaber, 1991, Mol. Cell. Biol. 11:6317-6327), and class II by
the human HDAC4, 5, 6 (Grozinger et al., 1999, Proc. Natl. Acad.
Sci. USA 96:4868-4873; Fischle, et al., 1999, J. Biol. Chem.
274:11713-11720), and yeast HDA1 (Rundlett et al., 1996, Proc.
Natl. Acad. Sci. USA 93:14503-14508). The two classes share a
.about.390 amino acid region of sequence similarity, comprising the
deacetylase core, but are divergent outside this region. The
histone deacetylase genes belong to an even larger superfamily
(Leipe & Landsman, 1997, Nucleic Acids Res. 25:3693-3697) that
contains the prokaryotic acetoin utilization proteins (AcuC; 28.1%
sequence identity to HDAC1), and the prokaryotic acetylpolyamine
amidohydrolases (APAH; 15.0% sequence identity to HDAC1). The
enzymatic activity of AcuC is not clear, but its disruption reduces
the ability of B. subtilis to breakdown acetoin and utilize it as a
carbon source. Grundy et al., 1993, Mol. Microbiol. 10:259-271.
APAHs catalyze the deacetylation of polyamines by cleaving a
non-peptide amide bond (reviewed in Leipe & Landsman, 1997,
Nucleic Acids Res. 25:3693-3697).
[0010] It is useful to address the questions of how HDACs and
HDAC-related proteins catalyze the deacetylation of histones and
how the above-referenced compounds, particularly those compounds
with antitumor activity, inhibit this activity in order to better
understand the mechanism of inhibition of HDACs and to facilitate
discovery of additional useful compounds which may inhibit this
activity. To this end, the present invention has determined the
three dimensional structure of a HDAC1-like protein from the
thermophilic bacterium Aquifex aeolicus, herein after HDLP. The
determination of the nucleic acid coding sequence of HDLP was
described by Deckert et al., 1998, Nature 392:353-358. The encoded
375 residue protein, whose sequence was determined from the nucleic
acid encoding sequence, shares 35.2% amino acid sequence identity
with HDAC1, deacetylates histones in vitro, and is inhibited by
TSA, SAHA and several other HDAC inhibitors. The determination of
the three-dimensional structure of HDLP is useful in the design,
identification and screening of new HDAC family inhibitory
compounds which are useful for the inhibition of cell growth both
in vivo and in vitro.
SUMMARY OF THE INVENTION
[0011] In general, it is the object of the present invention to
provide detailed three-dimensional structural information for a
family of proteins known as histone deacetylases (HDAC), and
particularly a homologue from the hyperthermophilic bacterium
Aquifex aeolicus HDLP (histone deacetylase-like protein) which
shares 35.2% sequence identity with human histone deacetylase
(HDAC1). It is also an object of the present invention to provide
three-dimensional structural information of an HDLP bound to an
inhibitory compound.
[0012] In one embodiment of the invention, three-dimensional
structure information is obtained from a crystal of wild-type HDLP
(SEQ ID NO:1) (the nucleic acid encoding wild-type HDLP is SEQ ID
NO:2). In a further embodiment of the invention, three-dimensional
information is obtained from a mutant HDLP comprising two mutations
(1) cysteine 75 to a serine and (2) cysteine 77 to a serine
(Cys75Ser/Cys77Ser double mutant; SEQ ID NO:3) (the nucleic acid
encoding HDLP Cys75Ser/Cys77Ser double mutant is SEQ ID NO:4). The
HDLP mutant of the present invention facilitates the determination
of three-dimensional structural information of HDLP bound to a zinc
atom at its zinc atom-binding site.
[0013] In a preferred embodiment of the invention, the
three-dimensional structural information is obtained from a
co-crystal of a protein-inhibitor compound complex that comprises
HDLP or HDLP Cys75Ser/Cys77Ser double mutant and trichostatin A
(TSA). In another preferred embodiment of the invention the
three-dimensional structural information is obtained from a
co-crystal of a protein-inhibitor compound complex that comprises
HDLP or HDLP. Cys7SSer/Cys77Ser double mutant and suberoylanilide
hydroxamic acid (SAHA). Any HDLP or HDLP-related protein (e.g.
HDAC) inhibitor compound that may be co-crystallized with HDLP may
be used to form a co-crystal of the present invention.
[0014] The protein crystals and protein-inhibitory complex
co-crystals of the present invention diffract to a high resolution
limit of at least equal to or greater than 4 angstrom (.ANG.). In a
preferred embodiment, the protein crystals and protein-inhibitory
complex co-crystals of the present invention diffract to a high
resolution limit of greater than 2.5 .ANG..
[0015] A crystal of the present invention may take a variety of
forms, all of which are contemplated by the present invention. In a
preferred embodiment, the crystal has a space group of C2 with one
molecule in the asymmetric unit and with unit dimensions of a =51.4
.ANG., b=93.8 .ANG., c=78.7 .ANG. and .beta.=96.9.degree. (see,
e.g., Example 2, below). In another preferred embodiment, the
crystal has a space group of P2.sub.12.sub.12.sub.1 with two
molecules in the asymmetric unit and with unit dimensions of a=53.4
.ANG., b=94.4 .ANG., c=156.3 .ANG. (see, e.g., Example 2, below).
The HDLP structure comprises a parallel .beta. sheet with .alpha.
helices packing against both faces. At one end of the .beta. sheet,
the HDLP has a narrow, tube-like pocket formed by several
well-ordered loops. The walls of the pocket are lined with
hydrophobic residues and there is a zinc binding site and several
polar side chains at the bottom of the pocket. The inhibitory
compounds of the present invention bind in the pocket.
[0016] The three-dimensional structural information obtained from
crystals of HDLP, HDLP Cys75Ser/Cys77Ser double mutant, HDLP
Cys75Ser/Cys77Ser double mutant comprising a zinc atom, HDLP
comprising an inhibitory compound such as TSA or SAHA, and HDLP
Cys75Ser/Cys77Ser double mutant comprising an inhibitor compound
such as TSA or SAHA may be employed to solve the structure of any
HDLP-related protein (e.g. HDAC) crystal, or any mutant
HDLP-related protein and particularly any wild type or mutant of
HDLP-related protein complexed with a ligand, including a substrate
or inhibitor compound. If the crystals are in a different space
group than the known structure, molecular replacement may be
employed to solve the structure, or if the crystals are in the same
space group, refinement and difference fourier methods may be
employed. The structure of HDLP-related proteins (e.g. HDAC1)
comprise no greater than a 2.0 .ANG. root mean square deviation
(rmsd) in the positions of the C.alpha. atoms for at least 50% or
more of the amino acids of the full-length HDLP structure.
[0017] The present invention also provides a nucleic acid molecule
encoding an HDLP Cys75Ser/Cys77Ser double mutant having the amino
acid sequence of SEQ ID NO:3 and the nucleic acid sequence of SEQ
ID NO:4. It is also contemplated by the invention that mutations be
made in HDLP-related proteins at cysteine residues, as with the
Cys75Ser/Cys77Ser double mutant, in order to facilitate the
determination of the structure of said proteins bound to a zinc
atom. Additionally, the present invention provides expression
vectors which comprise the nucleic acid molecule encoding an HDLP
Cys75Ser/Cys77Ser double mutant encoded by the sequence represented
by SEQ ID NO:4 operatively linked to expression control
sequences.
[0018] It is another object of the present invention to provide
methods for the design, identification and screening of potential
inhibitor compounds of the HDLP/HDAC family. In a preferred
embodiment the method for the rational design, identification and
screening of potential inhibitor compounds for HDLP and
HDLP-related proteins (e.g. HDACs) comprising deacetylase activity
comprises the steps of: (a) using a three-dimensional structure of
an HDLP as defined by the atomic coordinates of the present
invention; (b) employing said three-dimensional structure to design
or select said potential inhibitor compound; (c) synthesizing
and/or selecting said potential inhibitor; (d) contacting said
potential inhibitor compound with said enzyme in the presence of
acetylated substrate; and (e) determining the percent inhibition of
deacetylase activity to determine the inhibitory activity of said
potential inhibitor compound. In a further preferred embodiment,
the binding properties of said rationally designed inhibitory
compound may be determined by a method comprising the steps of: (a)
forming a complex comprising said inhibitory compound and HDLP or a
HDLP-related protein, (b) co-crystallizing said inhibitory
compound-HDLP complex; (c) determining said three-dimensional
structure of said co-crystal through molecular replacement or
refinement and difference fourier with the molecular coordinates of
HDLP as defined by the present invention; and (d) analyzing the
three-dimensional structure to determine the binding
characteristics of said potential inhibitor compound.
[0019] It is a further object of the present invention to identify
a defined class of HDLP/HDAC family inhibitor compounds. The
HDLP/HDAC family inhibitor compounds of the present invention are
represented by formula (I): ##STR1## wherein X comprises a cap
group which binds to at least one amino acid selected from the
group consisting of proline and leucine; Y comprises an aliphatic
chain group which binds to at least one amino acid selected from
the group consisting of leucine, phenylalanine and glycine; and Z
comprises and active site binding group which binds to at least one
amino acid selected from the group consisting of aspartic acid,
tyrosine and histidine and may further bind to a zinc atom.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1 is a table listing the statistics from the X-ray
crystallographic analysis of a HDLP crystal, a HDLP-TSA co-crystal,
and a HDLP-SAHA co-crystal.
[0021] FIG. 2 shows an alignment of various HDAC homologues with
percent sequence identity depicted.
[0022] FIG. 3 shows a graph indicating the histone deacetylase
activity of HDLP and HDAC1 and the inhibition of HDLP and HDAC1 by
the inhibitors TSA and HC-toxin.
[0023] FIG. 4 shows (A & B) a schematic representation of the
HDLP-Zn.sup.2+-TSA complex in two approximately orthogonal views,
(C) a topology diagram of HDLP indicating the regions of homology
with HDAC1, and (D) a close-up schematic representation of the
HDLP-Zn.sup.2+-SAHA complex.
[0024] FIG. 5 shows (A) a schematic representation of a slice
through a surface representation of HDLP with the pocket internal
cavities and position of the .beta. sheet indicated, (B) a
schematic representation of a close-up view of the active site
looking down into the pocket in an orientation similar to FIG.
4B.
[0025] FIG. 6 shows (A) a space-filling representation of TSA in
the active site pocket, (B) a closeup stereo view of the structure
of the HDLP-ZN.sup.2+-TSA complex in a similar orientation to FIG.
4B, and (C) a schematic representation of the HDLP-TSA
interactions.
[0026] FIG. 7 shows (A) a schematic representation of the regions
of homology shared between HDLP and HDAC1 in an orientation similar
to that of FIG. 4A, and (B) a detailed schematic representation of
the homology shared in the pocket and internal cavity between HDLP
and HDAC1 in an orientation similar to that of FIG. 4B.
[0027] FIG. 8 shows a schematic representation of the proposed
catalytic mechanism for the deacetylation of acetylated lysine.
[0028] FIG. 9 shows a schematic representation of a space filling
diagram showing the conserved amino acids in the active site and
nearby grooves.
[0029] FIG. 10 is the nucleic acid sequence of HDLP from Aquifex
aeolicus (SEQ ID NO. 2).
[0030] FIG. 11 is the amino acid sequence of full length HDLP from
Aquifex aeolicus (SEQ ID NO. 1).
[0031] FIG. 12 is the nucleic acid sequence of the HDLP active site
mutant Tyr297Phe (SEQ ID NO. 6).
[0032] FIG. 13 is the amino acid sequence of the HDLP active site
mutant Tyr297Phe (SEQ ID NO. 5).
[0033] FIG. 14 is the nucleic acid sequence of a double mutant of
HDLP from Aquifex aeolicus comprising a Cys75Ser and Cys77Ser
mutation (SEQ ID NO. 4).
[0034] FIG. 15 is the amino acid sequence of a double mutant of
HDLP from Aquifex aeolicus comprising a Cys75Ser and Cys77Ser
mutation (SEQ ID NO. 3).
[0035] FIG. 16-1 to 16-49 lists the atomic structure coordinates
for HDLP as derived by X-ray diffraction from a crystal of
HDLP.
[0036] FIG. 17-1 to 17-49 lists the atomic structure coordinates
for HDLP Cys75Ser/Cys77Ser double mutant comprising a zinc atom in
the active site as derived by X-ray diffraction from a crystal of
the HDLP Cys75Ser/Cys77Ser double mutant.
[0037] FIG. 18-1 to 18-99 lists the atomic structure coordinates
for HDLP Cys75Ser/Cys77Ser double mutant as derived by X-ray
diffraction from a co-crystal of HDLP complexed with TSA.
[0038] FIG. 19-1 to 19-48 lists the atomic structure coordinates
for HDLP Cys75Ser/Cys77Ser double mutant as derived by X-ray
diffraction from a co-crystal of HDLP complexed with SAHA.
[0039] The foregoing and other objects, features and advantages of
the invention will be apparent from the following more particular
description of preferred embodiments of the invention, as
illustrated in the accompanying drawings in which like reference
characters refer to the same parts throughout the different views.
The drawings are not necessarily to scale, emphasis instead being
placed upon illustrating the principles of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0040] The present invention provides crystals of a histone
deacetylase (HDAC) homologue grown in the presence and absence of a
compound capable of inhibiting the histone deacetylase activity of
said HDAC homologue. As referred to herein, a HDAC homologue (as
well as a HDLP-related protein) is any protein molecule having (a)
greater than 15% sequence identity to over the 375 amino acid
residues of HDLP; (b) having no more than twenty insertions or
deletions for a total of no more than 100 amino acids; and (c)
deacetylase activity. Sequence identity is calculated by the
program DNAstar.TM. using the identity matrix weighing scheme
clustal method (DNAstar program, Madison, Wis.).
[0041] A HDLP/HDAC inhibitor compound, as used herein, refers to
any compound represented by Formula (I): ##STR2## wherein X
comprises a cap group which binds to at least one amino acid
selected from the group consisting of tyrosine, proline and
leucine; Y comprises an aliphatic chain group from about 5 to about
10 .ANG., preferably 7 .ANG., which binds to at least one amino
acid selected from the group consisting of phenylalanine and
glycine; and Z comprises a active site binding group which binds to
at least one amino acid selected from the group consisting of
aspartic acid, tyrosine and histidine and which may further bind to
a zinc atom. The HDAC inhibitory compounds of the present invention
can inhibit greater than 50% of the histone deacetylase activity of
a HDAC homologue or a HDLP-related protein.
[0042] To grow the crystals of the present invention, the HDAC and
HDAC-inhibitory compound complex are purified to greater than 80%
total protein and more preferably purified to greater than 90%
total protein. For expression and purification purposes, the
full-length HDLP (Genbank accession number AE000719) may be
subcloned from Aquifex aeolicus chromosomal DNA preparation by the
polymerase chain reaction (PCR) and inserted into an expression
vector.
[0043] A large number of vector-host systems known in the art may
be used. Possible vectors include, but are not limited to, plasmids
or modified viruses, but the vector system must be compatible with
the host cell used. Examples of vectors include E. coli
bacteriophages such as lambda derivatives, or plasmids such as
pBR322 derivatives or pUC plasmid derivatives, e.g., pGEX vectors
(Amersham-Pharmacia, Piscataway, N.J.), pET vectors (Novagen,
Madison, Wis.), pmal-c vectors (Amersham-Pharmacia, Piscataway,
N.J.), pFLAG vectors (Chiang and Roeder, 1993, Pept. Res. 6:62-64),
baculovirus vectors (Invitrogen, Carlsbad, Calif.; Pharmingen, San
Diego, Calif.), etc. The insertion into a cloning vector can, for
example, be accomplished by ligating the DNA fragment into a
cloning vector which has complementary cohesive termini, by blunt
end ligation if no complementary cohesive termini are available or
by through nucleotide linkers using techniques standard in the art.
E.g., Ausubel et al. (eds.), Current Protocols in Molecular
Biology, (1992). Recombinant vectors comprising the nucleic acid of
interest may then be introduced into a host cell compatible with
the vector (e.g. E. coli, insect cells, mammalian cells, etc.) via
transformation, transfection, infection, electroporation, etc. The
nucleic acid may also be placed in a shuttle vector which may be
cloned and propagated to large quantities in bacteria and then
introduced into a eukaryotic cell host for expression. The vector
systems of the present invention may provide expression control
sequences and may allow for the expression of proteins in
vitro.
[0044] In a preferred embodiment, the full length HDLP (SEQ ID
NO:2) is subcloned from Aquifex aeolicus chromosomal DNA
preparation into pGEX4T3 (Amersham-Pharmacia, Piscataway, N.J.). In
order to construct a double mutant comprising a Cys75Ser and
Cys77Ser mutation (SEQ ID NO:4), and to construct the HDLP active
site mutant Tyr297Phe (SEQ ID NO:5 and SEQ ID NO:6), PCR site
directed mutagenesis may be employed with verification by DNA
sequencing by methods known to those skilled in the art (see, e.g.,
Example 1 below). The mutants of the present invention may be
subcloned into a suitable expression vector and introduced into a
host cell for protein production, as described above.
[0045] The HDLP nucleic acids of the present invention may be
subcloned into an expression vector to create an expression
construct such that the resultant HDLP molecule which is produced
comprises a fusion protein wherein said fusion protein comprises a
tag for ease of purification. As referred to herein, a "tag" is any
additional amino acids which are provided in a protein either
c-terminally, n-terminally or internally for the ease of
purification, for the improvement of production or for any other
purpose which may facilitate the goals of the present invention
(e.g. to achieve higher levels of production and/or purification).
Such tags include tags known to those skilled in the art to be
useful in purification such as, but not limited to, his tag,
glutathione-s-transferase tag, flag tag, mbp (maltose binding
protein) tag, etc. In a preferred embodiment, the wild-type and
mutant HDLPs of the present invention are tagged with
glutathione-s-transferase (see Example 1 below). In another
preferred embodiment, HDAC1 is flag tagged (see Example 1 below).
Such tagged proteins may also be engineered to comprise a cleavage
site, such as a thrombin, enterokinase or factor X cleavage site,
for ease of removal of the tag before, during or after
purification. Vector systems which provide a tag and a cleavage
site for removal of the tag are particularly useful to make the
expression constructs of the present invention.
[0046] The tagged HDLPs and HDACs of the present invention may be
purified by immuno-affinity or conventional chromatography,
including but not limited to, chromatography employing the
following: glutathione-sepharose.TM. (Amersham-Pharmacia,
Piscataway, N.J.) or an equivalent resin, nickel or
cobalt-purification resins, anion exchange chromatography, cation
exchange chromatography, hydrophobic resins, gel filtration,
antiflag epitope resin, reverse phase chromatography, etc. After
purification, the HDLP and HDLP-inhibitor compound complex may be
concentrated to greater than 1 mg/ml for crystallization purposes.
In a preferred embodiment HDLP and HDLP-inhibitor complexes are
concentrated to greater than to mg/ml for crystallization and in a
particularly preferred embodiment, HDLP and HDLP-inhibitor
complexes are concentrated to greater than 20 mg/ml.
[0047] In order to determine whether the purified HDLPs of the
present invention demonstrate histone deacetylase activity, the
purified HDLPs and also any HDLP-related protein may be assayed by
any method known to those skilled in the art for the determination
of said activity. In a preferred embodiment, the purified HDLPs of
the present invention are incubated in the presence of
[.sup.3H]acetyl-labeled histone substrate (Carmen et al., 1996, J.
Biol. Chem. 27:15837-15844) in a buffer suitable for detection of
histone deacetylase activity (see Example 3 below); stopping the
reaction; extracting the released acetate and measuring said
released acetate, as described by Henzel et al. (J. Biol. Chem.
266:21936-21942 (1991); Example 3 below). In a preferred
embodiment, the HDLPs of the present invention are inclubated in
the presence of ZnCl.sub.2 in order to obtain histone deacetylase
activity therefrom (Example 3 below).
[0048] In another embodiment, the crystals of the present invention
comprise purified wild-type HDLP (SEQ ID NO:1) and are grown at
room temperature by the hanging-drop vapor-diffusion method from a
crystallization solution comprising one or more precipitants
selected from the group consisting of isopropanol, polyethylene
glycol, and tert butanol (see Example 2 below). The crystallization
solution may further comprise one or more salts including salts
selected from the group consisting of NaCl and KCl, and one or more
buffers including buffers selected from the group consisting of
Tris (tris(hydroxymethyl)aminomethane and bis-tris propane-C1
(1,3-bis[tris(hydroxymethyl)methyl-amino]propane) (see Example 2
below). The pH of the crystallization solution is preferably
between pH 5 to 9, although other pH values are also contemplated
by the present invention (see Example 2 below).
[0049] Any crystallization technique known to those skilled in the
art may be employed to obtain the crystals of the present
invention, including, but not limited to, batch crystallization,
vapor diffusion (either by sitting drop or hanging drop) and micro
dialysis. Seeding of the crystals in some instances may be required
to obtain X-ray quality crystals. Standard micro and/or macro
seeding of crystals may therefore be used.
[0050] The crystals of the present invention may form in the space
group C2 with one molecule in the asymmetric unit and with unit
dimensions of a=51.4 .ANG., b=93.8 .ANG., c=78.7 .ANG. and
.beta.=96.9.degree. (see Example 2 below). The crystals of the
present invention may also form in the space group
P2.sub.12.sub.12.sub.1, with two molecules in the asymmetric unit
and with unit dimensions of a=53.4 .ANG., b=94.4 .ANG., c=156.3
.ANG. (see Example 2 below). However, the present invention
contemplates crystals which form in any space group including, but
not limited to, C2, P2.sub.1, P2.sub.12.sub.12.sub.1, P3.sub.121,
P4.sub.32.sub.12.sub.1, and C222.sub.1. The crystals diffract to a
resolution greater than 4 .ANG., preferably greater than 2.5
.ANG..
[0051] To collect diffraction data from the crystals of the present
invention, the crystals may be flash-frozen in the crystallization
buffer employed for the growth of said crystals, however with
preferably higher precipitant concentration (see, e.g., Example 2
below). For example, but not by way of limitation, if the
precipitant used was 28% PEG 1500, the crystals may be flash frozen
in the same crystallization solution employed for said crystal
growth wherein the concentration of the precipitant is increased to
35% (see Example 2 below). If the precipitant is not a sufficient
cryoprotectant (i.e. a glass is not formed upon flash-freezing),
cryoprotectants (e.g. glycerol, low molecular weight PEGs,
alcohols, etc) may be added to the solution in order to achieve
glass formation upon flash-freezing, providing the cryoprotectant
is compatible with preserving the integrity of the crystals. The
flash-frozen crystals are maintained at a temperature of less than
-110.degree. C. and preferably less than -150.degree. C. during the
collection of the crystallographic data by X-ray diffraction. The
X-ray diffraction data may be processed with DENZO and SCALEPACK
(Otwinowski & Minor, 1997, Method Ensemble. 276:307-326) but
any method known to those skilled in the art may be used to process
the X-ray diffraction data.
[0052] In order to determine the atomic structure of HDLP according
to the present invention, multiple isomorphous replacement (MIR)
analysis, model building and refinement may be performed. For MIR
analysis, the crystals may be soaked in heavy-atoms to produce
heavy atom derivatives necessary for MIR analysis. As used herein,
heavy atom derivative or derivitization refers to the method of
producing a chemically modified form of a protein or protein
complex crystal wherein said protein is specifically bound to a
heavy atom within the crystal. In practice a crystal is soaked in a
solution containing heavy metal atoms or salts, or organometallic
compounds, e.g., lead chloride, gold cyanide, thimerosal, lead
acetate, uranyl acetate, mercury chloride, gold chloride, etc,
which can diffuse through the crystal and bind specifically to the
protein. The location(s) of the bound heavy metal atom(s) or salts
can be determined by X-ray diffraction analysis of the soaked
crystal. This information is used to generate MIR phase information
which is used to construct the three-dimensional structure of the
crystallized HDLPs and HDLP-related proteins of the present
invention. In a preferred embodiment, the heavy atoms comprise
thimerosal, KAu(CN).sub.2 and Pb(Me).sub.3OAc (see Example 2
below). The MIR phases may be calculated by any program known to
those skilled in the art and preferably with the program MLPHARE
(The CCP4 suite: Programs for computational crystallography, 1994,
Acta Crystallogr. D. 50:760-763) and may also use the anomalous
diffraction signal from the thimerosal derivative. In a preferred
embodiment, the MIR phases were calculated at 2.5 .ANG. and have a
mean figure of merit of 0.55 (see FIG. 19 and Example 2 below). The
phases may be improved where necessary by solvent flattening by
methods known to those skilled in the art including, but not
limited to, through the use of the program DM (The CCP4 suite:
Programs for computational crystallography, 1994, Acta Crystallogr.
D 50:760-763).
[0053] Thereafter, an initial model of the three-dimensional
structure may be built using the program O (Jones et al., 1991,
Acta Crystallogr. A 47:110-119). The interpretation and building of
the structure may be further facilitated by use of the program CNS
(Brunger et al., 1998, Acta Crystallogr. D 54:9.05-921).
[0054] For the determination of the HDLP-inhibitor compound complex
structure, if the space group of the HDLP-inhibitor compound
complex crystal is different, molecular replacement may be employed
using a known structure of apo-HDLP (as referred to herein,
apo-HDLP or apo-HDAC is the enzyme which is not complexed with an
inhibitor compound) or any known HDLP/inhibitor complex structure
whose structure may be determined as described above and below in
Example 2. If the space group of the HDLP-inhibitor compound
crystals is the same, then rigid body refinement and difference
fourier may be employed to solve the structure using a known
structure of apo-HDLP (as referred to herein, apo-HDLP or apo-HDAC
is the enzyme which is not complexed with an inhibitor compound) or
any known HDLP/inhibitor complex structure.
[0055] The term "molecular replacement" refers to a method that
involves generating a preliminary model of the three-dimensional
structure of the HDLP crystals of the present invention whose
structure coordinates are unknown prior to the employment of
molecular replacement. Molecular replacement is achieved by
orienting and positioning a molecule whose structure coordinates
are known (in this case the previously determined apo-HDLP) within
the unit cell as defined by the X-ray diffraction pattern obtained
from an HDLP or HDLP-related protein crystal whose structure is
unknown so as to best account for the observed diffraction pattern
of the unknown crystal. Phases can then be calculated from this
model and combined with the observed amplitudes to give an
approximate Fourier synthesis of the structure whose coordinates
are unknown. This in turn can be subject to any of several forms of
refinement to provide a final, accurate structure.
[0056] Any method known to the skilled artisan may be employed to
determine the structure by molecular replacement. For example, the
program AMORE (The CCP4 suite: Programs for computational
crystallography, 1994, Acta Crystallogr. D. 50:760-763) may be
employed to determine the structure of an unknown histone
deacetylase +/- an inhibitor by molecular replacement using the
apo-HDLP coordinates (FIG. 16). For the structure determination of
the inhibitory compound TSA, the structure of TSA was obtained from
the Cambridge Structural Database (Refcode TRCHST,
<<http://www.ccdc.cam.ac.uk>>) may be employed to
define the stereochemical restraints used in the refinement with
the program CNS (Brunger et al., 1998, Acta Crystallogr. D
54:905-921).
[0057] The three-dimensional structural information and the atomic
coordinates associated with said structural information of HDLP are
useful for solving the structure of crystallized proteins which
belong to the HDAC family by molecular replacement. Similarly, any
structure of a crystallized protein which is thought to be similar
in structure based on function or sequence similarity or identity
to HDLP may be solved by molecular replacement with the HDLP
structural information of the present invention. The structure of
HDLP-related proteins as determined by molecular replacement as
described above and in Example 2 below, comprise a root mean square
deviation (rmsd) of no greater than 2.0 .ANG. in the positions of
C.alpha. atoms for at least 50% or more of the amino acids of the
structure over the 375 residues of full-length HDLP. Such a rmsd
may be expected based on the amino acid sequence identity. Chothia
& Lesk, 1986, Embo J. 5:823-826.
[0058] The refined three-dimensional HDLP structures of the present
invention, specifically apo-HDLP, Cys75Ser/Cys77Ser double mutant
HDLP comprising a zinc atom in the active site, HDLP/TSA complex
comprising a zinc atom in the active site, and HDLP/SAHA complex
comprising a zinc atom in the active site, are represented by the
atomic coordinates set forth in FIGS. 16 to 19 respectively. The
refined model for apo-HDLP comprising amino acids 1-375 consists of
wild-type HDLP residues 2 to 373 with residues 1, 374 and 375 not
modeled and presumed disordered and was determined to a resolution
of 1.8 .ANG.. Similarly, the refined model for Cys75Ser/Cys77Ser
double mutant HDLP comprising a zinc atom in the active site also
consists of residues 2 to 373 with residues 1, 374 and 375 not
modeled and presumed disordered and was determined to a resolution
of 2.0 .ANG.. The refined model for the HDLP/TSA complex comprising
a zinc atom in the active site consists of the Cys75Ser/Cys77Ser
double mutant HDLP residues 2 to 373 with residues 1, 374 and 375
not modeled and presumed disordered, has TSA in the binding pocket
and was determined to a resolution of 2.1 .ANG.. The HDLP/SAHA
complex is similar to the HDLP/TSA complex but has SAHA in the
binding pocket and was determined to a resolution of 2.5 .ANG..
[0059] For the purposes of further describing the structure of HDLP
and HDLP-related proteins, including, but not limited to, HDACs,
from the data obtained from the HDLP crystals of the present
invention, the definition of the following terms is provided:
[0060] The term ".beta. sheet" refers to two or more polypeptide
chains (or .beta. strands) that run alongside each other and are
linked in a regular manner by hydrogen bonds between the main chain
C.dbd.O and N--H groups. Therefore all hydrogen bonds in a
beta-sheet are between different segments of polypeptide. Most
.beta.-sheets in proteins are all-parallel (protein interiors) or
all-antiparallel (one side facing solvent, the other facing the
hydrophobic core). Hydrogen bonds in antiparallel sheets are
perpendicular to the chain direction and spaced evenly as pairs
between strands. Hydrogen bonds in parallel sheets are slanted with
respect to the chain direction and spaced evenly between
strands.
[0061] The term ".alpha. helix" refers to the most abundant helical
conformation found in globular proteins. The average length of an
.alpha. helix is 10 residues. In an .alpha. helix, all amide
protons point toward the N-terminus and all carbonyl oxygens point
toward the C-terminus. The repeating nature of the phi, psi pairs
ensure this orientation. Hydrogen bonds within an .alpha. helix
also display a repeating pattern in which the backbone C.dbd.O of
residue X (wherein X refers to any amino acid) hydrogen bonds to
the backbone HN of residue X+4. The .alpha. helix is a coiled
structure characterized by 3.6 residues per turn, and translating
along its axis 1.5 .ANG. per amino acid. Thus the pitch is
3.6.times.1.5 or 5.4 .ANG.. The screw sense of alpha helices is
always right-handed.
[0062] The term "loop" refers to any other conformation of amino
acids (i.e. not a helix, strand or sheet). Additionally, a loop may
contain bond interactions between amino acid side chains, but not
in a repetitive, regular fashion.
[0063] Amino acid residues in peptides shall herein after be
abbreviated as follows: Phenylalanine is Phe or F; Leucine is Leu
or L; Isoleucine is Ile or I; Methionine is Met or M; Valine is Val
or V; Serine is Ser or S; Proline is Pro or P; Threonine is Thr or
T; Alanine is Ala or A; Tyrosine is Tyr or Y; Histidine is His or
H; Glutamine is Gln or Q; Asparagine is Asn or N; Lysine is Lys or
K; Aspartic Acid is Asp or D; Glutamic Acid is Glu or E; Cysteine
is Cys or C; Tryptophan is Trp or W; Arginine is Arg or R; and
Glycine is Gly or G. For further description of amino acids, please
refer to Proteins: Structure and Molecular Properties by Creighton,
T. E., W.H. Freeman & Co., New York 1983.
[0064] The term "positively charged amino acid" refers to any amino
acid having a positively charged side chain under normal
physiological conditions. Examples of positively charged amino
acids are Arg, Lys and His. The term "negatively charged amino
acid" refers to any amino acid having a negatively charged side
chain under normal physiological conditions. Examples of negatively
charged amino acids are Asp and Glu. The term "hydrophobic amino
acid" refers to any amino acid having an uncharged, nonpolar side
chain that is relatively insoluble in water. Examples of
hydrophobic amino acids are Ala, Leu, Ile, Gly, Val, Pro, Phe, Trp
and Met. The term "hydrophilic amino acid" refers to any amino acid
having an uncharged, polar side chain that is relatively soluble in
water. Examples of hydrophilic amino acids are Ser, Thr, Tyr, Asp,
Gln, and Cys. The term "aromatic amino acid" refers to any amino
acid comprising a ring structure. Examples of aromatic amino acids
are His, Phe, Trp and Tyr.
[0065] The term "charge relay system" refers to a His-Asp
arrangement as described by Fersht & Sperling, 1973, J. Mol.
Biol. 74:137-149; Blow et al., 1969, Nature 221:337-340.
[0066] The information obtained from the three-dimensional
structures of the present invention reveal that HDLP has a
single-domain structure that belongs to the open .alpha./.beta.
class of folds (see, e.g., Branden, 1980, Q. Rev. Biophys.
13:317-38). Two orthogonal views of the overall three-dimensional
structure of HDLP are depicted in FIGS. 4A and 4B. The HDLP
structure has a central eight-stranded parallel .beta. sheet
(strands arranged as
.beta.2-.beta.1-.beta.3-.beta.8-.beta.7-.beta.4-.beta.5-.beta.6),
and sixteen .alpha. helices (labeled .alpha.1 through .alpha.16
respectively). See FIG. 4C. Four of the helices pack on either face
of the .beta. sheet (.alpha.7, .alpha.8, .alpha.9, .alpha.10 and
.alpha.11, .alpha.12, .alpha.13, .alpha.14) forming the core
.alpha./.beta. structure characteristic of this class of folds.
Most of the remaining eight helices are positioned near one side of
the .beta. sheet, near stands .beta.2-1-.beta.3-.beta.8. Large,
well defined loops (Loops L1-L7; FIG. 4C) originate from the
C-terminal ends of the .beta.-strands. The extra helices and the
large L1-L7 loops are associated with a significant extension of
the structure beyond the core .alpha./.beta. motif. This extension
of the structure gives rise to two prominent architectural
features: a deep, narrow pocket and an internal cavity adjacent to
the pocket. These two architectural features comprise the active
site (see FIG. 5A). The structure of HDLP-related proteins (e.g.
HDACs) may also comprise the conserved .alpha./.beta. structure
characteristic.
[0067] The term "active site" comprises any or all of the following
sites in HDLP, the substrate binding site, the site where the
cleavage of an acetyl group from a substrate occurs or the site
where an inhibitor of the HDAC family or, more particularly, HDLP
binds. The active site, as referred to herein, comprises Asp166,
Asp258, His170, Tyr297, His131, His132, Asp168, Asp173, Phe141,
Phe198, Leu265, Pro22 and Gly140, and also a metal bound at the
bottom of the pocket by Asp173, Asp168 and His defined by the
coordinates listed in FIGS. 16 to 19 with an rmsd of 2.0 .ANG.. The
metal which binds at the bottom of the pocket will be a divalent
cation selected from the group consisting of zinc, cobalt or
manganese.
[0068] The deep narrow pocket has a tube-like shape with a depth of
.about.11 .ANG.. The pocket opening constricts half way down to
.about.4.5 by 5.5 .ANG., and becomes wider at the bottom (see FIG.
5A). The pocket and its immediate surroundings are made up of
loops. L1 through L7.
[0069] The walls of the pocket are covered with side chains of
hydrophobic and aromatic residues (Pro22, Tyr91 near the entrance;
and Gly140, Phe141, Phe 198, Leu265 and Tyr297 further down; FIG.
5B). For numbering of amino acids please refer to SEQ ID NO:1. Of
particular interest are Phe141 and Phe198, whose phenyl groups face
each other in parallel at a distance of 7.5 .ANG., marking the most
slender portion of the pocket (see FIG. 5B). Of particular interest
is that only one pocket residue differs in HDAC1 when the sequences
are aligned (alignment may be accomplished using DNAstar.TM.
MegAlign.TM. program, Madison, Wis.), this residue is Glu98 of
HDAC1 which is Tyr91 in HDLP. The structure reveals that this
residue in HDLP is mostly solvent exposed.
[0070] Near the bottom of the pocket of the active site at its
narrowest point, is located a zinc ion (see FIG. 6A). In order to
obtain the zinc in the structure, the crystals may be soaked in
zinc (e.g. ZnCl.sub.2) or co-crystalized in the presence of zinc.
The zinc ion is coordinated by Asp168 (051, 2.1 .ANG.), His170
(N.delta.1, 2.1 .ANG.), Asp258 (P.delta., 1.9 .ANG.) and a water
molecule (2.5 .ANG.). See FIGS. 5B and 6B. The amino acid residues
that coordinate zinc are arranged in a tetrahedral geometry, but
the position of the water molecule, which is also hydrogen bonded
to His131, deviates from this geometry by .about.25.degree..
[0071] In addition to the zinc ligands, the bottom of the pocket
contains two histidine (His131 and His132), two aspartic acids
(Asp166 and Asp173) and a tyrosine (Tyr297). See FIGS. 5B and 10B.
Each of the histidines makes a hydrogen bond through its N.delta.1
to an aspartic acid carboxylate oxygen, with the oxygen located in
the plane of the imidizole ring (FIG. 5B). This His-Asp arrangement
is characteristic of the charge relay system present in the active
sites of serine proteases, where it serves to polarize the
imidizole Ne and increase its basicity. Fersht & Sperling,
1973, J. Mol. Biol. 74:137-149; Blow et al., 1969, Nature
221:337-340.
[0072] The Asp166-His131 charge pair relay (hereafter referred to
as "buried charged relay") is positioned even deeper in the pocket
and more buried compared to the Asp173-His132 charge relay
(hereafter referred to as "exposed charge relay") which is
partially solvent exposed. The buried charge relay makes a hydrogen
bond (2.6 .ANG.) to the zinc-bound water molecule referred to
above, and this hydrogen bond could contribute to the deviation of
the water-zinc coordination from ideal geometry (FIG. 5B). The
exposed charge relay is directed to a point .about.2.5 .ANG. away
from the water molecule and closer to the surface.
[0073] Tyr 297 is positioned next to the zinc, opposite from where
the two charge relay systems are located. The Tyr hydroxyl group
lies 4.4 .ANG. away from the zinc atom and has no interactions with
the rest of the protein (FIG. 5B). Next to Tyr297, there is an
opening in the pocket wall, which leads to the adjacent internal
cavity.
[0074] The floor of the internal cavity is made up of portions of
the L3 and L7 loops as they emerge from the .beta. strands, and the
roof is made up by the .alpha.1-L1-.alpha.2 segment. The L1 loop
appears more flexible than other loops in the structure.
[0075] This may allow the transient exchange of the cavity contents
with the bulk solvent. The cavity is lined primarily with
hydrophobic residues and is particularly rich in glycine residues
(Ala127, Gly128, Gly129, Met130, and Phe141 of L3; Gly293, Gly294,
Gly295 and Gly296 of L7; and Tyr17, Pro22 and Leu23 of L1). There
are only two charged residues in the cavity-(Arg27 and His 21) and
these are contributed by the L1 loop.
[0076] The cavity may provide space for the diffusion of the
acetate product away from the catalytic center, which may otherwise
be crowded and shielded during deacetylation from the solvent when
the substrate is bound. Such a role for the cavity is supported by
the observation that the cavity contains three water and two
isopropanol molecules (from the crystallization buffer) in the 1.8
.ANG. apo-protein structure. The cavity may also bind another
cofactor, in addition to zinc, for the facilitation of the
enzymatic activity of the HDLP. A proposed catalytic mechanism for
deacetylation is provided in FIG. 8.
[0077] The structure of HDLP as defined by the present invention,
in conjunction with the HDAC1 sequence homology, shows that the
375-amino acid HDLP protein corresponds to the histone deacetylase
catalytic core which is conserved across the HDAC family (see FIG.
2). The 35.2% HDLP-HDAC1 sequence identity predicts structural
similarity with a rmsd in Ca positions of .about.1.5 .ANG.. Chothia
and Lesk describe the relation between the divergence of sequence
and the structure of proteins in Embo J. 5:823-826 (1986). The
40-residue C-terminus of HDLP is likely to have a divergent
structure since this region has lower homology to HDAC1, although
the .alpha.16 helix in this region is part of the conserved open
.alpha./.beta. core fold and HDAC1 is likely to comprise a similar
helix. However divergent this C-terminal region may be, this region
is outside the active site and is likely to not effect the
structure of the active site. Beyond the C-terminus of the histone
deacetylase catalytic core, HDAC family members are divergent in
length and sequence. In the HDAC family, this region (amino acid
residues .about.390-482) is highly polar, populated with acidic
residues, and is likely to be flexible or loosely folded.
[0078] The HDLP-HDAC homology maps primarily to the hydrophobic
core and to the L1-L7 loops, with portions of the loops that make
up the pocket and adjacent cavity having the highest level of amino
acid residue sequence conservation (FIGS. 9A and 9B). Specifically,
all of the polar residues in the active site (the zinc ligands, the
two charge relay systems, and Tyr297) and the hydrophobic residues
that make up the walls of the pocket (Gly140, Phe141, Phe198 and
Leu265) are identical. Among the residues that make up the internal
cavity, the ones closest to the active site are either identical or
conservatively substituted (for example, Leu23.fwdarw.Met and
Met130.fwdarw.Leu). Surface residues around the pocket are
conserved to a lesser extent, but are still above 35% average
sequence identity.
[0079] The information obtained from the inhibitor-bound HDLP
complex crystal structures of the present invention reveal detailed
information which is useful in the design, isolation, screening and
determination of potential inhibitor compounds which may inhibit
HDLP/HDAC family members. As described above, the HDLP structure
consists of a parallel .beta. sheet with .alpha. helices packing
against both faces (FIGS. 4A, 4B, and 4C). At one end of the .beta.
sheet, 7 loops (L1-L7) form a narrow, tube-like pocket which are
lined with hydrophobic residues and which comprise a zinc binding
site, several polar side chains, including two Asp-His charge relay
systems. Mutation of the zinc ligands and other polar residues at
the pocket bottom reduces or eliminates the catalytic activity.
[0080] The present inventors found that mutation at the Tyr297Phe
site reduced activity. See also, Hassig et al., 1998, Proc. Natl.
Acad. Sci. USA 95:3519-3524; Kadosh & Struhl, 1998, Genes Dev.
12:797-805. The elimination of activity by mutation of these
residues indicates that this region is the enzyme active site.
Adjacent to the active site, there is an internal cavity that may
provide space for the diffusion of the acetate reaction product.
Homology at the active site between HDLP and HDAC1, as described
above, indicates that they share structural and functional
homology.
[0081] The inhibitor compound, trichostatin A (TSA) (Tsuji et al.,
1976, J. Antibiotics 29:1-6) binds HDLP by inserting its long
aliphatic chain, which has a hydroxamic acid group at one end, into
the pocket (FIGS. 6A, 6B and 6C). The aliphatic chain makes
multiple contacts in the well-like, hydrophobic portion of the
pocket. The hydroxamic acid reaches the polar bottom of the pocket,
where it coordinates the zinc in a bidentate fashion and also forms
hydrogen bonds with the polar residues in the active site,
including the two charge relay system histidines. The aromatic
dimethylamino-phenyl group at the other end of the TSA chain makes
contacts at the pocket entrance and serves to cap it. The amino
acid residues of HDLP which contact TSA are conserved in HDAC,
indicating that TSA binds and inhibits HDAC in a similar fashion to
HDLP.
[0082] In the complex, the hydroxamic acid, most of the aliphatic
chain and part of the dimethylamino-phenyl group of TSA are buried
(60% of TSA's surface area; FIG. 6A). The hydroxamic acid group
binds the zinc in a bidentite fashion forming bonds through its
carbonyl (2.4 .ANG.) and hydroxyl groups (2.2 .ANG.) resulting in a
penta-coordinated Zn.sup.2+ (FIGS. 6B and 6C). The hydroxamic acid
hydroxyl group replaces the water molecule that binds to the zinc
in the apo-HDLP structure described above. The hydroxamic acid also
hydrogen bonds with both charge relay system histidines
(hydroxyloxygen to His131 N.epsilon.2, 2.8 .ANG.; and nitrogen to
His132 N.epsilon.2, 2.8 .ANG.), and the Tyr297 hydroxyl group (2.4
.ANG.; FIGS. 6B and 6C).
[0083] The 5-carbon long branched alkene chain of TSA fits snugly
in the narrow portion of the pocket making multiple van der Waals
contacts with all of the hydrophobic groups lining the pocket
(FIGS. 6B and 6C). Near its center, the chain contains a methyl
substituted carbon-carbon double bond which is sandwiched between
the phenyl groups of the Phe141 and Phe98 at the tightest point of
the pocket (FIGS. 6A and 6B). The length of the alkene chain
appears optimal for spanning the length of the pocket, and allowing
contacts both at the bottom and at the entrance of the pocket,
although, the cap group of Formula (I) may provide length to span
the pocket allowing for a shorter alkene chain (aliphatic
chain).
[0084] At the entrance of the pocket, one face of the planar
structure formed by the dimethylamino-phenyl and adjacent carbonyl
groups of TSA makes contacts at the rim of the pocket (Pro22,
Tyr91, Phe141; FIGS. 6B and 6C). This packing is facilitated by the
roughly 110.degree. angle in the overall structure of TSA at the
junction of the aliphatic chain and the dimethylamino-phenyl group
(occurring at the sp.sup.3 hybridized C8 carbon). Upon TSA binding,
the side chain of Tyr91, which is mostly solvent exposed, changes
conformation to make space for the dimethylamino-phenyl group. This
is the only change near the active site observed upon TSA
binding.
[0085] The hydroxamic acid group is a common motif in zinc
metalloprotease inhibitors. See U.S. Pat. Nos. 5,919,940 and
5,917,090; See also, Grams et al., 1995, Biochemistry
34:14012-14020; Lovejoy et al., 1999, Nat. Struct. Biol. 6:217-221;
and Holmes & Matthews, 1981, Biochemisty 20:6912-6920. Like
TSA, these inhibitors also coordinate the active site zinc in a
bidentate fashion using their hydroxamate hyroxyl and carbonyl
oxygens, replace the nucleophilic water molecule with their
hydroxamate hydroxyl groups and form hydrogen bonds to the general
base (Grams et al., 1995, Biochemistry 34:14012-14020; Lovejoy et
al., 1999, Nat. Struct. Biol. 6:217-221; and Holmes & Matthews,
19.81, Biochemistry 20:6912-6920).
[0086] SAHA, which has a .about.30-fold weaker inhibitory activity
than TSA (Richon et al., 1998, Proc. Natl. Acad. Sci. USA
95:3003-3007), binds HDLP similarly to TSA (see, e.g., FIG. 4D).
The SAHA hydroxamic acid group makes the same contacts to the zinc
and active site residues, and the importance of these interactions
is underscored by the loss of activity of SAHA derivatives lacking
the hydroxamic group (Richon et al., 1998, Proc. Natl. Acad. Sci.
USA 95:3003-3007). The six-carbon long aliphatic chain of SAHA
packs in the tube-like hydrophobic portion of the pocket. Compared
to TSA however. SAHA's aliphatic chain packs less snugly and makes
fewer van der waals contacts, in part, because SAHA lacks TSA's C15
methyl group branch. SARA also lacks TSA's double bonds in this
region, and this may lead to increased flexibility of the aliphatic
chain. The cap group of SAHA consists of a phenyl-amino ketone
group. In the crystal structure, the phenyl group has weak electron
density, suggesting that it does not pack as well as the cap group
of TSA. This may be due to the larger separation between the
hydroxamic and cap groups of SAHA compared to TSA (compare TSA,
Formula (II) and SARA, Formula (III), below). ##STR3##
[0087] The determination of the structure of HDLP and HDLP bound to
an inhibitory compound has enabled, for the first time, the
identification of the active site of HDLP and of related HDLP
proteins, such as proteins belonging to the HDAC family.
[0088] The three-dimensional structural information and the atomic
coordinates associated with said structural information of HDLP
bound to an inhibitory compound is useful in rational drug design
providing for a method of identifying inhibitory compounds which
bind to and inhibit the enzymatic activity of HDLP, HDAC family
proteins and other histone deacetylase-like proteins related to
HDLP. Said method for identifying said potential inhibitor for an
enzyme comprising deacetylase activity comprises the steps of (a)
using a three-dimensional structure of HDLP as defined by its
atomic coordinates listed in FIG. 16 to 19; (b) employing said
three-dimensional structure to design or select said potential
inhibitor; (c) synthesizing said potential inhibitor; (d)
contacting said potential inhibitor with said enzyme in the
presence of an acetylated substrate; and (e) determining the
ability of said inhibitor to inhibit said deacetylase activity.
[0089] The potential HDLP and HDLP-related (e.g. HDAC) inhibitors
identified by the method of the present invention are represented
by formula (I) ##STR4## wherein X comprises a cap group which binds
to at least one amino acid selected from the group consisting of
proline and leucine; Y comprises an aliphatic chain group which
binds to at least one amino acid selected from the group consisting
of leucine, phenylalanine and glycine; and Z comprises an active
site binding group which binds to at least one amino acid selected
from the group consisting of aspartic acid, tyrosine and histidine
and wherein Z may further bind to a zinc atom and with the
provision that the compound of Formula (I) is not TSA, trapoxin,
SAHA, SAHA derivatives described in U.S. Pat. Nos. 5,608,108;
5,700,811; 5,773,474; 5,840,960 and 5,668,179.
[0090] The present invention permits the use of molecular design
techniques to design, identify and synthesize chemical entities and
compounds, including inhibitory compounds, capable of binding to
the active site of HDLP and HDLP-related proteins. The atomic
coordinates of apo-HDLP and inhibitor-bound HDLP may be used in
conjunction with computer modeling using a docking program such as
GRAM, DOCK, HOOK or AUTODOCK (Dunbrack et al., 1997, Folding &
Design 2:27-42) to identify potential inhibitors of HDLP and
HDLP-related proteins (e.g. HDAC1). This procedure can include
computer fitting of potential inhibitors to the active site of HDLP
to ascertain how well the shape and the chemical structure of the
potential inhibitor will complement the active site or to compare
the potential inhibitors with the binding of TSA or SAHA in the
active site. See Bugg et al, 1998, Scientific American
December:92-98; West et al., 1995, TIPS 16:67-74. The potential
inhibitors designed by modeling with a docking program conform to
the general formula (I) as described above. Computer programs may
also be employed to estimate the attraction, repulsion and stearic
hindrance of the HDLP and potential inhibitor compound. Generally,
the tighter the fit, the lower the stearic hindrances, the greater
the attractive forces, and the greater the specificity which are
important features for a specific inhibitory compound which is more
likely to interact with HDLP and HDLP-related proteins rather than
other classes of proteins. These features are desired particularly
where the inhibitory compound is a potential antitumor drug.
[0091] The compounds of the present invention may also be designed
by visually inspecting the three-dimensional structure to determine
more effective deacetylase inhibitors. This type of modeling may be
referred to as "manual" drug design. Manual drug design may employ
visual inspection and analysis using a graphics visualization
program such as "O" (Jones, T. A., Zhou, J. Y., Cowan, S. W., and
Kjeldgaard, M., Improved method for building protein models in
electron density maps and the location of errors in these models,
Acta Crystallog., A47, 110-119.
[0092] Initially potential inhibitor compounds can be selected for
their structural similarity to the X, Y and Z constituents of
formula (I) by manual drug design. The structural analog thus
designed can then be modified by computer modeling programs to
better define the most likely effective candidates. Reduction of
the number of potential candidates is useful as it may not be
possible to synthesize and screen a countless number of variations
compounds that may have some similarity to known inhibitory
molecules. Such analysis has been shown effective in the
development of HIV protease inhibitors (Lam et al., 1994, Science
263:380-384; Wlodawer et al., 1993, Ann. Rev. Biochem. 62:543-585;
Appelt, 1993 Perspectives in Drug Discovery and Design 1:23-48;
Erickson, 1993, Perspectives in Drug Discovery and Design
1:109-128. Alternatively, random screening of an small molecule
library could lead to potential inhibitors whose inhibitory
activity may then be analyzed by computer modeling as described
above to better determine their effectiveness as inhibitors.
[0093] The compounds designed using the information of the present
invention may be competitive or noncompetitive inhibitors. These
designed inhibitors may bind to all or a portion of the active site
of HDLP and may be more potent, more specific, less toxic and more
effective than known inhibitors for HDLP and HDLP-related proteins,
and particularly HDACs. The designed inhibitors may also be less
potent but have a longer half life in vivo and/or in vitro and
therefore be more effective at inhibiting histone deacetylase
activity in vivo and/or in vivo for prolonged periods of time. Said
designed inhibitors are useful to inhibit the histone deacetylase
activity of HDLP and HDLP-related proteins (e.g. HDAC1), to inhibit
cell growth in vitro and in vivo and may be particularly useful as
antitumor agents.
[0094] The present invention also permits the use of molecular
design techniques to computationally screen small molecule data
bases for chemical entities or compounds that can bind to HDLP in a
manner analogous to the TSA and SAHA as defined by the structure of
the present invention. Such computational screening may identify
various groups which may be defined as "X", "Y" or "Z" of formula
(I) above and may be employed to synthesize the potential
inhibitors of the present invention comprising formula (I). Such
potential inhibitors may be assayed for histone deacetylase
inhibitory activity in a histone deacetylase activity assay (see
Example 3 below), may be co-crystallized with HDLP to determine the
binding characteristics through X-ray crystallography techniques
defined above (e.g. said co-crystal structure may be determined by
molecular replacement to assess the binding characteristics of said
potential inhibitor), or may be assessed based on binding activity
by incubating said potential inhibitor with said HDLP, performing
gel filtration to separate any free potential inhibitor to
HDLP-bound inhibitor, and determining the amount of histone
deacetylase activity of the inhibitor-bound HDLP. To measure
binding constants (e.g., Kd), methods known to those in the art may
be employed such as Biacore.TM. analysis, isothermal titration
calorimetry, Elisa with a known drug on the plate to show
competitive binding, or by a deacetylase activity assay.
[0095] The design of potential inhibitors of the present invention
is further facilitated by reference to FIG. 9, which is a surface
representation figure that depicts the surface grooves. Analysis of
such grooves gives insight into the constituents of the cap group
of formula (I). The surface grooves are labeled groove A, groove A,
groove B and groove C, into which additional cap groups may bind.
The structure of HDLP bound to either TSA or SAHA shows that the
cap groups of TSA and SAHA bind in groove A. By analysis of the
amino acid sequence identity of HDLP and HDACs, Groove A is well
conserved in HDACs, has a significant hydrophobic component,
appears deep enough to allow for significant interactions and is
also the largest of the four grooves. In addition to the
dimethylamino phenyl group of the TSA, the A groove can fit
approximately 200 daltons worth of groups (e.g. groove A could
accommodate a naphthalene-like group after an appropriate spacer,
etc.). Groove. A, as referred to herein, is characterized by the
following conserved residues of HDLP: His 21, Pro22, Lys24, Phe141,
Leu265 and Phe335. The periphery of groove A comprises unconserved
residues. Additionally, Groove A', as referred to herein, comprises
primarily unconserved residues.
[0096] Groove B is immediately adjacent to the pocket. Of
significance is that the bottom of groove B comprises the N-epsilon
nitrogen of His170, which coordinates the zinc through its N-delta
nitrogen. Significant binding energy may be achieved by contacting
the Ne proton of His170 with a carboxylic acid or sulfate group. In
addition, groove B may be large enough to fit a phenyl group, the
face of which may comprise a partial negative charge which may pack
over the N-epsilon proton of His170. The conserved residues of
groove B, as referred to herein are: His170, Tyr196 and Leu265.
[0097] Groove C is not as well conserved as the other two grooves
and the amino acid residues which comprise groove C are mostly
polar and solvent exposed. Groove C, as referred to herein
comprises the following conserved residues: Asn87, Gly140 and
Phe198.
[0098] The compounds of the present invention are represented by
formula (I): ##STR5##
[0099] Examples for suitable X constituents wherein X comprises a
cap group may be described in three categories, depending upon
which surface of groove A, A', B and/or C they are targeted to. The
cap group may comprise all three categories on the same compound.
Of particular benefit may be replacing the cap group of TSA or SAHA
with a large, rigid structure. Nonlimiting examples for suitable
cap groups (X) of formula (I) which may bind in groove A are: (1)
attaching a 1-3 methyl linker followed by a phenyl or naphthalene
group from the para or meta position of SAHA's phenyl group
represented by formula (IV) ##STR6## (2) attaching a 2-3 methyl
linker followed by a phenyl or naphthalene group from the meta
position of TSA's phenyl cap group, or from TSA's dimethyl amino
group represented by formula (V): ##STR7## and which may bind in
groove B is a 1-3 methyl group spacer followed by a carboxylate,
sulfate or phenyl group as represented by formula (VI):
##STR8##
[0100] With respect to the aliphatic (Y) group, the diameter of the
pocket suggests that one more methyl "side chain" could fit, in
addition to the C15 methyl group on the C10 carbon. Nonlimiting
suitable examples for Y constituents wherein Y comprises an
aliphatic chain group are as follows: (1) add a methyl group to TSA
on the C12 carbon (with or without a methyl group on the C10 carbon
and with or without double bonds and with or without substituting
the X and/or Z constituents of formula (I) as represented by
formula (VII): ##STR9## (2) add a methyl group to TSA on the C9
carbon (with or without a methyl group on the C10 carbon; with or
without both or either of the double bonds, and with or without
substituting the X and/or Z constituents of formula (I) as
represented by formula (VIII): ##STR10## (3) replace the two
alkalene double bonds of TSA with only one between C10 and C11,
which may free the C11 and C12 torsion to allow for a better fit,
the X and/or Z groups may also be substituted as represented by
formula (IX): ##STR11## (4) cyclize C15 and C12 carbons of TSA
through a sulphur atom (or nitrogen atom), the X and/or Z groups
may also be substituted as represented by formula (X): ##STR12##
(5) extend from the C9 carbon of TSA such that the extension
approaches and/or enters groove B (see FIG. 9); making C9 sp3 so
that it can have some freedom; attach to C9 a 1-3 methyl group
spacer which may include a double bond and they attaching thereto a
sulfate, carboxylate, sulfate, hyroxyl, or phenyl group which may
make an interaction with the N-epsilon proton of His170 which may
coordinate the zinc atom as represented by formula (XI): ##STR13##
(6) extend off the C8 carbon (replacing C14) of TSA such that the
extension approaches or enters groove B; attach a 1-3 methyl group
spacer (which may include a double bond) and then link thereto a
carboxylate, sulfate, hydroxyl or phenyl group such that an
interaction is made with the N-epsilon proton of His170 that
coordinates the zinc atom; the X and/or Z constituents may also be
substituted as represented by formula (XII): ##STR14## (7)
substitute the C8 carbon at the end of the aliphatic chain such
that the substitution may contact groove A, A', B and or C, in such
an example, a cap group (X) may or may not be required and the X
and Z constituents may be substituted as well, as represented by
formula (XIII): ##STR15## (8) formulas VII through XIII above
wherein the aliphatic chain further comprises a methyl group
between the active site binding group (Z) and the C8 carbon, and
preferably just before the C8 carbon, increasing the distance
between X and Z, (9) make the connection between the aliphatic
chain and the cap group more rigid (e.g., by closing a 6-membered
ring which may or may not comprise oxygen, the X and Z group may
also be substituted as represented by formula (XIV): ##STR16## and
(10) combining two or more of the changes depicted by formulas
(VII-XIV).
[0101] Additionally, nonlimiting examples for suitable Z groups
wherein Z comprises an active site binding group are as follows:
(1) hydroxamic acid, (2) carboxylic acid, (3) sulfonamide, (4)
acetamide, (5) epoxyketone, (6) an ester with a methyl linker and a
hydroxyl of acetate ester group to lead into the cavity and
interact with a conserved arginine (Arg27) as represented by
formula (XV): ##STR17## and (7) an alphaketone as represented by
formula (XVI): ##STR18##
[0102] Additionally, other suitable X, Y and Z constituents may be
envisioned by the skilled artisan given the three-dimensional
structural information of the present invention.
[0103] After having determined potential suitable X, Y and Z
constituents, the constituents are combined to form a compound of
formula (I) using combinatorial chemistry techniques. This may be
achieved according to U.S. Pat. Nos. 5,608,108; 5,700,811;
5,773,474; 5,840,960 and 5,668,179, incorporated herein by
reference. Any methods known to one of skill in the art may be
employed to synthesize compounds of formula (I) comprising X, Y and
Z constituents as determined by the methods described above.
[0104] As mentioned above, the compounds of formula (I) are useful
to inhibit the histone deacetylase activity of HDLP and
HDAC-related proteins. Such inhibition may allow for a reduction or
cessation of cell growth in vitro and in vivo.
[0105] For in vitro use, such reduction or cessation of cell growth
is useful to study the role of histone deacetylation and
differentiation during the cell cycle and also to study other
mechanisms associated with cell cycle arrest and particularly how
the repression of transcription is involved in cell cycle
progression which may be studies in a yeast model system such as
that described by Kadosh & Struhl, 1998, Mol. Cell. Biol.
18:5121-5127. In vitro model systems which may be employed to study
the effects of potential inhibitors on cell cycle progression and
also tumor growth include those described by: Richon et al, 1998,
Proc. Natl. Acad. Sci. USA 95:3003-3007; Yoshida et al., 1995,
Bioessays 17:423-430; Kim et al., 1999, Oncogene 18:2461-2470;
Richon et al., 1996, Proc. Natl. Acad. Sci. USA 93:5705-5708; and
Yoshida et al., 1987, Cancer Res. A47:3688-3691.
[0106] For in vivo use, such a reduction or cessation of cell
growth is useful to study the effect of said inhibitor compounds in
non-human animal model systems of cancer and is also useful for the
treatment of cancer in a recipient in need of such treatment.
Non-limiting examples of animals which may serve as non-human
animal model systems include mice, rats, rabbits, chickens, sheep,
goats, cows, pigs, and non-human primates. See, e.g., Desai et al.,
1999, Proc. AACR 40: abstract #2396; Cohen et al., 1999, Cancer
Res., submitted. The compounds of the present invention may be
administered to a transgenic non-human animal wherein said animal
has developed cancer such as those animal models in which the
animal has a propensity for developing cancer (e.g. animal model
systems described in U.S. Pat. Nos. 5,777,193, 5,811,634,
5,709,844, 5,698,764, and 5,550,316). Such animal model systems may
allow for the determination of toxicity and tumor reduction
effectiveness of the compounds of the present invention.
[0107] A preferred compound of the present invention may comprise
high specific activity for HDLP and HDAC-related proteins, good
bioavailability when administered orally, activity in reducing or
ceasing cell growth in tumor cell lines, and activity in reducing
or ceasing tumor growth in animal models of various cancers.
[0108] Accordingly, another aspect of this invention is a method of
eradicating or managing cancer in a recipient, which may be an
animal and is preferably a human. Said method comprises
administering to said recipient a tumor reducing amount of a
compound as defined by formula (I) above, or a physiological
acceptable salt thereof.
[0109] In a further aspect of the invention, there is provided a
composition comprising the compound of formula, (I) and an
excipient or carrier. Administration of the foregoing agents may be
local or systemic. Such carriers include any suitable physiological
solutions or dispersant or the like. The physiologic solutions
include any acceptable solution or dispersion media, such as
saline, or buffered saline. The carrier may also include
antibacterial and antifungal agents, isotonic and absorption
delaying agents, and the like. Except insofar as any conventional
media, carrier or agent is incompatible with the active ingredient,
its use in the compositions is contemplated.
[0110] Routes of administration for the compositions containing the
delivery vehicle constructs of the present invention include any
conventional and physiologically acceptable routes, such as, for
example, oral, pulmonary, parenteral (intramuscular,
intraperitoneal, intravenous (IV) or subcutaneous injection),
inhalation (via a fine powder formulation or a fine mist),
transdermal, nasal, vaginal, rectal, or sublingual routes of
administration and can be formulated in dosage forms appropriate
for each route of administration.
[0111] The following examples are provided to more clearly
illustrate the aspects of the invention and are not intended to
limit the scope of the invention.
EXAMPLES
Example 1
Protein Production and Purification
[0112] Full-length wild-type HDLP (Genbank accession number
AE000719) was subcloned from an Aquifex aeolicus chromosomal DNA
preparation (provided by Robert Huber of Universitaet of
Regensburg, Germany) into the pGEX4T3 (Amersham-Pharmacia,
Piscataway, N.J.) vector using the polymerase chain reaction (PCR).
The cysteine-to-serine and active site mutants were constructed by
PCR site directed mutagenesis and were sequenced. The
HDLP-glutathione S-transferase (GST) fusion protein was produced in
Escherichia coli, purified by affinity chromatography using a
column of glutathione-sepharose resin (Amersham-Pharmacia,
Piscataway, N.J.), and by anion-exchange chromatography
(Q-sepharose.TM.; Amersham-Pharmacia, Piscataway, N.J.). HDLP was
cleaved from the fusion protein with thrombin at 4.degree. C., was
purified by anion-exchange (Q-sepharose.TM.; Amersham-Pharmacia,
Piscataway, N.J.) and gel filtration chromatography
(Superdex.TM.200; Amersham-Pharmacia, Piscataway, N.J.), and was
concentrated to typically 25 mg/ml in a buffer of 25 mM
bis-tris-propane (BTP), 500 mM NaCl, 5 mM dithiothrietiol (DTT), 2
isopropanol, pH 7.0.
[0113] Although, it is not known what metal cofactor HDLP contains
in vivo, it is presumed to be zinc because of the arrangement of
the ligands and the similarities in the active site to the zinc
proteases. The lack of metal in the purified HDLP is presumed due,
in part, to the use of DTT during purification. HDLP was
reconstituted with Zn.sup.2+ by mixing the Cys75Ser/Cys77Ser double
mutant at 10 mg/ml with a 5-fold molar excess of ZnCl.sub.2 in a
buffer of 25 mM bis-tris propane, 200 mM NaCl, 1% isopropanol, pH
7.0. Unbound ZnCl.sup.2 was removed by fractionating HDLP through a
G25 desalting column (Amersham-Pharmacia, Piscataway, N.J.). The
HDLP-Zn.sup.2+-TSA complex was, prepared by incubating the
Zn.sup.2+ reconstituted HDLP mutant with 1 mM TSA for 45 minutes,
followed by gel filtration chromatography (Superdex.TM.200;
Amersham-Pharmacia, Piscataway, N.J.) to remove excess TSA, and
concentration to typically 25 mg/ml in a buffer of 25 mM bis-tris
propane, 500 mM NaCl, 1% isopropanol, pH 7.0. FLAG epitope tagged
human HDAC1 was overexpressed using a baculovirus expression system
in Hi5 (Invitrogen, Carlsbad, Calif.) insect cells grown in
suspension in serum-free media (Sf900, Gibco, Grand Island, N.Y.).
The fusion protein was purified by anion exchange and affinity
chromatography using Anti-FLAG M2 affinity resin (Sigma, St. Louis,
Mo.) and FLAG Peptide (Sigma, St. Louis; MO).
Example 2
Crystallization and Data Collection
[0114] Crystals of apo-HDLP were grown at room temperature by the
hanging-drop vapor-diffusion method, from 7.5% isopropanol, 28% PEG
1500, 425 mM NaCl, 100 mM Tris-Cl, pH 7.0. They form in space group
C2 with a 51.4 .ANG., b=93.8 .ANG., c=78.7 .ANG., .beta.=96.9
.ANG., and contain one HDLP molecule in the asymmetric unit.
Diffraction data were collected with crystals flash-frozen in a
buffer of 7.5% isopropanol, 35% PEG 1500, 75 mM NaCl, 100 mM
Tris-Cl, pH 8.0, at -170.degree. C.
[0115] The structure of the HDLP-Zn.sup.2+ complex was determined
from HDLP Cys75Ser/Cys77Ser double mutant crystals grown from 23%
tert-butanol, 27% PEG 1500, 400 mM KCl, 100 mM bis-tris propane-Cl,
pH 6.8. Space group and cell dimensions were identical to the
apocrystals. The HDLP-Zn.sup.2+ crystals were harvested and frozen
in 27% tert-butanol, 22% PEG 1500, 50 mM KCl, 20 mM NaCl, 0.2 mM
ZnCl.sub.2, 100 mM bis-tris propane, pH 6.8, at -170.degree. C.
[0116] Crystals of the HDLP-Zn.sup.2+-TSA complex comprised HDLP
Cys75Ser/Cys77Ser double mutant and were grown from 23%
tert-butanol, 27% PEG 1500, 600 mM KCl, 100 mM bis-tris propane-Cl,
pH 6.8, by microseeding. The crystals were grown in the presence of
zinc. They form in space group P2.sub.12.sub.12.sub.1 with a=53.4
.ANG., b=94.4 .ANG., c=156.3 .ANG. and contain two
HDLP-Zn.sup.2+-TSA complexes in the asymmetric unit. The
HDLP-Zn.sup.2+-TSA crystals were harvested and frozen in the same
cryobuffer as the HDLP-Zn.sup.2+ crystals except that 0.5 mM TSA
was added. Data were processed with DENZO and SCALEPACK (Otwinowski
& Minor, 1997, Method. Ensemble. 276:307-326). MIR analysis,
model building and refinement.
[0117] The HDLP-Zn.sup.2+-SAHA complex crystals were grown and
evaluated the same as the HDLP-Zn.sup.2+-TSA crystals. However, the
restraints for the SAHA structure were constructed based on
stereochemical parameters from TSA. Like the apo-HDLP crystals, the
SAHA/HDLP co-crystals grew in space group C2.
[0118] Heavy-atom soaks were performed with the apo-HDLP crystals
in a buffer of 7.5% isopropanol, 30% PEG 1500, 75 mM NaCl, 100 mM
Tris-Cl, pH 8.0, supplemented with 1.0 mM thimerosal for 2 h, 5 mM
KAu(CN).sub.2 for 1 h, and 1 mM Pb(Me).sub.3OAc for 2 h. MIR phases
were calculated with the program MLPHARE (The CCP4 suite: Programs
for computational crystallography, 1994, Acta Crystallogr. D
50:760-763) at 2.5 .ANG. using the anomalous diffraction signal
from the thimerosal derivative, and had a mean figure of merit of
0.55. The phases were improved by solvent flattening with the
program DM (The CCP4 suite: Programs for computational
crystallography, 1994, Acta Crystallogr. D 50:760-763), and were
used to build the initial model with the program 0 (Jones et al.,
1991, Acta Crystallogr. A 47:110-109) Successive rounds of
rebuilding and simulated annealing refinement with the program CNS
(Brunger et al., 1998, Acta Crystallogr. D 54:905-921) allowed
interpretation of HDLP from residues 2 to 373. Residues 1, 374, and
375 were not modeled and are presumed to be disordered.
[0119] The structure of the HDLP-Zn.sup.2+-TSA and
HDLP-Zn.sup.2+-SAHA complex were determined by molecular
replacement with the program AMORE (The CCP4 suite: Programs for
computational crystallography, 1994, Acta Crystallogr. D
50:760-7.63) using the apo-HDLP structure as a search model. The
initial electron density maps had strong and continuous difference
density for the entire TSA molecule. However the SAHA molecule was
not as well ordered in the cap group region. The structure of TSA
was obtained from the Cambridge Structural Database (Refcode
TRCHST) and was used to define stereochemical restraints used in
the refinement with the program CNS. The restraints of SAHA were
constructed based on stereochemical parameters from TSA and
surrounding amino acid residues. The dimer interface in the
HDLP-Zn.sup.2+-TSA and HDLP-Zn.sup.2+-SARA crystals primarily
involves Phe200 on the protein surface. The Phe200 side chain
contacts Tyr91, whose side chain conformation changes on TSA
binding, and part of the dimethyl amino phenyl group of TSA from
the second protomer. The HDAC family does not contain a
phenylalanine residue at the equivalent position.
Example 3
Histone Deacetylase Assays
[0120] Purified proteins were assayed by incubating 10 .mu.g of
[.sup.3H]acetyl-labeled murine erythroleukemia histone substrate
and HDAC assay buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 10%
glycerol) for 30-60 minutes at 37.degree. C. in a total volume of
30 .mu.l. The final concentrations of HDLP and HDAC1-FLAG were 3.6
.mu.M and 0.24 .mu.M, respectively. Assays were performed in
duplicate. The reactions were stopped and the released acetate was
extracted and assayed as described (Hendzel et al., 1991, J. Biol.
Chem. 266:21936-21942). [3H]acetyl-labeled murine erythroleukemia
histones were prepared essentially as described (Carmen et al.,
1996, J. Biol. Chem. 271:15837-15844). Inhibitors were added in the
absence of substrate and incubated on ice for 20 minutes, substrate
was added, and the assay performed as described above. HDLP was
inclubated with 20 .mu.M ZnCl.sub.2 and 20 .mu.M MnCl.sub.2
(H2O).sub.4 in HDAC buffer and tested for activity.
[0121] Only HDLP dialyzed against ZnCl.sub.2 had activity.
HDAC1-FLAG was dialyzed against 20 .mu.M ZnCl.sub.2 in HDAC buffer
which had no effect on activity. Therefore, HDAC1-FLAG contains a
metal as purified.
[0122] The in vivo substrate of HDLP is not known. HDLP may have a
role in acetoin utilization like the B. subtilis AcuC gene product,
and it has been annotated as such in the genome sequence, but the
reaction catalyzed by AcuC is also not known. Furthermore, the A.
aeolicus genome appears to lack the acuA and acuB genes that are
part of the acuABC operon of B. subtilis (Deckert et al., 1998
Nature 392:353-358), and HDLP is as similar to human HDAC1 (35.2%
identity) as it is to B. subtilis AcuC (34.7% identity).
[0123] Throughout the application, various publications are
referenced by author, date and citation. The disclosures of these
publication in their entireties are hereby incorporated by
reference.
[0124] While this invention has been particularly shown and
described with references to preferred embodiments thereof, it will
be understood by those skilled in the art that various changes in
form and details may be made therein without departing from the
scope of the invention encompassed by the appended claims.
Sequence CWU 1
1
18 1 375 PRT Aquifex aeolicus 1 Met Lys Lys Val Lys Leu Ile Gly Thr
Leu Asp Tyr Gly Lys Tyr Arg 1 5 10 15 Tyr Pro Lys Asn His Pro Leu
Lys Ile Pro Arg Val Ser Leu Leu Leu 20 25 30 Arg Phe Leu Asp Ala
Met Asn Leu Ile Asp Glu Lys Glu Leu Ile Lys 35 40 45 Ser Arg Pro
Ala Thr Lys Glu Glu Leu Leu Leu Phe His Thr Glu Asp 50 55 60 Tyr
Ile Asn Thr Leu Met Glu Ala Glu Arg Cys Gln Cys Val Pro Lys 65 70
75 80 Gly Ala Arg Glu Lys Tyr Asn Ile Gly Gly Tyr Glu Asn Pro Val
Ser 85 90 95 Tyr Ala Met Phe Thr Gly Ser Ser Leu Ala Thr Gly Ser
Thr Val Gln 100 105 110 Ala Ile Glu Glu Phe Leu Lys Gly Asn Val Ala
Phe Asn Pro Ala Gly 115 120 125 Gly Met His His Ala Phe Lys Ser Arg
Ala Asn Gly Phe Cys Tyr Ile 130 135 140 Asn Asp Pro Ala Val Gly Ile
Glu Tyr Leu Arg Lys Lys Gly Phe Lys 145 150 155 160 Arg Ile Leu Tyr
Ile Asp Leu Asp Ala His His Cys Asp Gly Val Gln 165 170 175 Glu Ala
Phe Tyr Asp Thr Asp Gln Val Phe Val Leu Ser Leu His Gln 180 185 190
Ser Pro Glu Tyr Ala Phe Pro Phe Glu Lys Gly Phe Leu Glu Glu Ile 195
200 205 Gly Glu Gly Lys Gly Lys Gly Tyr Asn Leu Asn Ile Pro Leu Pro
Lys 210 215 220 Gly Leu Asn Asp Asn Glu Phe Leu Phe Ala Leu Glu Lys
Ser Leu Glu 225 230 235 240 Ile Val Lys Glu Val Phe Glu Pro Glu Val
Tyr Leu Leu Gln Leu Gly 245 250 255 Thr Asp Pro Leu Leu Glu Asp Tyr
Leu Ser Lys Phe Asn Leu Ser Asn 260 265 270 Val Ala Phe Leu Lys Ala
Phe Asn Ile Val Arg Glu Val Phe Gly Glu 275 280 285 Gly Val Tyr Leu
Gly Gly Gly Gly Tyr His Pro Tyr Ala Leu Ala Arg 290 295 300 Ala Trp
Thr Leu Ile Trp Cys Glu Leu Ser Gly Arg Glu Val Pro Glu 305 310 315
320 Lys Leu Asn Asn Lys Ala Lys Glu Leu Leu Lys Ser Ile Asp Phe Glu
325 330 335 Glu Phe Asp Asp Glu Val Asp Arg Ser Tyr Met Leu Glu Thr
Leu Lys 340 345 350 Asp Pro Trp Arg Gly Gly Glu Val Arg Lys Glu Val
Lys Asp Thr Leu 355 360 365 Glu Lys Ala Lys Ala Ser Ser 370 375 2
1127 DNA Aquifex aeolicus 2 atgaagaagg ttaaacttat cggaacttta
gactacggaa agtacagata tcccaaaaac 60 catcctctta aaataccaag
agtttcccta ctccttaggt ttttagatgc catgaacctt 120 atagatgaga
aggaattaat caagagcaga cccgcaacta aagaagaact ccttttattc 180
cacacggaag actacataaa cactttaatg gaagcggaaa ggtgtcagtg cgttccgaag
240 ggagctaggg aaaagtacaa cataggcgga tacgaaaacc ccgtatctta
cgcgatgttt 300 acaggctctt ctctcgcaac gggttcaaca gtgcaggcga
tagaggaatt tttaaaggga 360 aatgtagctt tcaatcccgc gggaggtatg
caccacgctt ttaaaagcag ggcaaacggc 420 ttttgctaca taaacgaccc
cgctgtggga attgagtact tgagaaaaaa aggctttaag 480 agaatactct
acatagacct tgatgcccac cactgcgacg gtgttcagga agccttttac 540
gatacagacc aggtgttcgt cctgtccctt caccagtcgc ccgagtacgc ctttcccttt
600 gagaagggct tcctggagga gataggagaa ggaaaaggaa agggctacaa
cctgaacatt 660 cccctgccaa agggcttgaa cgacaacgag ttcctctttg
ccctagaaaa atctctggaa 720 atagtcaaag aagtatttga gcccgaggtt
taccttcttc aactcggaac tgacccactc 780 cttgaagatt acctttccaa
gttcaacctc tcaaacgttg cctttttaaa agctttcaac 840 atcgttcgtg
aggttttcgg ggagggagta tacctcggag gaggcggata ccatccttac 900
gccctcgcaa gggcatggac cctaatctgg tgcgagcttt cgggaaggga agtgccggaa
960 aagctaaaca ataaagcaaa agagctttta aagagtatag actttgaaga
gtttgacgac 1020 gaggtggacc gctcgtacat gctcgaaacc ctaaaggacc
cctggagagg aggagaggta 1080 aggaaagaag taaaggatac gcttgaaaag
gcgaaagcct catctta 1127 3 375 PRT Aquifex aeolicus 3 Met Lys Lys
Val Lys Leu Ile Gly Thr Leu Asp Tyr Gly Lys Tyr Arg 1 5 10 15 Tyr
Pro Lys Asn His Pro Leu Lys Ile Pro Arg Val Ser Leu Leu Leu 20 25
30 Arg Phe Leu Asp Ala Met Asn Leu Ile Asp Glu Lys Glu Leu Ile Lys
35 40 45 Ser Arg Pro Ala Thr Lys Glu Glu Leu Leu Leu Phe His Thr
Glu Asp 50 55 60 Tyr Ile Asn Thr Leu Met Glu Ala Glu Arg Ser Gln
Ser Val Pro Lys 65 70 75 80 Gly Ala Arg Glu Lys Tyr Asn Ile Gly Gly
Tyr Glu Asn Pro Val Ser 85 90 95 Tyr Ala Met Phe Thr Gly Ser Ser
Leu Ala Thr Gly Ser Thr Val Gln 100 105 110 Ala Ile Glu Glu Phe Leu
Lys Gly Asn Val Ala Phe Asn Pro Ala Gly 115 120 125 Gly Met His His
Ala Phe Lys Ser Arg Ala Asn Gly Phe Cys Tyr Ile 130 135 140 Asn Asp
Pro Ala Val Gly Ile Glu Tyr Leu Arg Lys Lys Gly Phe Lys 145 150 155
160 Arg Ile Leu Tyr Ile Asp Leu Asp Ala His His Cys Asp Gly Val Gln
165 170 175 Glu Ala Phe Tyr Asp Thr Asp Gln Val Phe Val Leu Ser Leu
His Gln 180 185 190 Ser Pro Glu Tyr Ala Phe Pro Phe Glu Lys Gly Phe
Leu Glu Glu Ile 195 200 205 Gly Glu Gly Lys Gly Lys Gly Tyr Asn Leu
Asn Ile Pro Leu Pro Lys 210 215 220 Gly Leu Asn Asp Asn Glu Phe Leu
Phe Ala Leu Glu Lys Ser Leu Glu 225 230 235 240 Ile Val Lys Glu Val
Phe Glu Pro Glu Val Tyr Leu Leu Gln Leu Gly 245 250 255 Thr Asp Pro
Leu Leu Glu Asp Tyr Leu Ser Lys Phe Asn Leu Ser Asn 260 265 270 Val
Ala Phe Leu Lys Ala Phe Asn Ile Val Arg Glu Val Phe Gly Glu 275 280
285 Gly Val Tyr Leu Gly Gly Gly Gly Tyr His Pro Tyr Ala Leu Ala Arg
290 295 300 Ala Trp Thr Leu Ile Trp Cys Glu Leu Ser Gly Arg Glu Val
Pro Glu 305 310 315 320 Lys Leu Asn Asn Lys Ala Lys Glu Leu Leu Lys
Ser Ile Asp Phe Glu 325 330 335 Glu Phe Asp Asp Glu Val Asp Arg Ser
Tyr Met Leu Glu Thr Leu Lys 340 345 350 Asp Pro Trp Arg Gly Gly Glu
Val Arg Lys Glu Val Lys Asp Thr Leu 355 360 365 Glu Lys Ala Lys Ala
Ser Ser 370 375 4 1127 DNA Aquifex aeolicus 4 atgaagaagg ttaaacttat
cggaacttta gactacggaa agtacagata tcccaaaaac 60 catcctctta
aaataccaag agtttcccta ctccttaggt ttttagatgc catgaacctt 120
atagatgaga aggaattaat caagagcaga cccgcaacta aagaagaact ccttttattc
180 cacacggaag actacataaa cactttaatg gaagcggaaa ggagtcagag
cgttccgaag 240 ggagctaggg aaaagtacaa cataggcgga tacgaaaacc
ccgtatctta cgcgatgttt 300 acaggctctt ctctcgcaac gggttcaaca
gtgcaggcga tagaggaatt tttaaaggga 360 aatgtagctt tcaatcccgc
gggaggtatg caccacgctt ttaaaagcag ggcaaacggc 420 ttttgctaca
taaacgaccc cgctgtggga attgagtact tgagaaaaaa aggctttaag 480
agaatactct acatagacct tgatgcccac cactgcgacg gtgttcagga agccttttac
540 gatacagacc aggtgttcgt cctgtccctt caccagtcgc ccgagtacgc
ctttcccttt 600 gagaagggct tcctggagga gataggagaa ggaaaaggaa
agggctacaa cctgaacatt 660 cccctgccaa agggcttgaa cgacaacgag
ttcctctttg ccctagaaaa atctctggaa 720 atagtcaaag aagtatttga
gcccgaggtt taccttcttc aactcggaac tgacccactc 780 cttgaagatt
acctttccaa gttcaacctc tcaaacgttg cctttttaaa agctttcaac 840
atcgttcgtg aggttttcgg ggagggagta tacctcggag gaggcggata ccatccttac
900 gccctcgcaa gggcatggac cctaatctgg tgcgagcttt cgggaaggga
agtgccggaa 960 aagctaaaca ataaagcaaa agagctttta aagagtatag
actttgaaga gtttgacgac 1020 gaggtggacc gctcgtacat gctcgaaacc
ctaaaggacc cctggagagg aggagaggta 1080 aggaaagaag taaaggatac
gcttgaaaag gcgaaagcct catctta 1127 5 375 PRT Aquifex aeolicus 5 Met
Lys Lys Val Lys Leu Ile Gly Thr Leu Asp Tyr Gly Lys Tyr Arg 1 5 10
15 Tyr Pro Lys Asn His Pro Leu Lys Ile Pro Arg Val Ser Leu Leu Leu
20 25 30 Arg Phe Leu Asp Ala Met Asn Leu Ile Asp Glu Lys Glu Leu
Ile Lys 35 40 45 Ser Arg Pro Ala Thr Lys Glu Glu Leu Leu Leu Phe
His Thr Glu Asp 50 55 60 Tyr Ile Asn Thr Leu Met Glu Ala Glu Arg
Cys Gln Cys Val Pro Lys 65 70 75 80 Gly Ala Arg Glu Lys Tyr Asn Ile
Gly Gly Tyr Glu Asn Pro Val Ser 85 90 95 Tyr Ala Met Phe Thr Gly
Ser Ser Leu Ala Thr Gly Ser Thr Val Gln 100 105 110 Ala Ile Glu Glu
Phe Leu Lys Gly Asn Val Ala Phe Asn Pro Ala Gly 115 120 125 Gly Met
His His Ala Phe Lys Ser Arg Ala Asn Gly Phe Cys Tyr Ile 130 135 140
Asn Asp Pro Ala Val Gly Ile Glu Tyr Leu Arg Lys Lys Gly Phe Lys 145
150 155 160 Arg Ile Leu Tyr Ile Asp Leu Asp Ala His His Cys Asp Gly
Val Gln 165 170 175 Glu Ala Phe Tyr Asp Thr Asp Gln Val Phe Val Leu
Ser Leu His Gln 180 185 190 Ser Pro Glu Tyr Ala Phe Pro Phe Glu Lys
Gly Phe Leu Glu Glu Ile 195 200 205 Gly Glu Gly Lys Gly Lys Gly Tyr
Asn Leu Asn Ile Pro Leu Pro Lys 210 215 220 Gly Leu Asn Asp Asn Glu
Phe Leu Phe Ala Leu Glu Lys Ser Leu Glu 225 230 235 240 Ile Val Lys
Glu Val Phe Glu Pro Glu Val Tyr Leu Leu Gln Leu Gly 245 250 255 Thr
Asp Pro Leu Leu Glu Asp Tyr Leu Ser Lys Phe Asn Leu Ser Asn 260 265
270 Val Ala Phe Leu Lys Ala Phe Asn Ile Val Arg Glu Val Phe Gly Glu
275 280 285 Gly Val Tyr Leu Gly Gly Gly Gly Phe His Pro Tyr Ala Leu
Ala Arg 290 295 300 Ala Trp Thr Leu Ile Trp Cys Glu Leu Ser Gly Arg
Glu Val Pro Glu 305 310 315 320 Lys Leu Asn Asn Lys Ala Lys Glu Leu
Leu Lys Ser Ile Asp Phe Glu 325 330 335 Glu Phe Asp Asp Glu Val Asp
Arg Ser Tyr Met Leu Glu Thr Leu Lys 340 345 350 Asp Pro Trp Arg Gly
Gly Glu Val Arg Lys Glu Val Lys Asp Thr Leu 355 360 365 Glu Lys Ala
Lys Ala Ser Ser 370 375 6 1127 DNA Aquifex aeolicus 6 atgaagaagg
ttaaacttat cggaacttta gactacggaa agtacagata tcccaaaaac 60
catcctctta aaataccaag agtttcccta ctccttaggt ttttagatgc catgaacctt
120 atagatgaga aggaattaat caagagcaga cccgcaacta aagaagaact
ccttttattc 180 cacacggaag actacataaa cactttaatg gaagcggaaa
ggtgtcagtg cgttccgaag 240 ggagctaggg aaaagtacaa cataggcgga
tacgaaaacc ccgtatctta cgcgatgttt 300 acaggctctt ctctcgcaac
gggttcaaca gtgcaggcga tagaggaatt tttaaaggga 360 aatgtagctt
tcaatcccgc gggaggtatg caccacgctt ttaaaagcag ggcaaacggc 420
ttttgctaca taaacgaccc cgctgtggga attgagtact tgagaaaaaa aggctttaag
480 agaatactct acatagacct tgatgcccac cactgcgacg gtgttcagga
agccttttac 540 gatacagacc aggtgttcgt cctgtccctt caccagtcgc
ccgagtacgc ctttcccttt 600 gagaagggct tcctggagga gataggagaa
ggaaaaggaa agggctacaa cctgaacatt 660 cccctgccaa agggcttgaa
cgacaacgag ttcctctttg ccctagaaaa atctctggaa 720 atagtcaaag
aagtatttga gcccgaggtt taccttcttc aactcggaac tgacccactc 780
cttgaagatt acctttccaa gttcaacctc tcaaacgttg cctttttaaa agctttcaac
840 atcgttcgtg aggttttcgg ggagggagta tacctcggag gaggcggatt
ccatccttac 900 gccctcgcaa gggcatggac cctaatctgg tgcgagcttt
cgggaaggga agtgccggaa 960 aagctaaaca ataaagcaaa agagctttta
aagagtatag actttgaaga gtttgacgac 1020 gaggtggacc gctcgtacat
gctcgaaacc ctaaaggacc cctggagagg aggagaggta 1080 aggaaagaag
taaaggatac gcttgaaaag gcgaaagcct catctta 1127 7 375 PRT Aquifex
aeolicus 7 Met Lys Lys Val Lys Leu Ile Gly Thr Leu Asp Tyr Gly Lys
Tyr Arg 1 5 10 15 Tyr Pro Lys Asn His Pro Leu Lys Ile Pro Arg Val
Ser Leu Leu Leu 20 25 30 Arg Phe Leu Asp Ala Met Asn Leu Ile Asp
Glu Lys Glu Leu Ile Lys 35 40 45 Ser Arg Pro Ala Thr Lys Glu Glu
Leu Leu Leu Phe His Thr Glu Asp 50 55 60 Tyr Ile Asn Thr Leu Met
Glu Ala Glu Arg Cys Gln Cys Val Pro Lys 65 70 75 80 Gly Ala Arg Glu
Lys Tyr Asn Ile Gly Gly Tyr Glu Asn Pro Val Ser 85 90 95 Tyr Ala
Met Phe Thr Gly Ser Ser Leu Ala Thr Gly Ser Thr Val Gln 100 105 110
Ala Ile Glu Glu Phe Leu Lys Gly Asn Val Ala Phe Asn Pro Ala Gly 115
120 125 Gly Met His His Ala Phe Lys Ser Arg Ala Asn Gly Phe Cys Tyr
Ile 130 135 140 Asn Asp Pro Ala Val Gly Ile Glu Tyr Leu Arg Lys Lys
Gly Phe Lys 145 150 155 160 Arg Ile Leu Tyr Ile Asp Leu Asp Ala His
His Cys Asp Gly Val Gln 165 170 175 Glu Ala Phe Tyr Asp Thr Asp Gln
Val Phe Val Leu Ser Leu His Gln 180 185 190 Ser Pro Glu Tyr Ala Phe
Pro Phe Glu Lys Gly Phe Leu Glu Glu Ile 195 200 205 Gly Glu Gly Lys
Gly Lys Gly Tyr Asn Leu Asn Ile Pro Leu Pro Lys 210 215 220 Gly Leu
Asn Asp Asn Glu Phe Leu Phe Ala Leu Glu Lys Ser Leu Glu 225 230 235
240 Ile Val Lys Glu Val Phe Glu Pro Glu Val Tyr Leu Leu Gln Leu Gly
245 250 255 Thr Asp Pro Leu Leu Glu Asp Tyr Leu Ser Lys Phe Asn Leu
Ser Asn 260 265 270 Val Ala Phe Leu Lys Ala Phe Asn Ile Val Arg Glu
Val Phe Gly Glu 275 280 285 Gly Val Tyr Leu Gly Gly Gly Gly Tyr His
Pro Tyr Ala Leu Ala Arg 290 295 300 Ala Asn Thr Leu Ile Trp Cys Glu
Leu Ser Gly Arg Glu Val Pro Glu 305 310 315 320 Lys Leu Asn Asn Lys
Ala Lys Glu Leu Leu Lys Ser Ile Asp Phe Glu 325 330 335 Glu Phe Asp
Asp Glu Val Asp Arg Ser Tyr Met Leu Glu Thr Leu Lys 340 345 350 Asp
Pro Trp Arg Gly Gly Glu Val Arg Lys Glu Val Lys Asp Thr Leu 355 360
365 Glu Lys Ala Lys Ala Ser Ser 370 375 8 370 PRT Homo sapiens 8
Met Arg Lys Val Cys Tyr Tyr Tyr Asp Gly Asp Val Gly Asn Tyr Tyr 1 5
10 15 Tyr Gly Gln Gly His Pro Met Lys Pro His Arg Ile Arg Met Thr
His 20 25 30 Asn Leu Leu Leu Asn Tyr Gly Leu Tyr Arg Lys Met Glu
Ile Tyr Arg 35 40 45 Pro His Lys Ala Asn Ala Glu Glu Met Thr Lys
Tyr His Ser Asp Asp 50 55 60 Tyr Ile Lys Phe Leu Arg Ser Ile Arg
Pro Asp Asn His Ser Glu Ser 65 70 75 80 Lys Gln Met Gln Arg Phe Asn
Val Gly Glu Asp Cys Pro Val Phe Asp 85 90 95 Gly Leu Phe Glu Phe
Cys Gln Leu Ser Thr Gly Gly Ser Val Ala Ser 100 105 110 Ala Val Lys
Leu Asn Lys Gln Asp Ile Ala Val Asn Trp Ala Gly Gly 115 120 125 Leu
His His Ala Lys Lys Ser Glu Ala Ser Gly Phe Cys Tyr Val Asn 130 135
140 Asp Ile Val Leu Ala Ile Leu Glu Leu Leu Lys Tyr His Gln Arg Val
145 150 155 160 Leu Tyr Ile Asp Ile Asp Ile His His Gly Asp Gly Val
Glu Glu Ala 165 170 175 Phe Tyr Thr Thr Asp Arg Val Met Thr Val Ser
Phe His Lys Tyr Gly 180 185 190 Glu Tyr Phe Pro Gly Thr Gly Asp Leu
Arg Asp Ile Gly Ala Gly Lys 195 200 205 Gly Lys Tyr Tyr Ala Val His
Tyr Pro Leu Arg Asp Gly Ile Asp Asp 210 215 220 Glu Ser Tyr Glu Ala
Ile Phe Lys Pro Val Met Ser Lys Val Met Glu 225 230 235 240 Met Phe
Gln Pro Ser Ala Val Val Leu Gln Cys Gly Ser Asp Ser Leu 245 250 255
Ser Gly Asp Arg Leu Gly Cys Phe His Leu Thr Ile Lys Gly His Ala 260
265 270 Lys Cys Val Glu Phe Val Lys Ser Phe Asn Leu Pro Met Leu Met
Leu 275 280 285 Gly Gly Gly Gly Tyr Thr Ile Arg His Val Ala Arg Cys
Met Thr Tyr 290 295 300 Glu Thr Ala Val Ala Leu Asp Thr Glu Ile Pro
Asn Glu Leu Pro Asn 305 310 315 320 Asp Tyr Phe Glu Tyr Phe Gly Pro
Asp Phe Ser Asn His Thr Asn Gln 325 330 335 Asn Thr Asn Glu Tyr Leu
Glu Glu Asn Leu Arg Met Leu Pro His Ala 340 345 350 Pro Gly Val Gln
Met Gln Ile Pro Glu Asp Ala Ile Pro Glu Glu Ser 355
360 365 Gly Asp 370 9 363 PRT Homo sapiens 9 Leu Ala Gly Thr Gly
Leu Val Leu Asp Glu Gln Leu Asn Glu Phe His 1 5 10 15 Phe Pro Glu
Gly Pro Phe Arg Leu His Ala Ile Lys Glu Gln Leu Ile 20 25 30 Gln
Glu Gly Leu Leu Asp Val Ser Phe Gln Ala Arg Glu Ala Glu Lys 35 40
45 Glu Glu Leu Met Leu Val His Ser Leu Glu Tyr Ile Asp Leu Met Glu
50 55 60 Thr Thr Gln Tyr Met Asn Glu Gly Glu Arg Val Leu Ala Asp
Thr Tyr 65 70 75 80 Asp Ser Val Tyr Leu His Pro Asn Ser Tyr Ser Cys
Ala Cys Leu Ala 85 90 95 Ser Gly Ser Val Leu Arg Leu Val Asp Ala
Val Leu Gly Ala Glu Ala 100 105 110 Ile Ile Arg Pro Pro Gly His His
Ala Gln His Ser Leu Met Asp Gly 115 120 125 Tyr Cys Met Phe Ser His
Val Ala Val Ala Ala Arg Tyr Ala Gln Gln 130 135 140 Lys His Ile Arg
Arg Val Leu Ile Val Asp Trp Asp Val His His Gly 145 150 155 160 Gln
Gly Thr Gln Phe Thr Phe Asp Gln Asp Pro Ser Val Leu Tyr Phe 165 170
175 Ser Ile His Arg Tyr Glu Gln Gly Arg Phe Pro His Leu Lys Ala Ser
180 185 190 Trp Ser Thr Thr Gly Phe Gly Gln Gly Gln Gly Tyr Thr Ile
Asn Val 195 200 205 Pro Trp Asn Gln Gly His Arg Asp Ala Asp Tyr Ile
Ala Ala Phe Cys 210 215 220 His Val Leu Leu Pro Val Ala Leu Glu Phe
Gln Pro Gln Leu Val Leu 225 230 235 240 Val Ala Ala Gly Phe Asp Ala
Leu Gln Gly Asp Pro Lys Gly Glu Met 245 250 255 Ala Ala Thr Pro Ala
Gly Phe Ala Gln Leu Thr His Leu Leu Met Gly 260 265 270 Leu Ala Gly
Gly Lys Leu Ile Leu Ser Leu Gly Gly Tyr Asn Leu Arg 275 280 285 Ala
Leu Ala Glu Gly Val Ser Ala Ser Leu His Thr Leu Leu Gly Asp 290 295
300 Pro Cys Pro Met Leu Glu Ser Gly Ala Pro Cys Arg Ser Ala Gln Ala
305 310 315 320 Ser Val Ser Glu Pro Phe Trp Glu Val Leu Val Arg Ser
Thr Glu Thr 325 330 335 Glu Asp Asn Val Glu Pro Pro Val Leu Pro Ile
Leu Thr Trp Pro Leu 340 345 350 Gln Ser Arg Thr Gly Leu Val Tyr Asp
Gln Asn 355 360 10 7 PRT Homo sapiens 10 Ala Gln Thr Gln Gly Thr
Arg 1 5 11 7 PRT Homo sapiens 11 Asn Leu Glu Ala Glu Ala Leu 1 5 12
6 PRT Homo sapiens 12 Cys Leu Trp Asp Asp Ser 1 5 13 5 PRT Homo
sapiens 13 Ile Arg Asn Gly Met 1 5 14 6 PRT Homo sapiens 14 Lys Leu
His Ile Ser Pro 1 5 15 6 PRT Homo sapiens 15 Cys Ala Leu Glu Ala
Leu 1 5 16 7 PRT Homo sapiens 16 Lys Ile Lys Gln Arg Leu Phe 1 5 17
7 PRT Homo sapiens 17 Val Glu Arg Asp Asn Met Glu 1 5 18 9 PRT Homo
sapiens 18 Glu Ser Glu Glu Glu Gly Pro Trp Glu 1 5
* * * * *
References