U.S. patent application number 10/754929 was filed with the patent office on 2004-09-02 for crystallization and structure determination of staphylococcus aureus thymidylate kinase.
This patent application is currently assigned to PHARMACIA & UPJOHN COMPANY. Invention is credited to Benson, Timothy E..
Application Number | 20040171050 10/754929 |
Document ID | / |
Family ID | 30772478 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040171050 |
Kind Code |
A1 |
Benson, Timothy E. |
September 2, 2004 |
Crystallization and structure determination of staphylococcus
aureus thymidylate kinase
Abstract
An unliganded form of Staphylococcus aureus thymidylate kinase
(S. aureus TMK) has been crystallized, and the three dimensional
x-ray crystal structure has been solved to 2.3 .ANG. resolution.
The x-ray crystal structure is useful for solving the structure of
other molecules or molecular complexes, and designing inhibitors of
S. aureus TMK activity.
Inventors: |
Benson, Timothy E.;
(Chesterfield, MO) |
Correspondence
Address: |
MUETING, RAASCH & GEBHARDT, P.A.
P.O. BOX 581415
MINNEAPOLIS
MN
55458
US
|
Assignee: |
PHARMACIA & UPJOHN
COMPANY
301 Henrietta Street
Kalamazoo
MI
49001
|
Family ID: |
30772478 |
Appl. No.: |
10/754929 |
Filed: |
January 8, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10754929 |
Jan 8, 2004 |
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|
09632553 |
Aug 4, 2000 |
|
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6689595 |
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60147117 |
Aug 4, 1999 |
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Current U.S.
Class: |
435/5 ; 435/194;
435/252.3; 435/320.1; 435/6.19; 435/69.1; 702/19 |
Current CPC
Class: |
C12N 9/1229 20130101;
Y10S 530/825 20130101; Y10S 530/82 20130101; C07K 2299/00
20130101 |
Class at
Publication: |
435/006 ;
435/069.1; 435/194; 435/252.3; 435/320.1; 702/019 |
International
Class: |
C12Q 001/68; G06F
019/00; G01N 033/48; G01N 033/50; C12N 009/12 |
Claims
What is claimed is:
1. A molecule or molecular complex comprising at least a portion of
an S. aureus thymidylate kinase or thymidylate kinase-like TMP
binding pocket, wherein the TMP binding pocket comprises the amino
acids listed in Table 1, the TMP binding pocket being defined by a
set of points having a root mean square deviation of less than
about 2.1 .ANG. from points representing the backbone atoms of said
amino acids as represented by the structure coordinates listed in
FIG. 2.
2. The molecule or molecular complex of claim 1, wherein the TMP
binding pocket comprises the amino acids listed in Table 2.
3. The molecule or molecular complex of claim 1, wherein the TMP
binding pocket comprises the amino acids listed in Table 3.
4. A molecule or molecular complex comprising at least a portion of
an S. aureus thymidylate kinase TMP/ATP substrate binding pocket,
wherein the TMP substrate binding pocket comprises the amino acids
listed in Table 4, the substrate binding pocket being defined by a
set of points having a root mean square deviation of less than
about 2.1 .ANG. from points representing the backbone atoms of said
amino acids as represented by the structure coordinates listed in
FIG. 2.
5. The molecule or molecular complex of claim 4, wherein the
TMP/ATP binding pocket comprises the amino acids listed in Table
5.
6. The molecule or molecular complex of claim 4, wherein the
TMP/ATP binding pocket comprises the amino acids listed in Table
6.
7. A molecule or molecular complex that is structurally homologous
to an S. aureus thymidylate kinase molecule or molecular complex,
wherein the S. aureus thymidylate kinase molecule or molecular
complex is represented by at least a portion of the structure
coordinates listed in FIG. 2.
8. A scalable three dimensional configuration of points, at least a
portion of said points derived from structure coordinates of at
least a portion of an S. aureus thymidylate kinase molecule or
molecular complex listed in FIG. 2 comprising at least one of an S.
aureus thymidylate kinase or thymidylate kinase-like TMP or TMP/ATP
binding pocket.
9. The scalable three dimensional configuration of points of claim
8, wherein substantially all of said points are derived from
structure coordinates of an S. aureus thymidylate kinase molecule
or molecular complex listed in FIG. 2.
10. The scalable three dimensional configuration of points of claim
8 wherein at least a portion of the points derived from the S.
aureus thymidylate kinase structure coordinates are derived from
structure coordinates representing the locations of at least the
backbone atoms of amino acids defining an S. aureus thymidylate
kinase TMP binding pocket, the TMP binding pocket comprising the
amino acids listed in Table 1.
11. The scalable three dimensional configuration of points of claim
10, wherein the TMP binding pocket comprises the amino acids listed
in Table 2.
12. The scalable three dimensional configuration of points of claim
10, wherein the TMP binding pocket comprises the amino acids listed
in Table 3.
13. The scalable three dimensional configuration of points of claim
8 wherein at least a portion of the points derived from the S.
aureus thymidylate kinase structure coordinates are derived from
structure coordinates representing the locations of at least the
backbone atoms of amino acids defining an S. aureus thymidylate
kinase TMP/ATP binding pocket, the TMP/ATP substrate binding pocket
comprising the amino acids listed in Table 4.
14. The scalable three dimensional configuration of points of claim
13, wherein the TMP/ATP binding pocket comprises the amino acids
listed in Table 5.
15. The scalable three dimensional configuration of points of claim
13, wherein the TMP/ATP binding pocket comprises the amino acids
listed in Table 6.
16. The scalable three dimensional configuration of points of claim
8 displayed as a holographic image, a stereodiagram, a model or a
computer-displayed image.
17. A scalable three dimensional configuration of points, at least
a portion of the points derived from structure coordinates of at
least a portion of a molecule or a molecular complex that is
structurally homologous to an S. aureus thymidylate kinase molecule
or molecular complex and comprises at least one of an S. aureus
thymidylate kinase or thymidylate kinase-like TMP or TMP/ATP
binding pocket.
18. The scalable three-dimensional configuration of points of claim
17 displayed as a holographic image, a stereodiagram, a model or a
computer-displayed image
19. A machine-readable data storage medium comprising a data
storage material encoded with machine readable data which, when
using a machine programmed with instructions for using said data,
is capable of displaying a graphical three-dimensional
representation of at least one molecule or molecular complex
selected from the group consisting of: (i) a molecule or molecular
complex comprising at least a portion of an S. aureus thymidylate
kinase or thymidylate kinase-like TMP binding pocket comprising the
amino acids listed in Table 1, the TMP binding pocket defined by a
set of points having a root mean square deviation of less than
about 2.1 .ANG. from points representing the backbone atoms of said
amino acids as represented by structure coordinates listed in FIG.
2; (ii) a molecule or molecular complex comprising at least a
portion of an S. aureus thymidylate kinase or thymidylate
kinase-like TMP/ATP binding pocket comprising the amino acids
listed in Table 4, the TMP/ATP binding pocket defined by a set of
points having a root mean square deviation of less than about 2.1
.ANG. from points representing the backbone atoms of said amino
acids as represented by structure coordinates listed in FIG. 2; and
(iii) a molecule or molecular complex that is structurally
homologous to an S. aureus thymidylate kinase molecule or molecular
complex, wherein the S. aureus thymidylate kinase molecule or
molecular complex is represented by at least a portion of the
structure coordinates listed in FIG. 2.
20. A machine-readable data storage medium comprising a data
storage material encoded with a first set of machine readable data
which, when combined with a second set of machine readable data,
using a machine programmed with instructions for using said first
set of data and said second set of data, can determine at least a
portion of the structure coordinates corresponding to the second
set of machine readable data, wherein said first set of data
comprises a Fourier transform of at least a portion of the
structural coordinates for S. aureus thymidylate kinase listed in
FIG. 2; and said second set of data comprises an x-ray diffraction
pattern of a molecule or molecular complex of unknown
structure.
21. A method for obtaining structural information about a molecule
or a molecular complex of unknown structure comprising:
crystallizing the molecule or molecular complex; generating an
x-ray diffraction pattern from the crystallized molecule or
molecular complex; applying at least a portion of the structure
coordinates set forth FIG. 2 to the x-ray diffraction pattern to
generate a three-dimensional electron density map of at least a
portion of the molecule or molecular complex whose structure is
unknown.
22. A method for homology modeling an S. aureus thymidylate kinase
homolog comprising: aligning the amino acid sequence of an S.
aureus thymidylate kinase homolog with an amino acid sequence of S.
aureus thymidylate kinase and incorporating the sequence of the S.
aureus thymidylate kinase homolog into a model of S. aureus
thymidylate kinase derived from structure coordinates set forth in
FIG. 2 to yield a preliminary model of the S. aureus thymidylate
kinase homolog; subjecting the preliminary model to energy
minimization to yield an energy minimized model; remodeling regions
of the energy minimized model where stereochemistry restraints are
violated to yield a final model of the S. aureus thymidylate kinase
homolog.
23. A computer-assisted method for identifying an inhibitor of S.
aureus thymidylate kinase activity comprising: supplying a computer
modeling application with a set of structure coordinates of a
molecule or molecular complex, the molecule or molecular complex
comprising at least a portion of an S. aureus thymidylate kinase or
thymidylate kinase-like TMP binding pocket, the TMP binding pocket
comprising the amino acids listed in Table 1; supplying the
computer modeling application with a set of structure coordinates
of a chemical entity; and determining whether the chemical entity
is an inhibitor expected to bind to or interfere with the molecule
or molecular complex, wherein binding to or interfering with the
molecule or molecular complex is indicative of potential inhibition
of S. aureus thymidylate kinase activity.
24. A computer-assisted method for identifying an inhibitor of S.
aureus thymidylate kinase activity comprising: supplying a computer
modeling application with a set of structure coordinates of a
molecule or molecular complex, the molecule or molecular complex
comprising at least a portion of an S. aureus thymidylate kinase or
thymidylate kinase-like TMP/ATP binding pocket, the TMP/ATP binding
pocket comprising the amino acids listed in Table 4; supplying the
computer modeling application with a set of structure coordinates
of a chemical entity; and determining whether the chemical entity
is an inhibitor expected to bind to or interfere with the molecule
or molecular complex, wherein binding to or interfering with the
molecule or molecular complex is indicative of potential inhibition
of S. aureus thymidylate kinase activity.
25. The method of claim 23 wherein the TMP binding pocket comprises
the amino acids listed in Table 1, the TMP binding pocket being
defined by a set of points having a root mean square deviation of
less than about 2.1 .ANG. from points representing the backbone
atoms of said amino acids as represented by structure coordinates
listed in FIG. 2.
26. The method of claim 24 wherein the TMP/ATP binding pocket
comprises the amino acids listed in Table 4, the TMP/ATP binding
pocket being defined by a set of points having a root mean square
deviation of less than about 2.1 .ANG. from points representing the
backbone atoms of said amino acids as represented by structure
coordinates listed in FIG. 2.
27. The method of claim 23 or 24 wherein determining whether the
chemical entity is an inhibitor expected to bind to or interfere
with the molecule or molecular complex comprises performing a
fitting operation between the chemical entity and a binding pocket
of the molecule or molecular complex, followed by computationally
analyzing the results of the fitting operation to quantify the
association between the chemical entity and the binding pocket.
28. The method of claim 23 or 24 further comprising screening a
library of chemical entities.
29. A computer-assisted method for designing an inhibitor of S.
aureus thymidylate kinase activity comprising: supplying a computer
modeling application with a set of structure coordinates of a
molecule or molecular complex, the molecule or molecular complex
comprising at least a portion of an S. aureus thymidylate kinase or
thymidylate kinase-like TMP binding pocket, the TMP binding pocket
comprising the amino acids listed in Table 1; supplying the
computer modeling application with a set of structure coordinates
for a chemical entity; evaluating the potential binding
interactions between the chemical entity and substrate binding
pocket of the molecule or molecular complex; structurally modifying
the chemical entity to yield a set of structure coordinates for a
modified chemical entity; and determining whether the modified
chemical entity is an inhibitor expected to bind to or interfere
with the molecule or molecular complex, wherein binding to or
interfering with the molecule or molecular complex is indicative of
potential inhibition of S. aureus thymidylate kinase activity.
30. A computer-assisted method for designing an inhibitor of S.
aureus thymidylate kinase activity comprising: supplying a computer
modeling application with a set of structure coordinates of a
molecule or molecular complex, the molecule or molecular complex
comprising at least a portion of an S. aureus thymidylate kinase or
thymidylate kinase-like TMP/ATP binding pocket, the TMP/ATP binding
pocket comprising the amino acids listed in Table 4; supplying the
computer modeling application with a set of structure coordinates
for a chemical entity; evaluating the potential binding
interactions between the chemical entity and substrate binding
pocket of the molecule or molecular complex; structurally modifying
the chemical entity to yield a set of structure coordinates for a
modified chemical entity; and determining whether the modified
chemical entity is an inhibitor expected to bind to or interfere
with the molecule or molecular complex, wherein binding to or
interfering with the molecule or molecular complex is indicative of
potential inhibition of S. aureus thymidylate kinase activity.
31. The method of claim 29 wherein the TMP binding pocket comprises
the amino acids listed in Table 1, the TMP binding pocket being
defined by a set of points having a root mean square deviation of
less than about 2.1 .ANG. from points representing the backbone
atoms of said amino acids as represented by structure coordinates
listed in FIG. 2.
32. The method of claim 30 wherein the TMP/ATP binding pocket
comprises the amino acids listed in Table 1, the TMP/ATP binding
pocket being defined by a set of points having a root mean square
deviation of less than about 2.1 .ANG. from points representing the
backbone atoms of said amino acids as represented by structure
coordinates listed in FIG. 2.
33. The method of claim 29 or 30 wherein determining whether the
modified chemical entity is an inhibitor expected to bind to or
interfere with the molecule or molecular complex comprises
performing a fitting operation between the chemical entity and a
binding pocket of the molecule or molecular complex, followed by
computationally analyzing the results of the fitting operation to
quantify the association between the chemical entity and the
binding pocket.
34. The method of claim 29 or 30 wherein the set of structure
coordinates for the chemical entity is obtained from a chemical
fragment library
35. A computer-assisted method for designing an inhibitor of S.
aureus thymidylate kinase activity de novo comprising: supplying a
computer modeling application with a set of structure coordinates
of a molecule or molecular complex, the molecule or molecular
complex comprising at least a portion of an S. aureus thymidylate
kinase or thymidylate kinase-like TMP binding pocket, wherein the
TMP substrate binding pocket comprises the amino acids listed in
Table 1; computationally building a chemical entity represented by
set of structure coordinates; and determining whether the chemical
entity is an inhibitor expected to bind to or interfere with the
molecule or molecular complex, wherein binding to or interfering
with the molecule or molecular complex is indicative of potential
inhibition of S. aureus thymidylate kinase activity.
36. A computer-assisted method for designing an inhibitor of S.
aureus thymidylate kinase activity de novo comprising: supplying a
computer modeling application with a set of structure coordinates
of a molecule or molecular complex, the molecule or molecular
complex comprising at least a portion of an S. aureus thymidylate
kinase or thymidylate kinase-like TMP/ATP binding pocket, wherein
the TMP/ATP binding pocket comprises the amino acids listed in
Table 4; computationally building a chemical entity represented by
set of structure coordinates; and determining whether the chemical
entity is an inhibitor expected to bind to or interfere with the
molecule or molecular complex, wherein binding to or interfering
with the molecule or molecular complex is indicative of potential
inhibition of S. aureus thymidylate kinase activity.
37. The method of claim 35 wherein the TMP binding pocket comprises
the amino acids listed in Table 1, the TMP binding pocket being
defined by a set of points having a root mean square deviation of
less than about 2.1 .ANG. from points representing the backbone
atoms of said amino acids as represented by structure coordinates
listed in FIG. 2.
38. The method of claim 36 wherein the TMP/ATP binding pocket
comprises the amino acids listed in Table 4, the TMP/ATP binding
pocket being defined by a set of points having a root mean square
deviation of less than about 2.1 .ANG. from points representing the
backbone atoms of said amino acids as represented by structure
coordinates listed in FIG. 2.
39. The method of claim 35 or 36 wherein determining whether the
chemical entity is an inhibitor expected to bind to or interfere
with the molecule or molecular complex comprises performing a
fitting operation between the chemical entity and a binding pocket
of the molecule or molecular complex, followed by computationally
analyzing the results of the fitting operation to quantify the
association between the chemical entity and the binding pocket.
40. The method of any of claims 23, 24, 29, 30, 35, or 36 further
comprising supplying or synthesizing the potential inhibitor, then
assaying the potential inhibitor to determine whether it inhibits
S. aureus TMK activity.
41. A method for making an inhibitor of S. aureus TMK activity, the
method comprising chemically or enzymatically synthesizing a
chemical entity to yield an inhibitor of S. aureus TMK activity,
the chemical entity having been identified during a
computer-assisted process comprising supplying a computer modeling
application with a set of structure coordinates of a molecule or
molecular complex, the molecule or molecular complex comprising at
least a portion of at least one of a S. aureus thymidylate kinase
or thymidylate kinase-like TMP or TMP/AT binding pocket; supplying
the computer modeling application with a set of structure
coordinates of a chemical entity; and determining whether the
chemical entity is expected to bind to or interfere with the
molecule or molecular complex at a binding pocket, wherein binding
to or interfering with the molecule or molecular complex is
indicative of potential inhibition of S. aureus TMK activity.
42. A method for making an inhibitor of S. aureus TMK activity, the
method comprising chemically or enzymatically synthesizing a
chemical entity to yield an inhibitor of S. aureus TMK activity,
the chemical entity having been designed during a computer-assisted
process comprising supplying a computer modeling application with a
set of structure coordinates of a molecule or molecular complex,
the molecule or molecular complex comprising at least a portion of
at least one of a S. aureus thymidylate kinase or thymidylate
kinase-like TMP or TMP/ATP binding pocket; supplying the computer
modeling application with a set of structure coordinates for a
chemical entity; evaluating the potential binding interactions
between the chemical entity and a binding pocket of the molecule or
molecular complex; structurally modifying the chemical entity to
yield a set of structure coordinates for a modified chemical
entity; and determining whether the chemical entity is expected to
bind to or interfere with the molecule or molecular complex at the
binding pocket, wherein binding to or interfering with the molecule
or molecular complex is indicative of potential inhibition of S.
aureus TMK activity.
43. A method for making an inhibitor of S. aureus TMK activity, the
method comprising chemically or enzymatically synthesizing a
chemical entity to yield an inhibitor of S. aureus TMK activity,
the chemical entity having been designed during a computer-assisted
process comprising supplying a computer modeling application with a
set of structure coordinates of a molecule or molecular complex,
the molecule or molecular complex comprising at least a portion of
at least one of a S. aureus thymidylate kinase or thymidylate
kinase-like TMP or TMP/ATP binding pocket; computationally building
a chemical entity represented by set of structure coordinates; and
determining whether the chemical entity is expected to bind to or
interfere with the molecule or molecular complex at a binding
pocket, wherein binding to or interfering with the molecule or
molecular complex is indicative of potential inhibition of S.
aureus TMK activity.
44. An inhibitor of S. aureus thymidylate kinase activity
identified, designed or made according to the method of any of the
claims 23, 24, 29, 30, 35, 36, 41, 42, or 43.
45. A composition comprising an inhibitor of S. aureus thymidylate
kinase activity identified or designed according to the method of
any of the claims 23, 24, 29, 30, 35, 36, 41, 42, or 43.
46. A pharmaceutical composition comprising an inhibitor of S.
aureus thymidylate kinase activity identified or designed according
to the method of any of the claims 23, 24, 29, 30, 35, 36, 41, 42,
or 43 or a salt thereof, and pharmaceutically acceptable
carrier.
47. A method for crystallizing an S. aureus thymidylate kinase
molecule or molecular complex comprising: preparing purified S.
aureus thymidylate kinase at a concentration of about 1 mg/ml to
about 50 mg/ml; and crystallizing S. aureus thymidylate kinase from
a solution comprising about 5 wt. % to about 50 wt. % PEG, about
0.05 M to about 0.5 M MgCl.sub.2, and about 0 wt. % to about 20 wt.
% DMSO, wherein the solution is buffered to a pH of about 6 to
about 7.
48. A method for crystallizing an S. aureus thymidylate kinase
molecule or molecular complex comprising: preparing purified S.
aureus thymidylate kinase at a concentration of about 1 mg/ml to
about 50 mg/ml; and crystallizing S. aureus thymidylate kinase from
a solution comprising about 2 mM to about 20 mM
.beta.,.gamma.-difluoromethylene-bisphosphonate adenosine
monophosphate and about 0 wt. % to about 20 wt. % DMSO, wherein the
solution is buffered to a pH of about 6 to about 7.
49. A crystal of S. aureus thymidylate kinase.
50. The crystal of claim 49 having the trigonal space group
symmetry P2.sub.1.
51. The crystal of claim 49 comprising a unit cell having
dimensions of a, b, and c; wherein a is about 40 .ANG. to about 60
.ANG., b is about 80 .ANG. to about 100 .ANG., and c is about 40
.ANG. to about 60 .ANG.; and wherein .alpha.=.gamma.=90.degree. and
.beta. is about 80.degree. to about 120.degree..
52. The crystal of claim 49 comprising atoms arranged in a spatial
relationship represented by the structure coordinates listed in
FIG. 2.
53. The crystal of claim 49 having amino acid sequence SEQ ID
NO:1.
54. The crystal of claim 49 having amino acid sequence SEQ ID NO:1,
with the proviso that at least one methionine is replaced with
selenomethionine.
Description
[0001] This application claims the benefit of U.S. Provisional
Application Serial No. 60/147,117, filed 4 Aug. 1999, which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0002] This invention relates to the crystallization and structure
determination of thymidylate kinase (TMK) from Staphylococcus
aureus.
BACKGROUND
[0003] Thymidylate kinase (TMK) catalyzes the synthesis of
(deoxy)thymidine diphosphate (dTDP) from (deoxy)thymidine
monophosphate (dTMP) and ATP along the pathway leading to the
synthesis of (deoxy)thymidine triphosphate (dTTP) necessary for DNA
synthesis (FIG. 1). Since the phosphorylation of dTDP to dTDP is
conducted by a nonspecific diphosphate kinase, TMK is a key player
in the regulation of DNA synthesis and is a potential antibacterial
target. Interest in thymidylate kinase biochemistry increased when
it was recently discovered that this enzyme serves as one of the
activators for the AIDS drug, 3'-azido-3'-deoxythymidine (AZT) (L.
W. Frick et al., Biochem. Biophys. Res. Comm. 154:124-9 (1988); A.
Fridland et al., Mol. Pharmacol. 37:665-70 (1990)). Activation of
AZT to azidothymidine triphosphate (AZT-TP) proceeds along cellular
phosphorylation pathways to produce the species which is
incorporated into growing DNA chains by HIV reverse transcriptase.
Similar to its role in serving as a control point for the
production of dTTP, thymidylate kinase catalyzes the rate limiting
phosphorylation of AZT-monophosphate to AZT-diphosphate (AZT-DP).
AZT-DP phosphorylation to AZT-TP is then catalyzed by a nonspecific
diphosphate kinase.
SUMMARY OF THE INVENTION
[0004] In one aspect, the present invention provides a method for
crystallizing an S. aureus thymidylate kinase molecule or molecular
complex that includes preparing purified S. aureus thymidylate
kinase at a concentration of about 1 mg/ml to about 50 mg/ml and
crystallizing S. aureus thymidylate kinase from a solution
including about 5 wt. % to about 50 wt. % PEG (preferably having a
number average molecular weight between about 200 and about
20,000), about 0.05 M to about 0.5 M MgCl.sub.2, and about 0 wt. %
to about 20 wt. % DMSO, wherein the solution is buffered to a pH of
about 6 to about 7. In another aspect, the present invention
provides a method for crystallizing an S. aureus thymidylate kinase
molecule or molecular complex that includes preparing purified S.
aureus thymidylate kinase at a concentration of about 1 mg/ml to
about 50 mg/ml and crystallizing S. aureus thymidylate kinase from
a solution including about 2 mM to about 20 mM
.beta.,.gamma.-difluoromethy- lene-bisphosphonate adenosine
monophosphate and about 0 wt. % to about 20 wt. % DMSO, wherein the
solution is buffered to a pH of about 6 to about 7
[0005] In another aspect, the present invention provides
crystalline forms of an S. aureus thymidylate kinase molecule. In
one embodiment, a crystal of S. aureus thymidylate kinase is
provided having the trigonal space group symmetry P2.sub.1.
[0006] In another aspect, the present invention provides a scalable
three dimensional configuration of points derived from structure
coordinates of at least a portion of an S. aureus thymidylate
kinase molecule or molecular complex. In one embodiment, the
scalable three dimensional set of points is derived from structure
coordinates of at least the backbone atoms of the amino acids
representing a TMP and/or TMP/ATP substrate binding pocket of an S.
aureus thymidylate kinase molecule or molecular complex. In another
embodiment, the scalable three dimensional configuration of points
is derived from structure coordinates of at least a portion of a
molecule or a molecular complex that is structurally homologous to
an S. aureus thymidylate kinase molecule or molecular complex. On a
molecular scale, the configuration of points derived from a
homologous molecule or molecular complex have a root mean square
deviation of less than about 2.1 .ANG. from the structure
coordinates of the molecule or complex
[0007] In another aspect, the present invention provides a molecule
or molecular complex that includes at least a portion of an S.
aureus thymidylate kinase TMP and/or TMP/ATP substrate binding
pocket. In one embodiment, the S. aureus thymidylate kinase TMP
substrate binding pocket includes the amino acids listed in Table
1, preferably the amino acids listed in Table 2, and more
preferably the amino acids listed in Table 3, the substrate binding
pocket being defined by a set of points having a root mean square
deviation of less than about 2.1 .ANG., preferably less than about
1.5 .ANG., more preferably less than about 1.0 .ANG., and most
preferably less than about 0.5 .ANG. from points representing the
backbone atoms of the amino acids. In another embodiment, the S.
aureus thymidylate kinase TMP/ATP substrate binding pocket includes
the amino acids listed in Table 4, preferably the amino acids
listed in Table 5, and more preferably the amino acids listed in
Table 6, the substrate binding pocket being defined by a set of
points having a root mean square deviation of less than about 2.1
.ANG., preferably less than about 1.5 .ANG., more preferably less
than about 1.0 .ANG., and most preferably less than about 0.5 .ANG.
from points representing the backbone atoms of the amino acids.
1TABLE 1 Residues within about 4 .ANG. of the TMP binding pocket of
S. aureus TMK GLU 12 ARG 37 ILE 48 ARG 49 VAL 52 LEU 53 LEU 66 PHE
67 SER 70 ARG 71 ARG 93 SER 97 SER 98 TYR 101
[0008]
2TABLE 2 Residues within about 7 .ANG. of the TMP binding pocket of
S. aureus TMK GLY 10 GLU 12 ARG 37 GLU 38 PRO 39 GLY 45 GLU 46 GLU
47 ILE 48 ARG 49 LYS 50 ILE 51 VAL 52 LEU 53 GLU 63 MET 65 LEU 66
PHE 67 ALA 68 ALA 69 SER 70 ARG 71 ASP 92 ARG 93 TYR 94 ILE 95 ASP
96 SER 97 SER 98 LEU 99 ALA 100 TYR 101 GLN 102 ASN 117 PHE 160 TYR
168
[0009]
3TABLE 3 Residues within about 10 .ANG. of the TMP binding pocket
of S. aureus TMK PHE 8 GLU 9 GLY 10 PRO 11 GLU 12 GLY 13 SER 14 LYS
16 THR 17 ARG 37 GLU 38 PRO 39 GLY 40 GLY 41 VAL 42 PRO 43 THR 44
GLY 45 GLU 46 GLU 47 ILE 48 ARG 49 LYS 50 ILE 51 VAL 52 LEU 53 GLU
54 GLY 55 MET 58 ILE 60 THR 62 GLU 63 ALA 64 MET 65 LEU 66 PHE 67
ALA 68 ALA 69 SER 70 ARG 71 ARG 72 GLU 73 HIS 74 CYS 91 ASP 92 ARG
93 TYR 94 ILE 95 ASP 96 SER 97 SER 98 LEU 99 ALA 100 TYR 101 GLN
102 GLY 103 TYR 104 ALA 105 ARG 106 VAL 113 LEU 116 ASN 117 ILE 143
PHE 160 HIS 161 VAL 164 TYR 168
[0010]
4TABLE 4 Residues within about 4 .ANG. of the TMP/ATP binding
pocket of S. aureus TMK GLU 12 GLY 15 LYS 16 THR 17 THR 18 ARG 37
GLU 38 PHE 67 ARG 71 ASP 92 ARG 93 SER 97 SER 98 TYR 101 GLN 102
ARG 142 LEU 188
[0011]
5TABLE 5 Residues within about 7 .ANG. of the TMP/ATP binding
pocket of S. aureus TMK GLY 10 PRO 11 GLU 12 GLY 13 SER 14 GLY 15
LYS 16 THR 17 THR 18 VAL 19 ILE 20 ASN 21 ARG 37 GLU 38 ARG 49 GLU
63 ALA 64 PHE 67 ALA 68 ARG 71 ASP 92 ARG 93 TYR 94 ILE 95 ASP 96
SER 97 SER 98 LEU 99 ALA 100 TYR 101 GLN 102 ARG 106 ASN 117 LEU
132 GLU 141 ARG 142 ILE 143 PHE 160 ALA 184 GLN 186 PRO 187 LEU 188
GLU 189 VAL 191
[0012]
6TABLE 6 Residues within about 10 .ANG. of the TMP/ATP binding
pocket of S. aureus TMK PHE 8 GLU 9 GLY 10 PRO 11 GLU 12 GLY 13 SER
14 GLY 15 LYS 16 THR 17 THR 18 VAL 19 ILE 20 ASN 21 GLU 22 MET 35
THR 36 ARG 37 GLU 38 PRO 39 GLY 40 GLU 46 ARG 49 VAL 52 LEU 53 ILE
60 GLU 63 ALA 64 MET 65 LEU 66 PHE 67 ALA 68 ALA 69 SER 70 ARG 71
ARG 72 HIS 74 CYS 91 ASP 92 ARG 93 TYR 94 ILE 95 ASP 96 SER 97 SER
98 LEU 99 ALA 100 TYR 101 GLN 102 GLY 103 TYR 104 ALA 105 ARG 106
ILE 108 VAL 113 LEU 116 ASN 117 ALA 120 LEU 132 VAL 134 VAL 138 GLY
139 ARG 140 GLU 141 ARG 142 ILE 143 ASP 157 PHE 160 HIS 161 VAL 164
TYR 168 ASN 183 ALA 184 ASP 185 GLN 186 PRO 187 LEU 188 GLU 189 ASN
190 VAL 191 VAL 192
[0013] In another aspect, the present invention provides molecules
or molecular complexes that are structurally homologous to an S.
aureus thymidylate kinase molecule or molecular complex.
[0014] In another aspect, the present invention provides a machine
readable storage medium including the structure coordinates of all
or a portion of an S. aureus thymidylate kinase molecule, molecular
complex, a structurally homologous molecule or complex, including
structurally equivalent structures, as defined herein, particularly
a substrate binding pocket thereof, or a similarly shaped
homologous substrate binding pocket. A storage medium encoded with
these data is capable of displaying on a computer screen, or
similar viewing device, a three-dimensional graphical
representation of a molecule or molecular complex which comprises a
substrate binding pocket or a similarly shaped homologous substrate
binding pocket.
[0015] In another aspect, the present invention provides a method
for identifying inhibitors, ligands, and the like for an S. aureus
thymidylate kinase molecule by providing the coordinates of a
molecule of S. aureus thymidylate kinase to a computerized modeling
system; identifying chemical entities that are likely to bind to or
interfere with the molecule (e.g., screening a small molecule
library); and, optionally, procuring or synthesizing and assaying
the compounds or analogues derived therefrom for bioactivity. In
another aspect, the present invention provides methods for
designing inhibitors, ligands, and the like by providing the
coordinates of a molecule of S. aureus thymidylate kinase to a
computerized modeling system; designing a chemical entity that is
likely to bind to or interfere with the molecule; and optionally
synthesizing the chemical entity and assaying the chemical entity
for bioactivity. In another aspect, the present invention provides
inhibitors and ligands designed or identified by the above methods.
In one embodiment, a composition is provided that includes an
inhibitor or ligand designed or identified by the above method. In
another embodiment, the composition is a pharmaceutical
composition.
[0016] In another aspect, the present invention provides a method
involving molecular replacement to obtain structural information
about a molecule or molecular complex of unknown structure. The
method includes crystallizing the molecule or molecular complex,
generating an x-ray diffraction pattern from the crystallized
molecule or molecular complex, and applying at least a portion of
the structure coordinates set forth in FIG. 2 to the x-ray
diffraction pattern to generate a three-dimensional electron
density map of at least a portion of the molecule or molecular
complex.
[0017] In another aspect, the present invention provides a method
for homology modeling an S. aureus thymidylate kinase homolog.
[0018] Definitions
[0019] Two crystallographic data sets (with structure factors F)
are considered isomorphous if, after scaling, 1 F F = F 1 - F 2 F
1
[0020] is less than about 35% for the reflections between 8 .ANG.
and 4 .ANG..
[0021] Abbreviations
[0022] The following abbreviations are used throughout this
disclosure:
[0023] Staphylococcus aureus (S. aureus).
[0024] Thymidylate kinase (T. kinase or TMK).
[0025] Thymidine 5'-monophosphate (TMP).
[0026] Thymidine 5'-diphosphate (TDP).
[0027] Thymidine 5'-triphosphate (TTP).
[0028] Phospho(enol)pyruvate (PEP)
[0029] Reduced nicotinamide adenine dinucleotide (NADH)
[0030] Oxidized nicotinamide adenine dinucleotide (NAD.sup.+)
[0031] Pyruvate kinase (PK)
[0032] Lactate dehydrogenase (LDH)
[0033] Nucleoside-5'-diphosphate kinase (NDP-Kinase)
[0034] (Deoxy)thymidine monophosphate (dTMP).
[0035] (Deoxy)thymidine diphosphate (dTDP).
[0036] (Deoxy)thymidine triphosphate (dTTP).
[0037] Adenosine 5'-diphosphate (ADP).
[0038] Adenosine 5'-triphosphate (ATP).
[0039] Isopropylthio-.beta.-D-galactoside (IPTG).
[0040] Dithiothreitol (DTI).
[0041] Dimethyl sulfoxide (DMSO).
[0042] Polyethylene glycol (PEG).
[0043] Multiple anomalous dispersion (MAD).
[0044] The following amino acid abbreviations are used throughout
this disclosure:
7 A = Ala = Alanine T = Thr = Threonine V = Val = Valine C = Cys =
Cysteine L = Leu = Leucine Y = Tyr = Tyrosine I = Ile = Isoleucine
N = Asn = Asparagine P = Pro = Proline Q = Gln = Glutamine F = Phe
= Phenylalanine D = Asp = Aspartic Acid W = Trp = Tryptophan E =
Glu = Glutamic Acid M = Met = Methionine K = Lys = Lysine G = Gly =
Glycine R = Arg = Arginine S = Ser = Serine H = His = Histidine
BRIEF DESCRIPTION OF THE FIGURES
[0045] FIG. 1 shows the biosynthetic pathway for the synthesis of
thymidylate. The reaction catalyzed by thymidylate kinase is
boxed.
[0046] FIG. 2 lists the atomic structure coordinates for
recombinant S. aureus thymidylate kinase (with a His.sub.6 tag) as
derived by x-ray diffraction from a crystal of that complex. The
following abbreviations are used in FIG. 2:
[0047] "Atom" refers to the element whose coordinates are measured.
The second column defines the number of the atom in the structure.
The letters in the third column define the element. The fourth and
fifth columns define the amino acid and the number of the amino
acid in the structure, respectively.
[0048] "X, Y, Z" crystallographically define the atomic position of
the element measured.
[0049] "Occ" is an occupancy factor that refers to the fraction of
the molecules in which each atom occupies the position specified by
the coordinates. A value of "1" indicates that each atom has the
same conformation, i.e., the same position, in all molecules of the
crystal.
[0050] "B" is a thermal factor that measures movement of the atom
around its atomic center.
[0051] FIG. 3 depicts S. aureus thymidylate kinase using (a) a
ribbon diagram showing the backbone structure of the enzyme and (b)
a schematic diagram showing the secondary structure for a TMK
monomer Disordered loops are indicated by arrows.
[0052] FIG. 4 depicts a structural comparison of E. coli
TMK+AP.sub.5T and S. aureus TMK. The overall fold of the two
proteins is well-conserved, but note that the lid in the E. coli
TMK is not present in the S. aureus TMK due to the absence of a
ligand.
[0053] FIG. 5 depicts (a) a stereo view of a superposition of S.
aureus thymidylate kinase and E. coli thymidylate kinase and (b)
the amino acid sequence alignment of S. aureus thymidylate kinase
(SEQ ID NO:1) (capital letters, upper sequence) and E. coli
thymidylate kinase (SEQ ID NO:2) (lower sequence). Dots in the
sequences indicate gaps inserted in order to optimize the
alignment. Identical residues are indicated by .vertline. and
similar residues are indicated by . and : symbols.
[0054] FIG. 6 depicts (a) a stereo view of a superposition of S.
aureus thymidylate kinase and S. cerevisiae thymidylate kinase and
(b) the sequence alignment of S. aureus thymidylate kinase (SEQ ID
NO:1) (capital letters, upper sequence) and S. cerevisiae
thymidylate kinase (SEQ ID NO:3) (lower sequence). Dots in the
sequences indicate gaps inserted in order to optimize the
alignment. Identical residues are indicated by .vertline. and
similar residues are indicated by . and : symbols.
[0055] FIG. 7 depicts a) a substrate-based inhibitor (AP.sub.5T)
for thymidylate kinase with a K.sub.d of 20 nM for E. coli TMK (A.
Lavie et al., Biochemistry 37:3677-86 (1998); A. Lavie et al.,
Proc. Natl. Acad. Sci. USA, 95:14045-50 (1998)). b) protein ligand
interactions for E. coli TMK (shaded boxes, from A. Lavie et al.,
Proc. Natl. Acad. Sci. USA, 95:14045-50 (1998)) with the
corresponding residues from S. aureus TMK underlined (conservative
mutations are marked with an asterisk). Active site residues from
the S. cerevisiae are boxed (where no corresponding residue from E.
coli TMK is present, an arrow indicates the point of contact with
the substrate).
[0056] FIG. 8 depicts the anomalous difference Patterson maps at
(a) 2.7 .ANG. and (b) at 2.3 .ANG. resolution.
[0057] FIG. 9 depicts electron density maps of residues 76 to 82
from molecule 1 of S. aureus thymidylate kinase (SEQ ID NO:1) at
(a) 2.7 .ANG. and (b) at 2.3 .ANG. resolution.
[0058] FIG. 10 lists the structure factors and multiple anomalous
dispersion phases for the crystal structure of S. aureus
thymidylate kinase (SEQ ID NO:1). "INDE" refers to the indices h,
k, and l (columns 2, 3, and 4 respectively) of the lattice planes.
"FOBS" refers to the structure factor (F) of the observed
reflections. "SIGMA" is the standard deviation for the
observations. "PHAS" refers to the phase used for the observations.
"FOM" refers to the figure of merit.
[0059] FIG. 11 depicts a surface representation of a) E. coli TMK
with the inhibitor AP.sub.5T and b) S. aureus TMK with a
hypothetical positioning of AP.sub.5T based on a structural
alignment of C.sub..alpha. atoms from the E. coli TMK+AP.sub.5T
structure.
DETAILED DESCRIPTION OF THE INVENTION
[0060] Crystalline Form(s) and Method of Making
[0061] The three-dimensional structure of S. aureus thymidylate
kinase was solved using high resolution x-ray crystallography to
2.3 .ANG. resolution (see FIG. 2 and Example 1). Accordingly, the
invention includes a TMK crystal and/or a crystal with TMK
co-crystallized with a ligand, such as an inhibitor. Preferably,
the crystal has trigonal space group symmetry P2.sub.1. More
preferably, the crystal comprises rectangular shaped unit cells,
each unit cell having dimensions of a, b, and c; wherein a is about
40 .ANG. to about 60 .ANG., b is about 80 .ANG. to about 100 .ANG.,
and c is about 40 .ANG. to about 60 .ANG.; and wherein
.alpha.=.gamma.=90.degree. and .beta. is about 80.degree. to about
120.degree.. The crystallized enzyme is a dimer with a single dimer
in the asymmetric unit.
[0062] Purified S. aureus thymidylate kinase at a concentration of
about 1 mg/ml to about 50 mg/ml may be crystallized, for example,
by using a streak seeding procedure from a solution including about
5 wt. % to about 50 wt. % PEG (preferably having a number average
molecular weight between about 200 and about 20,000), about 0.05 M
to about 0.5 M MgCl.sub.2, and about 0 wt. % to about 20 wt. %
DMSO, wherein the solution is buffered to a pH of about 6 to about
7. Use of a buffer having a pK.sub.a of between about 5 and 8 is
preferred. Molecular complexes of purified S. aureus thymidylate
kinase at a concentration of about 1 mg/ml to about 50 mg/ml may
also be crystallized, for example, from a solution including about
2 mM to about 20 mM .beta.,.gamma.-difluoromethylene-bisphosphonate
adenosine monophosphate and about 0 wt. % to about 20 wt. % DMSO,
wherein the solution is buffered to a pH of about 6 to about 7. A
"molecular complex" means a protein in covalent or non-covalent
association with a chemical entity. A buffer having a pK.sub.a of
between about 5 and 8 is preferred for use in the crystallization
method. A particularly preferred buffer is about 0.4M to about 2.0M
sodium citrate. Variation in buffer and buffer pH as well as other
additives such as PEG is apparent to those skilled in the art and
may result in similar crystals.
[0063] The invention further includes an S. aureus thymidylate
kinase crystal or S. aureus thymidylate kinase/ligand crystal that
is isomorphous with an S. aureus thymidylate kinase crystal
characterized by a unit cell having dimensions of a, b, and c;
wherein a is about 40 .ANG. to about 60 .ANG., b is about 80 .ANG.
to about 100 .ANG., and c is about 40 .ANG. to about 60 .ANG.; and
wherein .alpha.=.gamma.=90.degree. and .beta. is about 80.degree.
to about 120.degree..
[0064] X-Ray Crystallographic Analysis
[0065] Each of the constituent amino acids of S. aureus thymidylate
kinase is defined by a set of structure coordinates as set forth in
FIG. 2. The term "structure coordinates" refers to Cartesian
coordinates derived from mathematical equations related to the
patterns obtained on diffraction of a monochromatic beam of x-rays
by the atoms (scattering centers) of an S. aureus thymidylate
kinase complex in crystal form. The diffraction data are used to
calculate an electron density map of the repeating unit of the
crystal. The electron density maps are then used to establish the
positions of the individual atoms of the S. aureus thymidylate
kinase protein or protein/ligand complex.
[0066] Slight variations in structure coordinates can be generated
by mathematically manipulating the S. aureus thymidylate kinase or
S. aureus thymidylate kinase/ligand structure coordinates. For
example, the structure coordinates set forth in FIG. 2 could be
manipulated by crystallographic permutations of the structure
coordinates, fractionalization of the structure coordinates,
integer additions or subtractions to sets of the structure
coordinates, inversion of the structure coordinates or any
combination of the above. Alternatively, modifications in the
crystal structure due to mutations, additions, substitutions,
and/or deletions of amino acids, or other changes in any of the
components that make up the crystal, could also yield variations in
structure coordinates. Such slight variations in the individual
coordinates will have little effect on overall shape. If such
variations are within an acceptable standard error as compared to
the original coordinates, the resulting three-dimensional shape is
considered to be structurally equivalent. Structural equivalence is
described in more detail below.
[0067] It should be noted that slight variations in individual
structure coordinates of the S. aureus thymidylate kinase would not
be expected to significantly alter the nature of chemical entities
such as ligands that could associate with the substrate binding
pockets. In this context, the phrase "associating with" refers to a
condition of proximity between a chemical entity, or portions
thereof, and an S. aureus thymidylate kinase molecule or portions
thereof. The association may be non-covalent, wherein the
juxtaposition is energetically favored by hydrogen bonding, van der
Waals forces, or electrostatic interactions, or it may be
covalent.
[0068] Thus, for example, a ligand that bound to a substrate
binding pocket of S. aureus thymidylate kinase would also be
expected to bind to or interfere with another substrate binding
pocket whose structure coordinates define a shape that falls within
the acceptable error.
[0069] It will be readily apparent to those of skill in the art
that the numbering of amino acids in other isoforms of S. aureus
thymidylate kinase may be different than that of S. aureus
thymidylate kinase expressed in E. coli.
[0070] Active Site and Other Structural Features
[0071] Applicants' invention has provided, for the first time,
information about the shape and structure of the substrate binding
pockets of S. aureus thymidylate kinase. The structures of both the
TMP and the TMP/ATP substrate binding pockets are elucidated. The
secondary structure of the S. aureus thymidylate kinase monomer
includes a five stranded parallel .beta. sheet surrounded by nine
.alpha. helices (FIG. 3). This solved crystal structure of S.
aureus thymidylate kinase does not contain any ligand which has
resulted in a disordered loop between helices .alpha.7 and .alpha.8
(FIG. 4). This loop has been called the "lid" in the structures of
thymidylate kinase homologs from E. coli and S. cerevisiae. In E.
coli the lid contains Arg 153 which is responsible for phosphate
binding of the ATP substrate as shown in the X-ray crystal
structure of the E. coli enzyme with the AP.sub.5T inhibitor, a
transition state analog of TMP/ATP (A. Lavie et al., Biochemistry
37:3677-86 (1998)). In contrast the analogous arginine in S.
cerevisiae comes from the P loop (Arg 15) between .beta.1 and
.alpha.1 (A. Lavie et al., Proc. Natl. Acad. Sci. USA, 95:14045-50
(1998)). This distinction as further manifested in sequence
differences between the P loop and lid regions has led to the
classification of the S. cerevisiae enzyme as a class I thymidylate
kinase (which also includes human thymidylate kinase) and the E.
coli enzyme as a class II thymidylate kinase (A. Lavie, Proc. Natl.
Acad. Sci. USA, 95:14045-50 (1998)). Fortunately, S. aureus (SEQ ID
NO:1) has greater sequence similarity to the E. coli enzyme (SEQ ID
NO:2, 38% identical, 59% similar) than the S. cerevisiae enzyme
(SEQ ID NO:3, 28% identical, 46% similar) and contains R148 in the
lid region suggesting it should be classified as a class II
thymidylate kinase (FIGS. 5 and 6). This classification suggests
that it might be possible to design inhibitors that are specific
for the S. aureus enzyme and not eukaryotic thymidylate
kinases.
[0072] Superposition of the S. aureus TMK with E. coli TMK gave a
r.m.s. deviation of 2.19 .ANG. for analogous residues (FIG. 5).
Similarly, superposition of S. aureus TMK with S. cerevisiae TMK
gave a r.m.s. deviation of 3.26 .ANG. (FIG. 6). Analysis of the
active site residues from E. coli TMK as observed in the AP.sub.5T
inhibitor complex shows at least eleven residues that make direct
hydrogen bonds to the inhibitor and another six residues make water
mediated or hydrophobic interactions. Analysis of the active site
residues from S. aureus TMK sequence reveals strong conservation of
these active site residues with the E. coli active site (FIG. 7b);
15 of the 17 residues involved in the protein-inhibitor complex are
identical while the two remaining residues are strongly conserved.
An analogous comparison for the S. cerevisiae TMK (FIG. 7b) shows
only four of 18 residues conserved within the active site
suggesting that specificity between the S. aureus and eukaryotic
thymidylate kinases might be attainable.
[0073] Comparing the liganded E. coli TMK structure with the
unliganded S. aureus structure, it is apparent that a significant
movement of the main chain around the active site (e.g. helix
.alpha.2 and helix .alpha.7) including the ordering of the
disordered residues must occur upon ligand binding. FIG. 11 shows
where the AP.sub.5T inhibitor would be expected in the S. aureus
TMK structure based on an alignment of the E. coli TMK-AP.sub.5T
inhibitor complex. There does appear to be a surface in the S.
aureus TMK structure which would complement the ATP and TMP
substrates, although it is clear from this surface view that an
important part of the structure, the lid, is missing from the S.
aureus TMK structure. FIG. 8 clearly illustrates the role for this
portion of the protein in completing the active site and closing
off the thymidylate moiety from solvent.
[0074] Binding pockets are of significant utility in fields such as
drug discovery. The association of natural ligands or substrates
with the binding pockets of their corresponding receptors or
enzymes is the basis of many biological mechanisms of action.
Similarly, many drugs exert their biological effects through
association with the binding pockets of receptors and enzymes. Such
associations may occur with all or any parts of the binding pocket.
An understanding of such associations helps lead to the design of
drugs having more favorable associations with their target, and
thus improved biological effects. Therefore, this information is
valuable in designing potential inhibitors of S. aureus thymidylate
kinase-like substrate binding pockets, as discussed in more detail
below.
[0075] The term "binding pocket," as used herein, refers to a
region of a molecule or molecular complex, that, as a result of its
shape, favorably associates with another chemical entity. Thus, a
binding pocket may include or consist of features such as cavities,
surfaces, or interfaces between domains. Chemical entities that may
associate with a binding pocket include, but are not limited to,
cofactors, substrates, inhibitors, agonists, and antagonists.
[0076] The amino acid constituents of an S. aureus thymidylate
kinase substrate binding pocket as defined herein are positioned in
three dimensions in accordance with the structure coordinates
listed in FIG. 2. In one aspect, the structure coordinates defining
a substrate binding pocket of S. aureus thymidylate kinase include
structure coordinates of all atoms in the constituent amino acids;
in another aspect, the structure coordinates of a substrate binding
pocket include structure coordinates of just the backbone atoms of
the constituent atoms.
[0077] The TMP substrate binding pocket of S. aureus thymidylate
kinase preferably includes the amino acids listed in Table 1, more
preferably the amino acids listed in Table 2, and most preferably
the amino acids listed in Table 3, as represented by the structure
coordinates listed in FIG. 2. Alternatively, the TMP substrate
binding pocket of S. aureus thymidylate kinase may be defined by
those amino acids whose backbone atoms are situated within about
3.5 .ANG., more preferably within about 5 .ANG., most preferably
within about 7 .ANG., of one or more constituent atoms of a bound
substrate or inhibitor. In yet another alternative, the TMP
substrate binding pocket may be defined by those amino acids whose
backbone atoms are situated within a sphere centered on the
coordinates representing the alpha carbon atom of residue Ser98,
the sphere having a radius of about 10 .ANG., preferably about 15
.ANG., and more preferably about 20 .ANG..
[0078] The TMP/ATP substrate binding pocket of S. aureus
thymidylate kinase preferably includes the amino acids listed in
Table 4, more preferably the amino acids listed in Table 5, and
most preferably the amino acids listed in Table 6, as represented
by the structure coordinates listed in FIG. 2. Alternatively, the
TMP/ATP substrate binding pocket of S. aureus thymidylate kinase
may be defined by those amino acids whose backbone atoms are
situated within about 3.5 .ANG., more preferably within about 5
.ANG., most preferably within about 7 .ANG., of one or more
constituent atoms of a bound substrate or inhibitor. In yet another
alternative, the TMP/ATP substrate binding pocket may be defined by
those amino acids whose backbone atoms are situated within a sphere
centered on the coordinates representing the alpha carbon atom of
residue Arg93, the sphere having a radius of about 10 .ANG.,
preferably about 15 .ANG., and more preferably about 20 .ANG..
[0079] The term "S. aureus thymidylate kinase-like substrate
binding pocket" refers to a portion of a molecule or molecular
complex whose shape is sufficiently similar to at least a portion
of a substrate binding pocket of S. aureus thymidylate kinase as to
be expected to bind related TMP and/or ATP structural analogues. A
structurally equivalent substrate binding pocket is defined by a
root mean square deviation from the structure coordinates of the
backbone atoms of the amino acids that make up substrate binding
pockets in S. aureus thymidylate kinase (as set forth in FIG. 2) of
at most about 2.1 .ANG.. How this calculation is obtained is
described below.
[0080] Accordingly, the invention provides molecules or molecular
complexes comprising an S. aureus thymidylate kinase substrate
binding pocket or S. aureus thymidylate kinase-like substrate
binding pocket, as defined by the sets of structure coordinates
described above.
[0081] Three-Dimensional Configurations
[0082] X-ray structure coordinates define a unique configuration of
points in space. Those of skill in the art understand that a set of
structure coordinates for protein or an protein/ligand complex, or
a portion thereof, define a relative set of points that, in turn,
define a configuration in three dimensions. A similar or identical
configuration can be defined by an entirely different set of
coordinates, provided the distances and angles between coordinates
remain essentially the same. In addition, a scalable configuration
of points can be defined by increasing or decreasing the distances
between coordinates by a scalar factor while keeping the angles
essentially the same.
[0083] The present invention thus includes the scalable
three-dimensional configuration of points derived from the
structure coordinates of at least a portion of an S. aureus
thymidylate kinase molecule or molecular complex, as listed in FIG.
2, as well as structurally equivalent configurations, as described
below. Preferably, the scalable three-dimensional configuration
includes points derived from structure coordinates representing the
locations of a plurality of the amino acids defining an S. aureus
thymidylate kinase substrate binding pocket.
[0084] In one embodiment, the scalable three-dimensional
configuration includes points derived from structure coordinates
representing the locations the backbone atoms of a plurality of
amino acids defining the S. aureus thymidylate kinase TMP substrate
binding pocket, preferably the amino acids listed in Table 1, more
preferably the amino acids listed in Table 2, and most preferably
the amino acids listed in Table 3. Alternatively, the scalable
three-dimensional configuration includes points derived from
structure coordinates representing the locations of the side chain
and the backbone atoms (other than hydrogens) of a plurality of the
amino acids defining the S. aureus thymidylate kinase TMP substrate
binding pocket, preferably the amino acids listed in Table 1, more
preferably the amino acids listed in Table 2, and most preferably
the amino acids listed in Table 3.
[0085] In another embodiment, the scalable three-dimensional
configuration includes points derived from structure coordinates
representing the locations the backbone atoms of a plurality of
amino acids defining the S. aureus thymidylate kinase TMP/ATP
substrate binding pocket, preferably the amino acids listed in
Table 4, more preferably the amino acids listed in Table 5, and
most preferably the amino acids listed in Table 6. Alternatively,
the scalable three-dimensional configuration includes points
derived from structure coordinates representing the locations of
the side chain and the backbone atoms (other than hydrogens) of a
plurality of the amino acids defining the S. aureus thymidylate
kinase TMP/ATP substrate binding pocket, preferably the amino acids
listed in Table 4, more preferably the amino acids listed in Table
5, and most preferably the amino acids listed in Table 6.
[0086] Likewise, the invention also includes the scalable
three-dimensional configuration of points derived from structure
coordinates of molecules or molecular complexes that are
structurally homologous to S. aureus thymidylate kinase, as well as
structurally equivalent configurations. Structurally homologous
molecules or molecular complexes are defined below. Advantageously,
structurally homologous molecules can be identified using the
structure coordinates of S. aureus thymidylate kinase according to
a method of the invention.
[0087] The configurations of points in space derived from structure
coordinates according to the invention can be visualized as, for
example, a holographic image, a stereodiagram, a model or a
computer-displayed image, and the invention thus includes such
images, diagrams or models.
[0088] Structurally Equivalent Crystal Structures
[0089] Various computational analyses can be used to determine
whether a molecule or a substrate binding pocket portion thereof is
"structurally equivalent," defined in terms of its
three-dimensional structure, to all or part of S. aureus
thymidylate kinase or its substrate binding pockets. Such analyses
may be carried out in current software applications, such as the
Molecular Similarity application of QUANTA (Molecular Simulations
Inc., San Diego, Calif.) version 4.1, and as described in the
accompanying User's Guide.
[0090] The Molecular Similarity application permits comparisons
between different structures, different conformations of the same
structure, and different parts of the same structure. The procedure
used in Molecular Similarity to compare structures is divided into
four steps: (1) load the structures to be compared; (2) define the
atom equivalences in these structures; (3) perform a fitting
operation; and (4) analyze the results.
[0091] Each structure is identified by a name. One structure is
identified as the target (i.e., the fixed structure); all remaining
structures are working structures (i.e., moving structures). Since
atom equivalency within QUANTA is defined by user input, for the
purpose of this invention equivalent atoms are defined as protein
backbone atoms (N, C.alpha., C, and O) for all conserved residues
between the two structures being compared. A conserved residue is
defined as a residue which is structurally or functionally
equivalent. Only rigid fitting operations are considered.
[0092] When a rigid fitting method is used, the working structure
is translated and rotated to obtain an optimum fit with the target
structure. The fitting operation uses an algorithm that computes
the optimum translation and rotation to be applied to the moving
structure, such that the root mean square difference of the fit
over the specified pairs of equivalent atom is an absolute minimum.
This number, given in angstroms, is reported by QUANTA.
[0093] For the purpose of this invention, any molecule or molecular
complex or substrate binding pocket thereof, or any portion
thereof, that has a root mean square deviation of conserved residue
backbone atoms (N, C.alpha., C, O) of less than about 2.1 .ANG.,
when superimposed on the relevant backbone atoms described by the
reference structure coordinates listed in FIG. 2, is considered
"structurally equivalent" to the reference molecule. That is to
say, the crystal structures of those portions of the two molecules
are substantially identical, within acceptable error. Particularly
preferred structurally equivalent molecules or molecular complexes
are those that are defined by the entire set of structure
coordinates listed in FIG. 2.+-.a root mean square deviation from
the conserved backbone atoms of those amino acids of not more than
2.1 .ANG.. More preferably, the root mean square deviation is less
than about 1.0 .ANG.. Another embodiment of this invention is a
molecular complex defined by the structure coordinates listed in
FIG. 2 for those amino acids listed in Table 1, .+-.a root mean
square deviation from the conserved backbone atoms of those amino
acids of not more than 2.1 .ANG., preferably less than about 1.0
.ANG.. Still another embodiment of this invention is a molecular
complex defined by the structure coordinates listed in FIG. 2 for
those amino acids listed in Table 4, .+-.a root mean square
deviation from the conserved backbone atoms of those amino acids of
not more than 2.1 .ANG., preferably less than about 1.0 .ANG..
[0094] The term "root mean square deviation" means the square root
of the arithmetic mean of the squares of the deviations. It is a
way to express the deviation or variation from a trend or object.
For purposes of this invention, the "root mean square deviation"
defines the variation in the backbone of a protein from the
backbone of S. aureus thymidylate kinase or a substrate binding
pocket portion thereof, as defined by the structure coordinates of
S. aureus thymidylate kinase described herein.
[0095] Machine Readable Storage Media
[0096] Transformation of the structure coordinates for all or a
portion of S. aureus thymidylate kinase or the S. aureus
thymidylate kinase/ligand complex or one of its substrate binding
pockets, for structurally homologous molecules as defined below, or
for the structural equivalents of any of these molecules or
molecular complexes as defined above, into three-dimensional
graphical representations of the molecule or complex can be
conveniently achieved through the use of commercially-available
software.
[0097] The invention thus further provides a machine-readable
storage medium comprising a data storage material encoded with
machine readable data which, when using a machine programmed with
instructions for using said data, is capable of displaying a
graphical three-dimensional representation of any of the molecule
or molecular complexes of this invention that have been described
above. In a preferred embodiment, the machine-readable data storage
medium comprises a data storage material encoded with machine
readable data which, when using a machine programmed with
instructions for using said data, is capable of displaying a
graphical three-dimensional representation of a molecule or
molecular complex comprising all or any parts of an S. aureus
thymidylate kinase substrate binding pocket or an S. aureus
thymidylate kinase-like substrate binding pocket, as defined above.
In another preferred embodiment, the machine-readable data storage
medium comprises a data storage material encoded with machine
readable data which, when using a machine programmed with
instructions for using said data, is capable of displaying a
graphical three-dimensional representation of a molecule or
molecular complex defined by the structure coordinates of all of
the amino acids listed in FIG. 2, .+-.a root mean square deviation
from the backbone atoms of said amino acids of not more than 2.1
.ANG..
[0098] In an alternative embodiment, the machine-readable data
storage medium comprises a data storage material encoded with a
first set of machine readable data which comprises the Fourier
transform of the structure coordinates set forth in FIG. 2, and
which, when using a machine programmed with instructions for using
said data, can be combined with a second set of machine readable
data comprising the x-ray diffraction pattern of a molecule or
molecular complex to determine at least a portion of the structure
coordinates corresponding to the second set of machine readable
data.
[0099] For example, a system for reading a data storage medium may
include a computer comprising a central processing unit ("CPU"), a
working memory which may be, e.g., RAM (random access memory) or
"core" memory, mass storage memory (such as one or more disk drives
or CD-ROM drives), one or more display devices (e.g., cathode-ray
tube ("CRT") displays, light emitting diode ("LED") displays,
liquid crystal displays ("LCDs"), electroluminescent displays,
vacuum fluorescent displays, field emission displays ("FEDs"),
plasma displays, projection panels, etc.), one or more user input
devices (e.g., keyboards, microphones, mice, track balls, touch
pads, etc.), one or more input lines, and one or more output lines,
all of which are interconnected by a conventional bidirectional
system bus. The system may be a stand-alone computer, or may be
networked (e.g., through local area networks, wide area networks,
intranets, extranets, or the internet) to other systems (e.g.,
computers, hosts, servers, etc.). The system may also include
additional computer controlled devices such as consumer electronics
and appliances.
[0100] Input hardware may be coupled to the computer by input lines
and may be implemented in a variety of ways. Machine-readable data
of this invention may be inputted via the use of a modem or modems
connected by a telephone line or dedicated data line. Alternatively
or additionally, the input hardware may comprise CD-ROM drives or
disk drives. In conjunction with a display terminal, a keyboard may
also be used as an input device.
[0101] Output hardware may be coupled to the computer by output
lines and may similarly be implemented by conventional devices. By
way of example, the output hardware may include a display device
for displaying a graphical representation of a binding pocket of
this invention using a program such as QUANTA as described herein.
Output hardware might also include a printer, so that hard copy
output may be produced, or a disk drive, to store system output for
later use.
[0102] In operation, a CPU coordinates the use of the various input
and output devices, coordinates data accesses from mass storage
devices, accesses to and from working memory, and determines the
sequence of data processing steps. A number of programs may be used
to process the machine-readable data of this invention. Such
programs are discussed in reference to the computational methods of
drug discovery as described herein. References to components of the
hardware system are included as appropriate throughout the
following description of the data storage medium.
[0103] Machine-readable storage devices useful in the present
invention include, but are not limited to, magnetic devices,
electrical devices, optical devices, and combinations thereof.
Examples of such data storage devices include, but are not limited
to, hard disk devices, CD devices, digital video disk devices,
floppy disk devices, removable hard disk devices, magneto-optic
disk devices, magnetic tape devices, flash memory devices, bubble
memory devices, holographic storage devices, and any other mass
storage peripheral device. It should be understood that these
storage devices include necessary hardware (e.g., drives,
controllers, power supplies, etc.) as well as any necessary media
(e.g., disks, flash cards, etc.) to enable the storage of data.
[0104] Structurally Homologous Molecules, Molecular Complexes, and
Crystal Structure
[0105] The structure coordinates set forth in FIG. 2 can be used to
aid in obtaining structural information about another crystallized
molecule or molecular complex. The method of the invention allows
determination of at least a portion of the three-dimensional
structure of molecules or molecular complexes which contain one or
more structural features that are similar to structural features of
S. aureus thymidylate kinase, These molecules are referred to
herein as "structurally homologous" to S. aureus thymidylate
kinase, Similar structural features can include, for example,
regions of amino acid identity, conserved active site or binding
site motifs, and similarly arranged secondary structural elements
(e.g., .alpha. helices and .beta. sheets). Optionally, structural
homology is determined by aligning the residues of the two amino
acid sequences to optimize the number of identical amino acids
along the lengths of their sequences; gaps in either or both
sequences are permitted in making the alignment in order to
optimize the number of identical amino acids, although the amino
acids in each sequence must nonetheless remain in their proper
order. Preferably, two amino acid sequences are compared using the
Blastp program, version 2.0.9, of the BLAST 2 search algorithm, as
described by Tatiana et al., FEMS Microbiol Lett 174, 247-50
(1999), and available at http://www.ncbi.nlm.nih.gov/gorf/bl2.html.
Preferably, the default values for all BLAST 2 search parameters
are used, including matrix=BLOSUM62; open gap penalty=11, extension
gap penalty=1, gap x_dropoff=50, expect=10, wordsize=3, and filter
on. In the comparison of two amino acid sequences using the BLAST
search algorithm, structural similarity is referred to as
"identity." Preferably, a structurally homologous molecule is a
protein that has an amino acid sequence sharing at least 65%
identity with a native or recombinant amino acid sequence of S.
aureus thymidylate kinase (for example, SEQ ID NO:1). More
preferably, a protein that is structurally homologous to S. aureus
thymidylate kinase includes at least one contiguous stretch of at
least 50 amino acids that shares at least 80% amino acid sequence
identity with the analogous portion of the native or recombinant S.
aureus thymidylate kinase (for example, SEQ ID NO:1). Methods for
generating structural information about the structurally homologous
molecule or molecular complex are well-known and include, for
example, molecular replacement techniques.
[0106] Therefore, in another embodiment this invention provides a
method of utilizing molecular replacement to obtain structural
information about a molecule or molecular complex whose structure
is unknown comprising the steps of:
[0107] (a) crystallizing the molecule or molecular complex of
unknown structure;
[0108] (b) generating an x-ray diffraction pattern from said
crystallized molecule or molecular complex; and
[0109] (c) applying at least a portion of the structure coordinates
set forth in FIG. 2 to the x-ray diffraction pattern to generate a
three-dimensional electron density map of the molecule or molecular
complex whose structure is unknown.
[0110] By using molecular replacement, all or part of the structure
coordinates of S. aureus thymidylate kinase or the S. aureus
thymidylate kinase/ligand complex as provided by this invention can
be used to determine the structure of a crystallized molecule or
molecular complex whose structure is unknown more quickly and
efficiently than attempting to determine such information ab
initio.
[0111] Molecular replacement provides an accurate estimation of the
phases for an unknown structure. Phases are a factor in equations
used to solve crystal structures that cannot be determined
directly. Obtaining accurate values for the phases, by methods
other than molecular replacement, is a time-consuming process that
involves iterative cycles of approximations and refinements and
greatly hinders the solution of crystal structures. However, when
the crystal structure of a protein containing at least a
structurally homologous portion has been solved, the phases from
the known structure provide a satisfactory estimate of the phases
for the unknown structure.
[0112] Thus, this method involves generating a preliminary model of
a molecule or molecular complex whose structure coordinates are
unknown, by orienting and positioning the relevant portion of S.
aureus thymidylate kinase or the S. aureus thymidylate
kinase/ligand complex according to FIG. 2 within the unit cell of
the crystal of the unknown molecule or molecular complex so as best
to account for the observed x-ray diffraction pattern of the
crystal of the molecule or molecular complex whose structure is
unknown. Phases can then be calculated from this model and combined
with the observed x-ray diffraction pattern amplitudes to generate
an electron density map of the structure whose coordinates are
unknown. This, in turn, can be subjected to any well-known model
building and structure refinement techniques to provide a final,
accurate structure of the unknown crystallized molecule or
molecular complex (E. Lattman, "Use of the Rotation and Translation
Functions," in Meth. Enzymol., 115, pp. 55-77 (1985); M. G.
Rossman, ed., "The Molecular Replacement Method," Int. Sci. Rev.
Ser., No. 13, Gordon & Breach, New York (1972)).
[0113] Structural information about a portion of any crystallized
molecule or molecular complex that is sufficiently structurally
homologous to a portion of S. aureus thymidylate kinase can be
resolved by this method. In addition to a molecule that shares one
or more structural features with S. aureus thymidylate kinase as
described above, a molecule that has similar bioactivity, such as
the same catalytic activity, substrate specificity or ligand
binding activity as S. aureus thymidylate kinase, may also be
sufficiently structurally homologous to S. aureus thymidylate
kinase to permit use of the structure coordinates of S. aureus
thymidylate kinase to solve its crystal structure.
[0114] In a preferred embodiment, the method of molecular
replacement is utilized to obtain structural information about a
molecule or molecular complex, wherein the molecule or molecular
complex comprises at least one S. aureus thymidylate kinase subunit
or homolog. A "subunit" of S. aureus thymidylate kinase is an S.
aureus thymidylate kinase molecule that has been truncated at the
N-terminus or the C-terminus, or both. In the context of the
present invention, a "homolog" of S. aureus thymidylate kinase is a
protein that contains one or more amino acid substitutions,
deletions, additions, or rearrangements with respect to the amino
acid sequence of S. aureus thymidylate kinase (SEQ ID NO:1), but
that, when folded into its native conformation, exhibits or is
reasonably expected to exhibit at least a portion of the tertiary
(three-dimensional) structure of S. aureus thymidylate kinase, For
example, structurally homologous molecules can contain deletions or
additions of one or more contiguous or noncontiguous amino acids,
such as a loop or a domain. Structurally homologous molecules also
include "modified" S. aureus thymidylate kinase molecules that have
been chemically or enzymatically derivatized at one or more
constituent amino acid, including side chain modifications,
backbone modifications, and N- and C-terminal modifications
including acetylation, hydroxylation, methylation, amidation, and
the attachment of carbohydrate or lipid moieties, cofactors, and
the like.
[0115] A heavy atom derivative of S. aureus thymidylate kinase is
also included as an S. aureus thymidylate kinase homolog. The term
"heavy atom derivative" refers to derivatives of S. aureus
thymidylate kinase produced by chemically modifying a crystal of S.
aureus thymidylate kinase, In practice, a crystal is soaked in a
solution containing heavy metal atom salts, or organometallic
compounds, e.g., lead chloride, gold thiomalate, thiomersal or
uranyl acetate, which can diffuse through the crystal and bind to
the surface of the protein. The location(s) of the bound heavy
metal atom(s) can be determined by x-ray diffraction analysis of
the soaked crystal. This information, in turn, is used to generate
the phase information used to construct three-dimensional structure
of the protein (T. L. Blundell and N. L. Johnson, Protein
Crystallography, Academic Press (1976)).
[0116] Because S. aureus thymidylate kinase can crystallize in more
than one crystal form, the structure coordinates of S. aureus
thymidylate kinase as provided by this invention are particularly
useful in solving the structure of other crystal forms of S. aureus
thymidylate kinase or S. aureus thymidylate kinase complexes.
[0117] The structure coordinates of S. aureus thymidylate kinase as
provided by this invention are particularly useful in solving the
structure of S. aureus thymidylate kinase mutants. Mutants may be
prepared, for example, by expression of S. aureus thymidylate
kinase cDNA previously altered in its coding sequence by
oligonucleotide-directed mutagenesis. Mutants may also be generated
by site-specific incorporation of unnatural amino acids into
thymidylate kinase proteins using the general biosynthetic method
of C. J. Noren et al., Science, 244:182-188 (1989). In this method,
the codon encoding the amino acid of interest in wild-type S.
aureus thymidylate kinase is replaced by a "blank" nonsense codon,
TAG, using oligonucleotide-directed mutagenesis. A suppressor tRNA
directed against this codon is then chemically aminoacylated in
vitro with the desired unnatural amino acid. The aminoacylated tRNA
is then added to an in vitro translation system to yield a mutant
S. aureus thymidylate kinase with the site-specific incorporated
unnatural amino acid.
[0118] Selenocysteine or selenomethionine may be incorporated into
wild-type or mutant S. aureus thymidylate kinase by expression of
S. aureus thymidylate kinase-encoding cDNAs in auxotrophic E. coli
strains (W. A. Hendrickson et al., EMBO J., 9(5):1665-1672 (1990)).
In this method, the wild-type or mutagenized S. aureus thymidylate
kinase cDNA may be expressed in a host organism on a growth medium
depleted of either natural cysteine or methionine (or both) but
enriched in selenocysteine or selenomethionine (or both).
Alternatively, selenomethionine analogues may be prepared by down
regulation methionine biosynthesis. (T. E. Benson et al., Nat.
Struct. Biol., 2:644-53 (1995); G. D. Van Duyne et al., J. Mol.
Biol. 229:105-24 (1993)).
[0119] The structure coordinates of S. aureus thymidylate kinase
listed in FIG. 2 are also particularly useful to solve the
structure of crystals of S. aureus thymidylate kinase, S. aureus
thymidylate kinase mutants or S. aureus thymidylate kinase homologs
co-complexed with a variety of chemical entities. This approach
enables the determination of the optimal sites for interaction
between chemical entities, including candidate S. aureus
thymidylate kinase inhibitors and S. aureus thymidylate kinase.
Potential sites for modification within the various binding site of
the molecule can also be identified. This information provides an
additional tool for determining the most efficient binding
interactions, for example, increased hydrophobic interactions,
between S. aureus thymidylate kinase and a chemical entity. For
example, high resolution x-ray diffraction data collected from
crystals exposed to different types of solvent allows the
determination of where each type of solvent molecule resides. Small
molecules that bind tightly to those sites can then be designed and
synthesized and tested for their S. aureus thymidylate kinase
inhibition activity.
[0120] All of the complexes referred to above may be studied using
well-known x-ray diffraction techniques and may be refined versus
1.5-3 .ANG. resolution x-ray data to an R value of about 0.20 or
less using computer software, such as X-PLOR (Yale University,
81992, distributed by Molecular Simulations, Inc.; see, e.g.,
Blundell & Johnson, supra; Meth. Enzymol., Vol. 114 & 115,
H. W. Wyckoff et al., eds., Academic Press (1985)). This
information may thus be used to optimize known S. aureus
thymidylate kinase inhibitors, and more importantly, to design new
S. aureus thymidylate kinase inhibitors.
[0121] The invention also includes the unique three-dimensional
configuration defined by a set of points defined by the structure
coordinates for a molecule or molecular complex structurally
homologous to S. aureus thymidylate kinase as determined using the
method of the present invention, structurally equivalent
configurations, and magnetic storage media comprising such set of
structure coordinates.
[0122] Further, the invention includes structurally homologous
molecules as identified using the method of the invention.
[0123] Homology Modeling
[0124] Using homology modeling, a computer model of an S. aureus
thymidylate kinase homolog can be built or refined without
crystallizing the homolog. First, a preliminary model of the S.
aureus thymidylate kinase homolog is created by sequence alignment
with S. aureus thymidylate kinase, secondary structure prediction,
the screening of structural libraries, or any combination of those
techniques. Computational software may be used to carry out the
sequence alignments and the secondary structure predictions.
Structural incoherences, e.g., structural fragments around
insertions and deletions, can be modeled by screening a structural
library for peptides of the desired length and with a suitable
conformation. For prediction of the side chain conformation, a side
chain rotamer library may be employed. If the S. aureus thymidylate
kinase homolog has been crystallized, the final homology model can
be used to solve the crystal structure of the homolog by molecular
replacement, as described above. Next, the preliminary model is
subjected to energy minimization to yield an energy minimized
model. The energy minimized model may contain regions where
stereochemistry restraints are violated, in which case such regions
are remodeled to obtain a final homology model. The homology model
is positioned according to the results of molecular replacement,
and subjected to further refinement comprising molecular dynamics
calculations.
[0125] Rational Drug Design
[0126] Computational techniques can be used to screen, identify,
select and/or design chemical entities capable of associating with
S. aureus thymidylate kinase or structurally homologous molecules.
Knowledge of the structure coordinates for S. aureus thymidylate
kinase permits the design and/or identification of synthetic
compounds and/or other molecules which have a shape complementary
to the conformation of the S. aureus thymidylate kinase binding
site. In particular, computational techniques can be used to
identify or design chemical entities, such as inhibitors, agonists
and antagonists, that associate with an S. aureus thymidylate
kinase substrate binding pocket or an S. aureus thymidylate
kinase-like substrate binding pocket. Inhibitors may bind to or
interfere with all or a portion of an active site of S. aureus
thymidylate kinase, and can be competitive, non-competitive, or
uncompetitive inhibitors; or interfere with dimerization by binding
at the interface between the two monomers. Once identified and
screened for biological activity, these
inhibitors/agonists/antagonists may be used therapeutically or
prophylactically to block S. aureus thymidylate kinase activity
and, thus, inhibit the growth of the bacteria or cause its death.
Structure-activity data for analogues of ligands that bind to or
interfere with S. aureus thymidylate kinase or S. aureus
thymidylate kinase-like substrate binding pockets can also be
obtained computationally.
[0127] The term "chemical entity," as used herein, refers to
chemical compounds, complexes of two or more chemical compounds,
and fragments of such compounds or complexes. Chemical entities
that are determined to associate with S. aureus thymidylate kinase
are potential drug candidates.
[0128] Data stored in a machine-readable storage medium that is
capable of displaying a graphical three-dimensional representation
of the structure of S. aureus thymidylate kinase or a structurally
homologous molecule, as identified herein, or portions thereof may
thus be advantageously used for drug discovery. The structure
coordinates of the chemical entity are used to generate a
three-dimensional image that can be computationally fit to the
three-dimensional image of S. aureus thymidylate kinase or a
structurally homologous molecule. The three-dimensional molecular
structure encoded by the data in the data storage medium can then
be computationally evaluated for its ability to associate with
chemical entities. When the molecular structures encoded by the
data is displayed in a graphical three-dimensional representation
on a computer screen, the protein structure can also be visually
inspected for potential association with chemical entities.
[0129] One embodiment of the method of drug design involves
evaluating the potential association of a known chemical entity
with S. aureus thymidylate kinase or a structurally homologous
molecule, particularly with an S. aureus thymidylate kinase
substrate binding pocket or S. aureus thymidylate kinase-like
substrate binding pocket. The method of drug design thus includes
computationally evaluating the potential of a selected chemical
entity to associate with any of the molecules or molecular
complexes set forth above. This method comprises the steps of: (a)
employing computational means to perform a fitting operation
between the selected chemical entity and a substrate binding pocket
or a pocket nearby the substrate binding pocket of the molecule or
molecular complex; and (b) analyzing the results of said fitting
operation to quantify the association between the chemical entity
and the substrate binding pocket.
[0130] In another embodiment, the method of drug design involves
computer-assisted design of chemical entities that associate with
S. aureus thymidylate kinase, its homologs, or portions thereof.
Chemical entities can be designed in a step-wise fashion, one
fragment at a time, or may be designed as a whole or "de novo."
[0131] To be a viable drug candidate, the chemical entity
identified or designed according to the method must be capable of
structurally associating with at least part of an S. aureus
thymidylate kinase or S. aureus thymidylate kinase-like substrate
binding pockets, and must be able, sterically and energetically, to
assume a conformation that allows it to associate with the S.
aureus thymidylate kinase or S. aureus thymidylate kinase-like
substrate binding pocket. Non-covalent molecular interactions
important in this association include hydrogen bonding, van der
Waals interactions, hydrophobic interactions, and electrostatic
interactions. Conformational considerations include the overall
three-dimensional structure and orientation of the chemical entity
in relation to the substrate binding pocket, and the spacing
between various functional groups of an entity that directly
interact with the S. aureus thymidylate kinase-like substrate
binding pocket or homologs thereof.
[0132] Optionally, the potential binding of a chemical entity to an
S. aureus thymidylate kinase or S. aureus thymidylate kinase-like
substrate binding pocket is analyzed using computer modeling
techniques prior to the actual synthesis and testing of the
chemical entity. If these computational experiments suggest
insufficient interaction and association between it and the S.
aureus thymidylate kinase or S. aureus thymidylate kinase-like
substrate binding pocket, testing of the entity is obviated.
However, if computer modeling indicates a strong interaction, the
molecule may then be synthesized and tested for its ability to bind
to or interfere with an S. aureus thymidylate kinase or S. aureus
thymidylate kinase-like substrate binding pocket. Binding assays to
determine if a compound actually interferes with S. aureus
thymidylate kinase can also be performed and are well known in the
art. Binding assays may employ kinetic or thermodynamic methodology
using a wide variety of techniques including, but not limited to,
microcalorimetry, circular dichroism, capillary zone
electrophoresis, nuclear magnetic resonance spectroscopy,
fluorescence spectroscopy, and combinations thereof.
[0133] One skilled in the art may use one of several methods to
screen chemical entities or fragments for their ability to
associate with an S. aureus thymidylate kinase or S. aureus
thymidylate kinase-like substrate binding pocket. This process may
begin by visual inspection of, for example, an S. aureus
thymidylate kinase or S. aureus thymidylate kinase-like substrate
binding pocket on the computer screen based on the S. aureus
thymidylate kinase structure coordinates listed in FIG. 2 or other
coordinates which define a similar shape generated from the
machine-readable storage medium. Selected fragments or chemical
entities may then be positioned in a variety of orientations, or
docked, within the substrate binding pocket. Docking may be
accomplished using software such as QUANTA and SYBYL, followed by
energy minimization and molecular dynamics with standard molecular
mechanics forcefields, such as CHARMM and AMBER.
[0134] Specialized computer programs may also assist in the process
of selecting fragments or chemical entities. Examples include GRID
(P. J. Goodford, J. Med. Chem. 28:849-857 (1985); available from
Oxford University, Oxford, UK); MCSS (A. Miranker et al., Proteins:
Struct. Funct. Gen., 11:29-34 (1991); available from Molecular
Simulations, San Diego, Calif.); AUTODOCK (D. S. Goodsell et al.,
Proteins: Struct. Funct. Genet. 8:195-202 (1990); available from
Scripps Research Institute, La Jolla, Calif.); and DOCK (I. D.
Kuntz et al., J. Mol. Biol. 161:269-288 (1982); available from
University of California, San Francisco, Calif.).
[0135] Once suitable chemical entities or fragments have been
selected, they can be assembled into a single compound or complex.
Assembly may be preceded by visual inspection of the relationship
of the fragments to each other on the three-dimensional image
displayed on a computer screen in relation to the structure
coordinates of S. aureus thymidylate kinase. This would be followed
by manual model building using software such as QUANTA or SYBYL
(Tripos Associates, St. Louis, Mo.).
[0136] Useful programs to aid one of skill in the art in connecting
the individual chemical entities or fragments include, without
limitation, CAVEAT (P. A. Bartlett et al., in Molecular Recognition
in Chemical and Biological Problems," Special Publ., Royal Chem.
Soc., 78:182-196 (1989); G. Lauri et al., J. Comput. Aided Mol.
Des. 8:51-66 (1994); available from the University of California,
Berkeley, Calif.); 3D database systems such as ISIS (available from
MDL Information Systems, San Leandro, Calif.; reviewed in Y. C.
Martin, J. Med. Chem. 35:2145-2154 (1992)); and HOOK (M. B. Eisen
et al., Proteins: Struc., Funct., Genet. 19:199-221 (1994);
available from Molecular Simulations, San Diego, Calif.).
[0137] S. aureus thymidylate kinase binding compounds may be
designed "de novo" using either an empty binding site or optionally
including some portion(s) of a known inhibitor(s). There are many
de novo ligand design methods including, without limitation, LUDI
(H.-J. Bohm, J. Comp. Aid. Molec. Design. 6:61-78 (1992); available
from Molecular Simulations Inc., San Diego, Calif.); LEGEND (Y.
Nishibata et al., Tetrahedron, 47:8985 (1991); available from
Molecular Simulations Inc., San Diego, Calif.); LeapFrog (available
from Tripos Associates, St. Louis, Mo.); and SPROUT (V. Gillet et
al., J. Comput. Aided Mol. Design 7:127-153 (1993); available from
the University of Leeds, UK).
[0138] Once a compound has been designed or selected by the above
methods, the efficiency with which that entity may bind to or
interfere with an S. aureus thymidylate kinase or S. aureus
thymidylate kinase-like substrate binding pocket may be tested and
optimized by computational evaluation. For example, an effective S.
aureus thymidylate kinase or S. aureus thymidylate kinase-like
substrate binding pocket inhibitor must preferably demonstrate a
relatively small difference in energy between its bound and free
states (i.e., a small deformation energy of binding). Thus, the
most efficient S. aureus thymidylate kinase or S. aureus
thymidylate kinase-like substrate binding pocket inhibitors should
preferably be designed with a deformation energy of binding of not
greater than about 10 kcal/mole; more preferably, not greater than
7 kcal/mole. S. aureus thymidylate kinase or S. aureus thymidylate
kinase-like substrate binding pocket inhibitors may interact with
the substrate binding pocket in more than one conformation that is
similar in overall binding energy. In those cases, the deformation
energy of binding is taken to be the difference between the energy
of the free entity and the average energy of the conformations
observed when the inhibitor binds to the protein.
[0139] An entity designed or selected as binding to or interfering
with an S. aureus thymidylate kinase or S. aureus thymidylate
kinase-like substrate binding pocket may be further computationally
optimized so that in its bound state it would preferably lack
repulsive electrostatic interaction with the target enzyme and with
the surrounding water molecules. Such non-complementary
electrostatic interactions include repulsive charge-charge,
dipole-dipole, and charge-dipole interactions.
[0140] Specific computer software is available in the art to
evaluate compound deformation energy and electrostatic
interactions. Examples of programs designed for such uses include:
Gaussian 94, revision C (M. J. Frisch, Gaussian, Inc., Pittsburgh,
Pa. 81995); AMBER, version 4.1 (P. A. Kollman, University of
California at San Francisco, 81995); QUANTA/CHARMM (Molecular
Simulations, Inc., San Diego, Calif. 81995); Insight II/Discover
(Molecular Simulations, Inc., San Diego, Calif. 81995); DelPhi
(Molecular Simulations, Inc., San Diego, Calif. 81995); and AMSOL
(Quantum Chemistry Program Exchange, Indiana University). These
programs may be implemented, for instance, using a Silicon Graphics
workstation such as an Indigo.sup.2 with "IMPACT" graphics. Other
hardware systems and software packages will be known to those
skilled in the art.
[0141] Another approach encompassed by this invention is the
computational screening of small molecule databases for chemical
entities or compounds that can bind in whole, or in part, to a S.
aureus thymidylate kinase or S. aureus thymidylate kinase-like
substrate binding pocket. In this screening, the quality of fit of
such entities to the binding site may be judged either by shape
complementarity or by estimated interaction energy (E. C. Meng et
al., J. Comp. Chem., 13, pp. 505-524 (1992)).
[0142] This invention also enables the development of chemical
entities that can isomerize to short-lived reaction intermediates
in the chemical reaction of a substrate or other compound that
interferes with or with S. aureus thymidylate kinase.
Time-dependent analysis of structural changes in S. aureus
thymidylate kinase during its interaction with other molecules is
carried out. The reaction intermediates of S. aureus thymidylate
kinase can also be deduced from the reaction product in co-complex
with S. aureus thymidylate kinase. Such information is useful to
design improved analogues of known S. aureus thymidylate kinase
inhibitors or to design novel classes of inhibitors based on the
reaction intermediates of the S. aureus thymidylate kinase and
inhibitor co-complex. This provides a novel route for designing S.
aureus thymidylate kinase inhibitors with both high specificity and
stability.
[0143] Yet another approach to rational drug design involves
probing the S. aureus thymidylate kinase crystal of the invention
with molecules comprising a variety of different functional groups
to determine optimal sites for interaction between candidate S.
aureus thymidylate kinase inhibitors and the protein. For example,
high resolution x-ray diffraction data collected from crystals
soaked in or co-crystallized with other molecules allows the
determination of where each type of solvent molecule sticks.
Molecules that bind tightly to those sites can then be further
modified and synthesized and tested for their thymidylate kinase
inhibitor activity (J. Travis, Science, 262:1374 (1993)).
[0144] In a related approach, iterative drug design is used to
identify inhibitors of S. aureus thymidylate kinase. Iterative drug
design is a method for optimizing associations between a protein
and a compound by determining and evaluating the three-dimensional
structures of successive sets of protein/compound complexes. In
iterative drug design, crystals of a series of protein/compound
complexes are obtained and then the three-dimensional structures of
each complex is solved. Such an approach provides insight into the
association between the proteins and compounds of each complex.
This is accomplished by selecting compounds with inhibitory
activity, obtaining crystals of this new protein/compound complex,
solving the three dimensional structure of the complex, and
comparing the associations between the new protein/compound complex
and previously solved protein/compound complexes. By observing how
changes in the compound affected the protein/compound associations,
these associations may be optimized.
[0145] A compound that is identified or designed as a result of any
of these methods can be obtained (or synthesized) and tested for
its biological activity, e.g., inhibition of thymidylate kinase
activity.
[0146] Pharmaceutical Compositions (Inhibitors)
[0147] Pharmaceutical compositions of this invention comprise an
inhibitor of S. aureus TMK activity identified according to the
invention, or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier, adjuvant, or vehicle. The term
"pharmaceutically acceptable carrier" refers to a carrier(s) that
is "acceptable" in the sense of being compatible with the other
ingredients of a composition and not deleterious to the recipient
thereof. Optionally, the pH of the formulation is adjusted with
pharmaceutically acceptable acids, bases, or buffers to enhance the
stability of the formulated compound or its delivery form.
[0148] Methods of making and using such pharmaceutical compositions
are also included in the invention. The pharmaceutical compositions
of the invention can be administered orally, parenterally, by
inhalation spray, topically, rectally, nasally, buccally,
vaginally, or via an implanted reservoir. Oral administration or
administration by injection is preferred. The term parenteral as
used herein includes subcutaneous, intracutaneous, intravenous,
intramuscular, intra-articular, intrasynovial, intrasternal,
intrathecal, intralesional, and intracranial injection or infusion
techniques.
[0149] Dosage levels of between about 0.01 and about 100 mg/kg body
weight per day, preferably between about 0.5 and about 75 mg/kg
body weight per day of the S. aureus TMK inhibitory compounds
described herein are useful for the prevention and treatment of S.
aureus TMK mediated disease. Typically, the pharmaceutical
compositions of this invention will be administered from about 1 to
about 5 times per day or alternatively, as a continuous infusion.
Such administration can be used as a chronic or acute therapy. The
amount of active ingredient that may be combined with the carrier
materials to produce a single dosage form will vary depending upon
the host treated and the particular mode of administration. A
typical preparation will contain from about 5% to about 95 active
compound (w/w). Preferably, such preparations contain from about
20% to about 80% active compound.
[0150] In order that this invention be more fully understood, the
following examples are set forth. These examples are for the
purpose of illustration only and are not to be construed as
limiting the scope of the invention in any way.
EXAMPLES
Example 1
Analysis of the Structure of S. aureus Thymidylate Kinase
[0151] A. Expression, Purification and Crystallization
[0152] The M15-1C Escherichia coli construct expressing S. aureus
thymidylate kinase was obtained as a strain in which the Qiagen
pREP4 vector was replaced with pREP4UX. Genes and polypeptides
derived from S. aureus, including S. aureus and thymidylate kinase,
are published in EP 786519 A2 and WO 0012678, both assigned to
Human Genome Sciences. This plasmid contains the argU gene which
codes for the AGA tRNA and prevents the lysine for arginine
substitution which occurred in the original construct from Human
Genome Sciences. For preparation of the selenomethionine analogue
of thymidylate kinase, the construct was grown in a minimal salts
medium, M9, which contained glucose and NH.sub.4Cl as the sources
of carbon and nitrogen. Endogenous methionine biosynthesis was then
inhibited while adding an excess of selenomethionine to the growth
medium just prior to IPTG induction of thymidylate kinase synthesis
(T. E. Benson et al., Nat. Struct. Biol., 2:644-53 (1995); G. D.
Van Duyne et al., J. Mol. Biol. 229:105-24 (1993)). The formulation
of basal M9 was Na.sub.2HPO.sub.4, 6 g; KH.sub.2PO.sub.4, 3 g;
NH.sub.4Cl, 1.0 g; and NaCl, 0.5 g per L of deionized water. The pH
was adjusted to 7.4 with concentrated KOH and the medium was
sterilized by autoclaving. Prior to inoculation, the following
filter sterilized solutions were added per L of basal medium: 1M
MgSO.sub.4, 1.0 mL; 1M CaCl.sub.2, 0.1 mL; trace metal salts
solution, 0.1 mL, 10 mM thiamin, 1.0 mL; and 20% glucose, 20 mL.
The trace metal salts solution contained per L of deionized water:
MgCl.sub.2.6H.sub.2O, 39.44 g; MnSO.sub.4.H.sub.2O, 5.58 g;
FeSO.sub.4.7H.sub.2O, 1.11 g; Na.sub.2MoO.sub.4.2H.sub.2O, 0.48 g;
CaCl.sub.2, 0.33 g; NaCl, 0.12 g; and ascorbic acid, 1.0 g. Filter
sterilized ampicillin and kanamycin were added to the medium at
final concentrations of 100 mg/mL and 30 mg/mL, respectively.
[0153] Fermentations were prepared in 100 mL volumes of M9 medium
contained in 500 mL wide mouth flasks. A 0.1 mL aliquot of the
stock culture was inoculated into the medium and allowed to grow at
30.degree. C. for 18-20 hours with a shaking rate of 200 rpm. The
seed culture was harvested by centrifugation and then resuspended
in an equal volume of M9 medium. The resuspended seed was used to
inoculate expression fermentations at a rate of 3%. For expression,
the culture was grown under the same conditions to an A600 of
.about.0.6. At this point, methionine biosynthesis was down
regulated by the addition of L-lysine, L-threonine, and
L-phenylalanine at a final concentration for each of 100 mg/mL and
L-leucine, L-isoleucine, and L-valine at 50 mg/mL each.
D,L-selenomethionine was added simultaneously to a final
concentration of 100 mg/mL. After 15-20 minutes, expression of
thymidylate kinase was induced by addition of IPTG (isopropyl
thio-.beta.-D-galactosidase, Gibco BRL) to 1 mM. Growth of the
culture was continued for an additional 3.5 hours until an A600 of
1.5-1.6. Cells were then harvested by centrifugation and frozen at
-80.degree. C. Under these conditions, the average yield of cell
paste was 3.0 to 3.5 g/L.
[0154] For protein purification, all buffers were chilled to
4.degree. C. prior to use and all procedures were performed at
4.degree. C. Cells (24.8 g wet weight) were resuspended in 125 mL
of lysis buffer (25 mM Tris (pH 7.8), 500 mM NaCl, 10% glycerol, 25
mM imidazole, 5 mM 2-mercaptoethanol, 0.2 mg/mL DNAse I) and
ruptured by using an American Instrument French Press at 16,000
PSI. The lysate was clarified by centrifugation at 39,200.times.g
for 60 minutes in a JA20 rotor. The supernatant was filtered by
using a Nalgene 0.2 .mu.m filter unit. The filtered supernatant was
applied at 74 cm/hr to a Qiagen NTA Superflow column (1.6 cm
i.d..times.11 cm (CV=22 mL)) charged with nickel that was
pre-equilibrated with EQ buffer (25 mM Tris (pH 7.8), 500 mM NaCl,
10% glycerol, 25 mM imidazole, 5 mM 2-mercaptoethanol). The column
was washed with 7.7 CV of EQ buffer, 12.5 CV of wash buffer (25 mM
Tris (pH 7.8), 500 mM NaCl, 10% glycerol, 50 mM imidazole, 5 mM
2-mercaptoethanol) and eluted with 1.4 CV of elution buffer (25 mM
Tris (pH 7.8), 500 mM NaCl, 10% glycerol, 300 mM imidazole, 5 mM
2-mercaptoethanol). During the elution the linear velocity was
decreased to 42 cm/hr. The eluted fraction was treated with DTT to
achieve a final concentration of 10 mM and dialyzed extensively
against nitrogen sparged dialysis buffer (25 mM Tris (pH 7.8), 500
mM NaCl, 10% glycerol, 10 mM DTT, pH 7.8).
[0155] The Mono Q analytical run was performed using 50 mL native
TMK (14 mg/mL) diluted to 200 mL with 20 mM Tris (pH 8.0). The
sample was loaded onto a Mono Q (Amersham Pharmacia Biotech) column
equilibrated with 20 mM Tris (pH 8.0) and run through a 20-40% (20
mM Tris (pH 8.0)+1.0M NaCl) gradient in 40 mL with a flow rate of
1.0 mL/min. The Mono P column run was performed using 50 mL TMK (14
mg/mL) diluted to 200 mL with 25 mM bis-Tris (pH 6.71). The sample
was injected onto a Mono P column (Amersham Pharmacia Biotech)
equilibrated with 25 mM bis-Tris (pH 6.71) and run through a step
gradient of 0-100-0% Polybuffer Mix 96/74 (20:1), pH 5.80. Gel
filtration studies were carried out on a Superose 200 column with a
500 mL sample of thymidylate kinase at a concentration of 4.2 mg/mL
using 50 mM Tris (pH 8.5), 500 mM NaCl, 5 mM 2-mercaptoethanol, and
0.5% glycerol at a flow rate of 1 mL/min. For dynamic light
scattering experiments, samples were mixed in 1.5 ml eppendorf
tubes, then sterile filtered through a 0.22 mm ceramic membrane
(Whatman). 20 mL of solution is read in a quartz cuvette in a Dyna
Pro Molecular Sizing Instrument (Protein Solutions, Inc.,
Charlottesville, Va.).
[0156] The native protein was exchanged into 50 mM Tris (pH 7.8), 5
mM 2-mercaptoethanol to a concentration of 15 mg/mL and screened
for crystallization conditions using Crystal Screen I, Crystal
Screen II, and MembFac Screen (Hampton Research, Laguna Niguel,
Calif.). The most encouraging lead was from Hampton Crystal Screen
I condition 23: 30% PEG 400, 0.1M Na HEPES pH 7.5, 0.2M MgCl.sub.2.
Follow up screens indicated that PIPES buffer was most conducive to
crystal formation.
[0157] The initial crystals of the thymidylate kinase were stacks
of small plates that were inseparable and unusable for diffraction
studies. Biochemical analysis of the protein revealed that the
sample was substantially pure by sodium dodecylsulfate
polyacrylamide-gel electrophoresis (SDS-PAGE) analysis, but
isoelectric focusing (IEF) gels revealed at least two distinct
isoelectric species. It is likely, although yet unproven, that
these isoelectric species were the cause of the morphology of the
thymidylate kinase crystals. Further efforts at purification with a
Mono Q column indicated that separation of these species would be
difficult and it was not clear that large scale isoelectrofocusing
using a Mono P column or preparative isoelectric focusing Would
improve the separation because of the small differences in pI. A
series experiments exploring the feasibility of preparative
isoelectric focusing experiments using PrIME (preparative
isoelectric membrane electrophoresis) was hampered due to
precipitation of the protein near its pI. Gel filtration did reveal
that thymidylate kinase behaves as a dimer in solution, confirming
earlier literature reports for the related E. coli and yeast TMK
The initial crystallization conditions contained 200 mM MgCl.sub.2
and later experiments showed that at least 150 mM MgCl.sub.2 was
required for crystal formation. Dynamic light scattering
experiments in the presence of MgCl.sub.2 revealed an interesting
phenomenon where protein aggregation was reduced in the presence of
MgCl.sub.2 over a number of hours leading to a monodisperse,
dimeric sample suitable for crystallization.
[0158] The stacked plates were eventually transformed into single
crystals through iterative streak seeding and crystallization on
hanging or sitting drops with thymidylate kinase in 0.1 M PIPES (pH
6.6), 14-19% PEG 400, 0.2 M MgCl2. This technique involved taking
the multinucleated crystals, crushing them into microcrystals, and
using a dilution series of this suspension of microcrystals for
seeding. It was observed that this second round of crystals were
usually less multinucleated than when crystal formation was allowed
to proceed via spontaneous nucleation. A second round of streak
seeding was usually necessary in order to obtain multiple single
crystals. Refinement of the streak seeding technique resulted in
native and selenomethionine TMK crystals on the order of about 100
.mu.m.times.about 100 .mu.m.times.about 20 .mu.m.
[0159] Subsequent crystallization experiments also indicated that a
protein concentration of 7 mg/mL was able to yield suitable
crystals. The crystallization solution was a cryoprotective agent
making it straightforward to freeze the crystals in liquid nitrogen
for data collection. Selenomethionine thymidylate kinase was
exchanged into 10 mM Tris (pH 7.8), 10 mM DTT and concentrated to 7
or 14 mg/mL for crystallization experiments.
[0160] B. X-Ray Diffraction Characterization
[0161] Thymidylate kinase crystals were generally too small for
useful data collection using standard x-ray diffraction equipment.
Therefore, all data collection was carried out at the Advanced
Photon Source (Argonne, Ill.). The structure of S. aureus
thymidylate kinase was determined by multiple anomalous dispersion
(MAD) using synchrotron radiation. Crystals were of the space group
P2.sub.1, with cell constants a=49.8 .ANG., b=90.1 .ANG., c=46.5
.ANG., .alpha.=.gamma.=90.degree. and .beta.=101.8.degree.. The
Matthews coefficient for these crystals assuming that there are two
molecules in the asymmetric unit is 2.1 .ANG./Da with 40% solvent.
Two MAD data sets were collected B one at 2.7 .ANG. and one at 2.3
.ANG..
[0162] Two selenomethionine multiple anomalous dispersion (MAD)
experiments were performed (2.7 .ANG. resolution and 2.3 .ANG.
resolution) using three different wavelengths (remote wavelength
1.0332 .ANG., 12000 eV, inflection point wavelength 0.979746 .ANG.,
12654.8 eV, and the peak wavelength 0.979617 .ANG., 12656.5
eV).
[0163] C. Heavy Atom Derivative
[0164] Selenomethionine thymidylate kinase was expressed using
downregulation of methionine biosynthesis (T. E. Benson et al.,
Nat. Struct. Biol., 2:644-53 (1995); G. D. Van Duyne et al., J.
Mol. Biol. 229:105-24 (1993)) and purified in order to obtain de
novo phases by multiple anomalous dispersion (W. A. Hendrickson,
Science 254:51-8 (1991)). Anomalous difference Patterson maps
revealed six selenium sites (three for each of the two monomers in
the asymmetric unit) (FIG. 9). Patterson maps at 2.7 .ANG. showed
that the atomic positions for the seleniums were not well resolved,
but maps at 2.3 .ANG. clearly defined the atomic positions of the
heavy atoms. Unfortunately, the MAD phases for data collected at
2.3 .ANG. were of lower quality than the phases at 2.7 .ANG., so
initial model building was performed using the MAD phased map to
2.7 .ANG. (FIG. 10). Subsequent refinement was conducted against
the 2.3 .ANG. data, and this higher resolution structure is the one
reported here.
[0165] D. Phase Combination
[0166] Each of these individual data sets was indexed and
integrated separately (see Tables 7 and 8 for integration
statistics). The data sets were scaled to each other using the
program SCALEIT in the CCP4 Program Suite (Collaborative
Computational Project N4, Acta Cryst. D50:760-3 (1994)). Patterson
maps revealed six selenium sites (three for each monomer in the
asymmetric unit) whose locations were determined by direct methods
using SHELX (G. M. Sheldrick & R. O. Gould, Acta Cryst.
B51:423-31 (1995)). Heavy atom refinement and phase calculations
were conducted using SHARP (E. La Fortelle et al., A
Maximum-Likelihood Heavy-Atom Parameter Refinement and Phasing
Program for the MIR and MAD Methods, P. Boume & K. Watenpaugh,
eds., Crystallographic Computing 7 (1997)). Phases calculated in
SHARP were solvent flattened using the program SOLOMON
(Collaborative Computational Project N4, Acta Cryst. D50:760-3
(1994)) and gave a significantly improved electron density map.
8TABLE 7 Data collection and phasing statistics for structure of S.
aureus TMK .lambda. 1.0332 .ANG. .lambda. 0.979746 .ANG. .lambda.
0.979617 .ANG. (12000 eV) (12654.8 eV) (12656.5 eV) Resolution 2.7
.ANG. 2.7 .ANG. 2.7 .ANG. No. observations 76,132 62,273 76,145 No.
unique refl. 10,901 10,941 10,928 % completeness 100% 100% 100%
R.sub.sym 0.085 0.103 0.106 R.sub.cullis acentrics -- 0.61 0.67
R.sub.cullis anomalous 0.98 0.78 0.69 Phasing power centrics --
1.28 1.21 acentrics -- 2.30 1.83 Mean figure of merit (to 2.7 .ANG.
resolution) before solvent 0.51 flattening after solvent flattening
0.94
[0167]
9TABLE 8 Data collection and phasing statistics for structure of S.
aureus TMK .lambda. 1.0332 .ANG. .lambda. 0.979746 .ANG. .lambda.
0.979617 .ANG. (12000 eV) (12654.8 eV) (12656.5 eV) Resolution 2.3
.ANG. 2.3 .ANG. 2.3 .ANG. No. Observations 76,712 123,553 123,372
No. unique refl. 17,661 17,887 17,991 % completeness 98.2% 99.4%
99.3% R.sub.sym 0.083 0.107 0.099 R.sub.cullis acentrics -- 0.56
0.61 R.sub.cullis anomalous 0.99 0.69 0.70 Phasing power centrics
-- 1.34 1.38 acentrics -- 2.22 2.04 Mean figure of merit (to 2.3
.ANG. resolution) before solvent 0.57 flattening after solvent
flattening 0.87
[0168] E. Model Building and Refinement
[0169] At this stage in the structure solution, the coordinates for
E. coli thymidylate kinase greatly aided the process of model
building for placement of the main chain backbone. Model building
was done using the program CHAIN (J. S. Sack, Journal of Molecular
Graphics 6:224-5 (1988)) and LORE (B. C. Finzel, Meth, Enzymol.
277:230-42 (1997)). Refinement was carried out with XPLOR98 (A. T.
Brunger, X-PLOR version 3.1: A system for X-ray Crystallography and
NMR, New Haven: Yale Univ. Press, (1992)) incorporating bulk
solvent correction during the refinement (J. S. Jiang & A. T.
Brunger, J. Mol. Biol. 243:100-15 (1994)). Progress of the
refinement was monitored by a decrease in both the R-factor and
Free R-factor.
10TABLE 9 Refinement Statistics for structure of S. aureus TMK
R-factor Free R-factor No. of reflections 20-2.3 .ANG. F .gtoreq.
2.sigma. 0.2366 0.3084 15,908 Bonds (.ANG.) Angles(.degree.) r.m.s
deviation from ideal geometry 0.008 1.32 Number of atoms Average
B-factor Protein 2978 27.2 Waters 174 38.9 Total 3152 27.81
[0170] Stereochemistry of the model was checked using PROCHECK (R.
A. Laskowski et al., J. Appl. Cryst. 26:283-91 (1993)) revealing
no-residues in disallowed regions of the Ramachandran plot. FIG. 9
was made using SETOR (S. V. Evans, J. Mol. Graphics 11:134-8
(1993)) and FIGS. 3a, 4 were produced in MOLSCRIPT (P. Kraulis, J.
Appl. Cryst. 24:946-50 (1991)) and Raster 3D (E. A. Merritt &
M. E. P. Murphy, Acta Cryst. D50:869-73 (1994)) while FIGS. 5a and
6a were produced in MOLSCRIPT (P. Kraulis, J. Appl., Cryst.
24:946-50 (1991)) alone.
[0171] F. Assays.
[0172] Binding assays to determine if a compound actually
interferes with S. aureus thymidylate kinase can also be performed.
For example, thymidylate kinase activity can be measured by
coupling the formation of ADP and TDP to the reactions catalyzed by
PD, LDH, and NDP-Kinase, as shown below. Oxidation of NADH is
accompanied by a decrease in absorbance at 340 nm, which is
measured spectrophotometrically. 1
[0173] The standard reaction conditions employed during the kinetic
characterization of the enzyme were: 50 mM HEPES, pH 8.0, 50 mM
KCl, 2 mM MgCl.sub.2, 4 U/ml PK, 5 U/ml LDH, 2 mM PEP, 1.5 mM ATP,
5 U/ml NDP-Kinase, 1.0 mM TMP, 0.22 mM NADH, and 0.8 .mu.g/ml T.
kinase. All of the reagents except the T. Kinase were added to a
cuvette and mixed, and the mixture was incubated at 24.5.degree. C.
for 2 minutes. To start the reaction, the T. Kinase was added, the
contents of the cuvette were mixed, and the decrease in absorbance
at 340 nm was monitored for 4-5 minutes.
Sequence Listing Free Text
[0174] SEQ ID NO:1 recombinant S. aureus thymidylate kinase (with
polyhistidine [his.sub.6] sequence tag)
[0175] SEQ ID NO:2 E. coli thymidylate kinase
[0176] SEQ ID NO:3 S. cerevisiae thymidylate kinase
Sequence CWU 1
1
3 1 214 PRT Artificial Sequence Description of Artificial Sequence
Recombinant Staphylococcus aureus thymidylate kinase with 6-His tag
1 Met Gly Ser Ala Phe Ile Thr Phe Glu Gly Pro Glu Gly Ser Gly Lys 1
5 10 15 Thr Thr Val Ile Asn Glu Val Tyr His Arg Leu Val Lys Asp Tyr
Asp 20 25 30 Val Ile Met Thr Arg Glu Pro Gly Gly Val Pro Thr Gly
Glu Glu Ile 35 40 45 Arg Lys Ile Val Leu Glu Gly Asn Asp Met Asp
Ile Arg Thr Glu Ala 50 55 60 Met Leu Phe Ala Ala Ser Arg Arg Glu
His Leu Val Leu Lys Val Ile 65 70 75 80 Pro Ala Leu Lys Glu Gly Lys
Val Val Leu Cys Asp Arg Tyr Ile Asp 85 90 95 Ser Ser Leu Ala Tyr
Gln Gly Tyr Ala Arg Gly Ile Gly Val Glu Glu 100 105 110 Val Arg Ala
Leu Asn Glu Phe Ala Ile Asn Gly Leu Tyr Pro Asp Leu 115 120 125 Thr
Ile Tyr Leu Asn Val Ser Ala Glu Val Gly Arg Glu Arg Ile Ile 130 135
140 Lys Asn Ser Arg Asp Gln Asn Arg Leu Asp Gln Glu Asp Leu Lys Phe
145 150 155 160 His Glu Lys Val Ile Glu Gly Tyr Gln Glu Ile Ile His
Asn Glu Ser 165 170 175 Gln Arg Phe Lys Ser Val Asn Ala Asp Gln Pro
Leu Glu Asn Val Val 180 185 190 Glu Asp Thr Tyr Gln Thr Ile Ile Lys
Tyr Leu Glu Lys Ile Arg Ser 195 200 205 His His His His His His 210
2 213 PRT Escherichia coli 2 Met Arg Ser Lys Tyr Ile Val Ile Glu
Gly Leu Glu Gly Ala Gly Lys 1 5 10 15 Thr Thr Ala Arg Asn Val Val
Val Glu Thr Leu Glu Gln Leu Gly Ile 20 25 30 Arg Asp Met Val Phe
Thr Arg Glu Pro Gly Gly Thr Gln Leu Ala Glu 35 40 45 Lys Leu Arg
Ser Leu Val Leu Asp Ile Lys Ser Val Gly Asp Glu Val 50 55 60 Ile
Thr Asp Lys Ala Glu Val Leu Met Phe Tyr Ala Ala Arg Val Gln 65 70
75 80 Leu Val Glu Thr Val Ile Lys Pro Ala Leu Ala Asn Gly Thr Trp
Val 85 90 95 Ile Gly Asp Arg His Asp Leu Ser Thr Gln Ala Tyr Gln
Gly Gly Gly 100 105 110 Arg Gly Ile Asp Gln His Met Leu Ala Thr Leu
Arg Asp Ala Val Leu 115 120 125 Gly Asp Phe Arg Pro Asp Leu Thr Leu
Tyr Leu Asp Val Thr Pro Glu 130 135 140 Val Gly Leu Lys Arg Ala Arg
Ala Arg Gly Glu Leu Asp Arg Ile Glu 145 150 155 160 Gln Glu Ser Phe
Asp Phe Phe Asn Arg Thr Arg Ala Arg Tyr Leu Glu 165 170 175 Leu Ala
Ala Gln Asp Lys Ser Ile His Thr Ile Asp Ala Thr Gln Pro 180 185 190
Leu Glu Ala Val Met Asp Ala Ile Arg Thr Thr Val Thr His Trp Val 195
200 205 Lys Glu Leu Asp Ala 210 3 216 PRT Saccharomyces cerevisiae
3 Met Met Gly Arg Gly Lys Leu Ile Leu Ile Glu Gly Leu Asp Arg Thr 1
5 10 15 Gly Lys Thr Thr Gln Cys Asn Ile Leu Tyr Lys Lys Leu Gln Pro
Asn 20 25 30 Cys Lys Leu Leu Lys Phe Pro Glu Arg Ser Thr Arg Ile
Gly Gly Leu 35 40 45 Ile Asn Glu Tyr Leu Thr Asp Asp Ser Phe Gln
Leu Ser Asp Gln Ala 50 55 60 Ile His Leu Leu Phe Ser Ala Asn Arg
Trp Glu Ile Val Asp Lys Ile 65 70 75 80 Lys Lys Asp Leu Leu Glu Gly
Lys Asn Ile Val Met Asp Arg Tyr Val 85 90 95 Tyr Ser Gly Val Ala
Tyr Ser Ala Ala Lys Gly Thr Asn Gly Met Asp 100 105 110 Leu Asp Trp
Cys Leu Gln Pro Asp Val Gly Leu Leu Lys Pro Asp Leu 115 120 125 Thr
Leu Phe Leu Ser Thr Gln Asp Val Asp Asn Asn Ala Glu Lys Ser 130 135
140 Gly Phe Gly Asp Glu Arg Tyr Glu Thr Val Lys Phe Gln Glu Lys Val
145 150 155 160 Lys Gln Thr Phe Met Lys Leu Leu Asp Lys Glu Ile Arg
Lys Gly Asp 165 170 175 Glu Ser Ile Thr Ile Val Asp Val Thr Asn Lys
Gly Ile Gln Glu Val 180 185 190 Glu Ala Leu Ile Trp Gln Ile Val Glu
Pro Val Leu Ser Thr His Ile 195 200 205 Asp His Asp Lys Phe Ser Phe
Phe 210 215
* * * * *
References