Claims
- 1. A crystal of a binding complex between β-ketoacyl-ACP synthase I (FabB) and thiolactomycin (TLM) that effectively diffracts X-rays for the determination of the atomic coordinates to a resolution of better than 3.5 Angstroms.
- 2. The crystal of claim 1, wherein the FabB is E. coli FabB
- 3. The crystal of claim 2 having space group of P212121 and a unit cell of dimensions of a=59.1 b=139 and c=211.9 Angstroms.
- 4. A crystal of a binding complex between β-ketoacyl-ACP synthase I (FabB) and cerulenin that effectively diffracts X-rays for the determination of the atomic coordinates to a resolution of better than 3.5 Angstroms.
- 5. The crystal of claim 4, wherein the FabB is E. coli FabB.
- 6. The crystal of claim 5 having space group of P212121 and a unit cell of dimensions of a=59.2 b=139.6 and c=212.2 Angstroms.
- 7. A method of obtaining a crystal of an inhibitor-FabB complex comprising growing a crystal of the inhibitor-FabB complex in a buffered solution containing 2.0 M ammonium sulfate, and 20% PEG 400.
- 8. The method of claim 7 wherein said growing is performed by a method selected from the group consisting of batch crystallization, vapor diffusion, and microdialysis.
- 9. A computer comprising within its memory a representation of the binding complex between FabB and cerulenin (the FabB-cerulenin binding complex) or a portion of said FabB-cerulenin binding complex, said computer comprising:
(a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises atomic coordinates from Table III; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into a three-dimensional representation of the FabB-cerulenin binding complex or a portion of said FabB-cerulenin binding complex; and (d) a display coupled to said central-processing unit for displaying said three-dimensional representation.
- 10. A computer comprising within its memory a representation of the binding complex between FabB and thiolactomycin (the FabB-TLM binding complex) or a portion of said FabB-TLM binding complex, said computer comprising:
(a) a machine-readable data storage medium comprising a data storage material encoded with machine-readable data, wherein said data comprises atomic coordinates from Table IV; (b) a working memory for storing instructions for processing said machine-readable data; (c) a central processing unit coupled to said working memory and to said machine-readable data storage medium for processing said machine readable data into a three-dimensional representation of the FabB-TLM binding complex or a portion of said FabB-TLM binding complex; and (d) a display coupled to said central-processing unit for displaying said three-dimensional representation.
- 11. A method of identifying an agent for use as an inhibitor of bacterial fatty acid synthesis using the crystal of claim 3 comprising:
(a) selecting a potential agent by performing rational drug design with the atomic coordinates determined from the crystal, wherein said selecting is performed in conjunction with computer modeling; (b) contacting the potential agent with a β-ketoacyl-(Acyl Carrier Protein) synthase; and (c) measuring the activity of the β-ketoacyl-(Acyl Carrier Protein) synthase; wherein a potential agent is identified as an agent that inhibits bacterial fatty acid synthesis when there is a decrease in the activity of the β-ketoacyl-(Acyl Carrier Protein) synthase.
- 12. A method of identifying an agent for use as an inhibitor of bacterial fatty acid synthesis using the crystal of claim 6 comprising:
(a) selecting a potential agent by performing rational drug design with the atomic coordinates determined from the crystal, wherein said selecting is performed in conjunction with computer modeling; (b) contacting the potential agent with a β-ketoacyl-(Acyl Carrier Protein) synthase; and (c) measuring the activity of the β-ketoacyl-(Acyl Carrier Protein) synthase; wherein a potential agent is identified as an agent that inhibits bacterial fatty acid synthesis when there is a decrease in the activity of the β-ketoacyl-(Acyl Carrier Protein) synthase.
- 13. A method of identifying an agent that inhibits bacterial growth using the atomic coordinates obtained from the crystal of claim 3 comprising:
(a) selecting a potential agent by performing rational drug design with the atomic coordinates determined for the crystal, wherein said selecting is performed in conjunction with computer modeling; (b) contacting the potential agent with a bacterial culture; and (c) measuring the growth of the bacterial culture; wherein a potential agent is identified as an agent that inhibits bacterial growth when there is a decrease in the growth of the bacterial culture.
- 14. The method of claim 13, further comprising:
(d) growing a supplemental crystal containing FabB formed in the presence of the potential agent, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates to a resolution of better than 5.0 Angstroms; (e) determining the atomic coordinates of the supplemental crystal with molecular replacement analysis; and (f) selecting a second generation agent by performing rational drug design with the atomic coordinates determined for the supplemental crystal, wherein said selecting is performed in conjunction with computer modeling.
- 15. The method of claim 14, further comprising:
(g) contacting the second generation agent with a eukaryotic cell; and (h) measuring the amount of proliferation of the eukaryotic cell; wherein a potential agent is identified as an agent for inhibiting bacterial growth when there is no change in the proliferation of the eukaryotic cell.
- 16. A method of identifying an agent that inhibits bacterial growth using the atomic coordinates obtained from the crystal of claim 6 comprising:
(a) selecting a potential agent by performing rational drug design with the atomic coordinates determined for the crystal, wherein said selecting is performed in conjunction with computer modeling; (b) contacting the potential agent with a bacterial culture; and (c) measuring the growth of the bacterial culture; wherein a potential agent is identified as an agent that inhibits bacterial growth when there is a decrease in the growth of the bacterial culture.
- 17. The method of claim 16, further comprising:
(d) growing a supplemental crystal containing FabB formed in the presence of the potential agent, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates to a resolution of better than 5.0 Angstroms; (e) determining the atomic coordinates of the supplemental crystal with molecular replacement analysis; and (f) selecting a second generation agent by performing rational drug design with the atomic coordinates determined for the supplemental crystal, wherein said selecting is performed in conjunction with computer modeling.
- 18. The method of claim 17, further comprising:
(g) contacting the second generation agent with a eukaryotic cell; and (h) measuring the amount of proliferation of the eukaryotic cell; wherein a potential agent is identified as an agent for inhibiting bacterial growth when there is no change in the proliferation of the eukaryotic cell.
- 19. A method of identifying an agent for use as an inhibitor of bacterial fatty acid synthesis comprising:
(a) selecting a potential agent by performing rational drug design with the set of atomic coordinates in Table III and/or Table IV, wherein said selecting is performed in conjunction with computer modeling; (b) contacting the potential agent with a bacterial β-ketoacyl-(Acyl Carrier Protein) synthase; and (c) measuring the activity of the bacterial β-ketoacyl-(Acyl Carrier Protein) synthase; wherein a potential agent is identified as an agent that inhibits bacterial fatty acid synthesis when there is a decrease in the activity of the bacterial β-ketoacyl-(Acyl Carrier Protein) synthase in the presence of the agent relative to in its absence.
- 20. The method of claim 19, further comprising:
(d) growing a crystal containing a bacterial Fab formed in the presence of the potential agent, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates to a resolution of better than 5.0 Angstroms; (e) determining the atomic coordinates of the crystal with molecular replacement analysis; and (f) selecting a second generation agent by performing rational drug design with the atomic coordinates determined for the crystal, wherein said selecting is performed in conjunction with computer modeling.
- 21. A method of identifying an agent that inhibits bacterial growth comprising:
(a) selecting a potential agent by performing rational drug design with the set of atomic coordinates in Table III and/or Table IV, wherein said selecting is performed in conjunction with computer modeling; (b) contacting the potential agent with a bacterial culture; and (c) measuring the growth of the bacterial culture; wherein a potential agent is identified as an agent that inhibits bacterial growth when there is a decrease in the growth of the bacterial culture.
- 22. The method of claim 21, further comprising:
(d) growing a supplemental crystal containing FabB formed in the presence of the potential agent, wherein the crystal effectively diffracts X-rays for the determination of the atomic coordinates to a resolution of better than 5.0 Angstroms; (e) determining the atomic coordinates of the supplemental crystal with molecular replacement analysis; and (f) selecting a second generation agent by performing rational drug design with the atomic coordinates determined for the supplemental crystal, wherein said selecting is performed in conjunction with computer modeling.
- 23. The method of claim 22, further comprising:
(g) contacting the second generation agent with a eukaryotic cell; and (h) measuring the amount of proliferation of the eukaryotic cell; wherein a potential agent is identified as an agent for inhibiting bacterial growth when there is no change in the proliferation of the eukaryotic cell.
- 24. A method of selecting a compound that potentially inhibits fatty acid synthesis comprising:
(a) defining the structure of the FabB-inhibitor complex by the atomic coordinates in Table III and/or Table IV; and (b) selecting a compound which potentially inhibits fatty acid synthesis; wherein said selecting is performed with the aid of the structure defined in step (a).
- 25. The method of claim 24, further comprising:
(c) contacting the compound with a bacterial β-ketoacyl-(Acyl Carrier Protein) synthase; and (d) measuring the activity of the bacterial β-ketoacyl-(Acyl Carrier Protein) synthase; wherein the compound is identified as an agent that inhibits bacterial β-ketoacyl-(Acyl Carrier Protein) synthase when there is a decrease in the activity of the bacterial β-ketoacyl-(Acyl Carrier Protein) synthase in the presence of the compound relative to in its absence.
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a non-provisional application claiming the priority of copending provisional U.S. Ser. No. 60/223,222 filed Aug. 4, 2000, the disclosure of which is hereby incorporated by reference in its entirety. Applicants claim the benefits of this application under 35 U.S.C. § 119(e).
RESEARCH SUPPORT
[0002] The research leading to the present invention was supported in part by the National Institutes of Health Grants GM34496, GM44973, the Cancer Center Support (CORE) grant CA-21765. The government may have certain rights in the present invention.
[0003] Support for this invention was also provided by the AMERICAN LEBANESE SYRIAN ASSOCIATED CHARITIES.
Provisional Applications (1)
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Number |
Date |
Country |
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60223222 |
Aug 2000 |
US |