Novel purified FabI polypeptides from fransicella tularensis

• US 20080108793A1
• Filed: 12/28/2006
• Published: 05/08/2008
• Est. Priority Date: 06/30/2004
• Status: Active Grant

• Current Assignee: Debiopharm SA (Debiopharm)
• Original Assignee: Affinium Pharmaceuticals, Inc.
• Inventors: Schmid, Molly B., Awrey, Donald E., Dorsey, Mandy, Beattie, Bryan, McGrath, Teresa Clarke, Berman, Judd M.
• Granted Patent: US 7,601,526 B2
• Time in Patent Office: Days
• US Class Current: 530/387.1
• CPC Class Codes: C07K 14/195 from bacteria
• CPC Class Codes: C07K 2299/00 Coordinates from 3D structures of peptides, e.g. proteins or enzymes

Abstract

The present invention relates to novel drug targets for pathogenic bacteria. Accordingly, the invention provides purified protein comprising the amino acid sequence set forth in SEQ ID NO: 2. The invention also provides biochemical and biophysical characteristics of the polypeptides of the invention.

Claims

1. A composition comprising an isolated, recombinant polypeptide, wherein the polypeptide comprises:

(a) an amino acid sequence set forth in SEQ ID NO;

2;

(b) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO;

2;

or (c) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO;

1 and has at least one biological activity of Fabi from F. tularensis; and

wherein the polypeptide of (a), (b) or (c) is at least about 90% pure in a sample of the composition.

2. The composition of claim 1, wherein the polypeptide is at least about 95% pure as determined by gel electrophoresis.

3. The composition of claim 1, wherein the polypeptide is purified to essential homogeneity.

4. The composition of claim 1, wherein at least about two-thirds of the polypeptide in the sample is soluble.

5. The composition of claim 1, wherein the polypeptide is fused to at least one heterologous polypeptide that increases the solubility or stability of the polypeptide.

6. The composition of claim 1, which further comprises a matrix suitable for mass spectrometry.

7. The composition of claim 6, wherein the matrix is a nicotinic acid derivative or a cinnamic acid derivative.

19. A method for designing a modulator for the prevention or treatment of F. tularensis related disease or disorder, comprising:

a. providing a three-dimensional structure for a crystallized, recombinant polypeptide of claim 13;

b. identifying a potential modulator for the prevention or treatment of F. tularensis related disease or disorder by reference to the three-dimensional structure;

c. contacting a polypeptide of the composition of claim 1 or F. tularensis with the potential modulator; and

d. assaying the activity of the polypeptide or determining the viability of F. tularensis after contact with the modulator, wherein a change in the activity of the polypeptide or the viability of F. tularensis indicates that the modulator may be useful for prevention or treatment of a F. tularensis related disease or disorder.

8. A sample comprising an isolated, recombinant polypeptide, wherein the polypeptide comprises:

(a) an amino acid sequence set forth in SEQ ID NO;

2;

(b) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO;

2;

or (c) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO;

1 and has at least one biological activity of FabI from F. tularensis; and

wherein the polypeptide of (a), (b) or (c) is labeled with a heavy atom.

9. The sample of claim 8, wherein the heavy atom is one of the following:

cobalt, selenium, krypton, bromine, strontium, molybdenum, ruthenium, rhodium, palladium, silver, cadmium, tin, iodine, xenon, barium, lanthanum, cerium, praseodymium, neodymium, samarium, europium, gadolinium, terbium, dysprosium, holmium, erbium, thulium, ytterbium, lutetium, tantalum, tungsten, rhenium, osmium, iridium, platinum, gold, mercury, thallium, lead, thorium and uranium.

10. The sample of claim 8, wherein the polypeptide is labeled with seleno-methionine.

11. The sample of claim 8, further comprising a cryo-protectant.

12. The sample of claim 11, wherein the cryo-protectant is one of the following:

methyl pentanediol, isopropanol, ethylene glycol, glycerol, formate, citrate, mineral oil and a low-molecular-weight polyethylene glycol.

13. A crystallized, recombinant polypeptide comprising:

(a) an amino acid sequence set forth in SEQ ID NO;

2;

(b) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO;

2;

or (c) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO;

1 and has at least one biological activity of FabI from F. tularensis;

wherein the polypeptide of (a), (b) or (c) is in crystal form.

14. A crystallized complex comprising the crystallized, recombinant polypeptide of claim 13 and a co-factor, wherein the complex is in crystal form.

15. A crystallized complex comprising the crystallized, recombinant polypeptide of claim 13 and a small organic molecule, wherein the complex is in crystal form.

16. The crystallized, recombinant polypeptide of claim 13, which diffracts x-rays to a resolution of about 3.5 Å

or better.

17. The crystallized, recombinant polypeptide of claim 13, wherein the polypeptide comprises at least one heavy atom label.

18. The crystallized, recombinant polypeptide of claim 17, wherein the polypeptide is labeled with seleno-methionine.

20. A sample comprising an isolated, recombinant polypeptide, wherein the polypeptide comprises:

(a) an amino acid sequence set forth in SEQ ID NO;

2;

(b) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO;

2;

or (c) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO;

1 and has at least one biological activity of FabI from F. tularensis; and

wherein the polypeptide of (a), (b) or (c) is enriched in at least one NMR isotope.

21. The sample of claim 20, wherein the NMR isotope is one of the following:

hydrogen-1 (¹H), hydrogen-2(²H), hydrogen-3(³H), phosphorous-31 (³¹ P), sodium-23 (²³Na), nitrogen-14 (¹⁴N), nitrogen-15 (¹⁵N), carbon-13 (¹³C) and fluorine-19 (¹⁹F).

22. The sample of claim 20, further comprising a deuterium lock solvent.

23. The sample of claim 22, wherein the deuterium lock solvent is one of the following:

acetone (CD₃COCD₃), chloroform (CDCl₃), dichloro methane (CD₂Cl₂), methylnitrile (CD₃CN), benzene (C₆D₆), water (D₂O), diethylether ((CD₃CD₂)₂O), dimethylether ((CD₃)₂O), N,N-dimethylformamide ((CD₃)₂NCDO), dimethyl sulfoxide (CD₃SOCD₃), ethanol (CD₃CD₂OD), methanol (CD₃OD), tetrahydrofuran (C₄D₈O), toluene

(C₆D₅CD₃), pyridine (C₅D₅N) and cyclohexane (C₆H₁₂).

24. The sample of claim 20, which is contained within an NMR tube.

25. Crystalline FabI from F. tularensis comprising a crystal having unit cell dimensions a=b=130.072 Å

, c=88.436 Å

, 0=γ

=90°

, p=120°

, with space group P6₂22.

26. A crystallized polypeptide comprising (1) an amino acid sequence set forth in SEQ ID NO:

2;

(2) an amino acid sequence having at least about 95% identity with the amino acid sequence set forth in SEQ ID NO;

2;

or (3) an amino acid sequence encoded by a polynucleotide that hybridizes under stringent conditions to the complementary strand of a polynucleotide having SEQ ID NO;

1 and has at least one biological activity of Fabi from F. tularensis;

wherein the crystal has a P6₂22 space group.

27. A crystallized polypeptide comprising a structure of a polypeptide that is defined by a substantial portion of the atomic coordinates set forth in FIG. 20.