Discovering high-affinity ligands for proteins: SAR by NMR

Science

Volumn 274, Issue 5292, 1996, Pages 1531-1534

  Shuker, Suzanne B ^a   Hajduk, Philip J ^a   Meadows, Robert P ^a   Fesik, Stephen W ^a  

^a ABBOTT LABORATORIES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ANILIDE; DRUG BINDING PROTEIN; IMMUNOSUPPRESSIVE AGENT; LIGAND; PIPECOLIC ACID DERIVATIVE; PROTEIN; TACROLIMUS;

ARTICLE; BINDING AFFINITY; DRUG RESEARCH; DRUG STRUCTURE; MOLECULAR WEIGHT; NUCLEAR MAGNETIC RESONANCE; NUCLEAR MAGNETIC RESONANCE SPECTROSCOPY; NUCLEAR OVERHAUSER EFFECT; PRIORITY JOURNAL; PROTEIN BINDING; PROTON NUCLEAR MAGNETIC RESONANCE; STRUCTURE ACTIVITY RELATION; SYNTHESIS; X RAY CRYSTALLOGRAPHY;

ANILIDES; BINDING SITES; CARRIER PROTEINS; DNA-BINDING PROTEINS; HEAT-SHOCK PROTEINS; LIGANDS; MAGNETIC RESONANCE SPECTROSCOPY; MODELS, MOLECULAR; PROTEINS; STRUCTURE-ACTIVITY RELATIONSHIP; TACROLIMUS; TACROLIMUS BINDING PROTEINS;

EID: 0029836953     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.274.5292.1531     Document Type: Article

References (36)

1. Our library of compounds consists of a diverse set of 10,000 small molecules (average molecular weight, 213) dissolved in perdeuterated dimethylsulfoxide (DMSO).
2. G. Bodenhausen and D. J. Ruben, Chem. Phys. Lett. 69, 185 (1980); L. E. Kay, P. Keifer, T. Saarinen, J. Am. Chem. Soc. 114, 10663 (1992).
3. G. Bodenhausen and D. J. Ruben, Chem. Phys. Lett. 69, 185 (1980); L. E. Kay, P. Keifer, T. Saarinen, J. Am. Chem. Soc. 114, 10663 (1992).
4. 15N-labeled protein can be acquired in about 10 minutes on a 500-MHz NMR spectrometer, allowing 100 2D spectra to be recorded in 1 day with an automatic sample changer. If compounds are tested in mixtures of 10 (as in our strategy), it is possible to robotically screen 1000 compounds per day.
5. J. Liu, J. D. Farmer, Jr., W. S. Lane, J. Friedman, I. Weissman, S. L. Schreiber, Cell 66, 807 (1991).
6. At the time of this screening our library consisted of approximately 1000 compounds.
7. G. T. Wang, B. Lane, S. W. Fesik, A. Petros, J. Luly, G. A. Krafft, Bioorg. Med. Chem. Lett. 4, 1161 (1994); S. Werner and M. Alfred. German Patent P33 17 356.7; J. R. Hauske, P. Dorff, S. Julin, J. DiBrino, R. Spencer, R. Williams, J. Med. Chem. 35, 4284 (1992); D. A. Holt et al., J. Am. Chem. Soc. 115, 9925 (1993).
8. G. T. Wang, B. Lane, S. W. Fesik, A. Petros, J. Luly, G. A. Krafft, Bioorg. Med. Chem. Lett. 4, 1161 (1994); S. Werner and M. Alfred. German Patent P33 17 356.7; J. R. Hauske, P. Dorff, S. Julin, J. DiBrino, R. Spencer, R. Williams, J. Med. Chem. 35, 4284 (1992); D. A. Holt et al., J. Am. Chem. Soc. 115, 9925 (1993).
9. G. T. Wang, B. Lane, S. W. Fesik, A. Petros, J. Luly, G. A. Krafft, Bioorg. Med. Chem. Lett. 4, 1161 (1994); S. Werner and M. Alfred. German Patent P33 17 356.7; J. R. Hauske, P. Dorff, S. Julin, J. DiBrino, R. Spencer, R. Williams, J. Med. Chem. 35, 4284 (1992); D. A. Holt et al., J. Am. Chem. Soc. 115, 9925 (1993).
10. G. T. Wang, B. Lane, S. W. Fesik, A. Petros, J. Luly, G. A. Krafft, Bioorg. Med. Chem. Lett. 4, 1161 (1994); S. Werner and M. Alfred. German Patent P33 17 356.7; J. R. Hauske, P. Dorff, S. Julin, J. DiBrino, R. Spencer, R. Williams, J. Med. Chem. 35, 4284 (1992); D. A. Holt et al., J. Am. Chem. Soc. 115, 9925 (1993).
11. R. P. Meadows et al., Biochemistry 32, 754 (1993). G. D. Van Duyne, R. F. Standaert, P. A. Karplus, S. L. Schreiber, J. Clardy, Science 252, 839 (1991); G. D. Van Duyne, R. F. Standaert, S. L. Schreiber, J. Clardy, J. Am. Chem. Soc. 113, 7433 (1991).
12. R. P. Meadows et al., Biochemistry 32, 754 (1993). G. D. Van Duyne, R. F. Standaert, P. A. Karplus, S. L. Schreiber, J. Clardy, Science 252, 839 (1991); G. D. Van Duyne, R. F. Standaert, S. L. Schreiber, J. Clardy, J. Am. Chem. Soc. 113, 7433 (1991).
13. R. P. Meadows et al., Biochemistry 32, 754 (1993). G. D. Van Duyne, R. F. Standaert, P. A. Karplus, S. L. Schreiber, J. Clardy, Science 252, 839 (1991); G. D. Van Duyne, R. F. Standaert, S. L. Schreiber, J. Clardy, J. Am. Chem. Soc. 113, 7433 (1991).
14. At the time of this screening, our library consisted of approximately 1600 compounds.
15. The binding of benzamide, N,N-dimethylbenzamide, 4-hydroxyacetanilide, and 4-hydroxyphenylbenzoate to FKBP was negligible at compound concentrations up to 10 mM.
16. p-Anisoyl chloride was treated with p-anisidine, and the methyl groups were removed with boron tribromide.
17. 13C-edited NMR experiments [G. M. Clore and A. M. Gronenborn, Methods Enzymol. 239, 349 (1994)]. A total of 17 intermolecular restraints were used to dock 9 to the known structure of FKBP (7). Compound 2 was placed in a location similar to that observed in the ascomycin complex, which was consistent with the chemical shift changes observed on binding of 2 (Fig. 3).
18. nOAc, n = 3 to 6], and the products were hydrolyzed with aqueous NaOH. The resulting benzoic acid derivatives were coupled with p-(t -butyldimethylsilyloxy)aniline. The primary alcohols were coupled with N-FMOC-L-pipecolinic acid, the FMOC group was removed with piperidine, and the resulting amines were treated with 3,4,5-trimethoxybenzoylformyl chloride. The silyl groups were then removed with tetrabutylammonium fluoride to provide the desired compounds 10 to 13.
19. Methyl-4-hydroxybenzoate was treated with base and allyl iodide, and the methyl ester was hydrolyzed with aqueous NaOH. The resulting benzoic acid derivative was coupled with p-(t -butyldimethylsityloxy)aniline, and the allyl ether was then converted to the corresponding allyl phenol via Claisen rearrangement. The resulting hydroxyl group was protected with t -butyldimethylsilyl chloride, and the terminal alkene was converted to the desired primary alcohol by hydroboration and oxidation. This alcohol was converted to the desired compound 14 as described for 10 to 13 (12).
20. Fluorescence measurements were performed on a SPEX Fluorolog 2-1-2 instrument. Titrations were carried out by following the decrease in intrinsic fluorescence of FKBP on sequential addition of the compounds as perdeuterated DMSO stock solutions DMSO concentrations did not exceed 1% of the total volume.
21. noe = 10.3 kcal/mol.
22. noe = 10.3 kcal/mol.
23. noe = 10.3 kcal/mol.
24. 1.
25. C. L. M, J. Verlinde, G. R. Rudenko, W. G. J. Hol, J. Comput. Aided Mol. Des. 6, 131 (1992); H.-J. Böhm, ibid., p. 61; S. H. Rotstein and M. A. Murcko, J. Med. Chem. 36, 1700 (1993); M. B. Eisen, D. C. Wiley, M. Karplus, R. E Hubbard, Proteins 19, 199 (1994).
26. C. L. M, J. Verlinde, G. R. Rudenko, W. G. J. Hol, J. Comput. Aided Mol. Des. 6, 131 (1992); H.-J. Böhm, ibid., p. 61; S. H. Rotstein and M. A. Murcko, J. Med. Chem. 36, 1700 (1993); M. B. Eisen, D. C. Wiley, M. Karplus, R. E Hubbard, Proteins 19, 199 (1994).
27. C. L. M, J. Verlinde, G. R. Rudenko, W. G. J. Hol, J. Comput. Aided Mol. Des. 6, 131 (1992); H.-J. Böhm, ibid., p. 61; S. H. Rotstein and M. A. Murcko, J. Med. Chem. 36, 1700 (1993); M. B. Eisen, D. C. Wiley, M. Karplus, R. E Hubbard, Proteins 19, 199 (1994).
28. C. L. M, J. Verlinde, G. R. Rudenko, W. G. J. Hol, J. Comput. Aided Mol. Des. 6, 131 (1992); H.-J. Böhm, ibid., p. 61; S. H. Rotstein and M. A. Murcko, J. Med. Chem. 36, 1700 (1993); M. B. Eisen, D. C. Wiley, M. Karplus, R. E Hubbard, Proteins 19, 199 (1994).
29. K. N. Allen, C. R. Bellamacina, X. Ding, C. J. Jeffrey, C. Mattos, G. A. Petsko, D. Ringe, J. Phys. Chem. 100, 2605 (1996).
30. M. A. Gallop, R. W. Barrett, W. J. Dower, S. P. A. Fodor, E. M. Gordon, J. Med. Chem. 37, 1233 (1994); D. J. Ecker and S. T. Crooke, Biotechnology 13, 351 (1995).
31. M. A. Gallop, R. W. Barrett, W. J. Dower, S. P. A. Fodor, E. M. Gordon, J. Med. Chem. 37, 1233 (1994); D. J. Ecker and S. T. Crooke, Biotechnology 13, 351 (1995).
32. w, where D is the number of fragments contained in the database, w is the number of adjacent, independent binding sites in the protein, and n is the number of linkers that could be used to join the fragments.
33. To reduce the amount of protein required by the technique, the protein is recycled by removing the ligands with dialysis after the NMR experiment.
34. The SAR by NMR method is even applicable to proteases, provided that the first ligand inhibits the enzyme.
35. M. Carson, J. Mol. Graphics 5, 103 (1987).
36. We thank G. Wang for the synthesis of 2 and trimethoxybenzoylformic acid; T. Holzman and J. Severin for the preparation of isotopically labeled FKBP; A. Phillips, A. Peterson, and L. Tucker-Garcia for sample preparation and aid in building our library of compounds; K. Swift and E. Matayoshi for assistance in performing the fluorescence measurements; and D. Norbeck for helpful comments on the manuscript.