Advertisement

Biophysical Methods for Identifying Fragment-Based Inhibitors of Protein-Protein Interactions

  • Samuel J. Pfaff
  • Michael S. Chimenti
  • Mark J. S. Kelly
  • Michelle R. Arkin
Part of the Methods in Molecular Biology book series (MIMB, volume 1278)

Abstract

Fragment-based lead discovery complements high-throughput screening and computer-aided drug design for the discovery of small-molecule inhibitors of protein-protein interactions. Fragments are molecules with molecular masses ca 280 Da or smaller, and are generally screened using structural or biophysical approaches. Several methods of fragment-based screening are feasible for any soluble protein that can be expressed and purified; specific techniques also have size limitations and/or require multiple milligrams of protein. This chapter describes some of the most common fragment-discovery methods, including surface plasmon resonance, nuclear magnetic resonance, differential scanning fluorimetry, and X-ray crystallography.

Key words

Fragment Ligand Discovery Biophysics SPR NMR DSF Crystallography Protein-protein Interaction 

References

  1. 1.
    Rees DC, Congreve M, Murray CW et al (2004) Fragment-based lead discovery. Nat Rev Drug Discov 3:660–672CrossRefPubMedGoogle Scholar
  2. 2.
    Hajduk PJ, Greer J (2007) A decade of fragment-based drug design: Strategic advances and lessons learned. Nat Rev Drug Discov 6:211–219CrossRefPubMedGoogle Scholar
  3. 3.
    Scott DE, Ehebauer MT, Pukala T et al (2013) Using a fragment-based approach to target protein-protein interactions. Chembiochem 14:332–342CrossRefPubMedCentralPubMedGoogle Scholar
  4. 4.
    Braisted AC, Oslob JD, Delano WL et al (2003) Discovery of a potent small molecule IL-2 inhibitor through fragment assembly. J Am Chem Soc 125:3714–3715CrossRefPubMedGoogle Scholar
  5. 5.
    Arkin MR, Randal M, Delano WL et al (2003) Binding of small molecules to an adaptive protein-protein interface. Proc Natl Acad Sci U S A 100:1603–1608CrossRefPubMedCentralPubMedGoogle Scholar
  6. 6.
    Petros AM, Huth JR, Oost T et al (2010) Discovery of a potent and selective bcl-2 inhibitor using SAR by NMR. Bioorg Med Chem Lett 20:6587–6591CrossRefPubMedGoogle Scholar
  7. 7.
    Fuller JC, Burgoyne NJ, Jackson RM (2009) Predicting druggable binding sites at the protein-protein interface. Drug Discov Today 14:155–161CrossRefPubMedGoogle Scholar
  8. 8.
    Lau WF, Withka JM, Hepworth D et al (2011) Design of a multi-purpose fragment screening library using molecular complexity and orthogonal diversity metrics. J Comput Aided Mol Des 25:621–636CrossRefPubMedGoogle Scholar
  9. 9.
    Na J, Hu Q (2011) Design of screening collections for successful fragment-based lead discovery. Methods Mol Biol 685:219–240CrossRefPubMedGoogle Scholar
  10. 10.
    Chen IJ, Hubbard RE (2009) Lessons for fragment library design: analysis of output from multiple screening campaigns. J Comput Aided Mol Des 23:603–620CrossRefPubMedGoogle Scholar
  11. 11.
    Erlanson DA, Wells JA, Braisted AC (2004) Tethering: fragment-based drug discovery. Annu Rev Biophys Biomol Struct 33:199–223CrossRefPubMedGoogle Scholar
  12. 12.
    Wilson CG, Arkin MR (2013) Probing structural adaptivity at PPI interfaces with small molecules Drug Discovery Today: Technologies 10 (4):e501–e508Google Scholar
  13. 13.
    Giannetti AM, Koch BD, Browner MF (2008) Surface plasmon resonance based assay for the detection and characterization of promiscuous inhibitors. J Med Chem 51:574–580CrossRefPubMedGoogle Scholar
  14. 14.
    Babaoglu K, Simeonov A, Irwin JJ et al (2008) Comprehensive mechanistic analysis of hits from high-throughput and docking screens against beta-lactamase. J Med Chem 51:2502–2511CrossRefPubMedCentralPubMedGoogle Scholar
  15. 15.
    Cimmperman P, Baranauskiene L, Jachimoviciute S et al (2008) A quantitative model of thermal stabilization and destabilization of proteins by ligands. Biophys J 95:3222–3231CrossRefPubMedCentralPubMedGoogle Scholar
  16. 16.
    Kranz JK, Schalk-Hihi C (2011) Protein thermal shifts to identify low molecular weight fragments. Methods Enzymol 493:277–298CrossRefPubMedGoogle Scholar
  17. 17.
    Rizo J, Rosen MK, Gardner KH (2012) Enlightening molecular mechanisms through study of protein interactions. J Mol Cell Biol 4:270–283CrossRefPubMedCentralPubMedGoogle Scholar
  18. 18.
    Pellecchia M, Bertini I, Cowburn D et al (2008) Perspectives on NMR in drug discovery: a technique comes of age. Nat Rev Drug Discov 7:738–745CrossRefPubMedCentralPubMedGoogle Scholar
  19. 19.
    Ito Y, Selenko P (2010) Cellular structural biology. Curr Opin Struct Biol 20:640–648CrossRefPubMedGoogle Scholar
  20. 20.
    Dalvit C, Fagerness PE, Hadden DT et al (2003) Fluorine-NMR experiments for high-throughput screening: theoretical aspects, practical considerations, and range of applicability. J Am Chem Soc 125:7696–7703CrossRefPubMedGoogle Scholar
  21. 21.
    Dalvit C, Flocco M, Veronesi M et al (2002) Fluorine-NMR competition binding experiments for high-throughput screening of large compound mixtures. Comb Chem High Throughput Screen 5:605–611CrossRefPubMedGoogle Scholar
  22. 22.
    Hajduk PJ, Meadows RP, Fesik SW (1999) NMR-based screening in drug discovery. Q Rev Biophys 32:211–240CrossRefPubMedGoogle Scholar
  23. 23.
    Hajduk PJ, Gerfin T, Boehlen JM et al (1999) High-throughput nuclear magnetic resonance-based screening. J Med Chem 42:2315–2317CrossRefPubMedGoogle Scholar
  24. 24.
    Murray CW, Blundell TL (2010) Structural biology in fragment-based drug design. Curr Opin Struct Biol 20:497–507CrossRefPubMedGoogle Scholar
  25. 25.
    Spurlino JC (2011) Fragment screening purely with protein crystallography. Methods Enzymol 493:321–356CrossRefPubMedGoogle Scholar
  26. 26.
    Bottcher J, Jestel A, Kiefersauer R et al (2011) Key factors for successful generation of protein-fragment structures requirement on protein, crystals, and technology. Methods Enzymol 493:61–89CrossRefPubMedGoogle Scholar
  27. 27.
    Prakash O, Eisenberg MA (1979) Biotinyl 5'-adenylate: corepressor role in the regulation of the biotin genes of Escherichia coli k-12. Proc Natl Acad Sci U S A 76:5592–5595CrossRefPubMedCentralPubMedGoogle Scholar
  28. 28.
    Giannetti AM (2011) From experimental design to validated hits a comprehensive walk-through of fragment lead identification using surface plasmon resonance. Methods Enzymol 493:169–218CrossRefPubMedGoogle Scholar
  29. 29.
    Zhang JH, Chung TD, Oldenburg KR (1999) A simple statistical parameter for use in evaluation and validation of high throughput screening assays. J Biomol Screen 4:67–73CrossRefPubMedGoogle Scholar
  30. 30.
    Niesen FH, Berglund H, Vedadi M (2007) The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat Protoc 2:2212–2221CrossRefPubMedGoogle Scholar
  31. 31.
    Matulis D, Kranz JK, Salemme FR et al (2005) Thermodynamic stability of carbonic anhydrase: measurements of binding affinity and stoichiometry using thermofluor. Biochemistry 44:5258–5266CrossRefPubMedGoogle Scholar
  32. 32.
    Maurer T (2011) Advancing fragment binders to lead-like compounds using ligand and protein-based NMR spectroscopy. Methods Enzymol 493:469–485CrossRefPubMedGoogle Scholar
  33. 33.
    Bertini I, Molinari H, Niccolai N (1991) NMR and biomolecular structure, vol xvii. VCH, Weinheim, 209 pGoogle Scholar
  34. 34.
    Dalvit C, Pevarello P, Tato M et al (2000) Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water. J Biomol NMR 18:65–68CrossRefPubMedGoogle Scholar
  35. 35.
    Gossert AD, Henry C, Blommers MJ et al (2009) Time efficient detection of protein-ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment. J Biomol NMR 43:211–217CrossRefPubMedGoogle Scholar
  36. 36.
    Shuker SB, Hajduk PJ, Meadows RP et al (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science 274:1531–1534CrossRefPubMedGoogle Scholar
  37. 37.
    Kabsch W (2010) Xds. Acta Crystallogr D Biol Crystallogr 66:125–132CrossRefPubMedCentralPubMedGoogle Scholar
  38. 38.
    Holton J, Alber T (2004) Automated protein crystal structure determination using ELVES. Proc Natl Acad Sci U S A 101:1537–1542CrossRefPubMedCentralPubMedGoogle Scholar
  39. 39.
    Adams PD, Afonine PV, Bunkoczi G et al (2010) Phenix: a comprehensive python-based system for macromolecular structure solution. Acta Crystallogr D Biol Crystallogr 66:213–221CrossRefPubMedCentralPubMedGoogle Scholar
  40. 40.
    Emsley P, Cowtan K (2004) Coot: model-building tools for molecular graphics. Acta Crystallogr D Biol Crystallogr 60:2126–2132CrossRefPubMedGoogle Scholar
  41. 41.
    Hamalainen MD, Zhukov A, Ivarsson M et al (2008) Label-free primary screening and affinity ranking of fragment libraries using parallel analysis of protein panels. J Biomol Screen 13:202–209CrossRefPubMedGoogle Scholar
  42. 42.
    Schrodinger, Llc (2010) The PyMOL molecular graphics system, version 1.3r1Google Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Samuel J. Pfaff
    • 1
    • 2
  • Michael S. Chimenti
    • 2
  • Mark J. S. Kelly
    • 2
  • Michelle R. Arkin
    • 1
    • 2
    • 3
  1. 1.Small Molecule Discovery CenterUniversity of California San FranciscoSan FranciscoUSA
  2. 2.Department of Pharmaceutical ChemistryUniversity of California San FranciscoSan FranciscoUSA
  3. 3.UCSFSan FranciscoUSA

Personalised recommendations