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Biophysical Methods in Drug Discovery from Small Molecule to Pharmaceutical

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Protein-Ligand Interactions

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1008))

Abstract

Biophysical methods have become established in many areas of drug discovery. Application of these methods was once restricted to a relatively small number of scientists using specialized, low throughput technologies and methods. Now, automated high-throughput instruments are to be found in a growing number of laboratories. Many biophysical methods are capable of measuring the equilibrium binding constants between pairs of molecules crucial for molecular recognition processes, encompassing protein–protein, protein–small molecule, and protein–nucleic acid interactions, and several can be used to measure the kinetic or thermodynamic components controlling these biological processes. For a full characterization of a binding process, determinations of stoichiometry, binding mode, and any conformational changes associated with such interactions are also required. The suite of biophysical methods that are now available represents a powerful toolbox of techniques which can effectively deliver this full characterization.

The aim of this chapter is to provide the reader with an overview of the drug discovery process and how biophysical methods, such as surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), nuclear magnetic resonance, mass spectrometry (MS), and thermal unfolding methods can answer specific questions in order to influence project progression and outcomes. The selection of these examples is based upon the experiences of the authors at AstraZeneca, and relevant approaches are highlighted where they have utility in a particular drug discovery scenario.

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References

  1. Kola I, Landis J (2004) Can the pharmaceutical industry reduce attrition rates? Nat Rev Drug Discov 8:711–715

    Article  CAS  Google Scholar 

  2. Hopkins AL, Groom CR (2002) The druggable genome. Nat Rev Drug Discov 1:727–730

    Article  PubMed  CAS  Google Scholar 

  3. Edfeldt FBN, Folmer RHA, Breeze AL (2011) Fragment screening to predict druggability (ligandability) and lead discovery success. Drug Discov Today 16:284–287

    Article  PubMed  CAS  Google Scholar 

  4. Pérot S et al (2010) Druggable pockets and binding site centric chemical space: a paradigm shift in drug discovery. Drug Discov Today 15:656–667

    Article  PubMed  CAS  Google Scholar 

  5. Egner U, Hillig RC (2008) A structural biology view of target druggability. Expert Opin Drug Discov 3:391–401

    Article  PubMed  CAS  Google Scholar 

  6. Hajduk PJ, Huth JR, Tse C (2005) Predicting protein druggability. Drug Discov Today 10:1675–1682

    Article  PubMed  CAS  Google Scholar 

  7. Cheng AC et al (2007) Structure-based maximal affinity model predicts small-molecule druggability. Nat Biotechnol 25:71–75

    Article  PubMed  CAS  Google Scholar 

  8. Hajduk PJ, Huth JR, Fesik SW (2005) Druggability indices for protein targets derived from NMR-based screening data. J Med Chem 48:2518–2525

    Article  PubMed  CAS  Google Scholar 

  9. Fink T, Reymond JL (2007) Virtual exploration of the chemical universe up to 11 atoms of C, N, O, F: assembly of 26.4 million structures (110.9 million stereoisomers) and analysis for new ring systems, stereochemistry, physicochemical properties, compound classes, and drug discovery. J Chem Inf Model 47:342–353

    Article  PubMed  CAS  Google Scholar 

  10. Medina-Franco JL et al (2007) A similarity-based data-fusion approach to the visual characterization and comparison of compound databases. Chem Biol Drug Des 70:393–412

    Article  PubMed  CAS  Google Scholar 

  11. Vivat Hannah V et al (2010) Native MS: an ‘ESI’ way to support structure- and fragment-based drug discovery. Future Med Chem 2:35–50

    Article  PubMed  Google Scholar 

  12. Copeland R (2003) A mechanistic considerations in high-throughput screening. Anal Biochem 320:1–12

    Article  PubMed  CAS  Google Scholar 

  13. Campos-Olivas R (2010) NMR screening and hit validation in fragment-based drug discovery. Curr Top Med Chem 11:43–67

    Article  Google Scholar 

  14. Pellecchia M et al (2008) Perspectives on NMR in drug discovery: a technique comes of age. Nat Rev Drug Discov 7:738–745

    Article  PubMed  CAS  Google Scholar 

  15. Billeter M, Wagner G, Wüthrich K (2008) Solution NMR structure determination of proteins revisited. J Biomol NMR 42:155–158

    Article  PubMed  CAS  Google Scholar 

  16. Seidler J et al (2003) Identification and prediction of promiscuous aggregating inhibitors among known drugs. J Med Chem 46:4477–4486

    Article  PubMed  CAS  Google Scholar 

  17. Dalvit C et al (2000) Identification of compounds with binding affinity to proteins via magnetisation transfer from bulk water. J Biomol NMR 18:65–68

    Article  PubMed  CAS  Google Scholar 

  18. Mayer M, Meyer B (1999) Characterisation of ligand binding by saturation transfer difference NMR spectroscopy. Angew Chem Int 38:1784–1788

    Article  CAS  Google Scholar 

  19. Hajduk PJ, Olejniczak ET, Fesik SW (1997) One-dimensional relaxation- and diffusion-edited NMR methods for screening compounds that bind to macromolecules. J Am Chem Soc 119:12257–12261

    Article  CAS  Google Scholar 

  20. Shuker SB et al (1996) Discovering high-affinity ligands for proteins: SAR by NMR. Science 274:1531–1534

    Article  PubMed  CAS  Google Scholar 

  21. Ludwig C, Guenther UL (2009) Ligand-based NMR methods for drug discovery. Front Biosci 14:4565–4574

    Article  PubMed  CAS  Google Scholar 

  22. Vanwetswinkel S et al (2005) TINS, target immobilised NMR screening: an efficient and sensitive method for ligand discovery. Chem Biol 12:207–216

    Article  PubMed  CAS  Google Scholar 

  23. Dalvit C et al (2005) Sensitivity improvement in 19F NMR-based screening experiments: theoretical considerations and experimental applications. J Am Chem Soc 127:13380–13385

    Article  PubMed  CAS  Google Scholar 

  24. Jahnke W, Rüdisser S, Zurini M (2001) Spin label enhanced NMR screening. J Am Chem Soc 123:3149–3150

    Article  PubMed  CAS  Google Scholar 

  25. Moy FJ et al (2010) Novel synthesis and structural characterisation of a high-affinity paramagnetic kinase probe for the identification of non-ATP site binders by nuclear magnetic resonance. J Med Chem 53:1238–1249

    Article  PubMed  CAS  Google Scholar 

  26. Becattini B et al (2006) Structure-activity relationships by interligand NOE-based design and synthesis of antiapoptotic compounds targeting Bid. Proc Natl Acad Sci U S A 103:12602–12606

    Article  PubMed  CAS  Google Scholar 

  27. Sledz P et al (2010) Optimization of the interligand Overhauser effect for fragment linking: application to inhibitor discovery against Mycobacterium tuberculosis pantothenate synthetase. J Am Chem Soc 132:4544–4545

    Article  PubMed  CAS  Google Scholar 

  28. Danielson UH (2009) Fragment library screening and lead characterization using SPR biosensors. Curr Top Med Chem 9:1725–1735

    Article  PubMed  CAS  Google Scholar 

  29. Huber W, Mueller F (2006) Biomolecular interaction analysis in drug discovery using surface plasmon resonance technology. Curr Pharm Des 12:3999–4021

    Article  PubMed  CAS  Google Scholar 

  30. Mukundan H (2009) Waveguide-based biosensors for pathogen detection. Sensors 9:5783–5809

    Article  PubMed  CAS  Google Scholar 

  31. Fang Y et al (2006) Resonant waveguide grating biosensor for living cell sensing. Biophys J 91:1925–1940

    Article  PubMed  CAS  Google Scholar 

  32. Cunningham BT (2004) Label-free assays on the BIND system. J Biomol Screen 9:481–490

    Article  PubMed  CAS  Google Scholar 

  33. Dodgson K et al (2009) 100K well screen for a muscarinic receptor using the Epic label-free system—a reflection on the benefits of the label-free approach to screening seven-transmembrane receptors. J Recept Signal Transduct Res 29:163–172

    Article  PubMed  CAS  Google Scholar 

  34. Chan LL et al (2009) A method for identifying small molecule aggregators using photonic crystal biosensor microplates. Conf Proc IEEE Eng Med Biol Soc 2009:788–791

    PubMed  Google Scholar 

  35. Heeres JT et al (2009) Identifying modulators of protein-protein interactions using photonic crystal biosensors. J Am Chem Soc 131:18202–18203

    Article  PubMed  CAS  Google Scholar 

  36. Chan LL et al (2008) A general method for discovering inhibitors of protein-DNA interactions using photonic crystal biosensors. ACS Chem Biol 3:437–448

    Article  PubMed  CAS  Google Scholar 

  37. Fenn JB et al (1989) Electrospray ionization for mass spectrometry of large biomolecules. Science 246:64–71

    Article  PubMed  CAS  Google Scholar 

  38. Bruylants G, Wouters J, Michaux C (2005) Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design. Curr Med Chem 12:2011–2020

    Article  PubMed  CAS  Google Scholar 

  39. Zhang R, Monsma F (2010) Fluorescence-based thermal shift assays. Curr Opin Drug Discov Devel 13:389–402

    PubMed  CAS  Google Scholar 

  40. Holdgate GA et al (2010) Affinity-based, biophysical methods to detect and analyze ligand binding to recombinant proteins: matching high information content with high throughput. J Struct Biol 172:142–157

    Article  PubMed  CAS  Google Scholar 

  41. Muckenschnabel I et al (2004) SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands. Anal Biochem 324:241–249

    Article  PubMed  CAS  Google Scholar 

  42. Patriksson A, Marklund E, van der Spoel D (2007) Protein structures under electrospray conditions. Biochemistry 46:933–945

    Article  PubMed  CAS  Google Scholar 

  43. van den Heuvel RH, Heck AJ (2004) Native protein mass spectrometry: from intact oligomers to functional machineries. Curr Opin Chem Biol 8:519–526

    Article  PubMed  CAS  Google Scholar 

  44. Hofstadler SA, Sannes-Lowery KA (2007) Interrogation of noncovalent complexes by ESI-MS: a powerful platform for high throughput drug discovery. In: Wanner KT, Höfner G (eds) Mass spectrometry in medicinal chemistry, 1st edn. Wiley, Weinheim, pp 321–338

    Chapter  Google Scholar 

  45. Coan KE, Shoichet BK (2008) Stoichiometry and physical chemistry of promiscuous aggregate-based inhibitors. J Am Chem Soc 130:9606–9612

    Article  PubMed  CAS  Google Scholar 

  46. Feng BY et al (2007) A high-throughput screen for aggregation-based inhibition in a large compound library. J Med Chem 50:2385–2390

    Article  PubMed  CAS  Google Scholar 

  47. Achyuthan KE, Whitten DG (2007) Design considerations for high throughput screening and in vitro diagnostic assays. Comb Chem High Throughput Screen 10:399–412

    Article  PubMed  CAS  Google Scholar 

  48. Renaud JP, Delsuc MA (2009) Biophysical technique for ligand screening and drug design. Curr Opin Pharmacol 9:622–628

    Article  PubMed  CAS  Google Scholar 

  49. Miller JR et al (2010) The use of biochemical and biophysical tools for triage of high-throughput sceening hits—a case study with Escherichia coli phosphopantetheine adenylyltransferase. Chem Biol Drug Des 75:444–454

    Article  PubMed  CAS  Google Scholar 

  50. Zhu Z, Cuozo J (2009) High-throughput affinity-based technologies for small-molecule drug discovery. J Biomol Screen 14:1157–1164

    Article  PubMed  CAS  Google Scholar 

  51. Holdgate G (2007) Thermodynamics of binding interactions in the rational drug design process. Expert Opin Drug Discov 2:1103–1114

    Article  PubMed  CAS  Google Scholar 

  52. Zehender H et al (2004) SpeedScreen: the “missing link” between genomics and lead discovery. J Biomol Screen 9:498–505

    Article  PubMed  CAS  Google Scholar 

  53. Giannetti AM, Koch BD, Browner MF (2008) Surface plasmon resonance based assay for the detection and characterization of promiscuous inhibitors. J Med Chem 51:574–580

    Article  PubMed  CAS  Google Scholar 

  54. Freyer MW, Lewis EA (2008) Isothermal titration calorimetry: experimental design, data analysis, and probing macromolecule/ligand binding and kinetic interactions. Methods Cell Biol 84:79–113

    Article  PubMed  CAS  Google Scholar 

  55. Velazquez-Campoy A et al (2004) Isothermal titration calorimetry. Curr Protoc Cell Biol Chapter 17, Unit 17.8

    Google Scholar 

  56. Breeze AL (2000) Isotope-filtered NMR methods for the study of biomolecular structure and interactions. Prog NMR Spectrosc 36:323–372

    Article  CAS  Google Scholar 

  57. Tugarinov V, Kay LE (2005) Methyl groups as probes of structure and dynamics in NMR studies of high-molecular-weight proteins. Chembiochem 6:1567–1577

    Article  PubMed  CAS  Google Scholar 

  58. Li D, DeRose EF, London RE (1999) The inter-ligand Overhauser effect: a powerful new NMR approach for mapping structural relationships of macromolecular ligands. J Biomol NMR 15:71–76

    Article  PubMed  CAS  Google Scholar 

  59. Becattini B, Pellecchia M (2006) SAR by ILOEs: an NMR-based approach to reverse chemical genetics. Chem Eur J 12:2658–2662

    Article  PubMed  CAS  Google Scholar 

  60. Sanchez-Pedregal VM et al (2005) The INPHARMA method: protein-mediated interligand NOEs for pharmacophore mapping. Angew Chem Int Ed 44:4172–4175

    Article  CAS  Google Scholar 

  61. Bartoschek S et al (2010) Drug design for G-protein-coupled receptors by a ligand-based NMR method. Angew Chem Int Ed 49:1426–1429

    Article  CAS  Google Scholar 

  62. Ladbury JE, Klebe G, Freire E (2010) Adding calorimetric data to decision making in lead discovery: a hot tip. Nat Rev Drug Discov 9:23–27

    Article  PubMed  CAS  Google Scholar 

  63. Freire E (2009) A thermodynamic approach to the affinity optimization of drug candidates. Chem Biol Drug Des 74:468–472

    Article  PubMed  CAS  Google Scholar 

  64. Ruben AJ, Kiso Y, Freire E (2006) Overcoming roadblocks in lead optimization: a thermodynamic perspective. Chem Biol Drug Des 67:2–4

    Article  PubMed  CAS  Google Scholar 

  65. Freire E (2008) Do enthalpy and entropy distinguish first in class from best in class? Drug Discov Today 13:869–874

    Article  PubMed  CAS  Google Scholar 

  66. Ferenczy GG, Keserű GM (2010) Thermodynamics guided lead discovery and optimization. Drug Discov Today 15:919–932

    Article  PubMed  CAS  Google Scholar 

  67. Adams H et al (2001) Quantitative determination of intermolecular interactions with fluorinated aromatic rings. Chemistry 7:3494–3503

    Article  PubMed  CAS  Google Scholar 

  68. Swinney DC (2009) The role of binding kinetics in therapeutically useful drug action. Curr Opin Drug Discov Devel 12:31–39

    PubMed  CAS  Google Scholar 

  69. Zhang R, Monsma F (2009) The importance of drug-target residence time. Curr Opin Drug Discov Devel 12:488–496

    PubMed  CAS  Google Scholar 

  70. Tummino PJ, Copeland RA (2008) Residence time of receptor-ligand complexes and its effect on biological function. Biochemistry 47:5481–5492

    Article  PubMed  CAS  Google Scholar 

  71. Copeland RA, Pompliano DL, Meek TD (2006) Drug-target residence time and its implications for lead optimization. Nat Rev Drug Discov 5:730–739

    Article  PubMed  CAS  Google Scholar 

  72. Williams JW, Morrison JF, Duggleby RG (1979) Methotrexate, a high-affinity pseudosubstrate of dihydrofolate reductase. Biochemistry 18:2567–2573

    Article  PubMed  CAS  Google Scholar 

  73. Magotti P et al (2009) Structure-kinetic relationship analysis of the therapeutic complement inhibitor compstatin. J Mol Recognit 22:495–505

    Article  PubMed  CAS  Google Scholar 

  74. Lu H et al (2009) Slow-onset inhibition of the FabI enoyl reductase from Francisella tularensis: residence time and in vivo activity. ACS Chem Biol 4:221–231

    Article  PubMed  CAS  Google Scholar 

  75. Wan H, Rehngren M (2006) High-throughput screening of protein binding by equilibrium dialysis combined with liquid chromatography and mass spectroscopy. J Chromatogr A 1102:125–134

    Article  PubMed  CAS  Google Scholar 

  76. Lee KJ et al (2003) Modulation of nonspecific binding in ultrafiltration protein binding studies. Pharm Res 20:1015–1021

    Article  PubMed  CAS  Google Scholar 

  77. Rich RL et al (2001) High-resolution and high-throughput protocols for measuring drug/human serum albumin interactions using biacore. Anal Biochem 296:197–207

    Article  PubMed  CAS  Google Scholar 

  78. Valko K et al (2003) Fast gradient HPLC method to determine compounds binding to human serum albumin. Relationships with octanol/water and immobilized artificial membrane lipophilicity. J Pharm Sci 92:2236–2248

    Article  PubMed  CAS  Google Scholar 

  79. Eddershaw PJ, Beresford AP, Baylis MK (2000) ADME/PK as part of a rational approach to drug discovery. Drug Discov Today 5:409–414

    Article  PubMed  CAS  Google Scholar 

  80. Hop CE et al (2008) High throughput ADME screening: practical considerations, impact on the portfolio and enabler of in silico ADME models. Curr Drug Metab 9:847–853

    Article  PubMed  CAS  Google Scholar 

  81. Hsieh Y (2008) HPLC-MS/MS in drug metabolism and pharmacokinetic screening. Expert Opin Drug Metab Toxicol 4:93–101

    Article  PubMed  CAS  Google Scholar 

  82. Pang KS (2003) Modeling of intestinal drug absorption: roles of transporters and metabolic enzymes (for the Gillette review series). Drug Metab Dispos 31:1507–1519

    Article  PubMed  CAS  Google Scholar 

  83. Muenster U et al (2008) Characterization of substrates and inhibitors for the in vitro assessment of bcrp mediated drug-drug interactions. Pharm Res 25:2320–2326

    Article  PubMed  CAS  Google Scholar 

  84. Iwatsubo T et al (1997) Prediction of in vivo drug metabolism in the human liver from in vitro metabolism data. Pharmacol Ther 73:147–171

    Article  PubMed  CAS  Google Scholar 

  85. Prakash C, Shaffer CL, Nedderman A (2007) Analytical strategies for identifying drug metabolites. Mass Spectrom Rev 26:340–369

    Article  PubMed  CAS  Google Scholar 

  86. Riley RJ, McGinnity DF, Austin RP (2005) A unified model for predicting human hepatic, metabolic clearance from in vitro intrinsic clearance data in hepatocytes and microsomes. Drug Metab Dispos 33:1304–1311

    Article  PubMed  CAS  Google Scholar 

  87. Ito K et al (1998) Prediction of pharmacokinetic alterations caused by drug-drug interactions: metabolic interaction in the liver. Pharmacol Rev 50:387–412

    PubMed  CAS  Google Scholar 

  88. Moody GC et al (1999) Fully automated analysis of activities catalysed by the major human liver cytochrome P450 (CYP) enzymes: assessment of human CYP inhibition potential. Xenobiotica 29:53–75

    Article  PubMed  CAS  Google Scholar 

  89. Trubetskoy OV, Gibson JR, Marks BD (2005) Highly miniaturized formats for in vitro drug metabolism assays using vivid fluorescent substrates and recombinant human cytochrome P450 enzymes. J Biomol Screen 10:56–66

    Article  PubMed  CAS  Google Scholar 

  90. Zientek M et al (2008) Development of an in vitro drug-drug interaction assay to simultaneously monitor five cytochrome P450 isoforms and performance assessment using drug library compounds. J Pharmacol Toxicol Methods 58:206–214

    Article  PubMed  CAS  Google Scholar 

  91. Wang RW et al (2000) Human cytochrome P-450 3A4: in vitro drug-drug interaction patterns are substrate-dependent. Drug Metab Dispos 28:360–366

    PubMed  CAS  Google Scholar 

  92. Grimm SW et al (2009) The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America. Drug Metab Dispos 37:1355–1370

    Article  PubMed  CAS  Google Scholar 

  93. Atkinson A, Kenny JR, Grime K (2005) Automated assessment of time-dependent inhibition of human cytochrome P450 enzymes using liquid chromatography-tandem mass spectrometry analysis. Drug Metab Dispos 33:1637–1647

    Article  PubMed  CAS  Google Scholar 

  94. Ma B, Prueksaritanont T, Lin JH (2000) Drug interactions with calcium channel blockers: possible involvement of metabolite-intermediate complexation with CYP3A. Drug Metab Dispos 28:125–130

    PubMed  CAS  Google Scholar 

  95. Grime KH et al (2009) Mechanism-based inhibition of cytochrome P450 enzymes: an evaluation of early decision making in vitro approaches and drug-drug interaction prediction methods. Eur J Pharm Sci 36:175–191

    Article  PubMed  CAS  Google Scholar 

  96. Tiller PR, Romanyshyn LA, Neue UD (2003) Fast LC/MS in the analysis of small molecules. Anal Bioanal Chem 375:788–802

    Article  CAS  Google Scholar 

  97. Hsieh Y, Korfmacher A (2006) Increasing speed and throughput when using HPLC-MS/MS systems for drug metabolism and pharmacokinetic screening. Curr Drug Metab 7:479–489

    Article  PubMed  CAS  Google Scholar 

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Authors and Affiliations

  1. AstraZeneca R&D, Macclesfield, UK

    Geoffrey Holdgate, Alex Breeze, Gareth Davies, Nicola Colclough, David Temesi & Lara Ward

  2. AstraZeneca R&D, Mölndal, Sweden

    Stefan Geschwindner

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  1. Geoffrey Holdgate
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  2. Stefan Geschwindner
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  3. Alex Breeze
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  4. Gareth Davies
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  5. Nicola Colclough
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  6. David Temesi
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  7. Lara Ward
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Editors and Affiliations

  1. Birbeck, University of London, ISMB Biophysics Centre, Institute of Str, Malet Street, London, WC1E 7HX, United Kingdom

    Mark A. Williams

  2. Birkbeck, University of London, School of Crystallography, ISMB Biophysics Centre, Institute of Str, Malet Street, London, WC1E 7HX, United Kingdom

    Tina Daviter

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Holdgate, G. et al. (2013). Biophysical Methods in Drug Discovery from Small Molecule to Pharmaceutical. In: Williams, M., Daviter, T. (eds) Protein-Ligand Interactions. Methods in Molecular Biology, vol 1008. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-398-5_12

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  • DOI: https://doi.org/10.1007/978-1-62703-398-5_12

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