The rapid development of sensitive biophysical methods is transforming drug discovery by providing early profiling of compound properties and insights into mechanisms of action.
Biophysical methods are used extensively in hit finding (fragment-based screening and high-throughput screening), hit validation, in depth characterization of compound binding and lead optimization.
A growing application of biophysical methods is to understand the relationship between the kinetics of binding, mode of action, and molecular structures and interactions. This has had substantial impact on recognizing the importance of binding kinetics and residence time for therapeutic action.
Additional information derived from biophysical methods ranges from protein quality control, quantitative binding data, and determination of ligand–target complex structures to complex mode-of-action studies.
The efficient use of these methods requires experience in experimental design and data analysis, and a strategic combination of orthogonal methods.
Newly emerging technologies such as cryo-electron microscropy (cryo-EM) for high-resolution determination of massive biological structures and more rapid methods for the characterization of binding kinetics and thermodynamics will further reinforce the importance of biophysics in drug discovery.
Over the past 25 years, biophysical technologies such as X-ray crystallography, nuclear magnetic resonance spectroscopy, surface plasmon resonance spectroscopy and isothermal titration calorimetry have become key components of drug discovery platforms in many pharmaceutical companies and academic laboratories. There have been great improvements in the speed, sensitivity and range of possible measurements, providing high-resolution mechanistic, kinetic, thermodynamic and structural information on compound–target interactions. This Review provides a framework to understand this evolution by describing the key biophysical methods, the information they can provide and the ways in which they can be applied at different stages of the drug discovery process. We also discuss the challenges for current technologies and future opportunities to use biophysical methods to solve drug discovery problems.
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Arkin, M. R., Tang, Y. & Wells, J. A. Small-molecule inhibitors of protein–protein interactions: progressing toward the reality. Chem. Biol. 21, 1102–1114 (2014).
Higueruelo, A. P., Jubb, H. & Blundell, T. L. Protein–protein interactions as druggable targets: recent technological advances. Curr. Opin. Pharmacol. 13, 791–796 (2013).
Holdgate, G. A. & Gill, A. L. Kinetic efficiency: the missing metric for enhancing compound quality? Drug Discov. Today 16, 910–913 (2011).
Copeland, R. A. The drug-target residence time model: a 10-year retrospective. Nat. Rev. Drug Discov. 15, 87–95 (2016). This paper explains the context and importance of the drug–target residence time concept in drug discovery. Progress in exploiting this concept in the past decade is summarized alongside opportunities for further advances.
Copeland, R. A., Pompliano, D. L. & Meek, T. D. Drug-target residence time and its implications for lead optimization. Nat. Rev. Drug Discov. 5, 730–739 (2006).
Jhoti, H., Williams, G., Rees, D. C. & Murray, C. W. The 'rule of three' for fragment-based drug discovery: where are we now? Nat. Rev. Drug Discov. 12, 644–645 (2013).
Silvestre, H. L., Blundell, T. L., Abell, C. & Ciulli, A. Integrated biophysical approach to fragment screening and validation for fragment-based lead discovery. Proc. Natl Acad. Sci. USA 110, 12984–12989 (2013). This paper describes the application of fragment screening using different biophysical techniques.
Erlanson, D. A. & Zartler, E. Fragments in the clinic: 2015 edition. Practical Fragments, http://practicalfragments.blogspot.fr/2015/01/fragments-in-clinic-2015-edition.html (2015).
Klebe, G. Applying thermodynamic profiling in lead finding and optimization. Nat. Rev. Drug Discov. 14, 95–110 (2015). In this paper, multiple factors influencing the thermodynamics of binding are described. Key issues in understanding the effects of these factors and the application of this this knowledge in drug discovery are presented.
Leeson, P. D. & Springthorpe, B. The influence of drug-like concepts on decision-making in medicinal chemistry. Nat. Rev. Drug Discov. 6, 881–890 (2007).
Blundell, T. L. & Patel, S. High-throughput X-ray crystallography for drug discovery. Curr. Opin. Pharmacol. 4, 490–496 (2004).
Cala, O., Guilliere, F. & Krimm, I. NMR-based analysis of protein-ligand interactions. Anal. Bioanal. Chem. 406, 943–956 (2014).
Pellecchia, M. et al. Perspectives on NMR in drug discovery: a technique comes of age. Nat. Rev. Drug Discov. 7, 738–745 (2008).
Śledź, P., Abell, C. & Ciulli, A. NMR of Biomolecules: Towards Mechanistic Systems Biology. 1st edn (eds Bertini, I., McGreevy, K. S. & Parigi, G.) 265–280 (Wiley-VCH Verlag GmbH & Co. KGaA, 2012).
Cooper, M. A. Optical biosensors in drug discovery. Nat. Rev. Drug Discov. 1, 515–528 (2002).
Geschwindner, S., Carlsson, J. F. & Knecht, W. Application of optical biosensors in small-molecule screening activities. Sensors (Basel) 12, 4311–4323 (2012).
Huber, W. & Mueller, F. Biomolecular interaction analysis in drug discovery using surface plasmon resonance technology. Curr. Pharm. Des. 12, 3999–4021 (2006).
Neumann, T., Junker, H. D., Schmidt, K. & Sekul, R. SPR-based fragment screening: advantages and applications. Curr. Top. Med. Chem. 7, 1630–1642 (2007).
Renaud, J. P., Neumann, T. & Van Hijfte, L. Small Molecule Medicinal Chemistry: Strategies and Technologies. (eds Czechtizky, W. & Hamley, P.) 221–249 (John Wiley & Sons Inc., 2015).
Lo, M. C. et al. Evaluation of fluorescence-based thermal shift assays for hit identification in drug discovery. Anal. Biochem. 332, 153–159 (2004).
Niesen, F. H., Berglund, H. & Vedadi, M. The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability. Nat. Protoc. 2, 2212–2221 (2007).
Ladbury, J. E., Klebe, G. & Freire, E. Adding calorimetric data to decision making in lead discovery: a hot tip. Nat. Rev. Drug Discov. 9, 23–27 (2010).
Chaires, J. B. Calorimetry and thermodynamics in drug design. Annu. Rev. Biophys. 37, 135–151 (2008).
Hofstadler, S. A. & Sannes-Lowery, K. A. Applications of ESI-MS in drug discovery: interrogation of noncovalent complexes. Nat. Rev. Drug Discov. 5, 585–595 (2006).
Vivat Hannah, V., Atmanene, C., Zeyer, D., Van Dorsselaer, A. & Sanglier-Cianferani, S. Native MS: an 'ESI' way to support structure- and fragment-based drug discovery. Future Med. Chem. 2, 35–50 (2010).
Annis, D. A., Nickbarg, E., Yang, X., Ziebell, M. R. & Whitehurst, C. E. Affinity selection-mass spectrometry screening techniques for small molecule drug discovery. Curr. Opin. Chem. Biol. 11, 518–526 (2007).
Chalmers, M. J., Busby, S. A., Pascal, B. D., West, G. M. & Griffin, P. R. Differential hydrogen/deuterium exchange mass spectrometry analysis of protein–ligand interactions. Expert Rev. Proteom. 8, 43–59 (2011).
Konermann, L., Pan, J. & Liu, Y. H. Hydrogen exchange mass spectrometry for studying protein structure and dynamics. Chem. Soc. Rev. 40, 1224–1234 (2011).
Seidel, S. A. et al. Microscale thermophoresis quantifies biomolecular interactions under previously challenging conditions. Methods 59, 301–315 (2013).
Seidel, S. A. et al. Label-free microscale thermophoresis discriminates sites and affinity of protein–ligand binding. Angew. Chem. Int. 51, 10656–10659 (2012).
Tuukkanen, A. T. & Svergun, D. I. Weak protein-ligand interactions studied by small-angle X-ray scattering. FEBS J. 281, 1974–1987 (2014).
Vestergaard, B. & Sayers, Z. Investigating increasingly complex macromolecular systems with small-angle X-ray scattering. IUCrJ 1, 523–529 (2014).
Hirst, E. R., Yuan, Y. J., Xu, W. L. & Bronlund, J. E. Bond-rupture immunosensors — a review. Biosens. Bioelectron. 23, 1759–1768 (2008).
Cooper, M. A. & Singleton, V. T. A survey of the 2001 to 2005 quartz crystal microbalance biosensor literature: applications of acoustic physics to the analysis of biomolecular interactions. J. Mol. Recognit 20, 154–184 (2007).
Björke, H. & Andersson, K. Measuring the affinity of a radioligand with its receptor using a rotating cell dish with in situ reference area. Appl. Radiat. Isot. 64, 32–37 (2006).
Danielson, U. H. Fragment library screening and lead characterization using SPR biosensors. Curr. Top. Med. Chem. 9, 1725–1735 (2009).
Giannetti, A. M. From experimental design to validated hits a comprehensive walk-through of fragment lead identification using surface plasmon resonance. Methods Enzymol. 493, 169–218 (2011).
Davis, B. J. & Erlanson, D. A. Learning from our mistakes: the 'unknown knowns' in fragment screening. Bioorg. Med. Chem. Lett. 23, 2844–2852 (2013).
Kitova, E. N., El-Hawiet, A., Schnier, P. D. & Klassen, J. S. Reliable determinations of protein-ligand interactions by direct ESI-MS measurements. Are we there yet? J. Am. Soc. Mass Spectrom. 23, 431–441 (2012).
Hubbard, R. E. & Murray, J. B. Experiences in fragment-based lead discovery. Methods Enzymol. 493, 509–531 (2011). This paper provides an overview of the application of biophysical techniques in fragment screening across a range of different targets.
Shuker, S. B., Hajduk, P. J., Meadows, R. P. & Fesik, S. W. Discovering high-affinity ligands for proteins: SAR by NMR. Science 274, 1531–1534 (1996).
Hajduk, P. J. & Greer, J. A decade of fragment-based drug design: strategic advances and lessons learned. Nat. Rev. Drug Discov. 6, 211–219 (2007).
Nienaber, V. L. et al. Discovering novel ligands for macromolecules using X-ray crystallographic screening. Nat. Biotechnol. 18, 1105–1108 (2000).
Sharff, A. & Jhoti, H. High-throughput crystallography to enhance drug discovery. Curr. Opin. Chem. Biol. 7, 340–345 (2003).
Murray, C. W., Verdonk, M. L. & Rees, D. C. Experiences in fragment-based drug discovery. Trends Pharmacol. Sci. 33, 224–232 (2012).
Hubbard, R. E., Davis, B., Chen, I. & Drysdale, M. J. The SeeDs approach: integrating fragments into drug discovery. Curr. Top. Med. Chem. 7, 1568–1581 (2007).
Nordström, H. et al. Identification of MMP-12 inhibitors by using biosensor-based screening of a fragment library. J. Med. Chem. 51, 3449–3459 (2008).
Kuo, L. C. Fragment-based drug design — tools, practical approaches, and examples. Preface. Methods Enzymol. 493, xxi–xxii (2011).
Schiebel, J. et al. One question, multiple answers: biochemical and biophysical screening methods retrieve deviating fragment hit lists. ChemMedChem. 10, 1511–1521 (2015). This is a study of how different biophysical filtering techniques result in poorly overlapping compound collections in fragment screening.
Hennig, M., Ruf, A. & Huber, W. Combining biophysical screening and X-ray crystallography for fragment-based drug discovery. Top. Curr. Chem. 317, 115–143 (2012).
Zhang, J. et al. Targeting Bcr–Abl by combining allosteric with ATP-binding-site inhibitors. Nature 463, 501–506 (2010).
Jahnke, W. et al. Allosteric non-bisphosphonate FPPS inhibitors identified by fragment-based discovery. Nat. Chem. Biol. 6, 660–666 (2010).
Darby, J. F. et al. Discovery of selective small-molecule activators of a bacterial glycoside hydrolase. Angew. Chem. Int. 53, 13419–13423 (2014).
Winter, A. et al. Biophysical and computational fragment-based approaches to targeting protein-protein interactions: applications in structure-guided drug discovery. Q. Rev. Biophys. 45, 383–426 (2012).
Iversen, P. W. et al. in Assay Guidance Manual (eds Sittampalam, G. S. et al.) (Bethesda (MD), 2004).
Folmer, R. H. Integrating biophysics with HTS-driven drug discovery projects. Drug Discov. Today 21, 491–498 (2016). This is a discussion of the impact of biophysical techniques for screening as well as their application as filters for HTS outcomes.
Genick, C. C. et al. Applications of biophysics in high-throughput screening hit validation. J. Biomol. Screen 19, 707–714 (2014).
Pantoliano, M. W. et al. High-density miniaturized thermal shift assays as a general strategy for drug discovery. J. Biomol. Screen 6, 429–440 (2001).
Roddy, T. P. et al. Mass spectrometric techniques for label-free high-throughput screening in drug discovery. Anal. Chem. 79, 8207–8213 (2007).
Baell, J. & Walters, M. A. Chemistry: chemical con artists foil drug discovery. Nature 513, 481–483 (2014).
Seidler, J., McGovern, S. L., Doman, T. N. & Shoichet, B. K. Identification and prediction of promiscuous aggregating inhibitors among known drugs. J. Med. Chem. 46, 4477–4486 (2003).
Holdgate, G. A., Anderson, M., Edfeldt, F. & Geschwindner, S. 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 (2010).
Elinder, M. et al. Inhibition of HIV-1 by non-nucleoside reverse transcriptase inhibitors via an induced fit mechanism-Importance of slow dissociation and relaxation rates for antiviral efficacy. Biochem. Pharmacol. 80, 1133–1140 (2010).
Geitmann, M. et al. Interaction kinetic and structural dynamic analysis of ligand binding to acetylcholine-binding protein. Biochemistry 49, 8143–8154 (2010).
Seeger, C., Gorny, X., Reddy, P. P., Seidenbecher, C. & Danielson, U. H. Kinetic and mechanistic differences in the interactions between caldendrin and calmodulin with AKAP79 suggest different roles in synaptic function. J. Mol. Recognit 25, 495–503 (2012).
Souers, A. J. et al. ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor activity while sparing platelets. Nat. Med. 19, 202–208 (2013).
Saalau-Bethell, S. M. et al. Discovery of an allosteric mechanism for the regulation of HCV NS3 protein function. Nat. Chem. Biol. 8, 920–925 (2012).
Friberg, A. et al. Discovery of potent myeloid cell leukemia 1 (Mcl-1) inhibitors using fragment-based methods and structure-based design. J. Med. Chem. 56, 15–30 (2013).
Maurer, T. et al. Small-molecule ligands bind to a distinct pocket in Ras and inhibit SOS-mediated nucleotide exchange activity. Proc. Natl Acad. Sci. USA 109, 5299–5304 (2012).
Sun, Q. et al. Discovery of small molecules that bind to K-Ras and inhibit Sos-mediated activation. Angew. Chem. Int. 51, 6140–6143 (2012).
Ostrem, J. M., Peters, U., Sos, M. L., Wells, J. A. & Shokat, K. M. K-Ras(G12C) inhibitors allosterically control GTP affinity and effector interactions. Nature 503, 548–551 (2013).
Pickhardt, M. et al. Identification of small molecule inhibitors of tau aggregation by targeting monomeric tau as a potential therapeutic approach for tauopathies. Curr. Alzheimer Res. 12, 814–828 (2015).
Bunnage, M. E. Getting pharmaceutical R&D back on target. Nat. Chem. Biol. 7, 335–339 (2011).
Martinez Molina, D. et al. Monitoring drug target engagement in cells and tissues using the cellular thermal shift assay. Science 341, 84–87 (2013).
Reinhard, F. B. et al. Thermal proteome profiling monitors ligand interactions with cellular membrane proteins. Nat. Methods 12, 1129–1131 (2015).
Savitski, M. M. et al. Tracking cancer drugs in living cells by thermal profiling of the proteome. Science 346, 1255784 (2014).
Martinez Molina, D. & Nordlund, P. The cellular thermal shift assay: a novel biophysical assay for in situ drug target engagement and mechanistic biomarker studies. Annu. Rev. Pharmacol. Toxicol. 56, 141–161 (2016).
Navratilova, I., Dioszegi, M. & Myszka, D. G. Analyzing ligand and small molecule binding activity of solubilized GPCRs using biosensor technology. Anal. Biochem. 355, 132–139 (2006).
Navratilova, I., Sodroski, J. & Myszka, D. G. Solubilization, stabilization, and purification of chemokine receptors using biosensor technology. Anal. Biochem. 339, 271–281 (2005).
Aristotelous, T. et al. Discovery of β2 adrenergic receptor ligands using biosensor fragment screening of tagged wild-type receptor. ACS Med. Chem. Lett. 4, 1005–1010 (2013).
Chu, R., Reczek, D. & Brondyk, W. Capture-stabilize approach for membrane protein SPR assays. Sci. Rep. 4, 7360 (2014).
Christopher, J. A. et al. Biophysical fragment screening of the β1-adrenergic receptor: identification of high affinity arylpiperazine leads using structure-based drug design. J. Med. Chem. 56, 3446–3455 (2013).
Errey, J. C., Dore, A. S., Zhukov, A., Marshall, F. H. & Cooke, R. M. Purification of stabilized GPCRs for structural and biophysical analyses. Methods Mol. Biol. 1335, 1–15 (2015).
Congreve, M. et al. Fragment screening of stabilized G-protein-coupled receptors using biophysical methods. Methods Enzymol. 493, 115–136 (2011).
Christopher, J. A. et al. Discovery of HTL6641, a dual orexin receptor antagonist with differentiated pharmacodynamic properties. MedChemComm 6, 947–955 (2015).
Egloff, P. et al. Structure of signaling-competent neurotensin receptor 1 obtained by directed evolution in Escherichia coli. Proc. Natl Acad. Sci. USA 111, E655–E662 (2014).
Chen, D. et al. Fragment screening of GPCRs using biophysical methods: identification of ligands of the adenosine A2A receptor with novel biological activity. ACS Chem. Biol. 7, 2064–2073 (2012).
Fruh, V. et al. Application of fragment-based drug discovery to membrane proteins: identification of ligands of the integral membrane enzyme DsbB. Chem. Biol. 17, 881–891 (2010).
Bocquet, N. et al. Real-time monitoring of binding events on a thermostabilized human A2A receptor embedded in a lipid bilayer by surface plasmon resonance. Biochim. Biophys. Acta 1848, 1224–1233 (2015).
Dawson, R. J. et al. Structure of the acid-sensing ion channel 1 in complex with the gating modifier Psalmotoxin 1. Nat. Commun. 3, 936 (2012).
Seeger, C. et al. Histaminergic pharmacology of homo-oligomeric β3 γ-aminobutyric acid type A receptors characterized by surface plasmon resonance biosensor technology. Biochem. Pharmacol. 84, 341–351 (2012).
Kesters, D. et al. Structural basis of ligand recognition in 5-HT3 receptors. EMBO Rep. 14, 49–56 (2013).
Spurny, R. et al. Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the alpha7 nicotinic acetylcholine receptor. Proc. Natl Acad. Sci. USA 112, E2543–2552 (2015).
Perspicace, S. et al. Isothermal titration calorimetry with micelles: thermodynamics of inhibitor binding to carnitine palmitoyltransferase 2 membrane protein. FEBS Open Bio. 3, 204–211 (2013).
Claridge, S. A., Schwartz, J. J. & Weiss, P. S. Electrons, photons, and force: quantitative single-molecule measurements from physics to biology. ACS Nano. 5, 693–729 (2011).
Kapanidis, A. N. & Strick, T. Biology, one molecule at a time. Trends Biochem. Sci. 34, 234–243 (2009).
Walter, N. G., Huang, C. Y., Manzo, A. J. & Sobhy, M. A. Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat. Methods 5, 475–489 (2008). This is a summary of the current state of the art in single-molecule tools, including fluorescence spectroscopy, tethered particle microscopy, optical and magnetic tweezers, and atomic force microscopy, including guidelines on when to apply each method and what outcome to expect from them.
Grohmann, D., Werner, F. & Tinnefeld, P. Making connections — strategies for single molecule fluorescence biophysics. Curr. Opin. Chem. Biol. 17, 691–698 (2013).
Smiley, R. D. & Hammes, G. G. Single molecule studies of enzyme mechanisms. Chem. Rev. 106, 3080–3094 (2006).
Roy, R., Hohng, S. & Ha, T. A practical guide to single-molecule FRET. Nat. Methods 5, 507–516 (2008).
Gunnarsson, A. et al. Drug discovery at the single molecule level: inhibition-in-solution assay of membrane-reconstituted beta-secretase using single-molecule imaging. Anal. Chem. 87, 4100–4103 (2015).
Zhao, Y. et al. Lab-on-a-chip technologies for single-molecule studies. Lab. Chip 13, 2183–2198 (2013).
Marx, V. Structural biology: 'seeing' crystals the XFEL way. Nat. Methods 11, 903–908 (2014).
Schlichting, I. Serial femtosecond crystallography: the first five years. IUCrJ 2, 246–255 (2015).
Bai, X. C., McMullan, G. & Scheres, S. H. How cryo-EM is revolutionizing structural biology. Trends Biochem. Sci. 40, 49–57 (2015). This is a good overview of recent advances in cryo-EM techniques and what can be achieved with these techniques regarding opportunities in structural biology (such as size of molecules and resolution).
Cheng, Y., Grigorieff, N., Penczek, P. A. & Walz, T. A primer to single-particle cryo-electron microscopy. Cell 161, 438–449 (2015).
Kern, J., Yachandra, V. K. & Yano, J. Metalloprotein structures at ambient conditions and in real-time: biological crystallography and spectroscopy using X-ray free electron lasers. Curr. Opin. Struct. Biol. 34, 87–98 (2015).
Liao, M., Cao, E., Julius, D. & Cheng, Y. Structure of the TRPV1 ion channel determined by electron cryo-microscopy. Nature 504, 107–112 (2013).
Bai, X. C. et al. An atomic structure of human gamma-secretase. Nature 525, 212–217 (2015).
Bartesaghi, A. et al. 2.2 Å resolution cryo-EM structure of beta-galactosidase in complex with a cell-permeant inhibitor. Science 348, 1147–1151 (2015).
Henderson, R. The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules. Q. Rev. Biophys. 28, 171–193 (1995).
Markgren, P. O., Hämäläinen, M. & Danielson, U. H. Kinetic analysis of the interaction between HIV-1 protease and inhibitors using optical biosensor technology. Anal. Biochem. 279, 71–78 (2000).
Gossas, T. et al. The advantage of biosensor analysis over enzyme inhibition studies for slow dissociating inhibitors — characterization of hydroxamate-based matrix metalloproteinase-12 inhibitors. MedChemComm 4, 432–442 (2013).
Talibov, V. O., Linkiviene, V., Matulis, D. & Danielson, U. H. Kinetically selective inhibitors of human carbonic anhydrase isoenzymes I, II, VII, IX, XII and XIII. J. Med. Chem. 59, 2083–2093 (2016).
Christopeit, T. et al. A surface plasmon resonance-based biosensor with full-length BACE1 in a reconstituted membrane. Anal. Biochem. 414, 14–22 (2011).
Dominguez, J. L. et al. Effect of the protonation state of the titratable residues on the inhibitor affinity to BACE-1. Biochemistry 49, 7255–7263 (2010).
Sussman, F., Villaverde, M. C., Dominguez, J. L. & Danielson, U. H. On the active site protonation state in aspartic proteases: implications for drug design. Curr. Pharm. Des. 19, 4257–4275 (2013).
Cusack, K. P. et al. Design strategies to address kinetics of drug binding and residence time. Bioorg. Med. Chem. Lett. 25, 2019–2027 (2015). In this paper, factors contributing to long residence time are introduced by the authors as well as how these factors could be useful to medicinal chemists in the design of compounds with long residence times. Case studies on structure–kinetic relationships are given.
Swinney, D. C. The role of binding kinetics in therapeutically useful drug action. Curr. Opin. Drug Discov. Devel 12, 31–39 (2009).
Dahl, G. & Åkerud, T. Pharmacokinetics and the drug-target residence time concept. Drug Discov. Today 18, 697–707 (2013). This article raises concerns about the utility of drug–target residence times for predicting the in vivo outcomes in terms of duration of effect of drugs when pharmacokinetics are not taken into account.
Pan, A. C., Borhani, D. W., Dror, R. O. & Shaw, D. E. Molecular determinants of drug-receptor binding kinetics. Drug Discov. Today 18, 667–673 (2013). This is a discussion of the challenges of using kinetics during lead optimization and a review of key factors that are thought to be important for drug–receptor binding kinetics.
Bradshaw, J. M. et al. Prolonged and tunable residence time using reversible covalent kinase inhibitors. Nat. Chem. Biol. 11, 525–531 (2015).
Ferenczy, G. G. & Keseru, G. M. Thermodynamics guided lead discovery and optimization. Drug Discov. Today 15, 919–932 (2010).
Holdgate, G. A. Thermodynamics of binding interactions in the rational drug design process. Expert Opin. Drug Discov. 2, 1103–1114 (2007).
Chodera, J. D. & Mobley, D. L. Entropy-enthalpy compensation: role and ramifications in biomolecular ligand recognition and design. Annu. Rev. Biophys. 42, 121–142 (2013).
Klebe, G. The use of thermodynamic and kinetic data in drug discovery: decisive insight or increasing the puzzlement? ChemMedChem. 10, 229–231 (2015).
Geschwindner, S., Ulander, J. & Johansson, P. Ligand binding thermodynamics in drug discovery: still a hot tip? J. Med. Chem. 58, 6321–6335 (2015). This article shows that the mere use of enthalpy and entropy data is of little help in lead optimization in the absence of structural information, owing to multiple factors affecting thermodynamic signatures. Several case studies are presented.
Schiele, F., Ayaz, P. & Fernandez-Montalvan, A. A universal homogeneous assay for high-throughput determination of binding kinetics. Anal. Biochem. 468, 42–49 (2014). This article introduces a method for high-throughput determination of kinetic parameters, exemplifying it using three different target classes: enzymes, protein–protein interactions and GPCRs.
Motulsky, H. J. & Mahan, L. C. The kinetics of competitive radioligand binding predicted by the law of mass action. Mol. Pharmacol. 25, 1–9 (1984).
Braissant, O. et al. Isothermal microcalorimetry accurately detects bacteria, tumorous microtissues, and parasitic worms in a label-free well-plate assay. Biotechnol. J. 10, 460–468 (2015).
Luchinat, E. & Banci, L. A. Unique tool for cellular structural biology: in-cell NMR. J. Biol. Chem. 291, 3776–3784 (2016).
Leake, M. C. The physics of life: one molecule at a time. Phil. Trans. R. Soc. B 368, 20120248 (2013).
Aristotelous, T., Hopkins, A. L. & Navratilova, I. Surface plasmon resonance analysis of seven-transmembrane receptors. Methods Enzymol. 556, 499–525 (2015). This is an overview of SPR technologies and their application to GPCRs.
Olaru, A., Bala, C., Jaffrezic-Renault, N. & Aboul-Enein, H. Y. Surface plasmon resonance (SPR) biosensors in pharmaceutical analysis. Crit. Rev. Anal. Chem. 45, 97–105 (2015).
Patching, S. G. Surface plasmon resonance spectroscopy for characterisation of membrane protein-ligand interactions and its potential for drug discovery. Biochim. Biophys. Acta 1838, 43–55 (2014).
Recht, M. I. et al. Identification and optimization of PDE10A inhibitors using fragment-based screening by nanocalorimetry and X-ray crystallography. J. Biomol. Screen 19, 497–507 (2014).
Oltersdorf, T. et al. An inhibitor of Bcl-2 family proteins induces regression of solid tumours. Nature 435, 677–681 (2005).
Brough, P. A. et al. 4,5-diarylisoxazole Hsp90 chaperone inhibitors: potential therapeutic agents for the treatment of cancer. J. Med. Chem. 51, 196–218 (2008).
Murray, J. B., Roughley, S. D., Matassova, N. & Brough, P. A. Off-rate screening (ORS) by surface plasmon resonance. An efficient method to kinetically sample hit to lead chemical space from unpurified reaction products. J. Med. Chem. 57, 2845–2850 (2014).
Brough, P. A. et al. Combining hit identification strategies: fragment-based and in silico approaches to orally active 2-aminothieno[2,3-d]pyrimidine inhibitors of the Hsp90 molecular chaperone. J. Med. Chem. 52, 4794–4809 (2009).
Marzinzik, A. L. et al. Discovery of novel allosteric non-bisphosphonate inhibitors of farnesyl pyrophosphate synthase by integrated lead finding. ChemMedChem. 10, 1884–1891 (2015).
Harner, M. J., Frank, A. O. & Fesik, S. W. Fragment-based drug discovery using NMR spectroscopy. J. Biomol. NMR 56, 65–75 (2013).
Wu, B. et al. HTS by NMR of combinatorial libraries: a fragment-based approach to ligand discovery. Chem. Biol. 20, 19–33 (2013).
Wu, B. et al. High-throughput screening by nuclear magnetic resonance (HTS by NMR) for the identification of PPIs antagonists. Curr. Top. Med. Chem. 15, 2032–2042 (2015).
Bruylants, G., Wouters, J. & Michaux, C. Differential scanning calorimetry in life science: thermodynamics, stability, molecular recognition and application in drug design. Curr. Med. Chem. 12, 2011–2020 (2005).
Concepcion, J. et al. Label-free detection of biomolecular interactions using BioLayer interferometry for kinetic characterization. Comb. Chem. High Throughput Screen. 12, 791–800 (2009).
Shah, N. B. & Duncan, T. M. Bio-layer interferometry for measuring kinetics of protein–protein interactions and allosteric ligand effects. J. Vis. Exp. 18, e51383 (2014).
Wartchow, C. A. et al. Biosensor-based small molecule fragment screening with biolayer interferometry. J. Comput. Aided Mol. Des. 25, 669–676 (2011).
Baksh, M. M., Kussrow, A. K., Mileni, M., Finn, M. G. & Bornhop, D. J. Label-free quantification of membrane-ligand interactions using backscattering interferometry. Nat. Biotechnol. 29, 357–360 (2011).
Bornhop, D. J. et al. Free-solution, label-free molecular interactions studied by back-scattering interferometry. Science 317, 1732–1736 (2007).
Heym, R. G., Hornberger, W. B., Lakics, V. & Terstappen, G. C. Label-free detection of small-molecule binding to a GPCR in the membrane environment. Biochim. Biophys. Acta 1854, 979–986 (2015).
Perpeet, M. et al. SAW sensor system for marker-free molecular interaction analysis. Anal. Lett. 39, 1747–1757 (2006).
Moree, B. et al. Protein conformational changes are detected and resolved site specifically by second-harmonic generation. Biophys. J. 109, 806–815 (2015).
Moree, B. et al. Small molecules detected by second-harmonic generation modulate the conformation of monomeric α-synuclein and reduce its aggregation in cells. J. Biol. Chem. 290, 27582–27593 (2015).
Salafsky, J. S. Detection of protein conformational change by optical second-harmonic generation. J. Chem. Phys. 125, 074701 (2006).
Kozma, P., Hamori, A., Kurunczi, S., Cottier, K. & Horvath, R. Grating coupled optical waveguide interferometer for label-free biosensing. Sens. Actuators B: Chem. 155, 446–450 (2011).
Kozma, P., Kehl, F., Ehrentreich-Forster, E., Stamm, C. & Bier, F. F. Integrated planar optical waveguide interferometer biosensors: a comparative review. Biosens. Bioelectron. 58, 287–307 (2014).
Langer, A. et al. Protein analysis by time-resolved measurements with an electro-switchable DNA chip. Nat. Commun. 4, 2099 (2013).
J.P.R. is a co-founder and a shareholder of NovAliX.
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Renaud, J., Chung, C., Danielson, U. et al. Biophysics in drug discovery: impact, challenges and opportunities. Nat Rev Drug Discov 15, 679–698 (2016). https://doi.org/10.1038/nrd.2016.123
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