Abstract
Small-molecule drug discovery involves the optimization of various physicochemical properties of a ligand, particularly its binding affinity for its target receptor (or receptors). In recent years, there has been growing interest in using thermodynamic profiling of ligand–receptor interactions in order to select and optimize those ligands that might be most likely to become drug candidates with desirable physicochemical properties. The thermodynamics of binding is influenced by multiple factors, including hydrogen bonding and hydrophobic interactions, desolvation, residual mobility, dynamics and the local water structure. This article discusses key issues in understanding the effects of these factors and applying this knowledge in drug discovery.
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References
Wermuth, C. G. in The Practice of Medicinal Chemistry Ch. 18 (Elsevier, 2003).
Blundell, T. L., Jhoti, H. & Abell, C. High-throughput crystallography for lead discovery in drug design. Nature Rev. Drug. Discov. 2, 45–53 (2002).
de Kloe, G. E., Bailey, D., Leurs, R. & de Esch, I. J. Transforming fragments into candidates: small becomes big in medicinal chemistry. Drug Discov. Today 14, 630–646 (2009).
Ajay & Murcko, M. A. Computational methods to predict binding free energy in ligand-receptor complexes. J. Med. Chem. 38, 4953–4967 (1995).
Cheng, Y.-C. & Prusoff, W. H. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973).
Klebe, G. Drug Design: Methodology, Concepts, and Mode-of-Action Ch. 4 (Springer Reference, 2013).
Chaires, J. B. Calorimetry and thermodynamics in drug design. Ann. Rev. Biophys. 37, 135–151 (2008).
Weber, I. T. & Agniswamy, J. HIV-1 protease: structural perspective on drug resistance. Viruses 1, 1110–1136 (2009).
Ali, A. et al. Molecular basis for drug resistance in HIV-1 protease. Viruses 2, 2509–2535 (2010).
Freire, E. Do enthalpy and entropy distinguish first in class from best in class? Drug Discov. Today 13, 869–874 (2008).
Ohtaka, H. & Freire, E. Adaptive inhibitors of the HIV-1 protease. Prog. Biophys. Mol. Biol. 88, 193–208 (2005).
Fernandez, A., Frazer, C. & Scott, L. R. Purposely engineered drug-target mismatches for entropy-based drug optimization. Trends Biotech. 30, 1–7 (2012).
Das, K., Lewi, P. J., Hughes, S. H. & Arnold, E. Crystallography and the design of anti-AIDS drugs: conformational flexibility and positional adaptability are important in the design of non-nucleoside HIV-1 reverse transcriptase inhibitors. Prog. Biophys. Mol. Biol. 88, 209–231 (2005).
Ladbury, J. & Chowdhry, B. Z. Sensing the heat: the application of isothermal titration calorimetry to thermodynamic studies of biomolecular interactions. Chem. Biol. 3, 791–801 (1996).
Ladbury, J. E. Isothermal titration calorimetry: application to structure-based drug design. Thermochim. Acta 380, 209–215 (2001).
Velazquez-Campoy, A. & Freire, E. ITC in the post-genomic era? Priceless. Biophys. Chem. 115, 115–124 (2005).
Jelesarov, I. & Bossard, H. R. Isothermal titration calorimetry and differential scanning calorimetry as complementary tools to investigate the energetic of biomolecular recognition. J. Mol. Recogn. 12, 3–18 (1999).
Baker, B. M. & Murphy, K. P. Evaluation of linked protonation effects in protein binding reactions using isothermal titration calorimetry. Biophys. J. 71, 2049–2055 (1996).
Falconer, R. J. & Collins, B. M. Survey of the year 2009: applications of isothermal titration calorimetry. J. Mol. Recogn. 24, 1–16 (2011).
Czodrowski, P., Sotriffer, C. A. & Klebe, G. Protonation changes upon ligand binding to trypsin and thrombin: structural interpretation based on pKa calculations and ITC experiments. J. Mol. Biol. 367, 1347–1356 (2007).
Baum, B. et al. Think twice: understanding the high potency of bis(phenyl)methane inhibitors of thrombin. J. Mol. Biol. 391, 552–564 (2009).
Goldberg, R. N., Kishore, N. & Lennen, R. M. Thermodynamic quantities for the reactions of buffers. J. Phys. Chem. Ref. Data 31, 231–370 (2002).
Simunec, J. Microcalorimetric Studies to Understand the Thermodynamic and Structural Properties of Inhibitors of the Blood Coagulation Cascade. Thesis, Univ. Marburg (2007).
Sharp, K. Entropy-enthalpy compensation: fact or artifact? Prot. Sci. 10, 661–667 (2001).
Olsson, T. S. G., Ladbury, J. E., Pitt, W. R. & Williams, M. A. Extent of enthalpy-entropy compensation in protein-ligand interactions. Prot. Sci. 20, 1607–1618 (2011).
Chodera, J. D. & Mobley, D. L. Entropy-enthalpy compensation: role and ramifications in biomolecular ligand recognition and design. Ann. Rev. Biophys. 42, 121–142 (2013).
Dunitz, J. D. Win some, lose some: enthalpy-entropy compensation in weak intermolecular interactions. Chem. Biol. 2, 709–712 (2003).
Page, M. I. & Jencks, W. P. Entropic contributions to rate accelerations in enzymic and intramolecular reactions and chelate effect. Proc. Natl Acad. Sci. USA 68, 1678–1683 (1971).
Murray, C. W. & Verdonk, M. L. The consequences of translational and rotational entropy lost by small molecules on binding to proteins. J. Comput. Aided Mol. Design 16, 741–753 (2002).
Nazare, M. et al. Fragment deconstruction of small, potent factor Xa inhibitors: exploring the superadditivity energetic of fragment linking in protein-ligand complexes. Angew. Chem. Int. Ed. 51, 905–911 (2012).
Borsi, V., Calderone, V., Fragai, M., Luchinat, C. & Sarti, N. Entropic contribution to the linking coefficient in fragment-based drug design: a case study. J. Med. Chem. 53, 4285–4289 (2010).
Olsson, T. S. G., Williams, M. A., Pitt, W. R. & Ladbury, J. E. The thermodynamics of protein-ligand interactions and solvation: insights for ligand design. J. Mol. Biol. 384, 1002–1017 (2008).
Hann, M. M. & Kerserü, G. M. Finding the sweet spot: the role of nature and nurture in medicinal chemistry. Nature Rev. Drug Discov. 11, 355–365 (2011).
Ferenczy, G. G. & Kerserü, G. M. Thermodynamics guided lead discovery and optimization. Drug Discov. Today 15, 919–932 (2010).
Reynolds, C. H. & Holloway, M. K. Thermodynamics of ligand binding and efficiency. ACS Med. Chem. Lett. 2, 433–437 (2011).
Ferenczy, G. G. & Keserü, G. M. Thermodynamics of fragment binding. J. Chem. Inf. Model. 52, 1039–1045 (2012).
Ladbury, J. E., Klebe, G. & Freire, E. Adding calorimetric data to decision making in lead discovery: a hot tip. Nature Rev. Drug Discov. 9, 23–27 (2010).
Freire, E. A thermodynamic approach to the affinity optimization of drug candidates. Chem. Biol. Drug Des. 74, 488–472 (2009).
Martin, S. F. & Clements, J. H. Correlating structure and energetics in protein-ligand interactions: paradigms and paradoxes. Annu. Rev. Biochem. 82, 267–293 (2013).
Glas, A. et al. Constrained peptides with target-adapted cross-inks as inhibitors of a pathogenic protein-protein interaction. Angew. Chem. Int. Ed. 53, 2489–2493 (2014).
Biela, A. et al. Ligand binding stepwise disrupts water network in thrombin: enthalpic and entropic changes reveal classical hydrophobic effect. J. Med. Chem. 55, 6094–6110 (2012).
Kyte, J. The basis of the hydrophobic effect. Biophys. Chem. 100, 193–203 (2003).
Chandler, D. Interfaces and the driving force of hydrophobic assembly. Nature 437, 640–647 (2005).
Dill, K. A., Truskett, T. M., Vlachy, V. & Hribar-Lee, B. Modeling water, the hydrophobic effect, and ion solvation. Annu. Rev. Biophys. Biomol. Struct. 34, 173–199 (2005).
Steuber, H., Heine, A. & Klebe, G. Structural and thermodynamic study on aldose reductase: nitro-substituted inhibitors with strong enthalpic binding contribution. J. Mol. Biol. 368, 618–638 (2007).
Rees, D. C., Congreve, M., Murray, C. W. & Carr, R. Fragment-based lead discovery. Nature Rev. Drug Discov. 3, 660–672 (2004).
Erlanson, D. A., McDowell, R. S. & O'Brien, T. Fragment-based drug discovery. J. Med. Chem. 47, 3463–3482 (2004).
Hopkins, A. L., Groom, C. R. & Alex, A. Ligand efficiency: a useful metric for lead selection. Drug Discov. Today 9, 430–431 (2004).
Ferenczy, G. G. & Keserü, G. M. How are fragments optimized? A retrospective analysis of 145 fragment optimizations. J. Med. Chem. 56, 2478–2486 (2013).
Mondal, M. et al. Structure-based design exploiting dynamic combinatorial chemistry to identify novel inhibitors for the aspartic protease endothiapepsin. Angew. Chem. Int. Ed. 53, 3259–3263 (2014).
Zhang, Y.-L. & Zhang, Z.-Y. Low-affinity binding determined by titration calorimetry using a high-affinity coupling ligand: a thermodynamic study of ligand binding to protein tyrosine phosphatase 1B. Analy. Biochem. 261, 139–148 (1998).
Valezques-Campoy, A. & Freire, E. Isothermal titration calorimetry to determine association constants for high-affinity ligands. Nature Protoc. 1, 186–191 (2006).
Steuber, H., Czodrowski, P., Sotriffer, C. A. & Klebe, G. Tracing changes in protonation: a prerequisite to factorize thermodynamic data of inhibitor binding to aldose reductase. J. Mol. Biol. 373, 1305–1320 (2007).
Baum, B. et al. Non-additivity of functional group contributions in protein-ligand binding: a comprehensive study by crystallography and isothermal titration calorimetry. J. Mol. Biol. 397, 1042–1057 (2010).
Baum, B. et al. More than a simple lipophilic contact: a detailed thermodynamic analysis of non-basic residues in the S1 pocket of thrombin. J. Mol. Biol. 390, 56–69 (2009).
Blum, A. Structure-Based Design and Synthesis of Pyrrolidines as Inhibitors of HIV-1 Protease. Thesis, Univ. Marburg (2007).
Homans, S. W. Water, water everywhere — except where it matters. Drug Discov. Today 12, 534–539 (2007).
Englert, L. et al. Displacement of disordered water molecules from the hydrophobic pocket creates enthalpic signature: binding of phosphonamidate to the S1′-pocket of thermolysin. Biochim. Biophys. Acta 1800, 1192–1202 (2010).
Snyder, P. W. et al. Mechanism of the hydrophobic effect in the biomolecular recognition of arylsulfonamides by carbonic anhydrase. Proc. Natl Acad. Sci. USA 108, 17889–17894 (2011).
Wang, J., Berne, B. J. & Friesner, R. A. Ligand binding to protein-binding pockets with wet and dry regions. Proc. Natl Acad. Sci. USA 108, 1326–1330 (2011).
Setny, P., Baron, R. & McCammon, J. A. How can hydrophobic association be enthalpy driven? J. Chem. Theory Comput. 6, 2866–2871 (2010).
Muley, L. et al. Enhancement of hydrophobic interactions and hydrogen bond strength by cooperativity: synthesis, modeling, and molecular dynamics simulations of a series of thrombin inhibitors. J. Med. Chem. 53, 2126–2135 (2010).
Sleigh, S. H., Seavers, P. R., Wilkingson, A. J., Ladbury, J. E. & Tame, J. R. H. Crystallographic and calorimetric analysis of peptide binding to OppA protein. J. Mol. Biol. 291, 393–415 (1999).
Davies, T. G., Hubbard, R. E. & Tame, J. R. H. Relating structures to thermodynamics: the crystal structures and binding affinity of eight OppA-peptide complexes. Protein Sci. 8, 1432–1444 (1999).
Brandt, T. et al. Congeneric but still distinct: how closely related trypsin ligands exhibit different thermodynamic and structural properties. J. Mol. Biol. 405, 1170–1187 (2011).
Petrova, T. et al. Factorizing selectivity determinants of inhibitor binding toward aldose and aldehyde reductases: structural and thermodynamic properties of the aldose reductase mutant Leu300Pro-fidarestat complex. J. Med. Chem. 48, 5659–5665 (2005).
Biela, A. et al. Impact of ligand and protein desolvation on ligand binding to the S1 pocket of thrombin. J. Mol. Biol. 418, 350–366 (2012).
Young, T., Abel, R., Kim, B., Berne, B. J. & Friesner, R. A. Motifs for molecular recognition exploiting hydrophobic enclosure in protein-ligand binding. Proc. Natl Acad. Sci. USA 104, 808–813 (2007).
Abel, R., Young, T., Farid, R., Berne, B. J. & Friesner, R. A. Role of the acive-site solvent in the thermodynamics of factor Xa ligand binding. J. Am. Chem. Soc. 130, 2817–2831 (2008).
Abel, R. et al. Contribution of explicit solvent effects to the binding affinity of small-molecule inhibitors in blood coagulation factor serine proteases. ChemMedChem. 6, 1049–1066 (2011).
Biela, A., Betz, M., Heine, A. & Klebe, G. Water makes the difference: rearrangement of water solvation layer triggers non-additivity of functional group contributions in protein-ligand binding. ChemMedChem. 7, 1423–1434 (2012).
Biela, A. et al. Dissecting the hydrophobic effect on the molecular level: the role of water, enthalpy, and entropy in ligand binding to thermolysin. Angew. Chem. Int. Ed. 52, 1822–1828 (2013).
Krimmer, S., Betz, M., Heine, A. & Klebe, G. Methyl, ethyl, propyl, butyl: futile but not for water, as the correlation of structure and thermodynamic signature shows in a congeneric series of thermolysin inhibitors. ChemMedChem. 9, 833–846 (2014).
Leung, C. S., Leung, S. S. F., Tirado-Rives, J., Jorgensen, W. L. Methyl effects on protein-ligand binding. J. Med. Chem. 55, 4489–4500 (2012).
MacRaild, C. A., Daranas, A. H., Bronowska, A. & Homans, S. W. Global changes in local protein dynamics reduce the entropic cost of carbohydrate binding in the arabinose binding protein. J. Mol. Biol. 368, 822–832 (2007).
Diehl, C., Genheden, S., Modig, K., Ryde, U. &, Akke, M. Conformational entropy changes upon lactose binding to the carbohydrate recognition domain of galectin-3. J. Biomol. NMR 45, 157–169 (2009).
Popovych, N., Sun, S., Ebright, R. H. & Kalodimos, C. G. Dynamically driven protein allostery. Nature Struct. Biol. 13, 831–838 (2006).
Zidek, L., Novotny, M. V. & Stone, M. J. Increased protein backbone conformational entropy upon hydrophobic ligand binding. Nature Struct. Biol. 6, 1118–1121 (1999).
Diehl, C. et al. Protein flexibility and conformational entropy in ligand design targeting the carbohydrate recognition domain of galectin-3. J. Am. Chem. Soc. 132, 14577–14589 (2010).
Stöckmann, H. et al. Residual ligand entropy in the binding of p-substituted benzenesulfonamide ligands to bovine carbonic anhydrase II. J. Am. Chem. Soc. 130, 12420–12426 (2008).
Syme, N. R., Dennis, C., Bronowska, A., Paesen, G. C. & Homans, S. W. Comparison of entropic contributions to binding in a “hydrophilic” versus “hydrophobic” ligand-protein interaction. J. Am. Chem. Soc. 132, 8682–8689 (2010).
Neeb, M. et al. Chasing protons: how ITC, mutagenesis and pKa calculations trace the locus of charge in ligand binding to a tRNA-binding enzyme. J. Med. Chem. 57, 5554–5565 (2014).
Neeb, M. et al. Beyond affinity: enthalpy-entropy factorization unravels complexity of a flat structure-activity relationship for inhibiton of tRNA-modifying enzyme. J. Med. Chem. 57, 5566–5578 (2014).
Rühmann, E. et al. Thermodynamic signatures of fragment binding: validation of direct versus displacement ITC titrations. Biochim. Biophys. Acta http://dx.doi.org/10.1016/j.bbagen.2014.12.007 (2015)
Acknowledgements
The author is grateful to the European Commission for a generous European Research Council (ERC) Advanced Grant (DrugProfilBind No. 268145), which made these systematic studies possible. Many members of the Marburg research team have contributed to the reported work. Their effort and engagement — in particular that of B. Baum, A. Biela, A. Heine and S. Krimmer — are highly appreciated.
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Supplementary information
Supplementary information S1 (box)
ITC versus van't Hoff data and determination of heat capacity changes ΔCp (PDF 1222 kb)
Supplementary information S2 (figure)
Congeneric series of thermolysin inhibitors with a terminal carboxylate group and with P2′ substituents of growing hydrophobicity (R= H to benzyl). (PDF 382 kb)
Supplementary information S3 (figure)
Congeneric series of thermolysin inhibitors without a terminal carboxylate group and with P2′ substituents of growing hydrophobicity. (PDF 1327 kb)
Glossary
- Bioisosteric groups
-
Chemical groups that have a different chemical composition but elicit the same biological response once they are attached to a basic scaffold and bound to the target protein.
- Classical hydrophobic effect
-
The tendency of non-polar molecules to aggregate and repel water. The effect is measured by the surface area of hydrophobic patches that is removed from solvent access, and it is usually explained as an entropy-driven process, as when the hydrophobic surface becomes buried it displaces water molecules.
- Congeneric
-
In a congeneric series of ligands, one property, such as the size of a hydrophobic substituent, is gradually increased from ligand to ligand.
- Difference electron density
-
X-rays are diffracted at the electrons of the molecules forming a crystal. Therefore, crystallography determines an electron density in the crystal to which a molecular model is fitted. Residual density — so-called 'difference electron density' — not explained by the atoms of the protein, indicates, for example, bound ligands or water molecules.
- Enthalpic efficiency
-
A ligand parameter that, similarly to ligand efficiency, relates the enthalpic binding component of a ligand to its molecular size. Accordingly, the enthalpic binding signal is divided by the number of non-hydrogen atoms that compose the ligand.
- Ligand efficiency
-
A parameter that relates the affinity of a ligand to its molecular size. Various equations to calculate ligand efficiency have been proposed: for example, dividing the Gibbs free energy of binding by the number of non-hydrogen atoms that compose the ligand.
- Scaffold
-
The central molecular core of a lead compound, onto which different substituents and functional groups are attached.
- Surface patch increment
-
The area of the hydrophobic surface of a ligand that becomes buried in the hydrophobic pocket of a protein upon the formation of a protein–ligand complex, contributing to affinity. This description follows the ideas of the classical hydrophobic effect.
- Thrombin's 60-loop
-
Unlike other members of the trypsin-like serine proteases, thrombin has an additional loop that covers the S2 pocket of the protease and has an important influence on the selective recognition of the substrate.
- Trajectory
-
A record along a molecular dynamics simulation of all the geometrical and conformational changes of the simulated system with time.
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Klebe, G. Applying thermodynamic profiling in lead finding and optimization. Nat Rev Drug Discov 14, 95–110 (2015). https://doi.org/10.1038/nrd4486
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DOI: https://doi.org/10.1038/nrd4486
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