87
General discussion
In this study, I showed that the use of a thermostable GPCR mutant acquisition platform dramatically improves the possibility of obtaining structural information on membrane proteins that are difficult to analyze in early stages of drug discovery research. I, along with my colleagues, show for the first time the successful analysis of crystal structure of the GPR40-fasiglifam complex using a four-point thermostable mutant obtained in this study (Figure 23) [58]. Other groups have reported GPR40 crystal structure analysis using this thermostable mutant with other ligands [113] [114].
Thermostable mutants generated by this platform can be exploited to obtain protein structure information with various ligands. Furthermore, the amino acid locus of mutant GPR40 in this study was thought to contribute to thermostability and was in close homology to the amino acid loci of other GPRs. Thus, the analysis of the acquired thermostable mutant information may allow for the efficient acquisition of other
mutants of various GPCRs. Furthermore, since the phylogenetic tree analysis of GPCRs confirmed the tendency for similar thermo-stabilizing mutation loci to be conserved within a single phylogenetic group, it is expected that the combination of molecular dynamics calculations with molecular evolutionary analyses will make it possible to efficiently discover novel thermo-stabilized GPCR variants.
Regarding the structural information of protein kinase, I showed that the information complementary to X-ray crystal structure analysis can be obtained from a FRET-based binding assay using fluorescent probes. The preparation of a platform for TR-FRET binding assay system specific for protein kinases would provide high-throughput structural information. These types of fluorescent probes can also be used to develop competitive binding assay systems and to evaluate the kinase selectivity of various
88
compounds in a kinase panel assay [101]. Thus, the TR-FRET binding assay platform for comprehensive kinases may be essential for generation of selective kinase inhibitors.
Drug discovery research is generally conducted as per the flow shown in Figure 24.
Once a compound that binds to a therapeutic target has been discovered, it is extremely important to perform lead generation and lead optimization to select a candidate
compound for evaluation in clinical trials. Protein structure analysis provides crucial information for compound optimization, as it reveals the structure of the interaction mode between ligands and proteins. It is often difficult to obtain protein structural information at the early stage of drug discovery and there are no reports of crystal structure analysis for membrane proteins.
In summary, it is important to obtain the necessary protein structure information at the right time for the right purpose. Through this research, I showed that complementary biochemical platforms, in addition to existing methods of protein structure analysis, can efficiently provide a new insight. The platforms constructed in this research are capable of revealing detailed structural information on GPCRs and protein kinases, and may be potentially applicable for drug discovery research.
89
Figure 23 Crystal structure of GPR40 with fasiglifam.
90
Figure 24 The process of drug discovery and development flow and the timing of requirements of the efforts of medicinal chemistry.
91
Acknowledgements
92
I am deeply grateful to Professor Tetsuo Hashimoto for supervising my work and valuable discussions through my doctoral program. I am also grateful to Professors Ryuhei Harada, Kaori Ishikawa, and Kisaburo Nagamune of University of Tsukuba for guiding my work and valuable discussions through my doctoral program.
I also thank Dr. Hidenori Kamiguchi and Mr. Hiroaki Atsuji and Mr. Hiroyuki Hirai, Takeda Pharmaceutical Company Limited, for their understanding and support on my doctoral program.
Finally, I would like to appreciate my wife and sons for supporting my life and this work in University of Tsukuba.
93
References
94
1. Sanger, F., The arrangement of amino acids in proteins. Adv Protein Chem, 1952.
7: p. 1-67.
2. Pauling, L., R.B. Corey, and H.R. Branson, The structure of proteins; two hydrogen-bonded helical configurations of the polypeptide chain. Proc Natl Acad Sci U S A, 1951. 37(4): p. 205-11.
3. Maveyraud, L. and L. Mourey, Protein X-ray Crystallography and Drug Discovery. Molecules, 2020. 25(5).
4. Lengyel, J., et al., Towards an integrative structural biology approach: combining Cryo-TEM, X-ray crystallography, and NMR. J Struct Funct Genomics, 2014.
15(3): p. 117-24.
5. Fernandez-Leiro, R. and S.H. Scheres, Unravelling biological macromolecules with cryo-electron microscopy. Nature, 2016. 537(7620): p. 339-46.
6. Kendrew, J.C., et al., A three-dimensional model of the myoglobin molecule obtained by x-ray analysis. Nature, 1958. 181(4610): p. 662-6.
7. Scapin, G., Structural biology and drug discovery. Curr Pharm Des, 2006. 12(17):
p. 2087-97.
8. Pandey, A., et al., Current strategies for protein production and purification enabling membrane protein structural biology. Biochem Cell Biol, 2016. 94(6):
p. 507-527.
9. Sachs, J.N. and D.M. Engelman, Introduction to the membrane protein reviews:
the interplay of structure, dynamics, and environment in membrane protein function. Annu Rev Biochem, 2006. 75: p. 707-12.
10. von Heijne, G., The membrane protein universe: what's out there and why bother?
J Intern Med, 2007. 261(6): p. 543-57.
11. Smith, S.M., Strategies for the Purification of Membrane Proteins. Methods Mol Biol, 2017. 1485: p. 389-400.
12. Lagerstrom, M.C. and H.B. Schioth, Structural diversity of G protein-coupled receptors and significance for drug discovery. Nat Rev Drug Discov, 2008. 7(4):
p. 339-57.
13. Lefkowitz, R.J., A brief history of G-protein coupled receptors (Nobel Lecture).
Angew Chem Int Ed Engl, 2013. 52(25): p. 6366-78.
14. Schioth, H.B. and R. Fredriksson, The GRAFS classification system of G-protein coupled receptors in comparative perspective. Gen Comp Endocrinol, 2005.
142(1-2): p. 94-101.
15. Latorraca, N.R., A.J. Venkatakrishnan, and R.O. Dror, GPCR Dynamics:
Structures in Motion. Chem Rev, 2017. 117(1): p. 139-155.
95
16. Chattopadhyay, A., B.D. Rao, and M. Jafurulla, Solubilization of G protein-coupled receptors: a convenient strategy to explore lipid-receptor interaction.
Methods Enzymol, 2015. 557: p. 117-34.
17. Palczewski, K., et al., Crystal structure of rhodopsin: A G protein-coupled receptor. Science, 2000. 289(5480): p. 739-45.
18. Cherezov, V., et al., High-resolution crystal structure of an engineered human beta2-adrenergic G protein-coupled receptor. Science, 2007. 318(5854): p. 1258-65.
19. Serrano-Vega, M.J., et al., Conformational thermostabilization of the beta1-adrenergic receptor in a detergent-resistant form. Proc Natl Acad Sci U S A, 2008.
105(3): p. 877-82.
20. Shibata, Y., et al., Thermostabilization of the neurotensin receptor NTS1. J Mol Biol, 2009. 390(2): p. 262-77.
21. Manning, G., et al., The protein kinase complement of the human genome. Science, 2002. 298(5600): p. 1912-34.
22. Wu, P., T.E. Nielsen, and M.H. Clausen, FDA-approved small-molecule kinase inhibitors. Trends Pharmacol Sci, 2015. 36(7): p. 422-39.
23. Zhang, J., et al., Cytotoxicity of 34 FDA approved small-molecule kinase inhibitors in primary rat and human hepatocytes. Toxicol Lett, 2018. 291: p. 138-148.
24. Kannaiyan, R. and D. Mahadevan, A comprehensive review of protein kinase inhibitors for cancer therapy. Expert Rev Anticancer Ther, 2018. 18(12): p. 1249-1270.
25. Congreve, M., et al., Discovery of 1,2,4-triazine derivatives as adenosine A(2A) antagonists using structure based drug design. J Med Chem, 2012. 55(5): p. 1898-903.
26. Kolb, P., et al., Structure-based discovery of beta2-adrenergic receptor ligands.
Proc Natl Acad Sci U S A, 2009. 106(16): p. 6843-8.
27. Venkatakrishnan, A.J., et al., Molecular signatures of G-protein-coupled receptors.
Nature, 2013. 494(7436): p. 185-94.
28. Ulrik Gether,. et al., Structural Instability of a Constitutively Active G Protein-coupled Receptor. AGONIST-INDEPENDENT ACTIVATION DUE TO CONFORMATIONAL FLEXIBILITY. Journal of Biological Chemistry, 1997.
272(5): p. 2587-2590.
29. Lebon, G., et al., Thermostabilisation of an agonist-bound conformation of the human adenosine A(2A) receptor. J Mol Biol, 2011. 409(3): p. 298-310.
96
30. Magnani, F., et al., Co-evolving stability and conformational homogeneity of the human adenosine A2a receptor. Proc Natl Acad Sci U S A, 2008. 105(31): p.
10744-9.
31. Miller, J.L. and C.G. Tate, Engineering an ultra-thermostable beta(1)-adrenoceptor. J Mol Biol, 2011. 413(3): p. 628-38.
32. Bertheleme, N., et al., Loss of constitutive activity is correlated with increased thermostability of the human adenosine A2A receptor. Br J Pharmacol, 2013.
169(5): p. 988-98.
33. Congreve, M., et al., Fragment screening of stabilized G-protein-coupled receptors using biophysical methods. Methods Enzymol, 2011. 493: p. 115-36.
34. Congreve, M., C. Langmead, and F.H. Marshall, The use of GPCR structures in drug design. Adv Pharmacol, 2011. 62: p. 1-36.
35. Dore, A.S., et al., Structure of the adenosine A(2A) receptor in complex with ZM241385 and the xanthines XAC and caffeine. Structure, 2011. 19(9): p. 1283-93.
36. Robertson, N., et al., The properties of thermostabilised G protein-coupled receptors (StaRs) and their use in drug discovery. Neuropharmacology, 2011.
60(1): p. 36-44.
37. zur Megede, J., et al., Increased expression and immunogenicity of sequence-modified human immunodeficiency virus type 1 gag gene. J Virol, 2000. 74(6): p.
2628-35.
38. Zemanova, L., et al., Endothelin receptor in virus-like particles: ligand binding observed by fluorescence fluctuation spectroscopy. Biochemistry, 2004. 43(28): p.
9021-8.
39. Ho, T.T., et al., Method for rapid optimization of recombinant GPCR protein expression and stability using virus-like particles. Protein Expr Purif, 2017. 133:
p. 41-49.
40. Muckenschnabel, I., et al., SpeedScreen: label-free liquid chromatography-mass spectrometry-based high-throughput screening for the discovery of orphan protein ligands. Anal Biochem, 2004. 324(2): p. 241-9.
41. Zehender, H., et al., SpeedScreen: The "missing link" between genomics and lead discovery. J Biomol Screen, 2004. 9(6): p. 498-505.
42. Annis, D.A., et al., Affinity selection-mass spectrometry screening techniques for small molecule drug discovery. Curr Opin Chem Biol, 2007. 11(5): p. 518-26.
43. Itoh, Y., et al., Free fatty acids regulate insulin secretion from pancreatic beta cells through GPR40. Nature, 2003. 422(6928): p. 173-6.
97
44. Araki, T., et al., GPR40-induced insulin secretion by the novel agonist TAK-875:
first clinical findings in patients with type 2 diabetes. Diabetes Obes Metab, 2012.
14(3): p. 271-8.
45. Burant, C.F., et al., TAK-875 versus placebo or glimepiride in type 2 diabetes mellitus: a phase 2, randomised, double-blind, placebo-controlled trial. Lancet, 2012. 379(9824): p. 1403-11.
46. Leifke, E., et al., A multiple-ascending-dose study to evaluate safety, pharmacokinetics, and pharmacodynamics of a novel GPR40 agonist, TAK-875, in subjects with type 2 diabetes. Clin Pharmacol Ther, 2012. 92(1): p. 29-39.
47. Naik, H., et al., Safety, tolerability, pharmacokinetics, and pharmacodynamic properties of the GPR40 agonist TAK-875: results from a double-blind, placebo-controlled single oral dose rising study in healthy volunteers. J Clin Pharmacol, 2012. 52(7): p. 1007-16.
48. Tsujihata, Y., et al., TAK-875, an orally available G protein-coupled receptor 40/free fatty acid receptor 1 agonist, enhances glucose-dependent insulin secretion and improves both postprandial and fasting hyperglycemia in type 2 diabetic rats. J Pharmacol Exp Ther, 2011. 339(1): p. 228-37.
49. Yashiro, H., et al., The effects of TAK-875, a selective G protein-coupled receptor 40/free fatty acid 1 agonist, on insulin and glucagon secretion in isolated rat and human islets. J Pharmacol Exp Ther, 2012. 340(2): p. 483-9.
50. Yabuki, C., et al., A novel antidiabetic drug, fasiglifam/TAK-875, acts as an ago-allosteric modulator of FFAR1. PLoS One, 2013. 8(10): p. e76280.
51. Muk, S., et al., Machine Learning for Prioritization of Thermostabilizing Mutations for G-Protein Coupled Receptors. Biophys J, 2019. 117(11): p. 2228-2239.
52. Zhu, B., et al., In-Fusion™ assembly: seamless engineering of multidomain fusion proteins, modular vectors, and mutations. BioTechniques, 2007. 43(3): p. 354-359.
53. Chun, E., et al., Fusion partner toolchest for the stabilization and crystallization of G protein-coupled receptors. Structure, 2012. 20(6): p. 967-76.
54. Tate, C.G., A crystal clear solution for determining G-protein-coupled receptor structures. Trends Biochem Sci, 2012. 37(9): p. 343-52.
55. Edgar, R.C., MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res, 2004. 32(5): p. 1792-7.
56. Gouy, M., S. Guindon, and O. Gascuel, SeaView version 4: A multiplatform graphical user interface for sequence alignment and phylogenetic tree building.
98
Mol Biol Evol, 2010. 27(2): p. 221-4.
57. Kobilka, B.K. and X. Deupi, Conformational complexity of G-protein-coupled receptors. Trends Pharmacol Sci, 2007. 28(8): p. 397-406.
58. Srivastava, A., et al., High-resolution structure of the human GPR40 receptor bound to allosteric agonist TAK-875. Nature, 2014. 513(7516): p. 124-7.
59. Garnier, L., et al., Incorporation of pseudorabies virus gD into human immunodeficiency virus type 1 Gag particles produced in baculovirus-infected cells. J Virol, 1995. 69(7): p. 4060-8.
60. Cheng, R.K.Y., et al., Structural insight into allosteric modulation of protease-activated receptor 2. Nature, 2017. 545(7652): p. 112-115.
61. Zhang, D., et al., Two disparate ligand-binding sites in the human P2Y1 receptor.
Nature, 2015. 520(7547): p. 317-21.
62. Zhang, K., et al., Structure of the human P2Y12 receptor in complex with an antithrombotic drug. Nature, 2014. 509(7498): p. 115-8.
63. Oswald, C., et al., Intracellular allosteric antagonism of the CCR9 receptor.
Nature, 2016. 540(7633): p. 462-465.
64. Wu, B., et al., Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science, 2010. 330(6007): p. 1066-71.
65. Qin, L., et al., Structural biology. Crystal structure of the chemokine receptor CXCR4 in complex with a viral chemokine. Science, 2015. 347(6226): p. 1117-22.
66. Tan, Q., et al., Structure of the CCR5 chemokine receptor-HIV entry inhibitor maraviroc complex. Science, 2013. 341(6152): p. 1387-90.
67. Ma, Y., et al., Structural Basis for Apelin Control of the Human Apelin Receptor.
Structure, 2017. 25(6): p. 858-866 e4.
68. Shihoya, W., et al., X-ray structures of endothelin ETB receptor bound to clinical antagonist bosentan and its analog. Nat Struct Mol Biol, 2017. 24(9): p. 758-764.
69. Shihoya, W., et al., Crystal structures of human ETB receptor provide mechanistic insight into receptor activation and partial activation. Nat Commun, 2018. 9(1):
p. 4711.
70. White, J.F., et al., Structure of the agonist-bound neurotensin receptor. Nature, 2012. 490(7421): p. 508-13.
71. Standfuss, J., et al., The structural basis of agonist-induced activation in constitutively active rhodopsin. Nature, 2011. 471(7340): p. 656-60.
72. Deupi, X., et al., Stabilized G protein binding site in the structure of constitutively active metarhodopsin-II. Proc Natl Acad Sci U S A, 2012. 109(1): p. 119-24.
73. Kang, Y., et al., Crystal structure of rhodopsin bound to arrestin by femtosecond
99
X-ray laser. Nature, 2015. 523(7562): p. 561-7.
74. Wang, S., et al., Structure of the D2 dopamine receptor bound to the atypical antipsychotic drug risperidone. Nature, 2018. 555(7695): p. 269-273.
75. Chien, E.Y., et al., Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist. Science, 2010. 330(6007): p. 1091-5.
76. Yin, W., et al., Crystal structure of the human 5-HT1B serotonin receptor bound to an inverse agonist. Cell Discov, 2018. 4: p. 12.
77. Rosenbaum, D.M., et al., Structure and function of an irreversible agonist-beta(2) adrenoceptor complex. Nature, 2011. 469(7329): p. 236-40.
78. Cheng, R.K.Y., et al., Structures of Human A1 and A2A Adenosine Receptors with Xanthines Reveal Determinants of Selectivity. Structure, 2017. 25(8): p. 1275-1285 e4.
79. Lebon, G., et al., Molecular Determinants of CGS21680 Binding to the Human Adenosine A2A Receptor. Mol Pharmacol, 2015. 87(6): p. 907-15.
80. Hua, T., et al., Crystal structures of agonist-bound human cannabinoid receptor CB1. Nature, 2017. 547(7664): p. 468-471.
81. Chrencik, J.E., et al., Crystal Structure of Antagonist Bound Human Lysophosphatidic Acid Receptor 1. Cell, 2015. 161(7): p. 1633-43.
82. Liu, W., et al., Serial femtosecond crystallography of G protein-coupled receptors.
Science, 2013. 342(6165): p. 1521-4.
83. Peng, Y., et al., 5-HT2C Receptor Structures Reveal the Structural Basis of GPCR Polypharmacology. Cell, 2018. 172(4): p. 719-730 e14.
84. Hollenstein, K., et al., Structure of class B GPCR corticotropin-releasing factor receptor 1. Nature, 2013. 499(7459): p. 438-43.
85. Song, G., et al., Human GLP-1 receptor transmembrane domain structure in complex with allosteric modulators. Nature, 2017. 546(7657): p. 312-315.
86. Jazayeri, A., et al., Crystal structure of the GLP-1 receptor bound to a peptide agonist. Nature, 2017. 546(7657): p. 254-258.
87. Jazayeri, A., et al., Extra-helical binding site of a glucagon receptor antagonist.
Nature, 2016. 533(7602): p. 274-7.
88. Dore, A.S., et al., Structure of class C GPCR metabotropic glutamate receptor 5 transmembrane domain. Nature, 2014. 511(7511): p. 557-62.
89. Byrne, E.F.X., et al., Structural basis of Smoothened regulation by its extracellular domains. Nature, 2016. 535(7613): p. 517-522.
90. Ferguson, F.M. and N.S. Gray, Kinase inhibitors: the road ahead. Nat Rev Drug Discov, 2018. 17(5): p. 353-377.
100
91. Omura, S., et al., A new alkaloid AM-2282 OF Streptomyces origin. Taxonomy, fermentation, isolation and preliminary characterization. J Antibiot (Tokyo), 1977. 30(4): p. 275-82.
92. Takahashi, I., et al., UCN-01 and UCN-02, new selective inhibitors of protein kinase C. I. Screening, producing organism and fermentation. J Antibiot (Tokyo), 1989. 42(4): p. 564-70.
93. Gani, O.A. and R.A. Engh, Protein kinase inhibition of clinically important staurosporine analogues. Nat Prod Rep, 2010. 27(4): p. 489-98.
94. Tanramluk, D., et al., On the origins of enzyme inhibitor selectivity and promiscuity: a case study of protein kinase binding to staurosporine. Chem Biol Drug Des, 2009. 74(1): p. 16-24.
95. Disney, A.J., B. Kellam, and L.V. Dekker, Alkylation of Staurosporine to Derive a Kinase Probe for Fluorescence Applications. ChemMedChem, 2016. 11(9): p.
972-9.
96. Shi, H., et al., Proteome profiling reveals potential cellular targets of staurosporine using a clickable cell-permeable probe. Chem Commun (Camb), 2011. 47(40): p. 11306-8.
97. Shomin, C.D., S.C. Meyer, and I. Ghosh, Staurosporine tethered peptide ligands that target cAMP-dependent protein kinase (PKA): optimization and selectivity profiling. Bioorg Med Chem, 2009. 17(17): p. 6196-202.
98. Cheng, X., et al., A tuned affinity-based staurosporine probe for in situ profiling of protein kinases. Chem Commun (Camb), 2014. 50(22): p. 2851-3.
99. Kawaguchi, M., et al., Development of a novel fluorescent probe for fluorescence correlation spectroscopic detection of kinase inhibitors. Bioorg Med Chem Lett, 2008. 18(13): p. 3752-5.
100. Lebakken, C.S., K. Hee Chol, and K.W. Vogel, A fluorescence lifetime based binding assay to characterize kinase inhibitors. J Biomol Screen, 2007. 12(6): p.
828-41.
101. Lebakken, C.S., et al., Development and applications of a broad-coverage, TR-FRET-based kinase binding assay platform. J Biomol Screen, 2009. 14(8): p. 924-35.
102. Wallace, A.C., R.A. Laskowski, and J.M. Thornton, LIGPLOT: a program to generate schematic diagrams of protein-ligand interactions. Protein Eng, 1995.
8(2): p. 127-34.
103. Laskowski, R.A. and M.B. Swindells, LigPlot+: multiple ligand-protein interaction diagrams for drug discovery. J Chem Inf Model, 2011. 51(10): p.
101
2778-86.
104. Karaman, M.W., et al., A quantitative analysis of kinase inhibitor selectivity. Nat Biotechnol, 2008. 26(1): p. 127-32.
105. Zhao, B., et al., Structural basis for Chk1 inhibition by UCN-01. J Biol Chem, 2002. 277(48): p. 46609-15.
106. Chu, X.J., et al., Discovery of [4-Amino-2-(1-methanesulfonylpiperidin-4-ylamino)pyrimidin-5-yl](2,3-difluoro-6- methoxyphenyl)methanone (R547), a potent and selective cyclin-dependent kinase inhibitor with significant in vivo antitumor activity. J Med Chem, 2006. 49(22): p. 6549-60.
107. Liu, F., et al., Discovery of a Highly Selective STK16 Kinase Inhibitor. ACS Chem Biol, 2016. 11(6): p. 1537-43.
108. Sakamoto, H., et al., CH5424802, a selective ALK inhibitor capable of blocking the resistant gatekeeper mutant. Cancer Cell, 2011. 19(5): p. 679-90.
109. Das, J., et al., 2-aminothiazole as a novel kinase inhibitor template. Structure-activity relationship studies toward the discovery of N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1- piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, BMS-354825) as a potent pan-Src kinase inhibitor. J Med Chem, 2006. 49(23): p. 6819-32.
110. Boschelli, D.H., et al., Optimization of 4-phenylamino-3-quinolinecarbonitriles as potent inhibitors of Src kinase activity. J Med Chem, 2001. 44(23): p. 3965-77.
111. Davis, M.I., et al., Comprehensive analysis of kinase inhibitor selectivity. Nat Biotechnol, 2011. 29(11): p. 1046-51.
112. Heerding, D.A., et al., Identification of 4-(2-(4-amino-1,2,5-oxadiazol-3-yl)-1-ethyl-7-{[(3S)-3-piperidinylmethyl]oxy}-1H- imidazo[4,5-c]pyridin-4-yl)-2-methyl-3-butyn-2-ol (GSK690693), a novel inhibitor of AKT kinase. J Med Chem, 2008. 51(18): p. 5663-79.
113. Lu, J., et al., Structural basis for the cooperative allosteric activation of the free fatty acid receptor GPR40. Nat Struct Mol Biol, 2017. 24(7): p. 570-577.
114. Ho, J.D., et al., Structural basis for GPR40 allosteric agonism and incretin stimulation. Nat Commun, 2018. 9(1): p. 1645.