Screening through Lead Optimization of High Affinity, Allosteric Cyclin-Dependent Kinase 2 (CDK2) Inhibitors as Male Contraceptives That Reduce Sperm Counts in Mice

Erik B. Faber; Nan Wang; Kristen John; Luxin Sun; Henry L. Wong; David Burban; Rawle Francis; Defeng Tian; Kwon H. Hong; An Yang; Liming Wang; Mazen Elsaid; Hira Khalid; Nicholas M. Levinson; Ernst Schönbrunn; Jon E. Hawkinson; Gunda I. Georg

doi:10.1021/acs.jmedchem.2c01731

Screening through Lead Optimization of High Affinity, Allosteric Cyclin-Dependent Kinase 2 (CDK2) Inhibitors as Male Contraceptives That Reduce Sperm Counts in Mice

Erik B. Faber, Nan Wang, Kristen John, Luxin Sun, Henry L. Wong, David Burban, Rawle Francis, Defeng Tian, Kwon H. Hong, An Yang, Liming Wang, Mazen Elsaid, Hira Khalid, Nicholas M. Levinson, Ernst Schönbrunn, Jon E. Hawkinson, Gunda I. Georg

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Abstract

Although cyclin-dependent kinase 2 (CDK2) is a validated target for both cancer and contraception, developing a CDK2 inhibitor with exquisite selectivity has been challenging due to the structural similarity of the ATP-binding site, where most kinase inhibitors bind. We previously discovered an allosteric pocket in CDK2 with the potential to bind a selective compound and then discovered and structurally confirmed an anthranilic acid scaffold that binds this pocket with high affinity. These allosteric inhibitors are selective for CDK2 over structurally similar CDK1 and show contraceptive potential. Herein, we describe the screening and optimization that led to compounds like EF-4-177 with nanomolar affinity for CDK2. EF-4-177 is metabolically stable, orally bioavailable, and significantly disrupts spermatogenesis, demonstrating this series’ therapeutic potential. This work details the discovery of the highest affinity allosteric CDK inhibitors reported and shows promise for this series to yield an efficacious and selective allosteric CDK2 inhibitor.

Original language	English (US)
Pages (from-to)	1928-1940
Number of pages	13
Journal	Journal of medicinal chemistry
Volume	66
Issue number	3
DOIs	https://doi.org/10.1021/acs.jmedchem.2c01731
State	Published - Feb 9 2023

Bibliographical note

Funding Information:
Funding for this project was provided by NICHD: 5 R01 HD080431, 1 R61 HD099743, and R01 GM121515. E.B.F. was supported by NIH/NIGMS (through training grants T32 GM008244 and T32 GM132029), as well as by an NIH/NCI fellowship (F30 CA232303). We acknowledge the support provided by the GM/CA beamlines at the Advanced Photon Source (APS), funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396, and S10OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract no. DE-AC02-06CH11357. We thank the Moffitt Chemical Biology Core for use of the crystallization and X-ray facilities (National Cancer Institute grant P30-CA076292). We thank Pharmaron for conducting liver microsome metabolic stability assays and thank Reaction Biology for conducting kinase activity data for Table S2. We thank Dr. Timothy Ward for his initial contributions to the project.

Funding Information:
Funding for this project was provided by NICHD: 5 R01 HD080431, 1 R61 HD099743, and R01 GM121515. E.B.F. was supported by NIH/NIGMS (through training grants T32 GM008244 and T32 GM132029), as well as by an NIH/NCI fellowship (F30 CA232303). We acknowledge the support provided by the GM/CA beamlines at the Advanced Photon Source (APS), funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396, and S10OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract no. DE-AC02-06CH11357. We thank the Moffitt Chemical Biology Core for use of the crystallization and X-ray facilities (National Cancer Institute grant P30-CA076292). We thank Pharmaron for conducting liver microsome metabolic stability assays and thank Reaction Biology for conducting kinase activity data for Table S2 . We thank Dr. Timothy Ward for his initial contributions to the project.

Publisher Copyright:
© 2023 American Chemical Society.

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Research Support, U.S. Gov't, Non-P.H.S.

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Faber, E. B., Wang, N., John, K., Sun, L., Wong, H. L., Burban, D., Francis, R., Tian, D., Hong, K. H., Yang, A., Wang, L., Elsaid, M., Khalid, H., Levinson, N. M., Schönbrunn, E., Hawkinson, J. E., & Georg, G. I. (2023). Screening through Lead Optimization of High Affinity, Allosteric Cyclin-Dependent Kinase 2 (CDK2) Inhibitors as Male Contraceptives That Reduce Sperm Counts in Mice. Journal of medicinal chemistry, 66(3), 1928-1940. https://doi.org/10.1021/acs.jmedchem.2c01731

Faber, EB, Wang, N, John, K, Sun, L, Wong, HL, Burban, D, Francis, R, Tian, D, Hong, KH, Yang, A, Wang, L, Elsaid, M, Khalid, H, Levinson, NM, Schönbrunn, E, Hawkinson, JE & Georg, GI 2023, 'Screening through Lead Optimization of High Affinity, Allosteric Cyclin-Dependent Kinase 2 (CDK2) Inhibitors as Male Contraceptives That Reduce Sperm Counts in Mice', Journal of medicinal chemistry, vol. 66, no. 3, pp. 1928-1940. https://doi.org/10.1021/acs.jmedchem.2c01731

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abstract = "Although cyclin-dependent kinase 2 (CDK2) is a validated target for both cancer and contraception, developing a CDK2 inhibitor with exquisite selectivity has been challenging due to the structural similarity of the ATP-binding site, where most kinase inhibitors bind. We previously discovered an allosteric pocket in CDK2 with the potential to bind a selective compound and then discovered and structurally confirmed an anthranilic acid scaffold that binds this pocket with high affinity. These allosteric inhibitors are selective for CDK2 over structurally similar CDK1 and show contraceptive potential. Herein, we describe the screening and optimization that led to compounds like EF-4-177 with nanomolar affinity for CDK2. EF-4-177 is metabolically stable, orally bioavailable, and significantly disrupts spermatogenesis, demonstrating this series{\textquoteright} therapeutic potential. This work details the discovery of the highest affinity allosteric CDK inhibitors reported and shows promise for this series to yield an efficacious and selective allosteric CDK2 inhibitor.",

author = "Faber, {Erik B.} and Nan Wang and Kristen John and Luxin Sun and Wong, {Henry L.} and David Burban and Rawle Francis and Defeng Tian and Hong, {Kwon H.} and An Yang and Liming Wang and Mazen Elsaid and Hira Khalid and Levinson, {Nicholas M.} and Ernst Sch{\"o}nbrunn and Hawkinson, {Jon E.} and Georg, {Gunda I.}",

note = "Funding Information: Funding for this project was provided by NICHD: 5 R01 HD080431, 1 R61 HD099743, and R01 GM121515. E.B.F. was supported by NIH/NIGMS (through training grants T32 GM008244 and T32 GM132029), as well as by an NIH/NCI fellowship (F30 CA232303). We acknowledge the support provided by the GM/CA beamlines at the Advanced Photon Source (APS), funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396, and S10OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract no. DE-AC02-06CH11357. We thank the Moffitt Chemical Biology Core for use of the crystallization and X-ray facilities (National Cancer Institute grant P30-CA076292). We thank Pharmaron for conducting liver microsome metabolic stability assays and thank Reaction Biology for conducting kinase activity data for Table S2. We thank Dr. Timothy Ward for his initial contributions to the project. Funding Information: Funding for this project was provided by NICHD: 5 R01 HD080431, 1 R61 HD099743, and R01 GM121515. E.B.F. was supported by NIH/NIGMS (through training grants T32 GM008244 and T32 GM132029), as well as by an NIH/NCI fellowship (F30 CA232303). We acknowledge the support provided by the GM/CA beamlines at the Advanced Photon Source (APS), funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396, and S10OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract no. DE-AC02-06CH11357. We thank the Moffitt Chemical Biology Core for use of the crystallization and X-ray facilities (National Cancer Institute grant P30-CA076292). We thank Pharmaron for conducting liver microsome metabolic stability assays and thank Reaction Biology for conducting kinase activity data for Table S2 . We thank Dr. Timothy Ward for his initial contributions to the project. Publisher Copyright: {\textcopyright} 2023 American Chemical Society.",

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doi = "10.1021/acs.jmedchem.2c01731",

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AU - Faber, Erik B.

AU - Wang, Nan

AU - John, Kristen

AU - Sun, Luxin

AU - Wong, Henry L.

AU - Burban, David

AU - Francis, Rawle

AU - Tian, Defeng

AU - Hong, Kwon H.

AU - Yang, An

AU - Wang, Liming

AU - Elsaid, Mazen

AU - Khalid, Hira

AU - Levinson, Nicholas M.

AU - Schönbrunn, Ernst

AU - Hawkinson, Jon E.

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N1 - Funding Information: Funding for this project was provided by NICHD: 5 R01 HD080431, 1 R61 HD099743, and R01 GM121515. E.B.F. was supported by NIH/NIGMS (through training grants T32 GM008244 and T32 GM132029), as well as by an NIH/NCI fellowship (F30 CA232303). We acknowledge the support provided by the GM/CA beamlines at the Advanced Photon Source (APS), funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396, and S10OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract no. DE-AC02-06CH11357. We thank the Moffitt Chemical Biology Core for use of the crystallization and X-ray facilities (National Cancer Institute grant P30-CA076292). We thank Pharmaron for conducting liver microsome metabolic stability assays and thank Reaction Biology for conducting kinase activity data for Table S2. We thank Dr. Timothy Ward for his initial contributions to the project. Funding Information: Funding for this project was provided by NICHD: 5 R01 HD080431, 1 R61 HD099743, and R01 GM121515. E.B.F. was supported by NIH/NIGMS (through training grants T32 GM008244 and T32 GM132029), as well as by an NIH/NCI fellowship (F30 CA232303). We acknowledge the support provided by the GM/CA beamlines at the Advanced Photon Source (APS), funded by the National Cancer Institute (ACB-12002) and the National Institute of General Medical Sciences (AGM-12006, P30GM138396, and S10OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract no. DE-AC02-06CH11357. We thank the Moffitt Chemical Biology Core for use of the crystallization and X-ray facilities (National Cancer Institute grant P30-CA076292). We thank Pharmaron for conducting liver microsome metabolic stability assays and thank Reaction Biology for conducting kinase activity data for Table S2 . We thank Dr. Timothy Ward for his initial contributions to the project. Publisher Copyright: © 2023 American Chemical Society.

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N2 - Although cyclin-dependent kinase 2 (CDK2) is a validated target for both cancer and contraception, developing a CDK2 inhibitor with exquisite selectivity has been challenging due to the structural similarity of the ATP-binding site, where most kinase inhibitors bind. We previously discovered an allosteric pocket in CDK2 with the potential to bind a selective compound and then discovered and structurally confirmed an anthranilic acid scaffold that binds this pocket with high affinity. These allosteric inhibitors are selective for CDK2 over structurally similar CDK1 and show contraceptive potential. Herein, we describe the screening and optimization that led to compounds like EF-4-177 with nanomolar affinity for CDK2. EF-4-177 is metabolically stable, orally bioavailable, and significantly disrupts spermatogenesis, demonstrating this series’ therapeutic potential. This work details the discovery of the highest affinity allosteric CDK inhibitors reported and shows promise for this series to yield an efficacious and selective allosteric CDK2 inhibitor.

AB - Although cyclin-dependent kinase 2 (CDK2) is a validated target for both cancer and contraception, developing a CDK2 inhibitor with exquisite selectivity has been challenging due to the structural similarity of the ATP-binding site, where most kinase inhibitors bind. We previously discovered an allosteric pocket in CDK2 with the potential to bind a selective compound and then discovered and structurally confirmed an anthranilic acid scaffold that binds this pocket with high affinity. These allosteric inhibitors are selective for CDK2 over structurally similar CDK1 and show contraceptive potential. Herein, we describe the screening and optimization that led to compounds like EF-4-177 with nanomolar affinity for CDK2. EF-4-177 is metabolically stable, orally bioavailable, and significantly disrupts spermatogenesis, demonstrating this series’ therapeutic potential. This work details the discovery of the highest affinity allosteric CDK inhibitors reported and shows promise for this series to yield an efficacious and selective allosteric CDK2 inhibitor.

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Screening through Lead Optimization of High Affinity, Allosteric Cyclin-Dependent Kinase 2 (CDK2) Inhibitors as Male Contraceptives That Reduce Sperm Counts in Mice

Abstract

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