Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding

Erik B. Faber; Luxin Sun; Jian Tang; Emily Roberts; Sornakala Ganeshkumar; Nan Wang; Damien Rasmussen; Abir Majumdar; Laura E. Hirsch; Kristen John; An Yang; Hira Khalid; Jon E. Hawkinson; Nicholas M. Levinson; Vargheese Chennathukuzhi; Daniel A. Harki; Ernst Schönbrunn; Gunda I. Georg

doi:10.1038/s41467-023-38732-x

Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding

Erik B. Faber, Luxin Sun, Jian Tang, Emily Roberts, Sornakala Ganeshkumar, Nan Wang, Damien Rasmussen, Abir Majumdar, Laura E. Hirsch, Kristen John, An Yang, Hira Khalid, Jon E. Hawkinson, Nicholas M. Levinson, Vargheese Chennathukuzhi, Daniel A. Harki, Ernst Schönbrunn, Gunda I. Georg

Research output: Contribution to journal › Article › peer-review

Abstract

Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception. However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. In this paper, we describe the development and mechanism of action of type III inhibitors that bind CDK2 with nanomolar affinity. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as contraceptive agents is seen by incubation with spermatocyte chromosome spreads from mouse testicular explants, where they recapitulate Cdk2 ^-/- and Spdya ^-/- phenotypes.

Original language	English (US)
Article number	3213
Journal	Nature communications
Volume	14
Issue number	1
DOIs	https://doi.org/10.1038/s41467-023-38732-x
State	Published - Dec 2023

Bibliographical note

Funding Information:
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). Funding for this project was provided by NICHD: R01 HD080431 (V.C.), R61 HD099743 (G.I.G. and V.C.), NIGMS: R01 GM121515 (N.M.L.), DOD: W81XWH-21-1-0674 (D.A.H.), W81XWH-19-1-0336 (J.T.), and Minnesota Ovarian Cancer Alliance (G.I.G.). Production of CDK1 was made possible by NIH HHSN27520180007I. We acknowledge support 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, P30 GM138396, S10 OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE-AC02-06CH11357. The Moffitt Chemical Biology Core is acknowledged for use of the crystallization and X-ray facilities (National Cancer Institute grant P30 CA076292). We thank Dr. Tim Ward for his initial contributions to this project, Madeline Honig for help with HRMS, and Todd Rappe for help with C NMR. We also thank Dr. Angel Nebreda for supplying the anti-SPY1 antibodies. Reaction Biology provided the data for kinase activity data in Supplementary Tables –, and Pharmaron provided data for Supplementary Fig. . 13

Funding Information:
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). Funding for this project was provided by NICHD: R01 HD080431 (V.C.), R61 HD099743 (G.I.G. and V.C.), NIGMS: R01 GM121515 (N.M.L.), DOD: W81XWH-21-1-0674 (D.A.H.), W81XWH-19-1-0336 (J.T.), and Minnesota Ovarian Cancer Alliance (G.I.G.). Production of CDK1 was made possible by NIH HHSN27520180007I. We acknowledge support 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, P30 GM138396, S10 OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE-AC02-06CH11357. The Moffitt Chemical Biology Core is acknowledged for use of the crystallization and X-ray facilities (National Cancer Institute grant P30 CA076292). We thank Dr. Tim Ward for his initial contributions to this project, Madeline Honig for help with HRMS, and Todd Rappe for help with13C NMR. We also thank Dr. Angel Nebreda for supplying the anti-SPY1 antibodies. Reaction Biology provided the data for kinase activity data in Supplementary Tables 2 –3 , and Pharmaron provided data for Supplementary Fig. 13.

Publisher Copyright:
© 2023, The Author(s).

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

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Faber, E. B., Sun, L., Tang, J., Roberts, E., Ganeshkumar, S., Wang, N., Rasmussen, D., Majumdar, A., Hirsch, L. E., John, K., Yang, A., Khalid, H., Hawkinson, J. E., Levinson, N. M., Chennathukuzhi, V., Harki, D. A., Schönbrunn, E., & Georg, G. I. (2023). Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding. Nature communications, 14(1), Article 3213. https://doi.org/10.1038/s41467-023-38732-x

Faber, EB, Sun, L, Tang, J, Roberts, E, Ganeshkumar, S, Wang, N, Rasmussen, D, Majumdar, A, Hirsch, LE, John, K, Yang, A, Khalid, H, Hawkinson, JE, Levinson, NM, Chennathukuzhi, V, Harki, DA, Schönbrunn, E & Georg, GI 2023, 'Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding', Nature communications, vol. 14, no. 1, 3213. https://doi.org/10.1038/s41467-023-38732-x

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abstract = "Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception. However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. In this paper, we describe the development and mechanism of action of type III inhibitors that bind CDK2 with nanomolar affinity. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as contraceptive agents is seen by incubation with spermatocyte chromosome spreads from mouse testicular explants, where they recapitulate Cdk2 -/- and Spdya -/- phenotypes.",

author = "Faber, {Erik B.} and Luxin Sun and Jian Tang and Emily Roberts and Sornakala Ganeshkumar and Nan Wang and Damien Rasmussen and Abir Majumdar and Hirsch, {Laura E.} and Kristen John and An Yang and Hira Khalid and Hawkinson, {Jon E.} and Levinson, {Nicholas M.} and Vargheese Chennathukuzhi and Harki, {Daniel A.} and Ernst Sch{\"o}nbrunn and Georg, {Gunda I.}",

note = "Funding Information: 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). Funding for this project was provided by NICHD: R01 HD080431 (V.C.), R61 HD099743 (G.I.G. and V.C.), NIGMS: R01 GM121515 (N.M.L.), DOD: W81XWH-21-1-0674 (D.A.H.), W81XWH-19-1-0336 (J.T.), and Minnesota Ovarian Cancer Alliance (G.I.G.). Production of CDK1 was made possible by NIH HHSN27520180007I. We acknowledge support 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, P30 GM138396, S10 OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE-AC02-06CH11357. The Moffitt Chemical Biology Core is acknowledged for use of the crystallization and X-ray facilities (National Cancer Institute grant P30 CA076292). We thank Dr. Tim Ward for his initial contributions to this project, Madeline Honig for help with HRMS, and Todd Rappe for help with C NMR. We also thank Dr. Angel Nebreda for supplying the anti-SPY1 antibodies. Reaction Biology provided the data for kinase activity data in Supplementary Tables –, and Pharmaron provided data for Supplementary Fig. . 13 Funding Information: 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). Funding for this project was provided by NICHD: R01 HD080431 (V.C.), R61 HD099743 (G.I.G. and V.C.), NIGMS: R01 GM121515 (N.M.L.), DOD: W81XWH-21-1-0674 (D.A.H.), W81XWH-19-1-0336 (J.T.), and Minnesota Ovarian Cancer Alliance (G.I.G.). Production of CDK1 was made possible by NIH HHSN27520180007I. We acknowledge support 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, P30 GM138396, S10 OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE-AC02-06CH11357. The Moffitt Chemical Biology Core is acknowledged for use of the crystallization and X-ray facilities (National Cancer Institute grant P30 CA076292). We thank Dr. Tim Ward for his initial contributions to this project, Madeline Honig for help with HRMS, and Todd Rappe for help with13C NMR. We also thank Dr. Angel Nebreda for supplying the anti-SPY1 antibodies. Reaction Biology provided the data for kinase activity data in Supplementary Tables 2 –3 , and Pharmaron provided data for Supplementary Fig. 13. Publisher Copyright: {\textcopyright} 2023, The Author(s).",

year = "2023",

month = dec,

doi = "10.1038/s41467-023-38732-x",

language = "English (US)",

volume = "14",

journal = "Nature communications",

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T1 - Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding

AU - Faber, Erik B.

AU - Sun, Luxin

AU - Tang, Jian

AU - Roberts, Emily

AU - Ganeshkumar, Sornakala

AU - Wang, Nan

AU - Rasmussen, Damien

AU - Majumdar, Abir

AU - Hirsch, Laura E.

AU - John, Kristen

AU - Yang, An

AU - Khalid, Hira

AU - Hawkinson, Jon E.

AU - Levinson, Nicholas M.

AU - Chennathukuzhi, Vargheese

AU - Harki, Daniel A.

AU - Schönbrunn, Ernst

AU - Georg, Gunda I.

N1 - Funding Information: 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). Funding for this project was provided by NICHD: R01 HD080431 (V.C.), R61 HD099743 (G.I.G. and V.C.), NIGMS: R01 GM121515 (N.M.L.), DOD: W81XWH-21-1-0674 (D.A.H.), W81XWH-19-1-0336 (J.T.), and Minnesota Ovarian Cancer Alliance (G.I.G.). Production of CDK1 was made possible by NIH HHSN27520180007I. We acknowledge support 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, P30 GM138396, S10 OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE-AC02-06CH11357. The Moffitt Chemical Biology Core is acknowledged for use of the crystallization and X-ray facilities (National Cancer Institute grant P30 CA076292). We thank Dr. Tim Ward for his initial contributions to this project, Madeline Honig for help with HRMS, and Todd Rappe for help with C NMR. We also thank Dr. Angel Nebreda for supplying the anti-SPY1 antibodies. Reaction Biology provided the data for kinase activity data in Supplementary Tables –, and Pharmaron provided data for Supplementary Fig. . 13 Funding Information: 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). Funding for this project was provided by NICHD: R01 HD080431 (V.C.), R61 HD099743 (G.I.G. and V.C.), NIGMS: R01 GM121515 (N.M.L.), DOD: W81XWH-21-1-0674 (D.A.H.), W81XWH-19-1-0336 (J.T.), and Minnesota Ovarian Cancer Alliance (G.I.G.). Production of CDK1 was made possible by NIH HHSN27520180007I. We acknowledge support 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, P30 GM138396, S10 OD012289). The APS is a U.S. Department of Energy Office of Science User Facility operated under Contract No. DE-AC02-06CH11357. The Moffitt Chemical Biology Core is acknowledged for use of the crystallization and X-ray facilities (National Cancer Institute grant P30 CA076292). We thank Dr. Tim Ward for his initial contributions to this project, Madeline Honig for help with HRMS, and Todd Rappe for help with13C NMR. We also thank Dr. Angel Nebreda for supplying the anti-SPY1 antibodies. Reaction Biology provided the data for kinase activity data in Supplementary Tables 2 –3 , and Pharmaron provided data for Supplementary Fig. 13. Publisher Copyright: © 2023, The Author(s).

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Y1 - 2023/12

N2 - Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception. However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. In this paper, we describe the development and mechanism of action of type III inhibitors that bind CDK2 with nanomolar affinity. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as contraceptive agents is seen by incubation with spermatocyte chromosome spreads from mouse testicular explants, where they recapitulate Cdk2 -/- and Spdya -/- phenotypes.

AB - Compared to most ATP-site kinase inhibitors, small molecules that target an allosteric pocket have the potential for improved selectivity due to the often observed lower structural similarity at these distal sites. Despite their promise, relatively few examples of structurally confirmed, high-affinity allosteric kinase inhibitors exist. Cyclin-dependent kinase 2 (CDK2) is a target for many therapeutic indications, including non-hormonal contraception. However, an inhibitor against this kinase with exquisite selectivity has not reached the market because of the structural similarity between CDKs. In this paper, we describe the development and mechanism of action of type III inhibitors that bind CDK2 with nanomolar affinity. Notably, these anthranilic acid inhibitors exhibit a strong negative cooperative relationship with cyclin binding, which remains an underexplored mechanism for CDK2 inhibition. Furthermore, the binding profile of these compounds in both biophysical and cellular assays demonstrate the promise of this series for further development into a therapeutic selective for CDK2 over highly similar kinases like CDK1. The potential of these inhibitors as contraceptive agents is seen by incubation with spermatocyte chromosome spreads from mouse testicular explants, where they recapitulate Cdk2 -/- and Spdya -/- phenotypes.

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Development of allosteric and selective CDK2 inhibitors for contraception with negative cooperativity to cyclin binding

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