Development of Allosteric Inhibitors against Cyclin-dependent Kinase 2 (CDK2)

Abstract

Cyclin-dependent kinase 2 (CDK2) is a validated therapeutic target for male nonhormonal contraception as well as various anticancer indications. Despite this, selectively targeting CDK2 has been challenging, largely due to the structural homology of the active site among kinases that most developed therapeutics bind. Our group previously discovered an unexplored allosteric site in CDK2 that binds the commercial dye 8-anilino-1-naphthalene sulfonic acid (ANS) with moderate affinity. In this work, I explored the cooperative effects of ANS with other ATP-site (i.e. orthosteric) ligands to enhance the affinity of ANS. Next, I developed various synthetic methods, including an Ullmann coupling method, to make ANS derivatives whereas this had been previously challenging. Using the fluorescent properties of ANS, we discovered a new chemical scaffold that binds this allosteric site. From there, we conducted a structure-activity relationship (SAR) campaign to improve the affinity of this compound ~5,000 fold from the original screening hit, leading to compounds with single-digit nanomolar affinity. We observed a negatively cooperative relationship with this series and cyclin binding, representing an underexplored mechanism of CDK2 inhibition.