Discovery of a Potential Allosteric Ligand Binding Site in CDK2

Abstract

Cyclin-dependent kinases (CDKs) are key regulatory enzymes in cell cycle progression and transcription. Aberrant activity of CDKs has been implicated in a number of medical conditions, and numerous small molecule CDK inhibitors have been reported as potential drug leads. However, these inhibitors exclusively bind to the ATP site, which is largely conserved among protein kinases, and clinical trials have not resulted in viable drug candidates, attributed in part to the lack of target selectivity. CDKs are unique among protein kinases, as their functionality strictly depends on association with their partner proteins, the cyclins. In an effort to identify potential target sites for disruption of the CDK-cyclin interaction, we probed the extrinsic fluorophore 8-anilino-1-naphthalene sulfonate (ANS) with human CDK2 and cyclin A using fluorescence spectroscopy and protein crystallography. ANS interacts with free CDK2 in a saturation-dependent manner with an apparent Kd of 37 μM, and cyclin A displaced ANS from CDK2 with an EC50 value of 0.6 μM. Co-crystal structures with ANS alone and in ternary complex with ATP site-directed inhibitors revealed two ANS molecules bound adjacent to one another, away from the ATP site, in a large pocket that extends from the DFG region above the C-helix. Binding of ANS is accompanied by substantial structural changes in CDK2, resulting in a C-helix conformation that is incompatible for cyclin A association. These findings indicate the potential of the ANS binding pocket as a new target site for allosteric inhibitors disrupting the interaction of CDKs and cyclins.

Structure-based discovery of the first allosteric inhibitors of cyclin-dependent kinase 2

“…Compounds 1 (EC 50 of 7 μM) and 2 (EC 50 of 3 μM) bound CDK2 with an affinity higher than ANS (K d = 37 μM), 15 while compounds 3 and 5-7 had similar affinity. To confirm the truly allosteric nature of these ligands, competition experiments between compounds 1-7 and ANS were repeated in the presence of a potent ATP-competitive type I inhibitor that blocks the ATP site, i.e., staurosporine (Fig.…”

“…Using the crystal structure of CDK2 in complex with the allosteric probe ANS 15 (PDB code 1PXF), a virtual screening of commercially available compounds (the Asinex collection, >600 000 compounds) in this newly discovered allosteric pocket was performed using a combination of docking (AutoDock 4) and post-docking (BEAR) 18,19 methods. BEAR has been extensively validated and used in prospective and retrospective drug discovery applications.…”

“…3 Recently, crystal structures of CDK2 bound with the extrinsic fluorophore 8-anilino-1-naphtalene sulfonate (ANS) have been reported. 15,16 In the crystals, 2 molecules of ANS bind a cavity formed by αC and the nearby strands β4 and β5 and induce a remarkable outward displacement of the αC helix, ultimately resulting in disruption of the recognition and binding site of Cyclin A. Ternary crystal structures of CDK2 in complex with ANS and type I inhibitors (JWS648, SU9516, and staurosporine) confirmed the truly allosteric nature of this fluorophore and resulted in CDK2 conformations nearly identical to that of the binary CDK2-ANS complex. 15,16 This is the first time that a completely allosteric ligand proved to be able to displace the αC helix and to inactivate a CDK kinase via an allosteric mechanism, prospecting a new strategy to design inhibitors with potentially improved selectivity.…”

“…However, ANS is readily displaced from CDK2 upon Cyclin binding, because its affinity for CDK2 (K d = 37 μM) is significantly lower than that of Cyclin A. 15 Therefore, the design of compounds able to bind this newly discovered allosteric pocket with higher affinity and with better drug-like properties is highly desirable.…”

Variability of the Cyclin-Dependent Kinase 2 Flexibility Without Significant Change in the Initial Conformation of the Protein or Its Environment; a Computational Study

“…There are several reports that have studied the flexibility of the hCDK2 protein, applying a comparative approach (18)(19)(20)(21)(22), that is studying the flexibility of this protein in significantly different conditions of the protein structure or environment, such as phosphorylation, ATP binding, protein binding etc. However, in our work we have studied variations and changes in the flexibility of hCDK2 protein with no significant changes in its initial environment, structure or even its conformation.…”