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.
Recent studies have shown that the DNA gyrase inhibitor, novobiocin, binds to a previously unrecognized ATP-binding site located at the C-terminus of Hsp90 and induces degradation of Hsp90-dependent client proteins at approximately 700 microM. As a result of these studies, several analogues of the coumarin family of antibiotics have been reported and shown to exhibit increased Hsp90 inhibitory activity; however, the monomeric species lacked the ability to manifest anti-proliferative activity against cancer cell lines at concentrations tested. In an effort to develop more efficacious compounds that produce growth inhibitory activity against cancer cell lines, structure-activity relationships were investigated surrounding the prenylated benzamide side chain of the natural product. Results obtained from these studies have produced the first novobiocin analogues that manifest anti-proliferative activity against several cancer cell lines.
Natural products have continued to drive the development of new chemotherapeutics and elucidation of new biological targets for the treatment of disease. Since Whitesell and Neckers' original discovery that geldanamycin does not directly inhibit v-Src, but instead manifests its biological activity through inhibition of the Hsp90 molecular chaperone, additional natural products and natural product derivatives have been identified and developed to inhibit the Hsp90 protein folding machinery. 17-AAG, a geldanamycin analogue, is currently in clinical trials for the treatment of several types of cancer. Recent work has produced improved radicicol analogues that show promising Hsp90 inhibitory activity in vitro. In addition, chimeric molecules of these two natural products are active in vitro and represent a novel class of Hsp90 inhibitors for cancer treatment. In addition to their chemotherapeutic uses, natural product inhibitors and their derivatives have been utilized to probe the biological mechanisms by which Hsp90 inhibition regulates tumor cell growth. As a consequence of these studies, the molecular chaperones have emerged as an exciting new class of therapeutic targets. This review will highlight the utility of the natural products, geldanamycin and radicicol, as well as improved analogues and the activities exhibited by these compounds against various cancer cell lines.
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