Hsp90 is a molecular chaperone of pivotal importance for multiple cell pathways. ATP-regulated internal dynamics are critical for its function and current anticancer pharmacological approaches block the chaperone by using ATP-competitive inhibitors. In this paper, we propose a general approach to perturb Hsp90 through design of new allosteric modulators that alter the functional dynamics of the protein. We rationally developed a library of 2-phenylbenzofurans that, rather than inhibiting, activate Hsp90 ATPase by targeting an allosteric site that we recently identified, located 65Å from the active site. Analysis of protein responses to first-generation activators was exploited to guide the design of second-generation derivatives with improved ability to stimulate ATP hydrolysis. The molecules’ effects on Hsp90 enzymatic, conformational, co-chaperone and client-binding properties were characterized through biochemical, biophysical and cellular approaches. The new, rationally designed probes act as allosteric activators of the chaperone and affect the viability of cancer cell lines for which proper functioning of Hsp90 is necessary.

Controlling biochemical pathways through chemically designed modulators may provide novel opportunities to develop therapeutic drugs and chemical tools. The underlying challenge is to design new molecular entities able to act as allosteric chemical switches that selectively turn on/off functions by modulating the conformational dynamics of their target protein. We examine the origins of the stimulation of ATPase and closure kinetics in the molecular chaperone Hsp90 by allosteric modulators through atomistic molecular dynamics (MD) simulations and analysis of protein-ligand interactions. In particular, we focus on the cross-talk between allosteric ligands and protein conformations and its effect on the dynamic properties of the chaperone’s active state. We examine the impact of different allosteric modulators on the stability, structural and internal dynamics properties of Hsp90 closed state. A critical aspect of this study is the development of a quantitative model that correlates Hsp90 activation to the presence of a certain compound, making use of information on the dynamic adaptation of protein conformations to the presence of the ligand, which allows to capture conformational states relevant in the activation process. We discuss the implications of considering the conformational dialogue between allosteric ligands and protein conformations for the design of new functional modulators.