Concluding Remarks Perspective

Autoinhibition is a regulatory strategy employed by many Rho GTPase effectors including enzymes and non-catalytic, scaffolding proteins. The conformational changes and binding events that activate and relieve autoinhibition can provide opportunities for small-molecule binding and intervention as illustrated by N-WASP and wiskostatin. In particular, the trapping of native, inactive conformations may be a broadly applicable approach to inhibiting proteins regulated in this way. In principle, this mechanism provides two key advantages over active site-directed inhibitors. First, inhibition by stabilization of autoinhibited states allows for the targeting of proteins, such as scaffolding proteins, which lack classical small molecule-binding active sites and have not been considered "druggable" targets. As such, the spectrum of proteins amenable to small-molecule inhibition may be significantly greater than previously appreciated, implying the existence of a larger number of potential targets for therapeutic intervention. Second, increased inhibitor specificity may be achieved since autoregulatory domains may not be as evolutionarily conserved in sequence as catalytic sites between closely related proteins. Indeed, the well-documented promiscuity of many ATP-competitive kinase inhibitors illustrates the challenge of achieving target specificity with active site-directed inhibitors.

Wiskostatin was identified in a phenotypic screen for inhibitors of a signaling cascade without preordination of target or mechanism of action. Nevertheless, inhibitors that act by a similar mechanism should, in principle, be identifiable by targeted screens using purified proteins that include the relevant autoinhibitory domains. Consequently, prior detailed biochemical and/or structural knowledge of the regulatory mechanism of the targeted protein facilitates the design of assays that specifically detect compounds that exploit that mechanism. Pakl and mDial are two Rho GTPase effectors for which a wealth of biochemical and structural data are available regarding their mechanisms of autoinhibition and activation. We used these examples here to illustrate how this knowledge can be applied to specifically identify inhibitors that stabilize their autoinhibited states. Furthermore, specific chemical inhibitors of these proteins have not been reported and would be novel and complementary tools to elucidate their biological functions.

Stabilization of autoinhibited conformations need not be limited to effectors of the Rho GTPases, however. Many other proteins are regulated by defined autoinhibitory domains and could be amenable to the screening strategies and allosteric small-molecule inhibition as described here. The example of wiskostatin hints that autoinhibited proteins, in general, could represent a hitherto largely unexploited class of small-molecule targets.

Acknowledgements The authors gratefully acknowledge Drs. Jonathan Chernoff and Harry Higgs for critical reading of the manuscript.

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