Computationally Driven Discovery and Characterization of SIRT3-Activating Compounds that Fully Recover Catalytic Activity under NAD^{+} Depletion

Abstract

Many chronic, age-related disorders could be mitigated by enhancing the activity of enzymes that regulate biochemical signaling pathways, but all known modes of enzyme activation rely on allosteric binding sites, which have only been identified in less than 10% of proteins. Sirtuins (SIRT1-7) are nicotinamide adenine dinucleotide (NAD^{+})-dependent deacylases playing critical roles in lifespan and age-related diseases. The physiological importance of sirtuins and the reduction in their catalytic activity with the age-related decline in NAD^{+} levels have stimulated intense interest in designing sirtuin-activating compounds; however, except for substrate-specific allosteric SIRT1 activators, methodologies for rational design of sirtuin-activating compounds are lacking. Here, we introduce methods for the activation of such enzymes that do not rely on allosteric binding sites, and we demonstrate their successful application to the discovery of first-in-class activators of sirtuin enzymes. We establish how all-atom simulations of an enzyme’s active site under the potential of a small molecule modulator can be used to identify molecular properties that achieve desired changes in local enzyme conformational degrees of freedom conducive to the enhancement of catalytic activity. We apply computational high-throughput screening based on this biophysical model for activation of sirtuin enzymes to the major mitochondrial sirtuin SIRT3, which plays a critical role in age-related disorders but does not have a known allosteric site; we thereby identify first-in-class, nonallosteric activators of this enzyme. These compounds are the first reported steady-state activators of the major mitochondrial sirtuin enzyme, and they operate according to a mode of action not shared by any existing drug. Two such compounds can almost double the catalytic efficiency of SIRT3 with respect to NAD^{+}, thus compensating for the loss in SIRT3 activity that occurs due to the age-related decline in NAD^{+}, and they may be developed for therapeutic applications to combat multiple types of age-related disorders. These discoveries establish a foundation for the development of a new class of drugs that function through the activation of enzymes by modulation of local conformational ensembles.