Targeting the longevity regulator PAPP-A with small molecule inhibitors

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Abstract: Aging can be slowed, lifespan extended, and diseases postponed in mammals by diet, mutations, and small molecules. The most potent longevity mutations in mice modulate levels or actions of growth hormone and/or its downstream mediator, IGF1. The PAPP-A protease activates IGF1 signaling in specific tissues by cleavage of IGF binding proteins (IGFBPs), principally IGFBP4 and IGFBP5. Pappa KO (PKO) mice are long-lived, and show protection from diverse aging pathologies, e.g. cancer, kidney disease, and atherosclerosis. PAPP-A deficiency induced in adult mice also provokes longevity, implying that pharmacologic inhibition of PAPP-A might represent an effective and well-tolerated anti-aging strategy. Although inhibitory antibodies against PAPP-A have been developed, and shown to be effective treatments in preclinical models of atherosclerosis and kidney disease, no small molecule inhibitors against PAPP-A have been described. This represents a critical knowledge gap in this field, since, for aging studies, it would be highly desirable to use small molecule PAPP-A inhibitors, rather than antibody reagents. The objective of this application is to design, synthesize and develop potent, selective, and bioavailable PAPP-A inhibitors, to elucidate their mechanism of action, and test them in vivo. Our preliminary data have shown that our lead PAPP-A inhibitor, P100, effectively inhibits proteolytic cleavage of IGFBP-4 and IGF signaling, the latter assessed by measuring IGF receptor phosphorylation. The proposed work is based on a large body of foundational data, and will be performed in the context of two Specific Aims. In Aim 1, we will design and synthesize new analogs based on our lead PAPP-A inhibitor, to improve their potency, selectivity, and drug-like properties, with the ultimate goal of developing compounds with in vivo activity. A series of assays will be used to characterize the new analogs at the biochemical, biophysical, structural and functionals level, and thus to establish structure-activity relationships of this new class of PAPP-A inhibitors. In Aim 2, optimized PAPP- A inhibitors will be evaluated using a battery of novel cellular and mouse assays of PAPP-A function. The activity of PAPP-A inhibitors in suppressing cellular IGF1 signaling will be tested. The ability of inhibitors to induce a reduction in nucleolar area, a novel phenotype of PKO cells we have uncovered, will be tested. These assays will all be performed in wild-type and PKO cells, to test for PAPP-A dependency of phenotypes observed. In vivo, the impact of PAPP-A inhibitors will be evaluated on PAPP-A-dependent gene expression changes we have uncovered in adipose, skeletal muscle, and thymus, by qRT-PCR and RNA-seq. The most promising small molecules will be evaluated for their ability to induce novel phenotypes of Pappa KO mice: “beiging” of white adipose tissue (WAT), altered ratio of M1/M2 macrophages in WAT, and increased expression of FNDC5, a pro- hormone with beneficial physiological effects. These studies will generate, for the first time, potent and selective small molecule PAPP-A inhibitors. Such molecules will serve as chemical tools to explore the biology of IGF1 signaling and PAPP-A function, and as the basis for future candidate anti-aging compounds.