PROJECT SUMMARY/ABSTRACT Cellular senescence is an important fundamental mechanism of aging and age-related diseases. It has been demonstrated that genetic elimination of senescent cells (SCs) can dramatically alleviate aging-related phenotypes and extend both healthspan and lifespan in mice. Senolytics, referring to small-molecules that can selectively induce apoptosis in SCs, thus have the potential to be developed as “anti-aging” drugs that extend human healthspan by preventing or treating age-related diseases. We and others have recently discovered a number of senolytics that target various SC anti-apoptotic pathways (SCAPs). Subsequent studies on these senolytics in animal models have shown that clearance of SCs can treat several age-related diseases and chemo-/radio-therapy caused adverse effects in mice, thus demonstrating the therapeutic potential of senolytics. However, most of the reported senolytics have potential on-target and off-target toxicities. In addition, SCs are not all the same; different SCs may use different SCAPs to resist apoptosis. Thus, it is important to continue the search of new SCAPs/protein targets and new senolytics. Piperlongumine (PL), a dietary natural product, was identified as a novel senolytic agent through our targeted screening campaign. PL appears to be safe, without observable toxicity in vivo and having a high maximum tolerated oral dose in mice. However, its mechanism of action (MOA) against SC is undefined, raising the potential of discovering novel molecular targets for senolytic drug development. Therefore, we hypothesize that PL can be used to identify new molecular targets in SCs, which can then be exploited for the development of novel and specific senolytics. Our preliminary structure–activity relationship (SAR) studies have identified PL analogues (piperlogues) with senolytic profile comparable to that of ABT263, one of the most potent and selective senolytics identified to date, and also designed and synthesized PL-based probe molecules for the pulldown of target proteins in SCs. With proteomics analyses and RNAi technology, we identified, validated, and characterized oxidation protein 1 (OXR1) as one of the protein targets of PL in SCs. The objectives of this application are to continue the discovery and evaluation of piperlogues as potent and selective senolytics, identification of their molecular targets, and elucidation of the MOA of these novel agents. Specifically, we will pursue the following specific aims: 1) design, synthesize, and evaluate novel piperlogues with improved senolytic activity and druglike properties; 2) identify and validate the protein targets of PL and piperlogues in SCs, and elucidate their MOA; and 3) evaluate the in vivo efficacy and safety of lead piperlogues. Success in these proposed aims will not only produce anti-aging drug development candidates but also identify and validate additional novel molecular target(s) to enable future design and target-based discov...