PROJECT SUMMARY / ABSTRACT The recent discovery that senescent cells (SnCs) play a causative role in aging and in many age-related diseases suggests that cellular senescence is a fundamental mechanism of aging. Selective elimination of SnCs with a small molecule, termed senolytic, has become a new “anti-aging” strategy that has the potential to extend human healthspan by preventing and treating age-related diseases. Although a few senolytics have been identified and characterized, the majority of the senolytics discovered to date are repurposed anticancer agents because SnCs use some of the same molecular mechanisms to evade apoptosis as cancer cells. These senolytics usually possess various on-target and/or off-target toxicities, which could preclude their clinical use as anti-aging agents because old people are more susceptible to adverse drug effects than young individuals and tolerate drug toxicity less well than cancer patients. ABT263, a selective BCL-2 and BCL-xL inhibitor, is one of the most potent and broad-spectrum repurposed senolytics discovered to date. We and others found that many different types of SnCs depend on the anti-apoptotic BCL-2 family proteins for survival, particularly BCL- xL. ABT263 can potently kill a variety of SnCs in cell culture and effectively clear SnCs in various murine tissues. Clearance of SnCs with ABT263 can also rejuvenate aged hematopoietic stem cells (HSCs) and the senescent hematopoietic system in both prematurely and naturally aged mice, and ameliorate several pathological conditions associated with aging. However, its on-target toxicity of thrombocytopenia prevents its clinical use even for cancer patients, because platelets also depend on BCL-xL for survival. We hypothesize that this on- target toxicity can be averted by converting ABT263 and other BCL-xl inhibitors into platelet-sparing BCL-xL proteolysis targeting chimeras (PROTACs) that target BCL-XL to an E3 ligase poorly expressed in platelets for ubiquitination and degradation. This hypothesis is supported by our preliminary findings that BCL-xL PROTACs are more potent against SnCs but less toxic to non-SnCs and platelets than ABT263 in vitro, and can clear SnCs as effectively as ABT263 in normally aged mice without causing thrombocytopenia. We expect that converting ABT263 into platelet-sparing BCL-xL PROTACs can also reduce systemic drug exposure to lower off-target toxicities of ABT263 because PROTACs can eliminate their target proteins upon binding to the targets (event- driven pharmacology) and mediate multiple rounds of target degradation (sub-stoichiometric activity), whereas the activity of an inhibitor depends on occupancy of a binding site that directly affects protein function (occupancy-driven pharmacology and stoichiometric activity). Based on these exciting preliminary data, we plan to pursue the following specific aims: 1) design and synthesize platelet-sparing BCL-xL PROTACs with optimal safety, potency, and in vivo efficacy as...