Develop BCL-xL proteolysis targeting chimeras as safer and better senolytics

项目摘要/摘要 最近发现，衰老细胞(SNC)在衰老和许多与年龄相关的疾病中起着致病作用 表明细胞衰老是衰老的基本机制。有选择地消除SNC 被称为感觉剂的小分子已经成为一种具有推广潜力的新的抗衰老策略。 人类通过预防和治疗与年龄有关的疾病来延长健康。尽管一些感冒药已经被 经过鉴定和表征，到目前为止发现的大多数感觉剂都是重新调整用途的抗癌剂。 因为SNCs使用一些与癌细胞相同的分子机制来逃避凋亡。这些 感觉剂通常具有各种靶上和/或靶外毒性，这可能会阻碍其临床应用。 作为抗衰老药物，因为老年人比年轻人更容易受到药物不良反应的影响 而且对药物毒性的耐受性不如癌症患者。ABT263是一种选择性的bcl2和bclxl抑制剂， 是迄今为止发现的最有效和最广谱的再利用增感物质之一。我们和其他人发现 许多不同类型的SNCs依赖于抗凋亡的bcl2家族蛋白生存，尤其是bcl2家族。 Xl.ABT263能有效杀伤细胞培养中的多种SNCs，并能有效清除小鼠各种组织中的SNCs。 ABT263清除SNCs也能使衰老的造血干细胞(HSCs)和 对早衰和自然衰老小鼠的造血系统的影响，并改善几种病理变化 与衰老相关的条件。然而，其靶向毒性血小板减少阻碍了其临床应用。 即使对癌症患者也是如此，因为血小板也依赖bclxl生存。我们假设这件事发生在- 通过将ABT263和其他bclxl抑制剂转化为血小板保护的bclxl可以避免靶向毒性 蛋白水解靶向嵌合体(PROTAC)靶向BCL-XL到E3连接酶在血小板中低表达 泛素化和降解。我们的初步发现支持这一假设，即bcl-xl PROTACs 体外抗SNCs作用强于ABT263，但对非SNCs和血小板的毒性较小，并能清除SNCs 在正常衰老的小鼠中与ABT263一样有效，而不会导致血小板减少。我们预计这一转变 ABT263进入保留血小板的BCL-XL PROTAC还可以减少全身药物暴露，降低非靶点 ABT263的毒性是因为PROTAC在与靶结合时可以消除其靶蛋白(Event- 驱动药理学)并调节多轮靶向降解(亚化学计量比活性)，而 抑制物的活性取决于直接影响蛋白质功能的结合部位的占有率。 (占有率驱动的药理学和化学计量活动)。基于这些令人兴奋的初步数据，我们计划 追求以下具体目标：1)设计和合成具有最佳血小板保护功能的bclxlPROTACs 作为感觉剂的安全性、有效性和体内疗效；2)选择铅-血小板保护的BCL-XL PROTAC用于 它们清除中性粒细胞及对老年和衰老者造血干细胞返老还童的疗效评价 小鼠的造血和免疫系统(HIS)；以及3)决定用铅血小板进行全身治疗是否- Sparing BCL-XL PROTAC联合应用可有效治疗老年小鼠骨关节炎(OA) 局部UBX101共裂解治疗。我们预计拟议的研究将导致发现更安全和 更有效的抗衰老药物，更有可能转化为临床使用，以预防和治疗OA和其他 与年龄有关的疾病。此外，我们的初步数据提供了概念证明，PROTAC技术可以 有助于将其他有毒的再利用增感剂转化为更安全、更有效的抗衰老药物。

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 senolytic agents; 2) select lead platelet-sparing BCL-xL PROTACs for evaluation of their therapeutic efficacy for clearance of SnC and rejuvenation of aged HSCs and the senescent hematopoietic and immune system (HIS) in mice; and 3) determine whether systemic therapy with lead platelet- sparing BCL-xL PROTACs can be used to effectively treat osteoarthritis (OA) in aged mice when combined with local UBX101 synolytic treatment. We anticipate that the proposed studies will lead to the discovery of safer and more potent senolytic agents that are more likely to translate into clinical use to prevent and treat OA and other age-related diseases. Furthermore, our preliminary data provide proof-of-concept that PROTAC technology may be useful to convert other toxic repurposed senolytic agents into safer and more effective anti-aging agents.