SELF-MASKED ALDEHYDES AS INHIBITORS OF THE CYSTEINE PROTEASES 3CL PROTEASE, CATHEPSIN L, AND CRUZAIN

项目总结 半胱氨酸蛋白酶在许多传染病的病原体中扮演着重要的角色， 包括疟疾、恰加斯病(CD，由克氏锥虫寄生虫引起)和COVID- 19(由冠状病毒SARS-CoV-2引起)。既没有疫苗，也没有很好的耐受性 可用于CD的治疗方法，在中美洲和南美洲每年导致5万人死亡， 据估计，美国有30万名感染者。克鲁赞是一种半胱氨酸蛋白酶 对于建立和维持克氏毛滴虫的人类感染是必不可少的。到2021年2月， 新冠肺炎疫情已导致全球1.03亿病例和220万人死亡。 尽管最近出现了有效的疫苗，但SARS-CoV-2新变种的出现 随着时间的推移，可能会使它们变得不那么有效。因此，新的治疗剂的发现 治疗新冠肺炎仍然是一个关键的、尚未得到满足的医疗需求。我们最近展示了K11777，一个 不可逆的克鲁扎因和组织蛋白酶共价灭活剂L有效地阻断了SARS-CoV-2 半胱氨酸蛋白酶组织蛋白L失活感染哺乳动物细胞 此后，K11777已进入治疗新冠肺炎的第二阶段临床试验。最近 据报道，组织蛋白酶L在中国人的肺部尸检样本中显著上调。 这表明该CP不仅在CoV-2细胞进入细胞中起关键作用，而且在 人类疾病的严重性。感染SARS-CoV-2需要冠状病毒的作用 蛋白酶3CL蛋白(3CL-PR)也是一种半胱氨酸蛋白酶，因此是一种重要的 新冠肺炎的毒品目标。我们的方法是确定形成半胱氨酸蛋白酶的抑制剂 与这些酶的活性部位不变的半胱氨酸形成可逆的共价键。理想情况下， 为这种抑制剂选择合适的多肽支架将提供一种化合物， 有效地抑制不止一个CP。醛是非常有效的，共价的，但可逆的， 半胱氨酸蛋白酶抑制剂，尽管与其反应性和代谢相关的风险 不稳定。在这里，我们试图通过以下方式将醛重新定位为有效的可逆酶抑制剂 将它们修饰为分子内乳醇，或“自掩蔽”醛(SMAI)。的目标是 这项提议是设计、合成和评估新的SMAI，包括前药形式，作为新的 这三种CP药物靶点的抑制剂，并评价其在细胞内的抗感染性能 CD和新冠肺炎的型号。我们将改善这些新抑制剂的类药物特性 药物动力学、代谢稳定性方面的条件，并确定其他系列的SMAI。我们会 利用这些抑制剂的前药形式，提供增强的代谢稳定性 活性抑制剂在酶修饰时应以细胞间的形式释放。

PROJECT SUMMARY Cysteine proteases play essential roles in the causative agents of numerous infectious diseases, including malaria, Chagas disease (CD, caused by the parasite Trypanosoma cruzi), and COVID- 19 (caused by the coronavirus SARS-CoV-2). There are neither vaccines nor well-tolerated therapies available for CD, which results in 50,000 annual deaths in Central and South America, with an estimated 300,000 infected individuals in the US. Cruzain is a cysteine protease that is essential to the establishment and maintenance of human infection by T. cruzi. By February 2021, the COVID-19 pandemic has resulted in 103 million cases worldwide and 2.2 million deaths. Despite the recent arrival of effective vaccines, the emergence of new variants of SARS-CoV-2 may render them less effective over time. Accordingly, the discovery of new therapeutic agents to treat COVID-19 remains a critical, unmet medical need. We recently showed that K11777, an irreversible, covalent inactivator of both cruzain and cathepsin L, potently blocked SARS-CoV-2 infection of mammalian cells by inactivation of the cysteine protease cathepsin L. Consequently, since then K11777 has advanced to Phase 2 clinical trials for the treatment of COVID-19. A recent publication reported that cathepsin L was significantly upregulated in lung autopsy samples from COVID-19 patients, suggesting that this CP not only has a key role in CoV-2 cell entry but also in the severity of human disease. Infection by SARS-CoV-2 requires the action of coronaviral protease 3CL protease (3CL-PR); also a cysteine protease, and consequently is an important drug target for COVID-19. Our approach is to identify inhibitors of cysteine proteases which form reversible covalent bonds with the invariant active-site cysteines of these enzymes. Ideally, selection of an appropriate peptide scaffold for such an inhibitor would provide a compound that potently inhibits more than one CP. Aldehydes are exceptionally potent, covalent, but reversible, inhibitors of cysteine proteases, despite the risk associated with their reactivity and metabolic instability. Here, we seek to re-address aldehydes as potent, reversible enzyme inhibitors by modifying them as intramolecular lactols, or “self-masked” aldehydes (SMAIs). The objective of this proposal is to design, synthesize, and evaluate new SMAIs, including prodrug forms, as novel inhibitors of these three CP drug targets, and evaluate their anti-infective properties in cellular models of CD and COVID-19. We will improve the drug-like properties of these new inhibitors in terms of pharmacokinetics, metabolic stability, and identify additional series of SMAIs. We will utilize prodrug forms of these inhibitors that provide enhanced metabolic stability from which active inhibitors should be released intercellularly upon enzymatic modification.