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

项目摘要 半胱氨酸蛋白酶在许多感染性疾病的病原体中起重要作用， 包括疟疾、恰加斯病（CD，由寄生虫克氏锥虫引起）和COVID- 19例（由冠状病毒SARS-CoV-2引起）。既没有疫苗也没有良好的耐受性 可用于CD的治疗，其在中美洲和南美洲每年导致50，000人死亡， 估计美国有30万人感染了这种病毒Cruzain是一种半胱氨酸蛋白酶， 对T.克鲁兹到2021年2月， COVID-19大流行已导致全球1.03亿病例和220万死亡。 尽管最近出现了有效的疫苗，但SARS-CoV-2的新变种的出现， 可能会使它们随着时间的推移而变得不那么有效。因此，新治疗剂的发现 治疗COVID-19仍然是一个关键的，未满足的医疗需求。我们最近发现，K11777，一个 cruzain和cathepsin L的不可逆共价灭活剂，有效阻断SARS-CoV-2 通过半胱氨酸蛋白酶组织蛋白酶L的失活感染哺乳动物细胞。因此，委员会认为， 此后，K11777已进入治疗COVID-19的2期临床试验。最近的一 一份出版物报道了组织蛋白酶L在来自于肺的尸检样品中显著上调， COVID-19患者，这表明这种CP不仅在CoV-2细胞进入中起关键作用，而且在 人类疾病的严重性。SARS-CoV-2的感染需要冠状病毒的作用， 蛋白酶3CL蛋白酶（3CL-PR）;也是一种半胱氨酸蛋白酶，因此是一种重要的 COVID-19的药物靶点。我们的方法是鉴定半胱氨酸蛋白酶的抑制剂， 与这些酶的不变活性位点半胱氨酸的可逆共价键。理想情况下， 为这种抑制剂选择合适的肽支架将提供一种化合物， 有效抑制一种以上的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，我们试图重新解决醛作为有效的，可逆的酶抑制剂， 将它们修饰为分子内的内乳醇或“自掩蔽”醛（SMAI）。的目标 该建议是设计、合成和评价新的SMAI，包括前药形式，作为新的 这三种CP药物靶点的抑制剂，并评估其在细胞中的抗感染特性 CD和COVID-19的模型。我们将改进这些新抑制剂的药物样特性， 药代动力学、代谢稳定性，并确定其他SMAI系列。我们将 利用这些抑制剂的前药形式，其提供增强的代谢稳定性， 活性抑制剂应该在酶修饰时在细胞间释放。

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.