Zhu Jiyun, Li Linfeng, Drelich Aleksandra, Chenna Bala C, Mellott Drake M, Taylor Zane W, Tat Vivian, Garcia Christopher Z, Katzfuss Ardala, Tseng Chien-Te K, Meek Thomas D
Department of Biochemistry and Biophysics, College of Agriculture and Life Sciences, Texas A&M University College Station, College Station, TX, United States.
Department of Microbiology and Immunology, John Sealy School of Medicine, University of Texas Medical Branch at Galveston, Galveston, TX, United States.
Front Chem. 2022 Jul 4;10:867928. doi: 10.3389/fchem.2022.867928. eCollection 2022.
Cysteine proteases comprise an important class of drug targets, especially for infectious diseases such as Chagas disease (cruzain) and COVID-19 (3CL protease, cathepsin L). Peptide aldehydes have proven to be potent inhibitors for all of these proteases. However, the intrinsic, high electrophilicity of the aldehyde group is associated with safety concerns and metabolic instability, limiting the use of aldehyde inhibitors as drugs. We have developed a novel class of compounds, self-masked aldehyde inhibitors (SMAIs) which are based on the dipeptide aldehyde inhibitor (Cbz-Phe-Phe-CHO, ), for which the P Phe group contains a 1'-hydroxy group, effectively, an -tyrosinyl aldehyde (Cbz-Phe--Tyr-CHO, ; (Li (2021) 11,267-11,287)). Compound and other SMAIs exist in aqueous mixtures as stable δ-lactols, and apparent catalysis by the cysteine protease cruzain, the major cysteine protease of , results in the opening of the lactol ring to afford the aldehydes which then form reversible thiohemiacetals with the enzyme. These SMAIs are also potent, time-dependent inhibitors of human cathepsin L ( = 11-60 nM), an enzyme which shares 36% amino acid identity with cruzain. As inactivators of cathepsin L have recently been shown to be potent anti-SARS-CoV-2 agents in infected mammalian cells (Mellott (2021) , 642-650), we evaluated SMAIs in VeroE6 and A549/ACE2 cells infected with SARS-CoV-2. These SMAIs demonstrated potent anti-SARS-CoV-2 activity with values of EC = 2-8 μM. We also synthesized pro-drug forms of the SMAIs in which the hydroxyl groups of the lactols were O-acylated. Such pro-drug SMAIs resulted in significantly enhanced anti-SARS-CoV-2 activity (EC = 0.3-0.6 μM), demonstrating that the O-acylated-SMAIs afforded a level of stability within infected cells, and are likely converted to SMAIs by the action of cellular esterases. Lastly, we prepared and characterized an SMAI in which the sidechain adjacent to the terminal aldehyde is a 2-pyridonyl-alanine group, a mimic of both phenylalanine and glutamine. This compound () inhibited both cathepsin L and 3CL protease at low nanomolar concentrations, and also exerted anti-CoV-2 activity in an infected human cell line.
半胱氨酸蛋白酶是一类重要的药物靶点，尤其对于恰加斯病（克氏锥虫蛋白酶）和新冠肺炎（3CL蛋白酶、组织蛋白酶L）等传染病而言。肽醛已被证明是所有这些蛋白酶的有效抑制剂。然而，醛基固有的高亲电性与安全性问题和代谢不稳定性相关，限制了醛类抑制剂作为药物的应用。我们开发了一类新型化合物，即自掩蔽醛抑制剂（SMAIs），其基于二肽醛抑制剂（Cbz-Phe-Phe-CHO），其中Phe基团含有一个1'-羟基，实际上是一个酪氨酰醛（Cbz-Phe-Tyr-CHO）；（Li（2021）11,267 - 11,287）。化合物及其他SMAIs在水性混合物中以稳定的δ-内酯形式存在，克氏锥虫的主要半胱氨酸蛋白酶克氏锥虫蛋白酶的明显催化作用会导致内酯环打开，生成醛，然后醛与该酶形成可逆的硫代半缩醛。这些SMAIs也是人组织蛋白酶L的有效、时间依赖性抑制剂（IC₅₀ = 11 - 60 nM），该酶与克氏锥虫蛋白酶有36%的氨基酸序列一致性。由于组织蛋白酶L的失活剂最近已被证明是受感染哺乳动物细胞中有效的抗SARS-CoV-2药物（Mellott（2021），642 - 650），我们在感染了SARS-CoV-2的VeroE6和A549/ACE2细胞中评估了SMAIs。这些SMAIs表现出强大的抗SARS-CoV-2活性，EC₅₀值为2 - 8 μM。我们还合成了SMAIs的前药形式，其中内酯的羟基被O-酰化。这种前药SMAIs导致抗SARS-CoV-2活性显著增强（EC₅₀ = 0.3 - 0.6 μM），表明O-酰化-SMAIs在受感染细胞内具有一定程度的稳定性，并且可能通过细胞酯酶的作用转化为SMAIs。最后，我们制备并表征了一种SMAI，其中与末端醛相邻的侧链是一个2-吡啶酮基丙氨酸基团，它模拟了苯丙氨酸和谷氨酰胺。该化合物（）在低纳摩尔浓度下同时抑制组织蛋白酶L和3CL蛋白酶，并且在受感染的人细胞系中也发挥了抗-CoV-2活性。
