PDF(Pubmed)
Abstract：
The main protease (Mpro) remains an essential therapeutic target for COVID-19 post infection intervention given its critical role in processing the majority of viral proteins encoded by the genome of severe acute respiratory syndrome related coronavirus 2 (SARS-CoV-2). Upon viral entry, the +ssRNA genome is translated into two long polyproteins (pp1a or the frameshift-dependent pp1ab) containing all the nonstructural proteins (nsps) required by the virus for immune modulation, replication, and ultimately, virion assembly. Included among these nsps is the cysteine protease Mpro (nsp5) which self-excises from the polyprotein, dimerizes, then sequentially cleaves 11 of the 15 cut-site junctions found between each nsp within the polyprotein. Many structures of Mpro (often bound to various small molecule inhibitors or peptides) have been detailed recently, including structures of Mpro bound to each of the polyprotein cleavage sequences, showing that Mpro can accommodate a wide range of targets within its active site. However, to date, kinetic characterization of the interaction of Mpro with each of its native cleavage sequences remains incomplete. Here, we present a robust and cost-effective FRET based system that benefits from a more consistent presentation of the substrate that is also closer in organization to the native polyprotein environment compared to previously reported FRET systems that use chemically modified peptides. Using this system, we were able to show that while each site maintains a similar Michaelis constant, the catalytic efficiency of Mpro varies greatly between cut-site sequences, suggesting a clear preference for the order of nsp processing.
摘要：
主要蛋白酶(Mpro)仍然是感染干预后COVID-19的重要治疗靶标，因为它在处理SARS-CoV-2基因组编码的大多数病毒蛋白中发挥了关键作用。病毒进入后，+ssRNA基因组被翻译成两个长的多蛋白（pp1a或移码依赖性pp1ab），包含病毒免疫调节所需的所有非结构蛋白（nsps），复制,最终,病毒体组装.这些nsp中包括半胱氨酸蛋白酶Mpro（nsp5），它从多蛋白中自我切除，二聚化，然后连续切割在多蛋白内每个nsp之间发现的15个切割位点连接中的11个。最近已经详细描述了Mpro的许多结构(通常与各种抑制剂或肽抑制剂结合),包括与每个多蛋白切割序列结合的Mpro结构，表明Mpro可以在其活性位点内适应广泛的靶标。然而,到目前为止,Mpro与其每个天然切割位点序列相互作用的动力学表征仍然不完整。在这里，我们提出了一种稳健且具有成本效益的基于FRET的系统，该系统受益于更一致的底物呈递，与以前报道的使用化学修饰肽的FRET系统相比，该底物在组织中也更接近天然多蛋白环境。使用这个系统，我们能够证明，虽然每个网站都保持着类似的迈克尔斯常数，Mpro的催化效率在切割位点序列之间变化很大，建议对nsp处理顺序有明确的偏好。
