Padmanabha Das Krishna M, Chen Jun, Charifson Paul S, Green Jeremy, Tang Henry, Panchal Sanjay, Pu Fan, Korepanova Alla, Dubey Abhinav, Afanador Gustavo, Stojkovic Vladimir, Nocek Boguslaw, Bigelow Lance, Stubbs Sarah H, Davey Robert A, DeGoey David A, Arthanari Haribabu, Namchuk Mark N
Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA 02115, USA.
Research and Development, AbbVie Inc, 1 N Waukegan Road, North Chicago, IL 60064, USA.
iScience. 2025 May 28;28(7):112773. doi: 10.1016/j.isci.2025.112773. eCollection 2025 Jul 18.
SARS-CoV-2 Mpro is a cysteine protease that acts as a symmetrical dimer and displays positive cooperativity for substrate turnover. A series of potent reversible covalent peptidomimetic aldehydes and nitriles was designed as Mpro inhibitors. To better understand the observed structure activity relationships (SAR), binding potency and mechanism was examined by enzyme activity assay, surface plasmon resonance, X-ray crystallography, matrix-assisted laser desorption electrospray ionization, and nuclear magnetic resonance (NMR). Potent aldehydes bind Mpro cooperativity but bind covalently to only one subunit of the dimer. The analogous nitriles do not bind cooperatively, and the degree of covalent binding observed varied depending on the assay method employed. The NMR studies support that potent inhibition of Mpro by the nitriles does not require covalent binding. The data highlight the caveats in using orthogonal assays to confirm compound mechanism, particularly in cooperative systems.
严重急性呼吸综合征冠状病毒2（SARS-CoV-2）的主蛋白酶（Mpro）是一种半胱氨酸蛋白酶，以对称二聚体形式发挥作用，对底物周转表现出正协同性。设计了一系列有效的可逆共价拟肽醛和腈作为Mpro抑制剂。为了更好地理解观察到的构效关系（SAR），通过酶活性测定、表面等离子体共振、X射线晶体学、基质辅助激光解吸电喷雾电离和核磁共振（NMR）来研究结合效力和作用机制。强效醛类与Mpro协同结合，但仅与二聚体的一个亚基共价结合。类似的腈类不协同结合，观察到的共价结合程度因所采用的测定方法而异。NMR研究表明，腈类对Mpro的强效抑制作用不需要共价结合。这些数据突出了使用正交测定法来确认化合物作用机制时的注意事项，特别是在协同系统中。
