Science Division, New York University Abu Dhabi, Abu Dhabi, United Arab Emirates.
Graduate School of Engineering and Natural Sciences, Istanbul Medipol University, Istanbul, Turkey.
FASEB J. 2021 Aug;35(8):e21774. doi: 10.1096/fj.202100994.
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for coronavirus disease 2019 (COVID-19), one of the most challenging global pandemics of the modern era. Potential treatment strategies against COVID-19 are yet to be devised. It is crucial that antivirals that interfere with the SARS-CoV-2 life cycle be identified and developed. 3-Chymotrypsin-like protease (3CLpro) is an attractive antiviral drug target against SARS-CoV-2, and coronaviruses in general, because of its role in the processing of viral polyproteins. Inhibitors of 3CLpro activity are screened in enzyme assays before further development of the most promising leads. Dimethyl sulfoxide (DMSO) is a common additive used in such assays and enhances the solubility of assay components. However, it may also potentially affect the stability and efficiency of 3CLpro but, to date, this effect had not been analyzed in detail. Here, we investigated the effect of DMSO on 3CLpro-catalyzed reaction. While DMSO (5%-20%) decreased the optimum temperature of catalysis and thermodynamic stability of 3CLpro, it only marginally affected the kinetic stability of the enzyme. Increasing the DMSO concentration up to 20% improved the catalytic efficiency and peptide-binding affinity of 3CLpro. At such high DMSO concentration, the solubility and stability of peptide substrate were improved because of reduced aggregation. In conclusion, we recommend 20% DMSO as the minimum concentration to be used in screens of 3CLpro inhibitors as lead compounds for the development of antiviral drugs against COVID-19.
严重急性呼吸系统综合症冠状病毒 2 型（SARS-CoV-2）是造成 2019 年冠状病毒病（COVID-19）的元凶，这是现代史上最具挑战性的全球大流行之一。目前仍在探索针对 COVID-19 的潜在治疗策略。鉴定和开发能够干扰 SARS-CoV-2 生命周期的抗病毒药物至关重要。3-糜蛋白酶样蛋白酶（3CLpro）是针对 SARS-CoV-2 及一般冠状病毒的有吸引力的抗病毒药物靶标，这是因为它在病毒多蛋白的加工过程中发挥作用。在进一步开发最有前途的先导化合物之前，会在酶分析中筛选 3CLpro 活性抑制剂。二甲基亚砜（DMSO）是此类分析中常用的常见添加剂，可提高分析成分的溶解度。然而，它也可能潜在地影响 3CLpro 的稳定性和效率，但迄今为止，尚未对此进行详细分析。在这里，我们研究了 DMSO 对 3CLpro 催化反应的影响。尽管 DMSO（5%-20%）降低了 3CLpro 催化的最适温度和热力学稳定性，但它仅略微影响了酶的动力学稳定性。将 DMSO 浓度提高到 20% 可提高 3CLpro 的催化效率和肽结合亲和力。在如此高的 DMSO 浓度下，由于减少了聚集，肽底物的溶解度和稳定性得到了提高。总之，我们建议将 20% DMSO 作为筛选 3CLpro 抑制剂的最低浓度，作为开发针对 COVID-19 的抗病毒药物的先导化合物。
