Novel small-molecule inhibitors of SARS-CoV-2 main protease with nanomolar antiviral potency
Authors: Haoran Zhang, Kangping Zhou, Fei Peng, Zhao Gao,, Guowei Song, Bing Hu, Sophia Chun, Junfeng Xiao, Mengfei Qian, Jin Wu, Kai Pan, Fan Gao, Meng Guo, Cheng Peng, Gang Zou, Jim Zhen Wu, Kun Cai, Yan Li
Journal: Journal of Infection (2024)

Dear Editor, Recently, Saeed et al. reported the in silico screening of inhibitor molecules targeting the SARS-CoV-2 main protease 3CLpro (also known as Nsp5 or Mpro) in this Journal1Saeed Aamir Ahmad Basharat Majaz Sidra Nouroz Faisal Ahmad Ashfaq Xie Yingqiu Targeting omicron and other reported SARS-CoV-2 lineages by potent inhibitors of main protease 3CL Mpro: Molecular simulation analysis.Journal of Infection. 2022; 84: e133-e136https://doi.org/10.1016/j.jinf.2022.02.012Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar. Despite this progress, the continuous emergence of new viral variants highlights the need for further antiviral drug development against SARS-CoV-2. After three years of circulation in the human population, the COVID-19 pandemic has evidently obtained heightened adaptability to humans. Although less virulent than the original strain, SARS-CoV-2 variants like Omicron sublineage EG.5 acquired greater transmissibility possibly via adaptive evolution2Scarpa Fabio Pascarella Stefano Ciccozzi Alessandra Giovanetti Marta Azzena Ilenia Locci Chiara et al.Genetic and structural analyses reveal the low potential of the SARS-CoV-2 EG.5 variant.Journal of Medical Virology. 2023; 95e29075https://doi.org/10.1002/jmv.29075Crossref Scopus (0) Google Scholar. In light of the potential risk of enhanced immune evasion by variants such as EG.53Zhang Lu Kempf Amy Nehlmeier Inga Cossmann Anne Dopfer-Jablonka Alexandra Stankov Metodi V. et al.Neutralisation sensitivity of SARS-CoV-2 lineages EG.5.1 and XBB.2.3.The Lancet Infectious Diseases. 2023; 23: e391-e392https://doi.org/10.1016/S1473-3099(23)00547-9Abstract Full Text Full Text PDF Scopus (3) Google Scholar and possible antigenic/virulent changes in the future, SARS-CoV-2 antiviral drug development remains an urgent need as a critical component of COVID-19 therapeutics. As a critical cysteine hydrolase, SARS-CoV-2 3CLpro cleaves viral polyproteins into functional non-structural proteins essential for viral replication. Additionally, 3CLpro interrupts the modulator of nuclear factor-κB in host brain endothelial cells, eliciting deleterious microvascular brain pathology4Wenzel Jan Lampe Josephine Müller-Fielitz Helge Schuster Raphael Zille Marietta Müller Kristin et al.The SARS-CoV-2 main protease Mpro causes microvascular brain pathology by cleaving NEMO in brain endothelial cells.Nat Neurosci. 2021; 24: 1522-1533https://doi.org/10.1038/s41593-021-00926-1Crossref PubMed Scopus (126) Google Scholar. Notably, the 3CLpro substrate-binding domain exhibits an ultralow mutation rate of 0.0015Gao Kaifu Wang Rui Chen Jiahui Tepe Jetze J. Huang Faqing Wei Guo-Wei Perspectives on SARS-CoV-2 Main Protease Inhibitors.J Med Chem. 2021; 64: 16922-16955https://doi.org/10.1021/acs.jmedchem.1c00409Crossref Scopus (56) Google Scholar, prospectively ensuring durable 3CLpro-targeting antiviral potency considering viral evolution. Collectively, targeting the conserved 3CLpro is a promising strategy for SARS-CoV-2 therapeutic development. Nirmatrelvir (also known as PF-07321332)6Owen Dafydd R. Allerton Charlotte M.N. Anderson Annaliesa S. Aschenbrenner Lisa Avery Melissa Berritt Simon et al.An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.Science. 2021; 374: 1586-1593https://doi.org/10.1126/science.abl4784Crossref PubMed Scopus (879) Google Scholar, the antiviral component of PaxlovidTM, is a highly potent covalent peptidomimetic inhibitor. By covalently binding to the catalytic Cys145 residue and enabling additional non-covalent interactions like hydrogen bonds and hydrophobic contacts, nirmatrelvir effectively blocks viral replication by suppressing 3CLpro proteolysis7Zhao Yao Fang Chao Zhang Qi Zhang Ruxue Zhao Xiangbo Duan Yinkai et al.Crystal structure of SARS-CoV-2 main protease in complex with protease inhibitor PF-07321332.Protein & Cell. 2022; 13: 689-693https://doi.org/10.1007/s13238-021-00883-2Crossref PubMed Scopus (99) Google Scholar. Combined with ritonavir that does not act as an antiviral but a Cyp P450 inhibitor to boost nirmatrelvir's exposure in host, nirmatrelvir treatment ameliorates patient symptoms and reduces viral load. However, concerning viral rebound with recurrent symptomatic infection is frequently reported following therapy discontinuation8Wang Yu Chen Xubo Xiao Wenying Zhao Danyang Feng Liuliu Rapid COVID-19 rebound in a severe COVID-19 patient during 20-day course of Paxlovid.Journal of Infection. 2022; 85: e134-e136https://doi.org/10.1016/j.jinf.2022.08.012Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar. Hence, optimizing this compound is imperative to improve efficacy. Herein, we report several novel binder molecules that effectively inhibit both SARS-CoV-2 3CLpro enzymatic activity and viral capability to infect cells with nanomolar inhibition potency. We identified six candidate compounds as potential 3CLpro inhibitors, namely AKEX0730, AKEX0736, AKEX0737, AKEX0763, AKEX0766, and AKEX0806 (Fig. 1A). Initially, we evaluated their capability to inhibit 3CLpro enzymatic activity. For comparison, the 3CLpro inhibitor nirmatrelvir6Owen Dafydd R. Allerton Charlotte M.N. Anderson Annaliesa S. Aschenbrenner Lisa Avery Melissa Berritt Simon et al.An oral SARS-CoV-2 Mpro inhibitor clinical candidate for the treatment of COVID-19.Science. 2021; 374: 1586-1593https://doi.org/10.1126/science.abl4784Crossref PubMed Scopus (879) Google Scholar served as a positive control. Relative to the potency of nirmatrelvir, which showed a half-maximal inhibitory concentration (IC50) of 26.10 nM, all six candidates exhibited low-nanomolar IC50 analogously or superior to nirmatrelvir (Fig. 1B). Subsequently, we characterized the cytopathic effects following HCoV-OC43 and SARS-CoV-2 infection of Vero E6 cells, and the viral inhibition by our 3CLpro inhibitor candidates and nirmatrelvir. As anticipated, nirmatrelvir showed antiviral activity (IC50 = 93.05 nM against HCoV-OC43 and 543.5 nM against SARS-CoV-2) and cytotoxicity, validating assay reliability. Outstandingly, AKEX0763 proved the most potent inhibitor against both viral infections (IC50 = 326.1 nM against SARS-CoV-2 and 5.352 nM against HCoV-OC43), surpassing nirmatrelvir in potency (Fig. 1C-D). Hence, AKEX0763 warrants further investigation as a promising 3CLpro-targeting antiviral candidate. In order to better understand the interaction between the inhibitor candidates and 3CLpro, we first set out to characterize the binding properties of the compounds to the SARS-CoV-2 3CLpro N-terminal truncation (3CLpro-NTD). This truncated protein contains the catalytic domains I and II, where the enzyme's active site is located. Through microscale thermophoresis (MST) experiments, we elucidated the interaction between AKEX0763 and SARS-CoV-2 3CLpro-NTD as a tight binding event (Fig. 1E). To probe the specifics of this interaction, we next performed nuclear magnetic resonance (NMR) assays, which can provide binding properties at the residue level. Notably, a recent study utilized direct transfer of amide 1H-15N chemical shift assignments from the SARS-CoV orthologous protease to SARS-CoV-2 3CLpro-NTD9Gao Jia Zhang Liang Liu Xiaodan Li Fudong Ma Rongsheng Zhu Zhongliang et al.Repurposing Low-Molecular-Weight Drugs against the Main Protease of Severe Acute Respiratory Syndrome Coronavirus 2.J Phys Chem Lett. 2020; 11: 7267-7272https://doi.org/10.1021/acs.jpclett.0c01894Crossref Google Scholar. This was enabled by the extremely high sequence identity of 96% between these two proteases. To experimentally validate these transferred assignments for SARS-CoV-2, we acquired 3D heteronuclear NMR spectra, including HNCA and 15N-edited NOESY-HSQC, on our SARS-CoV-2 3CLpro-NTD sample. This allowed us to achieve partial assignments of the 1H and 15N resonances of the SARS-CoV-2 3CLpro-NTD protein (Fig. 2A). As an important control, we compared the 1H-15N HSQC spectra of our 3CLpro-NTD construct with data reported for the full-length 3CLpro protein10Cantrelle François-Xavier Boll Emmanuelle Brier Lucile Moschidi Danai Belouzard Sandrine Landry Valérie et al.NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment.Angewandte Chemie International Edition. 2021; 60: 25428-25435https://doi.org/10.1002/anie.202109965Crossref Scopus (21) Google Scholar, which contains the domain III driving dimerization. The overall agreement between the 1H-15N cross-peak signals in these datasets verified the reliability of our assignments. With high confidence in the NMR spectra of 3CLpro-NTD, we then conducted titration experiments with 13C,15N-labeled 3CLpro-NTD protein and unlabeled AKEX0763 to both validate and build upon the MST results. Here, we observed an intensity reduction of free-state amide peaks and the emergence of bound-state peaks (Fig. 2A), verifying a tight binding in the nanomolar regime via slow chemical exchange kinetics. Further HNCA analysis of the bound state revealed three emerging peaks exclusive to the bound state matching a characteristic chemical shift pattern of triple sequential peptide fragment as Asp/Asn (49.2 ppm), Gly (42.3 ppm), and Ser/Thr (59.0 ppm). By cross-referencing tripeptide sequence segments in 3CLpro-NTD and validating the loss of peak intensity for the corresponding free-state peaks, this triplet was assigned to Asn142-Gly143-Ser144 (Fig. 2B). The sequentially adjacent Cys145, predicted to covalently react with AKEX0763 similarly to nirmatrelvir, could not be assigned in either free or bound state after iterating over unassigned peaks. This may indicate intricate dynamics for this critical active site residue which typically causes line broadening. Collectively, these NMR data characterize a specific binding event involving AKEX0763 to the solvent-exposed Asn142-Gly143-Ser144-Cys145 segment of the SARS-CoV-2 3CLpro N-terminal domain (Fig. 2B-2C). This residue-level mapping of the drug-target interface paves the way for rationale optimization of next-generation therapeutics. For instance, derivatization of the AKEX0763 scaffold to reinforce covalent contact with Cys145 and non-covalent ones with neighboring residues represents a viable tactic for enhancing inhibitory potency. Indeed, the continuous emergence of pathogenic variations across distinct geographical localities spotlights the imperative for identifying viral elements amenable to selective pharmacological interference that are relatively conserved. The 3CLpro protein presents an irresistible drug target in this regard owing to its functional indispensability in directing viral maturation and dissemination. However, the biochemical intricacies underlying drug-target interactions necessitate comprehensive elucidation across isolates for maximizing inhibitory efficacy. For example, while AKEX0763 shows remarkable pan-coronaviral inhibition via converged engagements with an identical binding hotspot, subtype-specific customizations may offer enhanced potency against individual viral strains. Considering the continuous threat posed by SARS-CoV-2 variants to public health, this structure-guided drug development effort leveraging biochemical, biophysical, and structural biology techniques has delivered valuable mechanistic insights into inhibitory modalities against this formidable pathogen. As exemplified by the quantitative assays herein, lead compounds such as AKEX0763 exhibit remarkable potency in blocking the enzymatic activity of the viral-encoded 3CLpro, which is functionally indispensable for effectuating SARS-CoV-2 maturation. Additionally, the tight, specific binding with the protease elucidated structurally paves the way for further rationale optimization. Although the latest dominant variants have undergone antigenic drifts progressively eroding vaccine efficacy, the extraordinarily high conservation of druggable hotspots on viral proteins ensures durable potency for structure-informed drug development. By continuing to illuminate the intricate molecular interplay through state-of-the-art analytical approaches, the repertoire of antiviral agents will be enhanced dramatically, accelerating the advent of efficacious, resistance-resilient therapeutic regimens against coronaviruses, including SARS-CoV-2. Yan Li, Kun Cai, Gang Zou, Cheng Peng and Jim Zhen Wu conceived the project and supervised the research. Fei Peng and Haoran Zhang expressed and purified the proteins. Guowei Song, Mengfei Qian, Jin Wu, and Fan Gao synthesized the inhibitory compounds. Zhao Gao performed the enzyme activity assays. Zhao Gao, Sophia Chun, Gang Zou, and analyzed the data. Kangping Zhou, Bing Hu, Kai Pan, Meng Guo, and Kun Cai performed the live virus neutralization experiments and analyzed the data. Haoran Zhang performed NMR experiments and analyzed the data. Haoran Zhang, Fei Peng, and Yan Li drafted and revised the manuscript. All authors contributed to data analysis and approved the manuscript. This work was funded by the National Key Research and Development Program of China (2022YFC2305500 and 2021YFC2600200 to Y.L.), the Hubei Provincial Key Research and Development Program (2021ACB004 to K.C.), the General Program of National Natural Science Foundation of China (82372223 to K.C.), the Hubei Provincial Public Health Outstanding Young Talents Project (to Y.L., and K.C.), the Hubei Province Natural Science Foundation for Distinguished Young Scholars (2022CFA068 to Y.L.).