Latest Publications
Profiling of coronaviral Mpro and enteroviral 3Cpro specificity provides a framework for the development of broad-spectrum antiviral compounds
Wioletta Rut, Katarzyna Groborz, Xinyuanyuan Sun, Rolf Hilgenfeld & Marcin Drag
August 2024, Protein Science
Abstract:
The main protease from coronaviruses and the 3C protease from enteroviruses play a crucial role in processing viral polyproteins, making them attractive targets for the development of antiviral agents. In this study, we employed a combinatorial chemistry approach—HyCoSuL—to compare the substrate specificity profiles of the main and 3C proteases from alphacoronaviruses, betacoronaviruses, and enteroviruses. The obtained data demonstrate that coronavirus Mpros exhibit overlapping substrate specificity in all binding pockets, whereas the 3Cpro from enterovirus displays slightly different preferences toward natural and unnatural amino acids at the P4-P2 positions. However, chemical tools such as substrates, inhibitors, and activity-based probes developed for SARS-CoV-2 Mpro can be successfully applied to investigate the activity of the Mpro from other coronaviruses as well as the 3Cpro from enteroviruses. Our study provides a structural framework for the development of broad-spectrum antiviral compounds.
Why is the Omicron main protease of SARS-CoV-2 less stable than its wild-type counterpart? A crystallographic, biophysical, and theoretical study of the free enzyme and its complex with inhibitor 13b-K
Mohamed Ibrahim, Xinyuanyuan Sun, Vinicius Martins de Oliveira, Ruibin Liu, Joseph Clayton, Haifa El Kilani, Jana Shen & Rolf Hilgenfeld
March 2024, bioRxiv
Abstract:
During the continuing evolution of SARS-CoV-2, the Omicron variant of concern emerged in the second half of 2021 and has been dominant since November that year. Along with its sublineages, it has maintained a prominent role ever since. The Nsp5 main protease (Mpro) of the Omicron virus is characterized by a single dominant mutation, P132H. Here we determined the X-ray crystal structures of the P132H mutant (or O-Mpro) as free enzyme and in complex with the Mpro inhibitor, the alpha-ketoamide 13b-K, and we conducted enzymology, biophysical as well as theoretical studies to characterize the O-Mpro. We found that O-Mpro has a similar overall structure and binding with 13b-K; however, it displays lower enzymatic activity and lower thermal stability compared to the WT-Mpro (with "WT" referring to the original Wuhan-1 strain). Intriguingly, the imidazole ring of His132 and the carboxylate plane of Glu240 are in a stacked configuration in the X-ray structures determined here. The empirical folding free energy calculations suggest that the O-Mpro dimer is destabilized relative to the WT-Mpro due to the less favorable van der Waals interactions and backbone conformation in the individual protomers. The all-atom continuous constant pH molecular dynamics (MD) simulations reveal that His132 and Glu240 display coupled titration. At pH 7, His132 is predominantly neutral and in a stacked configuration with respect to Glu240 which is charged. In order to examine whether the Omicron mutation eases the emergence of further Mpro mutations, we also determined crystal structures of the relatively frequent P132H+T169S double mutant but found little evidence for a correlation between the two sites.
Multiple redox switches of the SARS-CoV-2 main protease in vitro provide opportunities for drug design
Lisa-Marie Funk, Gereon Poschmann, Fabian Rabe von Pappenheim, Ashwin Chari, Kim M. Stegmann, Antje Dickmanns, Marie Wensien, Nora Eulig, Elham Paknia, Gabi Heyne, Elke Penka, Arwen R. Pearson, Carsten Berndt, Tobias Fritz, Sophia Bazzi, Jon Uranga, Ricardo A. Mata, Matthias Dobbelstein, Rolf Hilgenfeld, Ute Curth & Kai Tittmann
January 2024, Nature Communications
Abstract:
Besides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for preventing future COVID outbreaks. The SARS-CoV-2 main protease (Mpro), a cysteine protease with essential functions in viral replication, has been validated as an effective drug target. Here, we show that Mpro is subject to redox regulation in vitro and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include a disulfide-dithiol switch between the catalytic cysteine C145 and cysteine C117, and generation of an allosteric cysteine-lysine-cysteine SONOS bridge that is required for structural stability under oxidative stress conditions, such as those exerted by the innate immune system. We identify homo- and heterobifunctional reagents that mimic the redox switching and inhibit Mpro activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS-CoV and SARS-CoV, indicating their potential as common druggable sites.
doi:10.1038/s41467-023-44621-0
Ebselen derivatives inhibit SARS-CoV-2 replication by inhibition of its essential proteins: PLpro and Mpro proteases, and nsp14 guanine N7-methyltransferase
Mikolaj Zmudzinski, Wioletta Rut, Kamila Olech, Jarosław Granda, Mirosław Giurg, Małgorzata Burda-Grabowska, Rafał Kaleta, Michala Zgarbova, Renata Kasprzyk, Linlin Zhang, Xinyuanyuan Sun, Zongyang Lv, Digant Nayak, Malgorzata Kesik-Brodacka, Shaun K. Olsen, Jan Weber, Rolf Hilgenfeld, Jacek Jemielity & Marcin Drag
June 2023, Nature Scientific Reports
Abstract:
Proteases encoded by SARS-CoV-2 constitute a promising target for new therapies against COVID-19. SARS-CoV-2 main protease (Mpro, 3CLpro) and papain-like protease (PLpro) are responsible for viral polyprotein cleavage—a process crucial for viral survival and replication. Recently it was shown that 2-phenylbenzisoselenazol-3(2H)-one (ebselen), an organoselenium anti-inflammatory small-molecule drug, is a potent, covalent inhibitor of both the proteases and its potency was evaluated in enzymatic and antiviral assays. In this study, we screened a collection of 34 ebselen and ebselen diselenide derivatives for SARS-CoV-2 PLpro and Mpro inhibitors. Our studies revealed that ebselen derivatives are potent inhibitors of both the proteases. We identified three PLpro and four Mpro inhibitors superior to ebselen. Independently, ebselen was shown to inhibit the N7-methyltransferase activity of SARS-CoV-2 nsp14 protein involved in viral RNA cap modification. Hence, selected compounds were also evaluated as nsp14 inhibitors. In the second part of our work, we employed 11 ebselen analogues—bis(2-carbamoylaryl)phenyl diselenides—in biological assays to evaluate their anti-SARS-CoV-2 activity in Vero E6 cells. We present their antiviral and cytoprotective activity and also low cytotoxicity. Our work shows that ebselen, its derivatives, and diselenide analogues constitute a promising platform for development of new antivirals targeting the SARS-CoV-2 virus.
doi:10.1038/s41598-023-35907-w
Integrative Approach to Dissect the Drug Resistance Mechanism of the H172Y Mutation of SARS-CoV-2 Main Protease
Joseph Clayton, Vinícius Martins de Oliveira, Mohamed Fouad Ibrahim, Xinyuanyuan Sun, Paween Mahinthichaichan, Mingzhe Shen, Rolf Hilgenfeld, & Jana Shen
May 2023, Journal of Chemical Information and Modeling
Abstract:
Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of Paxlovid, a drug approved by the U.S. Food and Drug Administration for high-risk COVID-19 patients. Recently, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvir’s inhibitory activity. As the COVID-19 cases skyrocket in China and the selective pressure of antiviral therapy builds in the US, there is an urgent need to characterize and understand how the H172Y mutation confers drug resistance. Here, we investigated the H172Y Mpro’s conformational dynamics, folding stability, catalytic efficiency, and inhibitory activity using all-atom constant pH and fixed-charge molecular dynamics simulations, alchemical and empirical free energy calculations, artificial neural networks, and biochemical experiments. Our data suggest that the mutation significantly weakens the S1 pocket interactions with the N-terminus and perturbs the conformation of the oxyanion loop, leading to a decrease in the thermal stability and catalytic efficiency. Importantly, the perturbed S1 pocket dynamics weaken the nirmatrelvir binding in the P1 position, which explains the decreased inhibitory activity of nirmatrelvir. Our work demonstrates the predictive power of the combined simulation and artificial intelligence approaches, and together with biochemical experiments, they can be used to actively surveil continually emerging mutations of SARS-CoV-2 Mpro and assist the optimization of antiviral drugs. The presented approach, in general, can be applied to characterize mutation effects on any protein drug targets.
Therapeutic strategies for COVID-19: progress and lessons learned
Guangdi Li, Rolf Hilgenfeld, Richard Whitley & Erik De Clercq
April 2023, Nature Reviews Drug Discovery
Abstract:
The coronavirus disease 2019 (COVID-19) pandemic has stimulated tremendous efforts to develop therapeutic strategies that target severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and/or human proteins to control viral infection, encompassing hundreds of potential drugs and thousands of patients in clinical trials. So far, a few small-molecule antiviral drugs (nirmatrelvir–ritonavir, remdesivir and molnupiravir) and 11 monoclonal antibodies have been marketed for the treatment of COVID-19, mostly requiring administration within 10 days of symptom onset. In addition, hospitalized patients with severe or critical COVID-19 may benefit from treatment with previously approved immunomodulatory drugs, including glucocorticoids such as dexamethasone, cytokine antagonists such as tocilizumab and Janus kinase inhibitors such as baricitinib. Here, we summarize progress with COVID-19 drug discovery, based on accumulated findings since the pandemic began and a comprehensive list of clinical and preclinical inhibitors with anti-coronavirus activities. We also discuss the lessons learned from COVID-19 and other infectious diseases with regard to drug repurposing strategies, pan-coronavirus drug targets, in vitro assays and animal models, and platform trial design for the development of therapeutics to tackle COVID-19, long COVID and pathogenic coronaviruses in future outbreaks.
doi:10.1038/s41573-023-00672-y
Diastereomeric Resolution Yields Highly Potent Inhibitor of SARS-CoV-2 Main Protease
Mark S. Cooper, Linlin Zhang, Mohamed Ibrahim, Kaixuan Zhang, Xinyuanyuan Sun, Judith Röske, Matthias Göhl, Mark Brönstrup, Justin K. Cowell, Lucie Sauerhering, Stephan Becker, Laura Vangeel, Dirk Jochmans, Johan Neyts, Katharina Rox, Graham P. Marsh, Hannah J. Maple & Rolf Hilgenfeld
October 2022, Journal of Medicinal Chemistry
Abstract:
SARS-CoV-2 is the causative agent behind the COVID-19 pandemic. The main protease (Mpro, 3CLpro) of SARS-CoV-2 is a key enzyme that processes polyproteins translated from the viral RNA. Mpro is therefore an attractive target for the design of inhibitors that block viral replication. We report the diastereomeric resolution of the previously designed SARS-CoV-2 Mpro α-ketoamide inhibitor 13b. The pure (S,S,S)-diastereomer, 13b-K, displays an IC50 of 120 nM against the Mpro and EC50 values of 0.8–3.4 μM for antiviral activity in different cell types. Crystal structures have been elucidated for the Mpro complexes with each of the major diastereomers, the active (S,S,S)-13b (13b-K), and the nearly inactive (R,S,S)-13b (13b-H); results for the latter reveal a novel binding mode. Pharmacokinetic studies show good levels of 13b-K after inhalative as well as after peroral administration. The active inhibitor (13b-K) is a promising candidate for further development as an antiviral treatment for COVID-19.
doi:10.1021/acs.jmedchem.2c01131
Evaluation of the anti-SARS-CoV-2 properties of essential oils and aromatic extracts
Daniel Jan Strub, Michał Talma, Maria Strub, Wioletta Rut, Mikolaj Zmudzinski, Władysław Brud, Johan Neyts, Laura Vangeel, Linlin Zhang, Xinyuanyuan Sun, Zongyang Lv, Digant Nayak, Shaun K. Olsen, Rolf Hilgenfeld, Dirk Jochmans & Marcin Drąg
August 2022, Nature Scientific Reports
Abstract:
Essential oils and aromatic extracts (oleoresins, absolutes, concretes, resinoids) are often used as food flavorings and constituents of fragrance compositions. The flavor and fragrance industry observed significant growth in the sales of some natural materials during the COVID-19 outbreak. Some companies worldwide are making false claims regarding the effectiveness of their essential oils or blends (or indirectly point toward this conclusion) against coronaviruses, even though the available data on the activity of plant materials against highly pathogenic human coronaviruses are very scarce. Our exploratory study aimed to develop pioneering knowledge and provide the first experimental results on the inhibitory properties of hundreds of flavor and fragrance materials against SARS-CoV-2 main and papain-like proteases and the antiviral potential of the most active protease inhibitors. As essential oils are volatile products, they could provide an interesting therapeutic strategy for subsidiary inhalation in the long term.
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