Biology subjects

Hilgenfeld, R.

Publications and source records attributed to Hilgenfeld, R..

9 recordsLinked to original sources

bioRxiv2025

Orthorhombic coronavirus main protease crystals provide a higher success rate in fragment screening

In more and more drug discovery projects, crystallographic fragment screening (CFS) is employed as an early screening method. Here, we demonstrate that choosing the right crystal form has a profound influence on the hit rates and hence success and speed of downstream lead generation. Two CFS campaigns with the same fragment library and an almost identical experimental setup were carried out against the two crystal forms of the SARS-CoV-2 main protease.While both crystal forms exhibit similar diffraction properties, the observed hit rates in the two campaigns were vastly different. For the monoclinic crystals a hit rate of 3% was determined, while a hit rate of 16% was observed for the orthorhombic crystals. These findings align with the more open molecular packing in the orthorhombic crystals where the solvent channels leading to the active sites are about twice larger than in the monoclinic crystal form. Our results highlight the critical importance of the crystal system in a crystallographic fragment-screening campaign and identify this parameter as one of the most important ones to be optimized during preparation of a campaign.

biochemistry↗

bioRxiv2024

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

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.

biochemistry↗

bioRxiv2022

H172Y mutation perturbs the S1 pocket and nirmatrelvir binding of SARS CoV-2 main protease through a nonnative hydrogen bond

Nirmatrelvir is an orally available inhibitor of SARS-CoV-2 main protease (Mpro) and the main ingredient of PAXLOVID, a drug approved by FDA for high-risk COVID-19 patients. Recently, a rare natural mutation, H172Y, was found to significantly reduce nirmatrelvirs inhibitory activity. As the COVID-19 cases skyrocket in China and the selective pressure of antiviral therapy builds up in the US, there is an urgent need to characterize and understand how the H172Y mutation confers drug resistance. Here we investigated the H172Y Mpros 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 suggests 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 weakens the nirma-trelvir 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 intel-ligence approaches, and together with biochemical experiments they can be used to actively surveil continually emerging mutations of SARS-CoV-2 Mpro and assist the discovery of new antiviral drugs. The presented workflow can be applicable to characterize mutation effects on any protein drug targets.

biochemistry↗

bioRxiv2022

Redox regulation of the SARS-CoV-2 main protease provides new opportunities for drug design

AO_SCPLOWBSTRACTC_SCPLOWBesides vaccines, the development of antiviral drugs targeting SARS-CoV-2 is critical for stopping the current COVID-19 pandemic and preventing future 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 and reversibly switches between the enzymatically active dimer and the functionally dormant monomer through redox modifications of cysteine residues. These include sulfenylation, disulfide formation between the catalytic cysteine and a proximal cysteine, and generation of an allosteric 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 possess antiviral activity. The discovered redox switches are conserved in main proteases from other coronaviruses, e.g. MERS and SARS-CoV, indicating their potential as common druggable sites.

biochemistry↗

bioRxiv2020

Ebselen derivatives are very potent dual inhibitors of SARS-CoV-2 proteases - PLpro and Mpro in in vitro studies

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 anti-viral assays. In this study, we screened a collection of 23 ebselen 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. Our work shows that ebselen constitutes a promising platform for development of new antiviral agents targeting both SARS-CoV-2 PLpro and Mpro.

biochemistry↗

bioRxiv2020

Substrate specificity profiling of SARS-CoV-2 Mpro protease provides basis for anti-COVID-19 drug design

In December 2019, the first cases of infection with a novel coronavirus, SARS-CoV-2, were diagnosed in Wuhan, China. Due to international travel and human-to-human transmission, the virus spread rapidly inside and outside of China. Currently, there is no effective antiviral treatment for coronavirus disease 2019 (COVID-19); therefore, research efforts are focused on the rapid development of vaccines and antiviral drugs. The SARS-CoV-2 main protease constitutes one of the most attractive antiviral drug targets. To address this emerging problem, we have synthesized a combinatorial library of fluorogenic substrates with glutamine in the P1 position. We used it to determine the substrate preferences of the SARS-CoV and SARS-CoV-2 main proteases, using natural and a large panel of unnatural amino acids. On the basis of these findings, we designed and synthesized an inhibitor and two activity-based probes, for one of which we determined the crystal structure of its complex with the SARS-CoV-2 Mpro. Using this approach we visualized SARS-CoV-2 active Mpro within nasopharyngeal epithelial cells of a patient with active COVID-19 infection. The results of our work provide a structural framework for the design of inhibitors as antiviral agents or diagnostic tests.

biochemistry↗

bioRxiv2020

X-ray Structure of Main Protease of the Novel Coronavirus SARS-CoV-2 Enables Design of α-Ketoamide Inhibitors

A novel coronavirus has been identified as the causative agent of a massive outbreak of atypical pneumonia originating at Wuhan, Hubei province, China. Involved in the formation of the coronavirus replication complex, the viral main protease (Mpro, also called 3CLpro) represents an attractive target for therapy. We determined the crystal structure of the unliganded Mpro at 1.75 [A] resolution and used this structure to guide optimization of a series of alpha-ketoamide inhibitors. The main goal of the optimization efforts was improvement of the pharmacokinetic properties of the compounds. We further describe 1.95- and 2.20-[A] crystal structures of the complex between the enzyme and the most potent alpha-ketoamide optimized this way. These structures will form the basis for further development of these compounds to antiviral drugs.

biochemistry↗

bioRxiv2020

Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication

The main protease of coronaviruses and the 3C protease of enteroviruses share a similar active-site architecture and a unique requirement for glutamine in the P1 position of the substrate. Because of their unique specificity and essential role in viral polyprotein processing, these proteases are suitable targets for the development of antiviral drugs. In order to obtain near-equipotent, broad-spectrum antivirals against alphacoronaviruses, betacoronaviruses, and enteroviruses, we pursued structure-based design of peptidomimetic -ketoamides as inhibitors of main and 3C proteases. Six crystal structures of protease:inhibitor complexes were determined as part of this study. Compounds synthesized were tested against the recombinant proteases as well as in viral replicons and virus-infected cell cultures; most of them were not cell-toxic. Optimization of the P2 substituent of the -ketoamides proved crucial for achieving near-equipotency against the three virus genera. The best near-equipotent inhibitors, 11u (P2 = cyclopentylmethyl) and 11r (P2 = cyclohexylmethyl), display low-micromolar EC50 values against enteroviruses, alphacoronaviruses, and betacoronaviruses in cell cultures. In Huh7 cells, 11r exhibits three-digit picomolar activity against Middle East Respiratory Syndrome coronavirus.

biochemistry↗

bioRxiv2019

Processing of the SARS-CoV pp1a/ab nsp7-10 region

1.1.Severe acute respiratory syndrome coronavirus (SARS-CoV) is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex (RTC), essential steps include processing of the conserved polyprotein nsp7-10 region by the main protease Mpro and subsequent complex formation of the released nsps. Here, we analyzed processing of the coronavirus nsp7-10 region using native mass spectrometry showing consumption of substrate, rise and fall of intermediate products and complexation. Importantly, there is a clear order of cleavage efficiencies, which is influenced by the polyprotein tertiary structure. Furthermore, the predominant product is an nsp7+8(2:2) hetero-tetramer with nsp8 scaffold. In conclusion, native MS, opposed to other methods, can expose the processing dynamics of viral polyproteins and the landscape of protein interactions in one set of experiments. Thereby, new insights into protein interactions, essential for generation of viral progeny, were provided, with relevance for development of antivirals.

biophysics↗