Massive X-ray screening reveals two allosteric drug binding sites of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of repurposing drug libraries containing 5953 individual compounds against the SARS-CoV-2 main protease (Mpro), which is a potent drug target as it is essential for the virus replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. Interestingly, two compounds bind outside the active site to the native dimer interface in close proximity to the S1 binding pocket. Another compound binds in a cleft between the catalytic and dimerization domain of Mpro. Neither binding site is related to the enzymatic active site and both represent attractive targets for drug development against SARS-CoV-2. This X-ray screening approach thus has the potential to help deliver an approved drug on an accelerated time-scale for this and future pandemics

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (M^(pro)), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to M^(pro). In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

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

Severe acute respiratory syndrome coronavirus is the causative agent of a respiratory disease with a high case fatality rate. During the formation of the coronaviral replication/transcription complex, essential steps include processing of the conserved polyprotein nsp7–10 region by the main protease M$^{pro}$ and subsequent complex formation of the released nsp's. 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

Hallmarks of Alpha- And Betacoronavirus non-structural protein 7+8 complexes

Coronaviruses infect many different species including humans. The last two decades have seen three zoonotic coronaviruses, with SARS-CoV-2 (severe acute respiratory syndrome coronavirus 2) causing a pandemic in 2020. Coronaviral non-structural proteins (nsps) form the replication-transcription complex (RTC). Nsp7 and nsp8 interact with and regulate the RNA-dependent RNA-polymerase and other enzymes in the RTC. However, the structural plasticity of nsp7+8 complexes has been under debate. Here, we present the framework of nsp7+8 complex stoichiometry and topology based on native mass spectrometry and complementary biophysical techniques of nsp7+8 complexes from seven coronaviruses in the genera Alpha- and Betacoronavirus including SARS-CoV-2. Their complexes cluster into three groups, which systematically form either heterotrimers or heterotetramers or both, exhibiting distinct topologies. Moreover, even at high protein concentrations, SARS-CoV-2 nsp7+8 consists primarily of heterotetramers. From these results, the different assembly paths can be pinpointed to specific residues and an assembly model proposed.ISSN:2375-254

Structure and target interaction of a G-quadruplex RNA-aptamer

G-quadruplexes have recently moved into focus of research in nucleic acids, thereby evolving in scientific significance from exceptional secondary structure motifs to complex modulators of gene regulation. Aptamers (nucleic acid based ligands with recognition properties for a specific target) that form Gquadruplexes may have particular potential for therapeutic applications as they combine the characteristics of specific targeting and Gquadruplex mediated stability and regulation. We have investigated the structure and target interaction properties of one such aptamer: AIR-3 and its truncated form AIR-3A. These RNA aptamers are specific for human interleukin-6 receptor (hIL-6R), a key player in inflammatory diseases and cancer, and have recently been exploited for in vitro drug delivery studies. With the aim to resolve the RNA structure, global shape, RNA:protein interaction site and binding stoichiometry, we now investigated AIR-3 and AIR-3A by different methods including RNA structure probing, Small Angle X-ray scattering and microscale thermophoresis. Our findings suggest a broader spectrum of folding species than assumed so far and remarkable tolerance toward different modifications. Mass spectrometry based binding site analysis, supported by molecular modeling and docking studies propose a general Gquadruplex affinity for the target molecule hIL-6R

Catalytic cleavage of HEAT and subsequent covalent binding of the tetralone moiety by the SARS-CoV-2 main protease

Here we present the crystal structure of SARS-CoV-2 main protease (Mpro) covalently bound to 2-methyl-1-tetralone. This complex was obtained by co-crystallization of Mpro with HEAT (2-(((4-hydroxyphenethyl)amino)methyl)-3,4-dihydronaphthalen-1(2H)-one) in the framework of a large X-ray crystallographic screening project of Mpro against a drug repurposing library, consisting of 5632 approved drugs or compounds in clinical phase trials. Further investigations showed that HEAT is cleaved by Mpro in an E1cB-like reaction mechanism into 2-methylene-1-tetralone and tyramine. The catalytic Cys145 subsequently binds covalently in a Michael addition to the methylene carbon atom of 2-methylene-1-tetralone. According to this postulated model HEAT is acting in a pro-drug-like fashion. It is metabolized by Mpro, followed by covalent binding of one metabolite to the active site. The structure of the covalent adduct elucidated in this study opens up a new path for developing non-peptidic inhibitors

Inhibition of SARS-CoV-2 main protease by allosteric drug-binding

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous health problems and economical challenges for mankind. To date, no effective drug is available to directly treat the disease and prevent virus spreading. In a search for a drug against COVID-19, we have performed a massive X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for the virus replication and, thus, a potent drug target. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds binding to M$^{pro}$. In subsequent cell-based viral reduction assays, one peptidomimetic and five non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2

X-ray screening identifies active site and allosteric inhibitors of SARS-CoV-2 main protease

The coronavirus disease (COVID-19) caused by SARS-CoV-2 is creating tremendous human suffering. To date, no effective drug is available to directly treat the disease. In a search for a drug against COVID-19, we have performed a high-throughput X-ray crystallographic screen of two repurposing drug libraries against the SARS-CoV-2 main protease (Mpro), which is essential for viral replication. In contrast to commonly applied X-ray fragment screening experiments with molecules of low complexity, our screen tested already approved drugs and drugs in clinical trials. From the three-dimensional protein structures, we identified 37 compounds that bind to Mpro. In subsequent cell-based viral reduction assays, one peptidomimetic and six non-peptidic compounds showed antiviral activity at non-toxic concentrations. We identified two allosteric binding sites representing attractive targets for drug development against SARS-CoV-2