Kinetic Characterization of Cerium and Gallium Ions as Inhibitors of Cysteine Cathepsins L, K, and S.

Heavy metal ions can disrupt biological functions via multiple molecular mechanisms, including inhibition of enzymes. We investigate the interactions of human papain-like cysteine endopeptidases cathepsins L, K, and S with gallium and cerium ions, which are associated with medical applications. We compare these results with zinc and lead, which are known to inhibit thiol enzymes. We show that Ga3+, Ce3+, and Ce4+ ions inhibit all tested peptidases with inhibition constants in the low micromolar range (between 0.5 µM and 10 µM) which is comparable to Zn2+ ions, whereas inhibition constants of Pb2+ ions are one order of magnitude higher (30 µM to 150 µM). All tested ions are linear specific inhibitors of cathepsin L, but cathepsins K and S are inhibited by Ga3+, Ce3+, and Ce4+ ions via hyperbolic inhibition mechanisms. This indicates a mode of interaction different from that of Zn2+ and Pb2+ ions, which act as linear specific inhibitors of all peptidases. All ions also inhibit the degradation of insoluble elastin, which is a common target of these peptidases in various inflammatory diseases. Our results suggest that these ions and their compounds have the potential to be used as cysteine cathepsin inhibitors in vitro and possibly in vivo.

Ultralarge Virtual Screening Identifies SARS-CoV-2 Main Protease Inhibitors with Broad-Spectrum Activity against Coronaviruses.

Drugs targeting SARS-CoV-2 could have saved millions of lives during the COVID-19 pandemic, and it is now crucial to develop inhibitors of coronavirus replication in preparation for future outbreaks. We explored two virtual screening strategies to find inhibitors of the SARS-CoV-2 main protease in ultralarge chemical libraries. First, structure-based docking was used to screen a diverse library of 235 million virtual compounds against the active site. One hundred top-ranked compounds were tested in binding and enzymatic assays. Second, a fragment discovered by crystallographic screening was optimized guided by docking of millions of elaborated molecules and experimental testing of 93 compounds. Three inhibitors were identified in the first library screen, and five of the selected fragment elaborations showed inhibitory effects. Crystal structures of target-inhibitor complexes confirmed docking predictions and guided hit-to-lead optimization, resulting in a noncovalent main protease inhibitor with nanomolar affinity, a promising in vitro pharmacokinetic profile, and broad-spectrum antiviral effect in infected cells.

Direct Observation of Protonation State Modulation in SARS-CoV-2 Main Protease upon Inhibitor Binding with Neutron Crystallography.

The main protease (3CL Mpro) from severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus that causes COVID-19, is an essential enzyme for viral replication with no human counterpart, making it an attractive drug target. To date, no small-molecule clinical drugs are available that specifically inhibit SARS-CoV-2 Mpro. To aid rational drug design, we determined a neutron structure of Mpro in complex with the α-ketoamide inhibitor telaprevir at near-physiological (22 °C) temperature. We directly observed protonation states in the inhibitor complex and compared them with those in the ligand-free Mpro, revealing modulation of the active-site protonation states upon telaprevir binding. We suggest that binding of other α-ketoamide covalent inhibitors can lead to the same protonation state changes in the Mpro active site. Thus, by studying the protonation state changes induced by inhibitors, we provide crucial insights to help guide rational drug design, allowing precise tailoring of inhibitors to manipulate the electrostatic environment of SARS-CoV-2 Mpro.

Structure-Guided Design of Potent Inhibitors of SARS-CoV-2 3CL Protease: Structural, Biochemical, and Cell-Based Studies.

The COVID-19 pandemic is having a major impact on public health worldwide, and there is an urgent need for the creation of an armamentarium of effective therapeutics, including vaccines, biologics, and small-molecule therapeutics, to combat SARS-CoV-2 and emerging variants. Inspection of the virus life cycle reveals multiple viral- and host-based choke points that can be exploited to combat the virus. SARS-CoV-2 3C-like protease (3CLpro), an enzyme essential for viral replication, is an attractive target for therapeutic intervention, and the design of inhibitors of the protease may lead to the emergence of effective SARS-CoV-2-specific antivirals. We describe herein the results of our studies related to the application of X-ray crystallography, the Thorpe-Ingold effect, deuteration, and stereochemistry in the design of highly potent and nontoxic inhibitors of SARS-CoV-2 3CLpro.

Pan-3C Protease Inhibitor Rupintrivir Binds SARS-CoV-2 Main Protease in a Unique Binding Mode.

Rupintrivir targets the 3C cysteine proteases of the picornaviridae family, which includes rhinoviruses and enteroviruses that cause a range of human diseases. Despite being a pan-3C protease inhibitor, rupintrivir activity is extremely weak against the homologous 3C-like protease of SARS-CoV-2. In this study, the crystal structures of rupintrivir were determined bound to enterovirus 68 (EV68) 3C protease and the 3C-like main protease (Mpro) from SARS-CoV-2. While the EV68 3C protease-rupintrivir structure was similar to previously determined complexes with other picornavirus 3C proteases, rupintrivir bound in a unique conformation to the active site of SARS-CoV-2 Mpro splitting the catalytic cysteine and histidine residues. This bifurcation of the catalytic dyad may provide a novel approach for inhibiting cysteine proteases.

Masitinib is a broad coronavirus 3CL inhibitor that blocks replication of SARS-CoV-2.

There is an urgent need for antiviral agents that treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. We screened a library of 1900 clinically safe drugs against OC43, a human beta coronavirus that causes the common cold, and evaluated the top hits against SARS-CoV-2. Twenty drugs significantly inhibited replication of both viruses in cultured human cells. Eight of these drugs inhibited the activity of the SARS-CoV-2 main protease, 3CLpro, with the most potent being masitinib, an orally bioavailable tyrosine kinase inhibitor. X-ray crystallography and biochemistry show that masitinib acts as a competitive inhibitor of 3CLpro. Mice infected with SARS-CoV-2 and then treated with masitinib showed >200-fold reduction in viral titers in the lungs and nose, as well as reduced lung inflammation. Masitinib was also effective in vitro against all tested variants of concern (B.1.1.7, B.1.351, and P.1).

A head-to-head comparison of the inhibitory activities of 15 peptidomimetic SARS-CoV-2 3CLpro inhibitors.

The COVID-19 pandemic caused by SARS-CoV-2 has created an unprecedented global health emergency. As of July 2021, only three antiviral therapies have been approved by the FDA for treating infected patients, highlighting the urgent need for more antiviral drugs. The SARS-CoV-2 3CL protease (3CLpro) is deemed an attractive drug target due to its essential role in viral polyprotein processing and pathogenesis. Indeed, a number of peptidomimetic 3CLpro inhibitors armed with electrophilic warheads have been reported by various research groups that can potentially be developed for treating COVID-19. However, it is currently impossible to compare their relative potencies due to the different assays employed. To solve this, we conducted a head-to-head comparison of fifteen reported peptidomimetic inhibitors in a standard FRET-based SARS-CoV-2 3CLpro inhibition assay to compare and identify potent inhibitors for development. Inhibitor design and the suitability of various warheads are also discussed.

Combining High-Throughput Synthesis and High-Throughput Protein Crystallography for Accelerated Hit Identification.

Protein crystallography (PX) is widely used to drive advanced stages of drug optimization or to discover medicinal chemistry starting points by fragment soaking. However, recent progress in PX could allow for a more integrated role into early drug discovery. Here, we demonstrate for the first time the interplay of high throughput synthesis and high throughput PX. We describe a practical multicomponent reaction approach to acrylamides and -esters from diverse building blocks suitable for mmol scale synthesis on 96-well format and on a high-throughput nanoscale format in a highly automated fashion. High-throughput PX of our libraries efficiently yielded potent covalent inhibitors of the main protease of the COVID-19 causing agent, SARS-CoV-2. Our results demonstrate, that the marriage of in situ HT synthesis of (covalent) libraires and HT PX has the potential to accelerate hit finding and to provide meaningful strategies for medicinal chemistry projects.

In Silico Characterization of Masitinib Interaction with SARS-CoV-2 Main Protease.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a global health problem. Despite the current implementation of COVID-19 vaccination schedules, identifying effective antiviral drug treatments for this disease continues to be a priority. A recent study showed that masitinib (MST), a tyrosine kinase inhibitor, blocks the proteolytic activity of SARS-CoV-2 main protease (Mpro ). Although MST is a potential candidate for COVID-19 treatment, a comprehensive analysis of its interaction with Mpro has not been done. In this work, we performed molecular dynamics simulations of the MST-Mpro complex crystal structure. The effect of the protonation states of Mpro H163 residue and MST titratable groups were studied. Furthermore, we identified the MST substituents and Mpro mutations that affect the stability of the complex. Our results provide valuable insights into the design of new MST analogs as potential treatments for COVID-19.

Improved SARS-CoV-2 Mpro inhibitors based on feline antiviral drug GC376: Structural enhancements, increased solubility, and micellar studies.

Replication of SARS-CoV-2, the coronavirus causing COVID-19, requires a main protease (Mpro) to cleave viral proteins. Consequently, Mpro is a target for antiviral agents. We and others previously demonstrated that GC376, a bisulfite prodrug with efficacy as an anti-coronaviral agent in animals, is an effective inhibitor of Mpro in SARS-CoV-2. Here, we report structure-activity studies of improved GC376 derivatives with nanomolar affinities and therapeutic indices >200. Crystallographic structures of inhibitor-Mpro complexes reveal that an alternative binding pocket in Mpro, S4, accommodates the P3 position. Alternative binding is induced by polar P3 groups or a nearby methyl. NMR and solubility studies with GC376 show that it exists as a mixture of stereoisomers and forms colloids in aqueous media at higher concentrations, a property not previously reported. Replacement of its Na+ counter ion with choline greatly increases solubility. The physical, biochemical, crystallographic, and cellular data reveal new avenues for Mpro inhibitor design.

SARS-CoV-2 main protease inhibition by compounds isolated from Luffa cylindrica using molecular docking.

In this study, chemical investigation of methanol extract of the air-dried fruits of Luffa cylindrica led to the identification of a new δ-valerolactone (1), along with sixteen known compounds (2-17). Their chemical structures including the absolute configuration were elucidated by extensive spectroscopic analysis and electronic circular dichroism analysis, as well as by comparison with those reported in the literature. For the first time in literature, we have examined the binding potential of the isolated compounds to highly conserved protein, Mpro of SARS-CoV-2 using the molecular docking technique. We found that the isolated saponins (14-17) bind to the substrate-binding pocket of SARS-CoV-2 Mpro with docking energy scores of -7.13, -7.29, -7.47, and -7.54 kcal.mol-1, respectively, along with binding abilities equivalent to an already claimed N3 protease inhibitor (-7.51 kcal.mol-1).

Protease targeted COVID-19 drug discovery: What we have learned from the past SARS-CoV inhibitors?

The fascinating similarity between the SARS-CoV and SARS-CoV-2, inspires scientific community to investigate deeper into the SARS-CoV proteases such as main protease (Mpro) and papain-like protease (PLpro) and their inhibitors for the discovery of SARS-CoV-2 protease inhibitors. Because of the similarity in the proteases of these two corona viruses, there is a greater chance for the previous SARS-CoV Mpro and PLpro inhibitors to provide effective results against SARS-CoV-2. In this context, the molecular fragments from the SARS-CoV protease inhibitors through the fragment-based drug design and discovery technique can be useful guidance for COVID-19 drug discovery. Here, we have focused on the structure-activity relationship studies of previous SARS-CoV protease inhibitors and discussed about crucial fragments generated from previous SARS-CoV protease inhibitors important for the lead optimization of SARS-CoV-2 protease inhibitors. This study surely offers different strategic options of lead optimization to the medicinal chemists to discover effective anti-viral agent against the devastating disease, COVID-19.

Protease targeted COVID-19 drug discovery and its challenges: Insight into viral main protease (Mpro) and papain-like protease (PLpro) inhibitors.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) brutally perils physical and mental health worldwide. Unavailability of effective anti-viral drug rendering global threat of COVID-19 caused by SARS-CoV-2. In this scenario, viral protease enzymes are crucial targets for drug discovery. This extensive study meticulously focused on two viral proteases such as main protease (Mpro) and papain-like protease (PLpro), those are essential for viral replication. This review provides a detail overview of the targets (Mpro and PLpro) from a structural and medicinal chemistry point of view, together with recently reported protease inhibitors. An insight into the challenges in the development of effective as well as drug like protease inhibitors is discussed. Peptidomimetic and/or covalent coronavirus protease inhibitors possessed potent and selective active site inhibition but compromised in pharmacokinetic parameters to be a drug/drug like molecule. Lead optimization of non-peptidomimetic and/or low molecular weight compounds may be a better option for oral delivery. A masterly combination of adequate pharmacokinetic properties with coronavirus protease activity as well as selectivity will provide potential drug candidates in future. This study is a part of our endeavors which surely dictates medicinal chemistry efforts to discover effective anti-viral agent for this devastating disease.

Structure-Activity Relationships of Benzamides and Isoindolines Designed as SARS-CoV Protease Inhibitors Effective against SARS-CoV-2.

Inhibition of coronavirus (CoV)-encoded papain-like cysteine proteases (PLpro ) represents an attractive strategy to treat infections by these important human pathogens. Herein we report on structure-activity relationships (SAR) of the noncovalent active-site directed inhibitor (R)-5-amino-2-methyl-N-(1-(naphthalen-1-yl)ethyl) benzamide (2 b), which is known to bind into the S3 and S4 pockets of the SARS-CoV PLpro . Moreover, we report the discovery of isoindolines as a new class of potent PLpro inhibitors. The studies also provide a deeper understanding of the binding modes of this inhibitor class. Importantly, the inhibitors were also confirmed to inhibit SARS-CoV-2 replication in cell culture suggesting that, due to the high structural similarities of the target proteases, inhibitors identified against SARS-CoV PLpro are valuable starting points for the development of new pan-coronaviral inhibitors.

A Ligand Selection Strategy Identifies Chemical Probes Targeting the Proteases of SARS-CoV-2.

Activity-based probes are valuable tools for chemical biology. However, finding probes that specifically target the active site of an enzyme remains a challenging task. Herein, we present a ligand selection strategy that allows to rapidly tailor electrophilic probes to a target of choice and showcase its application for the two cysteine proteases of SARS-CoV-2 as proof of concept. The resulting probes were specific for the active site labeling of 3CLpro and PLpro with sufficient selectivity in a live cell model as well as in the background of a native human proteome. Exploiting the probes as tools for competitive profiling of a natural product library identified salvianolic acid derivatives as promising 3CLpro inhibitors. We anticipate that our ligand selection strategy will be useful to rapidly develop customized probes and discover inhibitors for a wide range of target proteins also beyond corona virus proteases.

Structural plasticity of SARS-CoV-2 3CL Mpro active site cavity revealed by room temperature X-ray crystallography.

The COVID-19 disease caused by the SARS-CoV-2 coronavirus has become a pandemic health crisis. An attractive target for antiviral inhibitors is the main protease 3CL Mpro due to its essential role in processing the polyproteins translated from viral RNA. Here we report the room temperature X-ray structure of unliganded SARS-CoV-2 3CL Mpro, revealing the ligand-free structure of the active site and the conformation of the catalytic site cavity at near-physiological temperature. Comparison with previously reported low-temperature ligand-free and inhibitor-bound structures suggest that the room temperature structure may provide more relevant information at physiological temperatures for aiding in molecular docking studies.

In silico analysis of selected alkaloids against main protease (Mpro) of SARS-CoV-2.

In the present situation, COVID-19 has become the global health concern due to its high contagious nature. It initially appeared in December 2019 in Wuhan, China and now affected more than 190 countries. As of now preventive measures are the sole solution to stop this disease for further transmission from person to person transmissions as there is no effective treatment or vaccine available to date. Research and development of new molecule is a laborious process; therefore, drug repurposing can be an alternative solution that involves the identification of potential compounds from the already available data. Alkaloids are potential source of therapeutic agents which might be able to treat novel COVID-19. Therefore, in the present study, twenty potential alkaloid molecules that possess antiviral activity against different viral diseases have taken into consideration and scrutinized using Lipinski's rule. Then out of twenty compounds seventeen were further selected for docking study. Docking study was performed using Autodock software and the best four molecule which provides maximum negative binding energy was selected for further analysis. Two alkaloids namely thalimonine and sophaline D showed potential activity to inhibit the Mpro but to confirm the claim further in-vitro studies are required.

A Quick Route to Multiple Highly Potent SARS-CoV-2 Main Protease Inhibitors*.

The COVID-19 pathogen, SARS-CoV-2, requires its main protease (SC2MPro ) to digest two of its translated long polypeptides to form a number of mature proteins that are essential for viral replication and pathogenesis. Inhibition of this vital proteolytic process is effective in preventing the virus from replicating in infected cells and therefore provides a potential COVID-19 treatment option. Guided by previous medicinal chemistry studies about SARS-CoV-1 main protease (SC1MPro ), we have designed and synthesized a series of SC2MPro inhibitors that contain ß-(S-2-oxopyrrolidin-3-yl)-alaninal (Opal) for the formation of a reversible covalent bond with the SC2MPro active-site cysteine C145. All inhibitors display high potency with Ki values at or below 100 nM. The most potent compound, MPI3, has as a Ki value of 8.3 nM. Crystallographic analyses of SC2MPro bound to seven inhibitors indicated both formation of a covalent bond with C145 and structural rearrangement from the apoenzyme to accommodate the inhibitors. Virus inhibition assays revealed that several inhibitors have high potency in inhibiting the SARS-CoV-2-induced cytopathogenic effect in both Vero E6 and A549/ACE2 cells. Two inhibitors, MPI5 and MPI8, completely prevented the SARS-CoV-2-induced cytopathogenic effect in Vero E6 cells at 2.5-5 µM and A549/ACE2 cells at 0.16-0.31 µM. Their virus inhibition potency is much higher than that of some existing molecules that are under preclinical and clinical investigations for the treatment of COVID-19. Our study indicates that there is a large chemical space that needs to be explored for the development of SC2MPro inhibitors with ultra-high antiviral potency.

2-Pyridone natural products as inhibitors of SARS-CoV-2 main protease.

The disease, COVID-19, is caused by the severe acute respiratory coronavirus 2 (SARS-CoV-2) for which there is currently no treatment. The SARS-CoV-2 main protease (Mpro) is an important enzyme for viral replication. Small molecules that inhibit this protease could lead to an effective COVID-19 treatment. The 2-pyridone scaffold was previously identified as a possible key pharmacophore to inhibit SARS-CoV-2 Mpro. A search for natural, antimicrobial products with the 2-pyridone moiety was undertaken herein, and their calculated potency as inhibitors of SARS-CoV-2 Mpro was investigated. Thirty-three natural products containing the 2-pyridone scaffold were identified from the literature. An in silico methodology using AutoDock was employed to predict the binding energies and inhibition constants (Ki values) for each 2-pyridone-containing compound with SARS-CoV-2 Mpro. This consisted of molecular optimization of the 2-pyridone compound, docking of the compound with a crystal structure of SARS-CoV-2 Mpro, and evaluation of the predicted interactions and ligand-enzyme conformations. All compounds investigated bound to the active site of SARS-CoV-2 Mpro, close to the catalytic dyad (His-41 and Cys-145). Thirteen molecules had predicted Ki values <1 µM. Glu-166 formed a key hydrogen bond in the majority of the predicted complexes, while Met-165 had some involvement in the complex binding as a close contact to the ligand. Prominent 2-pyridone compounds were further evaluated for their ADMET properties. This work has identified 2-pyridone natural products with calculated potent inhibitory activity against SARS-CoV-2 Mpro and with desirable drug-like properties, which may lead to the rapid discovery of a treatment for COVID-19.