Rhenium(V) Complexes as Cysteine-Targeting Coordinate Covalent Warheads.

Interest in covalent enzyme inhibitors as therapeutic agents has seen a recent resurgence. Covalent enzyme inhibitors typically possess an organic functional group that reacts with a key feature of the target enzyme, often a nucleophilic cysteine residue. Herein, the application of small, modular ReV complexes as inorganic cysteine-targeting warheads is described. These metal complexes were found to react with cysteine residues rapidly and selectively. To demonstrate the utility of these ReV complexes, their reactivity with SARS-CoV-2-associated cysteine proteases is presented, including the SARS-CoV-2 main protease and papain-like protease and human enzymes cathepsin B and L. As all of these proteins are cysteine proteases, these enzymes were found to be inhibited by the ReV complexes through the formation of adducts. These findings suggest that these ReV complexes could be used as a new class of warheads for targeting surface accessible cysteine residues in disease-relevant target proteins.

Acriflavine and proflavine hemisulfate as potential antivirals by targeting Mpro.

The evolving SARS-CoV-2 epidemic buffets the world, and the concerted efforts are needed to explore effective drugs. M^pro is an intriguing antiviral target for interfering with viral RNA replication and transcription. In order to get potential anti-SARS-CoV-2 agents, we established an enzymatic assay using a fluorogenic substrate to screen the inhibitors of M^pro. Fortunately, Acriflavine (ACF) and Proflavine Hemisulfate (PRF) with the same acridine scaffold were picked out for their good inhibitory activity against M^pro with IC₅₀ of 5.60 ± 0.29 µM and 2.07 ± 0.01 µM, respectively. Further evaluation of MST assay and enzymatic kinetics experiment in vitro showed that they had a certain affinity to SARS-CoV-2 M^pro and were both non-competitive inhibitors. In addition, they inhibited about 90 % HCoV-OC43 replication in BHK-21 cells at 1 µM. Both compounds showed nano-molar activities against SARS-CoV-2 virus, which were superior to GC376 for anti-HCoV-43, and equivalent to the standard molecule remdesivir. Our study demonstrated that ACF and PRF were inhibitors of M^pro, and ACF has been previously reported as a PL^pro inhibitor. Taken together, ACF and PRF might be dual-targeted inhibitors to provide protection against infections of coronaviruses.

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.

Covalent Reversible Inhibitors of Cysteine Proteases Containing the Nitrile Warhead: Recent Advancement in the Field of Viral and Parasitic Diseases.

In the field of drug discovery, the nitrile group is well represented among drugs and biologically active compounds. It can form both non-covalent and covalent interactions with diverse biological targets, and it is amenable as an electrophilic warhead for covalent inhibition. The main advantage of the nitrile group as a warhead is mainly due to its milder electrophilic character relative to other more reactive groups (e.g., -CHO), reducing the possibility of unwanted reactions that would hinder the development of safe drugs, coupled to the ease of installation through different synthetic approaches. The covalent inhibition is a well-assessed design approach for serine, threonine, and cysteine protease inhibitors. The mechanism of hydrolysis of these enzymes involves the formation of a covalent acyl intermediate, and this mechanism can be exploited by introducing electrophilic warheads in order to mimic this covalent intermediate. Due to the relevant role played by the cysteine protease in the survival and replication of infective agents, spanning from viruses to protozoan parasites, we will review the most relevant and recent examples of protease inhibitors presenting a nitrile group that have been introduced to form or to facilitate the formation of a covalent bond with the catalytic cysteine active site residue.

Neoechinulin A as a Promising SARS-CoV-2 Mpro Inhibitor: In Vitro and In Silico Study Showing the Ability of Simulations in Discerning Active from Inactive Enzyme Inhibitors.

The COVID-19 pandemic and its continuing emerging variants emphasize the need to discover appropriate treatment, where vaccines alone have failed to show complete protection against the new variants of the virus. Therefore, treatment of the infected cases is critical. This paper discusses the bio-guided isolation of three indole diketopiperazine alkaloids, neoechinulin A (1), echinulin (2), and eurocristatine (3), from the Red Sea-derived Aspergillus fumigatus MR2012. Neoechinulin A (1) exhibited a potent inhibitory effect against SARS-CoV-2 Mpro with IC50 value of 0.47 µM, which is comparable to the reference standard GC376. Despite the structural similarity between the three compounds, only 1 showed a promising effect. The mechanism of inhibition is discussed in light of a series of extensive molecular docking, classical and steered molecular dynamics simulation experiments. This paper sheds light on indole diketopiperazine alkaloids as a potential structural motif against SARS-CoV-2 Mpro. Additionally, it highlights the potential of different molecular docking and molecular dynamics simulation approaches in the discrimination between active and inactive structurally related Mpro inhibitors.

Structure-Based Optimization of ML300-Derived, Noncovalent Inhibitors Targeting the Severe Acute Respiratory Syndrome Coronavirus 3CL Protease (SARS-CoV-2 3CLpro).

Starting from the MLPCN probe compound ML300, a structure-based optimization campaign was initiated against the recent severe acute respiratory syndrome coronavirus (SARS-CoV-2) main protease (3CLpro). X-ray structures of SARS-CoV-1 and SARS-CoV-2 3CLpro enzymes in complex with multiple ML300-based inhibitors, including the original probe ML300, were obtained and proved instrumental in guiding chemistry toward probe compound 41 (CCF0058981). The disclosed inhibitors utilize a noncovalent mode of action and complex in a noncanonical binding mode not observed by peptidic 3CLpro inhibitors. In vitro DMPK profiling highlights key areas where further optimization in the series is required to obtain useful in vivo probes. Antiviral activity was established using a SARS-CoV-2-infected Vero E6 cell viability assay and a plaque formation assay. Compound 41 demonstrates nanomolar activity in these respective assays, comparable in potency to remdesivir. These findings have implications for antiviral development to combat current and future SARS-like zoonotic coronavirus outbreaks.

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.

Identification of potent small molecule inhibitors of SARS-CoV-2 entry.

The severe acute respiratory syndrome coronavirus 2 responsible for COVID-19 remains a persistent threat to mankind, especially for the immunocompromised and elderly for which the vaccine may have limited effectiveness. Entry of SARS-CoV-2 requires a high affinity interaction of the viral spike protein with the cellular receptor angiotensin-converting enzyme 2. Novel mutations on the spike protein correlate with the high transmissibility of new variants of SARS-CoV-2, highlighting the need for small molecule inhibitors of virus entry into target cells. We report the identification of such inhibitors through a robust high-throughput screen testing 15,000 small molecules from unique libraries. Several leads were validated in a suite of mechanistic assays, including whole cell SARS-CoV-2 infectivity assays. The main lead compound, calpeptin, was further characterized using SARS-CoV-1 and the novel SARS-CoV-2 variant entry assays, SARS-CoV-2 protease assays and molecular docking. This study reveals calpeptin as a potent and specific inhibitor of SARS-CoV-2 and some variants.

Apigenin analogues as SARS-CoV-2 main protease inhibitors: In-silico screening approach.

The COVID-19 new variants spread rapidly all over the world, and until now scientists strive to find virus-specific antivirals for its treatment. The main protease of SARS-CoV-2 (Mpro) exhibits high structural and sequence homology to main protease of SARS-CoV (93.23% sequence identity), and their sequence alignment indicated 12 mutated/variant residues. The sequence alignment of SARS-CoV-2 main protease led to identification of only one mutated/variant residue with no significant role in its enzymatic process. Therefore, Mpro was considered as a high-profile drug target in anti-SARS-CoV-2 drug discovery. Apigenin analogues to COVID-19 main protease binding were evaluated. The detailed interactions between the analogues of Apigenin and SARS-CoV-2 Mpro inhibitors were determined as hydrogen bonds, electronic bonds and hydrophobic interactions. The binding energies obtained from the molecular docking of Mpro with Boceprevir, Apigenin, Apigenin 7-glucoside-4'-p-coumarate, Apigenin 7-glucoside-4'-trans-caffeate and Apigenin 7-O-beta-d-glucoside (Cosmosiin) were found to be -6.6, -7.2, -8.8, -8.7 and -8.0 kcal/mol, respectively. Pharmacokinetic parameters and toxicological characteristics obtained by computational techniques and Virtual ADME studies of the Apigenin analogues confirmed that the Apigenin 7-glucoside-4'-p-coumarate is the best candidate for SARS-CoV-2 Mpro inhibition.

A Patent Review on SARS Coronavirus Main Protease (3CLpro ) Inhibitors.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic is an unprecedented global health emergency causing more than 4.2 million fatalities as of 30 July 2021. Only three antiviral therapies have been approved or granted emergency use authorization by the FDA. The SARS-CoV-2 3CL protease (3CLpro ) is deemed an attractive drug target as it plays an essential role in viral polyprotein processing and pathogenesis, although no inhibitors have been approved. This patent review discusses SARS coronavirus 3CLpro inhibitors that have been filed up to 30 July 2021, giving an overview on the types of inhibitors that have generated commercial interest, especially amongst drug companies. Insights into the common structural motifs required for active site binding is also discussed.

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.

All-Trans Retinoic Acid Exhibits Antiviral Effect against SARS-CoV-2 by Inhibiting 3CLpro Activity.

The pandemic of COVID-19 caused by SARS-CoV-2 continues to spread despite the global efforts taken to control it. The 3C-like protease (3CLpro), the major protease of SARS-CoV-2, is one of the most interesting targets for antiviral drug development because it is highly conserved among SARS-CoVs and plays an important role in viral replication. Herein, we developed high throughput screening for SARS-CoV-2 3CLpro inhibitor based on AlphaScreen. We screened 91 natural product compounds and found that all-trans retinoic acid (ATRA), an FDA-approved drug, inhibited 3CLpro activity. The 3CLpro inhibitory effect of ATRA was confirmed in vitro by both immunoblotting and AlphaScreen with a 50% inhibition concentration (IC50) of 24.7 ± 1.65 µM. ATRA inhibited the replication of SARS-CoV-2 in VeroE6/TMPRSS2 and Calu-3 cells, with IC50 = 2.69 ± 0.09 µM in the former and 0.82 ± 0.01 µM in the latter. Further, we showed the anti-SARS-CoV-2 effect of ATRA on the currently circulating variants of concern (VOC); alpha, beta, gamma, and delta. These results suggest that ATRA may be considered as a potential therapeutic agent against SARS-CoV-2.

Potent Anti-SARS-CoV-2 Activity by the Natural Product Gallinamide A and Analogues via Inhibition of Cathepsin L.

Cathepsin L is a key host cysteine protease utilized by coronaviruses for cell entry and is a promising drug target for novel antivirals against SARS-CoV-2. The marine natural product gallinamide A and several synthetic analogues were identified as potent inhibitors of cathepsin L with IC50 values in the picomolar range. Lead molecules possessed selectivity over other cathepsins and alternative host proteases involved in viral entry. Gallinamide A directly interacted with cathepsin L in cells and, together with two lead analogues, potently inhibited SARS-CoV-2 infection in vitro, with EC50 values in the nanomolar range. Reduced antiviral activity was observed in cells overexpressing transmembrane protease, serine 2 (TMPRSS2); however, a synergistic improvement in antiviral activity was achieved when combined with a TMPRSS2 inhibitor. These data highlight the potential of cathepsin L as a COVID-19 drug target as well as the likely need to inhibit multiple routes of viral entry to achieve efficacy.

NMR Spectroscopy of the Main Protease of SARS-CoV-2 and Fragment-Based Screening Identify Three Protein Hotspots and an Antiviral Fragment.

The main protease (3CLp) of the SARS-CoV-2, the causative agent for the COVID-19 pandemic, is one of the main targets for drug development. To be active, 3CLp relies on a complex interplay between dimerization, active site flexibility, and allosteric regulation. The deciphering of these mechanisms is a crucial step to enable the search for inhibitors. In this context, using NMR spectroscopy, we studied the conformation of dimeric 3CLp from the SARS-CoV-2 and monitored ligand binding, based on NMR signal assignments. We performed a fragment-based screening that led to the identification of 38 fragment hits. Their binding sites showed three hotspots on 3CLp, two in the substrate binding pocket and one at the dimer interface. F01 is a non-covalent inhibitor of the 3CLp and has antiviral activity in SARS-CoV-2 infected cells. This study sheds light on the complex structure-function relationships of 3CLp and constitutes a strong basis to assist in developing potent 3CLp inhibitors.

Design of SARS-CoV-2 PLpro Inhibitors for COVID-19 Antiviral Therapy Leveraging Binding Cooperativity.

Antiviral agents that complement vaccination are urgently needed to end the COVID-19 pandemic. The SARS-CoV-2 papain-like protease (PLpro), one of only two essential cysteine proteases that regulate viral replication, also dysregulates host immune sensing by binding and deubiquitination of host protein substrates. PLpro is a promising therapeutic target, albeit challenging owing to featureless P1 and P2 sites recognizing glycine. To overcome this challenge, we leveraged the cooperativity of multiple shallow binding sites on the PLpro surface, yielding novel 2-phenylthiophenes with nanomolar inhibitory potency. New cocrystal structures confirmed that ligand binding induces new interactions with PLpro: by closing of the BL2 loop of PLpro forming a novel "BL2 groove" and by mimicking the binding interaction of ubiquitin with Glu167 of PLpro. Together, this binding cooperativity translates to the most potent PLpro inhibitors reported to date, with slow off-rates, improved binding affinities, and low micromolar antiviral potency in SARS-CoV-2-infected human cells.

Macrolactin A as a Novel Inhibitory Agent for SARS-CoV-2 Mpro: Bioinformatics Approach.

COVID-19 is a disease that puts most of the world on lockdown and the search for therapeutic drugs is still ongoing. Therefore, this study used in silico screening to identify natural bioactive compounds from fruits, herbaceous plants, and marine invertebrates that are able to inhibit protease activity in SARS-CoV-2 (PDB: 6LU7). We have used extensive screening strategies such as drug likeliness, antiviral activity value prediction, molecular docking, ADME, molecular dynamics (MD) simulation, and MM/GBSA. A total of 17 compounds were shortlisted using Lipinski's rule in which 5 compounds showed significant predicted antiviral activity values. Among these 5, only 2 compounds, Macrolactin A and Stachyflin, showed good binding energy of -9.22 and -8.00 kcal/mol, respectively, within the binding pocket of the Mpro catalytic residues (HIS 41 and CYS 145). These two compounds were further analyzed to determine their ADME properties. The ADME evaluation of these 2 compounds suggested that they could be effective in developing therapeutic drugs to be used in clinical trials. MD simulations showed that protein-ligand complexes of Macrolactin A and Stachyflin with the target receptor (6LU7) were stable for 100 nanoseconds. The MM/GBSA calculations of Mpro-Macrolactin A complex indicated higher binding free energy (-42.58 ± 6.35 kcal/mol). Dynamic cross-correlation matrix (DCCM) and principal component analysis (PCA) on the residual movement in the MD trajectories further confirmed the stability of Macrolactin A bound state with 6LU7. In conclusion, this study showed that marine natural compound Macrolactin A could be an effective therapeutic inhibitor against SARS-CoV-2 protease (6LU7). Additional in vitro and in vivo validations are strongly needed to determine the efficacy and therapeutic dose of Macrolactin A in biological systems.

Discovery and Mechanism of SARS-CoV-2 Main Protease Inhibitors.

The emergence of a new coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents an urgent public health crisis. Without available targeted therapies, treatment options remain limited for COVID-19 patients. Using medicinal chemistry and rational drug design strategies, we identify a 2-phenyl-1,2-benzoselenazol-3-one class of compounds targeting the SARS-CoV-2 main protease (Mpro). FRET-based screening against recombinant SARS-CoV-2 Mpro identified six compounds that inhibit proteolysis with nanomolar IC50 values. Preincubation dilution experiments and molecular docking determined that the inhibition of SARS-CoV-2 Mpro can occur by either covalent or noncovalent mechanisms, and lead E04 was determined to inhibit Mpro competitively. Lead E24 inhibited viral replication with a nanomolar EC50 value (844 nM) in SARS-CoV-2-infected Vero E6 cells and was further confirmed to impair SARS-CoV-2 replication in human lung epithelial cells and human-induced pluripotent stem cell-derived 3D lung organoids. Altogether, these studies provide a structural framework and mechanism of Mpro inhibition that should facilitate the design of future COVID-19 treatments.

Bioguided Isolation of Cyclopenin Analogues as Potential SARS-CoV-2 Mpro Inhibitors from Penicillium citrinum TDPEF34.

SARS-CoV-2 virus mutations might increase its virulence, and thus the severity and duration of the ongoing pandemic. Global drug discovery campaigns have successfully developed several vaccines to reduce the number of infections by the virus. However, finding a small molecule pharmaceutical that is effective in inhibiting SARS-CoV-2 remains a challenge. Natural products are the origin of many currently used pharmaceuticals and, for this reason, a library of in-house fungal extracts were screened to assess their potential to inhibit the main viral protease Mpro in vitro. The extract of Penicillium citrinum, TDPEF34, showed potential inhibition and was further analysed to identify potential Mpro inhibitors. Following bio-guided isolation, a series of benzodiazepine alkaloids cyclopenins with good-to-moderate activity against SARS-CoV-2 Mpro were identified. The mode of enzyme inhibition of these compounds was predicted by docking and molecular dynamic simulation. Compounds 1 (isolated as two conformers of S- and R-isomers), 2, and 4 were found to have promising in vitro inhibitory activity towards Mpro, with an IC50 values range of 0.36-0.89 µM comparable to the positive control GC376. The in silico investigation revealed compounds to achieve stable binding with the enzyme active site through multiple H-bonding and hydrophobic interactions. Additionally, the isolated compounds showed very good drug-likeness and ADMET properties. Our findings could be utilized in further in vitro and in vivo investigations to produce anti-SARS-CoV-2 drug candidates. These findings also provide critical structural information that could be used in the future for designing potent Mpro inhibitors.

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.