AI-Driven De Novo Design and Molecular Modeling for Discovery of Small-Molecule Compounds as Potential Drug Candidates Targeting SARS-CoV-2 Main Protease.

Over the past three years, significant progress has been made in the development of novel promising drug candidates against COVID-19. However, SARS-CoV-2 mutations resulting in the emergence of new viral strains that can be resistant to the drugs used currently in the clinic necessitate the development of novel potent and broad therapeutic agents targeting different vulnerable spots of the viral proteins. In this study, two deep learning generative models were developed and used in combination with molecular modeling tools for de novo design of small molecule compounds that can inhibit the catalytic activity of SARS-CoV-2 main protease (Mpro), an enzyme critically important for mediating viral replication and transcription. As a result, the seven best scoring compounds that exhibited low values of binding free energy comparable with those calculated for two potent inhibitors of Mpro, via the same computational protocol, were selected as the most probable inhibitors of the enzyme catalytic site. In light of the data obtained, the identified compounds are assumed to present promising scaffolds for the development of new potent and broad-spectrum drugs inhibiting SARS-CoV-2 Mpro, an attractive therapeutic target for anti-COVID-19 agents.

Why All the Fury over Furin?

Analysis of the SARS-CoV-2 sequence revealed a multibasic furin cleavage site at the S1/S2 boundary of the spike protein distinguishing this virus from SARS-CoV. Furin, the best-characterized member of the mammalian proprotein convertases, is an ubiquitously expressed single pass type 1 transmembrane protein. Cleavage of SARS-CoV-2 spike protein by furin promotes viral entry into lung cells. While furin knockout is embryonically lethal, its knockout in differentiated somatic cells is not, thus furin provides an exciting therapeutic target for viral pathogens including SARS-CoV-2 and bacterial infections. Several peptide-based and small-molecule inhibitors of furin have been recently reported, and select cocrystal structures have been solved, paving the way for further optimization and selection of clinical candidates. This perspective highlights furin structure, substrates, recent inhibitors, and crystal structures with emphasis on furin's role in SARS-CoV-2 infection, where the current data strongly suggest its inhibition as a promising therapeutic intervention for SARS-CoV-2.

Unravelling lead antiviral phytochemicals for the inhibition of SARS-CoV-2 Mpro enzyme through in silico approach.

A new SARS coronavirus (SARS-CoV-2) belonging to the genus Betacoronavirus has caused a pandemic known as COVID-19. Among coronaviruses, the main protease (Mpro) is an essential drug target which, along with papain-like proteases catalyzes the processing of polyproteins translated from viral RNA and recognizes specific cleavage sites. There are no human proteases with similar cleavage specificity and therefore, inhibitors are highly likely to be nontoxic. Therefore, targeting the SARS-CoV-2 Mpro enzyme with small molecules can block viral replication. The present study is aimed at the identification of promising lead molecules for SARS-CoV-2 Mpro enzyme through virtual screening of antiviral compounds from plants. The binding affinity of selected small drug-like molecules to SARS-CoV-2 Mpro, SARS-CoV Mpro and MERS-CoV Mpro were studied using molecular docking. Bonducellpin D was identified as the best lead molecule which shows higher binding affinity (-9.28 kcal/mol) as compared to the control (-8.24 kcal/mol). The molecular binding was stabilized through four hydrogen bonds with Glu166 and Thr190 as well as hydrophobic interactions via eight residues. The SARS-CoV-2 Mpro shows identities of 96.08% and 50.65% to that of SARS-CoV Mpro and MERS-CoV Mpro respectively at the sequence level. At the structural level, the root mean square deviation (RMSD) between SARS-CoV-2 Mpro and SARS-CoV Mpro was found to be 0.517 Å and 0.817 Å between SARS-CoV-2 Mpro and MERS-CoV Mpro. Bonducellpin D exhibited broad-spectrum inhibition potential against SARS-CoV Mpro and MERS-CoV Mpro and therefore is a promising drug candidate, which needs further validations through in vitro and in vivo studies.

Emerging therapeutic opportunities for integrin inhibitors.

Integrins are cell adhesion and signalling proteins crucial to a wide range of biological functions. Effective marketed treatments have successfully targeted integrins αIIbß3, α4ß7/α4ß1 and αLß2 for cardiovascular diseases, inflammatory bowel disease/multiple sclerosis and dry eye disease, respectively. Yet, clinical development of others, notably within the RGD-binding subfamily of αv integrins, including αvß3, have faced significant challenges in the fields of cancer, ophthalmology and osteoporosis. New inhibitors of the related integrins αvß6 and αvß1 have recently come to the fore and are being investigated clinically for the treatment of fibrotic diseases, including idiopathic pulmonary fibrosis and nonalcoholic steatohepatitis. The design of integrin drugs may now be at a turning point, with opportunities to learn from previous clinical trials, to explore new modalities and to incorporate new findings in pharmacological and structural biology. This Review intertwines research from biological, clinical and medicinal chemistry disciplines to discuss historical and current RGD-binding integrin drug discovery, with an emphasis on small-molecule inhibitors of the αv integrins.

Virtual Combinatorial Chemistry and Pharmacological Screening: A Short Guide to Drug Design.

Traditionally, drug development involved the individual synthesis and biological evaluation of hundreds to thousands of compounds with the intention of highlighting their biological activity, selectivity, and bioavailability, as well as their low toxicity. On average, this process of new drug development involved, in addition to high economic costs, a period of several years before hopefully finding a drug with suitable characteristics to drive its commercialization. Therefore, the chemical synthesis of new compounds became the limiting step in the process of searching for or optimizing leads for new drug development. This need for large chemical libraries led to the birth of high-throughput synthesis methods and combinatorial chemistry. Virtual combinatorial chemistry is based on the same principle as real chemistry-many different compounds can be generated from a few building blocks at once. The difference lies in its speed, as millions of compounds can be produced in a few seconds. On the other hand, many virtual screening methods, such as QSAR (Quantitative Sturcture-Activity Relationship), pharmacophore models, and molecular docking, have been developed to study these libraries. These models allow for the selection of molecules to be synthesized and tested with a high probability of success. The virtual combinatorial chemistry-virtual screening tandem has become a fundamental tool in the process of searching for and developing a drug, as it allows the process to be accelerated with extraordinary economic savings.

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.

Screening of Botanical Drugs against SARS-CoV-2 Entry Reveals Novel Therapeutic Agents to Treat COVID-19.

An escalating pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has severely impacted global health. There is a severe lack of specific treatment options for diseases caused by SARS-CoV-2. In this study, we used a pseudotype virus (pv) containing the SARS-CoV-2 S glycoprotein to screen a botanical drug library containing 1037 botanical drugs to identify agents that prevent SARS-CoV-2 entry into the cell. Our study identified four hits, including angeloylgomisin O, schisandrin B, procyanidin, and oleanonic acid, as effective SARS-CoV-2 S pv entry inhibitors in the micromolar range. A mechanistic study revealed that these four agents inhibited SARS-CoV-2 S pv entry by blocking spike (S) protein-mediated membrane fusion. Furthermore, angeloylgomisin O and schisandrin B inhibited authentic SARS-CoV-2 with a high selective index (SI; 50% cytotoxic concentration/50% inhibition concentration). Our drug combination studies performed in cellular antiviral assays revealed that angeloylgomisin O has synergistic effects in combination with remdesivir, a drug widely used to treat SARS-CoV-2-mediated infections. We also showed that two hits could inhibit the newly emerged alpha (B.1.1.7) and beta (B.1.351) variants. Our findings collectively indicate that angeloylgomisin O and schisandrin B could inhibit SARS-CoV-2 efficiently, thereby making them potential therapeutic agents to treat the coronavirus disease of 2019.

Discovery of Highly Potent Fusion Inhibitors with Potential Pan-Coronavirus Activity That Effectively Inhibit Major COVID-19 Variants of Concern (VOCs) in Pseudovirus-Based Assays.

We report the discovery of several highly potent small molecules with low-nM potency against severe acute respiratory syndrome coronavirus (SARS-CoV; lowest half-maximal inhibitory concentration (IC50: 13 nM), SARS-CoV-2 (IC50: 23 nM), and Middle East respiratory syndrome coronavirus (MERS-CoV; IC50: 76 nM) in pseudovirus-based assays with excellent selectivity index (SI) values (>5000), demonstrating potential pan-coronavirus inhibitory activities. Some compounds showed 100% inhibition against the cytopathic effects (CPE; IC100) of an authentic SARS-CoV-2 (US_WA-1/2020) variant at 1.25 µM. The most active inhibitors also potently inhibited variants of concern (VOCs), including the UK (B.1.1.7) and South African (B.1.351) variants and the Delta variant (B.1.617.2) originally identified in India in pseudovirus-based assay. Surface plasmon resonance (SPR) analysis with one potent inhibitor confirmed that it binds to the prefusion SARS-CoV-2 spike protein trimer. These small-molecule inhibitors prevented virus-mediated cell-cell fusion. The absorption, distribution, metabolism, and excretion (ADME) data for one of the most active inhibitors, NBCoV1, demonstrated drug-like properties. An in vivo pharmacokinetics (PK) study of NBCoV1 in rats demonstrated an excellent half-life (t1/2) of 11.3 h, a mean resident time (MRT) of 14.2 h, and oral bioavailability. We expect these lead inhibitors to facilitate the further development of preclinical and clinical candidates.

Inhibitors of bacterial H2S biogenesis targeting antibiotic resistance and tolerance.

Emergent resistance to all clinical antibiotics calls for the next generation of therapeutics. Here we report an effective antimicrobial strategy targeting the bacterial hydrogen sulfide (H2S)-mediated defense system. We identified cystathionine γ-lyase (CSE) as the primary generator of H2S in two major human pathogens, Staphylococcus aureus and Pseudomonas aeruginosa, and discovered small molecules that inhibit bacterial CSE. These inhibitors potentiate bactericidal antibiotics against both pathogens in vitro and in mouse models of infection. CSE inhibitors also suppress bacterial tolerance, disrupting biofilm formation and substantially reducing the number of persister bacteria that survive antibiotic treatment. Our results establish bacterial H2S as a multifunctional defense factor and CSE as a drug target for versatile antibiotic enhancers.

Dasabuvir Inhibits Human Norovirus Infection in Human Intestinal Enteroids.

Human noroviruses (HuNoVs) are acute viral gastroenteritis pathogens that affect all age groups, yet no approved vaccines and drugs to treat HuNoV infection are available. In this study, we screened an antiviral compound library to identify compound(s) showing anti-HuNoV activity using a human intestinal enteroid (HIE) culture system in which HuNoVs are able to replicate reproducibly. Dasabuvir (DSB), which has been developed as an anti-hepatitis C virus agent, was found to inhibit HuNoV infection in HIEs at micromolar concentrations. Dasabuvir also inhibited severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and human rotavirus A (RVA) infection in HIEs. To our knowledge, this is the first study to screen an antiviral compound library for HuNoV using HIEs, and we successfully identified dasabuvir as a novel anti-HuNoV inhibitor that warrants further investigation. IMPORTANCE Although there is an urgent need to develop effective antiviral therapy directed against HuNoV infection, compound screening to identify anti-HuNoV drug candidates has not been reported so far. Using a human HIE culture system, our compound screening successfully identified dasabuvir as a novel anti-HuNoV inhibitor. Dasabuvir's inhibitory effect was also demonstrated in the cases of SARS-CoV-2 and RVA infection, highlighting the usefulness of the HIE platform for screening antiviral agents against various viruses that target the intestines.

A novel class of TMPRSS2 inhibitors potently block SARS-CoV-2 and MERS-CoV viral entry and protect human epithelial lung cells.

The host cell serine protease TMPRSS2 is an attractive therapeutic target for COVID-19 drug discovery. This protease activates the Spike protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and of other coronaviruses and is essential for viral spread in the lung. Utilizing rational structure-based drug design (SBDD) coupled to substrate specificity screening of TMPRSS2, we have discovered covalent small-molecule ketobenzothiazole (kbt) TMPRSS2 inhibitors which are structurally distinct from and have significantly improved activity over the existing known inhibitors Camostat and Nafamostat. Lead compound MM3122 (4) has an IC50 (half-maximal inhibitory concentration) of 340 pM against recombinant full-length TMPRSS2 protein, an EC50 (half-maximal effective concentration) of 430 pM in blocking host cell entry into Calu-3 human lung epithelial cells of a newly developed VSV-SARS-CoV-2 chimeric virus, and an EC50 of 74 nM in inhibiting cytopathic effects induced by SARS-CoV-2 virus in Calu-3 cells. Further, MM3122 blocks Middle East respiratory syndrome coronavirus (MERS-CoV) cell entry with an EC50 of 870 pM. MM3122 has excellent metabolic stability, safety, and pharmacokinetics in mice, with a half-life of 8.6 h in plasma and 7.5 h in lung tissue, making it suitable for in vivo efficacy evaluation and a promising drug candidate for COVID-19 treatment.

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.

Probing the Allosteric Inhibition Mechanism of a Spike Protein Using Molecular Dynamics Simulations and Active Compound Identifications.

The receptor recognition of the novel coronavirus SARS-CoV-2 relies on the "down-to-up" conformational change in the receptor-binding domain (RBD) of the spike (S) protein. Therefore, understanding the process of this change at the molecular level facilitates the design of therapeutic agents. With the help of coarse-grained molecular dynamic simulations, we provide evidence showing that the conformational dynamics of the S protein are globally cooperative. Importantly, an allosteric path was discovered that correlates the motion of the RBD with the motion of the junction between the subdomain 1 (SD1) and the subdomain 2 (SD2) of the S protein. Building on this finding, we designed non-RBD binding modulators to inhibit SARS-CoV-2 by prohibiting the conformational change of the S protein. Their inhibition effect and function stages at inhibiting SARS-CoV-2 were evaluated experimentally. In summary, our studies establish a molecular basis for future therapeutic agent design through allosteric effects.

Bioactive recombinant human oncostatin M for NMR-based screening in drug discovery.

Oncostatin M (OSM) is a pleiotropic, interleukin-6 family inflammatory cytokine that plays an important role in inflammatory diseases, including inflammatory bowel disease, rheumatoid arthritis, and cancer progression and metastasis. Recently, elevated OSM levels have been found in the serum of COVID-19 patients in intensive care units. Multiple anti-OSM therapeutics have been investigated, but to date no OSM small molecule inhibitors are clinically available. To pursue a high-throughput screening and structure-based drug discovery strategy to design a small molecule inhibitor of OSM, milligram quantities of highly pure, bioactive OSM are required. Here, we developed a reliable protocol to produce highly pure unlabeled and isotope enriched OSM from E. coli for biochemical and NMR studies. High yields (ca. 10 mg/L culture) were obtained in rich and minimal defined media cultures. Purified OSM was characterized by mass spectrometry and circular dichroism. The bioactivity was confirmed by induction of OSM/OSM receptor signaling through STAT3 phosphorylation in human breast cancer cells. Optimized buffer conditions yielded 1H, 15N HSQC NMR spectra with intense, well-dispersed peaks. Titration of 15N OSM with a small molecule inhibitor showed chemical shift perturbations for several key residues with a binding affinity of 12.2 ± 3.9 µM. These results demonstrate the value of bioactive recombinant human OSM for NMR-based small molecule screening.

In quest of small-molecules as potent non-competitive inhibitors against influenza.

A series of scaffolds namely aurones, 3-indolinones, 4-quinolones and cinnamic acid-piperazine hybrids, was designed, synthesized and investigated in vitro against influenza A/H1N1pdm09 virus. Designed molecules adopted different binding mode i.e., in 430-cavity of neuraminidase, unlike sialic acid and oseltamivir in molecular docking studies. All molecules reduced the viral titer and exhibited non-cytotoxicity along with cryo-protective property towards MDCK cells. Molecules (Z)-2-(3'-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2f), (Z)-2-(4'-Chloro-benzylidene)-1,2-dihydro-indol-3-one (2g) and 2-(2'-Methoxy-phenyl)-1H-quinolin-4-one (3a) were the most interesting molecules identified in this research, endowed with robust potencies showing low-nanomolar EC50 values of 4.0 nM, 6.7 nM and 4.9 nM, respectively, compared to reference competitive and non-competitive inhibitors: oseltamivir (EC50 = 12.7 nM) and quercetin (EC50 = 0.56 µM), respectively. Besides, 2f, 2g and 3a exhibited good neuraminidase inhibitory activity in sub-micromolar range (IC50 = 0.52 µM, 3.5 µM, 1.3 µM respectively). Moreover, these molecules were determined as non-competitive inhibitors similar to reference non-competitive inhibitor quercetin unlike reference competitive inhibitor oseltamivir in kinetics studies.

A Solid Supported Membrane-Based Technology for Electrophysical Screening of B0AT1-Modulating Compounds.

Classical high-throughput screening (HTS) technologies for the analysis of ionic currents across biological membranes can be performed using fluorescence-based, radioactive, and mass spectrometry (MS)-based uptake assays. These assays provide rapid results for pharmacological HTS, but the underlying, indirect analytical character of these assays can be linked to high false-positive hit rates. Thus, orthogonal and secondary assays using more biological target-based technologies are indispensable for further compound validation and optimization. Direct assay technologies for transporter proteins are electrophysiology-based, but are also complex, time-consuming, and not well applicable for automated profiling purposes. In contrast to conventional patch clamp systems, solid supported membrane (SSM)-based electrophysiology is a sensitive, membrane-based method for transporter analysis, and current technical developments target the demand for automated, accelerated, and sensitive assays for transporter-directed compound screening. In this study, the suitability of the SSM-based technique for pharmacological compound identification and optimization was evaluated performing cell-free SSM-based measurements with the electrogenic amino acid transporter B0AT1 (SLC6A19). Electrophysiological characterization of leucine-induced currents demonstrated that the observed signals were specific to B0AT1. Moreover, B0AT1-dependent responses were successfully inhibited using an established in-house tool compound. Evaluation of current stability and data reproducibility verified the robustness and reliability of the applied assay. Active compounds from primary screens of large compound libraries were validated, and false-positive hits were identified. These results clearly demonstrate the suitability of the SSM-based technique as a direct electrophysiological method for rapid and automated identification of small molecules that can inhibit B0AT1 activity.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of nsp13 helicase.

The coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is a global public health challenge. While the efficacy of vaccines against emerging and future virus variants remains unclear, there is a need for therapeutics. Repurposing existing drugs represents a promising and potentially rapid opportunity to find novel antivirals against SARS-CoV-2. The virus encodes at least nine enzymatic activities that are potential drug targets. Here, we have expressed, purified and developed enzymatic assays for SARS-CoV-2 nsp13 helicase, a viral replication protein that is essential for the coronavirus life cycle. We screened a custom chemical library of over 5000 previously characterized pharmaceuticals for nsp13 inhibitors using a fluorescence resonance energy transfer-based high-throughput screening approach. From this, we have identified FPA-124 and several suramin-related compounds as novel inhibitors of nsp13 helicase activity in vitro. We describe the efficacy of these drugs using assays we developed to monitor SARS-CoV-2 growth in Vero E6 cells.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp5 main protease.

The coronavirus 2019 (COVID-19) pandemic, caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), spread around the world with unprecedented health and socio-economic effects for the global population. While different vaccines are now being made available, very few antiviral drugs have been approved. The main viral protease (nsp5) of SARS-CoV-2 provides an excellent target for antivirals, due to its essential and conserved function in the viral replication cycle. We have expressed, purified and developed assays for nsp5 protease activity. We screened the nsp5 protease against a custom chemical library of over 5000 characterised pharmaceuticals. We identified calpain inhibitor I and three different peptidyl fluoromethylketones (FMK) as inhibitors of nsp5 activity in vitro, with IC50 values in the low micromolar range. By altering the sequence of our peptidomimetic FMK inhibitors to better mimic the substrate sequence of nsp5, we generated an inhibitor with a subnanomolar IC50. Calpain inhibitor I inhibited viral infection in monkey-derived Vero E6 cells, with an EC50 in the low micromolar range. The most potent and commercially available peptidyl-FMK compound inhibited viral growth in Vero E6 cells to some extent, while our custom peptidyl FMK inhibitor offered a marked antiviral improvement.

Identifying SARS-CoV-2 antiviral compounds by screening for small molecule inhibitors of Nsp3 papain-like protease.

The COVID-19 pandemic has emerged as the biggest life-threatening disease of this century. Whilst vaccination should provide a long-term solution, this is pitted against the constant threat of mutations in the virus rendering the current vaccines less effective. Consequently, small molecule antiviral agents would be extremely useful to complement the vaccination program. The causative agent of COVID-19 is a novel coronavirus, SARS-CoV-2, which encodes at least nine enzymatic activities that all have drug targeting potential. The papain-like protease (PLpro) contained in the nsp3 protein generates viral non-structural proteins from a polyprotein precursor, and cleaves ubiquitin and ISG protein conjugates. Here we describe the expression and purification of PLpro. We developed a protease assay that was used to screen a custom compound library from which we identified dihydrotanshinone I and Ro 08-2750 as compounds that inhibit PLpro in protease and isopeptidase assays and also inhibit viral replication in cell culture-based assays.