Mycolactone: A Broad Spectrum Multitarget Antiviral Active in the Picomolar Range for COVID-19 Prevention and Cure.

We have previously shown computationally that Mycolactone (MLN), a toxin produced by Mycobacterium ulcerans, strongly binds to Munc18b and other proteins, presumably blocking degranulation and exocytosis of blood platelets and mast cells. We investigated the effect of MLN on endocytosis using similar approaches, and it bound strongly to the N-terminal of the clathrin protein and a novel SARS-CoV-2 fusion protein. Experimentally, we found 100% inhibition up to 60 nM and 84% average inhibition at 30 nM in SARS-CoV-2 live viral assays. MLN was also 10× more potent than remdesivir and molnupiravir. MLN's toxicity against human alveolar cell line A549, immortalized human fetal renal cell line HEK293, and human hepatoma cell line Huh7.1 were 17.12%, 40.30%, and 36.25%, respectively. The cytotoxicity IC50 breakpoint ratio versus anti-SARS-CoV-2 activity was more than 65-fold. The IC50 values against the alpha, delta, and Omicron variants were all below 0.020 µM, and 134.6 nM of MLN had 100% inhibition in an entry and spread assays. MLN is eclectic in its actions through its binding to Sec61, AT2R, and the novel fusion protein, making it a good drug candidate for treating and preventing COVID-19 and other similarly transmitted enveloped viruses and pathogens.

In Silico Binding of 2-Aminocyclobutanones to SARS-CoV-2 Nsp13 Helicase and Demonstration of Antiviral Activity.

The landscape of viral strains and lineages of SARS-CoV-2 keeps changing and is currently dominated by Delta and Omicron variants. Members of the latest Omicron variants, including BA.1, are showing a high level of immune evasion, and Omicron has become a prominent variant circulating globally. In our search for versatile medicinal chemistry scaffolds, we prepared a library of substituted ɑ-aminocyclobutanones from an ɑ-aminocyclobutanone synthon (11). We performed an in silico screen of this actual chemical library as well as other virtual 2-aminocyclobutanone analogs against seven SARS-CoV-2 nonstructural proteins to identify potential drug leads against SARS-CoV-2, and more broadly against coronavirus antiviral targets. Several of these analogs were initially identified as in silico hits against SARS-CoV-2 nonstructural protein 13 (Nsp13) helicase through molecular docking and dynamics simulations. Antiviral activity of the original hits as well as ɑ-aminocyclobutanone analogs that were predicted to bind more tightly to SARS-CoV-2 Nsp13 helicase are reported. We now report cyclobutanone derivatives that exhibit anti-SARS-CoV-2 activity. Furthermore, the Nsp13 helicase enzyme has been the target of relatively few target-based drug discovery efforts, in part due to a very late release of a high-resolution structure accompanied by a limited understanding of its protein biochemistry. In general, antiviral agents initially efficacious against wild-type SARS-CoV-2 strains have lower activities against variants due to heavy viral loads and greater turnover rates, but the inhibitors we are reporting have higher activities against the later variants than the wild-type (10-20X). We speculate this could be due to Nsp13 helicase being a critical bottleneck in faster replication rates of the new variants, so targeting this enzyme affects these variants to an even greater extent. This work calls attention to cyclobutanones as a useful medicinal chemistry scaffold, and the need for additional focus on the discovery of Nsp13 helicase inhibitors to combat the aggressive and immune-evading variants of concern (VOCs).

Protein structure-based in-silico approaches to drug discovery: Guide to COVID-19 therapeutics.

With more than 5 million fatalities and close to 300 million reported cases, COVID-19 is the first documented pandemic due to a coronavirus that continues to be a major health challenge. Despite being rapid, uncontrollable, and highly infectious in its spread, it also created incentives for technology development and redefined public health needs and research agendas to fast-track innovations to be translated. Breakthroughs in computational biology peaked during the pandemic with renewed attention to making all cutting-edge technology deliver agents to combat the disease. The demand to develop effective treatments yielded surprising collaborations from previously segregated fields of science and technology. The long-standing pharmaceutical industry's aversion to repurposing existing drugs due to a lack of exponential financial gain was overrun by the health crisis and pressures created by front-line researchers and providers. Effective vaccine development even at an unprecedented pace took more than a year to develop and commence trials. Now the emergence of variants and waning protections during the booster shots is resulting in breakthrough infections that continue to strain health care systems. As of now, every protein of SARS-CoV-2 has been structurally characterized and related host pathways have been extensively mapped out. The research community has addressed the druggability of a multitude of possible targets. This has been made possible due to existing technology for virtual computer-assisted drug development as well as new tools and technologies such as artificial intelligence to deliver new leads. Here in this article, we are discussing advances in the drug discovery field related to target-based drug discovery and exploring the implications of known target-specific agents on COVID-19 therapeutic management. The current scenario calls for more personalized medicine efforts and stratifying patient populations early on for their need for different combinations of prognosis-specific therapeutics. We intend to highlight target hotspots and their potential agents, with the ultimate goal of using rational design of new therapeutics to not only end this pandemic but also uncover a generalizable platform for use in future pandemics.

Data structuring may prevent ambiguity and improve personalized medical prognosis.

Topics expected to influence personalized medicine (PM), where medical decisions, practices, and treatments are tailored to the individual patient, are reviewed. Lack of discrimination due to different biological conditions that express similar values of numerical variables (ambiguity) is regarded to be a major potential barrier for PM. This material explores possible causes and sources of ambiguity and offers suggestions for mitigating the impacts of uncertainties. Three causes of ambiguity are identified: (1) delayed adoption of innovations, (2) inadequate emphases, and (3) inadequate processes used when new medical practices are developed and validated. One example of the first problem is the relative lack of medical research on "compositional data" -the type that characterizes leukocyte data. This omission results in erroneous use of data abundantly utilized in medicine, such as the blood cell differential. Emphasis on data output ‒not biomedical interpretation that facilitates the use of clinical data‒ exemplifies the second type of problems. Reliance on tools generated in other fields (but not validated within biomedical contexts) describes the last limitation. Because reductionism is associated with these problems, non-reductionist alternatives are reviewed as potential remedies. Data structuring (converting data into information) is considered a key element that may promote PM. To illustrate a process that includes data-information-knowledge and decision-making, previously published data on COVID-19 are utilized. It is suggested that ambiguity may be prevented or ameliorated. Provided that validations are grounded on biomedical knowledge, approaches that describe certain criteria - such as non-overlapping data intervals of patients that experience different outcomes, immunologically interpretable data, and distinct graphic patterns - can inform, at personalized bases, earlier and/or with fewer observations.

Multi-Cellular Immunological Interactions Associated With COVID-19 Infections.

To rapidly prognosticate and generate hypotheses on pathogenesis, leukocyte multi-cellularity was evaluated in SARS-CoV-2 infected patients treated in India or the United States (152 individuals, 384 temporal observations). Within hospital (<90-day) death or discharge were retrospectively predicted based on the admission complete blood cell counts (CBC). Two methods were applied: (i) a "reductionist" one, which analyzes each cell type separately, and (ii) a "non-reductionist" method, which estimates multi-cellularity. The second approach uses a proprietary software package that detects distinct data patterns generated by complex and hypothetical indicators and reveals each data pattern's immunological content and associated outcome(s). In the Indian population, the analysis of isolated cell types did not separate survivors from non-survivors. In contrast, multi-cellular data patterns differentiated six groups of patients, including, in two groups, 95.5% of all survivors. Some data structures revealed one data point-wide line of observations, which informed at a personalized level and identified 97.8% of all non-survivors. Discovery was also fostered: some non-survivors were characterized by low monocyte/lymphocyte ratio levels. When both populations were analyzed with the non-reductionist method, they displayed results that suggested survivors and non-survivors differed immunologically as early as hospitalization day 1.

Iron dysregulation in COVID-19 and reciprocal evolution of SARS-CoV-2: Natura nihil frustra facit.

After more than a year of the COVID-19 pandemic, SARS-CoV-2 infection rates with newer variants continue to devastate much of the world. Global healthcare systems are overwhelmed with high positive patient numbers. Silent hypoxia accompanied by rapid deterioration and some cases with septic shock is responsible for COVID-19 mortality in many hospitalized patients. There is an urgent need to further understand the relationships and interplay with human host components during pathogenesis and immune evasion strategies. Currently, acquired immunity through vaccination or prior infection usually provides sufficient protection against the emerging variants of SARS-CoV-2 except Omicron variant requiring recent booster. New strains have shown higher viral loads and greater transmissibility with more severe disease presentations. Notably, COVID-19 has a peculiar prognosis in severe patients with iron dysregulation and hypoxia which is still poorly understood. Studies have shown abnormally low serum iron levels in severe infection but a high iron overload in lung fibrotic tissue. Data from our in-silico structural analysis of the spike protein sequence along with host proteolysis processing suggests that the viral spike protein fragment mimics Hepcidin and is resistant to the major human proteases. This functional spike-derived peptide dubbed "Covidin" thus may be intricately involved with host ferroportin binding and internalization leading to dysregulated host iron metabolism. Here, we propose the possible role of this potentially allogenic mimetic hormone corresponding to severe COVID-19 immunopathology and illustrate that this molecular mimicry is responsible for a major pathway associated with severe disease status. Furthermore, through 3D molecular modeling and docking followed by MD simulation validation, we have unraveled the likely role of Covidin in iron dysregulation in COVID-19 patients. Our meta-analysis suggests the Hepcidin mimetic mechanism is highly conserved among its host range as well as among all new variants to date including Omicron. Extensive analysis of current mutations revealed that new variants are becoming alarmingly more resistant to selective human proteases associated with host defense.

A novel compound active against SARS-CoV-2 targeting uridylate-specific endoribonuclease (NendoU/NSP15): in silico and in vitro investigations.

NendoU (NSP15) is an Mn(2+)-dependent, uridylate-specific enzyme, which leaves 2'-3'-cyclic phosphates 5' to the cleaved bond. Our in-house library was subjected to high throughput virtual screening (HTVS) to identify compounds with potential to inhibit NendoU enzyme, high-rank compounds (those that bound to multiple target structures) were further subjected to 100 nanoseconds MD simulations. Among these, one was found to be bound highly stable within the active site of the NendoU protein structure. Here, we are reporting a derivative of piperazine based '(2S,3S)-3-amino-1-(4-(4-(tert-butyl)benzyl)piperazin-1-yl)-4-phenylbutan-2-ol' (IV) from our in-house libraries having potential efficacy against SARS-CoV-2 in in vitro assays. This compound demonstrated inhibition of viral replication at the same level as Ivermectin, a known SARS-CoV-2 inhibitor, which is not used due to its toxicity at a higher than the currently approved dosage. Compound IV was not toxic to the cell lines up to a 50 µM concentration and exhibited IC50s of 4.97 µM and 8.46 µM in viral entry and spread assay, respectively. Therefore, this novel class of NendoU inhibitor could provide new insights for the development of treatment options for COVID-19.

Antiviral evaluation of hydroxyethylamine analogs: Inhibitors of SARS-CoV-2 main protease (3CLpro), a virtual screening and simulation approach.

The continued toll of COVID-19 has halted the smooth functioning of civilization on a global scale. With a limited understanding of all the essential components of viral machinery and the lack of structural information of this new virus, initial drug discovery efforts had limited success. The availability of high-resolution crystal structures of functionally essential SARS-CoV-2 proteins, including 3CLpro, supports the development of target-specific therapeutics. 3CLpro, the main protease responsible for the processing of viral polypeptide, plays a vital role in SARS-CoV-2 viral replication and translation and is an important target in other coronaviruses. Additionally, 3CLpro is the target of repurposed drugs, such as lopinavir and ritonavir. In this study, target proteins were retrieved from the protein data bank (PDB IDs: 6 M03, 6LU7, 2GZ7, 6 W63, 6SQS, 6YB7, and 6YVF) representing different open states of the main protease to accommodate macromolecular substrate. A hydroxyethylamine (HEA) library was constructed from harvested chemical structures from all the series being used in our laboratories for screening against malaria and Leishmania parasites. The database consisted of ∼1000 structure entries, of which 70% were new to ChemSpider at the time of screening. This in-house library was subjected to high throughput virtual screening (HTVS), followed by standard precision (SP) and then extra precision (XP) docking (Schrodinger LLC 2021). The ligand strain and complex energy of top hits were calculated by Molecular Mechanics Generalized Born Surface Area (MM/GBSA) method. Promising hit compounds (n = 40) specifically binding to 3CLpro with high energy and average MM/GBSA scores were then subjected to (100-ns) MD simulations. Using this sequential selection followed by an in-silico validation approach, we found a promising HEA-based compound (N,N'-((3S,3'S)-piperazine-1,4-diylbis(3-hydroxy-1-phenylbutane-4,2-diyl))bis(2-(5-methyl-1,3-dioxoisoindolin-2-yl)-3-phenylpropanamide)), which showed high in vitro antiviral activity against SARS-CoV-2. Further to reduce the size of the otherwise larger ligand, a pharmacophore-based predicted library of âˆ¼42 derivatives was constructed, which were added to the previous compound library and rescreened virtually. Out of several hits from the predicted library, two compounds were synthesized, tested against SARS-CoV-2 culture, and found to have markedly improved antiviral activity.

Heparin: A simplistic repurposing to prevent SARS-CoV-2 transmission in light of its in-vitro nanomolar efficacy.

The world is currently facing a novel coronavirus (SARS-CoV-2) pandemic. The greatest threat that is disrupting the normal functioning of society is the exceptionally high species independent transmission. Drug repurposing is understood to be the best strategy to immediately deploy well-characterized agents against new pathogens. Several repurposable drugs are already in evaluation for determining suitability to treat COVID-19. One such promising compound includes heparin, which is widely used in reducing thrombotic events associated with COVID-19 induced pathology. As part of identifying target-specific antiviral compounds among FDA and world-approved libraries using high-throughput virtual screening (HTVS), we previously evaluated top hits for anti-SARS-CoV-2 activity. Here, we report results of highly efficacious viral entry blocking properties of heparin (IC50 = 12.3 nM) in the complete virus assay, and further, propose ways to use it as a potential transmission blocker. Exploring further, our in-silico analysis indicated that the heparin interacts with post-translational glycoconjugates present on spike proteins. The patterns of accessible spike-glycoconjugates in open and closed states are completely contrasted by one another. Heparin-binding to the open conformation of spike structurally supports the state and may aid ACE2 binding as reported with cell surface-bound heparan sulfate. We also studied spike protein mutant variants' heparin interactions for possible resistance. Based on available data and optimal absorption properties by the skin, heparin could potentially be used to block SARS-CoV-2 transmission. Studies should be designed to exploit its nanomolar antiviral activity to formulate heparin as topical or inhalation-based formulations, particularly on exposed areas and sites of primary viremia e.g. ACE2 rich epithelia of the eye (conjunctiva/lids), nasal cavity, and mouth.

Multi-targeting approach for nsp3, nsp9, nsp12 and nsp15 proteins of SARS-CoV-2 by Diosmin as illustrated by molecular docking and molecular dynamics simulation methodologies.

Novel coronavirus SARS-CoV-2continues tospread rapidly worldwide and causing serious health and economic loss. In the absence of any effective treatment, various in-silico approaches are being explored towards the therapeutic discovery against COVID-19. Targeting multiple key enzymes of SARS-CoV-2 with a single potential drug could be an important in-silico strategy to tackle the therapeutic emergency. A number of Food and Drug Administration (FDA) approved drugs entered into clinical stages were originated from multi-target approaches with an increased rate, 16-21% between 2015 and 2017. In this study, we selected an FDA-approved library (Prestwick Chemical Library of 1520 compounds) and implemented in-silico virtual screening against multiple protein targets of SARS-CoV-2 on the Glide module of Schrödinger software (release 2020-1). Compounds were analyzed for their docking scores and the top-ranked against each targeted protein were further subjected to Molecular Dynamics (MD) simulations to assess the binding stability of ligand-protein complexes. A multi-targeting approach was optimized that enabled the analysis of several compounds' binding efficiency with more than one protein targets. It was demonstrated that Diosmin (6) showed the highest binding affinity towards multiple targets with binding free energy (kcal/mol) values of -63.39 (nsp3); -62.89 (nsp9); -31.23 (nsp12); and -65.58 (nsp15). Therefore, our results suggests that Diosmin (6) possesses multi-targeting capability, a potent inhibitor of various non-structural proteins of SARS-CoV-2, and thus it deserves further validation experiments before using as a therapeutic against COVID-19 disease.

Molecular Insights into Severe Acute Respiratory Syndrome Coronavirus 2 Pathobiology: Dissecting the Interplay between Cellular Innate Immunity and Immune Evasion.

In December 2019, outbreak of a novel coronavirus flared in Wuhan, the capital city of Hubei province, China. The identified pathogen was an enveloped RNA betacoronavirus called severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The outbreak was declared a pandemic by the World Health Organization (WHO), because the continual spread of this deadly and highly infectious virus is a health emergency for all world nations. SARS-CoV-2 is associated with severe atypical pneumonia coronavirus disease-19. Typical symptoms of this disease include fever, malaise, cough, shortness of breath, and in severe cases, death. As the virus continues to invade host cells deep into alveoli, infection severity mostly depends on the undeterred immune response that is triggered by elevated levels of inflammation-inducing cytokines, called a cytokine storm. In this article, we provide a comprehensive review of the viral life cycle and immunological responses associated with the SARS-CoV-2 infection.

Bisindolylmaleimide IX: A novel anti-SARS-CoV2 agent targeting viral main protease 3CLpro demonstrated by virtual screening pipeline and in-vitro validation assays.

SARS-CoV-2, the virus that causes COVID-19 consists of several enzymes with essential functions within its proteome. Here, we focused on repurposing approved and investigational drugs/compounds. We targeted seven proteins with enzymatic activities known to be essential at different stages of the viral cycle including PLpro, 3CLpro, RdRP, Helicase, ExoN, NendoU, and 2'-O-MT. For virtual screening, energy minimization of a crystal structure of the modeled protein was carried out using the Protein Preparation Wizard (Schrodinger LLC 2020-1). Following active site selection based on data mining and COACH predictions, we performed a high-throughput virtual screen of drugs and investigational molecules (n = 5903). The screening was performed against viral targets using three sequential docking modes (i.e., HTVS, SP, and XP). Virtual screening identified ∼290 potential inhibitors based on the criteria of energy, docking parameters, ligand, and binding site strain and score. Drugs specific to each target protein were further analyzed for binding free energy perturbation by molecular mechanics (prime MM-GBSA) and pruning the hits to the top 32 candidates. The top lead from each target pool was further subjected to molecular dynamics simulation using the Desmond module. The resulting top eight hits were tested for their SARS-CoV-2 anti-viral activity in-vitro. Among these, a known inhibitor of protein kinase C isoforms, Bisindolylmaleimide IX (BIM IX), was found to be a potent inhibitor of SARS-CoV-2. Further, target validation through enzymatic assays confirmed 3CLpro to be the target. This is the first study that has showcased BIM IX as a COVID-19 inhibitor thereby validating our pipeline.

A Review of the Preclinical and Clinical Efficacy of Remdesivir, Hydroxychloroquine, and Lopinavir-Ritonavir Treatments against COVID-19.

In December of 2019, an outbreak of a novel coronavirus flared in Wuhan, the capital city of the Hubei Province, China. The pathogen has been identified as a novel enveloped RNA beta-coronavirus named severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The virus SARS-CoV-2 is associated with a disease characterized by severe atypical pneumonia known as coronavirus 2019 (COVID-19). Typical symptoms of this disease include cough, fever, malaise, shortness of breath, gastrointestinal symptoms, anosmia, and, in severe cases, pneumonia.1 The high-risk group of COVID-19 patients includes people over the age of 60 years as well as people with existing cardiovascular disease and/or diabetes mellitus. Epidemiological investigations have suggested that the outbreak was associated with a live animal market in Wuhan. Within the first few months of the outbreak, cases were growing exponentially all over the world. The unabated spread of this deadly and highly infectious virus is a health emergency for all nations in the world and has led to the World Health Organization (WHO) declaring a pandemic on March 11, 2020. In this report, we consolidate and review the available clinically and preclinically relevant results emanating from in vitro animal models and clinical studies of drugs approved for emergency use as a treatment for COVID-19, including remdesivir, hydroxychloroquine, and lopinavir-ritonavir combinations. These compounds have been frequently touted as top candidates to treat COVID-19, but recent clinical reports suggest mixed outcomes on their efficacies within the current clinical protocol frameworks.

Discovery of New Hydroxyethylamine Analogs against 3CLpro Protein Target of SARS-CoV-2: Molecular Docking, Molecular Dynamics Simulation, and Structure-Activity Relationship Studies.

The novel coronavirus, SARS-CoV-2, has caused a recent pandemic called COVID-19 and a severe health threat around the world. In the current situation, the virus is rapidly spreading worldwide, and the discovery of a vaccine and potential therapeutics are critically essential. The crystal structure for the main protease (Mpro) of SARS-CoV-2, 3-chymotrypsin-like cysteine protease (3CLpro), was recently made available and is considerably similar to the previously reported SARS-CoV. Due to its essentiality in viral replication, it represents a potential drug target. Herein, a computer-aided drug design (CADD) approach was implemented for the initial screening of 13 approved antiviral drugs. Molecular docking of 13 antivirals against the 3-chymotrypsin-like cysteine protease (3CLpro) enzyme was accomplished, and indinavir was described as a lead drug with a docking score of -8.824 and a XP Gscore of -9.466 kcal/mol. Indinavir possesses an important pharmacophore, hydroxyethylamine (HEA), and thus, a new library of HEA compounds (>2500) was subjected to virtual screening that led to 25 hits with a docking score more than indinavir. Exclusively, compound 16 with a docking score of -8.955 adhered to drug-like parameters, and the structure-activity relationship (SAR) analysis was demonstrated to highlight the importance of chemical scaffolds therein. Molecular dynamics (MD) simulation analysis performed at 100 ns supported the stability of 16 within the binding pocket. Largely, our results supported that this novel compound 16 binds with domains I and II, and the domain II-III linker of the 3CLpro protein, suggesting its suitability as a strong candidate for therapeutic discovery against COVID-19.