Ferrocenoyl-substituted quinolinone and coumarin as organometallic inhibitors of SARS-CoV-2 3CLpro main protease.

The 3-chymotrypsin-like protease 3CLpro from severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) is a potential target for antiviral drug development. In this work, three organometallic ferrocene-modified quinolinones and coumarins were compared to their benzoic acid ester analogues with regard to inhibition of 3CLpro using an HPLC-based assay with a 15mer model peptide as the substrate. In contrast to FRET-based assays, this allows direct identification of interference of buffer constituents with the inhibitors, as demonstrated by the complete abolishment of ebselen inhibitory activity in the presence of dithiothreitol as a redox protectant. The presence of the organometallic ferrocene moiety significantly increased the stability of the title compounds towards hydrolysis. Among the studied compounds, 4-ferrocenyloxy-1-methyl-quinol-2-one was identified as the most stable and potent inhibitor candidate. IC50 values determined for ebselen and this sandwich complex compound are (0.40 ± 0.07) and (2.32 ± 0.21) µM, respectively.

Clinical Trial Subgroup Analyses to Investigate Clinical and Immunological Outcomes of Convalescent Plasma Therapy in Severe COVID-19.

Objective: To assess the clinical and immunological benefits of passive immunization using convalescent plasma therapy (CPT). Materials and Methods: A series of subclass analyses were performed on the previously published outcome data and accompanying clinical metadata from a completed randomized controlled trial (RCT) (Clinical Trial Registry of India, number CTRI/2020/05/025209). The subclass analyses were performed on the outcome data and accompanying clinical metadata from a completed RCT (patient recruitment between May 15, 2020 and October 31, 2020). Data on the plasma abundance of a large panel of cytokines from the same cohort of patients were also used to characterize the heterogeneity of the putative anti-inflammatory function of convalescent plasma (CP) in addition to passively providing neutralizing antibodies. Results: Although the primary clinical outcomes were not significantly different in the RCT across all age groups, significant immediate mitigation of hypoxia, reduction in hospital stay, and significant survival benefit were registered in younger (<67 years in our cohort) patients with severe coronavirus disease 2019 and acute respiratory distress syndrome on receiving CPT. In addition to neutralizing the antibody content of CP, its anti-inflammatory proteome, by attenuation of the systemic cytokine deluge, significantly contributed to the clinical benefits of CPT. Conclusion: Subgroup analyses revealed that clinical benefits of CPT in severe coronavirus disease 2019 are linked to the anti-inflammatory protein content of CP apart from the anti-severe acute respiratory syndrome coronavirus 2 neutralizing antibody content.

Essentials of COVID-19 and treatment approaches

The coronavirus family is as old as the 1930s when it first showed symptoms in chicken. The virus thereafter kept evolving and it has significantly taken over a large percentage of people worldwide in the form of this new pandemic. As of the present day, there is no treatment available for coronavirus disease 2019 (COVID-19) (caused by the severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2]), although supportive therapy and preventive measures have shown a tremendous control rate among certain patients. Drugs like remdesivir, camostat, nafamostat, ritonavir/lopinavir, several monoclonal antibodies, and CPs are in their early phases of trials. There are approved by the WHO under an emergency use authorization program. Favipiravir has entered its phase 3 clinical trial and is supported by evidence to show no or less adverse effects in patients infected with SARS-CoV-2. Vaccine development is accelerating its pace, and vaccines will probably become available by the end of the year 2020. © 2022 Elsevier Inc.

In Silico Molecular Characterization of Human TMPRSS2 Protease Polymorphic Variants and Associated SARS-CoV-2 Susceptibility.

The 2019 coronavirus disease (COVID-19) pandemic continues to challenge health care systems worldwide. Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been responsible for the cause of global pandemic. Type 2 transmembrane serine protease (TMPRSS2) is important in the cell entry and spread of SARS-CoV-2 and plays a crucial role in the proteolytic cleavage of SARS-CoV-2 spike (S) glycoprotein. Here, using reported structural data, we analyzed the molecular complex of TMPRSS2 and the S glycoprotein and further examined intermolecular interactions of natural TMPRSS2 polymorphic variants. We identified several TMPRSS2 variants that could possibly alter host susceptibility to the SARS-CoV-2 infection. Molecular docking analysis revealed that G462D/G462S variants were predicted to be protective variants, whereas Q438E and S339F variants were predicted to increase susceptibility. In addition, we examined intermolecular interactions between TMPRSS2 and its two potential serine protease inhibitors, camostat mesylate and nafamostat. Further, we investigated the effect of TMPRSS2 variants on these interactions. Our structural analysis revealed that G462D, C297S and S460R variants had possibly altered the interactions with the protease inhibitors. Our results identified important TMPRSS2 variations that could be useful to develop high affinity and personalized drugs for treating COVID-19 patients.

Approaches to the Potential Therapy of COVID-19: A General Overview from the Medicinal Chemistry Perspective.

In spite of advances in vaccination, control of the COVID-19 pandemic will require the use of pharmacological treatments against SARS-CoV2. Their development needs to consider the existence of two phases in the disease, namely the viral infection and the inflammatory stages. The main targets for antiviral therapeutic intervention are: (a) viral proteins, including the spike (S) protein characteristic of the viral cover and the viral proteases in charge of processing the polyprotein arising from viral genome translation; (b) host proteins, such as those involved in the processes related to viral entry into the host cell and the release of the viral genome inside the cell, the elongation factor eEF1A and importins. The use of antivirals targeted at host proteins is less developed but it has the potential advantage of not being affected by mutations in the genome of the virus and therefore being active against all its variants. Regarding drugs that address the hyperinflammatory phase of the disease triggered by the so-called cytokine storm, the following strategies are particularly relevant: (a) drugs targeting JAK kinases; (b) sphingosine kinase 2 inhibitors; (c) antibodies against interleukin 6 or its receptor; (d) use of the traditional anti-inflammatory corticosteroids.

Complement Factor D as a Strategic Target for Regulating the Alternative Complement Pathway.

The complement system is central to first-line defense against invading pathogens. However, excessive complement activation and/or the loss of complement regulation contributes to the development of autoimmune diseases, systemic inflammation, and thrombosis. One of the three pathways of the complement system, the alternative complement pathway, plays a vital role in amplifying complement activation and pathway signaling. Complement factor D, a serine protease of this pathway that is required for the formation of C3 convertase, is the rate-limiting enzyme. In this review, we discuss the function of factor D within the alternative pathway and its implication in both healthy physiology and disease. Because the alternative pathway has a role in many diseases that are characterized by excessive or poorly mediated complement activation, this pathway is an enticing target for effective therapeutic intervention. Nonetheless, although the underlying disease mechanisms of many of these complement-driven diseases are quite well understood, some of the diseases have limited treatment options or no approved treatments at all. Therefore, in this review we explore factor D as a strategic target for advancing therapeutic control of pathological complement activation.

The TMPRSS2 Inhibitor Nafamostat Reduces SARS-CoV-2 Pulmonary Infection in Mouse Models of COVID-19.

The coronavirus disease 2019 (COVID-19) pandemic has caused significant morbidity and mortality on a global scale. The etiologic agent, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), initiates host cell entry when its spike protein (S) binds to its receptor, angiotensin-converting enzyme 2 (ACE2). In airway epithelia, the spike protein is cleaved by the cell surface protease TMPRSS2, facilitating membrane fusion and entry at the cell surface. This dependence on TMPRSS2 and related proteases suggests that protease inhibitors might limit SARS-CoV-2 infection in the respiratory tract. Here, we tested two serine protease inhibitors, camostat mesylate and nafamostat mesylate, for their ability to inhibit entry of SARS-CoV-2 and that of a second pathogenic coronavirus, Middle East respiratory syndrome coronavirus (MERS-CoV). Both camostat and nafamostat reduced infection in primary human airway epithelia and in the Calu-3 2B4 cell line, with nafamostat exhibiting greater potency. We then assessed whether nafamostat was protective against SARS-CoV-2 in vivo using two mouse models. In mice sensitized to SARS-CoV-2 infection by transduction with human ACE2, intranasal nafamostat treatment prior to or shortly after SARS-CoV-2 infection significantly reduced weight loss and lung tissue titers. Similarly, prophylactic intranasal treatment with nafamostat reduced weight loss, viral burden, and mortality in K18-hACE2 transgenic mice. These findings establish nafamostat as a candidate for the prevention or treatment of SARS-CoV-2 infection and disease pathogenesis. IMPORTANCE The causative agent of COVID-19, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), requires host cell surface proteases for membrane fusion and entry into airway epithelia. We tested the hypothesis that inhibitors of these proteases, the serine protease inhibitors camostat and nafamostat, block infection by SARS-CoV-2. We found that both camostat and nafamostat reduce infection in human airway epithelia, with nafamostat showing greater potency. We then asked whether nafamostat protects mice against SARS-CoV-2 infection and subsequent COVID-19 lung disease. We performed infections in mice made susceptible to SARS-CoV-2 infection by introducing the human version of ACE2, the SARS-CoV-2 receptor, into their airway epithelia. We observed that pretreating these mice with nafamostat prior to SARS-CoV-2 infection resulted in better outcomes, in the form of less virus-induced weight loss, viral replication, and mortality than that observed in the untreated control mice. These results provide preclinical evidence for the efficacy of nafamostat in treating and/or preventing COVID-19.

Exposure of human intestinal epithelial cells and primary human hepatocytes to trypsin-like serine protease inhibitors with potential antiviral effect.

Human intestinal epithelial cell line-6 (HIEC-6) cells and primary human hepatocytes (PHHs) were treated with 3-amidinophenylalanine-derived inhibitors of trypsin-like serine proteases for 24 hours. It was proven that treatment with MI-1900 and MI-1907 was tolerated up to 50 µM in HIEC-6. These inhibitors did not cause elevations in extracellular H2O2 levels and in the concentrations of interleukin (IL)-6 and IL-8 and did not alter occludin distribution in HIEC-6. It was also found that MI-1900 and MI-1907 up to 50 µM did not affect cell viability, IL-6 and IL-8 and occludin levels of PHH. Based on our findings, these inhibitors could be safely applicable at 50 µM in HIEC-6 and in PHH; however, redox status was disturbed in case of PHH. Moreover, it has recently been demonstrated that MI-1900 prevents the replication and spread of the new SARS-CoV-2 in infected Calu-3 cells, most-likely via an inhibition of the membrane-bound host protease TMPRSS2.

The past, present and future of RNA respiratory viruses: influenza and coronaviruses.

Influenza virus and coronaviruses continue to cause pandemics across the globe. We now have a greater understanding of their functions. Unfortunately, the number of drugs in our armory to defend us against them is inadequate. This may require us to think about what mechanisms to address. Here, we review the biological properties of these viruses, their genetic evolution and antiviral therapies that can be used or have been attempted. We will describe several classes of drugs such as serine protease inhibitors, heparin, heparan sulfate receptor inhibitors, chelating agents, immunomodulators and many others. We also briefly describe some of the drug repurposing efforts that have taken place in an effort to rapidly identify molecules to treat patients with COVID-19. While we put a heavy emphasis on the past and present efforts, we also provide some thoughts about what we need to do to prepare for respiratory viral threats in the future.

Inhibiting fusion with cellular membrane system: therapeutic options to prevent severe acute respiratory syndrome coronavirus-2 infection.

Severe acute respiratory syndrome coronavirus (SARS-CoV), an enveloped virus with a positive-sense single-stranded RNA genome, facilitates the host cell entry through intricate interactions with proteins and lipids of the cell membrane. The detailed molecular mechanism involves binding to the host cell receptor and fusion at the plasma membrane or after being trafficked to late endosomes under favorable environmental conditions. A crucial event in the process is the proteolytic cleavage of the viral spike protein by the host's endogenous proteases that releases the fusion peptide enabling fusion with the host cellular membrane system. The present review details the mechanism of viral fusion with the host and highlights the therapeutic options that prevent SARS-CoV-2 entry in humans.