Azafuramidines as potential anticancer Agents: Pro-apoptotic profile and cell cycle arrest.

The current study aimed to test the antiproliferative activity of three azafuramidines (X, Y, and Z) against three different human cell lines; liver HepG2, breast MCF-7, and bone U2OS. And to explore the molecular mechanism(s) of the antiproliferative activity of these derivatives. The three new azafuramidines demonstrated a potent cytotoxicity at < 2 µM against the three cell lines investigated. The azafuramidines were highly selective with selectivity index âˆ¼ 47 - 61 folds indicating safety to the normal cells. In the scratch assay, azafuramidines significantly reduced the percentage of wound healing indicating ability to prevent or reduce metastasis. Derivatives X and Z arrested the HepG2 cells at S and G2/M phases detected by the flow cytometry. Derivatives X, Y, and Z elevated the apoptosis of HepG2 cells by âˆ¼ 71 %, 66 %, and 59 %, respectively. Derivatives X and Z were superior to derivative Y. The potent antiproliferative, cell cycle arrest, and pro-apoptotic efficacy of these chlorophenyl derivatives could be attributed to their ability of inducing the overexpression of p53, p21, and p27. These derivatives had the potential to act as anticancer agents and merit further investigations.

In Silico and In Vitro Evaluation of Some Amidine Derivatives as Hit Compounds towards Development of Inhibitors against Coronavirus Diseases.

Coronaviruses, including SARS-CoV-2, SARS-CoV, MERS-CoV and influenza A virus, require the host proteases to mediate viral entry into cells. Rather than targeting the continuously mutating viral proteins, targeting the conserved host-based entry mechanism could offer advantages. Nafamostat and camostat were discovered as covalent inhibitors of TMPRSS2 protease involved in viral entry. To circumvent their limitations, a reversible inhibitor might be required. Considering nafamostat structure and using pentamidine as a starting point, a small set of structurally diverse rigid analogues were designed and evaluated in silico to guide selection of compounds to be prepared for biological evaluation. Based on the results of in silico study, six compounds were prepared and evaluated in vitro. At the enzyme level, compounds 10-12 triggered potential TMPRSS2 inhibition with low micromolar IC50 concentrations, but they were less effective in cellular assays. Meanwhile, compound 14 did not trigger potential TMPRSS2 inhibition at the enzyme level, but it showed potential cellular activity regarding inhibition of membrane fusion with a low micromolar IC50 value of 10.87 µM, suggesting its action could be mediated by another molecular target. Furthermore, in vitro evaluation showed that compound 14 inhibited pseudovirus entry as well as thrombin and factor Xa. Together, this study presents compound 14 as a hit compound that might serve as a starting point for developing potential viral entry inhibitors with possible application against coronaviruses.

Investigation of the effect of structure modification of furamidine on the DNA minor groove binding and antiprotozoal activity.

New analogs of the antiprotozoal agent Furamidine were prepared utilizing Stille coupling reactions and amidation of the bisnitrile intermediate using lithium bis-trimethylsilylamide. Both the phenyl groups and the furan moiety of furamidine were replaced by heterocycles including thiophene, selenophene, indole or benzimidazole. Based upon the ΔTm and the CD results, the new compounds showed strong binding to the DNA minor groove. The new analogues are also more active both in vitro and in vivo than furamidine. Compounds 7a, 7b, and 7f showed the highest activity in vivo by curing 75% of animals, and this merits further evaluation.

Multivalent Benzamidine Molecules for Plasmin Inhibition: Effect of Valency and Linker Length.

There is an emerging interest in utilizing synthetic multivalent inhibitors that comprise of multiple inhibitor moieties linked on a common scaffold to achieve strong and selective enzyme inhibition. As multivalent inhibition is impacted by valency and linker length, in this study, we explore the effect of multivalent benzamidine inhibitors of varying valency and linker length on plasmin inhibition. Plasmin is an endogenous enzyme responsible for digesting fibrin present in blood clots. Monovalent plasmin(ogen) inhibitors are utilized clinically to treat hyperfibrinolysis-associated bleeding events. Benzamidine is a reversible inhibitor that binds to plasmin's active site. Herein, multivalent benzamidine inhibitors of varying valencies (mono-, bi- and tri-valent) and linker lengths (∼1-12 nm) were synthesized to systematically study their effect on plasmin inhibition. Inhibition assays were performed using a plasmin substrate (S-2251) to determine inhibition constants (Ki). Pentamidine (shortest bivalent) and Tri-AMB (shortest trivalent) were the strongest inhibitors with Ki values of 2.1±0.8 and 3.9±1.7 µM, respectively. Overall, increasing valency and decreasing linker length, increases effective local concentration of the inhibitor and therefore, resulted in stronger inhibition of plasmin via statistical rebinding. This study aids in the design of multivalent inhibitors that can achieve desired enzyme inhibition by means of modulating valency and linker length.

Nafamostat mesylate as a broad-spectrum candidate for the treatment of flavivirus infections by targeting envelope proteins.

Epidemics caused by flaviviruses occur globally; however, no antiviral drugs treating flaviviruses infections have yet been developed. Nafamostat (NM) is a protease inhibitor approved for pancreatitis and anti-coagulation. The anti-flavivirus potential of NM has yet to be determined. Here, utilizing in vitro and in vivo infection assays, we present that NM effectively inhibits Zika virus (ZIKV) and other flaviviruses in vitro. NM inhibited the production of ZIKV viral RNA and proteins originating from Asia and African lineage in human-, mouse- and monkey-derived cell lines and the in vivo anti-ZIKV efficacy of NM was verified. Mode-of-action analysis using time-of-drug-addition assay, infectivity inhibition assay, surface plasmon resonance assay, and molecular docking revealed that NM interacted with viral particles and blocked the early stage of infection by targeting the domain III of ZIKV envelope protein. Analysing the anti-flavivirus effects of NM-related compounds suggested that the antiviral effect depended on the unique structure of NM. These findings suggest the potential use of NM as an anti-flavivirus candidate, and a novel drug design approach targeting the flavivirus envelope protein.

In Silico Analysis and Synthesis of Nafamostat Derivatives and Evaluation of Their Anti-SARS-CoV-2 Activity.

Inhibition of transmembrane serine protease 2 (TMPRSS2) is expected to block the spike protein-mediated fusion of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Nafamostat, a potent TMPRSS2 inhibitor as well as a candidate for anti-SARS-CoV-2 drug, possesses the same acyl substructure as camostat, but is known to have a greater antiviral effect. A unique aspect of the molecular binding of nafamostat has been recently reported to be the formation of a covalent bond between its acyl substructure and Ser441 in TMPRSS2. In this study, we investigated crucial elements that cause the difference in anti-SARS-CoV-2 activity of nafamostat and camostat. In silico analysis showed that Asp435 significantly contributes to the binding of nafamostat and camostat to TMPRSS2, while Glu299 interacts strongly only with nafamostat. The estimated binding affinity for each compound with TMPRSS2 was actually consistent with the higher activity of nafamostat; however, the evaluation of the newly synthesized nafamostat derivatives revealed that the predicted binding affinity did not correlate with their anti-SARS-CoV-2 activity measured by the cytopathic effect (CPE) inhibition assay. It was further shown that the substitution of the ester bond with amide bond in nafamostat resulted in significantly weakened anti-SARS-CoV-2 activity. These results strongly indicate that the ease of covalent bond formation with Ser441 in TMPRSS2 possibly plays a major role in the anti-SARS-CoV-2 effect of nafamostat and its derivatives.

Mechanisms of NMDA receptor inhibition by nafamostat, gabexate and furamidine.

N-methyl-D-aspartate (NMDA) receptors are affected by many pharmaceuticals. In this work, we studied the action of the serine protease inhibitors nafamostat, gabexate and camostat, and an antiprotozoal compound, furamidine, on native NMDA receptors in rat hippocampal pyramidal neurons. Nafamostat, furamidine and gabexate inhibited these receptors with IC50 values of 0.20 ± 0.04, 0.64 ± 0.13 and 16 ± 3 µM, respectively, whereas camostat was ineffective. Nafamostat and furamidine showed voltage-dependent inhibition, while gabexate showed practically voltage-independent inhibition. Nafamostat and furamidine demonstrated tail currents, implying a 'foot-in-the-door' mechanism of action; gabexate did not demonstrate any signs of 'foot-in-the-door' or trapping channel block. Gabexate action was also not competitive, suggesting allosteric inhibition of NMDA receptors. Furamidine and nafamostat are structurally similar to the previously studied diminazene and all three demonstrated a 'foot-in-the-door' mechanism. They have a rather rigid, elongated structures and cannot fold into more compact forms. By contrast, the gabexate molecule can fold, but its folded structure differs drastically from that of typical NMDA receptor blockers, in agreement with its voltage-independent inhibition. These findings provide a better understanding of the structural determinants of NMDA receptor antagonism, while also supporting the potential clinical repurposing of these drugs as neuroprotectors for glaucoma and other neurodegenerative diseases.

The blood-brain barrier is dysregulated in COVID-19 and serves as a CNS entry route for SARS-CoV-2.

Neurological complications are common in COVID-19. Although SARS-CoV-2 has been detected in patients' brain tissues, its entry routes and resulting consequences are not well understood. Here, we show a pronounced upregulation of interferon signaling pathways of the neurovascular unit in fatal COVID-19. By investigating the susceptibility of human induced pluripotent stem cell (hiPSC)-derived brain capillary endothelial-like cells (BCECs) to SARS-CoV-2 infection, we found that BCECs were infected and recapitulated transcriptional changes detected in vivo. While BCECs were not compromised in their paracellular tightness, we found SARS-CoV-2 in the basolateral compartment in transwell assays after apical infection, suggesting active replication and transcellular transport of virus across the blood-brain barrier (BBB) in vitro. Moreover, entry of SARS-CoV-2 into BCECs could be reduced by anti-spike-, anti-angiotensin-converting enzyme 2 (ACE2)-, and anti-neuropilin-1 (NRP1)-specific antibodies or the transmembrane protease serine subtype 2 (TMPRSS2) inhibitor nafamostat. Together, our data provide strong support for SARS-CoV-2 brain entry across the BBB resulting in increased interferon signaling.

Nafamostat reduces systemic inflammation in TLR7-mediated virus-like illness.

BACKGROUND: The serine protease inhibitor nafamostat has been proposed as a treatment for COVID-19, by inhibiting TMPRSS2-mediated viral cell entry. Nafamostat has been shown to have other, immunomodulatory effects, which may be beneficial for treatment, however animal models of ssRNA virus infection are lacking. In this study, we examined the potential of the dual TLR7/8 agonist R848 to mimic the host response to an ssRNA virus infection and the associated behavioural response. In addition, we evaluated the anti-inflammatory effects of nafamostat in this model. METHODS: CD-1 mice received an intraperitoneal injection of R848 (200 µg, prepared in DMSO, diluted 1:10 in saline) or diluted DMSO alone, and an intravenous injection of either nafamostat (100 µL, 3 mg/kg in 5% dextrose) or 5% dextrose alone. Sickness behaviour was determined by temperature, food intake, sucrose preference test, open field and forced swim test. Blood and fresh liver, lung and brain were collected 6 h post-challenge to measure markers of peripheral and central inflammation by blood analysis, immunohistochemistry and qPCR. RESULTS: R848 induced a robust inflammatory response, as evidenced by increased expression of TNF, IFN-γ, CXCL1 and CXCL10 in the liver, lung and brain, as well as a sickness behaviour phenotype. Exogenous administration of nafamostat suppressed the hepatic inflammatory response, significantly reducing TNF and IFN-γ expression, but had no effect on lung or brain cytokine production. R848 administration depleted circulating leukocytes, which was restored by nafamostat treatment. CONCLUSIONS: Our data indicate that R848 administration provides a useful model of ssRNA virus infection, which induces inflammation in the periphery and CNS, and virus infection-like illness. In turn, we show that nafamostat has a systemic anti-inflammatory effect in the presence of the TLR7/8 agonist. Therefore, the results indicate that nafamostat has anti-inflammatory actions, beyond its ability to inhibit TMPRSS2, that might potentiate its anti-viral actions in pathologies such as COVID-19.

Nafamostat protects against early brain injury after subarachnoid hemorrhage in mice.

This study aimed to evaluate the effects of nafamostat, a serin protease inhibitor, in the management of subarachnoid hemorrhage (SAH). SAH was induced by endovascular perforation in male mice. Nafamostat was administered intraperitoneally four times immediately after SAH induction. Cerebral blood flow, neurological behavior tests, SAH grade and protein expression were evaluated at 24 h after SAH induction. In the in vitro model, human brain microvascular endothelial cells (HBMVECs), HBVECs were exposed to thrombin and hypoxia for 24 h; nafamostat was administered and the protein expression was evaluated. Eighty-eight mice were included in the in vivo study. Fifteen mice (17%) were excluded because of death or procedure failure. Nafamostat exerted no significant effect on the SAH grade or cerebral blood flow; however, it improved the neurological behavior and suppressed the thrombin and MMP-9 expression. In addition, nafamostat suppressed the ICAM-1 expression and p38 phosphorylation in the in vitro study. Nafamostat has a protective effect against HBMVEC after exposure to thrombin and hypoxia, suggesting its role in improving the neurological outcomes after SAH. These findings indicate that nafamostat has the potential to be a novel therapeutic drug in the management of SAH.

Nafamostat-Interferon-α Combination Suppresses SARS-CoV-2 Infection In Vitro and In Vivo by Cooperatively Targeting Host TMPRSS2.

SARS-CoV-2 and its vaccine/immune-escaping variants continue to pose a serious threat to public health due to a paucity of effective, rapidly deployable, and widely available treatments. Here, we address these challenges by combining Pegasys (IFNα) and nafamostat to effectively suppress SARS-CoV-2 infection in cell culture and hamsters. Our results indicate that Serpin E1 is an important mediator of the antiviral activity of IFNα and that both Serpin E1 and nafamostat can target the same cellular factor TMPRSS2, which plays a critical role in viral replication. The low doses of the drugs in combination may have several clinical advantages, including fewer adverse events and improved patient outcome. Thus, our study may provide a proactive solution for the ongoing pandemic and potential future coronavirus outbreaks, which is still urgently required in many parts of the world.

Clinically Relevant Influenza Virus Evolution Reconstituted in a Human Lung Airway-on-a-Chip.

Human-to-human transmission of viruses, such as influenza viruses and coronaviruses, can promote virus evolution and the emergence of new strains with increased potential for creating pandemics. Clinical studies analyzing how a particular type of virus progressively evolves new traits, such as resistance to antiviral therapies, as a result of passing between different human hosts are difficult to carry out because of the complexity, scale, and cost of the challenge. Here, we demonstrate that spontaneous evolution of influenza A virus through both mutation and gene reassortment can be reconstituted in vitro by sequentially passaging infected mucus droplets between multiple human lung airway-on-a-chip microfluidic culture devices (airway chips). Modeling human-to-human transmission of influenza virus infection on chips in the continued presence of the antiviral drugs amantadine or oseltamivir led to the spontaneous emergence of clinically prevalent resistance mutations, and strains that were resistant to both drugs were identified when they were administered in combination. In contrast, we found that nafamostat, an inhibitor targeting host serine proteases, did not induce viral resistance. This human preclinical model may be useful for studying viral evolution in vitro and identifying potential influenza virus variants before they appear in human populations, thereby enabling preemptive design of new and more effective vaccines and therapeutics. IMPORTANCE The rapid evolution of viruses, such as influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), is challenging the use and development of antivirals and vaccines. Studies of within-host viral evolution can contribute to our understanding of the evolutionary and epidemiological factors that shape viral global evolution as well as development of better antivirals and vaccines. However, little is known about how viral evolution of resistance to antivirals occurs clinically due to the lack of preclinical models that can faithfully model influenza infection in humans. Our study shows that influenza viral evolution through mutation or gene reassortment can be recapitulated in a human lung airway-on-a-chip (airway chip) microfluidic culture device that can faithfully recapitulate the influenza infection in vitro. This approach is useful for studying within-host viral evolution, evaluating viral drug resistance, and identifying potential influenza virus variants before they appear in human populations, thereby enabling the preemptive design of new and more effective vaccines and therapeutics.

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.

Interrogating the Inhibition Mechanisms of Human Aldehyde Oxidase by X-ray Crystallography and NMR Spectroscopy: The Raloxifene Case.

Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.

Synergistic Block of SARS-CoV-2 Infection by Combined Drug Inhibition of the Host Entry Factors PIKfyve Kinase and TMPRSS2 Protease.

Repurposing FDA-approved inhibitors able to prevent infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) could provide a rapid path to establish new therapeutic options to mitigate the effects of coronavirus disease 2019 (COVID-19). Proteolytic cleavages of the spike (S) protein of SARS-CoV-2, mediated by the host cell proteases cathepsin and TMPRSS2, alone or in combination, are key early activation steps required for efficient infection. The PIKfyve kinase inhibitor apilimod interferes with late endosomal viral traffic and through an ill-defined mechanism prevents in vitro infection through late endosomes mediated by cathepsin. Similarly, inhibition of TMPRSS2 protease activity by camostat mesylate or nafamostat mesylate prevents infection mediated by the TMPRSS2-dependent and cathepsin-independent pathway. Here, we combined the use of apilimod with camostat mesylate or nafamostat mesylate and found an unexpected ∼5- to 10-fold increase in their effectiveness to prevent SARS-CoV-2 infection in different cell types. Comparable synergism was observed using both a chimeric vesicular stomatitis virus (VSV) containing S of SARS-CoV-2 (VSV-SARS-CoV-2) and SARS-CoV-2. The substantial ∼5-fold or higher decrease of the half-maximal effective concentrations (EC50s) suggests a plausible treatment strategy based on the combined use of these inhibitors. IMPORTANCE Infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is causing the coronavirus disease 2019 (COVID-2019) global pandemic. There are ongoing efforts to uncover effective antiviral agents that could mitigate the severity of the disease by controlling the ensuing viral replication. Promising candidates include small molecules that inhibit the enzymatic activities of host proteins, thus preventing SARS-CoV-2 entry and infection. They include apilimod, an inhibitor of PIKfyve kinase, and camostat mesylate and nafamostat mesylate, inhibitors of TMPRSS2 protease. Our research is significant for having uncovered an unexpected synergism in the effective inhibitory activity of apilimod used together with camostat mesylate or nafamostat mesylate.

TMPRSS2 Activates Hemagglutinin-Esterase Glycoprotein of Influenza C Virus.

Influenza C virus (ICV) has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein. HE functions similarly to hemagglutinin (HA) and neuraminidase of the influenza A and B viruses (IAV and IBV, respectively). It has a monobasic site, which is cleaved by some host enzymes. The cleavage is essential to activating the virus, but the enzyme or enzymes in the respiratory tract have not been identified. This study investigated whether the host serine proteases, transmembrane protease serine S1 member 2 (TMPRSS2) and human airway trypsin-like protease (HAT), which reportedly cleave HA of IAV/IBV, are involved in HE cleavage. We established TMPRSS2- and HAT-expressing MDCK cells (MDCK-TMPRSS2 and MDCK-HAT). ICV showed multicycle replication with HE cleavage without trypsin in MDCK-TMPRSS2 cells as well as IAV did. The HE cleavage and multicycle replication did not appear in MDCK-HAT cells infected with ICV without trypsin, while HA cleavage and multistep growth of IAV appeared in the cells. Amino acid sequences of the HE cleavage site in 352 ICV strains were completely preserved. Camostat and nafamostat suppressed the growth of ICV and IAV in human nasal surface epithelial (HNE) cells. Therefore, this study revealed that, at least, TMPRSS2 is involved in HE cleavage and suggested that nafamostat could be a candidate for therapeutic drugs for ICV infection. IMPORTANCE Influenza C virus (ICV) is a pathogen that causes acute respiratory illness, mostly in children, but there are no anti-ICV drugs. ICV has only one kind of spike protein, the hemagglutinin-esterase (HE) glycoprotein on the virion surface, which possesses receptor-binding, receptor-destroying, and membrane fusion activities. The HE cleavage is essential for the virus to be activated, but the enzyme or enzymes in the respiratory tract have not been identified. This study revealed that transmembrane protease serine S1 member 2 (TMPRSS2), and not human airway trypsin-like protease (HAT), is involved in HE cleavage. This is a novel study on the host enzymes involved in HE cleavage, and the result suggests that the host enzymes, such as TMPRSS2, may be a target for therapeutic drugs of ICV infection.

SARS-CoV-2 proteins and anti-COVID-19 drugs induce lytic reactivation of an oncogenic virus.

An outbreak of the novel coronavirus SARS-CoV-2, the causative agent of Coronavirus Disease-2019 (COVID-19), a respiratory disease, has infected almost one hundred million people since the end of 2019, killed over two million, and caused worldwide social and economic disruption. Because the mechanisms of SARS-CoV-2 infection of host cells and its pathogenesis remain largely unclear, there are currently no antiviral drugs with proven efficacy. Besides severe respiratory and systematic symptoms, several comorbidities increase risk of fatal disease outcome. Therefore, it is required to investigate the impacts of COVID-19 on pre-existing diseases of patients, such as cancer and other infectious diseases. In the current study, we report that SARS-CoV-2 encoded proteins and some currently used anti-COVID-19 drugs are able to induce lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV), one of major human oncogenic viruses, through manipulation of intracellular signaling pathways. Our data indicate that those KSHV + patients especially in endemic areas exposure to COVID-19 or undergoing the treatment may have increased risks to develop virus-associated cancers, even after they have fully recovered from COVID-19.

CAG RNAs induce DNA damage and apoptosis by silencing NUDT16 expression in polyglutamine degeneration.

DNA damage plays a central role in the cellular pathogenesis of polyglutamine (polyQ) diseases, including Huntington's disease (HD). In this study, we showed that the expression of untranslatable expanded CAG RNA per se induced the cellular DNA damage response pathway. By means of RNA sequencing (RNA-seq), we found that expression of the Nudix hydrolase 16 (NUDT16) gene was down-regulated in mutant CAG RNA-expressing cells. The loss of NUDT16 function results in a misincorporation of damaging nucleotides into DNAs and leads to DNA damage. We showed that small CAG (sCAG) RNAs, species generated from expanded CAG transcripts, hybridize with CUG-containing NUDT16 mRNA and form a CAG-CUG RNA heteroduplex, resulting in gene silencing of NUDT16 and leading to the DNA damage and cellular apoptosis. These results were further validated using expanded CAG RNA-expressing mouse primary neurons and in vivo R6/2 HD transgenic mice. Moreover, we identified a bisamidinium compound, DB213, that interacts specifically with the major groove of the CAG RNA homoduplex and disfavors the CAG-CUG heteroduplex formation. This action subsequently mitigated RNA-induced silencing complex (RISC)-dependent NUDT16 silencing in both in vitro cell and in vivo mouse disease models. After DB213 treatment, DNA damage, apoptosis, and locomotor defects were rescued in HD mice. This work establishes NUDT16 deficiency by CAG repeat RNAs as a pathogenic mechanism of polyQ diseases and as a potential therapeutic direction for HD and other polyQ diseases.

Strong Binding of Leupeptin with TMPRSS2 Protease May Be an Alternative to Camostat and Nafamostat for SARS-CoV-2 Repurposed Drug: Evaluation from Molecular Docking and Molecular Dynamics Simulations.

The unprecedented coronavirus SARS-CoV-2 outbreak at Wuhan, China, caused acute respiratory infection to humans. There is no precise vaccine/therapeutic agents available to combat the COVID-19 disease. Some repurposed drugs are saving the life of diseased, but the complete cure is relatively less. Several drug targets have been reported to inhibit the SARS-CoV-2 virus infection, in that TMPRSS2 (transmembrane protease serine 2) is one of the potential targets; inhibiting this protease stops the virus entry into the host human cell. Camostat mesylate, nafamostat, and leupeptin are the drugs, in which the first two drugs are being used for COVID-19 and leupeptin also tested. To consider these drugs as the repurposed drug for COVID-19, it is essential to understand their binding affinity and stability with TMPRSS2. In the present study, we performed the molecular docking and molecular dynamics (MD) simulation of these molecules with the TMPRSS2. The docking study reveals that leupeptin molecule strongly binds with TMPRSS2 protein than the other two drug molecules. The RMSD and RMSF values of MD simulation confirm that leupeptin and the amino acids of TMPRSS2 are very stable than the other two molecules. Furthermore, leupeptin forms interactions with the key amino acids of TMPRSS2 and the same have been maintained during the MD simulations. This structural and dynamical information is useful to evaluate these drugs to be used as repurposed drugs, however, the strong binding profile of leupeptin with TMPRSS2, suggests, it may be considered as a repurposed drug for COVID-19 disease after clinical trial.

The clinically used serine protease inhibitor nafamostat reduces influenza virus replication and cytokine production in human airway epithelial cells and viral replication in mice.

The effects of the clinically used protease inhibitor nafamostat on influenza virus replication have not been well studied. Primary human tracheal (HTE) and nasal (HNE) epithelial cells were pretreated with nafamostat and infected with the 2009 pandemic [A/Sendai-H/108/2009/(H1N1) pdm09] or seasonal [A/New York/55/2004(H3N2)] influenza virus. Pretreatment with nafamostat reduced the titers of the pandemic and seasonal influenza viruses and the secretion of inflammatory cytokines, including interleukin-6 and tumor necrosis factor-α, in the supernatants of the cells infected with the pandemic influenza virus. HTE and HNE cells exhibited mRNA and/or protein expression of transmembrane protease serine 2 (TMPRSS2), TMPRSS4, and TMPRSS11D. Pretreatment with nafamostat reduced cleavage of the precursor protein HA0 of the pandemic influenza virus into subunit HA1 in HTE cells and reduced the number of acidic endosomes in HTE and HNE cells where influenza virus RNA enters the cytoplasm. Additionally, nafamostat (30 mg/kg/day, intraperitoneal administration) reduced the levels of the pandemic influenza virus [A/Hyogo/YS/2011 (H1N1) pdm09] in mouse lung washes. These findings suggest that nafamostat may inhibit influenza virus replication in human airway epithelial cells and mouse lungs and reduce infection-induced airway inflammation by modulating cytokine production.