The emergence of the SARS-CoV-2 pandemic led to the spread of highly transmissible variants, such as the Delta variant, which originated in India, underscoring the urgent need to develop new antivirals, therapeutics, and vaccines. In our previous study, we showed that Membrane-Envelope Virus-like Particles exhibit antigenicity and neutralization activity. Hence, our present study was conducted to evaluate whether the M protein alone can form VLPs that elicit an immune response. Using computational methods, we identified key interacting residues in M-protein that contribute to VLP formation and interact with other structural proteins, including Spike (S), Nucleocapsid (N), and Envelope (E). The SARS-CoV-2-M protein was expressed in Sf-21 insect cells, and the resulting VLPs were purified, analyzed for shape and size, and characterized using DLS, FESEM, and TEM. The purified VLPs were injected into BALB/c mice to evaluate their immune response compared with uninfected controls. The biophysical analysis confirms that the particles are round and have a size of 180–200 nm. The serum levels of IgG, IgM, and IgA were found to be higher in immunized mice than in uninfected mice. Further qRT-PCR analysis demonstrated the levels of IFN-γ, IL-2, and IL-12, indicating a TH1-biased immune response against the M protein. Our study demonstrates that the highly conserved M protein can self-assemble into VLPs and elicit humoral and cellular immune response. Furthermore, our study indicates that while M-protein VLPs elicit significant antibodies and cytokine responses, they do not induce detectable neutralizing activity when given alone.

RdRp is a critical component of an RNA virus life cycle. Among coronaviruses, NSP12, along with one copy of NSP7 and two copies of NSP8, forms the RdRp holoenzyme and exhibits polymerase activity. While coronavirus RNA replication is sufficiently understood, the interplay among these NSPs and its influence on RNA binding and nascent strand synthesis remains poorly understood. Here, we reconstituted a functional RdRp holoenzyme using recombinant SARS-CoV-2 NSP12, NSP7, and NSP8 in vitro. Molecular interactions among NSPs and their effect on the polymerase activity were investigated, wherein NSP12 alone exhibited notable activity, which was further enhanced by the presence of both NSP7 and NSP8. The presence of only one cofactor, either NSP7 or NSP8, completely inhibited NSP12 activity and led to RNA template detachment. Computational analyses of different NSP12 complexes suggested that binding of NSP7 or NSP8 alone to NSP12 constricts the RNA entry channel, which was higher in the presence of NSP8, making it inappropriate for RNA entry/binding. We conclude that NSP7 and NSP8 together synergize to enhance the NSP12 activity, but antagonize when alone. These findings have implications for novel drug development, and compounds inhibiting NSP7 or NSP8 interactions with NSP12 can be lethal to coronavirus replication.

Hepatitis E virus (HEV) is a leading cause of acute viral hepatitis. Our earlier study reported the presence of an 87-nucleotide-long internal ribosome entry-site-like element (IRESl) in the genotype 1 (g1)-HEV genome, which mediated cap-independent translation of the viral ORF4 protein. RNA-protein interactome analysis of the HEV IRESl revealed its association with multiple host proteins, including translation regulatory proteins, which controlled its function. Role of HEV-encoded proteins in modulating the viral IRESl activity remains unknown. In the present study, we investigated the role of viral proteins in modulating the activity of the HEV IRESl element. Luciferase-reporter assay using a bicistronic vector revealed the ability of viral Helicase and MeT-Y-domain proteins in upregulating the HEV IRESl activity. Further studies confirmed direct interaction of the viral Helicase and MeT-Y-domain with the HEV IRESl. Collectively, these findings unravel the positive role of HEV Helicase and MeT-Y-domain proteins in modulating the viral IRESl activity.

The emergence of new coronavirus variants and concerns about vaccine effectiveness against these novel variants emphasize the need for broad-spectrum therapeutics targeting conserved coronaviral non-structural proteins. Accordingly, a virtual library of 178 putative inhibitors targeting SARS-CoV-2 Papain-like protease (PLpro) was compiled through a systematic review of published literature and subsequently screened using molecular docking. Selected hits were analyzed for protease inhibitory activities, binding strength, and antiviral activities against HCoV229E-based surrogate system and subsequently against SARS-CoV-2 for validation. Differences in potential modes of action were investigated using an HCoV229E-based system, combined with in silico and biophysical methods against SARS-CoV-2 system. Of the 178 hits, 13 molecules showed superior docking scores against PLpro and met the inclusion criteria for further investigations. Of these, seven showed notable inhibitory activities against PLpro. Particularly, both Psoralidin and Corylifol-A exhibited superior and, importantly, dual activities against SARS-CoV-2 Mpro. Both molecules were found to be biologically active against HCoV229E and SARS-CoV-2; however, Psoralidin exhibited more consistent effects and was relatively well-tolerated. Detailed in silico analyses of their interactions with the two proteases identified differences in their modes of action, primarily due to differences in their binding of PLpro. Based on these findings, we propose Psoralidin as a potential candidate for further development as a broad-spectrum antiviral and Corylifol-A as an ideal candidate for lead optimization.

In late December 2019, SARS-CoV-2 emerged in Wuhan, China. Since then, several variants have been identified. The delta variant, a variant of concern, originated in Lucknow, India. Since then, there has been an urgent need to develop effective therapeutics and vaccine candidates against SARS-CoV-2. Virus-Like Particles (VLPs) are promising vaccine candidates. The advantage of VLP-based vaccines is that they resemble viral structures. In this study, we developed SARS-CoV-2 Virus-Like Particles. The docking and MD simulations analysis confirmed the presence of strong interactions between M and E proteins. The purified VLPs were confirmed by TEM and FESEM, with a size range of 100-120 nm. Immunization of BALB/c mice with purified VLPs elicited a strong immune response, as measured by ELISA. The immunized mice serum showed high titers of IgG, IgM, and IFN-γ. qRT-PCR cytokine analysis showed a Th1/Th2 response. The virus-neutralization assay confirmed the presence of neutralizing antibodies in the sera of immunized mice. These results show that VLP-based vaccines can be effective against SARS-CoV-2 and constitute a scalable, safe, and effective vaccine platform.

Lung cancer, the second leading cause of cancer mortality, requires the development of novel therapeutic strategies due to emerging drug resistance and toxicity. With this objective, the present work explored the therapeutic potential of R. graveolens leaf extracts against EGFR_T790M-mediated drug resistance in NSCLC. To this end, we evaluated the functional and therapeutic potential of a panel of polar and non-polar solvent extracts using various in vitro assay systems. Among the extracts tested, EAE exhibited superior kinase inhibitory activity, which was more pronounced against the EGFR_T790M mutant phenotype. Accordingly, EAE exhibited a favorable cytotoxicity profile and potent growth inhibition of EGFR_T790M-positive NSCLC cells, as evident from its superior IC50 values in this cell type. Flow cytometry analysis further validated its inhibitory effects on the cell cycle and, well-supported by the data from the TUNEL assay, suggested induction of apoptosis in EAE-treated cells in a dose-dependent manner. Finally, mechanistic studies in EAE-treated cells showed that these outcomes were due to concentration-dependent inhibition of EGFR phosphorylation at Tyr1068 and Tyr1173. Importantly, this inhibition was consistently more pronounced in H1975 cells expressing the EGFR_T790M mutant phenotype. Further, pull-down assays, followed by mass spectrometry analysis, identified the most promising molecules within EAE. Together, the study highlighted the therapeutic potential of EAE from the leaves of Ruta graveolens for treating EGFR_T790M-mediated drug resistance in lung cancer.

OBJECTIVES:The rapid evolution of SARS-CoV-2 and the emergence of new variants have resulted in mutations in the Spike protein's receptor-binding domain (RBD), enhancing its binding affinity to the ACE2 receptor and increasing viral transmissibility. This study aims to identify inhibitors that can disrupt the Spike-ACE2 interaction, potentially preventing viral entry and immune evasion. The selected compounds may emerge as putative drugs following further studies. METHODS:FDA-approved compounds were screened using molecular docking and molecular dynamics (MD) simulations to identify potential inhibitors of the Spike-ACE2 interaction. The most promising candidates were further validated through in vitro assays, including sandwich ELISA and Microscale Thermophoresis, to assess their ability to reduce Spike-ACE2 complex formation. The shortlisted compounds were tested in a cell-based viral culture system to evaluate their impact on SARS-CoV-2 replication. RESULTS:Ten compounds were initially identified as potential inhibitors. Further in vitro validation narrowed the selection to five compounds that significantly reduced the formation of the Spike-ACE2 complex. Among them, Chrysin (a flavonoid) and Prednisolone (a corticosteroid) demonstrated the highest efficacy in suppressing the interaction, with IC50 values of 1.93 μM and 13.27 μM, respectively. Compounds could inhibit virus replication in the culture. CONCLUSIONS:Chrysin and Prednisolone emerged as the most effective inhibitors of the Spike-ACE2 interaction. Their potential to suppress SARS-CoV-2 replication suggests they could be valuable therapeutic candidates. Further studies, including animal model evaluations, are needed to explore their clinical applicability.

Entamoeba histolytica, a protozoan parasite, is the causative agent of amoebiasis, which is a significant global health concern. The virulence mechanisms underlying its pathogenicity are multifaceted and complex. However, endocytic processes and motility are well accepted virulence determinants. As previously reported, an AGCK family kinase, EhAGCK1 to be involved in trogocytosis exclusively while another one from same family named EhAGCK2 participates in all actin dependent endocytic processes. As the kinase dead mutants of EhAGCK1 showed significant defect in destruction of live host cells and also the localisation pattern of same is distinguishable from EhAGCK2. From observations so far, it appears that former initiates a distinguishable signaling cascade. In this work, we have demonstrated distinct biochemical properties of kinases involved in related yet distinguishable endocytic processes for the first time. Our biochemical characterization highlights distinct ion dependency of EhAGCK1 along with substrate specificity. We also show upstream activator of these kinases, 3-phosphoinositide dependent kinase 1 (PDK1) activity and its role in activating the kinase activity. The kinases exhibit property of autophosphorylation, and which may regulate the kinase activity subsequently. Summarily, these studies show that EhAGCK1 and EhAGCK2 show distinct biochemical properties which further confirm their unique role in related endocytic processes of trogocytosis and phagocytosis.

Discovering an alternative therapy with a long‐lasting effect on symptoms caused by chikungunya virus (CHIKV) infection is prompted by the lack of a vaccine and the absence of safe, effective and non‐toxic medications. One potential strategy is synthesizing or identifying small compounds that can specifically target the active site of an essential enzyme and prevent virus replication. Previous site‐directed mutagenesis studies have demonstrated the crucial role of the macrodomain, which is a part of non‐structural protein 3 (nsP3), in virus replication. Exploiting this fact, the macrodomain can be targeted to discover a natural substance that can inhibit its function and thereby impede virus replication. With this aim, the present study focused on potential CHIKV nsP3 macrodomain (nsP3 MD ) inhibitors through in silico , in vitro and cell‐based methods. Through virtual screening of the natural compound library, nine nsP3 MD inhibitors were initially identified. Molecular dynamics (MD) simulations were employed to evaluate these nine compounds based on the stability of their ligand–receptor complexes and energy parameters. Target analysis and ADMET (i.e. absorption, distribution, metabolism, excretion and toxicity) prediction of the selected compounds revealed their drug‐like characteristics. Subsequent in vitro investigation allowed us to narrow the selection down to one compound, N ‐[2‐(5‐methoxy‐1H‐indol‐3‐yl) ethyl]‐2‐oxo‐1,2‐dihydroquinoline‐4‐carboxamide, which exhibited potent inhibition of CHIKV growth. This molecule effectively inhibited CHIKV replication in the stable embryonal rhabdomyosarcoma cell line capable of producing CHIKV. Our findings demonstrate that the selected compound possesses substantial anti‐CHIKV nsP3 MD activity both in vitro and in vivo . This work provides a promising molecule for further preclinical studies to develop a potential drug against the CHIKV.

Hepatitis E Virus (HEV) is a positively oriented RNA virus having a 7.2 kb genome. HEV consists of three open reading frames (ORF1-3). Of these, ORF1 codes for the enzymes Methyltransferase (Mtase), Papain-like cysteine protease (PCP), RNA helicase, and RNA-dependent RNA polymerase (RdRp). Unavailability of a vaccine or effective drug against HEV and considering the side effects associated with the off-label use of ribavirin (RBV) and pegylated interferons, an alternative approach is required by the modulation of specific enzymes to prevent the infection. HEV helicase is involved in unwinding the double-stranded RNA, RNA processing, transcriptional regulation, and pre-mRNA processing. Therefore, we screened FDA-approved compounds from the ZINC15 database against the modelled 3D structure of HEV helicase and found that methotrexate and compound A (Pubchem ID BTB07890) inhibit the NTPase and dsRNA unwinding activity leading to inhibition of HEV RNA replication. This may be further authenticated by in vivo study.

ABSTRACT RNA-dependent RNA polymerase (RdRP) is a critical component of the RNA virus life cycle, including SCoV-2. Among the Coronavirus-encoded proteins, non-structural protein 12 ( NSP12 ) exhibits polymerase activity in collaboration with one unit of NSP7 and two units of NSP8 , constituting the RdRp holoenzyme. While there is abundant information on SCoV-2 RdRp-mediated RNA replication, the influence of interplay among NSP12, NSP7 , and NSP8 on template RNA binding and primer extension activity remains relatively unexplored and poorly understood. Here, we recreated a functional RdRp holoenzyme in vitro using recombinant SCoV-2 NSP12, NSP7 , and NSP8 , and established its functional activity. Subsequently, molecular interactions among the NSP s in the presence of a variety of templates and their effects on polymerase activity were studied, wherein we found that NSP12 alone exhibited notable polymerase activity that increased significantly in the presence of NSP7 and NSP8 . However, this activity was completely shut down, and the template RNA-primer complex was detached from NSP12 when one of the two cofactors was present. Through computational analysis, we found that the template RNA entry channel was more constricted in the presence of one of the two cofactors, which was relatively more constricted in the presence of NSP8 compared to that in the presence of NSP7 . In conclusion, we report that NSP7 and NSP8 together synergise to enhance the activity of NSP12 , but antagonise when present alone. Our findings have implications for novel drug development, and compounds that obstruct the binding of NSP7 or NSP8 to NSP12 can have lethal effects on viral RNA replication.

Hepatitis E virus (HEV) is primarily a hepatotropic virus that is responsible for acute hepatitis E in the general population and for chronic hepatitis in immunocompromised individuals. In the absence of a globally accessible vaccine, pegylated interferon-α and ribavirin are the only antiviral agents available for the treatment of chronic patients. As viral RNA-dependent RNA polymerases (RdRps) are indispensable for RNA replication, they are considered potential drug targets. In this study, we screened some well-known RdRp inhibitor molecules, notably, favipiravir, sofosbuvir, remdesivir, filibuvir, and tegobuvir. Of these, monotherapy with favipiravir and sofosbuvir inhibited the RdRp activity with an IC50 value of 10.2 ± 4.9 and 5.2 ± 2.9 μM, respectively, compared to the reference drug ribavirin (3.5 ± 1.6 μM). Further investigation of the combination therapy showed a reduction in viral RNA copy numbers by approximately 90%. Therefore, favipiravir has an additive effect when used with sofosbuvir. Therefore, we propose that favipiravir is a promising anti-HEV drug that can be used in combination with sofosbuvir.

The unavailability of a suitable treatment for human Hepatitis E virus (HEV) infection necessitate the development of anti HEV drugs. The HEV papain-like cysteine proteases (HEV PCP) is a crucial target to prevent viral replication and progression. E64 is a known HEV PCP inhibitor; however, its molecular mechanism of inhibition is not yet known. Since the crystal structure of HEV PCP is not available, the primary focuses of the present study was to refine the predicted HEV PCP structural model by molecular dynamics (MD) simulation. Further, we performed a 200 ns MD simulation to understand the structural complexity of HEV PCP and the effect of E64 binding with HEV PCP. The E64 binding with active site residues Gln48, Thr51, Gln55, Cys52, Ser81, Gln 98, Cys 132, Arg158, His159, Asn 160 and Ala96 leads to reduced fluctuations in the residue at N-terminal (18-41) that include the CHC motif (26-28). However, most of the other non interacting residues, including the inter-domain linker region (46-87), showed increased fluctuations in the HEV PCP-E64 complex. The residue Asp21 and Ala96 are involved in the formation of interdomain interactions in the HEV PCP apo enzyme. While in the PCP-E64 complex, E64 binds to Ala96 and creates a steric hindrance to prevent interdomain interactions. Thus, the E64 binding reduces interdomain interactions and restrict domain movements in the HEV PCP-E64 complex. This information will be important for the chemically designing more effective derivatives of E64 developing HEV PCP specific inhibitors. Communicated by Ramaswamy H. Sarma

The recurrent nature of coronavirus outbreaks, severity of the COVID‐19 pandemic, rapid emergence of novel variants, and concerns over the effectiveness of existing vaccines against novel variants have highlighted the need to develop therapeutic interventions. Targeted efforts to identify inhibitors of crucial viral proteins are the preferred strategy. In this study, we screened FDA‐approved and natural product libraries using in silico approach for potential hits against the SARS‐CoV‐2 main protease (Mpro) and experimentally validated their potency using in vitro biochemical and cell‐based assays. Seven potential hits were identified through in silico screening and were subsequently evaluated in SARS‐CoV‐2‐based cell‐free assays, followed by testing in the HCoV‐229E‐based culture system. Of the tested compounds, 4‐(3,4‐dihydroxyphenyl)‐6,7‐dihydroxy‐1‐isopropyl‐1H‐benzofuro[3,2‐b]pyrazolo[4,3‐e]pyridin‐3(2H)‐one (PubChem CID:71755304, hereafter referred to as STL522228) exhibited significant antiviral activity. Subsequently, its potential as a novel COVID therapeutic molecule was validated in the SARS‐CoV‐2‐culture system, where STL522228 demonstrated superior antiviral activity (EC 50 = 0.44 μ m ) compared to Remdesivir (EC 50 = 0.62 μ m ). Based on these findings, we report the strong anti‐coronavirus activity of STL522228, and propose that it as a promising pan‐ coronavirus Mpro inhibitor for further experimental and preclinical validation.

Hepatitis E Virus (HEV) is a quasi-enveloped virus having a single-stranded, positive-sense RNA genome (~7.2 kb), flanked with a 5′ methylated cap and a 3′ polyadenylated tail. The HEV open reading frame 1 (ORF1) encodes a 186-kDa polyprotein speculated to get processed and produce Methyltransferase (MTase), one of the four essential replication enzymes. In this study, we report the identification of the MTase inhibitor, which may potentially deplete its enzymatic activity, thus causing the cessation of viral replication. Using in silico screening through docking, we identified ten putative compounds, which were tested for their anti-MTase activity. This resulted in the identification of 3-(4-Hydroxyphenyl)propionic acid (HPPA), with an IC50 value of 0.932 ± 0.15 μM, which could be perceived as an effective HEV inhibitor. Furthermore, the compound was tested for inhibition of HEV replication in the HEV culture system. The viral RNA copies were markedly decreased from ~3.2 × 106 in untreated cells to ~4.3 × 102.8 copies in 800 μM HPPA treated cells. Therefore, we propose HPPA as a potential drug-like inhibitor against HEV-MTase, which would need further validation through in vivo analysis using animal models and the administration of Pharmacokinetic and Pharmacodynamic (PK/PD) studies.

Chikungunya Virus (CHIKV) is having a major impact on humans with potentially life-threatening and debilitating arthritis. The lack of a specific antiviral drug against the CHIKV disease has created an alarming situation to identify or develop potent chemical molecules for its remedial measures. Antiviral therapies for viral diseases are generally expensive and have adverse side effects. Plant-based antiviral natural compounds are the most suitable and best alternative of current antiviral drugs because of less toxicity. In the present study, non-structural protein 3 macrodomain (nsP3MD) of the CHIKV that is essential for virus replication has been selected for anti CHIKV drug target. The compounds were identified using molecular docking, virtual screening and further evaluated by molecular dynamics (MD) simulation studies. The binding mechanism of each compound was analyzed considering the stability and energetic parameter. We have found six plant-based natural antiviral compounds Baicalin, Rutaecarpine, Amentoflavone, Apigetrin, Luteoloside, and Baloxavir as strong inhibitors of nsP3MD of CHIKV. ADMET prediction and target analysis of the selected compounds showed drug likeliness of these compounds. MD simulation studies indicated energetically favorable complex formation between nsP3MD and the selected antiviral compounds. Furthermore, the structural effects on these substitutions were analyzed using the principles of each trajectory, which validated the interaction studies. Our analysis suggests a very high probability of these compounds to inhibit nsP3MD of CHIKV and could be evaluated for Chikungunya fever drug development. Communicated by Ramaswamy H. Sarma.