Biology subjects

Tomar, S.

Publications and source records attributed to Tomar, S..

At least 19 recordsLinked to original sources

bioRxiv2026

Projected habitat loss and spatial redistribution of two alpine and subalpine species in Himachal Pradesh, Western Himalaya under CMIP6 scenarios

Climate change will alter habitat suitability for plant communities in mountain ecosystems of the Western Himalaya. There will be contrasting magnitudes and spatial configurations of these changes for different plant species, and it is an important question how to translate these contrasts into effective climate-proof multi-species conservation planning. This study assessed current habitat associations and projected changes in suitable habitat for two contrasting species, Meconopsis aculeata and Rhododendron campanulatum, in Himachal Pradesh, India. Field surveys and spatially filtered occurrence records were combined with MaxEnt modelling using 10 non-collinear environmental predictors. Habitat suitability was projected to 2050 and 2070 under Shared Socioeconomic Pathways; SSP1-2.6, SSP2-4.5 and SSP5-8.5 using HadGEM3-GC31-LL. Field observations showed contrasting habitat associations, with M. aculeata occurring mainly in open alpine herbaceous communities and R. campanulatum in subalpine woody communities. Elevation was an important predictor for both species while climatic predictors contributed differently to their relative importance and response patterns. Future projections indicated reductions in suitable habitat of approximately 25% for M. aculeata and 27-28% for R. campanulatum. Habitat-change maps showed heterogeneous patterns of loss, gain and persistence rather than uniform contraction, and centroid displacement remained small (7.7-8.5 km). We performed proximity analyses to show which currently suitable areas are most close to areas of future gain, in order to support the design of climate-proof protected areas. We discuss how interspecific differences in response are relevant to multi-species conservation planning that considers the spatial relationship between current high-suitability areas and the gain, loss and redistribution of future suitable habitat.

ecology↗

bioRxiv2026

Structural Insights and Inhibitor Discovery for Kyasanur Forest Disease Virus NS5 Methyltransferase

Kyasanur Forest Disease Virus (KFDV) NS5 methyltransferase (MTase) protein is the essential enzyme that is involved in the cap methylation of viral RNA, viral replication, and immune evasion, and therefore it is an important protein of interest for antiviral research and drug design. In the present work, we successfully resolved the three-dimensional crystal structures of KFDV NS5 MTase co-crystallised with SAH and GTP at resolutions of 2.2 [A] and 2.6 [A], respectively. In previous studies, HC (Herbacetin) and CAPE (Caffeic acid phenethyl ester) have shown inhibitory activity against SAM-dependent viral MTase. To evaluate the inhibitory potential of HC and CAPE against KFDV NS5 MTase, we have performed isothermal titration calorimetry (ITC) and tryptophan fluorescence spectroscopy (TFS) to validate protein interaction with target compounds. MTase inhibition assay was performed using capillary electrophoresis (CE) assays. Additionally, fluorescence polarisation (FP) confirmed RNA binding inhibition by CAPE and HC. Together, these experiments suggest that HC and CAPE are promising inhibitors against KFDV NS5 MTase and could potentially act as lead compounds to design broad-spectrum anti-Orthoflavivirus drugs.

molecular biology↗

bioRxiv2026

Structural basis of substrate recognition and conformational gating in the bacteriophage M15 metalloendopeptidase LysPH

The rapid emergence of antimicrobial resistance has led to a surge in multidrug-resistant strains, jeopardising the efficacy of frontline and last-resort antibiotics and thereby aggravating the antimicrobial resistance crisis. Bacteriophage-derived endolysins represent a promising class of next-generation antimicrobials. Here, we report the isolation of a novel bacteriophage, PA_Ganga_001, targeting multidrug-resistant Pseudomonas aeruginosa. Genomic sequencing of this phage identified a previously uncharacterised endolysin LysPH, a zinc-dependent globular endolysin that exhibits antibacterial activity against multiple multidrug-resistant Gram-negative pathogens, including Pseudomonas aeruginosa, Acinetobacter baumannii, and Klebsiella pneumoniae. Site-directed mutagenesis of the endolysin LysPH suggests that His77, Asp84, His159, Arg41, and Asp156 are essential for catalysis and substrate accommodation. To understand the structural basis of substrate binding and catalysis, we determined the crystal structures of the apo-enzyme (1.8 [A]) and the catalytically attenuated D156A mutant in complex with a synthetic pentapeptide stem (PGX) of peptidoglycan (2.0 [A]). The pentapeptide-bound complex revealed a defined substrate-binding groove, and a pronounced displacement of the Thr42-Ser75 loop was observed. This led to an approximately six-fold volumetric expansion of the catalytic cleft, indicative of a substrate-induced transition to an open, catalytically competent conformation. Notably, a distinctive C-terminal helical element, which diverges sequentially from characterised M15 homologues, is predicted to interact with the NAG-NAM scaffold and may contribute to the positioning of the stem pentapeptide for Zn2+ dependent catalysis. These distinct structural features establish the molecular basis for L-Ala-D-Glu hydrolysis by M15 bacteriophage endolysins and provide a foundational framework for the rational design and engineering of the next generation of antimicrobial enzymes.

biochemistry↗

bioRxiv2025

An Oral Combination therapy against SARS-CoV-2 based on Synergistic Action of Auranofin and Remdesivir.

The combination of direct-acting and host-directed antivirals targeting SARS-CoV-2 represents an attractive treatment strategy to combat COVID-19. In our previous work, we showed that the FDA-approved anti-arthritis drug Auranofin restricts SARS-CoV-2 replication and pathology in an animal model. Here, we report that Auranofin inhibits SARS-CoV-2 by targeting viral entry and main protease (Mpro) activity without affecting viral transcription. Time-of-addition studies combined with functional assays of viral entry, protease activity, and cell-cell fusion delineated its inhibitory effects at both early and late stages of the viral life cycle. Molecular docking and isothermal titration calorimetry analyses of Auranofin and the known Mpro inhibitor Nirmatrelvir indicated competitive binding within the Mpro active-site pocket. In addition, Auranofin attenuated NF-{kappa}B-dependent signalling and suppressed proinflammatory cytokine production. Combination studies with SARS-CoV-2 targeting nucleoside analogues, revealed the strongest synergistic antiviral activity with remdesivir in vitro. Comparable synergy was observed between auranofin and GS-621763, the orally bioavailable derivative of remdesivir, and was further validated in a preclinical animal model. Collectively, these findings provide a rationale for the further development of auranofin-nucleoside analog combinations targeting SARS-CoV-2. FundingThis research has been supported by ICMR (IIRPIG-2023-0000978) and BIRAC grant (BT/CS0070/06/22) to ST. We acknowledge the infrastructure and research support provided to IISc by the Crypto Relief Fund, L&T Trust, DST-FIST program, Institute of Eminence Fund, Ministry of Education, and the DBT-IISc partnership program (Phase II). RN acknowledges DBT-RA fellowship, SK acknowledges PMRF fellowship, and RS acknowledges FICCI-PMRF fellowship. Research in contextO_ST_ABSEvidence before this studyC_ST_ABSMultiple previous studies have provided evidence showing that auranofin is an antiviral agent targeting SARS-CoV-2 through a host-targeting mechanism, involving the inhibition of thioredoxin reductase and redox homeostasis. Mixed results have been reported regarding the effect of this drug on inhibiting virus-induced syncytia. A prior study from our lab demonstrated the drugs effectiveness in reducing SARS-CoV-2 viral loads in both cell and animal models. Added value of the studyOur study firmly establishes the synergistic use of Auranofin and Remdesivir GS 621763 in the treatment of SARS-CoV-2 infection.

microbiology↗

bioRxiv2025

Oral Cavity Serves as Long-Term COVID-19 Reservoir with Increased Periodontal and Viral Disease Risk

BackgroundSARS-CoV-2 infection can lead to long-term health problems affecting multiple body systems termed long COVID. Currently, limited information exists about long-term oral health manifestations in COVID-19 patients with limited healthcare access. MethodsWe conducted a sequential, cross-sectional study (December 2020-March 2024) to assess how racial/ethnic differences (Black/Hispanic vs White/Asian) and health disparities affect oral and non-oral long COVID symptoms and their relationship with COVID-19 vaccination. We retrospectively reviewed patients oral health record from University of Illinois Chicago dental clinics before vaccination (December 2020; N=1150; Covid+/- N=575/group) and after vaccination (December 2021; N=592; Covid+/- N=292/group). Participants were recruited in two separate prospective groups of COVID-19 positive subjects (February-April 2021; pre-vaccination: N=158; January-March 2024; post-vaccination: N=171), we examined clinical indicators of oral (periodontal and salivary glands) and non-oral (neurologic) sequalae 3-6 months after initial exposure. We measured viral S protein by flow cytometry and quantified inflammatory markers, viral entry receptors, and oral viral load to correlate molecular, and cellular changes in COVID-19 positive subjects before and after vaccination. ResultsOur results identified racial disparity indicating oral associated post-acute sequelae (PASC) primarily manifested as periodontal (gum) disease (COVID-19 positive: 73.1{+/-}18.9% vs COVID-19 negative: 33.1{+/-}14.3%) and correlated with higher rates of dry mouth (57.5%), taste disturbance (47%), and smell loss (20%). Vaccination reduced oral PASC in COVID-19 positive subjects; however, periodontal disease indicators persisted compared to the COVID-19 negative group. Notably, 3-6 months post-infection, while SARS-CoV-2 Spike (S) transcript was rarely detected in saliva ([~]6%), its protein was commonly detected ([~]70%) in the COVID-19 positive subjects indicating incomplete viral clearance. This correlates with significantly higher salivary expression of viral entry receptors (ACE2, and TRMPSS2), and inflammatory mediators (IL-6, IL-8 and MMP-8), in COVID-19 positive subjects. This finding was further supported by higher prevalence of other oral viruses including Epstein-Barr Virus (70.5%), Herpes Simplex Virus (8.1%), and Human Papillomavirus (17.5%) in COVID-19 positive subjects. InterpretationCOVID-19 history significantly correlates with severe oral health complications in predominantly Black communities, while vaccination reduced but did not eliminate these issues. The oral cavity serves as a long-term viral reservoir, and periodontal inflammation with increased oral viral presence in COVID-positive patients may increase susceptibility to oral and non-oral viral diseases and identify risk for long COVID.

microbiology↗

bioRxiv2024

Disruption of molecular interactions between G3BP1 stress granule host protein and nucleocapsid (NTD-N) protein impedes SARS-CoV-2 virus replication

The Ras GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) serves as a formidable barrier to viral replication by generating stress granules (SGs) in response to viral infections. Interestingly, viruses, including SARS-CoV-2, have evolved defensive mechanisms to hijack SG proteins like G3BP1 for the dissipation of SGs that lead to the evasion of hosts immune responses. Previous research has demonstrated that the interaction between the NTF2-like domain of G3BP1 (G3BP1NTF-2) and the intrinsically disordered N-terminal domain (NTD-N1-25) of the N protein plays a crucial role in regulating viral replication and pathogenicity. Interestingly, the current study identified an additional upstream stretch of residues (128KDGIIWVATEG138) (N128-138) within the N-terminal domain of the N protein (NTD-N41-174) that also forms molecular contacts with the G3BP1 protein, as revealed through in silico analysis, site-directed mutagenesis and biochemical analysis. Remarkably, WIN-62577, and fluspirilene, the small molecules targeting the conserved peptide binding pocket in G3BP1NTF-2, not only disrupted the protein-protein interactions (PPIs) between the NTD-N41-174 and G3BP1NTF-2 but also exhibited significant antiviral efficacy against SARS-CoV-2 replication with EC50 values of [~]1.8 {micro}M and [~]1.3 {micro}M, respectively. The findings of this study, validated by biophysical thermodynamics and biochemical investigations, advance the potential of developing therapeutics targeting the SG host protein against SARS-CoV-2, which may also serve as a broad-spectrum antiviral target.

biochemistry↗

bioRxiv2024

Mechanistic insights into the inhibition of dengue virus NS5 methyltransferase by herbacetin

Herbacetin (HC) is a naturally occurring flavonoid compound with a dual antiviral mechanism. It inhibits the polyamine biosynthetic pathway and targets the methyltransferase (MTase) enzyme of the dengue and chikungunya virus. However, understanding the detailed inhibition mechanism of DENV MTase by HC remains unclear. The crystal structure of DENV3 nonstructural protein 5 (NS5) MTase in complex with HC and S-adenosyl-L-homocysteine presented in this study gives structural insights into the inhibition mechanism. Structural analysis revealed that HC binds to the cap 0 RNA site near the GTP binding site in the DENV3 NS5 MTase, and fluorescence polarization assay demonstrated HC inhibits GTP binding with an inhibition constant Ki value of [~] 0.43 {micro}M. This is the first study that identified an inhibitor that targets the conserved RNA-binding region of NS5 MTase, suggesting its potential as a highly effective scaffold for broad-spectrum antiviral agents against orthoflaviviruses. ImportanceDengue virus (DENV) is a vector-borne disease affecting global health, and no antiviral treatment is available for Dengue fever. DENV NS5 methyltransferase (DENV NS5 MTase) is a viral RNA capping enzyme essential for the virus replication. This study demonstrated that herbacetin (HC), a natural flavonoid, blocked the binding of GTP to NS5 MTase and inhibited the enzyme. In addition to DENV3, HC effectively inhibited the replication of DENV1,2, and 4 serotypes. The crystal structure of the DENV3 NS5 MTase in complex with HC revealed that HC binds at the cap 0 RNA binding site, which is close to the GTP binding site and thereby perturbs the spatial arrangement of GTP binding site residues. HC, as the first identified inhibitor that targets the conserved RNA-binding site of DENV3 NS5 MTase, presents a potential scaffold for the development of new antiviral drugs.

microbiology↗

bioRxiv2024

Structural insights into the RNA binding inhibitors of the C-terminal domain of the SARS-CoV-2 nucleocapsid

The SARS-CoV-2 nucleocapsid (N) protein is an essential structural element of the virion, playing a crucial role in enclosing the viral genome into a ribonucleoprotein (RNP) assembly, as well as viral replication and transmission. The C-terminal domain of the N-protein (N-CTD) is essential for encapsidation, contributing to the stabilization of the RNP complex. In a previous study, three inhibitors (ceftriaxone, cefuroxime, and ampicillin) were screened for their potential to disrupt the RNA packaging process by targeting the N-protein. However, the binding efficacy, mechanism of RNA binding inhibition, and molecular insights of binding with N-CTD remain unclear. In this study, we evaluated the binding efficacy of these inhibitors using isothermal titration calorimetry (ITC), revealing the affinity of ceftriaxone (18 {+/-} 1.3 M), cefuroxime (55 {+/-} 4.2 M), and ampicillin (28 {+/-} 1.2 M) with the N-CTD. Further inhibition assay and fluorescence polarisation assay demonstrated RNA binding inhibition, with IC50 ranging from 10.4 to 12.4 M and KD values between 24 and 32 M for the inhibitors. Additionally, we also determined the inhibitor-bound complex crystal structures of N-CTD-Ceftriaxone (2.0 [A]) and N-CTD-Ampicillin (2.2 [A]), along with the structure of apo N-CTD (1.4 [A]). These crystal structures revealed previously unobserved interaction sites involving residues K261, K266, R293, Q294, and W301 at the oligomerization interface and the predicted RNA-binding region of N-CTD. These findings provide valuable molecular insights into the inhibition of N-CTD, highlighting its potential as an underexplored but promising target for the development of novel antiviral agents against coronaviruses. HighlightsO_LIThe inhibitors ceftriaxone, cefuroxime, and ampicillin-demonstrated high-affinity binding to the C-terminal domain (N-CTD) of the SARS-CoV-2 nucleocapsid (N) protein, effectively disrupting the formation of the N-CTD-RNA complex. C_LIO_LIComplex crystal structures of N-CTD with ceftriaxone and ampicillin revealed previously unobserved distinct binding sites. C_LIO_LIStructures reveal how the selected inhibitors disrupt the oligomerization of N-CTD and hinder the RNA packaging process of the virus. C_LI

biophysics↗

bioRxiv2024

Designing and bioengineering of CDRs with higher affinity against receptor-binding domain (RBD) of SARS-CoV-2 Omicron variant

The emergence of the SARS-CoV-2 Omicron variant highlights the need for innovative strategies to address evolving viral threats. This study bioengineered three nanobodies H11-H4, C5, and H3 originally targeting the Wuhan RBD, to bind more effectively to the Omicron RBD. A structure-based in silico affinity maturation pipeline was developed to enhance their binding affinities. The pipeline consists of three key steps: high-throughput in silico mutagenesis of complementarity-determining regions (CDRs), protein-protein docking for screening, and molecular dynamics (MD) simulations for assessment of the complex stability. A total of 741, 551, and 684 mutations were introduced in H11-H4, C5, and H3 nanobodies, respectively. Protein-protein docking and MD simulations shortlisted high-affinity mutants for H11-H4(6), C5(5), and H3(6). Further, recombinant production of H11-H4 mutants and Omicron RBD enabled experimental validation through Isothermal Titration Calorimetry (ITC). The H11-H4 mutants R27E, S57D, S107K, D108W, and A110I exhibited improved binding affinities with dissociation constant (KD) values ranging from [~]8.8 to [~]27 {micro}M, compared to the H11-H4 nanobody KD of [~]32 {micro}M, representing a three-fold enhancement. This study demonstrates the potential of the developed in silico affinity maturation pipeline as a rapid, cost-effective method for repurposing nanobodies, aiding the development of robust prophylactic strategies against evolving SARS-CoV-2 variants and other pathogens. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=75 SRC="FIGDIR/small/613860v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@1ea8ff7org.highwire.dtl.DTLVardef@91d567org.highwire.dtl.DTLVardef@51629corg.highwire.dtl.DTLVardef@1212752_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2024

Unveiling Mechanistic and Structural Insights of EstS1 Esterase: A Potent Broad-Spectrum Phthalate Diester Degrading Enzyme

The ubiquitous presence of plastics and plasticizers around the globe has raised an alarming condition. Phthalate diesters are high-priority pollutants that mimic natural hormones and act as endocrine disruptors upon entering living systems. While certain bacterial esterases have been identified for their role in phthalate diester degradation, their structural and mechanistic characteristics remain largely unexplored. A thermostable and pH-tolerant EstS1 esterase from Sulfobacillus acidophilus catalyzes the conversion of low molecular weight phthalate diesters to monoesters. This study highlights the unique potential of EstS1 to degrade high molecular weight bis(2-ethylhexyl) phthalate (DEHP) by employing biophysical and biochemical approaches along with in-depth structural analysis utilizing high-resolution crystal structures in both apo and complex forms, with various substrates, products, and their analogs to elucidate mechanistic details. The catalytic tunnel mediating entry and exit of the substrate and product, respectively, centralized the Ser-His-Asp triad performing catalysis by bi-bi ping-pong mechanism, forming a tetrahedral intermediate. Additionally, structural analysis of the polypropylene analog jeffamine with EstS1 revealed effective covalent binding, demonstrating its multifunctional capability. Mutation analysis showed that the Met207Ala mutation abolished DEHP binding at the active site, confirming its essential role in supporting catalysis. These findings underscore the potential of EstS1 as a key tool for advancing technologies aimed at phthalate diesters biodegradation.

biophysics↗

bioRxiv2024

Viral methyltransferase inhibitors: berbamine, venetoclax, and ponatinib as efficacious antivirals against Chikungunya virus

Chikungunya virus (CHIKV), transmitted by mosquitoes, poses a significant global health threat. Presently, no effective treatment options are available to reduce the disease burden. The lack of approved therapeutics against CHIKV and the complex spectrum of chronic musculoskeletal and neurological manifestations raise significant concerns, and repurposing drugs could offer swift avenues in the development of effective treatment strategies. RNA capping is a crucial step meditated by non-structural protein 1 (nsP1) in CHIKV replication. In this study, FDA-approved antivirals targeting CHIKV nsP1 methyltransferase (MTase) have been identified by structure-based virtual screening. Berbamine Hydrochloride (BH), ABT199/Venetoclax (ABT), and Ponatinib (PT) were the top hits, which exhibited robust binding energies. Tryptophan fluorescence spectroscopy-based assay confirmed binding of BH-, ABT-, and PT to purified nsP1 with KD values [~]5.45 M, [~]161.3 M, and [~]3.83M, respectively. A dose-dependent decrease in CHIKV nsP1 MTase activity was observed in a capillary electrophoresis-based assay. Treatment with BH, ABT, and PT lead to a dose-dependent reduction in the virus titer with IC50 <100, [~]3.46, and <3.9 nM, respectively, and reduced viral mRNA levels. The nsP1 MTases are highly conserved among alphaviruses; therefore, BH, ABT, and PT, as expected, inhibited replication machinery in Sindbis virus (SINV) replicon assay with IC50 [~]1.94, [~]0.23, and >1.25 M, respectively. These results underscore the efficacy and promise of repurposing drugs as rapid and effective antiviral therapeutics against CHIKV. HighlightsO_LIBerbamine Hydrochloride (BH), ABT199/Venetoclax (ABT), and Ponatinib (PT) identified as inhibitors of Chikungunya virus (CHIKV) nsP1 C_LIO_LIMolecules BH, ABT and PT make moelcular interactions with nsP1: KD values [~]5.45, [~]161.3, and [~]3.83 in M, respectively and effectively inhibit methyltransferase activity. C_LIO_LIBH, ABT, and PT exhibited potent antiviral activity against CHIKV in cell-based assays, with IC50 <100, [~]3.46, and <3.9 nM. C_LIO_LIBH, ABT, and PT also inhibit Sindbis virus, the prototype alphavirus with IC50 [~]1.94, [~]0.23, and >1.25 M respectively demonstrating broad spectrum antiviral efficacy. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/594723v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@1af985dorg.highwire.dtl.DTLVardef@13c14fforg.highwire.dtl.DTLVardef@1868785org.highwire.dtl.DTLVardef@12b690a_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2024

Enhancing affinity of neutralizing SARS-CoV-2 nanobody through facile structure-guided mutations in CDRs

The optimization of antibodies to attain the desired levels of affinity and specificity holds great promise for development of the next generation therapeutics. This study delves into the refinement and engineering of CDRs through in silico affinity maturation followed by binding validation using ITC and pseudovirus-based neutralization assays. Specifically, it focuses on engineering CDRs targeting the epitopes of RBD of the spike protein of SARS-CoV-2. A structure-guided virtual library of 112 single mutations in CDRs was generated and screened against RBD to select the potential affinity-enhancing mutations. Subsequent biophysical studies using ITC provided insights into binding affinity and key thermodynamic parameters. Consistent with in silico findings, seven single mutations resulted in enhanced affinity. The mutants were further tested for neutralization activity against SARS-CoV-2 pseudovirus. L106T, L106Q, S107R, and S107Q generated mutants were more effective in virus-neutralizing with IC50 values of [~]0.03 {micro}M, [~]0.13 {micro}M, [~]0.14 {micro}M, and [~]0.14 {micro}M, respectively as compared to the native nanobody (IC50 [~]0.77 {micro}M). Thus, in this study, the developed computational pipeline guided by structure-aided interface profiles and thermodynamic analysis holds promise for the streamlined development of antibody-based therapeutic interventions against emerging variants of SARS-CoV-2 and other infectious pathogens.

bioengineering↗

bioRxiv2023

Repurposing HIV antiretroviral drug efavirenz against chikungunya virus

Chikungunya virus (CHIKV) has frequently recurred in recent decades, causing outbreaks worldwide in tropical and subtropical regions. The re-emergence of CHIKV poses a substantial risk to human health as no efficacious drugs are currently available to curb new outbreaks. Here, the anti-CHIKV activity of efavirenz was investigated by in vitro cell culture-based antiviral assay, immunofluorescence assay (IFA), and quantitative reverse transcription polymerase chain reaction (qRT-PCR). Efavirenz is a non-nucleoside reverse transcriptase inhibitor (NNRTI) used for treatment of acquired immunodeficiency syndrome (AIDS), and it has good oral bioavailability, long half-life and affordable low cost. This study demonstrated dose-dependent robust anti-CHIKV activity of efavirenz at low micromolar concentration (EC50 = 1.33 {micro}M). To determine potential broad anti-alphavirus activity of efavirenz, its inhibitory activity against Sindbis virus (SINV) was detected. Interestingly, efavirenz also inhibited the replication of SINV at a low micromolar range (EC50 = 0.7 {micro}M). Time of addition assay, direct transfection of virus replicon RNA and minus-sense specific RT-PCR elucidated that efavirenz hinders the viral replication at an early stage after the virus entry by inhibiting the viral RNA synthesis. Efavirenz showed a binding affinity with purified CHIKV capsid protein (CHIKV CP) and it is known that CHIKV CP plays a novel role in the early phase of viral protein synthesis, suggesting CP might be one of the potential protein targets in addition to viral or host proteins involved in viral genome replication. The present study underscores the repurposing of efavirenz for antiviral therapy against CHIKV to curb future viral outbreaks. HighlightsIdentification of in vitro anti-CHIKV activity of efavirenz. Efavirenz disrupts the early phase of virus replication by interfering in the CHIKV minus-sense RNA synthesis. Efavirenz and tomatidine also inhibit SINV replication indicating potential broad spectrum anti-alphavirus activity. Efavirenz holds potential as therapeutic treatment for clinical infections of Chikungunya.

molecular biology↗

bioRxiv2023

Revealing and evaluation of antivirals targeting multiple druggable sites of RdRp complex in SARS-CoV-2

SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) complex consisting of nsp12, nsp7, and nsp8 as the key enzyme for viral genome replication and is a proven antiviral drug target. In this study, molecular interactions of nsp7 and nsp8 with nsp12 and the active site of nsp12 were coterminously targeted using in-silico screening of small molecule libraries to identify potential antivirals. Surface plasmon resonance (SPR) based assay using purified nsp7 and nsp8 proteins was developed, and the binding of identified molecules to targets was validated. The antiviral efficacy of identified small molecules was evaluated using cell-based assays, and potent antiviral effect with EC50 values of 0.56 M, 0.73 M, and 2.8 M was demonstrated by fangchinoline, cepharanthine, and sennoside B, respectively. Further in vivo, investigation using hACE2 mice is being conducted. This is the first study that targets multiple sites in the RdRp complex of SARS-CoV-2 using a structure-based molecular repurposing approach and suggests potential therapeutic options for emerging variants of SARS-CoV-2.

molecular biology↗

bioRxiv2023

Unraveling antiviral efficacy of multifunctional immunomodulatory triterpenoids against SARS-COV-2 targeting virus-specific enzymes

The coronavirus disease 2019 (COVID-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) may be over, but its variants continue to emerge, and patients with mild symptoms having long COVID is still under investigation. SARS-CoV-2 infection leading to elevated cytokine levels and suppressed immune responses set off cytokine storm, fatal systemic inflammation, tissue damage, and multi-organ failure. Thus, drug molecules targeting the SARS-CoV-2 virus-specific proteins or capable of suppressing the host inflammatory responses to viral infection would provide an effective antiviral therapy against emerging variants of concern. Evolutionarily conserved papain-like protease (PLpro) and main protease (Mpro) play an indispensable role in the virus life cycle and immune evasion. Direct-acting antivirals targeting both these viral proteases represent an attractive antiviral strategy that is also expected to reduce viral inflammation. The present study has evaluated the antiviral and anti-inflammatory potential of natural triterpenoids: azadirachtin, withanolide_A, and isoginkgetin. These molecules inhibit the Mpro and PLpro proteolytic activities with half-maximal inhibitory concentrations (IC50) values ranging from 1.42 to 32.7 {micro}M. Isothermal titration calorimetry (ITC) analysis validated the binding of these compounds to Mpro and PLpro. As expected, the two compounds, withanolide_A and azadirachtin, exhibit potent anti-SARS-CoV-2 activity in cell-based assays, with half- maximum effective concentration (EC50) values of 21.73 {micro}M and 31.19 {micro}M, respectively. The anti-inflammatory role of azadirachtin and withanolide_A when assessed using HEK293T cells were found to significantly reduce the levels of CXCL10, TNF, IL6, and IL8 cytokines, which are elevated in severe cases of COVID-19. Interestingly, azadirachtin and withanolide_A were also found to rescue the decreased type-I interferon response (IFN-1). The results of this study clearly highlight the role of triterpenoids as effective antiviral molecules that target SARS-CoV-2 specific enzymes and also host immune pathways involved in virus mediated inflammation.

molecular biology↗

bioRxiv2023

Chimeric chikungunya virus-like particles with surface exposed SARS-CoV-2 RBD elicits potent immunogenic responses in mice.

The SARS-CoV-2 pandemic has reinforced efforts for developing effective vaccination strategy for existing and emerging viruses. Currently there are various vaccine technology available for treating viral diseases, however it is imperative to develop and investigate second-generation vaccines such as chimeric virus-like particles (chi-VLPs) vaccine for increased immunogenicity, ease of production and scalability to supplement the worldwide vaccine supply. Intriguingly, chi-VLPs expresses more than one antigenic epitope on its surface, hence it is expected to be a more effective vaccine candidate. Hereby, this study reports, a novel bivalent vaccine design of chimeric alphavirus coronavirus virus-like particles (ChAC-VLPs), displaying fusion glycoproteins of CHIKV and receptor binding domain (RBD) of SARS-CoV-2 on its surface. Uniqueness and versatility of ChAC-VLPs has been demonstrated via a various techniques including Western blot, Immunofluorescence, cryoEM, and dynamic light scattering (DLS). The multimeric epitope display of immunogenic antigens, i.e CHIKV envelop glycoprotein and SARS-CoV-2 RBD was validated by cell-based assays. ChAC-VLP immunized mice has shown substantial neutralization titres for CHIKV (PRNT50 of 1:25) from the serum collected after 2nd booster doses. Similarly, serum antibodies were detected for SARS-CoV2 RBD as observed by antigen specific ELISA and validated using surface plasmon resonance (SPR). SPR binding response was detected to be >200 RU for anti-RBD antibody in post-immunized mice sera. In conclusion, present study proposes ChAC-VLPs as a potential hybrid vaccine candidate for CHIKV and SARS-CoV-2 infection and contributes valuable insights in chi-VLPs domain.

molecular biology↗

bioRxiv2022

Crystal structure and activity profiling of deubiquitinating inhibitors-bound to SARS-CoV-2 papain like protease revealed new allosteric sites for antiviral therapies

SARS-CoV-2 papain-like protease (PLpro) is a key antiviral target as it plays a dual role in viral replication and in modulation of innate immune responses by deubiquitinating or deISGylating host proteins. Thus, therapeutic targeting of PLpro serves as a two-pronged approach to abate SARS-CoV-2. Interestingly, PLpro shares structural and functional similarities with the cellular deubiquitinating enzymes (DUBs) and in this study this fact has been exploited to identify DUBs inhibitors that target the Ubiquitin/ISG15 binding site and the known catalytic substrate binding pocket of PLpro. Among these identified compounds, flupenthixol, lithocholic acid, teneligliptin, and linagliptin markedly inhibited the proteolytic activity of purified PLpro and demonstrated potent antiviral efficacies against SARS-CoV-2 infection in a dose dependent manner. Treatment with lithocholic acid and linagliptin suppressed the expression levels of inflammatory mediators, thereby, restoring immune responses. Crystal structures of SARS-CoV-2 PLpro in complex with linagliptin and with lithocholic acid determined in this study, revealed insights into the inhibition mechanism with unique interactions within the Ubiquitin/ISG15 binding site (S2 site; Phe69, His73, Asn128, His175) and the substrate binding cleft. Additionally, oral and intraperitoneal treatments with linagliptin increased survival, reduced lung viral load, and ameliorated histopathological damage in mouse-adapted model of SARS-CoV-2 infection. The study for the first time demonstrates a two-pronged strategy using DUB inhibitors that target the proteolytic activity of PLpro and simultaneously reinstates the hosts immune response against SARS-CoV-2.

molecular biology↗

bioRxiv2022

Targeting host protein G3BP1 for the discovery of novel antiviral inhibitors against the Chikungunya virus

Molecular interactions of Chikungunya virus (CHIKV) nsP3 with the host G3BP1 stress granule (SG) protein is crucial for CHIKV replication. NsP3 binds to the nuclear transport factor 2 (NTF2)-like domain of G3BP1 via its two FGDF motifs, unsettling SGs formation. The present study identified seven small molecules targeting the FGDF binding pocket of G3BP1 using structure-based computer-aided drug discovery. All seven molecules exhibited good binding affinities in the range of [~]3.4 to [~]98M towards NTF2-like G3BP1 domain. Furthermore, identified molecules demonstrate dose-dependent inhibition in CHIKV infected Vero cells (EC50: 0.40 to 7.39{micro}M), and reduced SGs formation in these cells. These results highlight G3BP1 protein as a potential CHIKV therapeutic target and offer potential prospective for developing treatment for CHIKV disease.

microbiology↗