Biology subjects

Bhutkar, M.

Publications and source records attributed to Bhutkar, M..

6 recordsLinked to original sources

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↗

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

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

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↗

bioRxiv2022

Elucidation of the antiviral mechanism of natural therapeutic molecules Herbacetin and Caffeic acid phenethyl ester against Chikungunya and Dengue virus

Chikungunya (CHIKV) and dengue (DENV) viruses pose a public health risk and lack antiviral treatment. Structure-based virtual screening of natural MTase substrates library identified herbacetin (HC) and caffeic acid phenethyl ester (CAPE) as potential CHIKV nsP1 and DENV NS5 MTase inhibitors. Binding affinities and MTase inhibition were confirmed using purified proteins. Crystal structure of DENV3 NS5 MTase and CAPE complex revealed CAPE binding at GTP and cap 0 RNA sites. Interestingly, HC and CAPE depleted polyamines, which are crucial for RNA virus replication, and effectively diminished replication with IC50 values of [~]13.44 {micro}M and [~]0.57 {micro}M against CHIKV, and [~]7.24 {micro}M and [~]1.01 {micro}M against DENV, respectively. Polyamine addition did not reverse the antiviral effects, suggesting a dual inhibition mechanism. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=110 SRC="FIGDIR/small/494145v5_ufig1.gif" ALT="Figure 1"> View larger version (41K): org.highwire.dtl.DTLVardef@1f579eforg.highwire.dtl.DTLVardef@11862eborg.highwire.dtl.DTLVardef@67276org.highwire.dtl.DTLVardef@144c7f8_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗