Biology subjects

Sharma, G. K.

Publications and source records attributed to Sharma, G. K..

7 recordsLinked to original sources

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↗

bioRxiv2023

The histidine kinase NahK regulates pyocyanin production through the PQS system

Many bacterial histidine kinases work in two-component systems that combine into larger multi-kinase networks. NahK is one of the kinases in the GacS Multi-Kinase Network (MKN), which is the MKN that controls biofilm regulation in the opportunistic pathogen Pseudomonas aeruginosa (P. aeruginosa). This network has also been associated with regulating many virulence factors P. aeruginosa secretes to cause disease. However, the individual role of each kinase is unknown. In this study, we identify NahK as a novel regulator of the phenazine pyocyanin (PYO). Deletion of nahK led to a four-fold increase in PYO production, almost exclusively through upregulation of phenazine operon two (phz2). We determined that this upregulation is due to mis-regulation of all P. aeruginosa quorum sensing systems, with a large upregulation of the Pseudomonas quinolone signal (PQS) system and a decrease in production of the acyl-homoserine lactone-producing system, las. In addition, we see differences in expression of quorum sensing inhibitor proteins that align with these changes. Together, this data contributes to understanding how the GacS MKN modulates QS and virulence. ImportancePseudomonas aeruginosa is a Gram-negative bacterium that establishes biofilms as part of its pathogenicity. P. aeruginosa infections are associated with nosocomial infections. As the prevalence of multi-drug resistant P. aeruginosa increases, it is essential to understand underlying virulence molecular mechanisms. Histidine kinase NahK is one of several kinases in P. aeruginosa implicated in biofilm formation and dispersal. Previous work has shown that the nitric oxide sensor, NosP, triggers biofilm dispersal by inhibiting NahK. The data presented here demonstrates that NahK plays additional important roles in the P. aeruginosa lifestyle, including regulating bacterial communication mechanisms such as quorum sensing. These effects have larger implications in infection as they affect toxin production and virulence.

microbiology↗

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↗

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

Discovery of anti-SARS-CoV-2 molecules using structure-assisted repurposing approach targeting N-protein

The N-terminal (NTD) and the C-terminal (CTD) domains comprises the structure of the SARS-CoV-2 Nucleocapsid (N) protein. Crystal structure of the SARS-CoV-2 N protein determined by Kang et al, 2020, reveals the N-terminal RNA binding domain as a unique drug binding site. The present study targets this unique pocket with identified antivirals using structure-based drug repurposing approach. The high-affinity binding of potential molecules was characterised thermodynamically using Isothermal titration calorimetry. The selected molecules showed an inhibitory RNA binding potential between 8.8 M and 15.7 M IC50 when evaluated with a fluorescent-based assay. Furthermore, in an in vitro cell-based antiviral assay, these ten antiviral molecules demonstrated high effectiveness in halting SARS-CoV-2 replication. Telmisartan and BMS-189453, the two highly potent antivirals, have [~]0.98M and 1.02 M EC50 values with the selective index of >102, and >98, respectively. For the first time, this study presents drug molecules specifically targeting the NTD of SARS-CoV-2, offering essential insights for the development of therapeutic interventions against this virus, which is still a potential global threat to public health.

molecular biology↗

bioRxiv2022

Whole-Genome Sequence of African Swine Fever Virus isolate from India provides insights into diversity and evolution

African swine fever (ASF) was first reported in 1921, and since then has posed a major threat to the world pig industry and still remains a major challenge as there is no vaccine or therapy available. In May 2020, the first incidence of ASF was recorded in India, followed by a number of outbreaks in the north eastern part of India. In this study, we report the first whole genome of an Indian isolate of ASF virus (ASF/IND/20/CAD/543) using next generation sequencing and compared with the other ASFV complete genome. On phylogenetic analysis, the virus was assigned to genotype II on the basis of p72 genotyping. However, the whole genome based phylogeny distinguished it from other genotype II isolates of clade 1.1.1. This study adds to our understanding of ASFVs genetic diversity and molecular evolution.

microbiology↗