Biology subjects

Kumar, P.

Publications and source records attributed to Kumar, P..

At least 145 records · Page 8Linked to original sources

bioRxiv2022

Investigating the folding dynamics of NS2B protein of Zika virus

NS2B protein of the Zika virus acts as a co-factor for NS3 protease where only the cytosolic domain of NS2B is sufficient for the protease activity. At the same time, NS2B also involves in remodeling the NS3 protease structure. In isolation, we previously proved the NS2B cytosolic domain (residues 49-95) as a disordered type peptide conformation. Further, this study investigated the overall dynamics of NS2B full-length protein. Our Alphafold2 structure modeling system revealed surprising similarities between selected flavivirus NS2B proteins. This similarity reflects that the NS2B protein across flavivirus is conserved fold-wise. The MD simulation of Zika virus NS2B full-length protein shows that the cytosolic domain as a part of full-length protein is a disorder region supporting our previous experimental finding, which suggests the disordered nature of the cytosolic domain in isolation. Since the cytosolic domain of NS2B is essential for protease activity, we have also investigated the folding and dynamics of the NS2B cytosolic domain (residues 49-95) that shows the disorder to alpha helix transition in TFE. On the other hand, in the presence of SDS, macromolecular crowder like ficoll and PEG do not induce secondary structural change. This dynamics study could have implications for some unknown folds of the NS2B protein.

biophysics↗

bioRxiv2022

Red cell enzyme polymorphisms in Muslim population of Eastern UP, India

BackgroundRed cell enzyme polymorphisms have been used to study genetic variations in several human populations/countries worldwide. Owing to considerable ethnic and cultural heterogeneity in the Indian population, it is imperative to study different caste and religion of different regions. Aims and ObjectiveTo determine allele frequency of five Red Cell Enzymes (ADA, AK1, ESD, GLO1) in Muslim population of Eastern Uttar Pradesh Materials and methodsBlood samples were collected from 200 unrelated individuals belonging to Muslim community of eastern Uttar Pradesh. The phenotypes of ADA, AK1, ESD and GLO1 systems were determined by agarose/starch gel electrophoresis. The allele frequencies were calculated by gene count method. ResultsThe calculated frequencies of the alleles are as follows: ADA*1= 0.907, ADA*2= 0.092; AK*1= 0.92, AK*2= 0.08; ESD*1= 0.765, ESD*2= 0.235; GLO*1= 0.27,GLO*2 =0.73 ConclusionThe comparison of the allele frequencies of the four RBC enzymes studied in the present report with those of Asian populations showed that the allele frequencies are close to other Asian populations.

genomics↗

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

Origin and dynamics of Mycobacterium tuberculosis subpopulations that predictably generate drug tolerance and resistance

Initial responses to tuberculosis treatment are poor predictors of final therapeutic outcomes in drug-susceptible disease suggesting that treatment success depends on features that are hidden within a small minority of the overall infecting Mycobacterium tuberculosis (Mtb) population. We developed a multi-transwell robotic system to perform numerous parallel cultures of genetically barcoded Mtb exposed to steady-state concentrations of rifampicin to uncover these difficult to eliminate minority populations. We found that tolerance repeatedly emerged from at least two subpopulations of barcoded cells, one that could not grow on solid agar media and a second that could form colonies, but whose kill curves diverged from the general bacterial population within 4 and 16 days of drug exposure, respectively. These tolerant subpopulations reproducibly passed through a phase characterized by multiple unfixed resistance mutations followed by emergent drug resistance in some cultures. Barcodes associated with drug resistance identified an especially privileged subpopulation that was rarely eliminated despite 20 days of drug treatment even in cultures that did not contain any drug resistant mutants. The association of this evolutionary scenario with a defined subset of barcodes across multiple independent cultures suggested a transiently heritable phenotype, and indeed glpK phase variation mutants were associated with up to 16 % of the resistant cultures. Drug tolerance and resistance were eliminated in{Delta} ruvA mutant consistent with the importance of bacterial stress responses. This work provides a window into the origin and dynamics of bacterial drug tolerant subpopulations whose elimination may be critical to developing rapid and resistance free cures. ImportanceTuberculosis is unusual among bacterial diseases in that treatments which can rapidly resolve symptoms do not predictably lead to a durable cure unless treatment is continued for months after all clinical and microbiological signs of disease have been eradicated. Using a novel steady-state antibiotic exposure system combined with chromosomal barcoding, we identified small hidden Mycobacterium tuberculosis subpopulations that repeatedly enter into a state of drug tolerance with a predisposition to develop fixed drug resistance after first developing a cloud of unfixed resistance mutations. The existence of these difficult to eradicate subpopulations may explain the need for extended treatment regimen for tuberculosis. Their identification provides opportunities to test genetic and therapeutic approaches that may result in shorter and more effective TB treatments.

microbiology↗

bioRxiv2022

Flavonols contrary affect the interconnected glucosinolate and camalexin biosynthesis pathway in Arabidopsis thaliana

Flavonols are structurally and functionally diverse molecules playing roles in plant biotic and abiotic stress tolerance, auxin transport inhibition, pollen development, etc. Despite their ubiquitous occurrence in land plants and multifunctionality, the effect of perturbation of flavonol biosynthesis over global gene expression and pathways other than flavonoid biosynthesis has not been studied in detail. To understand the signaling role of different flavonol metabolites, herein, we used the flavonol deficient Arabidopsis thaliana loss-of-function mutant flavonol synthase1 (fls1-3) as object of study. Comparative transcriptome and metabolic profiling were used to study the effects of genetic flavonol deficiency and exogenous supplementation with flavonol derivatives (kaempferol, quercetin and rutin) on different cellular processes in the seedling. Various flavonol biosynthesis-related regulatory and structural genes were found to be up-regulated in the fls1-3 mutant which could be reversed by exogenous flavonol feeding. Our manifold comparative studies indicated the modulation of various biological processes and metabolic pathways by flavonols. Camalexin biosynthesis was found to be negatively regulated by flavonols. Interestingly, flavonols appeared to promote the accumulation of aliphatic glucosinolate through transcription factor-mediated up-regulation of biosynthesis genes. Overall, this study provides new insights into molecular mechanisms by which flavonols interfere with the relevant signal chains and their molecular targets and adds new knowledge to the expanding plethora of biological activity of flavonols in plants. SignificanceComparative transcriptome and metabolomic profiling of genetic flavonol deficiency and exogenous flavonol supplementation in A. thaliana seedlings, for the first-time revealed the inverse regulation of interconnected specialized metabolite pathways by flavonol aglycones, and -glycosides. Flavonols negatively regulate camalexin biosynthesis, while promoting the accumulation of aliphatic glucosinolates. Our study adds new insights into the expanding plethora of biological activity of flavonols in plants and will help to uncover the molecular mechanisms by which flavonols interfere with the relevant signal chains and their molecular targets.

plant biology↗

bioRxiv2022

30-Minute Highly Multiplexed VaxArray Immunoassay for Pneumococcal Vaccine Antigen Characterization

Pneumonia accounts for over 20% of deaths worldwide in children aged 1 to 5 years, disproportionately affecting lower- and middle-income countries. Effective, highly multivalent pneumococcal vaccines are available to decrease disease burden, with numerous new vaccines currently under development to serve a variety of worldwide markets. However, pneumococcal conjugate vaccines are among the hardest biologics to manufacture and characterize due to their complexity and heterogeneity. Current characterization methods are often inherently singleplex, requiring separate tests for each serotype present. In addition, identity and quantity are often determined with separate methods. We have developed the VaxArray Pneumococcal Assay for applications in identity, quantity, and stability testing of pneumococcal polysaccharide and pneumococcal conjugate vaccines. The VaxArray Pneumococcal Assay has a time to result of less than 30 minutes and is an off-the-shelf multiplexed, microarray-based immunoassay kit that can identify and simultaneously quantify 23 pneumococcal polysaccharide serotypes common to many on-market and in-development vaccines. Here, we highlight the potential of the assay for identity testing by showing high reactivity and serotype-specificity to a wide variety of native polysaccharides, CRM197-conjugated polysaccharides, and drug product. The assay also has vaccine-relevant lower limits of quantification in the low to mid ng/mL range and can be used for accurate quantification even in adjuvanted vaccines. Excellent correlation to the anthrone assay is demonstrated, with VaxArray resulting in significantly improved precision over this antiquated chemical method.

immunology↗

bioRxiv2022

Cellular proteomic profiling using proximity labelling by TurboID-NES in microglial and neuronal cell lines.

1.0Different brain cell types play distinct roles in brain development and disease. Molecular characterization of cell-specific mechanisms using cell type-specific approaches at the protein (proteomic) level, can provide biological and therapeutic insights. To overcome the barriers of conventional isolation-based methods for cell type-specific proteomics, in vivo proteomic labeling with proximity dependent biotinylation of cytosolic proteins using biotin ligase TurboID, coupled with mass spectrometry (MS) of labeled proteins, has emerged as a powerful strategy for cell type-specific proteomics in the native state of cells without need for cellular isolation. To complement in vivo proximity labeling approaches, in vitro studies are needed to ensure that cellular proteomes using the TurboID approach are representative of the whole cell proteome, and capture cellular responses to stimuli without disruption of cellular processes. To address this, we generated murine neuroblastoma (N2A) and microglial (BV2) lines stably expressing cytosolic TurboID to biotinylate the cellular proteome for downstream purification and analysis using MS. TurboID-mediated biotinylation captured 59% of BV2 and 65% of N2A proteomes under homeostatic conditions. TurboID expression and biotinylation minimally impacted homeostatic cellular proteomes of BV2 and N2A cells, and did not affect cytokine production or mitochondrial respiration in BV2 cells under resting or lipopolysaccharide (LPS)-stimulated conditions. These included endo-lysosome, translation, vesicle and signaling proteins in BV2 microglia, and synaptic, neuron projection and microtubule proteins in N2A neurons. The effect of LPS treatment on the microglial proteome was captured by MS analysis of biotinylated proteins (>500 differentially-abundant proteins) including increased canonical pro-inflammatory proteins (Il1a, Irg1, Oasl1) and decrease anti-inflammatory proteins (Arg1, Mgl2).

neuroscience↗

bioRxiv2022

ReproTox-KG: Toxicology Knowledge Graph for Structural Birth Defects

Birth defects are functional and structural abnormalities that impact 1 in 33 births in the United States. Birth defects have been attributed to genetic as well as other factors, but for most birth defects there are no known causes. Small molecule drugs, cosmetics, foods, and environmental pollutants may cause birth defects when the mother is exposed to them during pregnancy. These molecules may interfere with the process of normal fetal development. To characterize associations between small molecule compounds and their potential to induce specific birth abnormalities, we gathered knowledge from multiple sources to construct a reproductive toxicity Knowledge Graph (ReproTox-KG) with an initial focus on associations between birth defects, drugs, and genes. Specifically, to construct ReproTox-KG we gathered data from drug/birth-defect associations from co-mentions in published abstracts, gene/birth-defect associations from genetic studies, drug- and preclinical-compound-induced gene expression data, known drug targets, genetic burden scores for all human genes, and placental crossing scores for all small molecules in ReproTox-KG. Using the data stored within ReproTox-KG, we scored 30,000 preclinical small molecules for their potential to induce birth defects. Querying the ReproTox-KG, we identified over 500 birth-defect/gene/drug cliques that can be used to explain molecular mechanisms for drug-induced birth defects. The ReproTox-KG is provided as curated tables and via a web-based user interface that can enable users to explore the associations between birth defects, approved and preclinical drugs, and human genes.

bioinformatics↗

bioRxiv2022

SIC50: Determination of IC50 by an optimized Sobel operator and a vision transformer

As a measure of cytotoxic potency, half-maximal inhibitory concentration (IC50) is the concentration at which a drug exerts half of its maximal inhibitory effect against target cells. It can be determined by various methods that require applying additional reagents or lysing the cells. Here, we describe a label-free Sobel-edge-based method, which we name SIC50, for the evaluation of IC50. SIC50 classifies pre-processed phase-contrast images with a state-of-art vision transformer and allows for the continuous assessment of IC50 in a faster and more cost-efficient manner. We have validated this method using four drugs and 1536-well plates, as well as built a first-of-its-kind web application. We anticipate this method will assist in the high-throughput screening of chemical libraries (e.g., small molecule drugs, siRNA, and microRNA and drug discovery.

bioengineering↗

bioRxiv2022

Molecular basis for antiviral activity of pediatric neutralizing antibodies targeting SARS-CoV-2 Spike receptor binding domain

Neutralizing antibodies (NAbs) hold great promise for clinical interventions against SARS-CoV- 2 variants of concern (VOCs). Understanding NAb epitope-dependent antiviral mechanisms is crucial for developing vaccines and therapeutics against VOCs. Here we characterized two potent NAbs, EH3 and EH8, isolated from an unvaccinated pediatric patient with exceptional plasma neutralization activity. EH3 and EH8 cross-neutralize the early VOCs and mediate strong Fc-dependent effector activity in vitro. Structural analyses of EH3 and EH8 in complex with the receptor-binding domain (RBD) revealed the molecular determinants of the epitope-driven protection and VOC-evasion. While EH3 represents the prevalent IGHV3-53 NAb whose epitope substantially overlaps with the ACE2 binding site, EH8 recognizes a narrow epitope exposed in both RBD-up and RBD-down conformations. When tested in vivo, a single-dose prophylactic administration of EH3 fully protected stringent K18-hACE2 mice from lethal challenge with Delta VOC. Our study demonstrates that protective NAbs responses converge in pediatric and adult SARS-CoV-2 patients.

microbiology↗

bioRxiv2022

A comprehensive update to the Mycobacterium tuberculosis H37Rv reference genome

H37Rv is the most widely used M. tuberculosis strain. Its genome is globally used as the M. tuberculosis reference sequence. We developed Bact-Builder, a pipeline that leverages consensus building to generate complete and highly accurate gap-closed bacterial genomes and applied it to three independently sequenced cultures of a parental H37Rv laboratory stock. Two of the 4,417,942 base-pair long H37Rv assemblies were 100% identical, with the third differing by a single nucleotide. Compared to the existing H37Rv reference, the new sequence contained approximately 6.4 kb additional base pairs encoding ten new regions. These regions included insertions in PE/PPE genes and new paralogs of esxN and esxJ, which were differentially expressed compared to the reference genes. Additional sequencing and assembly with Bact-Builder confirmed that all 10 regions were also present in widely accepted strains of H37Rv: NR123 and TMC102. Bact-builder shows promise as an improved method to perform extremely accurate and reproducible de novo assemblies of bacterial genomes. Furthermore, our findings provide important updates to the primary tuberculosis reference genome.

microbiology↗

bioRxiv2022

Melanoma cells appropriate pericyte:endothelial cell crosstalk during perivascular invasion in the brain

Post brain colonization, cancer cells may adhere to and spread along the abluminal surface of the vasculature providing advantageous access to oxygen, nutrients, and vessel-derived paracrine factors. For example, brain-tropic melanoma cells are well-known to invade along or within blood vessels at the invasive front, but the molecular mechanisms that guide this process are not well-characterized. We have used melanoma cells of different phenotypic states (melanocytic versus mesenchymal) to characterize different modes of perivascular invasion in the brain. We find that Sox9hi mesenchymal state melanoma cells undergo pericyte-like spreading along brain blood vessels via a Snail1-dependent process; in contrast, Sox10hi melanocytic state melanoma form proliferative, perivascular clusters. Snai1 deletion in mesenchymal-state melanoma cells diminishes Tgf{beta}-induced expression of Pdgfr{beta} which impairs Tgf{beta}/Pdgf ligand-driven motility along the brain microvasculature and dramatically reduces perivascular dispersal of melanoma cells throughout the brain post-colonization. These data suggest that, depending on their transcriptional/phenotypic state, some melanoma cells may appropriate signals that typically mediate endothelial cell:pericyte cross talk as an adaptive mechanism for maintaining vessel proximity and motility within the brain microenvironment.

cancer biology↗

bioRxiv2022

Development of amidase-dependent pyrazinoic acid prodrugs with activity against pyrazinamide resistant Mycobacterium tuberculosis

Rapid emergence of drug resistance in Mycobacterium tuberculosis (Mtb) is one of the most significant healthcare challenges of our time. The cause of drug resistance is multifactorial, with the long course anti-tubercular therapy required to treat tuberculosis (TB) constituting a major contributing factor. Introduction of pyrazinamide (PZA) resulted in shortening of TB treatment from twelve to six months and consequently played a critical role in curbing drug resistance that developed over long course therapy. Nevertheless, because PZA is a prodrug activated by a nonessential amidase, PncA, resistance to PZA develops and frequently results in treatment failure. Here, we leveraged a whole cell drug screening approach to identify anti-tuberculars with unconventional mechanisms of action or activation that could be further developed into compounds effective at killing Mtb resistant to PZA. We discovered an amide containing prodrug, DG160, that was activated by the amidase, Rv2888c (AmiC). This amidase was capable of metabolizing a variety of amide containing compounds including a novel pyrazinoic acid-isoquinolin-1-amine prodrug, JSF-4302, which we developed as a potential PncA-independent replacement for PZA. As predicted, AmiC activation of JSF-4302 led to the generation of POA in Mtb including in a PZA resistant clinical isolate, thereby successfully delivering the active component of PZA while bypassing the need for activation by PncA. This work provides a framework for a new approach to drug development and prodrug activation in Mtb. SIGNIFICANCEPyrazinamide (PZA) is a vital component of Mycobacterium tuberculosis (Mtb) treatment since its inclusion shortened tuberculosis therapy by six months. However, PZA is a prodrug and resistance develops at a high frequency due to mutations in its activator PncA. Here, we present the discovery of amide-containing anti-tubercular prodrugs that are activated intracellularly by the Mtb amidase, AmiC. Taking advantage of this finding, we successfully designed and synthesized pyrazinoic acid (POA) prodrugs that were activated by AmiC and found that these compounds delivered intracellular POA to PZA- resistant Mtb isolates that contained a nonfunctional PncA. This new approach to prodrug development provides a method for delivering conjugated drugs into Mtb with the potential to overcome clinical drug resistance.

microbiology↗

bioRxiv2022

Multifunctional fluorophores for live-cell imaging and affinity capture of proteins

Enzyme-based self-labeling tags enable covalent attachment of synthetic molecules to proteins inside living cells. A frontier of this field is designing multifunctional ligands that contain both fluorophores and affinity tags or pharmacological agents and can still efficiently enter cells. Self-labeling tag ligands with short linkers can enter cells readily but often show less activity due to steric issues; ligands with long linkers can be more potent but show lower cell permeability. Here, we overcome this tug-of-war between efficacy and cell-permeability by devising a rational strategy for making cell permeable multifunctional ligands for labeling HaloTag fusions. We found that the lactone-zwitterion equilibrium sconstant (KL-Z) of rhodamines inversely correlates with their distribution coefficients (logD7.4), suggesting that ligands based on dyes exhibiting low KL-Z and high logD7.4 values, such as Si-rhodamines, would efficiently enter cells. We designed cell-permeable multifunctional HaloTag ligands with a biotin moiety to purify mitochondria or a JQ1 appendage to translocate BRD4 from euchromatin to the nucleolus or heterochromatin. We discovered that translocation of BRD4 to constitutive heterochromatin in cells expressing HaloTag-HP1a fusion proteins can lead to apparent increases in transcriptional activity. These new reagents enable affinity capture and translocation of intracellular proteins in living cells and the use of Si-rhodamines and other low KL- Z/high logD7.4 dye scaffolds will facilitate the design of new multifunctional chemical tools for biology. SIGNIFICANCE STATEMENTUnderstanding cellular processes requires tools to measure and manipulate proteins in living cells. Self- labeling tags, such as the HaloTag and SNAP-tag, enable modification of cellular proteins with synthetic molecules. Creating ligands for these systems that have more than one chemical motif remains challenging, however, due to competing demands between cell permeability and functionality. We discovered that multifunctional ligands based on Si-rhodamines efficiently entered cells and enabled affinity purification of mitochondria or translocation of nuclear proteins; the performance of these molecules could be verified by fluorescence microscopy. These compounds should be useful for a variety of biological experiments and our general framework will allow the design of other multifunctional ligands to study living systems.

cell biology↗

bioRxiv2022

Losartan controls immune checkpoint blocker-induced edema and improves survival in glioblastoma

Immune checkpoint blockers (ICBs) have failed in all Phase III glioblastoma trials. Here, we found that ICBs induce cerebral edema in some patients and mice with glioblastoma. Through single-cell RNA sequencing, intravital imaging, and T cell blocking studies in mice, we demonstrated that this edema results from an inflammatory response following anti-PD1 antibody treatment that disrupts the blood-tumor-barrier. Used in lieu of immunosuppressive corticosteroids, the angiotensin receptor blocker losartan prevented this ICB-induced edema and reprogrammed the tumor microenvironment, curing 20% of mice which increased to 40% in combination with standard of care treatment. Using a bihemispheric tumor model, we identified a "hot" tumor immune signature prior to losartan+anti-PD1 therapy that predicted long-term survival. Our findings provide the rationale and associated biomarkers to test losartan with ICBs in glioblastoma patients. One-Sentence SummaryLosartan prevents immunotherapy-associated edema and enhances the outcome of immunotherapy in glioblastoma.

cancer biology↗

bioRxiv2022

Simultaneous Multisheet Multichannel Imaging Cytometry (SMMIC) System Powered by Adaptive Vertically-Aligned Multi-Sheet Array (VAMSA) PSF

Volumetric interrogating of a large population of live specimens at high throughput is a challenging task that necessitates new technology. We propose vertical-aligned multi-sheet array (VAMSA) illumination PSF that enables interrogation of specimens flowing simultaneously through multiple microfluidic channels. The very geometry of PSF enables high quality cross-sectional imaging, and facilitates volumetric interrogation of specimens flowing through commercial microfluidic chip (consists of multiple flow-channels), which is a step towards large population screening. The SMMIC technique employs a unique combination of transmission grating, beam-expander and high NA objective system in a specific optical configuration to generate diffraction-limited illumination PSF (VAMSA-PSF). However, the detection is accomplished by a large field-of-view widefield 4f-system that consists of low NA objective lens, high performance fluorescence filters, and tube lens. Studies show high quality sectional images (resolution [~] 2.5m, and SBR [~] 4.8dB) of HeLa cancerous cells at high flow throughput (flow-rate of, 2500 nl/min). A cell count of > 1k and volume reconstruction efficiency of [~] 121 cells/min is noteworthy. In addition, SMMIC system demonstrate organelle-level resolution with a SBR comparable to that of confocal especially at low flow-rates. It is hoped that the proposed system may accelerates drug-treatment studies for a large population of live specimens to advance the evolving field of translational medicine and health-care.

biophysics↗

bioRxiv2022

Epistasis between promoter activity and coding mutations shapes gene evolvability

The evolution of protein-coding genes proceeds as mutations act on two main dimensions: regulation of transcription level and the coding sequence. The extent and impact of the connection between these two dimensions are largely unknown because they have generally been studied independently. By measuring the fitness effects of all possible mutations on a protein complex at various levels of promoter activity, we show that expression at the optimal level for the WT protein masks the effects of both deleterious and beneficial coding mutations. Mutants that are deleterious at low expression but masked at optimal expression are slightly destabilizing for individual subunits and binding interfaces. Coding mutations that increase protein abundance are beneficial at low expression but incur a fitness cost at high expression. We thereby demonstrate that expression level can dictate which coding mutations are beneficial or deleterious.

evolutionary biology↗

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↗