Biology subjects

Jha, S.

Publications and source records attributed to Jha, S..

28 records · Page 2Linked to original sources

bioRxiv2021

High Throughput Virtual Screening and Validation of a SARS-CoV-2 Main Protease Non-Covalent Inhibitor

Despite the recent availability of vaccines against the acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the search for inhibitory therapeutic agents has assumed importance especially in the context of emerging new viral variants. In this paper, we describe the discovery of a novel non-covalent small-molecule inhibitor, MCULE-5948770040, that binds to and inhibits the SARS-Cov-2 main protease (Mpro) by employing a scalable high throughput virtual screening (HTVS) framework and a targeted compound library of over 6.5 million molecules that could be readily ordered and purchased. Our HTVS framework leverages the U.S. supercomputing infrastructure achieving nearly 91% resource utilization and nearly 126 million docking calculations per hour. Downstream biochemical assays validate this Mpro inhibitor with an inhibition constant (Ki) of 2.9 {micro}M [95% CI 2.2, 4.0]. Further, using room-temperature X-ray crystallography, we show that MCULE-5948770040 binds to a cleft in the primary binding site of Mpro forming stable hydrogen bond and hydrophobic interactions. We then used multiple {micro}s-timescale molecular dynamics (MD) simulations, and machine learning (ML) techniques to elucidate how the bound ligand alters the conformational states accessed by Mpro, involving motions both proximal and distal to the binding site. Together, our results demonstrate how MCULE-5948770040 inhibits Mpro and offers a springboard for further therapeutic design. O_TEXTBOXSignificance StatementThe ongoing novel coronavirus pandemic (COVID-19) has prompted a global race towards finding effective therapeutics that can target the various viral proteins. Despite many virtual screening campaigns in development, the discovery of validated inhibitors for SARS-CoV-2 protein targets has been limited. We discover a novel inhibitor against the SARS-CoV-2 main protease. Our integrated platform applies downstream biochemical assays, X-ray crystallography, and atomistic simulations to obtain a comprehensive characterization of its inhibitory mechanism. Inhibiting Mpro can lead to significant biomedical advances in targeting SARS-CoV-2 treatment, as it plays a crucial role in viral replication. C_TEXTBOX

biophysics↗

bioRxiv2021

A highly thermotolerant, trimeric SARS-CoV-2 receptor binding domain derivative elicits high titers of neutralizing antibodies

The Receptor Binding Domain (RBD) of SARS-CoV-2 is the primary target of neutralizing antibodies. We designed a trimeric, highly thermotolerant glycan engineered RBD by fusion to a heterologous, poorly immunogenic disulfide linked trimerization domain derived from cartilage matrix protein. The protein expressed at a yield of [~]80-100 mg/liter in transiently transfected Expi293 cells, as well as CHO and HEK293 stable cell lines and formed homogeneous disulfide-linked trimers. When lyophilized, these possessed remarkable functional stability to transient thermal stress of upto 100 {degrees}C and were stable to long term storage of over 4 weeks at 37 {degrees}C unlike an alternative RBD-trimer with a different trimerization domain. Two intramuscular immunizations with a human-compatible SWE adjuvanted formulation, elicited antibodies with pseudoviral neutralizing titers in guinea pigs and mice that were 25-250 fold higher than corresponding values in human convalescent sera. Against the beta (B.1.351) variant of concern (VOC), pseudoviral neutralization titers for RBD trimer were [~] three-fold lower than against wildtype B.1 virus. RBD was also displayed on a designed ferritin-like Msdps2 nanoparticle. This showed decreased yield and immunogenicity relative to trimeric RBD. Replicative virus neutralization assays using mouse sera demonstrated that antibodies induced by the trimers neutralized all four VOC to date, namely B.1.1.7, B.1.351, P.1 and B.1.617.2 without significant differences. Trimeric RBD immunized hamsters were protected from viral challenge. The excellent immunogenicity, thermotolerance, and high yield of these immunogens suggest that they are a promising modality to combat COVID-19, including all SARS-CoV-2 VOC to date.

microbiology↗

bioRxiv2020

AI-Driven Multiscale Simulations Illuminate Mechanisms of SARS-CoV-2 Spike Dynamics

We develop a generalizable AI-driven workflow that leverages heterogeneous HPC resources to explore the time-dependent dynamics of molecular systems. We use this workflow to investigate the mechanisms of infectivity of the SARS-CoV-2 spike protein, the main viral infection machinery. Our workflow enables more efficient investigation of spike dynamics in a variety of complex environments, including within a complete SARS-CoV-2 viral envelope simulation, which contains 305 million atoms and shows strong scaling on ORNL Summit using NAMD. We present several novel scientific discoveries, including the elucidation of the spikes full glycan shield, the role of spike glycans in modulating the infectivity of the virus, and the characterization of the flexible interactions between the spike and the human ACE2 receptor. We also demonstrate how AI can accelerate conformational sampling across different systems and pave the way for the future application of such methods to additional studies in SARS-CoV-2 and other molecular systems. ACM Reference FormatLorenzo Casalino1{dagger}, Abigail Dommer1{dagger}, Zied Gaieb1{dagger}, Emilia P. Barros1, Terra Sztain1, Surl-Hee Ahn1, Anda Trifan2,3, Alexander Brace2, Anthony Bogetti4, Heng Ma2, Hyungro Lee5, Matteo Turilli5, Syma Khalid6, Lillian Chong4, Carlos Simmerling7, David J. Hardy3, Julio D. C. Maia3, James C. Phillips3, Thorsten Kurth8, Abraham Stern8, Lei Huang9, John McCalpin9, Mahidhar Tatineni10, Tom Gibbs8, John E. Stone3, Shantenu Jha5, Arvind Ramanathan2*, Rommie E. Amaro1*. 2020. AI-Driven Multiscale Simulations Illuminate Mechanisms of SARS-CoV-2 Spike Dynamics. In Supercomputing 20: International Conference for High Performance Computing, Networking, Storage, and Analysis. ACM, New York, NY, USA, 14 pages. https://doi.org/finalDOI

biophysics↗

bioRxiv2020

The oncogenic E3 ligase TRIP12 suppresses epithelial-mesenchymal transition (EMT) and metastasis-related processes through ZEB1/2

Thyroid hormone receptor interactor 12 (TRIP12) is an E3 ligase most notably involved in the proteolytic degradation of the tumor suppressor p14ARF. Through this process, it is proposed that TRIP12 plays an oncogenic role in tumor initiation and growth. However, its role in other cancer processes such as metastasis is unknown. In this study, using publicly available cancer patient datasets, we found TRIP12 to be associated with distant metastasis-free survival in breast cancer, suggesting a contrary cancer process inhibitory role in metastasis. Following TRIP12 depletion in the MCF10A breast epithelial cell model, an epithelial-mesenchymal transition (EMT) shift occurred with concomitant changes in EMT cell adhesion markers identified through RNA-seq. In line with EMT changes, TRIP12-depleted cells lose polarity and dislodge from bulk cells at a higher frequency. Furthermore, ectopic TRIP12 expression sensitized cells to anoikis, a major barrier against metastasis. Mechanistically, TRIP12 suppresses EMT through inhibiting ZEB1/2 gene expression and ZEB1/2 depletion rescues EMT markers and cellular behavior. Overall, our study delineates TRIP12s role in inhibition of EMT and metastasis-related processes, and implies a suppression role in breast cancer metastasis.

cancer biology↗

bioRxiv2020

TIP60 acetylates H2AZ and regulates doxorubicin-induced DNA damage sensitivity through RAD51 transcription

TIP60, a lysine acetyltransferase and H2AZ, a histone H2A variant are involved in transcription and DNA repair. Recent studies suggest that H2AZ acetylation is dependent on TIP60. Here, we show that TIP60 acetylates both isoforms of H2AZ in vitro and in cells. Utilizing ChIP-seq and RNA-seq to identify the genes regulated by TIP60-dependent acetylation of H2AZ, we find that TIP60-dependent acetylation of H2AZ correlates with the expression of genes involved in DNA damage repair, amongst several other pathways. In line with this, TIP60-depleted cells exhibit increased sensitivity to the DNA damage-inducing drug doxorubicin. Restoring the expression level of RAD51, one of the genes involved in the DNA damage repair pathway, partially rescues the doxorubicin sensitivity due to TIP60 depletion. Overall, our study uncovers a role for TIP60 in regulating doxorubicin-induced DNA damage sensitivity in a manner dependent on RAD51 transcription.

molecular biology↗

bioRxiv2019

Novel insights into expansion and functional diversification of MIR169 family in tomato

MIR169 family is an evolutionarily conserved miRNA family in plants. A systematic in-depth analysis of MIR169 family in tomato is lacking. We report eighteen miR169 precursors, annotating new loci for MIR169a, b and d, as well as four novel mature isoforms (MIR169f/g/h/i). The family has expanded by both tandem- and segmental-duplication events during evolution. A tandem-pair MIR169b/b-1 and MIR169b-2/h is polycistronic in nature coding for three MIR169b isoforms and a new variant miR169h, that is evidently absent in the wild relatives S. pennellii and S. pimpinellifolium. Seven novel miR169 targets including RNA-binding-protein, protein-phosphatase, aminotransferase, chaperone, tetratricopeptide-repeat-protein, and transcription factors ARF-9B and SEPELLATA-3 were established by efficient target cleavage in presence of specific precursors as well as increased target abundance upon miR169 chelation by short-tandem-target-mimic construct in transient assays. Comparative antagonistic expression profiles of MIR169:target pairs suggest MIR169 family as ubiquitous regulator of various abiotic stresses (heat, cold, dehydration and salt) and developmental pathways. This regulation is partly brought about by acquisition of new promoters as demonstrated by promoterMIR169:GUS-reporter assays as well as differential processivity of different precursors and miRNA cleavage efficiencies. Thus, the current study augments the functional horizon of MIR169 family with applications for stress tolerance in crops.\n\nHighlightExpansion of MIR169 members by duplication and new mature forms, acquisition of new promoters, differential precursor-miRNA processivity and engaging novel targets increases the functional diversification of MIR169 in tomato. (29/30)

genomics↗

bioRxiv2019

Characterization of novel regulators for heat stress tolerance in tomato from Indian sub-continent

The footprint of tomato cultivation, a cool region crop that exhibits heat stress (HS) sensitivity, is increasing in the tropics/sub-tropics. Knowledge of novel regulatory hot-spots from varieties growing in the Indian sub-continent climatic zones could be vital for developing HS-resilient crops. Comparative transcriptome-wide signatures of a tolerant (CLN1621L) and sensitive (CA4) cultivar-pair short-listed from a pool of varieties exhibiting variable thermo-sensitivity using physiological, survival and yield-related traits revealed redundant to cultivar-specific HS-regulation with more up-regulated genes for CLN1621L than CA4. The anatgonisiticly-expressing genes include enzymes; have roles in plant defense and response to different abiotic stresses. Functional characterization of three antagonistic genes by overexpression and TRV-VIGS silencing established Solyc09g014280 (Acylsugar acyltransferase) and Solyc07g056570 (Notabilis), that are up-regulated in tolerant cultivar, as positive regulators of HS-tolerance and Solyc03g020030 (Pin-II proteinase inhibitor), that is down-regulated in CLN1621L, as negative regulator of thermotolerance. Transcriptional assessment of promoters of these genes by SNPs in stress-responsive cis-elements and promoter swapping experiments in opposite cultivar background showed inherent cultivar-specific orchestration of transcription factors in regulating transcription. Moreover, overexpression of three ethylene response transcription factors (ERF.C1/F4/F5) also improved HS-tolerance in tomato. This study identifies several novel HS-tolerance genes and provides proof of their utility in tomato-thermotolerance.\n\nHighlightNovel heat stress regulatory pathways uncovered by comparative transcriptome profiling between contrasting tomato cultivars from Indian sub-continent for improving thermotolerance. (20/30)

genomics↗

bioRxiv2019

Boosting with ALVAC-HIV and AIDSVAX B/E enhances Env constant region 1 and 2 antibody-dependent cellular cytotoxicity

Induction of protective antibodies is a critical goal of HIV-1 vaccine development. One strategy is to induce non-neutralizing antibodies that kill virus-infected cells as these antibody specificities have been implicated in slowing HIV-1 disease progression and in protection. HIV-1 Env constant region 1 and 2 (C1C2) antibodies frequently contain potent antibody dependent cellular cytotoxicity (ADCC) making them a vaccine target. Here we explore the effect of delayed and repetitive boosting of RV144 vaccinee recipients with ALVAC/AIDSVAX B/E on the C1C2-specific antibody repertoire. It was found that boosting increased clonal lineage specific ADCC breadth and potency. A ligand crystal structure of a vaccine-induced broad and potent ADCC-mediating C1C2-specific antibody showed that it bound a highly conserved Env gp120 epitope. Thus, rationally designed boosting strategies to affinity mature these type of IgG C1C2-specific antibody responses may be one method by which to make an improved HIV vaccine with higher efficacy than seen in the RV144 trial.\n\nSignificanceOver one million people become infected with HIV-1 each year making the development of an efficacious HIV-1 vaccine an important unmet medical need. The RV144 human HIV-1 vaccine-regimen is the only HIV-1 clinical trial to date to demonstrate vaccine-efficacy. An area of focus has been on identifying ways by which to improve upon RV144 vaccine-efficacy. The RV305 HIV-1 vaccine-regimen was a follow-up boost of RV144 vaccine-recipients that occurred 6-8 years after the conclusion of RV144. Our studies focused on the effect of delayed boosting in humans on the vaccine-induced antibody repertoire. It was found that boosting with a HIV-1 Env vaccine increased antibody-mediated effector function potency and breadth.

immunology↗

bioRxiv2019

ZBTB33 (Kaiso) methylated binding sites are associated with primed heterochromatin

BackgroundZBTB33, also known as Kaiso, is a member of the zinc finger and BTB/POZ family. In contrast to many transcription factors, ZBTB33 has the ability to bind both a sequence-specific consensus and methylated DNA. Although these dual binding preferences enable ZBTB33 to function as an active as well as repressive regulator of gene expression, little is known about the underlining molecular mechanisms.\n\nResultsIn this study, we aimed to investigate the role of ZBTB33 as a methylated DNA binding factor. We took advantage of the latest releases of the ENCODE sequencing datasets, including ZBTB33 ChIP- seq, whole genome bisulfite sequencing (WGBS), histone mark ChIP-seq and sequencing assays determining the chromatin states, to characterize the chromatin landscapes surrounding methylated ZBTB33 binding sites. Interestingly, our integrative analyses demonstrated that the majority of methylated ZBTB33 binding sites were located within condensed chromatin, which are inaccessible to DNase I and Tn5 transposase. Moreover, these sites were carrying a newly revealed histone post-translational modification signature, with significant enrichment of mono-methylation at lysine 4 of histone 3 (H3K4me1) and a complete absence of other active or expected repressive histone marks.\n\nConclusionsOverall, our analyses revealed that ZBTB33 has the unique ability to bind methylated DNA across heterochromatin in a transition state, suggesting a potential role for ZBTB33 in heterochromatin priming.

genomics↗

bioRxiv2018

PIM-related kinases selectively regulate sensory functions in C. elegans

The mammalian PIM family of serine/threonine kinases regulate several cellular functions, such as cell survival and motility. Since we have observed PIM expression in the olfactory epithelium and other sensory organs of mice, this has raised the question of whether PIM kinases regulate also sensory cell functions. As our model organism to investigate this question, we used the Caenorhabditis elegans nematodes, which express two PIM-related kinases, PRK-1 and PRK-2. We demonstrated them to be true PIM orthologs with similar substrate specificity as well as sensitivity to PIM-inhibitory compounds. Furthermore, we obtained evidence to indicate that PRKs are selectively involved in regulation of olfactory sensations via AWB or AWCON neurons to volatile attractants or repellants, but do not affect gustatory sensations.

neuroscience↗