Biology subjects

Rai, G.

Publications and source records attributed to Rai, G..

13 recordsLinked to original sources

bioRxiv2026

SELECTIVE ACTIVATION OF GIRK POTASSIUM CHANNELS REDUCES BEHAVIORAL AND BRAIN RESPONSES TO ETHANOL IN MICE

Alcohol use disorder (AUD) is a chronic relapsing condition with limited pharmacological treatments. Ethanol modulates neuronal excitability in part through activation of G-protein-gated inwardly rectifying potassium (GIRK/Kir3) channels, which dampen neuronal activity in reward- and stress-related circuits implicated in AUD pathophysiology. In this study, we investigated the therapeutic potential of targeting activation of GIRK channels in mouse models of ethanol intoxication. GiGA1 (G protein-independent GIRK activator type 1) is a selective activator of GIRK1/GIRK2 channels and has good brain bioavailability. Systemic GiGA1 administration prevented acquisition of ethanol-induced conditioned place preference (CPP) in both male and female mice. GiGA1 also significantly reduced voluntary ethanol intake and decreased blood alcohol concentrations, when administered to mice after they developed high preference and consumption of ethanol. Similarly, Baclofen, a GABAB receptor agonist that leads to activation of GIRK channels also decreased ethanol consumption. However, systemic Baclofen did not prevent acquisition of ethanol-dependent CPP, suggesting a broader efficacy of direct GIRK1/GIRK2 activation by GiGA1. Whole-brain c-Fos mapping as a proxy for neuronal activity revealed that GiGA1 blunted ethanol-induced neuronal activation in several AUD-relevant brain regions, including the central amygdala, paraventricular thalamus, paraventricular hypothalamus, and Edinger-Westphal nucleus. These findings demonstrate that pharmacological activation of GIRK channels modulates key neural circuits involved in ethanol reward and intake, supporting GiGA1 as a promising lead compound for targeted AUD therapy.

neuroscience↗

bioRxiv2025

Interactions Between the Frequency of the Duffy Antigenand the Dynamics of P. vivax Malaria Infections

The malarial parasite Plasmodium vivax has infected humans for millennia. As such, alleles like the FY*O allele in the Duffy Antigen Receptor for Chemokines (DARC) and the sickle cell allele (HbS) have been naturally selected for in malaria-endemic regions because they confer resistance to malaria, thereby increasing the survival and reproductive success of individuals carrying these protective alleles. As resistance becomes more common in a population, malaria incidence is expected to decline, reducing the evolutionary pressure for additional resistance. In this work, we explore the interaction between these processes. We construct a model with seasonality that tracks the frequency of Duffy genotypes and leverages fast/slow dynamics to analyze the coupled dynamics of malaria transmission and changes in the gene frequency of the DARC genotype. Specifically, we investigate how the burden of malaria changes with the fractions of people with the various DARC genotypes. We derive the basic reproduction number as the threshold condition for the stability of the disease-free equilibrium and interpret the R0 as a weighted sum of the cases generated by infected individuals of each genotype. Additionally, we calibrate our model using data from the Amazonas region in Brazil, which has a polymorphic population with respect to DARC, and still reports a substantial number of P. vivax cases. Analysis of our model determines the proportion of the population that must be Duffy-negative in order for the entire population to be protected against P. vivax without any further interventions. Furthermore, we assess how different proportions of Duffy-negative individuals influence the monthly incidence of P. vivax cases.

ecology↗

bioRxiv2025

Uncovering the isoform-resolution kinetic landscape of nonsense-mediated mRNA decay with EZbakR

Cellular RNA levels are a product of synthesis and degradation kinetics, which can differ among transcripts of the same gene. An important cause of isoform-specific decay is the nonsense-mediated mRNA decay (NMD) pathway, which degrades transcripts with premature termination codons (PTCs) and other features. Understanding NMD functions requires strategies to quantify isoform kinetics; however, current approaches remain limited. Methods like nucleotide-recoding RNA-seq (NR-seq) enable insights into RNA kinetics, but existing bioinformatic tools do not provide robust, isoform-specific degradation rate constant estimates. We extend the EZbakR-suite by implementing a strategy to infer isoform-level kinetics from short-read NR-seq data. This approach uncovers unexpected variability in NMD efficiency among transcripts with conserved PTC-containing exons and rapid decay of a subset of mRNAs lacking PTCs. Our findings highlight the effects of competition between NMD and other decay pathways, provide mechanistic insights into established NMD efficiency correlates, and identify transcript features promoting efficient decay.

molecular biology↗

bioRxiv2024

Autoimmunity and clinical pathology amelioration in SLE by Dexamethasone primed Mesenchymal Stem Cell derived conditioned media

This study proposes a novel approach, utilizing cell-free dexamethasone (Dex) primed Whartons jelly mesenchymal stem cells derived conditioned media (DW), offering improved efficacy, simplicity, and alternative medicine for addressing complications associated with systemic lupus erythematosus (SLE). This study explores the immunomodulatory effects of DW treatment on immune cell populations in SLE patients and a pristane-induced lupus (PIL) mouse model. DW induces significant expansion of Tregs, Bregs, suppressing Th17, double-negative T cells, and inflammatory neutrophils through modulating IL-10 and IL-17A production. Comparisons with the standard drug hydroxychloroquine reveal similar effects, suggesting TGF beta; pathway mediation in DWs actions. Compared with the immunosuppressive drug Dex, DW better attenuated autoantibody production, increased anti-inflammatory cytokines and maintained a balanced Th17/Treg ratio. In the preclinical in vivo studies, DW exhibits therapeutic efficacy, reducing mortality, preventing proteinuria, and reversing limb inflammation, seizure and alopecia. Organ specific evaluations using advanced live imaging or histopathological analysis highlighted DWs protective effects on kidneys, liver, lungs, heart, and spleen. This provided insight into the immunomodulatory benefits of DW at various levels and suggested that it could be a potential therapeutic avenue for managing complications related to SLE.

immunology↗

bioRxiv2023

Macrophage differentiation requires DNA damage caused by Caspase-Activated DNase

Phagocytic macrophages are crucial for innate immunity and tissue homeostasis. Most macrophages develop from embryonic precursors that populate every organ before birth to self-renew lifelong. However, the mechanisms for macrophage differentiation remain unknown. Using in vivo genetic analysis of the Drosophila larval hematopoietic organ, the lymph gland, we show that the developmentally regulated transient activation of Caspase-Activated DNase (CAD)-mediated DNA breaks in intermediate progenitors is essential for macrophage differentiation. Insulin receptor-mediated PI3K/Akt signaling triggers apoptotic signaling and causes DNA breaks during macrophage development. However, the same Akt signaling attenuates Apoptosis signal-regulating kinase 1 (Ask1) to control apoptotic and JNK activity in differentiating macrophages. DNA-damaged differentiating cells display autophagy activity as a survival strategy. Furthermore, caspase activity is required for embryonic-origin macrophage development and efficient phagocytosis. This study reveals a previously unknown relationship between developmental signals and caspase-activated DNA breaks necessary for multifunctional macrophage differentiation.

developmental biology↗

bioRxiv2022

Application of Temperature-Responsive HIS-Tag Fluorophores to Differential Scanning Fluorimetry Screening of Small Molecule Libraries

Differential scanning fluorimetry is a rapid and economical biophysical technique used to monitor perturbations to protein structure during a thermal gradient most often by detecting protein unfolding events through an environment-sensitive fluorophore. By employing an NTA-complexed fluorophore that is sensitive to nearby structural changes in 6xHIS-tagged protein, a robust and sensitive differential scanning fluorimetry (DSF) assay is established with the specificity of an affinity tag-based system. We developed, optimized, and miniaturized this histidine-tag DSF assay (HIS-DSF) into a 1536-well high-throughput biophysical platform using the Borrelial high temperature requirement A protease (BbHtrA) as a proof of concept for the workflow. A production run of the BbHtrA HIS-DSF assay showed a tight negative control group distribution of Tm values with an average coefficient of variation of 0.51% and median coefficient of variation of compound Tm of 0.26%. The HIS-DSF platform will provide an additional assay platform for future drug discovery campaigns with applications in buffer screening and optimization, target engagement screening, and other biophysical assay efforts.

biochemistry↗

bioRxiv2022

Small Molecule Antiviral Compound Collection (SMACC): a database to support the discovery of broad-spectrum antiviral drug molecules.

Diseases caused by new viruses costs thousands if not millions of human lives and trillions of dollars in damage to the global economy. Despite the rapid development of vaccines for SARS-CoV-2, the lack of small molecule antiviral drugs that work against multiple viral families (broad-spectrum antivirals; BSAs) has left the entire worlds human population vulnerable to the infection between the beginning of the outbreak and the widespread availability of vaccines. Developing BSAs is an attractive, yet challenging, approach that could prevent the next, inevitable, viral outbreak from becoming a global catastrophe. To explore whether historical medicinal chemistry efforts suggest the possibility of discovering novel BSAs, we (i) identified, collected, curated, and integrated all chemical bioactivity data available in ChEMBL for molecules tested in respective assays for 13 emerging viruses that, based on published literature, hold the greatest potential threat to global human health; (ii) identified and solved the challenges related to data annotation accuracy including assay description ambiguity, missing cell or target information, and incorrect BioAssay Ontology (BAO) annotations; (iii) developed a highly curated and thoroughly annotated database of compounds tested in both phenotypic (21,392 entries) and target-based (11,123 entries) assays for these viruses; and (iv) identified a subset of compounds showing BSA activity. For the latter task, we eliminated inconclusive and annotated duplicative entries by checking the concordance between multiple assay results and identified eight compounds active against 3-4 viruses from the phenotypic data, 16 compounds active against two viruses from the target-based data, and 35 compounds active in at least one phenotypic and one target-based assay. The pilot version of our SMACC (Small Molecule Antiviral Compound Collection) database contains over 32,500 entries for 13 viruses. Our analysis indicates that previous research yielded very small number of BSA compounds. We posit that focused and coordinated efforts strategically targeting the discovery of such agents must be established and maintained going forward. The SMACC database publicly available at https://smacc.mml.unc.edu may serve as a reference for virologists and medicinal chemists working on the development of novel BSA agents in preparation for future viral outbreaks.

bioinformatics↗

bioRxiv2022

Bioluminescence-based reporters for characterizing inhibitors and activators of human Sonic Hedgehog protein autoprocessing in live cells at high throughput.

The Sonic hedgehog (SHh) precursor protein undergoes biosynthetic autoprocessing to cleave off and cholesterylate the SHh signaling ligand, a vital morphogen and oncogenic effector protein. Autoprocessing is self-catalyzed by SHhC, the SHh precursors enzymatic domain. Here we describe the development and validation of the first cellular reporter to monitor human SHhC autoprocessing non-invasively in high-throughput compatible plates. The assay couples intracellular SHhC autoprocessing to the extracellular secretion of the bioluminescent nanoluciferase enzyme. We developed a wild-type (WT) SHhC reporter line for evaluating potential autoprocessing inhibitors by concentration response-dependent suppression of extracellular bioluminescence. A conditional mutant SHhC (D46A) reporter line was developed for identifying potential autoprocessing activators by a concentration response-dependent gain of extracellular bioluminescence. The D46A mutation removes a conserved general base that is critical for the substrate activity of cholesterol. Inducibility of the D46A reporter was established using a synthetic sterol, 2- carboxy cholestanol, designed to bypass the defect through intra-molecular general base catalysis. To facilitate direct nanoluciferase detection in the cell culture media of 1536-well plates, we designed a novel membrane-impermeable nanoluciferase substrate, CLZ-2P. This new reporter system offers a long-awaited resource for small molecule discovery for cancer and for developmental disorders where SHh ligand biosynthesis is dysregulated. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/497760v1_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@63c641org.highwire.dtl.DTLVardef@6cee38org.highwire.dtl.DTLVardef@18bd310org.highwire.dtl.DTLVardef@cdfdc7_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2022

Allosteric binders of ACE2 are promising anti-SARS-CoV-2 agents

The COVID-19 pandemic has had enormous health, economic, and social consequences. Vaccines have been successful in reducing rates of infection and hospitalization, but there is still a need for an acute treatment for the disease. We investigate whether compounds that bind the human ACE2 protein can interrupt SARS-CoV-2 replication without damaging ACE2s natural enzymatic function. Initial compounds were screened for binding to ACE2 but little interruption of ACE2 enzymatic activity. This set of compounds was extended by application of quantitative structure-activity analysis, which resulted in 512 virtual hits for further confirmatory screening. A subsequent SARS-CoV-2 replication assay revealed that five of these compounds inhibit SARS-CoV-2 replication in human cells. Further effort is required to completely determine the antiviral mechanism of these compounds, but they serve as a strong starting point for both development of acute treatments for COVID-19 and research into the mechanism of infection. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=98 SRC="FIGDIR/small/484484v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@173d7c9org.highwire.dtl.DTLVardef@5c0021org.highwire.dtl.DTLVardef@c9caaorg.highwire.dtl.DTLVardef@18d23_HPS_FORMAT_FIGEXP M_FIG TOC Graphic: Overall study design. C_FIG

bioinformatics↗

bioRxiv2022

A comprehensive phenotypic screening strategy to identify modulators of cargo translocation by the bacterial Type IVB secretion system

Bacterial type IV secretion systems (T4SSs) are macromolecular machines that translocate effector proteins across multiple membranes into infected host cells. Loss of function mutations in genes encoding protein components of the T4SS render bacteria avirulent, highlighting the attractiveness of T4SSs as drug targets. Here, we designed an automated high-throughput screening approach for the identification of compounds that interfere with the delivery of a reporter-effector fusion protein from Legionella pneumophila into RAW264.7 mouse macrophages. Using a fluorescence resonance energy transfer (FRET)-based detection assay in a bacteria/macrophage co-culture format, we screened a library of over 18,000 compounds and, upon vetting compound candidates in a variety of in vitro and cell-based secondary screens, isolated several hits that efficiently interfered with biological processes that depend on a functional T4SS, such as intracellular bacterial proliferation or lysosomal avoidance, but had no detectable effect on L. pneumophila growth in culture medium, conditions under which the T4SS is dispensable. Notably, the same hit compounds also attenuated, to varying degrees, effector delivery by the closely related T4SS from Coxiella burnetii, notably without impacting growth of this organism within synthetic media. Together, these results support the idea that interference with T4SS function is a possible therapeutic intervention strategy, and the emerging compounds provide tools to interrogate at a molecular level the regulation and dynamics of these virulence-critical translocation machines. ImportanceMulti-drug-resistant pathogens are an emerging threat to human health. Since conventional antibiotics target not only the pathogen but also eradicate the beneficial microbiota, they often cause additional clinical complications. Thus, there is an urgent need for the development of "smarter" therapeutics that selectively target pathogens without affecting beneficial commensals. The bacterial type IV secretion system (T4SS) is essential for the virulence of a variety of pathogens but dispensable for bacterial viability in general and can, thus, be considered a pathogens Achilles heel. By identifying small molecules that interfere with cargo delivery by the T4SS from two important human pathogens, Legionella pneumophila and Coxiella burnetii, our study represents the first step in our pursuit towards precision medicine by developing pathogen-selective therapeutics capable of treating the infections without causing harm to commensal bacteria.

microbiology↗

bioRxiv2022

A real-time cellular thermal shift assay (RT-CETSA) to monitor target engagement

Determining a molecules mechanism of action is paramount during chemical probe development and drug discovery. The cellular thermal shift assay (CETSA) is a valuable tool to confirm target engagement in cells for a small molecule that demonstrates a pharmacological effect. CETSA directly detects biophysical interactions between ligands and protein targets, which can alter a proteins unfolding and aggregation properties in response to thermal challenge. In traditional CETSA experiments, each temperature requires an individual sample, which restricts throughput and requires substantial optimization. To capture the full aggregation profile of a protein from a single sample, we developed a prototype real-time CETSA (RT-CETSA) platform by coupling a real-time PCR instrument with a CCD camera to detect luminescence. A thermally stable Nanoluciferase variant (ThermLuc) was bioengineered that withstood unfolding at temperatures greater than 90 degrees Celsius and was compatible with monitoring target engagement events when fused to diverse targets. Utilizing well-characterized inhibitors of lactate dehydrogenase alpha, RT-CETSA showed significant correlation with enzymatic, biophysical, and other cell-based assays. A data analysis pipeline was developed to enhance the sensitivity of RT-CETSA to detect on-target binding. The RT-CETSA technology advances capabilities of the CETSA method and facilitates the identification of ligand-target engagement in cells, a critical step in assessing the mechanism of action of a small molecule. SignificanceValidating target engagement is a critical step when characterizing a small molecule modulator. The cellular thermal shift assay (CETSA) is a common approach to examine target engagement, as alterations in the thermal stability of a protein can be conferred by ligand binding. An advantage of CETSA is that it does not require modification of the protein target or small molecule. Major limitations are the throughput and ease-of-use, as the traditional detection method uses western blots, which limits the number of samples that can be processed. Higher-throughput CETSA methods have been developed but are performed at a single temperature and require target-specific optimization. We developed a high-throughput real-time CETSA to circumvent these challenges, providing a rapid and cost-effective strategy to assess on-target activity of a small molecule in living cells.

pharmacology and toxicology↗

bioRxiv2021

A hybrid in silico approach reveals novel inhibitors of multiple SARS-CoV-2 variants

The National Center for Advancing Translational Sciences (NCATS) has been actively generating SARS-CoV-2 high-throughput screening data and disseminates it through the OpenData Portal (https://opendata.ncats.nih.gov/covid19/). Here, we provide a hybrid approach that utilizes NCATS screening data from the SARS-CoV-2 cytophatic effect reduction assay to build predictive models, using both machine learning and pharmacophore-based modeling. Optimized models were used to perform two iterative rounds of virtual screening to predict small molecules active against SARS-CoV-2. Experimental testing with live virus provided 100 (~16% of predicted hits) active compounds (Efficacy > 30%, IC50 [≤] 15 M). Systematic clustering analysis of active compounds revealed three promising chemotypes which have not been previously identified as inhibitors of SARS-CoV-2 infection. Further analysis identified allosteric binders to host receptor angiotensin-converting enzyme 2, which were able to inhibit the entry of pseudoparticles bearing spike protein of wild type SARS-CoV-2 as well as South African B.1.351 and UK B.1.1.7 variants.

cell biology↗

bioRxiv2018

Discovery and optimization of piperazine-1-thiourea-based human phosphoglycerate dehydrogenase inhibitors

Proliferating cells, including cancer cells, obtain serine both exogenously and via the metabolism of glucose. By catalyzing the first, rate-limiting step in the synthesis of serine from glucose, phosphoglycerate dehydrogenase (PHGDH) controls flux through the biosynthetic pathway for this important amino acid and represents a putative target in oncology. To discover inhibitors of PHGDH, a coupled biochemical assay was developed and optimized to enable high-throughput screening for inhibitors of human PHGDH. Feedback inhibition was minimized by coupling PHGDH activity to two downstream enzymes (PSAT1 and PSPH), providing a significant improvement in enzymatic turnover. Further coupling of NADH to a diaphorase/resazurin system enabled a red-shifted detection readout, minimizing interference due to compound autofluorescence. With this protocol, over 400,000 small molecules were screened for PHGDH inhibition, and following hit validation and triage work, a piperazine-1-thiourea was identified. Following rounds of medicinal chemistry and SAR exploration, two probes (NCT-502 and NCT-503) were identified. These molecules demonstrated improved target activity and encouraging ADME properties, enabling both in vitro and in vivo assessment of the biological importance of PHGDH, and its role in the fate of serine in PHGDH-dependent cancer cells.

cancer biology↗