Biology subjects

Upadhyay, S.

Publications and source records attributed to Upadhyay, S..

17 recordsLinked to original sources

bioRxiv2026

Stabilizing the CHI3L1-Gal3BP complex remodels the glioblastoma immune microenvironment and synergizes with PD-1 blockade

CHI3L1 (YKL-40) is associated with mesenchymal, treatment-resistant glioblastoma (GBM) and shapes macrophage programs through interactions with galectin-3 or galectin-3-binding protein (Gal3BP). However, the CHI3L1-Gal3BP interaction has not been previously targeted. A surface plasmon resonance screen identified LZ6. Ternary complex formation was assessed by fluorescence-based, biophysical, and mass spectrometry methods. Activity was evaluated in five patient-derived GBM lines, myeloid cultures from eight donors, CRISPR interference with guide-resistant rescue, and a four-arm factorial study of orthotopic GL261 tumors. LZ6 increased Gal3BP engagement 3.4-fold while reducing galectin-3 in vitro. In GBM-conditioned cultures, LZ6 raised phagocytosis 22% to 44% and MHC class II 24% to 39% and lowered CD206 45% to 31% and IL-10 105 to 67 pg/mL, with concordant transcripts. Gal3BP knockdown reduced the response 41%; guide-resistant rescue restored it. Oral LZ6 raised the intratumoral Gal3BP:galectin-3 ratio from 1.02 to 1.56 at unchanged CHI3L1. In tumor tissue LZ6 shifted 20 of 24 immune endpoints, lowering CD206 and IL-10 and raising MHC class II and CD8 effector function. Median survival with LZ6 was 52.4 versus 26.3 days (P = 0.0012), and in combination with anti-PD-1 most animals survived to the end of follow-up (median not reached), with a significant factorial interaction (hazard ratio 0.24, 95% CI 0.10-0.57, P = 0.0014). LZ6 is a molecular glue of the CHI3L1-Gal3BP complex that engages its target in brain tumor after oral dosing and extends survival alone and synergistically with PD-1 blockade.

pharmacology and toxicology↗

bioRxiv2026

Resistance-Mutant Rescue Establishes On-Target CHI3L1 Inhibition by CHI-DEL, a Brain-Penetrant Small Molecule with In Vivo Efficacy in Glioblastoma

CHI3L1 (YKL-40) drives glioblastoma progression and immunosuppressive neuroimmune signaling, but as a non-enzymatic secreted glycoprotein it has lacked a genetic tool to confirm that small molecule effects are truly on-target. From a DNA-encoded small molecule library we identify CHI-DEL, a potent CHI3L1 ligand, and solve a 2.81 [A] co-crystal structure (PDB 10JV) revealing a binding trajectory centered on Trp99. Structure-guided mutagenesis yields W99F, a compound-resistant CHI3L1 allele that retains native secretion, basal signaling, and thermal stability, unlike the destabilizing W99A control, establishing a clean chemical-genetic separation of biology from drug sensitivity. In isogenic knockout/rescue cells, wild-type rescue restores both phenotype and CHI-DEL sensitivity across invasion, proliferation, and a multi-node signaling program (pSTAT3 and NF-{kappa}B), while W99F rescue restores phenotype but confers resistance to CHI-DEL that is complete for some readouts and partial for others. This same epistasis was reproduced in human iPSC-derived microglia, where CHI-DEL reverses a GBM-conditioned-medium-induced phenotype in a CHI3L1- and Trp99-dependent manner. In vivo, CHI-DEL achieves brain and tumor exposure, engages its target and disrupts CHI3L1-CD44 signaling in a dose-dependent manner, remodels the tumor immune microenvironment, and confers a dose-dependent survival benefit that is specific to the active compound. Together, these data extend CHI3L1 small molecule inhibitor development beyond in vitro optimization, providing genetic proof of on-target pharmacology and a brain-penetrant therapeutic strategy for glioblastoma.

neuroscience↗

bioRxiv2026

Discovery of a brain-penetrant CHI3L1 inhibitor as an oral modulator of neuroinflammation in Alzheimers disease

Chitinase-3-like protein 1 (CHI3L1) is elevated in Alzheimers disease (AD), but its role in glial dysfunction and therapeutic tractability remain unclear. Here, screening a 4.2-billion-compound DNA-encoded library identified DELC2, a brain-penetrant CHI3L1 modulator. Biophysical and crystallographic studies showed that DELC2 binds the CHI3L1 glycan-recognition groove within an aromatic-hydrophobic pocket centered on Trp99 and Trp352. DELC2 inhibited CHI3L1-RAGE association, suppressed CHI3L1-induced inflammatory signaling, reduced Gal-3-positive microglia, and restored amyloid-{beta} uptake in glial cultures. In human cerebrospinal fluid, CHI3L1 was elevated in AD and correlated with astrocytic NF-{kappa}B activation. DELC2 phenocopied CHI3L1 immunodepletion, whereas CHI3L1 add-back restored inflammatory and amyloid-{beta}-handling defects. Oral DELC2 achieved sustained unbound brain exposure in 5xFAD mice, improved cognitive performance, reduced multiple amyloid-{beta} species, and attenuated astrocytic and microglial inflammatory programs. These findings establish the CHI3L1 glycan-recognition groove as a druggable regulator of RAGE-associated glial dysfunction and identify DELC2 as a chemical scaffold for CHI3L1-directed therapy in AD.

pharmacology and toxicology↗

bioRxiv2026

A small molecule inhibitor of CD28 costimulation restrains pathogenic T-cell responses in inflammatory bowel disease

CD28 costimulation contributes to pathogenic T cell responses in inflammatory bowel disease (IBD), but current B7-directed blockade also limits CTLA-4 signaling. Using a sensitive NanoBiT split-luciferase screening platform, we identified and optimized CA-23, a small molecule antagonist that directly binds human and mouse CD28 without measurable binding to CD80, CD86, or CTLA-4. CA-23 inhibited CD28-B7 engagement and CD28-dependent T cell activation without agonist activity in human whole blood and peripheral blood mononuclear cells. CA-23 achieved exposure in the colon and mesenteric lymph nodes and reduced disease severity, histologic injury, and pathogenic Th1 and Th17 responses in a T cell transfer model of colitis. In PBMCs from donors with ulcerative colitis or Crohns disease, CA-23 suppressed inflammatory cytokine production and T cell activation to a degree matching or exceeding Abatacept. In human intestinal epithelial-PBMC co-cultures, CA-23 preserved Treg suppressive activity and epithelial barrier integrity, whereas Abatacept reduced Treg function. CA-23 did not alter CD80 or CD86 expression on autologous antigen-presenting cells and showed no substantial off-target activity in the tested selectivity panel. These findings support direct CD28 antagonism as a mechanistically differentiated alternative to B7-directed co-stimulation blockade for suppressing pathogenic T cell responses in preclinical models of IBD. One Sentence SummaryA CD28-selective small molecule blocks pathogenic T cell activation and preserves Treg function unlike Abatacept in IBD models.

pharmacology and toxicology↗

bioRxiv2026

Reversible CD28 checkpoint modulation by cyclic peptides outperforms biologic blockade under exposure-limited conditions

CD28 co-stimulatory blockade is an established therapeutic strategy in autoimmune disease, yet every clinical-stage agent shares a structural limitation: high-affinity, long-lived receptor occupancy that precludes dynamic control of immune suppression. In chronic inflammatory conditions, where prolonged immunosuppression carries infection risk and necessitates treatment interruptions, no existing agent permits rapid restoration of immune function. We report CP8, a disulfide-constrained cyclic peptide antagonist that matches the inhibitory potency of clinical-stage CD28 biologics (FR104, Acazicolcept, and Lulizumab) across primary human immune cells from healthy and ulcerative colitis donors, suppressing IL-2 and IFN-{gamma} production without agonist activity. Unlike these biologics, CP8 enables rapid and near-complete restoration of T-cell function upon compound removal, a property mechanistically inaccessible to antibody-based therapeutics and demonstrated here for the first time for any CD28-targeting agent. In a T-cell transfer colitis model, CP8 maintains efficacy under intermittent dosing and outperforms Acazicolcept, a dual CD28/ICOS inhibitor, under exposure-limited conditions, achieving superior disease suppression, tissue preservation, and cytokine reduction. These results demonstrate that potency and pharmacological persistence are decoupled properties, and reframe cyclic peptides as a superior modality for immune checkpoints where temporal control of signaling is essential to balance efficacy with the risks of chronic immune suppression.

pharmacology and toxicology↗

bioRxiv2026

AI-Enforced Ultra-Large Virtual Screening Discovers Potent CD28 Binders

Targeting protein-protein interactions (PPIs) with small molecules is historically challenging due to shallow, solvent-exposed interfaces that lack classical binding pockets. Furthermore, employing traditional structure-based virtual screening (SBVS) across ultra-large chemical spaces to find novel chemotypes imposes prohibitive computational bottlenecks. Here, we report the first prospective, real-world application of the PyRMD2Dock platform, an AI-enforced SBVS workflow that integrates machine learning and standard docking available within the PyRMD Studio suite. To target the structurally demanding immune receptor CD28, a chemically diverse subset of 2.4 million molecules from the Enamine REAL Diversity Space was docked into a cleft adjacent to the canonical ligand interface. These data were used to train 672 classification models, and the optimized model rapidly screened the remaining [~]46 million compounds. Following interaction filtering and clustering, 232 highly prioritized ligands were identified. Experimental validation of 150 purchased candidates yielded a remarkable hit rate, identifying multiple direct CD28 binders. Lead compounds 100 and 104 exhibited submicromolar affinity (Kd = 343.8 nM and 407.1 nM, respectively), potent CD28-CD80 disruption, and functional blockade in cellular reporter assays. Furthermore, these compounds successfully reduced cytokine secretion in primary human tumor-PBMC and epithelial tissue co-culture models. This study validates PyRMD2Dock as a highly scalable, effective protocol for mining massive chemical libraries to discover small-molecule modulators of challenging immune receptor interfaces.

pharmacology and toxicology↗

bioRxiv2026

AI-designed cyclic peptides enable controllable modulation of the CD28 immune checkpoint

Immune checkpoint therapies have transformed immunotherapy but remain dominated by biologic agents characterized by prolonged receptor occupancy and limited pharmacologic controllability. Synthetic modalities capable of targeting protein-protein interaction interfaces while enabling tunable immune regulation remain largely unexplored. Here, we report an AI-guided strategy for discovering cyclic peptide antagonists of the costimulatory receptor CD28. The lead peptide, CIP-3, binds the CD28 extracellular domain with nanomolar affinity and competitively disrupts CD28-ligand interactions. In primary human immune systems, CIP-3 suppresses CD28-dependent T-cell activation without intrinsic agonist activity and exhibits rapid pharmacologic reversibility, enabling exposure-dependent control of immune signaling. In a T-cell transfer model of chronic colitis, CIP-3 confers dose-dependent therapeutic efficacy and reduces systemic inflammatory cytokines. CIP-3 also suppresses cytokine production across independent healthy donors and patient-derived PBMCs from individuals with ulcerative colitis with efficacy comparable to a benchmark anti-CD28 biologic. Together, these findings establish AI-designed cyclic peptides as a controllable synthetic modality for immune checkpoint modulation.

pharmacology and toxicology↗

bioRxiv2025

HTS-Oracle: Experimentally validated AI-enabled prioritization for generalizable small molecule hit discovery

High-throughput screening (HTS) remains a central pillar of small molecule discovery yet routinely fails for immune receptors and protein-protein interaction-driven targets. Here, we introduce HTS-Oracle, an experimentally validated AI system for prospective hit discovery that integrates molecular language modeling with cheminformatics to prioritize bioactive compounds at scale. We deploy HTS-Oracle across three clinically validated yet historically intractable immune targets, TREM2, CHI3L1, and CD28, representing cryptic binding pockets, intrinsically disordered proteins, and protein-protein interaction-driven immune checkpoint, respectively. Across the tested targets, HTS-Oracle reduces experimental screening requirements by up to >99% while increasing hit rates by up to 176-fold relative to traditional HTS. Notably, the platform remains predictive under extreme data sparsity, achieving an eightfold improvement for CD28 despite fewer than 2% actives in training. By consistently enriching for experimentally validated hits, HTS-Oracle establishes a new performance benchmark for hit discovery and unlocks small molecule access to immune targets long regarded as chemically inaccessible.

bioinformatics↗

bioRxiv2025

Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation

CD28 is the prototypical costimulatory receptor that integrates with TCR signaling to sustain T-cell activation, proliferation, and survival. While indispensable for adaptive immunity, persistent CD28 signaling drives autoimmunity, graft-versus-host disease, and inflammatory pathology. Despite its therapeutic relevance, CD28 has long been regarded as an undruggable target due to its flat, solvent-exposed dimer interface, restricting modulation to biologics. Here, we describe a structure-activity relationship (SAR) campaign to optimize a small molecule CD28 inhibitor. Guided by biophysical profiling and functional assays, derivatives of the 8VS and 22VS chemotypes were evaluated, leading to the identification of BPU11 as a chemically tractable lead with improved pharmacokinetic stability, aqueous solubility, and plasma persistence. BPU11 consistently disrupted CD28-B7 interactions across biochemical and cellular systems, and potently suppressed T-cell activation in both a tumor-PBMC co-culture and a human PBMC- mucosal tissue model, functionally mimicking the biologic antagonist FR104. Molecular docking and dynamics simulations revealed engagement of the lipophilic canyon of CD28 through stabilizing hydrogen-bonding and hydrophobic interactions. These findings expand the pharmacological space of immune checkpoint blockade beyond antibodies and position BPU11 as a foundation for next-generation immunotherapies.

pharmacology and toxicology↗

bioRxiv2025

As48, a First-in-Class Dual-Function TREM2 Modulator: Receptor Activation and Shedding Inhibition

Triggering receptor expressed on myeloid cells 2 (TREM2) dysfunction contributes to Alzheimers disease pathogenesis, yet current therapeutics cannot prevent ADAM-mediated receptor shedding that diminishes signaling efficacy. Using Affinity Selection-Mass Spectrometry (AS-MS) screening, we identified As48, a novel small molecule that binds TREM2 with high affinity. Biophysical validation confirmed s 7-fold selectivity over TREM1. Cellular assays demonstrated that As48 functions as a TREM2 agonist, activating SYK phosphorylation and enhancing microglial phagocytosis. Molecular docking and molecular dynamics simulations revealed that As48 binds near the cleavage region, establishing hydrogen bonds with Gly68 and reducing conformational flexibility in regions 58-102. Based on this structural insight, we investigated the effect of As48 on TREM2 ectodomain shedding and discovered inhibition of receptor shedding without affecting ADAM10/17 protease activities, representing the first small molecule with anti-shedding properties through conformational restriction of protease accessibility. Importantly, As48 displayed favorable pharmacokinetics with potential for brain permeability, supporting its translational relevance. Through its dual and simultaneous promotion of receptor activation and prevention of shedding, As48 represents a paradigm shift in TREM2 modulation and neuroinflammatory drug discovery. Abstract figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/671919v1_ufig1.gif" ALT="Figure 1"> View larger version (38K): org.highwire.dtl.DTLVardef@1b67c28org.highwire.dtl.DTLVardef@1913034org.highwire.dtl.DTLVardef@f3bf58org.highwire.dtl.DTLVardef@97448a_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Discovery of CD28-Targeted Small Molecule Inhibitors of T Cell Co-stimulation Using Affinity Selection-Mass Spectrometry (AS-MS) and Ex Vivo Validation

CD28 is a key T cell co-stimulatory receptor implicated in antitumor immunity and immune-related disorders, yet no small molecule modulators of CD28 have reached clinical development. Here, we report the discovery and characterization of small molecule CD28 antagonists identified through affinity selection-mass spectrometry (AS-MS). Subsequent catalog-based structure-activity relationship (SAR) optimization led to the identification of two lead compounds, 5MS-5 and 19MS-5, which exhibit direct CD28 binding and potent inhibition of CD28-B7 interactions in cellular reporter assays. In vitro pharmacokinetic profiling demonstrated favorable solubility, metabolic stability, and permeability, alongside low off-target liabilities. Functionally, both compounds suppressed cytokine production in primary human T cells co-cultured with tumor spheroids and human epithelial tissues, validating their ability to inhibit CD28-driven immune activation in physiologically relevant models. These findings establish 5MS-5 and 19MS-5 as promising CD28 inhibitors and provide a foundation for developing orally bioavailable immunomodulators targeting T cell co-stimulation. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=58 SRC="FIGDIR/small/667814v1_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@2d0ab7org.highwire.dtl.DTLVardef@11a985forg.highwire.dtl.DTLVardef@109a13corg.highwire.dtl.DTLVardef@1388451_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

HTS-Oracle: A Retrainable AI Platform for High-Confidence Hit Identification Across Difficult-to-Drug Targets

Despite rapid advances in computational drug discovery, high-throughput screening (HTS) remains the primary method for identifying initial hits, particularly for targets with limited tractability to small molecules. Yet conventional HTS campaigns are costly and inefficient, often yielding hit rates below 2% and discarding valuable negative data. Here we present HTS-Oracle, a retrainable, deep learning-based platform that integrates transformer-derived molecular embeddings (ChemBERTa) with classical cheminformatics features in a multi-modal ensemble framework for hit prediction. We applied HTS-Oracle to the immune co-stimulatory receptor CD28, a prototypical difficult-to-drug target, and prioritized 345 candidates from a chemically diverse library of 1,120 small molecules. Experimental screening via temperature-related intensity change (TRIC) identified 29 hits (8.4% hit rate), representing an eightfold improvement over conventional methods such as surface plasmon resonance (SPR), TRIC, and affinity selection mass spectrometry (ASMS)-based HTS. By enriching true positives and filtering out non-binders upfront, HTS-Oracle streamlines the discovery pipeline and enables more focused, cost-effective screening. Two hit compounds disrupted the CD28-B7.1 interaction, with orthogonal validation provided by MST, ELISA, and molecular dynamics simulations. HTS-Oracle reduces screening burden and improves discovery efficiency, offering a powerful, scalable, and experimentally validated AI framework for accelerating hit identification across difficult-to-drug targets.

bioinformatics↗

bioRxiv2025

Small Molecule-Based Blockade of CD28 Suppresses T Cell Costimulation Across Cellular and Mucosal Co-culture Models

Background and PurposeCD28 is a pivotal costimulatory receptor that governs T cell activation through interaction with B7 ligands (CD80/CD86). While antibody-based inhibitors of CD28 signaling have advanced clinically, the development of small molecule modulators remains limited due to the receptors shallow, flexible surface. We sought to discover small-molecule modulators with favorable pharmacokinetic properties capable of disrupting CD28-B7 interactions in translational models of T cell activation. Experimental ApproachStructure-based virtual screening was conducted using pharmacophore filtering and consensus docking against the human CD28 ectodomain. Hit compounds were validated using temperature-related intensity change (TRIC) and microscale thermophoresis (MST). Functional antagonism was assessed through ELISA, NanoBit luciferase complementation, and a CD28 Blockade Bioassay. In vitro ADME and safety pharmacology profiling were performed, and immunosuppressive activity was evaluated in tumor-PBMC and mucosal-PBMC co-culture assays. Key ResultsLead compound 22VS bound CD28 in biophysical screening, targeting a lipophilic canyon anchored by K24, Q25, and P27. 22VS inhibited CD28-CD80/CD86 interactions in ELISA and cell-based assays with submicromolar potency. 22VS robustly suppressed T cell activation markers in both tumor- PBMC and human mucosal epithelial-PBMC co-culture models, phenocopying the anti-CD28 biologic FR104. It showed no cytotoxicity up to 300 {micro}M and exhibited high solubility, low clearance, strong membrane permeability, and minimal off-target effects in pharmacokinetic screens. Conclusion and ImplicationsThis study identifies a novel druggable site on CD28 and validates 22VS as a selective, non-toxic small molecule inhibitor with translational potential for immune modulation in autoimmunity, transplantation, and cancer. Bullet Point SummaryO_ST_ABSWhat is already knownC_ST_ABSO_LICD28 is a key T cell costimulatory receptor essential for immune activation. C_LIO_LISmall-molecule inhibitors of CD28 are largely unexplored compared to biologics. C_LI What this study addsO_LIIdentifies a novel druggable pocket on CD28 via structure-based virtual screening. C_LIO_LIDiscovers 22VS, a selective small molecule CD28 inhibitor with cellular activity. C_LIO_LIDemonstrates that 22VS suppresses T cell activation in tumor-PBMC and mucosal-PBMC co- culture assays, phenocopying a benchmark biologic (FR104). C_LIO_LIEstablishes 22VS as a drug-like compound with favorable in vitro pharmacokinetic properties, including metabolic stability, permeability, and low off-target toxicity. C_LI Clinical significanceO_LIHighlights the potential of 22VS as a lead for immunomodulatory therapeutic development. C_LIO_LISupports small-molecule targeting of CD28-B7 interactions in T cell-driven diseases. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/665260v1_ufig1.gif" ALT="Figure 1"> View larger version (36K): org.highwire.dtl.DTLVardef@f1d2forg.highwire.dtl.DTLVardef@451c98org.highwire.dtl.DTLVardef@158446corg.highwire.dtl.DTLVardef@1f42ef1_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Surface Plasmon Resonance (SPR)-Based Workflow for High-Throughput Discovery of CD28-Targeted Small Molecules

CD28 is a critical costimulatory receptor involved in T cell activation and immune regulation, making it a compelling target for immunomodulatory therapies. Despite its therapeutic relevance, small molecule CD28 inhibitors remain largely underexplored. To address this gap, we developed a high-throughput screening (HTS) workflow using surface plasmon resonance (SPR) to identify novel CD28-targeted small molecules. To our knowledge, this work represents the first SPR-based HTS platform applied to the discovery of small molecules targeting a stimulatory immune checkpoint receptor. A chemical library composed of diverse 1,056 small molecules was screened using a 384-well format. Compounds were evaluated based on level of occupancy (LO), binding response, and dissociation kinetics, resulting in 12 primary hits (1.14% hit rate). Follow-up dose-response SPR screening confirmed micromolar-range affinities for three compounds. Molecular docking and 100 ns molecular dynamics (MD) simulations of the top hit, DDS5, revealed a stable complex with CD28, maintained by hydrogen bonding and a persistent interaction with Phe93. Functional validation using a competitive ELISA confirmed that DDS5 inhibited the CD28-CD80 interaction. These results demonstrate that our SPR-based HTS platform is a robust and efficient strategy for discovering CD28-targeted small molecules. The integration of computational evaluation and orthogonal validation further underscores the potential of DDS5 as an early-stage immunomodulatory agent.

pharmacology and toxicology↗

bioRxiv2024

Novel Nuclear Roles for Testis-Specific ACTL7A and ACTL7B Supported by In Vivo Characterizations and AI Facilitated In Silico Mechanistic Modeling with Implications for Epigenetic Regulation in Spermiogenesis.

A mechanistic role for nuclear function of testis-specific actin related proteins (ARPs) is proposed here through contributions of ARP subunit swapping in canonical chromatin regulatory complexes. This is significant to our understanding of both mechanisms controlling regulation of spermiogenesis, and the expanding functional roles of the ARPs in cell biology. Among these roles, actins and ARPs are pivotal not only in cytoskeletal regulation, but also in intranuclear chromatin organization, influencing gene regulation and nucleosome remodeling. This study focuses on two testis-specific ARPs, ACTL7A and ACTL7B, exploring their intranuclear activities and broader implications utilizing combined in vivo, in vitro, and in silico approaches. ACTL7A and ACTL7B, previously associated with structural roles, are hypothesized here to serve in chromatin regulation during germline development. This study confirms the intranuclear presence of ACTL7B in spermatocytes and round spermatids, revealing a potential role in intranuclear processes, and identifies a putative nuclear localization sequence conserved across mammalian ACTL7B, indicating a potentially unique mode of nuclear transport which differs from conventional actin. Ablation of ACTL7B leads to varied transcriptional changes reported here. Additionally, in the absence of ACTL7A or ACTL7B there is a loss of intranuclear localization of HDAC1 and HDAC3, which are known regulators of epigenetic associated acetylation changes that in turn regulate gene expression. Thus, these HDACs are implicated as contributors to the aberrant gene expression observed in the KO mouse testis transcriptomic analysis. Furthermore, this study employed and confirmed the accuracy of in silico models to predict ARP interactions with Helicase-SANT-associated (HSA) domains, uncovering putative roles for testis-specific ARPs in nucleosome remodeling complexes. In these models, ACTL7A and ACTL7B were found capable of binding to INO80 and SWI/SNF nucleosome remodeler family members in a manner akin to nuclear actin and ACTL6A. These models thus implicate germline-specific ARP subunit swapping within chromatin regulatory complexes as a potential regulatory mechanism for chromatin and associated molecular machinery adaptations in nuclear reorganizations required during spermiogenesis. These results hold implications for male fertility and epigenetic programing in the male-germline that warrant significant future investigation. In summary, this study reveals that ACTL7A and ACTL7B play intranuclear gene regulation roles in male gametogenesis, adding to the multifaceted roles identified also spanning structural, acrosomal, and flagellar stability. ACTL7A and ACTL7B unique nuclear transport, impact on HDAC nuclear associations, impact on transcriptional processes, and proposed mechanism for involvement in nucleosome remodeling complexes supported by AI facilitated in silico modeling contribute to a more comprehensive understanding of the indispensable functions of ARPs broadly in cell biology, and specifically in male fertility.

cell biology↗

bioRxiv2023

Sida cordifolia, a medicinal plant is efficacious in models of Huntingtons disease, by reducing ER stress

Background and aimHuntingtons Disease is a severe neurodegenerative disorder caused by misfolded mutant huntingtin proteins with expanded stretches of polyglutamines aggregating and destroying cells in the nervous system. Sida cordifolia and Acorus calamus are medicinal plants used in traditional Ayurvedic medicine to treat neurological disorders. Here, we tested the effectiveness of extracts of both medicinal plants in decreasing aggregation of mutant huntingtin protein in models of Huntingtons Disease and explored the mode of action. Experimental procedureWe used two models, the nematode Caenorhabditis elegans and a transgenic mouse neuroblastoma cell line, both expressing mutant huntingtin proteins with elongated polyglutamines. We assessed the effect of Sida cordifolia and Acorus calamus on mutant huntingtin protein aggregation in both models, and additionally used the cell line for mechanistic studies to identify cellular pathways underlying the effects of treatment. Results and conclusionHere, we show that an extract of Sida cordifolia inhibits aggregation of mutant huntingtin proteins. In the C. elegans model, the extract prolonged life span and improved motility of the nematode by reducing aggregation of the mutant huntingtin protein. Acorus calamus did not exhibit these effects. In the transgenic mouse neuroblastoma cell line, the extract decreased aggregation of the mutant huntingtin protein by suppressing key pathways in the ER stress response caused by the mutant protein. Our results highlight the potential therapeutic value of Sida cordifolia and its promise as a source for novel medications. HighlightsO_LISida cordifolia extract reduces aggregates in HD model of transgenic worms C_LIO_LIReduction in aggregates leads to improved motility and longevity C_LIO_LISida cordifolia extract reduces ER stress in cells expressing mHTT protein C_LIO_LIFirst report on the pharmacology of Sida cordifolia in neurodegeneration C_LI

pharmacology and toxicology↗

bioRxiv2021

Developing selective FPR2 agonists can be a potential approach to treat moderate to severe asthma.

Formyl peptide receptor (FPR) family members have been reported to play important role in the resolution of inflammation. A few FPR2/FPR1 dual agonists are reported in the public domain for their anti-inflammatory properties. None of these molecules, however, have been successful as a therapy yet. Recent reports bring forward the ambiguous role of FPR1 in inflammation. These include both positive and negative outcomes. We, therefore, aimed to develop selective FPR2 agonists and evaluated their potential in mitigating the non-resolving inflammation in mouse models of moderate to severe asthma. Extensive structure-activity-relationship (SAR) studies were conducted on the imidazole and benzimidazole chemotype series to identify potent and selective FPR2 agonists. A few molecules were shortlisted based on their in vitro profile and absorption, distribution, metabolism and excretion (ADME) properties and were further evaluated in mouse models of asthma. We report herewith identification of 3 RCI compounds with low nanomolar potency for FPR2 agonism and >10,000 fold selectivity over FPR1 in Ca2+ release assay. These molecules also showed potency in other in vitro assays and potent efficacy in three distinct animal models of asthma. Our data suggest that FPR2 agonism can be a potential therapeutic approach to treat asthma. Our findings also propose that FPR1 can be spared to achieve the desired pharmacological activity.

pharmacology and toxicology↗