Biology subjects

Gabr, M.

Publications and source records attributed to Gabr, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2026

Phage Display-Derived Cyclic Peptides Target TREM2 and Modulate Microglial Responses under Amyloid Stress

Triggering receptor expressed on myeloid cells 2 (TREM2) is a key regulator of microglial function and a promising therapeutic target in Alzheimers disease. While current strategies have largely focused on antibody-based agonists, alternative modalities capable of modulating TREM2 signaling remain underexplored. Here, we report the discovery of TREM2-binding cyclic peptides using a disulfide-constrained phage display library. Screening and biophysical validation identified multiple binders, with TREM2-6 and TREM2-12 exhibiting micromolar affinity. Both peptides modulated microglial responses in human iPSC-derived model of amyloid stress and in neuron-microglia co-cultures. Molecular dynamics simulations supported stable peptide-TREM2 interactions, with TREM2-12 displaying a more constrained binding mode. In vitro pharmacokinetic profiling revealed favorable plasma and intestinal stability but limited permeability, consistent with cyclic peptide scaffolds. Together, these findings establish cyclic peptides as a viable modality for targeting TREM2 and provide a foundation for the development of tunable neuroimmune therapeutics. Insert Table of Contents artwork here O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=135 SRC="FIGDIR/small/720287v1_ufig1.gif" ALT="Figure 1"> View larger version (43K): org.highwire.dtl.DTLVardef@fbe978org.highwire.dtl.DTLVardef@9cd946org.highwire.dtl.DTLVardef@17cb644org.highwire.dtl.DTLVardef@a2669e_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

LigandForge: A Web Server for Structure-Guided De Novo Drug Design

Despite advances in computational drug discovery, de novo drug design remains hindered by high licensing costs and the need for specialized programming expertise. We present LigandForge, a webserver for structure-guided de novo ligand generation. LigandForge integrates structural validation and binding-site characterization; voxel-based property grid construction for spatial mapping of electrostatics and hydrophobicity; chemistry-aware fragment assembly; multi-objective lead optimization; and retrosynthetic feasibility analysis. The platform utilizes a structure-guided framework to assemble molecules from curated fragment libraries while enforcing physicochemical constraints, including molecular weight, LogP, and hybridization states. Generated molecules are refined via reinforcement learning and genetic algorithms which are subsequently evaluated using composite metrics such as the quantitative estimate of drug-likeness. By leveraging RDKit for cheminformatics and NGL viewer for real-time 3D visualization, LigandForge provides a synthesis-aware environment that bridges the gap between macromolecular structural data and experimentally feasible lead compounds without requiring local software installation.

bioinformatics↗

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↗

bioRxiv2026

Aβ-Overlapping Ectodomain Binding of the Clinical-Stage TREM2 Agonist VG-3927

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglial immune receptor genetically and functionally linked to Alzheimers disease (AD). VG-3927, the first clinical-stage small-molecule TREM2 agonist, has been proposed to function as a transmembrane molecular glue and positive allosteric modulator (PAM). Whether it directly engages the extracellular ligand-recognition surface of TREM2 remains unknown. Here, we used a deep learning-based blind docking algorithm to map potential VG-3927 binding sites across TREM2 and identified a binding site within the ectodomain hydrophobic groove, a ligand-recognition surface previously implicated in A{beta} and apoE binding. Microscale thermophoresis (MST) confirmed direct interaction of VG-3927 with TREM2 under optimized PEG-400 buffer conditions and independently demonstrated binding of A{beta}1-42 to the receptor. Co-incubation with A{beta} reduced the VG-3927 thermophoretic response, consistent with interference at an overlapping ectodomain binding surface. Consistently, A{beta} induced a rightward shift in the VG-3927 dose-response curve in a Jurkat TREM2-DAP12 NFAT reporter assay and attenuated VG-3927-induced phospho-SYK signaling. Together, these findings support the presence of a previously unrecognized ectodomain interaction mode for VG-3927 and suggest that amyloid-associated ligand occupancy may modulate TREM2 agonist activity within the AD microenvironment.

pharmacology and toxicology↗

bioRxiv2026

Discovery and Optimization of Small Molecule Inhibitors of the SLIT2/ROBO1 Protein-Protein Interaction Using DNA-Encoded Libraries

O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=63 SRC="FIGDIR/small/707154v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1e81c3borg.highwire.dtl.DTLVardef@1958c6borg.highwire.dtl.DTLVardef@1360015org.highwire.dtl.DTLVardef@3f9388_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO C_FIG Protein-protein interactions (PPIs) mediated by extracellular ligands remain challenging targets for small molecule intervention due to their large and dynamic interfaces. The interaction between SLIT2 and its receptor ROBO1 plays a critical role in cell migration and tumor progression, yet remains largely unexplored. Here, we report the discovery and optimization of small molecule inhibitors of the SLIT2/ROBO1 interaction enabled by DNA-encoded library (DEL) screening. Affinity selection against SLIT2 identified four structurally diverse hit compounds, which were subsequently validated using orthogonal biophysical assays. Among these, one hit exhibited measurable SLIT2 binding and functional inhibition of the SLIT2/ROBO1 interaction in a time-resolved FRET assay. Guided by physicochemical considerations, a solubility-optimized analog was designed, resulting in a [~]50-fold improvement in binding affinity and an [~]9-fold enhancement in functional potency. Molecular dynamics simulations and induced-fit docking revealed a stable binding mode within the SLIT2 LRR2 domain and suggested that a benzothiophene substituent was dispensable for target engagement. Fragment-based experimental validation confirmed this prediction, leading to the identification of a minimal azaindole-based pharmacophore that retained nanomolar binding affinity. Collectively, this study demonstrates how DEL-enabled hit discovery combined with rational optimization and fragment deconstruction can yield potent small molecule modulators of a challenging extracellular PPI, providing a foundation for further development of SLIT2/ROBO1 pathway inhibitors.

pharmacology and toxicology↗

bioRxiv2026

Discovery of MA48, a Small Molecule Inhibitor of CAPON (NOS1AP)-NOS1 Protein-Protein Interaction

CAPON (also known as NOS1AP) is an adaptor protein of neuronal nitric oxide synthase (nNOS) that has been implicated in the progression of multiple neurodegenerative diseases, making it an attractive but largely unexplored therapeutic target. To identify small molecule CAPON modulators, we screened a library of 10,000 compounds for CAPON binding using affinity selection-mass spectrometry (AS-MS), which led to the identification of compound MA48 as a potential CAPON binder. Subsequent biophysical validation using microscale thermophoresis (MST) confirmed direct binding, with MA48 exhibiting a dissociation constant (Kd) of 11.9 {micro}M. Structure-activity relationship (SAR) analysis combined with molecular docking was performed to elucidate key pharmacophoric features underlying the MA48/CAPON interaction. To determine whether MA48 disrupts the CAPON-nNOS interaction in a cellular context, we conducted a NanoBRET assay, which demonstrated that MA48 significantly inhibited this interaction in living cells. Collectively, these findings suggest that MA48 represents the first reported small molecule inhibitor of CAPON and provides a foundation for further development of CAPON-targeted therapeutics.

biophysics↗

bioRxiv2026

Exploiting NMR Ensemble Heterogeneity Enables Small Molecule Discovery Against Dynamic Protein-Protein Interfaces

Protein-protein interactions governed by conformationally heterogeneous domains remain difficult to drug because ligand-competent states are often absent from single static structures. Here, we present AtlasNMR, a statistical framework that transforms multi-model NMR ensembles into screening-ready conformational hypotheses for small molecule discovery. Using the neuronal nitric oxide synthase (nNOS) PDZ domain that engages the adaptor protein CAPON (NOS1AP) as a model system, AtlasNMR identified two representative conformational states capturing the dominant and minor populations of the NMR ensemble. Ensemble-based virtual screening followed by consensus ranking yielded MC-3, a small molecule modulator that disrupts the NOS1-NOS1AP interaction in live cells and directly engages the nNOS PDZ domain. MC-3 produced convergent neuroprotective effects in disease-relevant neuronal models by reducing amyloid-{beta}-induced cytotoxicity, suppressing NMDA-driven nitrosative stress, and attenuating pathological tau phosphorylation, while exhibiting a balanced early lead-like ADME and safety profile. Together, this work establishes a generalizable strategy for exploiting NMR ensemble heterogeneity to enable small molecule discovery against dynamic protein-protein interfaces.

pharmacology and toxicology↗

bioRxiv2026

Structural Optimization of CHI3L1 Inhibitors with Improved Pharmacokinetics and Functional Activity in 3D Glioblastoma Models

Chitinase-3-like protein 1 (CHI3L1) is a key driver of glioblastoma (GBM) progression and an emerging therapeutic target. Building on the CHI3L1 inhibitor 11g, we optimized the scaffold through medicinal chemistry to assess structure-property relationships and improve pharmacokinetics. Using microscale thermophoresis (MST) and computational studies, we validated 10p, which exhibits a CHI31 binding affinity (Kd) of 13.22 {micro}M. Notably, 10p overcomes previous developability hurdles by achieving a kinetic solubility of 758 {micro}M, a five-fold improvement over 11g. It further demonstrates high metabolic stability across species and no hERG inhibition. In 3D GBM spheroid models, 10p significantly reduced tumor viability, mass, and migration, exceeding the efficacy of prior analogues. Collectively, these findings establish 10p as a potent CHI3L1 inhibitor with a superior pharmacokinetic profile and robust functional activity, marking it as a promising candidate for further GBM drug development. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=124 SRC="FIGDIR/small/702243v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@dff733org.highwire.dtl.DTLVardef@1de4e56org.highwire.dtl.DTLVardef@1e910dcorg.highwire.dtl.DTLVardef@51e9d4_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2026

Development of a NanoBiT based high throughput screening assay for discovery of NOS1-CAPON interaction inhibitors

The interaction between neuronal nitric oxide synthase (NOS1) and its adaptor protein CAPON (NOS1AP) plays a critical role in various neurological processes and has been implicated in cardiovascular and neuropsychiatric disorders. Disruption of this protein-protein interaction represents a potential therapeutic strategy, yet identifying small molecule inhibitors has been challenging. Here, we present the development and validation of a NanoBiT-based luminescence complementation assay optimized for high-throughput screening (HTS) of NOS1-NOS1AP interaction inhibitors. We engineered NOS1 and NOS1AP fusion proteins with HiBiT and LgBiT complementary subunits, respectively, and established stable CHO-K1 cell lines for robust signal generation. The assay demonstrated excellent performance characteristics with a signal-to-background ratio exceeding 240-fold, and was validated using TAT-GESV, a known peptide inhibitor that showed time- and dose-dependent inhibition. We successfully screened a diverse library of 10,240 compounds and identified 19 validated hits with IC50 values ranging from 2.54 to greater than 30 M, with the majority exhibiting IC50 values below 30 M. The top three compounds exhibited potent inhibitory activity with IC50 values of less than 5 M. This NanoBiT-based assay provides a reliable and efficient platform for discovering novel NOS1-NOS1AP interaction inhibitors and can be adapted for other protein-protein interaction studies.

pharmacology and toxicology↗

bioRxiv2026

Small Molecule Agonists of TREM2 Reprogram Microglia and Protect Synapses in Human Alzheimer's Models

Triggering receptor expressed on myeloid cells-2 (TREM2) is a key immune receptor in the central nervous system that regulates microglial phagocytosis, survival, and neuroinflammatory responses. TRME2 variants have been established as genetic risk factors for Alzheimers disease (AD). However, the therapeutic development of TREM2 modulators has been limited to antibody-based approaches that face limitations in blood-brain barrier penetration and manufacturing scalability. Furthermore, there are no FDA approved TREM2 therapeutics available to date marking an unmet therapeutic gap. Herein, we report the identification of the first TREM2 small molecule submicromolar binders as a result of optimizing compound 4a to yield S9 with TREM2 binding affinity of 0.95 {micro}M. S9 demonstrated robust TREM2 agonism in cellular assays where it induced proximal Syk phosphorylation, activated downstream NFAT transcriptional signaling, enhanced APOE internalization and microglial phagocytic capacity. Pharmacokinetic profiling of the optimized hits revealed S9 to exhibit improved drug-likeness compared to 4a with 7-fold enhanced aqueous solubility, superior metabolic stability, reduced intrinsic clearance and a 9-fold improved hERG safety margin. Functional validation in human iPSC-derived microglia confirmed that S9 suppresses amyloid-beta (A{beta})-induced IL-1{beta} secretion through a TREM2-dependent mechanism. In human neuron-microglia co-culture models exposed to amyloid stress, S9 treatment preserved synaptic integrity as measured by PSD95 expression that indicates promising neuroprotective activity. Together, these findings establish S9 as a first-TREM2 submicromolar small molecule TREM2 agonist which is orally bioavailable with favorable pharmacokinetic properties and promising therapeutic potential for the treatment of Alzheimers disease.

pharmacology and toxicology↗

bioRxiv2026

Targeting CHI3L1 in Alzheimers Disease: Optimization of G721-0282 and Functional Evaluation in Astrocyte Models

Alzheimers disease (AD) involves astrocytic dysfunction characterized by impaired lysosomal activity, defective amyloid clearance, and neuroinflammation, processes strongly regulated by the inflammatory effector CHI3L1. G721-0282, a reported CHI3L1-binding small molecule with demonstrated modulation of downstream signaling pathways including MAPK and STAT3, provides a validated chemical starting point for targeting CHI3L1-driven astrocytic pathology in AD, but exhibits suboptimal potency and drug-like properties that limit its translational potential. We therefore performed a virtual screening of commercially available analogues of G721-0282 to enable structure-guided optimization, generating a detailed structure-activity map and prioritizing 24 derivatives. Biophysical analyses identified compound G721-0377 as the most promising candidate, with optimized substitutions improving CHI3L1 binding affinity. Compound G721-0377 also exhibited favorable pharmacokinetic properties, including improved solubility, balanced permeability, reduced microsomal clearance, and an enhanced cardiac safety margin. Functionally, G721-0377 uniquely reversed CHI3L1-induced astrocytic dysfunction, restoring amyloid uptake, lysosomal proteolysis and acidification, suppressing CHI3L1 and IL-6 secretion, and inhibiting NF-{kappa}B activation to levels comparable to a neutralizing anti-CHI3L1 antibody. In contrast, compounds G721-0179 and G857-1069 showed minimal activity. Collectively, these findings establish G721-0377 as a next-generation CHI3L1 inhibitor with improved affinity, safety, and robust functional efficacy, supporting its further development as a disease-modifying therapeutic for AD.

pharmacology and toxicology↗

bioRxiv2026

Bile Acid Scaffold Engineering Reveals an Androstane-Triol Derivative as a Potent Immunomodulator with Therapeutic Efficacy in EAE

BackgroundNeuroinflammation driven by dysregulated adaptive and innate immune responses plays a central role in the pathogenesis of multiple sclerosis and related autoimmune disorders of the central nervous system. While bile acids are increasingly recognized as endogenous immunomodulators, their therapeutic exploitation has been limited by modest potency and incomplete mechanistic understanding. Here, we report the rational engineering of a bile acid-derived scaffold that yields a potent small molecule immunomodulator with therapeutic efficacy in experimental autoimmune encephalomyelitis, a preclinical model of multiple sclerosis. MethodsA focused series of bile acid-based compounds was established, leading to the identification of an androstane-triol derivative, BA59. The immunomodulatory activity of BA59 was evaluated using in vitro T cell differentiation assays, antigen-presenting cell phenotyping, and ex vivo immune profiling. Therapeutic efficacy was assessed in mice with established experimental autoimmune encephalomyelitis. Flow cytometry was used to characterize peripheral and central nervous system immune populations, including effector T cells, regulatory T cells, and antigen-presenting cells. Disease progression was monitored using clinical scoring and cumulative disease burden analyses. ResultsBA59 treatment significantly attenuated disease severity and cumulative disease burden when administered therapeutically after disease onset. Immunophenotyping revealed a reduction in pro-inflammatory T helper 17 cells accompanied by an increase in regulatory T cells expressing the ectonucleotidase CD39. BA59 also reprogrammed antigen-presenting cells toward a tolerogenic phenotype, characterized by enhanced programmed death-ligand 1 expression. These immune changes were observed both in peripheral lymphoid tissues and within the central nervous system. Importantly, BA59 did not induce broad immunosuppression but instead reshaped immune checkpoint signaling and regulatory pathways associated with neuroinflammatory resolution. ConclusionsThis study identifies BA59 as a first-in-class androstane-triol immunomodulator that ameliorates experimental autoimmune encephalomyelitis through coordinated regulation of T cell balance, immune checkpoints, and antigen-presenting cell function. Our findings highlight bile acid scaffold engineering as a viable strategy for developing small molecule therapeutics that reprogram neuroinflammatory immune circuits, offering a promising translational approach for multiple sclerosis and related neuroinflammatory diseases.

pharmacology and toxicology↗

bioRxiv2026

First-in-Class Small Molecule ROBO2 Binders Identified through Integrated Virtual Screening and Biophysical Validation

Roundabout homolog 2 (ROBO2) is a transmembrane receptor implicated in glioblastoma progression through its interaction with Slit2-mediated signaling pathways. Dysregulated Slit2-ROBO2 signaling enhances tumor cell migration, invasion, and tissue infiltration, while elevated ROBO2 levels contribute to an immunosuppressive tumor microenvironment supporting GBM aggressiveness and highlighting ROBO2 as a therapeutic target. Despite its therapeutic relevance, no ROBO2-targeted small molecules have been reported. To address this gap, we performed a structure-based virtual screening campaign targeting ROBO2, followed by experimental validation with Dianthus TRIC platform and microscale thermophoresis (MST). Fifteen compounds were screened for ROBO2 binding, from which four candidates exhibited robust TRIC signals. Subsequent affinity measurements revealed that two small molecules, Z1334432986 and Z1692774161, bind ROBO2 in a reproducible concentration-dependent manner, with dissociation constants (Kd) of 40.8{+/-}4.8 M and 25.8{+/-}16.95 M, respectively. Molecular docking with validated hits revealed a shared ROBO2 binding pocket defined by conserved anchor residues (ASN354, SER366, ASP385) and accommodation of distinct ligand conformations within the ROBO2 binding pocket. This work establishes a screening pipeline for identifying ROBO2-targeted small molecules and lays the foundation for developing therapeutics aimed at disrupting Slit-ROBO2 signaling in GBM. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=140 SRC="FIGDIR/small/697640v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@2fa804org.highwire.dtl.DTLVardef@bfa3d5org.highwire.dtl.DTLVardef@19d1522org.highwire.dtl.DTLVardef@1d6774a_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Ligandability Assessment of the LAG-3 D1 Domain Enables Discovery of a Small-Molecule Inhibitor

LAG-3 is an emerging immune checkpoint whose extracellular D1 domain engages MHC class II through a broad protein-protein interface traditionally considered difficult to modulate with small molecules. To evaluate the ligandability of this region, we combined 100-ns molecular dynamics (MD) simulations, structure-based virtual screening, and biophysical and biochemical assays. MD sampling of the isolated D1 domain revealed a recurrent, transient cavity adjacent to the MHCII-binding surface. A representative pocket-open conformation was used to screen a [~]10,240-compound diversity library, yielding a single validated hit, N05. N05 bound the D1 domain with micromolar affinity measured by microscale thermophoresis (Kd = 59.2 {micro}M, TRIC/MST channel) and by spectral-shift detection (Kd = 56.1 {micro}M), and it partially inhibited the LAG-3/MHCII interaction (EC50 = 42.9 {micro}M; maximal inhibition [~]76%). A 30-ns MD simulation of the LAG-3-N05 complex showed stable ligand engagement within the MD-identified cavity and consistent stabilizing interactions with residues forming the pocket. These results demonstrate that the LAG-3 D1 domain possesses an accessible, dynamically formed binding site capable of accommodating small molecules, providing a structural and biophysical foundation for future exploration of LAG-3 ligandability. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/692800v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@177b446org.highwire.dtl.DTLVardef@1dc9efdorg.highwire.dtl.DTLVardef@14a2d6aorg.highwire.dtl.DTLVardef@ebca69_HPS_FORMAT_FIGEXP M_FIG C_FIG Overview of the computational and experimental workflow used to identify and validate a small-molecule binder of the LAG-3 D1 domain.

bioinformatics↗

bioRxiv2025

Restoring Amyloid Clearance via Astrocytes: Z17 Is a Selective Inhibitor of CHI3L1 in Alzheimers Disease

Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by cognitive decline, accumulation of hallmark protein aggregates and substantial neuroinflammation. Chitinase-3-like protein 1 (CHI3L1) which is predominantly produced by activated astrocytes in the central nervous system (CNS). Overexpression of CHI3L1 has been implicated with AD progression and worsening of symptoms. Herein, we report the identification of Z17 as a novel and selective CHI3L1 inhibitor which directly bind to CHI3L1 an equilibrium dissociation constant (KD) of 6.0 {micro}M. In human iPSC-derived astrocytes, Z17 acted as a dual-action regulator by reinstating astrocytic function and suppressing inflammation. Additionally, Z17 rescued CHI3L1-induced impairment by dose-dependently restoring A{beta} uptake and normalizing lysosomal proteolytic activity and pH. Furthermore, Z17 effectively blocked CHI3L1-driven activation of the NF-{kappa}B pathway in human astrocytes, providing a mechanistic explanation for the functional rescue. The in vitro pharmacokinetic (PK) profiling of Z17 demonstrated favorable drug-like properties for CNS development. These findings support the advancement of Z17 as a selective CHI3L1 inhibitor capable of simultaneously mitigating neuroinflammation and restoring astrocytic clearance mechanisms, making it a highly promising therapeutic candidate for Alzheimers disease. Abstract Figure O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=101 SRC="FIGDIR/small/692801v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@5cfc38org.highwire.dtl.DTLVardef@b2e291org.highwire.dtl.DTLVardef@38d243org.highwire.dtl.DTLVardef@746330_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

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↗