Biology subjects

Gabr, M.

Publications and source records attributed to Gabr, M..

At least 19 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

DNA-Encoded Library-Guided Discovery of a First-in-Class Molecular Glue of the NOS1AP-SCRIB Complex in Alzheimer's Disease Patient-Derived Neurons

NOS1AP is an adaptor for neuronal nitric oxide synthase that engages several partner proteins and is implicated in Alzheimer's disease. Efforts to drug NOS1AP have sought to disrupt these; we instead report pharmacological stabilization of one interaction. Affinity selection of a 1.4-billion-member DNA-encoded library identified a benzimidazole-5-carboxamide chemotype, optimized by fragment-directed SAR to NOSTRA with submicromolar NOS1AP binding affinity. Of six interactions, NOSTRA enhanced only SCRIB binding, with a cooperativity factor of 30, predominantly through a 14-fold slowing of dissociation; native mass spectrometry resolved a NOS1AP:NOSTRA:SCRIB ternary complex. In healthy human iPSC-derived cortical neurons, NOSTRA engaged the endogenous complex and activated Rac1 without cytotoxicity; in Alzheimer's patient-derived neurons, it lowered the A{beta}42/A{beta}40 ratio and p-tau217, reduced neurofilament light and improved network activity. With an in vitro ADME profile consistent with CNS exposure, NOSTRA is the first molecular glue for a NOS1AP complex, establishing interaction stabilization as a viable NOS1AP pharmacology.

pharmacology and toxicology↗

bioRxiv2026

A First-in-Class, Brain-Penetrant Small Molecule Inhibitor of the NOS1AP-Tau Interaction Rescues Tau Pathology In Vivo and Improves Cognition

NOS1AP is a genetically validated driver of tau pathology in Alzheimer's disease (AD), yet the NOS1AP-tau interaction itself has remained pharmacologically untargeted. We report CAPTOR-1, a first-in-class small molecule that disrupts this interaction by binding a pocket in the N-terminal region of NOS1AP (residues 1-126). A drug-resistant NOS1AP Y16A/F42A variant that retained tau binding but exhibited a 78-fold reduction in CAPTOR-1 affinity provides genetic evidence supporting an on-target mechanism. In human iPSC-derived neurons, CAPTOR-1 dose-dependently reduces endogenous NOS1AP-tau association, tau seeding activity, and pathogenic tau phosphorylation, phenocopying genetic NOS1AP knockdown; these effects extend to neurons from patients with sporadic AD. Orally dosed CAPTOR-1 crosses the blood-brain barrier and engages NOS1AP in brain dose-dependently. Chronic oral dosing rescues pathology, synaptic function, and memory in a tauopathy model (PS19) and an amyloidosis model (APP/PS1), sparing wild-type littermates in both. This work establishes pharmacological disruption of the NOS1AP-tau interaction as a viable, disease-modifying strategy for AD and positions CAPTOR-1 as a biomarker-ready chemical probe (pTau217, neurofilament light) for translational studies.

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

ClinOracle: Hierarchical AI Prediction of Target Binding and Patient-Derived Functional Activity Across Diverse Therapeutic Targets

AI platforms for drug discovery routinely achieve high hit rates against biochemical targets, yet the central translational challenge remains predicting whether a compound will be functionally active in patient-derived human cells. Here, we present ClinOracle, a hierarchical graph neural network that jointly predicts target binding and patient-derived functional activity by modeling functional activity as conditional on target engagement. Applied across five therapeutic targets spanning oncology, autoimmune, and neuroinflammatory diseases, ClinOracle ranked candidates using a Priority Score integrating translational probability with a developability score based on ADME and drug-likeness, advancing prioritized compounds through multistage prospective validation from biophysical binding to in vivo efficacy. Compounds with the highest Priority Scores consistently outperformed lower-ranked candidates across prospective experimental validation, demonstrating that hierarchical AI can prioritize compounds with patient-derived functional activity directly from molecular structure, rather than biochemical activity alone.

pharmacology and toxicology↗

bioRxiv2026

HTS-Oracle X: AI-Guided Prospective Discovery of Small Molecule Immune Checkpoint Binders

Targeting immune checkpoint protein-protein interactions (PPIs) using small molecules remains limited by the shallow, featureless binding surfaces of co-stimulatory and co-inhibitory receptors and the characteristically low hit rates of conventional high-throughput screening against these interfaces. Here we report HTS-Oracle X, a multimodal deep learning platform that integrates bidirectional cross-attention fusion of ChemBERTa SMILES embeddings with extended RDKit descriptors, trains on continuous biophysical binding signals rather than binary labels, and employs Monte Carlo Dropout uncertainty quantification for uncertainty-adjusted compound selection. Trained on 45,760 Dianthus TRIC-screened compounds per target under scaffold-aware cross-validation, HTS-Oracle X was applied prospectively to a 100,160-compound Enamine library against CD28, TIM-3, and VISTA. From 150 model-selected compounds, 45 dose-response confirmed binders were identified (30.0% overall hit rate), yielding enrichment factors of 234-408x over experimentally established random prospective baselines and 16 sub-micromolar hits. The top hits, HX-CD28-1 (KD = 233 nM), HX-TIM3-1 (KD = 249 nM), and HX-VISTA-1 (KD = 345 nM), demonstrated on-target functional activity in immune cell and tumor co-culture assays. HTSOracle X represents a scalable AI-guided framework for small molecule discovery against non-enzymatic immune checkpoint targets. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/732853v1_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@187fac7org.highwire.dtl.DTLVardef@6150d7org.highwire.dtl.DTLVardef@10266f0org.highwire.dtl.DTLVardef@b436e1_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2026

OracleScreen-LILRB4: Machine Learning-Guided Discovery of Myeloid Immune Checkpoint Binders Validated in Patient-Derived Cells

The identification of small molecule modulators of immune checkpoint proteins remains a significant challenge in drug discovery due to the flat, featureless nature of protein-protein interaction interfaces and the characteristically low hit rates observed in conventional high-throughput screening campaigns. Here we report OracleScreen-LILRB4, an ensemble machine learning framework trained on quantitative biophysical screening data from two structurally diverse compound libraries (19,800 compounds total) screened against the myeloid immune checkpoint leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3). By formulating binding prediction as a regression task targeting continuous {Delta}Fnorm values rather than binary hit classifications, OracleScreen-LILRB4 achieved a mean Spearman R of 0.61 and ROC-AUC of 0.86 under scaffold-aware cross-validation. Prospective virtual screening of a 45,760-member compound library and experimental validation of the top 200 predictions yielded a 28.5% hit rate, representing a 15.0-fold enrichment over baseline, with 16 compounds demonstrating nanomolar-affinity LILRB4 (ILT3) engagement. Lead compounds ORS-22 and ORS-14 restored anti-tumor immune activity across patient-derived colorectal cancer and acute myeloid leukemia co-culture systems, reversing SCG2-mediated immunosuppression and recovering cytotoxic T-cell function. These findings establish OracleScreen-LILRB4 as an effective computational framework for accelerating small molecule discovery against non-enzymatic immune checkpoint targets. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=99 SRC="FIGDIR/small/732859v1_ufig1.gif" ALT="Figure 1"> View larger version (48K): org.highwire.dtl.DTLVardef@1ef70a9org.highwire.dtl.DTLVardef@cd976dorg.highwire.dtl.DTLVardef@1907ebforg.highwire.dtl.DTLVardef@1716aec_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioinformatics↗

bioRxiv2026

HTS-Oracle v2: Prospective AI-Guided Discovery and Experimental Validation of Small Molecule Modulators Across Multiple Targets

High-throughput screening (HTS) remains the cornerstone of early-phase small molecule discovery yet consistently underperforms against immunotherapy targets, yielding validated hit rates below 0.1%. Here we introduce HTS-Oracle v2, which features rigorous cross-validation that ensures honest performance estimates. HTS-Oracle v2 was trained and validated across four clinically significant immune checkpoint targets (CD28, ICOS, LAG-3, and TIGIT) achieving ROC-AUC values of 0.968, 0.969, 0.875, 0.928 respectively under rigorous cross-validation. For prospective experimental validation, HTS-Oracle v2 was applied to an 8,960-compound Enamine Protein Mimetic Library, selecting only 25 compounds per target for experimental testing using temperature-related intensity change (TRIC) technology, a 99.7% reduction in screening burden. HTS-Oracle v2 identified 4, 5, 4, and 6 validated binders from 25 prospectively selected compounds per target, corresponding to validated hit rates of 16%, 20%, 16%, and 24%, respectively. Notably, 67-80% of all experimentally confirmed hits across the full 8,960-compound library were captured within just 25 model-selected compounds per target. For CD28, this represents a 28-fold improvement over HTS-Oracle v1 (239x versus 8.4x), establishing HTS-Oracle v2 as an efficient platform for AI-guided prospective hit discovery across immunotherapy targets.

bioinformatics↗

bioRxiv2026

AI-Guided Discovery of Small Molecule LILRB4 (ILT3) Inhibitors Reprograms Microglia and Reduces Amyloid Pathology

The inhibitory microglial receptor LILRB4 (ILT3) suppresses amyloid-beta clearance in Alzheimers disease (AD) through ApoE-dependent signaling but remains undrugged by small molecules. Here, we report the AI-guided discovery of small molecule inhibitors that directly disrupt the LILRB4-ApoE interaction. Ultralarge-scale screening of [~]500 million compounds identified small molecules that bind LILRB4 with nanomolar affinity and competitively block ApoE engagement, as validated across orthogonal biophysical assays. Structural and mutational analyses define a tractable interdomain pocket that mediates ligand recognition. In human iPSC-derived microglia, LILRB4 inhibition suppresses SHP1/2-dependent signaling, attenuates NF-{kappa}B activation, and restores A{beta} uptake. The lead compound exhibits favorable pharmacokinetics with robust brain penetration and, upon oral administration, improves cognitive performance, reduces amyloid burden, and dampens neuroinflammation in the 5xFAD mouse model. These findings establish LILRB4 as a druggable neuroimmune checkpoint and demonstrate that small molecule disruption of inhibitory microglial signaling can restore disease-relevant function in vivo.

pharmacology and toxicology↗

bioRxiv2026

From DNA-Encoded Library (DEL) Screening to In Vivo Validation: LILRB4 (ILT3)-Targeted Small Molecules Reprograms Myeloid Immune Suppression

Alzheimers disease (AD) remains a major unmet clinical challenge, with limited therapeutic strategies capable of effectively modulating neuroimmune dysfunction. Leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3) has recently emerged as an inhibitory microglial immune checkpoint implicated in ApoE-mediated suppression of amyloid-{beta} (A{beta}) clearance and inflammatory signaling, supporting its potential as a therapeutic target in AD. Here, we applied DNA-encoded library (DEL) screening of approximately 3.6 billion compounds to identify small molecule binders of LILRB4. Biophysical validation identified APX1 as a direct LILRB4 ligand with submicromolar affinity, which was further confirmed by cellular thermal shift assay (CETSA). Docking-guided mutagenesis studies defined a discrete ligand-binding interface involving key hotspot residues required for stable target engagement. Functionally, APX1 disrupted the LILRB4-ApoE interaction in orthogonal ELISA and biolayer interferometry assays. In human iPSC-derived microglia, APX1 suppressed SHP1/2 phosphorylation, attenuated NF-{kappa}B activation and IL-1{beta} secretion, and restored A{beta}42 uptake under ApoE-driven inflammatory conditions. APX1 further demonstrated favorable in vitro developability, metabolic stability, and CNS exposure properties. In the 5xFAD mouse model of AD, oral administration of APX1 improved cognitive performance, reduced cortical and hippocampal A{beta}42 burden, suppressed neuroinflammatory cytokines, and decreased activated microglial populations. Collectively, these findings establish APX1 as a promising small molecule modulator of the LILRB4-ApoE signaling axis and support pharmacological targeting of neuroimmune checkpoints as a therapeutic strategy for AD.

pharmacology and toxicology↗

bioRxiv2026

Direct Small Molecule Modulation of LILRB4 (ILT3) Restores Anti-Tumor Immunity In Vivo and in Patient-Derived Cells

Small molecule targeting of suppressive myeloid immune checkpoints remains a major challenge in cancer immunotherapy, particularly for non-enzymatic receptors lacking conventional druggable active sites. Leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3) is an immunosuppressive myeloid checkpoint implicated in tumor immune evasion, T-cell dysfunction, and resistance to immunotherapy across both solid and hematologic malignancies. Here, we report the discovery and characterization of GL-4512, a direct small molecule modulator of LILRB4 identified through a Dianthus-based temperature-related intensity change (TRIC) screening platform. Orthogonal biophysical studies, including microscale thermophoresis, surface plasmon resonance, and cellular thermal shift assays, confirmed direct target engagement with nanomolar affinity. Extensive microsecond molecular dynamics simulations combined with site-directed mutagenesis identified a previously unrecognized ligandable pocket within the flexible extracellular domain of LILRB4. Functionally, GL-4512 disrupted the immunosuppressive LILRB4-SCG2 signaling axis and suppressed downstream SHP1/SHP2 and STAT3 signaling. In patient-derived colorectal cancer and acute myeloid leukemia co-culture systems, pharmacological inhibition of LILRB4 restored anti-tumor immune activity, enhanced IFN-{gamma} and IL-2 production, increased cytotoxic T-cell activation, and reduced tumor-cell viability. GL-4512 additionally demonstrated favorable pharmacokinetic and safety properties supporting oral in vivo administration. In immunocompetent CT26 syngeneic colorectal tumors, once-daily oral treatment significantly suppressed tumor growth and enhanced intratumoral immune activation. Collectively, these findings establish LILRB4 as a tractable target for direct small molecule immunomodulation and support therapeutic targeting of suppressive myeloid immune checkpoints for cancer using non-biologic modalities.

pharmacology and toxicology↗

bioRxiv2026

Discovery of ILT3 (LILRB4) Small Molecule Inhibitors by Affinity Se-lection-Mass Spectrometry Reveals Druggability of a Neuroimmune Checkpoint in Alzheimers Disease

Leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3) is an emerging neuroimmune checkpoint that restricts microglial activation and amyloid clearance in Alzheimers disease (AD) through ApoE-dependent signaling. Here, we establish ILT3 as a tractable small molecule target using affinity selection-mass spectrometry (AS-MS) to identify direct binders. Biophysical validation confirmed high-affinity engagement, with LT12 exhibiting nanomolar binding by MST and SPR. Computational modeling and mutagenesis defined a discrete ILT3 binding pocket, revealing a distributed interaction network critical for ligand engagement. Targeting ILT3 disrupted the ILT3-ApoE interaction, with LT12 showing potent inhibition in orthogonal biochemical assays. In human iPSC-derived microglia, ILT3 modulation attenuated SHP1/2 signaling, suppressed NF-{kappa}B activation, reduced IL-1{beta} secretion, and restored A{beta} uptake. In vivo, pharmacological targeting of ILT3 improved cognition, reduced amyloid burden, and attenuated neuroinflammation in 5xFAD mice. Together, these findings validate ILT3 as a druggable neuroimmune checkpoint and support its therapeutic targeting in AD.

pharmacology and toxicology↗

bioRxiv2026

Targeting the Myeloid Immune Checkpoint ILT3 (LILRB4) with Small Molecules Enables Reprogramming of Suppressive Tumor Immunity

Cancer immunotherapy has transformed cancer treatment; however, durable responses remain limited by suppressive myeloid populations within the tumor microenvironment. Leukocyte immunoglobulin-like receptor B4 (LILRB4/ILT3) is an emerging myeloid immune checkpoint implicated in immune evasion and resistance to immunotherapy, yet small molecule targeting of ILT3 remains largely unexplored. Here, we report the discovery of small molecule ILT3 modulators identified using a Dianthus-based temperature-related intensity change (TRIC) screening platform. Screening of an 8,961-member Enamine Library identified multiple direct ILT3 binders, with lead compound ICB-7 demonstrating high-affinity binding to recombinant human ILT3 by microscale thermophoresis and robust cellular target engagement in CETSA assays. Molecular docking and molecular dynamics simulations revealed a stable hydrophobic binding pocket within the D2 domain of ILT3. Functionally, ICB-7 disrupted the ILT3-SCG2 interaction and inhibited downstream SHP1, SHP2, and STAT3 signaling. In patient-derived colorectal cancer and acute myeloid leukemia co-culture models, ICB-7 enhanced cytotoxic T-cell activity, and reduced tumor-cell viability. The compound also demonstrated favorable pharmacokinetic and safety properties together with significant anti-tumor efficacy in the CT26 syngeneic colorectal carcinoma model. Collectively, these findings establish ILT3 as a tractable target for small-molecule immunomodulation and support pharmacological targeting of suppressive myeloid checkpoints as a promising cancer immunotherapy strategy.

pharmacology and toxicology↗

bioRxiv2026

Development of a High-Throughput TR-FRET Assay for Identification of Small Molecule Inhibitors of the LILRB4 (ILT3)-SCG2 Immune Checkpoint Interaction

Leukocyte immunoglobulin-like receptor B4 (LILRB4, ILT3) is an inhibitory immune checkpoint expressed on myeloid cells, where it contributes to immunosuppression within the tumor microenvironment. Secretogranin 2 (SCG2) has recently been identified as a functional ligand of LILRB4, yet small molecule modulators of this interaction remain unexplored. Here, we report the development of a high-throughput time-resolved fluorescence resonance energy transfer (TR-FRET) assay to interrogate the LILRB4 (ILT3)-SCG2 interaction. The assay demonstrated robust performance and was validated using a blocking anti-LILRB4 antibody, consistent with orthogonal ELISA measurements. Pilot screening of chemical libraries identified 23 primary hits, of which two compounds, BMS-813160 and PSB-603, showed reproducible, dose-dependent inhibition with TR-FRET IC50 values of 26.7 {+/-} 1.03 {micro}M and 37.2 {+/-} 2.14 {micro}M, respectively. Activity was confirmed by ELISA, supporting the robustness of the assay. This platform enables high-throughput discovery of first-in-class small molecule modulators of the LILRB4-SCG2 immune checkpoint and provides a foundation for targeting myeloid-driven immunosuppression.

biophysics↗

bioRxiv2026

High-Throughput CETSA Identifies Small Molecule Modulators of ILT3 (LILRB4) with Functional Activity in Human iPSC-Derived Microglia for Alzheimers Disease

Immune inhibitory signaling in microglia contributes to impaired amyloid clearance and neuroinflammation in Alzheimers disease (AD), yet small molecule modulators targeting these pathways remain largely unexplored. Here, we report the development of a high-throughput cellular thermal shift assay (HT-CETSA) platform for identification of small molecule binders targeting the inhibitory immune receptor ILT3 (LILRB4). Screening of [~]40,000 compounds yielded multiple validated hits, including IB15C, a submicromolar ILT3 binder identified through preliminary structure-activity relationship optimization. Orthogonal validation by microscale thermophoresis, surface plasmon resonance, docking, and site-directed mutagenesis confirmed direct and target-specific ILT3 engagement. Functionally, IB15C disrupted the ILT3-ApoE interaction and restored microglial activity in human iPSC-derived microglia, reducing SHP1/2, suppressing cytokine secretion, and enhancing amyloid uptake. IB15C also demonstrated favorable in vitro pharmacokinetic and safety properties, supporting further development of ILT3-targeted neuroimmune therapeutics.

pharmacology and toxicology↗

bioRxiv2026

Cyclic Peptides Target CAPON and Modulate Cellular Responses under Alzheimers Disease-Relevant Stress

CAPON (NOS1AP) is an adaptor protein involved in neuronal nitric oxide synthase (nNOS) signaling and has been implicated in Alzheimers disease (AD), excitotoxicity, and tau-associated neurodegeneration. Here, we report the identification of cyclic peptide ligands targeting CAPON using phage display screening of a disulfide-constrained peptide library. Phage enrichment, ELISA validation, microscale thermophoresis (MST), and biolayer interferometry (BLI) identified CAP1 as the lead peptide, exhibiting low micromolar binding affinity toward CAPON. Computational studies further supported stable CAPON-CAP1 interactions through complementary hydrophobic and electrostatic contacts. Functionally, CAP1 attenuated A{beta}42-induced neuronal toxicity, suppressed NMDA-driven nitric oxide production, and reduced pathological tau phosphorylation in neuronal models under AD-relevant stress conditions. In addition, CAP1 demonstrated favorable preliminary pharmacokinetic properties, including good aqueous solubility, plasma stability, and measurable membrane permeability. Collectively, these findings establish the first cyclic peptide ligands targeting CAPON and identify CAP1 as a promising scaffold for modulation of CAPON-dependent neurodegenerative signaling.

pharmacology and toxicology↗