Biology subjects

Gabr, M.

Publications and source records attributed to Gabr, M..

At least 55 records · Page 3Linked to original sources

bioRxiv2025

High-Throughput Fragment Screening Identifies a New Small Molecule Scaffold that Modulates TREM2 Signaling

Fragment-based drug discovery (FBDD) remains a powerful tool in drug development for targeting a wide range of proteins and identifying new small molecule-based scaffolds. Here, we explored a fragment library of 3,200 compounds using a temperature-related intensity change (TRIC)-based high-throughput screening (HTS) approach, and successfully identified new scaffolds that bind to triggering receptor expressed on myeloid cells 2 (TREM2), a relevant target in neurodegenerative diseases and cancer immunotherapy. We first validated the hits with dose-dependent assays, then chose the three most promising compounds (2M06, 6B10, 7G19) with binding affinities in the low to medium micromolar range for a "SAR by catalog" study. We screened 29 selected derivatives and subsequently evaluated them with dose-dependent experiments, a thermal shift assay (TSA), selectivity studies with off-targets (LAG-3, TREM1), and finally, in vitro TREM2 activation assays. In this SAR study, derivative 6B10-9 emerged as the lead compound with moderate TREM2 binding affinity (KD = 68.3 {micro}M) and significant effects on TREM2-dependent phosphorylation of SYK and DAP12 in HEK cells as wells as on microglial phagocytosis in HMC3 cells. Additionally, an in silico analysis revealed that 6B10-9 forms a stable complex with TREM2 via hydrogen bonding, which maintains its structural integrity during extended molecular dynamics (MD) simulations. These results suggest that 6B10-9 could serve as a promising lead for future optimizations in the development of small molecule-based TREM2 modulators.

pharmacology and toxicology↗

bioRxiv2025

An Integrated Biophysical Fragment Screening Approach Identifies Novel Binders of the CD28 Immune Receptor

CD28 is an essential costimulatory receptor required for full T cell activation, and its dysregulation contributes to multiple immune-mediated pathologies. Despite its central immunological role, CD28 remains largely unexplored as a target for small-molecule modulation, primarily due to the shallow and large interface of its ligand-binding site. Here, we applied a fragment-based high-throughput screening (HTS) strategy to identify low molecular weight chemotypes capable of engaging with human CD28. A 3,200-member library composed of structurally diverse fragments, enriched for scaffolds designed to target protein-protein interaction (PPI) interfaces, was screened in single-dose format using temperature-related intensity change (TRIC) technology, yielding 36 primary hits (1.13% hit rate). Follow-up surface plasmon resonance (SPR) validation confirmed two fragments as direct CD28 binders. Molecular docking analysis revealed a plausible binding orientation for PPIF3 within the CD28 extracellular domain, suggesting potential interaction hotspots that may be exploited during future optimization. Together, these findings provide the first demonstration that fragment-based screening can successfully identify chemotypes capable of engaging with the CD28 PPI interface. This work establishes a scalable, biophysics-driven workflow for CD28 ligand discovery and lays the foundation for subsequent hit-to-lead development of small molecule CD28 modulators.

biophysics↗

bioRxiv2025

Identification of a First-In-Class Small Molecule CAPON Binder Using Affinity Selection-Mass Spectrometry Screening

NOS1AP (CAPON) is an adaptor protein of neuronal nitric oxide synthase (nNOS) correlated with Alzheimers disease progression, making it an attractive yet unexplored therapeutic target. To assess its chemical tractability, we employed affinity selection-mass spectrometry (AS-MS) to screen approximately 10,000 small molecules for CAPON binding, identifying 52 initial hits. These compounds were further evaluated for true binding interactions and potential autofluorescence or quenching effects using the Dianthus platform. Five compounds were selected for quantitative affinity determination by microscale thermophoresis (MST). Among these, compound MA32 exhibited a dissociation constant (Kd) of 74 M. MA32 thus represents the first AS-MS-identified small-molecule binder of CAPON. This study establishes a workflow for small-molecule discovery against novel, previously uncharacterized targets.

biophysics↗

bioRxiv2025

Computational Design and Biophysical Validation of Macrocyclic Peptides as Inhibitors of SLIT2/ROBO1 interaction

The SLIT2/ROBO1 signaling axis regulates cellular migration and angiogenesis but also contributes to tumor progression and immune evasion in glioblastoma. Targeting this pathway with small molecules or antibodies remains challenging due to the shallow and extended nature of the SLIT2/ROBO1 interface. Here, we report the first computational design and experimental validation of macrocyclic peptides that inhibit SLIT2/ROBO1 binding. Twenty peptides were generated through a structure-guided interface mapping approach (Des3PI 2.0) and ranked using a contact-based scoring function. The top candidates were synthesized and evaluated using time-resolved fluorescence resonance energy transfer (TR-FRET) and biolayer interferometry (BLI) assays. Among the SLIT2-targeting peptides, SP4 and SP3 showed the most pronounced inhibition in TR-FRET and BLI, confirming direct binding to the SLIT2/ROBO1 interface. The lead peptide SP4 also demonstrated favorable in vitro pharmacokinetic properties, including strong stability in simulated intestinal fluid, high plasma integrity, and moderate metabolic stability in rat liver microsomes. Collectively, this work establishes a computational-to-experimental pipeline for discovering macrocyclic peptides that disrupt challenging protein-protein interactions and provides a foundation for developing next-generation SLIT2/ROBO1 modulators for cancer and neuroimmune disorders. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=54 SRC="FIGDIR/small/684696v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@e011f7org.highwire.dtl.DTLVardef@bb8e5aorg.highwire.dtl.DTLVardef@17ec46corg.highwire.dtl.DTLVardef@1919666_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Discovery of a CHI3L1-Targeted Small Molecule Modulating Neuroinflammation in Alzheimers Disease via DNA-Encoded Library (DEL) Screening

Chitinase-3-like protein 1 (CHI3L1, also known as YKL-40) has emerged as a central effector of astrocyte-mediated neuroinflammation and a promising biomarker for Alzheimers disease (AD). However, small molecule CHI3L1 inhibitors that modulate neuroinflammation are limited. Here, we report the discovery of a CHI3L1-targeted small molecule, DEL-C1, identified through DNA-encoded library (DEL) screening and validated using orthogonal biophysical, computational, and cellular approaches. DEL-C1 demonstrated direct CHI3L1 binding in microscale thermophoresis (MST) and surface plasmon resonance (SPR) assays, with reversible and concentration-dependent association. Molecular docking and 100-ns molecular dynamics simulations revealed a stable binding mode within the CHI3L1 substrate groove, anchored by Tyr206 and flanked by Trp99 and Trp352, supporting a thermodynamically favorable interaction. In vitro ADME profiling indicated a balanced physicochemical profile, permeability, and metabolic stability, consistent with CNS drug-like properties. Functionally, DEL-C1 reversed CHI3L1-induced astrocyte dysfunction by restoring A{beta} uptake, lysosomal acidification, and proteolytic activity, while reducing CHI3L1 and IL-6 secretion. DEL-C1 also suppressed CHI3L1-driven NF-{kappa}B transcriptional activation, highlighting its anti-inflammatory potential. Collectively, this study establishes DEL-C1 as a promising small molecule modulator of CHI3L1 and a chemical tool to interrogate astrocyte-driven neuroinflammation in AD.

biophysics↗

bioRxiv2025

Affinity Selection-Mass Spectrometry Coupled with Biophysical Validation Enables Proof-of-Concept Discovery of CHI3L1 Binders

Chitinase-3-like protein 1 (CHI3L1) is a multifunctional extracellular glycoprotein implicated in tumor progression, immune suppression, and fibrosis, making it an attractive but challenging therapeutic target. To explore its chemical tractability, we applied an affinity selection-mass spectrometry (AS-MS) workflow to screen 10,000 small molecules for CHI3L1 binding. The screen yielded 124 initial hits with a hit rate of 1.24%, which were prioritized based on chemical suitability, and six candidates were advanced for validation using microscale thermophoresis (MST). Among these, compound A9 exhibited a clear, dose-dependent binding response in MST with a Kd of 182 {+/-} 18 {micro}M. Molecular docking supported these findings, revealing that A9 forms hydrophobic and hydrogen-bonding interactions within a defined pocket of the CHI3L1 structure. Although modest in affinity, A9 represents the first small molecule binder of CHI3L1 identified through AS-MS. This study provides a proof-of-concept demonstration that CHI3L1 can be chemically engaged using AS-MS, establishing a foundation for future medicinal chemistry optimization and the development of chemical probes targeting this previously undruggable extracellular protein. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/681114v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@12959c2org.highwire.dtl.DTLVardef@1c3e3a4org.highwire.dtl.DTLVardef@1962a04org.highwire.dtl.DTLVardef@1a0a2c4_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

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

AI-Guided Design of Cyclic Peptide Binders Targeting TREM2 Using CycleRFdiffusion and Experimental Validation

Triggering receptor expressed on myeloid cells 2 (TREM2) plays a central role in regulating microglial function in the central nervous system and has emerged as a promising therapeutic target for Alzheimers disease. Despite advances in antibody-based therapeutics, small molecules and peptides capable of modulating TREM2 remain limited. Here, we present a cyclic peptide design pipeline that integrates CycleRFdiffusion, ProteinMPNN for sequence design, and HighFold for structural prediction and screening. Using the TREM2 structure as input, we generated and screened 1,500 peptide-target complexes, prioritizing four candidates that met structural and energetic criteria. Subsequent biophysical evaluation identified TP4 as a TREM2 binder, demonstrating consistent binding in spectral shift, microscale thermophoresis, and surface plasmon resonance. Pharmacokinetic profiling indicated that TP4 possesses favorable plasma stability and moderate metabolic stability, supporting its tractability for further optimization. This study establishes a generalizable framework for AI-driven cyclic peptide discovery and provides the first proof-of-concept demonstration of TREM2-targeted cyclic peptide binders.

pharmacology and toxicology↗

bioRxiv2025

AI-Assisted Discovery and Optimization of Small Molecule TREM2 Agonists with Functional Microglial Activity

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor whose loss-of-function variants increase Alzheimers disease (AD) risk by impairing plaque compaction, survival, and protective microglial programming. While antibody-based agonists have shown promise, their translation is hindered by poor brain penetration and high cost. Here, we report the discovery and optimization of small molecule TREM2 agonists through an AI-assisted virtual screening strategy. Deep Docking of over five million purchasable compounds identified a structurally novel hit, T2K-014, which engaged TREM2 with modest affinity. A SAR-by-catalog campaign led to the identification of T2M-010 as a potent binder. T2M-010 demonstrated favorable in vitro PK properties, including high solubility, passive BBB permeability, moderate metabolic stability, and minimal safety liabilities. Functionally, T2M-010 activated receptor-proximal signaling, inducing SYK phosphorylation in TREM2-expressing cells, and promoted microglial phagocytosis. Together, these findings establish T2M-010 as the most potent small molecule TREM2 binder reported to date capable of driving protective microglial responses relevant to AD.

pharmacology and toxicology↗

bioRxiv2025

Structure-Based Virtual Screening Identifies TREM2-Targeted Small Molecules that Enhance Microglial Phagocytosis

Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor whose activation promotes phagocytosis and neuroprotection in Alzheimers disease (AD) and related neurodegenerative disorders. While therapeutic efforts have largely focused on antibodies, small molecule TREM2 modulators remain limited. Here, we applied a structure- based virtual screening workflow targeting a putative allosteric site on TREM2, guided by PyRod-derived pharmacophores from molecular dynamics simulations. Screening of the Enamine Collection yielded 20 candidate compounds, three of which demonstrated binding in TRIC assays. The top hit, EN020, exhibited a KD of 14.2 {micro}M (MST) and 35.9 {micro}M (SPR), and significantly enhanced microglial phagocytosis in BV2 cells outperforming the known TREM2 agonist VG-3927. A preliminary structure-activity relationship (SAR) study, including synthetic and catalog-derived analogs, highlighted a narrow tolerance for scaffold modifications, with only T2V002 retaining partial TREM2 binding affinity. This work identifies EN020 as a novel small molecule TREM2 modulator with functional activity, providing a framework for rational optimization toward potential AD therapeutics.

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 Small Molecule CHI3L1 Inhibitors by SPR-Based High-Throughput Screening

Chitinase-3-like 1 (CHI3L1) is a secreted glycoprotein implicated in carcinogenesis and tumor immune evasion. Elevated CHI3L1 expression is frequently detected in cancer patients, highlighting it as a promising therapeutic target. To overcome the limited availability of small molecule CHI3L1 inhibitors, we established a surface plasmon resonance (SPR)-based high-throughput screening platform and applied it to a focused chemical library of small molecules. Primary screening identified seven hits, with compounds 1-4 and 1-7 validated as CHI3L1 binders (Kd = 10.4 {+/-} 1.0 M and 7.40 {+/-} 0.78 M, respectively). Both compounds disrupted the CHI3L1-galectin-3 interaction in AlphaLISA assays and engaged the CHI3L1 binding pocket in docking and molecular dynamics (MD) simulations. Importantly, functional evaluation in a multicellular 3D glioblastoma (GBM) spheroid model demonstrated that compound 1-7 potently reduced spheroid viability and inhibited STAT3 phosphorylation, outperforming both compound 1-4 and the known CHI3L1-STAT3 disruptor hygromycin B (HB). These findings validate SPR as a robust primary screening platform for CHI3L1 and demonstrate that the identified small molecule binders exert functional activity in a physiologically relevant multicellular GBM spheroid model.

pharmacology and toxicology↗

bioRxiv2025

Temperature-Related Intensity Change (TRIC)-based High Throughput Screening Platform for the Discovery of CHI3L1-Targeted Small Molecules

Chitinase-3-like 1 (CHI3L1) protein is a secreted glycoprotein involved in various normal physiological processes, while its abnormal elevation is closely associated to carcinogenesis. CHI3L1 promotes tumor progression by recruiting and polarizing immune cells to sustain an immunosuppressive microenvironment, while directly stimulating cancer cell proliferation and migration. Although CHI3L1 deletion has shown therapeutic potential in animal models, the development of small molecule modulators remains limited. To address this gap, we developed a TRIC-based high-throughput screening (HTS) platform for CHI3L1 binding and applied it to a 5,280-compound library, yielding 11 binders (hit rate: 0.21%). Of these, three compounds (9N05, 11C19, and 3C13) were validated by surface plasmon resonance (SPR). Among the validated hits, 9N05 showed the strongest binding affinity to CHI3L1 (Kd = 202.3 {+/-} 76.6 M) but only weak inhibition of CHI3L1/galectin-3 interaction. In contrast, 11C19 significantly disrupted this interaction (73.9 {+/-} 7.9% inhibition at 100 M; IC50 = 188.6 {+/-} 0.18 M). This proof-of-concept study establishes TRIC-based screening as a viable approach for identifying CHI3L1-targeted small molecules and supports its use in future modulator discovery.

biophysics↗

bioRxiv2025

Design and Validation of the First-in-Class PROTACs for Targeted Degradation of the Immune Checkpoint LAG-3

Lymphocyte activation gene-3 (LAG-3) is an inhibitory immune checkpoint receptor that plays a central role in T cell exhaustion and immune evasion in cancer. While monoclonal antibodies targeting LAG-3 have entered clinical development, small molecule approaches remain largely unexplored. Here, we report the design and validation of the first-in-class PROTACs for targeted degradation of LAG-3. In this study, we repurposed a LAG-3-binding small molecule identified through DNA-encoded library (DEL) screening as the targeting ligand for a series of CRL4CRBN-based PROTACs designed with varied linker lengths. Western blot analysis in Raji-LAG3 cells demonstrated that LAG-3 PROTAC-1 and LAG-3 PROTAC-3 induce potent, dose-dependent degradation of LAG-3, with DC50 values of 274 nM and 421 nM, respectively. Molecular docking and molecular dynamics (MD) simulations revealed the LAG-3 binding mode of designed PROTACs and provided structural insights into PROTAC-mediated ternary complex formation. Collectively, this work establishes a proof-of-concept for chemical degradation of LAG-3 for the first time and paves the way for novel immunotherapeutic strategies.

pharmacology and toxicology↗

bioRxiv2025

Structure-Guided Discovery of CHI3L1 Inhibitors from Ultralarge Chemical Spaces for Glioblastoma Therapy

Glioblastoma (GBM) is the most aggressive primary brain tumor, with a median survival of approximately one year and limited therapeutic options. Chitinase 3-like 1 (CHI3L1) is increasingly recognized as a promising target in GBM due to its role in tumor progression and immune modulation. In this study, we employed an in-house structure-based screening strategy (SpaceDock) to explore a virtual chemical space of 377 billion compounds for potential CHI3L1 inhibitors. Using a reaction-aware ligand design approach, 60 top-scoring virtual hits were synthesized, and 45 were obtained with sufficient purity for experimental testing. Primary screening by microscale thermophoresis (MST) identified nine hits. Compounds showing dose-dependent binding were subsequently analyzed using surface plasmon resonance (SPR), leading to the identification of compound 9e with a dissociation constant (Kd) of 19.11 {micro}M. Importantly, 9e demonstrated robust, dose-dependent efficacy in a multicellular 3D GBM spheroid model, significantly reducing spheroid viability and downstream STAT3 signaling. These results highlight 9e as a promising drug candidate for modulating the CHI3L1-STAT3 axis and underscore the potential of structure-guided, reactivity-aware virtual screening of ultralarge chemical spaces to target non-enzymatic, conformationally dynamic proteins in complex cancer models.

pharmacology and toxicology↗

bioRxiv2025

Discovery and Optimization of LAG-3-Targeted Small Molecules via DNA-Encoded Chemical Library (DEL) Screening for Cancer Immunotherapy

Lymphocyte activation gene-3 protein (LAG-3) is an immune checkpoint receptor that promotes T cell exhaustion and immune evasion in cancer. While antibody-based LAG-3 inhibitors have reached the clinic, small molecule modulators remain unexplored. Here, we report compound 11, the most potent small molecule LAG-3 inhibitor to date. Identified via a 4.2-billion compound DNA-encoded chemical library (DEL) screen, compound 11 binds LAG-3 with submicromolar affinity and disrupts the LAG-3/MHCII interaction. Molecular modeling suggests direct antagonism at the LAG-3/MHCII interface with potential allosteric effects. In functional assays, compound 11 enhances IFN-{gamma} secretion and promotes tumor cell killing in co-cultures of PBMCs and cancer cells. Importantly, compound 11 also exhibits favorable pharmacokinetics. These findings support the development of small molecule LAG-3 inhibitors as immunotherapeutic agents and provide a foundation for further optimization. Table of Contents artwork O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=106 SRC="FIGDIR/small/668839v1_ufig1.gif" ALT="Figure 1"> View larger version (29K): org.highwire.dtl.DTLVardef@248c84org.highwire.dtl.DTLVardef@185510forg.highwire.dtl.DTLVardef@18cce50org.highwire.dtl.DTLVardef@13cfd8c_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2025

Targeting the CAPON-NOS Axis: A Computational Strategy for Small Molecule Modulator Discovery

The carboxy-terminal PDZ ligand of neuronal nitric oxide synthase (CAPON) serves as a critical regulatory protein controlling nitric oxide (NO) signaling across multiple physiological and pathological processes which encompass neurological, cardiac and metabolic functions. These diverse physiological roles of CAPON marks it as a key therapeutic target for conditions associated with its dysregulation. Despite this therapeutic potential there are no specific CAPON or nNOS/CAPON modulators which have been developed to date, highlighting a significant gap in targeted drug discovery. Herein, we report the first strategy specifically focused on disrupting the nNOS/CAPON protein-protein interface. Through screening of chemical libraries composed of 4.6 million compounds and eight molecular dynamics simulations, two potential hit compounds were identified. Beyond identifying these promising hits, our approach introduces two novel computational tools: a freely available Python-based toolset for NMR structural analysis and visualization and a second toolkit for accelerated ligand preparation. These tools significantly accelerate data preparation timelines while reducing computational costs, providing the research community with accessible resources for structure-based drug discovery efforts. Together, these tools represent a substantial contribution to the computational chemistry toolkit, enabling researchers to conduct high-throughput virtual screening campaigns more efficiently and with greater reproducibility. This work represents a foundational step toward developing targeted therapies for CAPON-mediated disorders and provides a scalable computational framework for future protein-protein interaction drug discovery efforts. HighlightsO_LIA novel structure-based strategy developed to target the CAPON/nNOS protein-protein interaction. C_LIO_LIA Python-based toolset for protein conformation analysis, identification, visualization and separation. C_LIO_LIPython pipeline enables efficient ligand preparation for ultra-large chemical libraries. C_LIO_LIVirtual screening identified 6 promising small-molecule candidates. C_LIO_LIA total of 8*100ns molecular dynamics (MD) simulations performed using DESMOND. C_LIO_LIMM/GBSA and contact-time analysis were conducted to assess binding stability and affinity. C_LI

bioinformatics↗

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↗