Biology subjects

Nada, H.

Publications and source records attributed to Nada, H..

At least 19 recordsLinked to original sources

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

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

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

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

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↗

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

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

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

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↗