Biology subjects

Kuncewicz, K.

Publications and source records attributed to Kuncewicz, K..

8 recordsLinked to original sources

bioRxiv2026

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

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

pharmacology and toxicology↗

bioRxiv2025

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

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 an Achiral Small Molecule TREM2 Agonist with Im-proved Pharmacokinetic Profile and Validated Target Engagement

The triggering receptor expressed on myeloid cells 2 (TREM2) is a lipid-sensing immunoreceptor on microglia that has emerged as a therapeutic target for Alzheimers disease. Here, we report the discovery of C1, an achiral structural analog of VG-3927--the first small molecule TREM2 agonist to enter clinical development. C1 was synthesized via a modular and enantioselective-free route using sequential Suzuki couplings, enabling rapid scaffold diversification. Compared to VG-3927, the stereochemically simplified derivative (C1) exhibits superior microglial phagocytosis and validated target engagement. C1 induces TREM2 activation in HEK293-hTREM2/DAP12 cells, and its direct binding to TREM2 was confirmed using both microscale thermophoresis (MST) and surface plasmon resonance (SPR). Importantly, C1 also demonstrates a superior pharmacokinetic profile to VG-3927, including enhanced metabolic stability in human and mouse microsomes, favorable PAMPA permeability, and a LogD7.4 compatible with CNS penetration. Docking studies suggested a potential binding mode of C1 at the extracellular domain of TREM2, revealing key polar and hydrophobic interactions. These findings position C1 as a synthetically accessible and pharmacokinetically favorable lead for the development of TREM2-targeted therapies

pharmacology and toxicology↗

bioRxiv2025

Discovery of a First-in-Class SLIT2 Binder Disrupting the SLIT2/ROBO1 Axis via DNA-Encoded Library (DEL) Screening

The SLIT2/ROBO1 signaling axis plays a critical role in neural development, immune regulation, and tumor progression, including glioblastoma. However, small molecule inhibitors targeting this protein-protein interaction remain unexplored. Herein, we report the discovery and validation of DEL-S1, a first-in-class small molecule that binds to SLIT2 and disrupts its interaction with ROBO1. Using a DNA-encoded library (DEL) screen of 4.2 billion compounds, DEL-S1 was identified and confirmed to bind SLIT2 via temperature-related intensity change (TRIC) assay. Functional inhibition of the SLIT2/ROBO1 complex by DEL-S1 was demonstrated using a Time-Resolved Fluorescence Resonance Energy Transfer (TR-FRET) assay, yielding an IC50 of 68.8 {+/-} 12.5 {micro}M. Molecular docking and molecular dynamics (MD) simulations revealed key interaction hotspots at the SLIT2 binding interface and confirmed that DEL-S1 impairs SLIT2/ROBO1 complex formation by inducing conformational rearrangements. DEL-S1 exhibited favorable ADME properties, including satisfactory plasma and microsomal stability, low cytotoxicity, and minimal hERG liability. To facilitate structure-activity relationship (SAR) exploration, we designed and implemented a modular, one-pot synthetic route leveraging cyanuric chloride reactivity, enabling rapid derivatization of the triazine scaffold of DEL-S1. This strategy yielded structurally diverse analogs, including water-soluble carboxylate derivatives with preserved SLIT2/ROBO1 inhibitory activity. Together, this work establishes a novel chemical scaffold targeting SLIT2 and introduces a flexible synthetic platform to support further optimization toward therapeutic development.

biochemistry↗

bioRxiv2025

TREM2 Hit Discovery Using TRIC Technology: A Proof-of-Concept High-Throughput Screening Approach

Triggering receptor expressed on myeloid cells 2 (TREM2) is an immunomodulatory receptor implicated in both neurodegenerative diseases and cancer. Depending on the context, TREM2 agonists or inhibitors hold therapeutic potential. To date, the majority of TREM2-targeted strategies have centered on monoclonal antibodies (mAbs), which face limitations such as poor tissue penetration and potential immunogenic side effects. To overcome these challenges and expand the chemical space for TREM2-targeting agents, we developed a high-throughput screening (HTS) platform to identify novel small molecule TREM2 binders. Using temperature-related intensity change (TRIC) technology in a 384-well plate format (NanoTemper Dianthus), we screened two focused compound libraries comprising over 1,200 molecules. From this screen, 18 preliminary hits (1.44% hit rate) were identified and subsequently validated by dose-response binding studies using microscale thermophoresis (MST), yielding four validated hits (0.32% hit rate) with binding affinities in the high to medium micromolar range (e.g., T2337, KD = 22.4 M). The binding of the top hit, T2337, was further validated using surface plasmon resonance (SPR). Additionally, we assessed the functional activity of all four validated hits in a cellular assay measuring TREM2-mediated Syk phosphorylation in HEK293 cells co-expressing human TREM2 and its adaptor protein DAP12. These findings establish a robust and scalable platform for the discovery of small molecule TREM2 modulators and serve as a proof-of-concept for broader HTS campaigns targeting TREM2.

pharmacology and toxicology↗

bioRxiv2025

TREM2 Activation by First-in-Class Direct Small Molecule Agonists: DEL Screening, Optimization, Biophysical Validation, and Functional Characterization

Triggering receptor expressed on myeloid cells 2 (TREM2) is a key regulator of microglial function, and its loss-of-function variants are linked to Alzheimers disease (AD) and neurodegenerative disorders. While TREM2 activation is a promising therapeutic strategy, no small molecule agonists acting via direct TREM2 binding have been reported to date. Here, we describe the discovery of first-in-class, direct small molecule TREM2 agonists identified through DNA-encoded library (DEL) screening. The DEL hit (4a) demonstrated TREM2 binding affinity, as validated by three biophysical screening platforms (TRIC, MST, and SPR), induced Syk phosphorylation, and enhanced microglial phagocytosis. Preliminary optimization yielded 4i, which maintained TREM2 engagement with improved selectivity over TREM1 and no cytotoxicity. Molecular dynamics simulations revealed that 4a stabilizes a transient binding pocket on TREM2, suggesting a novel mechanism for receptor activation. These findings provide the first proof-of-concept for direct pharmacological TREM2 agonism, offering a foundation for developing therapeutics against AD and related disorders. Table of Contents graphic O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/655617v1_ufig1.gif" ALT="Figure 1"> View larger version (13K): org.highwire.dtl.DTLVardef@14a3d24org.highwire.dtl.DTLVardef@f423deorg.highwire.dtl.DTLVardef@5ee108org.highwire.dtl.DTLVardef@107e6df_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗