Biology subjects

Garcia-Vazquez, N.

Publications and source records attributed to Garcia-Vazquez, N..

8 recordsLinked to original sources

bioRxiv2026

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

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

pharmacology and toxicology↗

bioRxiv2025

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

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

bioinformatics↗

bioRxiv2025

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

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

Virtual Screening-Guided Discovery of Small Molecule CHI3L1 Inhibitors with Functional Activity in Glioblastoma Spheroids

Chitinase-3-like protein 1 (CHI3L1), a glycoprotein implicated in inflammation, fibrosis, and cancer, has emerged as a potential therapeutic target for glioblastoma (GBM). CHI3L1 contributes to tumor progression and immune evasion by promoting STAT3 signaling and mesenchymal transition. To identify small molecule CHI3L1 inhibitors, a structure-based 3D pharmacophore model was developed and applied to virtually screen over 4.4 million compounds from the Enamine collection. Following multi-tiered filtering, 35 candidates were selected for experimental evaluation. Binding validation via microscale thermophoresis (MST) confirmed dose-dependent CHI3L1 interactions for two compounds, 8 and 39, with dissociation constants (Kd) of 6.8 {micro}M and 22 {micro}M, respectively. These affinities were further supported by surface plasmon resonance (SPR), which yielded Kd values of 5.69 {micro}M for compound 8 and 17.09 {micro}M for compound 39. In 3D GBM spheroid models, compound 8 significantly reduced spheroid viability and attenuated phospho-STAT3 levels, consistent with CHI3L1 pathway disruption. These findings identify two promising scaffolds and support the utility of pharmacophore-guided virtual screening for discovering functionally active ligands targeting CHI3L1 in GBM. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/667816v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@291464org.highwire.dtl.DTLVardef@f9d224org.highwire.dtl.DTLVardef@1535eeaorg.highwire.dtl.DTLVardef@7c596e_HPS_FORMAT_FIGEXP M_FIG Table of Contents artwork C_FIG

pharmacology and toxicology↗

bioRxiv2025

TRIC Coupled with TR-FRET as a High-Throughput Screening Platform for the Discovery of SLIT2 Binders: A Proof-of-Concept Approach

SLIT2, a secreted glycoprotein involved in axon guidance, immune modulation, and tumor progression, remains largely unexplored as a pharmacological target due to the absence of small-molecule modulators. Here, we present a proof-of-concept high-throughput screening platform that integrates Temperature-Related Intensity Change (TRIC) technology with time-resolved Forster resonance energy transfer (TR-FRET) to identify small molecules capable of disrupting the SLIT2/ROBO1 interaction. Screening a lipid metabolism-focused compound library (653 molecules) yielded bexarotene, as the most potent small molecule SLIT2 binder reported to date, with a dissociation constant (KD) of 2.62 {micro}M. Follow-up TR-FRET assays demonstrated dose-dependent inhibition of SLIT2/ROBO1 interaction, with an IC50 value of [~]22.8 {micro}M and maximal inhibition of [~]15- 25%. These findings suggest a novel extracellular activity of bexarotene and validate the combined use of TRIC and TR-FRET as a scalable screening strategy for SLIT2-targeted small molecules. This platform lays the groundwork for future high-throughput discovery efforts against SLIT2 and its signaling axis. Graphical abstractTRIC-based small molecule screening platform protocol steps with implementation of TR-FRET for the identification of SLIT2 inhibitors. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/663693v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@e585fborg.highwire.dtl.DTLVardef@bf3de5org.highwire.dtl.DTLVardef@178910dorg.highwire.dtl.DTLVardef@7b2086_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

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↗

bioRxiv2024

Stabilization of GTSE1 by cyclin D1-CDK4/6 promotes cell proliferation: relevance in cancer prognosis

In healthy cells, cyclin D1 is expressed during the G1 phase of the cell cycle, where it activates CDK4 and CDK6. Its dysregulation is a well-established oncogenic driver in numerous human cancers. The cancer-related function of cyclin D1 has been primarily studied by focusing on the phosphorylation of the retinoblastoma (RB) gene product. Here, using an integrative approach combining bioinformatic analyses and biochemical experiments, we show that GTSE1 (G-Two and S phases expressed protein 1), a protein positively regulating cell cycle progression, is a previously unrecognized substrate of cyclin D1-CDK4/6 in tumor cells overexpressing cyclin D1 during G1 and subsequent phases. The phosphorylation of GTSE1 mediated by cyclin D1-CDK4/6 inhibits GTSE1 degradation, leading to high levels of GTSE1 across all cell cycle phases. Functionally, the phosphorylation of GTSE1 promotes cellular proliferation and is associated with poor prognosis within a pan-cancer cohort. Our findings provide insights into cyclin D1s role in cell cycle control and oncogenesis beyond RB phosphorylation.

biochemistry↗