{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"institution":[{"name":"bioRxiv"}],"indexed":{"date-parts":[[2026,1,16]],"date-time":"2026-01-16T00:26:50Z","timestamp":1768523210159,"version":"3.49.0"},"posted":{"date-parts":[[2025,7,21]]},"group-title":"Pharmacology and Toxicology","reference-count":42,"publisher":"openRxiv","license":[{"start":{"date-parts":[[2025,7,21]],"date-time":"2025-07-21T00:00:00Z","timestamp":1753056000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/www.biorxiv.org\/about\/FAQ#license"}],"funder":[{"DOI":"10.13039\/100000062","name":"National Institute of Diabetes and Digestive and Kidney Diseases","doi-asserted-by":"crossref","award":["R01DK137299"],"award-info":[{"award-number":["R01DK137299"]}],"id":[{"id":"10.13039\/100000062","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":[],"accepted":{"date-parts":[[2025,7,21]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                <jats:sec>\n                  <jats:title>Background and Purpose<\/jats:title>\n                  <jats:p>CD28 is a pivotal costimulatory receptor that governs T cell activation through interaction with B7 ligands (CD80\/CD86). While antibody-based inhibitors of CD28 signaling have advanced clinically, the development of small molecule modulators remains limited due to the receptor\u2019s shallow, flexible surface. We sought to discover small-molecule modulators with favorable pharmacokinetic properties capable of disrupting CD28\u2013B7 interactions in translational models of T cell activation.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Experimental Approach<\/jats:title>\n                  <jats:p>Structure-based virtual screening was conducted using pharmacophore filtering and consensus docking against the human CD28 ectodomain. Hit compounds were validated using temperature-related intensity change (TRIC) and microscale thermophoresis (MST). Functional antagonism was assessed through ELISA, NanoBit luciferase complementation, and a CD28 Blockade Bioassay. In vitro ADME and safety pharmacology profiling were performed, and immunosuppressive activity was evaluated in tumor\u2013PBMC and mucosal-PBMC co-culture assays.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Key Results<\/jats:title>\n                  <jats:p>Lead compound 22VS bound CD28 in biophysical screening, targeting a lipophilic canyon anchored by K24, Q25, and P27. 22VS inhibited CD28\u2013CD80\/CD86 interactions in ELISA and cell-based assays with submicromolar potency. 22VS robustly suppressed T cell activation markers in both tumor\u2013 PBMC and human mucosal epithelial\u2013PBMC co-culture models, phenocopying the anti-CD28 biologic FR104. It showed no cytotoxicity up to 300 \u00b5M and exhibited high solubility, low clearance, strong membrane permeability, and minimal off-target effects in pharmacokinetic screens.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Conclusion and Implications<\/jats:title>\n                  <jats:p>This study identifies a novel druggable site on CD28 and validates 22VS as a selective, non-toxic small molecule inhibitor with translational potential for immune modulation in autoimmunity, transplantation, and cancer.<\/jats:p>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Bullet Point Summary<\/jats:title>\n                  <jats:sec>\n                    <jats:title>What is already known<\/jats:title>\n                    <jats:list list-type=\"bullet\">\n                      <jats:list-item>\n                        <jats:p>CD28 is a key T cell costimulatory receptor essential for immune activation.<\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Small-molecule inhibitors of CD28 are largely unexplored compared to biologics.<\/jats:p>\n                      <\/jats:list-item>\n                    <\/jats:list>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>What this study adds<\/jats:title>\n                    <jats:list list-type=\"bullet\">\n                      <jats:list-item>\n                        <jats:p>Identifies a novel druggable pocket on CD28 via structure-based virtual screening.<\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Discovers 22VS, a selective small molecule CD28 inhibitor with cellular activity.<\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Demonstrates that 22VS suppresses T cell activation in tumor\u2013PBMC and mucosal-PBMC co- culture assays, phenocopying a benchmark biologic (FR104).<\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Establishes 22VS as a drug-like compound with favorable in vitro pharmacokinetic properties, including metabolic stability, permeability, and low off-target toxicity.<\/jats:p>\n                      <\/jats:list-item>\n                    <\/jats:list>\n                  <\/jats:sec>\n                  <jats:sec>\n                    <jats:title>Clinical significance<\/jats:title>\n                    <jats:list list-type=\"bullet\">\n                      <jats:list-item>\n                        <jats:p>Highlights the potential of 22VS as a lead for immunomodulatory therapeutic development.<\/jats:p>\n                      <\/jats:list-item>\n                      <jats:list-item>\n                        <jats:p>Supports small-molecule targeting of CD28\u2013B7 interactions in T cell-driven diseases.<\/jats:p>\n                      <\/jats:list-item>\n                    <\/jats:list>\n                  <\/jats:sec>\n                <\/jats:sec>\n                <jats:sec>\n                  <jats:title>Graphical Abstract<\/jats:title>\n                  <jats:fig id=\"ufig1\" position=\"float\" orientation=\"portrait\" fig-type=\"figure\">\n                    <jats:graphic xmlns:xlink=\"http:\/\/www.w3.org\/1999\/xlink\" xlink:href=\"665260v1_ufig1\" position=\"float\" orientation=\"portrait\"\/>\n                  <\/jats:fig>\n                <\/jats:sec>","DOI":"10.1101\/2025.07.16.665260","type":"posted-content","created":{"date-parts":[[2025,7,21]],"date-time":"2025-07-21T14:25:10Z","timestamp":1753107910000},"source":"Crossref","is-referenced-by-count":0,"title":["Small Molecule-Based Blockade of CD28 Suppresses T Cell Costimulation Across Cellular and Mucosal Co-culture 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