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bioRxiv · 10.1101/2025.10.06.680836

Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation

Upadhyay, S.·Nada, H.·Cho, S.·Gabr, M.
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Abstract

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.

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Upadhyay, S., Nada, H., Cho, S., Gabr, M.. 2025-10-07. Structure-Guided Optimization and Functional Characterization of Small Molecule Antagonists Targeting CD28 Costimulation. https://doi.org/10.1101/2025.10.06.680836

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