Structure-based virtual screening identifies potent CD28 inhibitors that suppress T cell co-stimulation in cellular and mucosal models

Resistance to immune checkpoint inhibitors targeting PD-1 and CTLA-4 remains a major barrier to effective Cancer Immunotherapy, often arising from compensatory CD28-mediated costimulation. Here, we report the discovery and biological validation of small molecule CD28 antagonists identified through a structure-based virtual screening pipeline. Molecular dynamics and Pyrod-based water mapping revealed a cryptic lipophilic canyon on CD28 enriched in druggable features. A pharmacophore-based screen of over 7 million compounds yielded several candidates, of which compound 22VS emerged as a lead based on biophysical binding (TRIC and MST), structure-activity insights, and functional inhibition in ELISA and NanoBit assays. 22VS demonstrated potent and selective blockade of CD28-B7 interactions, with submicromolar IC₅₀ values in cellular assays and minimal cytotoxicity. Importantly, 22VS suppressed proinflammatory cytokines (IFN-γ, IL-2, TNF-α) in human tumor-PBMC and mucosal-PBMC co-culture systems, closely mimicking the biological activity of the CD28-targeting biologic FR104. Pharmacokinetic profiling revealed favorable solubility, metabolic stability, low CYP inhibition, and excellent safety in human fibroblasts. These findings establish CD28 as a druggable immunotherapeutic checkpoint and validate 22VS as a promising lead candidate for modulating T cell responses. This small-molecule approach offers a viable pharmacological strategy to overcome resistance mechanisms associated with PD-1 and CTLA-4 blockade, with implications for autoimmune disease, transplantation, and Cancer Immunotherapy.

Pharmacophore modeling; Protein–protein interaction inhibitors; Structure-based drug design; T cell co-stimulation; Virtual screening.