# An integrated biophysical fragment screening approach identifies novel binders of the CD28 immune receptor

## TL;DR

Researchers used a fragment-based screening method to find new molecules that bind to the CD28 immune receptor, opening new possibilities for drug development.

## Contribution

This is the first successful identification of small molecule binders for the CD28 receptor using fragment-based screening.

## Key findings

- Fragment-based screening identified two direct CD28 binders using TRIC and SPR technologies.
- PPIF3 showed measurable affinity and a unique binding mode within the CD28 extracellular domain.
- The fragments bind to interface features not previously targeted by small molecules.

## Abstract

CD28 is an essential costimulatory receptor required for full T cell activation and its dysregulation contributes to multiple immune-mediated pathologies. Despite its central immunological role, CD28 remains largely unexplored as a target for small-molecule modulation, primarily due to the shallow and large interface of its ligand-binding site. Here, we applied a fragment-based high-throughput screening (HTS) strategy to identify low molecular weight chemotypes capable of engaging with human CD28. A 3200-member library composed of structurally diverse fragments, enriched for scaffolds designed to target protein-protein interaction (PPI) interfaces, was screened in single-dose format using temperature-related intensity change (TRIC) technology, yielding 36 primary hits (1.13% hit rate). Follow-up surface plasmon resonance (SPR) validation confirmed two fragments as direct CD28 binders. Molecular docking analysis revealed a plausible binding orientation for PPIF3 within the CD28 extracellular domain, suggesting potential interaction hotspots that may be exploited during future optimization. Together, these findings provide the first demonstration that fragment-based screening can successfully identify chemotypes capable of engaging with the CD28 PPI interface. This work establishes a scalable, biophysics-driven workflow for CD28 ligand discovery and lays the foundation for subsequent hit-to-lead development of small molecule CD28 modulators.

•Fragment-based TRIC-SPR screening identifies direct CD28 binders.•First fragment hits reported for the CD28 immune receptor.•PPIF3 shows measurable affinity and a unique binding mode.•Fragments engage interface features not used by previous small molecules.•Provides new starting points for CD28-focused drug discovery.

Fragment-based TRIC-SPR screening identifies direct CD28 binders.

First fragment hits reported for the CD28 immune receptor.

PPIF3 shows measurable affinity and a unique binding mode.

Fragments engage interface features not used by previous small molecules.

Provides new starting points for CD28-focused drug discovery.