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Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
Small Molecule-Based Blockade of CD28 Suppresses T Cell Costimulation Across Cellular and Mucosal Co-culture Models
Background And Purpose:
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's shallow, flexible surface. We sought to discover small-molecule modulators with favorable pharmacokinetic properties capable of disrupting CD28-B7 interactions in translational models of T cell activation.
Experimental Approach:
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-PBMC and mucosal-PBMC co-culture assays.
Key Results:
Lead compound 22VS bound CD28 in biophysical screening, targeting a lipophilic canyon anchored by K24, Q25, and P27. 22VS inhibited CD28-CD80/CD86 interactions in ELISA and cell-based assays with submicromolar potency. 22VS robustly suppressed T cell activation markers in both tumor- PBMC and human mucosal epithelial-PBMC co-culture models, phenocopying the anti-CD28 biologic FR104. It showed no cytotoxicity up to 300 µM and exhibited high solubility, low clearance, strong membrane permeability, and minimal off-target effects in pharmacokinetic screens.
Conclusion And Implications:
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.
Bullet Point Summary:
What is already known: CD28 is a key T cell costimulatory receptor essential for immune activation.Small-molecule inhibitors of CD28 are largely unexplored compared to biologics.What this study adds: Identifies a novel druggable pocket on CD28 via structure-based virtual screening.Discovers 22VS, a selective small molecule CD28 inhibitor with cellular activity.Demonstrates that 22VS suppresses T cell activation in tumor-PBMC and mucosal-PBMC co- culture assays, phenocopying a benchmark biologic (FR104).Establishes 22VS as a drug-like compound with favorable in vitro pharmacokinetic properties, including metabolic stability, permeability, and low off-target toxicity.Clinical significance: Highlights the potential of 22VS as a lead for immunomodulatory therapeutic development.Supports small-molecule targeting of CD28-B7 interactions in T cell-driven diseases.
Insights
Researchers discovered 22VS, a novel small molecule that inhibits CD28 signaling by targeting a unique pocket. This drug-like compound effectively suppresses T cell activation, offering potential for treating immune-related diseases.
Area of Science:
- Immunology
- Pharmacology
- Drug Discovery
Background:
- CD28 is a critical costimulatory receptor for T cell activation, interacting with B7 ligands (CD80/CD86).
- While antibody-based CD28 inhibitors exist, small molecule development is limited due to the receptor's challenging surface.
- Targeting CD28 with small molecules offers a promising avenue for immunomodulation.
Purpose of the Study:
- To discover small-molecule modulators of CD28 signaling with favorable pharmacokinetic properties.
- To identify compounds capable of disrupting CD28-B7 interactions in translational models.
- To explore novel druggable sites on the CD28 receptor.
Main Methods:
- Structure-based virtual screening and pharmacophore filtering of the human CD28 ectodomain.
- Biophysical validation using TRIC and MST, followed by functional assays (ELISA, NanoBit complementation, Bioassay).
- In vitro ADME profiling and evaluation of immunosuppressive activity in co-culture assays (tumor-PBMC, mucosal-PBMC).
Main Results:
- Lead compound 22VS identified, binding CD28 in a lipophilic canyon and inhibiting CD28-B7 interactions with submicromolar potency.
- 22VS demonstrated robust suppression of T cell activation markers in co-culture models, mirroring the anti-CD28 biologic FR104.
- 22VS exhibited excellent drug-like properties: high solubility, low clearance, strong permeability, and minimal off-target effects.
Conclusions:
- A novel druggable site on CD28 was identified, enabling the discovery of small molecule inhibitors.
- 22VS is a selective, non-toxic small molecule inhibitor of CD28 with significant translational potential.
- 22VS represents a promising lead compound for immunomodulatory therapies in autoimmunity, transplantation, and cancer.
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