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Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries
Published on: January 22, 2019
Structure-based virtual screening identifies potent CD28 inhibitors that suppress T cell co-stimulation in cellular
Saurabh Upadhyay1, Valerij Talagayev2, Sungwoo Cho1
1Department of Radiology, Molecular Imaging Innovations Institute (MI3), Weill Cornell Medicine, New York, NY, 10065, USA.
Abstract:
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.
Insights
Researchers discovered a small molecule, 22VS, that blocks CD28 costimulation, a key mechanism of resistance to cancer immunotherapies like PD-1 and CTLA-4 inhibitors. This offers a new strategy to enhance cancer treatment effectiveness.
Area of Science:
- Immunology
- Pharmacology
- Structural Biology
Background:
- Immune checkpoint inhibitors (ICIs) targeting PD-1 and CTLA-4 are crucial cancer therapies.
- Resistance to ICIs often involves compensatory CD28 costimulation, limiting treatment efficacy.
- Novel strategies are needed to overcome ICI resistance and improve patient outcomes.
Purpose of the Study:
- To discover and validate small molecule antagonists of CD28.
- To develop a pharmacological approach to overcome resistance to PD-1 and CTLA-4 blockade.
- To identify a lead compound for modulating T cell responses in cancer immunotherapy.
Main Methods:
- Structure-based virtual screening of over 7 million compounds.
- Molecular dynamics and Pyrod-based water mapping to identify druggable sites on CD28.
- Biophysical binding assays (TRIC, MST) and functional assays (ELISA, NanoBit) for compound validation.
- Cellular assays to assess inhibition of CD28-B7 interactions and cytokine production.
- Pharmacokinetic and safety profiling of lead compounds.
Main Results:
- Identification of a cryptic lipophilic canyon on CD28 as a druggable target.
- Discovery of compound 22VS as a potent and selective small molecule CD28 antagonist.
- 22VS demonstrated submicromolar IC50 values and suppressed key proinflammatory cytokines (IFN-γ, IL-2, TNF-α).
- 22VS mimicked the activity of the CD28-targeting biologic FR104 and showed favorable safety and pharmacokinetic profiles.
- CD28 was validated as a druggable immunotherapeutic checkpoint.
Conclusions:
- Small molecule CD28 antagonists, exemplified by 22VS, represent a promising strategy to overcome resistance to PD-1 and CTLA-4 inhibitors.
- This approach has potential applications in enhancing cancer immunotherapy, as well as in treating autoimmune diseases and managing transplantation.
- 22VS is a validated lead candidate for modulating T cell responses and improving therapeutic outcomes.
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