Artificial intelligence-powered discovery of small molecules inhibiting CTLA-4 in cancer

Background/Objectives Checkpoint inhibitors, which generate durable responses in many cancer patients, have revolutionized cancer immunotherapy. However, their therapeutic efficacy is limited, and immune-related adverse events are severe, especially for monoclonal antibody treatment directed against cytotoxic T-lymphocyte–associated protein 4 (CTLA-4), which plays a pivotal role in preventing autoimmunity and fostering anticancer immunity by interacting with the B7 proteins CD80 and CD86. Small molecules impairing the CTLA-4/CD80 interaction have been developed; however, they directly target CD80, not CTLA-4. Subjects/Methods In this study, we performed artificial intelligence (AI)-powered virtual screening of approximately ten million compounds to identify those targeting CTLA-4. We validated the hits molecules with biochemical, biophysical, immunological, and experimental animal assays. Results The primary hits obtained from the virtual screening were successfully validated in vitro and in vivo. We then optimized lead compounds and obtained inhibitors (inhibitory concentration, 1 micromole) that disrupted the CTLA-4/CD80 interaction without degrading CTLA-4. Conclusions Several compounds inhibited tumor development prophylactically and therapeutically in syngeneic and CTLA–4–humanized mice. Our findings support using AI-based frameworks to design small molecules targeting immune checkpoints for cancer therapy. Schematic representation of AI-assisted anti-CTLA-4 drug discovery . (a) The extracellular domain of CTLA-4 with residues around the binding site is shown in yellow ( 26 PGKAT) and other colors ( 98 MYPPPYY 104 ). (b) The interactions between B7 (CD80 and CD86) and CTLA-4 or CD28 occurring with varying affinities are represented by the thicknesses of the arrows. AI, artificial intelligence; APC, antigen-presenting cell; CTLA-4, cytotoxic T-lymphocyte–associated protein 4; IFN-γ, interferon γ; MHC II-Ag, major histocompatibility class 2 antigen; PD-1, programmed cell death protein-1; PD-L1, programmed death-ligand 1; Tconv, T conventional; TCR, T-cell receptor; Treg, T regulatory cell.