Discovery of Potent, Dual-Inhibitors of Diacylglycerol Kinases Alpha and Zeta Guided by Phenotypic Optimization

Abstract

We describe a phenotypic screening and optimization strategy to discover compounds that block intracellular checkpoint signaling in T-cells. We identified dual DGKα and ζ inhibitors notwithstanding the modest similarity between α and ζ relative to other DGK isoforms. Optimized compounds produced cytokine release and T-cell proliferation consistent with DGK inhibition and potentiated an immune response in human and mouse T-cells. Additionally, lead inhibitor BMS-502 demonstrated dose-dependent immune stimulation in the mouse OT-1 model, setting the stage for a drug discovery program.

Figure 1

(A) Diacylglycerol phosphorylation. (B) TcR engagement leads to IL2 production. DGK interrupts this pathway by depletion of DAG.

Figure 2

(A) Assay format using the SEB-mediated T-cell interaction with Raji cells to stimulate IL2 production. (B) Counter-screen without PD-L1 presentation. .

Figure 3

(A) BMS-502 pharmacokinetic profile in C57 black mice. (B) BMS-502 dose response in the OT1 model. %CD69⁺ T-cells = 100(CD69⁺ CD8s)/total CD8s in the spleen. (C) Pharmacokinetic parameters for BMS-502.

Figure 4

Percent identity of DGK isoforms in catalytic and accessory domains grouped by type.