Phosphinophosphonates and Their Tris-pivaloyloxymethyl Prodrugs Reveal a Negatively Cooperative Butyrophilin Activation Mechanism

Butyrophilin 3A1 (BTN3A1) binds small phosphorous-containing molecules, which initiates transmembrane signaling and activates butyrophilin-responsive cells. We synthesized several phosphinophosphonates and their corresponding tris-pivaloyloxymethyl prodrugs and examined their effects on BTN3A1. An analog of (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) bound to BTN3A1 with intermediate affinity, which was enthalpy-driven. Docking studies revealed binding to the basic surface pocket and interactions between the allylic hydroxyl group and the BTN3A1 backbone. The phosphinophosphonate stimulated proliferation of Vγ9Vδ2 T cells with moderate activity (EC50 = 26 µM). Cellular potency was enhanced >600-fold in the tris-POM prodrug (EC50 = 0.041 µM). The novel prodrug also induced T cell mediated leukemia cell lysis. Analysis of dose response data reveals HMBPP-induced Hill coefficients of 0.69 for target cell lysis and 0.68 in interferon secretion. Together, tris-POM prodrugs enhance the cellular activity of phosphinophosphonates, reveal structure-activity relationships of butyrophilin ligands, and support a negatively cooperative model of cellular butyrophilin activation.