Phosphinophosphonates and Their Tris-pivaloyloxymethyl Prodrugs Reveal a Negatively Cooperative Butyrophilin Activation Mechanism ShippyRebekah R. LinXiaochen AgabitiSherry S. LiJin ZangariBrendan M. FoustBenjamin J. PoeMichael M. HsiaoChia-Hung Christine VinogradovaOlga WiemerDavid F. WiemerAndrew J. 2017 Butyrophilin 3A1 (BTN3A1) binds small phosphorus-containing molecules, which initiates transmembrane signaling and activates butyrophilin-responsive cells. We synthesized several phosphinophosphonates and their corresponding tris-pivaloyloxymethyl (tris-POM) prodrugs and examined their effects on BTN3A1. An analog of (<i>E</i>)-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 (EC<sub>50</sub> = 26 μM). Cellular potency was enhanced >600-fold in the tris-POM prodrug (EC<sub>50</sub> = 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.