Synthesis and Metabolism of BTN3A1 Ligands: Studies on Diene Modifications to the Phosphoantigen Scaffold

Abstract

Phosphoantigens (pAgs) are small organophosphorus compounds such as (E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) that trigger an immune response. These molecules bind to butyrophilin 3A1 (part of the HMBPP receptor) and activate Vγ9Vδ2 T cells. To explore the structure–activity relationships underlying this process, we evaluated a series of novel diene analogs of HMBPP. Here we report that prodrug forms of [(1E)-4-methylpenta-1,3-dien-1-yl] phosphonic acid that lack the allylic alcohol of HMBPP but instead contained a diene scaffold exhibit mid-nanomolar potency for the activation of Vγ9Vδ2 T cells. The compounds also trigger the production of T-cell interferon γ upon exposure to loaded K562 cells. Although both the allylic alcohol and the diene scaffold boost pAg activity, the combination of the two decreases the activity and results in glutathione conjugation. Together, these data show that the diene scaffold results in intermediate pAgs that may have implications for the mechanisms regulating the HMBPP receptor.

Diester Prodrugs of a Phosphonate Butyrophilin Ligand Display Improved Cell Potency, Plasma Stability, and Payload Internalization

“…The cellular metabolism of the prodrugs was determined using LC–MS post incubation with K562 cells. , The K562 cells were resuspended at a concentration of 2.5 × 10 6 cells in 500 μL of the K562 media. These cells were treated with 100 μM of the compound for 1 h. Post incubation, the cells were pelleted by centrifugation for 3 min at 600 rcf, and the media was aspirated.…”

Effective γδ T‐cell clinical therapies: current limitations and future perspectives for cancer immunotherapy

“…Until recently, it was thought that the transmembrane protein BTN3A1 would bind pAgs and directly present them to Vδ2Vγ9 TCRs 33 . More recently it has been shown that BTN3A1 binds pAgs intracellularly through their B30.2 domain, which likely induces extracellular conformational changes in the transmembrane protein 34–36 . As shown in Figure 1, BTN2A1 has also recently been shown to interact directly with the Vδ2Vγ9 TCR as well as with intracellular pAgs, also through their B30.2 intracellular domain, leading to the hypothesis that a butyrophilin heterodimer forms upon pAg‐induced conformational change and is required for Vδ2Vγ9 TCR binding and activation 34,37–39 .…”

“… 33 More recently it has been shown that BTN3A1 binds pAgs intracellularly through their B30.2 domain, which likely induces extracellular conformational changes in the transmembrane protein. 34 , 35 , 36 As shown in Figure 1 , BTN2A1 has also recently been shown to interact directly with the Vδ2Vγ9 TCR as well as with intracellular pAgs, also through their B30.2 intracellular domain, leading to the hypothesis that a butyrophilin heterodimer forms upon pAg‐induced conformational change and is required for Vδ2Vγ9 TCR binding and activation. 34 , 37 , 38 , 39 Upon activation by pAgs, Vδ2Vγ9 T cells tend to express a Th 1 ‐like phenotype and secrete the proinflammatory cytokines IFN‐γ and TNF‐α.…”

The Multifaceted MEP Pathway: Towards New Therapeutic Perspectives

“…More recently, modifications at the allylic alcohol of several monophosphonate prodrugs highlighted that the removal of the allylic alcohol abrogates activity; its replacement with an aldoxime simply decreases it, while an aldehyde (compound 66 ) is well tolerated as it is reduced intracellularly into the corresponding alcohol. Furthermore, using a diene scaffold instead of the classical isoprene unit, even without the hydroxyl group at C4 as in compound 67 , turned out to be sufficient to induce stimulation of the Vγ9Vδ2 T cells [ 95 , 118 ].…”