Synthesis and Metabolism of BTN3A1 Ligands: Studies on Modifications of the Allylic Alcohol

Abstract

(E)-4-Hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP) and its phosphonate analogs are potent phosphoantigens. HMBPP contains an (E)-allylic alcohol which interacts with the molecular target BTN3A1 giving an antigenic signal to activate Vγ9Vδ2 T cells. As probes of BTN3A1 function, we prepared prodrug derivatives of the HMBPP analog C-HMBP that lack the (E)-allylic alcohol or have modified it to an aldehyde or aldoxime and evaluated their biological activity. Removal of the alcohol completely abrogates phosphoantigenicity in these compounds while the aldoxime modification decreases potency relative to the (E)-allylic alcohol form. However, homoprenyl derivatives oxidized to an aldehyde stimulate Vγ9Vδ2 T cells at nanomolar concentrations. Selection of phosphonate protecting groups (i.e., prodrug forms) impacts the potency of phosphoantigen aldehydes, with mixed aryl acyloxyalkyl forms exhibiting superior activity relative to aryl amidate forms. The activity correlates with the cellular reduction of the aldehyde to the alcohol form. Thus, the functionality on this ligand framework can be altered concurrently with phosphonate protection to promote cellular transformation to highly potent phosphoantigens.

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

“…This is because in cells treated with acetate 13 , a significant m / z of 712.2507 consistent with [M+307-H] − of deacetylated 13 was found, indicative of glutathione conjugation. Similar glutathione conjugates were also observed in our prior study on pAg aldehydes . In the present study, glutathione conjugates were not observed with other dienes such as 9 , 21 , or 24 .…”

“…Similar glutathione conjugates were also observed in our prior study on pAg aldehydes. 25 In the present study, glutathione conjugates were not observed with other dienes such as 9, 21, or 24. Therefore, the presence of the oxygen on the diene scaffold presents a metabolism barrier to the activity of compounds such as 13.…”

“…At the same time, sodium salt 25 was inactive at concentrations below 100 μM. The activity of the diene aryl amidate 21 (11 μM), while not that potent, is in contrast with that of the DMAPP analog 4-methylpent-3-ene phosphonate, the latter of which was inactive up to 100 μM in a similar aryl-phosphonamidate form . Taken together, three novel diene prodrugs ( 8 , 9 , and 24 ) have pAg activity at mid-nanomolar concentrations.…”

“…The activity of the diene aryl amidate 21 (11 μM), while not that potent, is in contrast with that of the DMAPP analog 4methylpent-3-ene phosphonate, the latter of which was inactive up to 100 μM in a similar aryl-phosphonamidate form. 25 Taken together, three novel diene prodrugs (8, 9, and 24) have pAg activity at mid-nanomolar concentrations. Whereas this is less potent than the picomolar activity of the best prodrugs, 20,26,31 these dienes still have significant activity.…”

“…The expectation was that such compounds could be prepared more readily than alcohols, and the hope was that they would afford more information about the process of Vγ9Vδ2 T-cell activation. We evaluated the activation of Vγ9Vδ2 T cells by a series of phosphonate prodrugs that carry a novel (1 E )-4-methylpenta-1,3-diene scaffold 4 , a diene scaffold. − We used flow cytometry, enzyme-linked immunosorbent assay (ELISA), and liquid chromatography–mass spectrometry (LCMS) to understand the potency and metabolism of the novel scaffold. These findings expand the knowledge of the structure–activity relationships underlying Vγ9Vδ2 T-cell activation with implications for the design of synthetic pAgs and for understanding the molecular basis of pAg detection.…”