Phosphonamidate Prodrugs of a Butyrophilin Ligand Display Plasma Stability and Potent Vγ9 Vδ2 T Cell Stimulation

Abstract

Small organophosphorus compounds stimulate Vγ9 Vδ2 T cells if they serve as ligands of butyrophilin 3A1. Because the most potent natural ligand is ( E)-4-hydroxy-3-methyl-but-2-enyl diphosphate (HMBPP), which is the last intermediate in bacterial biosynthesis of isoprenoids that is not found in mammalian metabolism, activation of these T cells represents an important component of the immune response to bacterial infections. To identify butyrophilin ligands that may have greater plasma stability, and clinical potential, we have prepared a set of aryl phosphonamidate derivatives (9a-i) of the natural ligand. Testing of these new compounds in assays of T cell response has revealed that this strategy can provide compounds with high potency for expansion of Vγ9 Vδ2 T cells (9f, EC₅₀ = 340 pM) and interferon γ production in response to loaded K562 cells (9e, EC₅₀ = 62 nM). Importantly, all compounds of this class display extended plasma stability ( t_1/2 > 24 h). These findings increase our understanding of metabolism of butyrophilin ligands and the structure-activity relationships of phosphonamidate prodrugs.

Figure 1.

A natural phosphoantigen (1) and some phosphonate prodrug analogs (2 and 3).

Figure 2.

Plasma stability of phosphonamidate prodrugs. A) POM₂-C-HMBP was exposed to 50% pooled human plasma in PBS for indicated time points. The graph indicates the mean fraction remaining and error bars represent standard deviations. Each data point was evaluated 2–4 independent times. B) The stability of the indicated phosphonamidates at 2 or 24 hour time points. Each data point was evaluated 2 independent times.

Figure 3.

Expansion of Vγ9Vδ2 T cells from PBMCs by phosphonamidate prodrugs. A) Following 3 days of compound exposure and 11 days of proliferation, the number of Vγ9Vδ2 T cells was assessed. Data are representative of 3 independent experiments using a concentration of 100 nM of each positive control and each test compound. B) Compounds were assessed for activity in dose response experiments, in comparison to non-stimulated cells (NS) and the positive controls HMBPP (HM) and POM₂-C-HMBP (POM2) at 100 nM. Data shown is from three independent experiments each using a minimum of two different human donors.

Figure 4.

K562 cell toxicity of phosphonamidate prodrugs. K562 cells were treated with test compounds for 72 hours and viability was assessed. Each compound was assessed in three independent experiments.

Figure 5.

K562 cells loaded with phosphonamidate prodrugs stimulate production of interferon γ by Vγ9Vδ2 T cells. Each compound was assessed in three independent experiments using a minimum of two different human donors.

Figure 6.

A potent mixed aryl acyloxyalkyl butyrophilin ligand (13) and some control compounds.

Scheme 1.

Synthesis of aryloxy phosphonamidate derivatives of a BTN3A1 ligand. Reagents and conditions: (a) (COCl)₂, DMF (5 mol %), CH₂Cl₂, 0 °C to rt, overnight; (b) ArOH, Et₃N, THF or toluene, 0 °C to rt; (c) NaI, H₃CCN, reflux, overnight; (d) GlyOR·HCl, PPh₃, 2,2’-dithiodipyridine, pyridine, 60 °C, overnight; (e) SeO₂, 70% aqueous t-BuOOH, pyridine, methanol, 0 °C to rt, overnight.