Aryloxy Diester Phosphonamidate Prodrugs of Phosphoantigens (ProPAgens) as Potent Activators of Vγ9/Vδ2 T-Cell Immune Responses

Abstract

Vγ9/Vδ2 T-cells are activated by pyrophosphate-containing small molecules known as phosphoantigens (PAgs). The presence of the pyrophosphate group in these PAgs has limited their drug-like properties because of its instability and polar nature. In this work, we report a novel and short Grubbs olefin metathesis-mediated synthesis of methylene and difluoromethylene monophosphonate derivatives of the PAg ( E )-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBP) as well as their aryloxy diester phosphonamidate prodrugs, termed ProPAgens. These prodrugs showed excellent stability in human serum ( t 1/2 > 12 h) and potent activation of Vγ9/Vδ2 T-cells (EC 50 ranging from 5 fM to 73 nM), which translated into sub-nanomolar γδ T-cell-mediated eradication of bladder cancer cells in vitro . Additionally, a combination of in silico and in vitro enzymatic assays demonstrated the metabolism of these phosphonamidates to release the unmasked PAg monophosphonate species. Collectively, this work establishes HMBP monophosphonate ProPAgens as ideal candidates for further investigation as novel cancer immunotherapeutic agents.

Prodrugs of a 1-Hydroxy-2-oxopiperidin-3-yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers

“…Our in vitro results showing inefficient bioactivation of 17 and 18 compared to bis-ester prodrugs of 5 contrast previous studies showing superior bioactivation of McGuigan prodrugs in target cell lines/typesepitomized by pharmacodynamic differences between tenofovir disoproxil and TAF in lymphoid cells. , Screening 17 and 18 in the NCI-60 cell line panel revealed no significant growth inhibition after a 10 μM incubation for 24 h despite seeing growth-inhibitory activity by their bis-ester counterparts (Figures , S6). With the exception of the fructose 1,6-bisphosphatase inhibitor “compound 35l” described by Dang and colleagues, all other reports investigating the McGuigan prodrug strategy have been on mono-substituted Cα phosphonates and, in these cases, efficient bioactivation was observed. ,,, Our experience with 17 and 18 further underscores the role of Cα substitution level in modulating the rate of the intramolecular nucleophilic phosphoryl substitution required to displace the phenolic promoiety (Figures ; S7). It can be reasonably concluded that inefficient bioactivation stems from the intramolecular cyclization step rather than the subsequent ring-opening hydrolysis, based on observations of efficient ring-opening with 30 and previous work by Meier and colleagues on cycloSal and cycloAmb (phosphonoamidate counterparts) prodrugs of adefovir. , …”

“…With the exception of the fructose 1,6-bisphosphatase inhibitor "compound 35l" described by Dang and colleagues, all other reports investigating the McGuigan prodrug strategy have been on mono-substituted Cα phosphonates and, in these cases, efficient bioactivation was observed. 38,39,63,66 Our experience with 17 and 18 further underscores the role of Cα substitution level in modulating the rate of the intramolecular nucleophilic phosphoryl substitution required to displace the phenolic promoiety (Figures 4; S7). It can be reasonably concluded that inefficient bioactivation stems from the intramolecular cyclization step rather than the subsequent ring-opening hydrolysis, based on observations of efficient ring-opening with 30 and previous work by Meier and colleagues on cycloSal and cycloAmb (phosphonoamidate counterparts) prodrugs of adefovir.…”

“…Higher-order substitution at Cα on 5 likely impedes both the rate of intramolecular cyclization required to displace the phenol and the subsequent ring-opening hydrolysis. Beyond nucleotide analogues, efficient bioactivation of McGuigan phosphonoamidate prodrugs has also been observed on phospho­(no)­antigens containing the phosph­(on)­ates on mono-substituted Cα, supporting the influence of sterics on the bioactivation of McGuigan prodrugs. , The proposed causes for inefficient bioactivation are further substantiated by the uniformly poor growth inhibition observed for both 17 and 18 in the NCI-60 cell line panel, which suggests that part of their bioactivation is non-enzymatic and independent of cell line-specific expression of potential phosphonoamidate-hydrolyzing enzymes (Figures , S6). The intracellular metabolism of 20o is distinct from McGuigan prodrugs 17 and 18 , which likely explains the more efficient prodrug bioactivation and the resulting lower IC 50 value in D423 cells.…”

“…This is the first report of an expansive prodrug screening on a non-nucleotide phosphonate pharmacophore and is also the first report of a broad prodrug screen on a di-substituted Cα phosphonate. Other instances where phosph­(on)­ate prodrugs have been evaluated on non-nucleotide analogues include cyclophosphamide, TH-302, fructose 1,6-bisphosphatase inhibitor (MB06322), fosmetpantotenate (RE-024), organophosph­(on)­ate ligands of the butyrophilin 3A, N -acetyl glucosamine, anti-STAT3 phosphotyrosine prodrugs, and fosmidomycin analogues; however, these studies have focused exclusively on either the McGuigan or bis-ester/amidate prodrug strategies. ,,,,− Most of these studies have also been conducted on phosphates, which exhibit different metabolic susceptibilities compared to phosphonates. By applying precedented and novel prodrug strategies onto 5 , several important insights can be gleaned from this study.…”

The Multifaceted MEP Pathway: Towards New Therapeutic Perspectives

“…The same strategy was tested on the derivatives of the monophosphonate C-HMBP containing (i) different aryloxy groups, (ii) either glycine or l -alanine ester moieties, and (iii) a CH 2 –P or CF 2 –P bond (compounds 63 and 64 , Figure 10 ). These prodrugs are highly stable in human plasma and exhibit potent activation of Vγ9Vδ2 T-cell proliferation with EC 50 values from the mid-picomolar to the femtomolar range, thus displaying an efficiency similar to or even significantly greater than HMBPP [ 115 , 116 ].…”