Acyclic nucleoside phosphonates with adenine nucleobase inhibit Trypanosoma brucei adenine phosphoribosyltransferase in vitro

Abstract

All medically important unicellular protozoans cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Therefore, purine derivatives have been considered as a promising source of anti-parasitic compounds since they can act as inhibitors of the PSP enzymes or as toxic products upon their activation inside of the cell. Here, we characterized a Trypanosoma brucei enzyme involved in the salvage of adenine, the adenine phosphoribosyl transferase (APRT). We showed that its two isoforms (APRT1 and APRT2) localize partly in the cytosol and partly in the glycosomes of the bloodstream form (BSF) of the parasite. RNAi silencing of both APRT enzymes showed no major effect on the growth of BSF parasites unless grown in artificial medium with adenine as sole purine source. To add into the portfolio of inhibitors for various PSP enzymes, we designed three types of acyclic nucleotide analogs as potential APRT inhibitors. Out of fifteen inhibitors, four compounds inhibited the activity of the recombinant APRT1 with Ki in single µM values. The ANP phosphoramidate membrane-permeable prodrugs showed pronounced anti-trypanosomal activity in a cell-based assay, despite the fact that APRT enzymes are dispensable for T. brucei growth in vitro. While this suggests that the tested ANP prodrugs exert their toxicity by other means in T. brucei, the newly designed inhibitors can be further improved and explored to identify their actual target(s).

Characterization of adenine phosphoribosyltransferase (APRT) activity in Trypanosoma brucei brucei: Only one of the two isoforms is kinetically active

“…The reduction in APRT activity is explained by the 1:1 dilution of APRT1, thereby indicating a catalytically active APRT1:APRT2 heterodimer is not formed. A recent study revealed APRT1 and APRT2 do not interact in vivo, with APRT1 displaying homodimeric properties both in vivo and in vitro [41], in agreement to results showed in Fig 3 . The quaternary structure of APRT1 was evaluated in vitro by treating APRT1 and APRT2 with DMS, an agent that chemically crosslinks two lysine residues within a monomer and also between lysine residues found in two or more associated monomers (Fig 4) [46]. Analysis of crosslinked proteins by denaturing polyacrylamide electrophoresis will show homodimers and other multimers, indicating quaternary structure, which will still be observed after significant protein dilution.…”

“…When the same RNAi cell line was tested as bloodstream stage cells grown in medium containing adenine as the sole available purine, a significant growth phenotype was observed suggesting the APRTs are the only enzymes that can incorporate adenine into the purine salvage pathway. The same publication reported another RNAi cell line, silencing APRT1 activity only, which displayed a growth phenotype similar to the bloodstream stage cells grown in medium containing only adenine, suggesting APRT2 is not able to compensate for the loss of APRT1 even when media contained up to 50 µM adenine [41].…”

“…An ordered sequential mechanism has been previously described for LdAPRT [59]. The cytosolic LdAPRT [56][57][58] has higher sequence conservation to the cytosolic T. brucei brucei [35,41] APRT1 (52%) than to the glycosomal APRT2 (27%) [35,41]. Interestingly, GlAPRT was described to follow a unique reaction mechanism among phosphoribosyltransferases, in which there is a random addition of substrates (PRPP and adenine) followed by catalysis and an ordered release of the products, where PPi is the first product to leave the enzyme-products complex followed by AMP.…”

“…Type 1 PRTases share highly conserved structures, but low levels of sequence identity except for the PRPP binding motif [61]. This motif is conserved in both APRT1 A recent publication showed that RNAi silencing of both APRT1 and APRT2 activity in T. brucei bloodstream stage cells did not produce a major growth phenotype, indicating the HGXPRT activity is adequate to maintain cell growth under normal growth conditions [41]. When the same RNAi cell line was tested as bloodstream stage cells grown in medium containing adenine as the sole available purine, a significant growth phenotype was observed suggesting the APRTs are the only enzymes that can incorporate adenine into the purine salvage pathway.…”

“…Other reports on APRT were performed using total cell extract activity [39], detection of mRNA [24], co-immunoprecipitation [40], or proteomic analyses [26]. The APRTs have been evaluated as potential drug targets, however previous work has been unsuccessful in evaluating APRT1 and APRT2 independently [41]. In this study, we focus on the catalytic activity of each putative APRT individually.…”