Search

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). [ABSTRACT FROM AUTHOR]

Due to toxicity and compliance issues and the emergence of resistance to current medications new drugs for the treatment of Human African Trypanosomiasis are needed. A potential approach to developing novel anti-trypanosomal drugs is by inhibition of the 6-oxopurine salvage pathways which synthesise the nucleoside monophosphates required for DNA/RNA production. This is in view of the fact that trypanosomes lack the machinery for de novo synthesis of the purine ring. To provide validation for this approach as a drug target, we have RNAi silenced the three 6-oxopurine phosphoribosyltransferase (PRTase) isoforms in the infectious stage of Trypanosoma brucei demonstrating that the combined activity of these enzymes is critical for the parasites’ viability. Furthermore, we have determined crystal structures of two of these isoforms in complex with several acyclic nucleoside phosphonates (ANPs), a class of compound previously shown to inhibit 6-oxopurine PRTases from several species including Plasmodium falciparum. The most potent of these compounds have Ki values as low as 60 nM, and IC50 values in cell based assays as low as 4 μM. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery. [ABSTRACT FROM AUTHOR]

A series of novel 7-aryl-7-deazaadenine-based N -branched acyclic nucleoside phosphonates (aza-ANPs) has been prepared using the optimized Suzuki cross-coupling reaction as the key synthetic step. The final free phosphonates 15a-h were inactive, due to their inefficient transport across cell membranes, but they inhibited Trypanosoma brucei adenine phosphoribosyltransferase (Tbr APRT1) with K i values of 1.7–14.1 μM. The corresponding phosphonodiamidate prodrugs 14a-h exhibited anti-trypanosomal activity in the Trypanosoma brucei brucei cell-based assay with EC 50 values in the range of 0.58–6.8 μM. 7-(4-Methoxy)phenyl-7-deazapurine derivative 14h , containing two phosphonate moieties, was the most potent anti-trypanosomal agent from the series, with EC 50 = 0.58 μM and SI = 16. Finally, phosphonodiamidate prodrugs 14a-h exerted low micromolar cytotoxicity against leukemia and/or cancer cell lines tested. [Display omitted] • Synthesis of novel aza-ANPs with 7-aryl-7-deazapurine nucleobase. • Anti-trypanosomal activity with EC 50 values 0.58–6.8 μM. • Improved selectivity index of the most potent compound (SI = 16.4) compared to tubercidin (SI = 0.40). • Tbr APRT1 inhibitors with low micromolar K i values. [ABSTRACT FROM AUTHOR]

Some pathogens, including parasites of the genus Trypanosoma causing Human and Animal African Trypanosomiases, cannot synthesize purines de novo and they entirely rely on the purine salvage pathway (PSP) for their nucleotide generation. Thus, their PSP enzymes are considered as promising drug targets, sparsely explored so far. Recently, a significant role of acyclic nucleoside phosphonates (ANPs) as inhibitors of key enzymes of PSP, namely of 6-oxopurine phosphoribosyltransferases (PRTs), has been discovered. Herein, we designed and synthesized two series of new ANPs branched at the C1′ position as mimics of adenosine monophosphate. The novel ANPs efficaciously inhibited Trypanosoma brucei adenine PRT (Tbr APRT1) activity in vitro and it was shown that the configuration on the C1′ chiral centre strongly influenced their activity: the (R)-enantiomers proved to be more potent compared to the (S)-enantiomers. Two ANPs, with K i values of 0.39 μM and 0.57 μM, represent the most potent Tbr APRT1 inhibitors reported to date and they are an important tool to further study purine metabolism in various parasites. [Display omitted] • Synthesis of novel ANPs branched at the C1′ position as adenosine monophosphate mimics • The most potent Tbr APRT1 inhibitors to date, with submicromolar K i values • Inhibitory activity strongly dependent on configuration on the C1′ chiral centre, (R)-enantiomers are substantially more potent than (S)-enantiomers [ABSTRACT FROM AUTHOR]