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Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery.

ABSTRACT: 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 ?M. This data provides a solid platform for further investigations into the use of this pathway as a target for anti-trypanosomal drug discovery.

SUBMITTER: Dolezelova E

PROVIDER: S-EPMC5843355 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery.

Doleželová Eva E Terán David D Gahura Ondřej O Kotrbová Zuzana Z Procházková Michaela M Keough Dianne D Špaček Petr P Hocková Dana D Guddat Luke L Zíková Alena A

PLoS neglected tropical diseases 20180226 2

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 thi ...[more]

PMID: 29481567

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Project description:BackgroundTrypanosomes can synthesize polyunsaturated fatty acids. Previously, we have shown that they possess stearoyl-CoA desaturase (SCD) and oleate desaturase (OD) to convert stearate (C18) into oleate (C18:1) and linoleate (C18:2), respectively. Here we examine if OD is essential to these parasites.MethodologyCultured procyclic (insect-stage) form (PCF) and bloodstream-form (BSF) Trypanosoma brucei cells were treated with 12- and 13-thiastearic acid (12-TS and 13-TS), inhibitors of OD, and the expression of the enzyme was knocked down by RNA interference. The phenotype of these cells was studied.Principal findingsGrowth of PCF T. brucei was totally inhibited by 100 µM of 12-TS and 13-TS, with EC(50) values of 40±2 and 30±2 µM, respectively. The BSF was more sensitive, with EC(50) values of 7±3 and 2±1 µM, respectively. This growth phenotype was due to the inhibitory effect of thiastearates on OD and, to a lesser extent, on SCD. The enzyme inhibition caused a drop in total unsaturated fatty-acid level of the cells, with a slight increase in oleate but a drastic decrease in linoleate level, most probably affecting membrane fluidity. After knocking down OD expression in PCF, the linoleate content was notably reduced, whereas that of oleate drastically increased, maintaining the total unsaturated fatty-acid level unchanged. Interestingly, the growth phenotype of the RNAi-induced cells was similar to that found for thiastearate-treated trypanosomes, with the former cells growing twofold slower than the latter ones, indicating that the linoleate content itself and not only fluidity could be essential for normal membrane functionality. A similar deleterious effect was found after RNAi in BSF, even with a mere 8% reduction of OD activity, indicating that its full activity is essential.Conclusions/significanceAs OD is essential for trypanosomes and is not present in mammalian cells, it is a promising target for chemotherapy of African trypanosomiasis.

Dataset Information

Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery.

Publications

Evaluation of the Trypanosoma brucei 6-oxopurine salvage pathway as a potential target for drug discovery.

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