2,4-Diaminopyrimidines as potent inhibitors of Trypanosoma brucei and identification of molecular targets by a chemical proteomics approach

Abstract

Background: There is an urgent need to develop new, safe and effective treatments for human African trypanosomiasis (HAT) because current drugs have extremely poor safety profiles and are difficult to administer. Here we report the discovery of 2,4-diaminopyrimidines, exemplified by 4-[4-amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid phenylamide (SCYX-5070), as potent inhibitors of Trypanosoma brucei and the related trypanosomatid protozoans Leishmania spp.

Methodology/principal findings: In this work we show that loss of T. brucei viability following SCYX-5070 exposure was dependent on compound concentration and incubation time. Pulse incubation of T. brucei with SCYX-5070 demonstrates that a short period of exposure (10-12 hrs) is required to produce irreversible effects on survival or commit the parasites to death. SCYX-5070 cured an acute trypanosomiasis infection in mice without exhibiting signs of compound related acute or chronic toxicity. To identify the molecular target(s) responsible for the mechanism of action of 2,4-diaminopyrimidines against trypanosomatid protozoa, a representative analogue was immobilized on a solid matrix (sepharose) and used to isolate target proteins from parasite extracts. Mitogen-activated protein kinases (MAPKs) and cdc2-related kinases (CRKs) were identified as the major proteins specifically bound to the immobilized compound, suggesting their participation in the pharmacological effects of 2,4-diaminopyrimidines against trypanosomatid protozoan parasites.

Conclusions/significance: Results show that 2,4-diaminopyrimidines have a good in vitro and in vivo pharmacological profile against trypanosomatid protozoans and that MAPKs and CRKs are potential molecular targets of these compounds. The 2,4-diminipyrimidines may serve as suitable leads for the development of novel treatments for HAT.

Figure 1. Chemical structures of SCYX-5070 and SCYX-1120.

Figure 2. Time-to-kill plots demonstrating concentration-dependent and irreversibility of SCYX-5070 effects on T. b. brucei in vitro.

Parasite survival was measured at various times in the presence on continuous drug (A) or after compound wash-out at specified times and viability assessment at 72 h (B). Experiments were conducted in triplicate and plots are mean survival of treated parasites expressed as percentages of control untreated parasites.

Figure 3. Chemical structure of free and immobilized 4-[4-Amino-5-(2-methoxy-benzoyl)-pyrimidin-2-ylamino]-piperidine-1-carboxylic acid (4-hydroxy-phenyl)-amide (SCYX-7434).

SCYX-7434 was coupled to activated sepharose 6B via the carbodiimide reaction, as described in materials and methods. Control capped sepharose 6B was produced by the addition of ethanolamine to block the reactive groups.

Figure 4. Trypanosomatid protozoan proteins interacting with immobilized diaminopyridine.

Soluble extracts from different stages of T. brucei and Leishmania spp. were loaded onto compound-immobilized or control sepharose beads. Proteins bound to each matrix were eluted and resolved on SDS-PAGE followed by visualization with silver staining. Protein samples are: molecular weight markers (lane 1); L. donovani promastigotes (lane 2, 6); L. major promastigotes (lane 3, 7); T. brucei procyclics (lane 4, 8) and T. brucei blood stage (lane 5, 9).

Figure 5. Analysis and identification of proteins retained by immobilized SCYX-7434.

Lysates from blood stage T. brucei or L. major were loaded onto SCYX-7434-immobilized sepharose beads, eluted and analyzed on silver stained SDS-PAGE gels. (A) Bound T. brucei proteins were eluted with SCYX-5070 and ATP (lane 1) or by boiling in sample buffer (lane 2). (B) L. major proteins bound to immobilized SCYX-7434 (lane 1) or capped control beads (lane 2) were eluted with SCYX-5070 and ATP.

Figure 6. Competitive lysate binding in the presence of free SCYX-5070.

Procyclic T. brucei lysates were bound in the presence of varying concentrations of SCYX-5070. No SCYX-5070 (lane 1), 2.5 µM SCYX-5070 (lane 2), 40 µM SCYX-5070 (lane 3).