High-throughput decoding of antitrypanosomal drug efficacy and resistance

Abstract

SummaryThe concept of specific chemotherapy was developed a century ago by Paul Ehrlich and others. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work 1,2, and the drugs that emerged remain in use for treating Human African Trypanosomiasis (HAT) 3. Ehrlich recognised the importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies, but these mechanisms have remained largely mysterious. Here, we use all five current HAT drugs for genome-scale RNA interference (RNAi) target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate anti-trypanosomal drug action. RIT-seq profiling identifies both known drug importers 4,5 and the only known pro-drug activator 6, and links more than fifty additional genes to drug action. A specific bloodstream stage invariant surface glycoprotein (ISG75) family mediates suramin uptake while the AP-1 adaptin complex, lysosomal proteases and major lysosomal transmembrane protein, as well as spermidine and N-acetylglucosamine biosynthesis all contribute to suramin action. Further screens link ubiquinone availability to nitro-drug action, plasma membrane P-type H+-ATPases to pentamidine action, and trypanothione and multiple putative kinases to melarsoprol action. We also demonstrate a major role for aquaglyceroporins in pentamidine and melarsoprol cross-resistance. These advances in our understanding of mechanisms of anti-trypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented levels of molecular insight into the mode of action of anti-trypanosomal drugs.

Vacuolar ATPase depletion affects mitochondrial ATPase function, kinetoplast dependency, and drug sensitivity in trypanosomes

“…1A). Genome-scale RNA interference (RNAi) library screens previously revealed mechanisms of resistance to anti-trypanosomal compounds used in humans (14). As illustrated in the schematic (Fig.…”

Aquaglyceroporin 2 controls susceptibility to melarsoprol and pentamidine in African trypanosomes

“…Subcellular fractionation by hypotonic lysis was carried out as described (5). All protein samples were stored in the presence of a protease inhibitor mixture (Roche) and were not boiled.…”

“…A recent set of highthroughput, loss-of-function RNAi screens linked AT1 and several additional membrane-spanning transporters to melarsoprol or pentamidine resistance (5). In particular, the plasma membrane H + -ATPases, HA1-3, were linked to pentamidine resistance, suggesting that at least one of the relevant pentamidine transporters is a proton symporter.…”

“…Cells lacking both AQP2 and AQP3 were resistant to melarsoprol and pentamidine (5). Because the analysis above indicated major differences between AQP2 and AQP3 that may be of functional significance, we wanted to determine what contribution each protein makes to the drug susceptibility phenotype.…”

Synthesis and antiprotozoal activity of oligomethylene- and p-phenylene-bis(methylene)-linked bis(+)-huprines

“…These include high-throughput screening of large compound libraries, new strategies to functionally validate novel druggable targets involved in key steps of the parasite life-cycle, [7][8][9][10] or the simultaneous inhibition of two or more key biological targets with combination therapies or multitarget-directed ligands. [11][12][13] Increasingly, the search for novel antiprotozoal agents also involves the repositioning of existing drugs registered for other applications 14 or the synthesis of new chemical entities endowed with antiprotozoal activity.…”