The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited and beset by toxicity and emergent resistance. Furthermore, our understanding of drug mode of action and potential routes to resistance is limited. Forward genetic approaches have revolutionized our understanding of drug mode of action in the related kinetoplastid parasite Trypanosoma brucei. Therefore, we screened our genome-scale T. brucei RNA interference (RNAi) library against the current antileishmanial drugs sodium stibogluconate (antimonial), paromomycin, miltefosine, and amphotericin B. Identification of T. brucei orthologues of the known Leishmania antimonial and miltefosine plasma membrane transporters effectively validated our approach, while a cohort of 42 novel drug efficacy determinants provides new insights and serves as a resource. Follow-up analyses revealed the antimonial selectivity of the aquaglyceroporin TbAQP3. A lysosomal major facilitator superfamily transporter contributes to paromomycin-aminoglycoside efficacy. The vesicle-associated membrane protein TbVAMP7B and a flippase contribute to amphotericin B and miltefosine action and are potential cross-resistance determinants. Finally, multiple phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania miltefosine transporter, a putative β-subunit/CDC50 cofactor, and additional membrane-associated hits, affect amphotericin B efficacy, providing new insights into mechanisms of drug uptake and action. The findings from this orthology-based chemogenomic profiling approach substantially advance our understanding of antileishmanial drug action and potential resistance mechanisms and should facilitate the development of improved therapies as well as surveillance for drug-resistant parasites.

217; importance, 135 23 Main text (intro, results, discussion), 4953. 24 Abstract 25The arsenal of drugs used to treat leishmaniasis, caused by Leishmania spp., is limited 26 and beset by toxicity and emergent resistance. Furthermore, our understanding of drug 27 mode-of-action and potential routes to resistance is limited. Forward genetic approaches 28 have revolutionised our understanding of drug mode-of-action in the related kinetoplastid 29 parasite, Trypanosoma brucei. Therefore, we screened our genome-scale T. brucei RNAi 30 library in the current anti-leishmanial drugs, sodium stibogluconate (antimonial), 31 paromomycin, miltefosine and amphotericin-B. Identification of T. brucei orthologues of the 32 known Leishmania antimonial and miltefosine plasma membrane transporters effectively 33 validated our approach, while a cohort of 42 novel drug efficacy determinants provides 34 new insights and serves as a resource. Follow-up analyses revealed the antimonial 35 selectivity of the aquaglyceroporin, TbAQP3. A lysosomal major facilitator superfamily 36 transporter contributes to paromomycin/aminoglycoside efficacy. The vesicle-associated 37 membrane protein, TbVAMP7B, and a flippase contribute to amphotericin-B and 38 miltefosine action, and are potential cross-resistance determinants. Finally, multiple 39 phospholipid-transporting flippases, including the T. brucei orthologue of the Leishmania 40 miltefosine transporter, a putative β-subunit/CDC50 co-factor, and additional membrane-41 associated hits, affect amphotericin-B efficacy, providing new insights into mechanisms of 42 drug uptake and action. The findings from this orthology-based chemogenomic profiling 43 approach substantially advance our understanding of anti-leishmanial drug action and 44 potential resistance mechanisms, and should facilitate the development of improved 45 therapies, as well as surveillance for drug-resistant parasites.46 Importance 47 Leishmaniasis is a devastating disease caused by the Leishmania parasites and is 48 endemic to a wide swathe of the tropics and sub-tropics. While there are drugs available 49 for the treatment of leishmaniasis, these suffer from various challenges, including the 50 spread of drug resistance. Our understanding of anti-leishmanial drug action and the 51 modes of drug resistance in Leishmania is limited. The development of genetic screening 52 tools in the related parasite, Trypanosoma brucei, has revolutionised our understanding of 53 these processes in this parasite. Therefore, we applied these tools to the anti-leishmanial 54 drugs, identifying T. brucei orthologues of known Leishmania proteins that drive drug 55 uptake, as well as a panel of novel proteins not previously associated with anti-leishmanial 56 drug action. Our findings substantially advance our understanding of anti-leishmanial 57 mode-of-action and provide a valuable starting point for further research.58 101 efficacy (reviewed in (14)). In addition, the generation of drug resistant Leishmania in the 102 laboratory and various 'omi...