High-throughput decoding of antitrypanosomal drug efficacy and resistance

Abstract

The concept of disease-specific chemotherapy was developed a century ago. Dyes and arsenical compounds that displayed selectivity against trypanosomes were central to this work, and the drugs that emerged remain in use for treating human African trypanosomiasis (HAT). The importance of understanding the mechanisms underlying selective drug action and resistance for the development of improved HAT therapies has been recognized, but these mechanisms have remained largely unknown. Here we use all five current HAT drugs for genome-scale RNA interference target sequencing (RIT-seq) screens in Trypanosoma brucei, revealing the transporters, organelles, enzymes and metabolic pathways that function to facilitate antitrypanosomal drug action. RIT-seq profiling identifies both known drug importers and the only known pro-drug activator, and links more than fifty additional genes to drug action. A bloodstream stage-specific invariant surface glycoprotein (ISG75) family mediates suramin uptake, and the AP1 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 several 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 antitrypanosomal drug efficacy and resistance will aid the rational design of new therapies and help to combat drug resistance, and provide unprecedented molecular insight into the mode of action of antitrypanosomal drugs.

Figure 1. Identification of drug efficacy determinants in T. brucei

a, The schematic illustrates the RNAi library screening approach. Expected outcomes are illustrated for RNAi targets that fail to affect drug resistance (black), increase resistance to drug A (blue), drug B (orange) or both (green). b, Each screen yielded a population displaying Tet-inducible (RNAi-dependent) drug-resistance; see Supplementary Fig. 1. The plot indicates the proportion of the resistance phenotype that is Tet-inducible. c, Genome-wide RIT-seq profiles. Each map represents a non-redundant set of 7,435 protein-coding sequences. Red bars represent ‘primary’ read-density signatures. Black bars represent all other signatures of >50 reads (see Supplementary data File 1). All three expected ‘hits’, AAT6, AT1 and NTR, are indicated. d, Selected signatures. Each peak represents a unique RIT-seq tag. ‘+’, numbers of additional genes identified in each category. See Supplementary Figure 2 for details and additional signatures.

Figure 2. A network of proteins link ISG75, endocytosis and lysosomal functions to suramin action

a, Western blots demonstrate knockdown; Coomassie stains serve as loading controls. See Supplementary Fig. 3 for growth curves. b, Endosomal/lysosomal factors and ISG75 contribute to suramin action. Error bars, s.d. from independent RNAi strains; see Supplementary Fig. 4. c, MFST and EMP70 are membrane-associated. The western blots show supernatant (S), wash (W) and pellet (P; membrane-fraction). d, MFST co-localises with lysosomal protein, p67, but not recycling endosomes (Rab11). e, Knockdown of UbH1 specifically decreases ISG75 expression. f, ISG75 mediates suramin binding. Error bars, s.d. from duplicate experiments. P value from Student’s t-test. ISG75 knockdown is shown. Scale bar, 5 μm. g, CatL/CatB and ODC inhibitors, FMK024 and eflornithine, respectively, antagonise suramin action. Isobolograms showing 50% fractional inhibitory concentrations (FICs). The solid lines indicate antagonism. The dashed lines indicate expected outcomes for no interaction.

Figure 3. aqp2/3 null cells are melarsoprol, pentamidine cross-resistant

a, Analysis of read-density for all (74,350) possible pair-wise comparisons of a non-redundant T. brucei gene set; E, eflornithine; M, melarsoprol; N, nifurtimox; P, pentamidine; S, suramin; ^X and ^Y, axes representing each dataset. The box on the right shows the read-density signatures for this locus (Tb927.10.14160-70). b, AQP2/3 knockout was confirmed by Southern blotting. S, SacII; Δ, the region deleted; bars indicate probes. c, EC₅₀ analysis indicates melarsoprol, pentamidine cross-resistance in aqp2/3 null cells. Error bars, s.d. from triplicate assays and independent null strains.

Figure 4. Determinants of drug efficacy in African trypanosomes

Proteins and metabolites linked to drug action are identified by red and green text, respectively. a, The schematic summarises the findings from the RIT-seq screens. In the case of suramin, we propose that ISG75 binds the drug at the cell surface. ISG75 trafficking then delivers the complex, via the flagellar pocket (FP), to the endosomal system, leading to accumulation in the lysosome where the drug is liberated by proteases. The MFST may deliver the drug to the cytosol. HAPT/LAPT, high/low-affinity pentamidine transporters; TS₂, oxidised trypanothione; T[SH]₂, reduced trypanothione. b, Biosynthetic pathways linked to drug action. See text and Supplementary data File 1 for acronyms and further details.