UDP-galactose and acetyl-CoA transporters as Plasmodium multidrug resistance genes

Abstract

A molecular understanding of drug resistance mechanisms enables surveillance of the effectiveness of new antimicrobial therapies during development and deployment in the field. We used conventional drug resistance selection as well as a regime of limiting dilution at early stages of drug treatment to probe two antimalarial imidazolopiperazines, KAF156 and GNF179. The latter approach permits isolation of low-fitness mutants that might otherwise be out-competed during selection. Whole-genome sequencing of 24 independently-derived resistant P. falciparum clones revealed four parasites with mutations in the known cyclic amine resistance locus (pfcarl), and a further 20 with mutations in two previously unreported P. falciparum drug resistance genes, an acetyl-CoA transporter (pfact) and a UDP-galactose transporter (pfugt). Mutations were validated both in vitro by CRISPR editing in P. falciparum, and in vivo by evolution of resistant P. berghei mutants. Both PfACT and PfUGT were localized to the endoplasmic reticulum by fluorescence microscopy. As mutations in pfact and pfugt conveyed resistance against additional unrelated chemical scaffolds, these genes are likely to be involved in broad mechanisms of antimalarial drug resistance.

Genetic diversity of next generation antimalarial targets: A baseline for drug resistance surveillance programmes

“…The pfact gene was recently identified to be the target, together with the UDP-galactose transporter gene-target ( Pfugt), of a variety of imidazolopiperazine compounds. One of these compounds (KAF156) has potent activity against gametocytes and parasite liver stages, and is currently in Phase II clinical trial (Lim et al., 2016). Rab11A is a molecular target for aminopyridine class compounds (McNamara et al., 2013), and eEF2 is the target for quinoline-4-carboxamide (DDD107498) compounds, both with activity against multiple lifecycle parasite stages (Baragaña et al., 2015).…”

“…Here we consider eleven gene-targets of key investigated compounds that due to their efficiency might become the next antimalarial drugs, and for which mutations conferring resistance have been identified in in vitro studies (Baragaña et al., 2015, Dong et al., 2011, Flannery et al., 2015, Herman et al., 2015, Kato et al., 2016, LaMonte et al., 2016, Lim et al., 2016, McNamara et al., 2013, Ross et al., 2014). These 11 genes were also selected because they are gene-targets for a range of new antimalarial compounds already under evaluation in clinical trials.…”

Regulation and Essentiality of the StAR-related Lipid Transfer (START) Domain-containing Phospholipid Transfer Protein PFA0210c in Malaria Parasites

“…Schizonts of the 1G5DC strain were transfected according to standard protocol (38) with pBLD529 (the plasmid that introduces the LoxP sites, see below) and the Cas9-expressing pDC2-cam-Cas9-U6-hDHFR (42) plasmid to which the gene encoding the yeast cytosine deaminase-uracil phosphoribosyltransferase had been added, as well as a sequence encoding guide RNAs specific for PFA0210c. Transfectants were initially selected with 2.5 n m WR99210 and were subsequently treated with 1 μ m ancotil to select against parasites carrying the pDC-based plasmid.…”

Antimalarial drug resistance: linking Plasmodium falciparum parasite biology to the clinic

“…Resistance to KAF156 and related imidazolopiperazines in ABS can be mediated by point mutations in the P. falciparum cyclic amine resistance locus ( pfcarl ), which encodes a conserved protein that localizes in the cis -Golgi apparatus, where it might contribute to protein sorting or membrane trafficking 111,114 . Resistance to these compounds can also be mediated by mutations in an acetyl-CoA transporter (PfACT) or a UDP-galactose transporter (PfUGT), which are localized to the endoplasmic reticulum (ER) 115 (Supplementary Table 2). The subcellular locations of these resistance mediators suggest that imidazolopiperazines might interfere with the transport of parasite biomolecules into the ER or Golgi 115 .…”

“…Resistance to these compounds can also be mediated by mutations in an acetyl-CoA transporter (PfACT) or a UDP-galactose transporter (PfUGT), which are localized to the endoplasmic reticulum (ER) 115 (Supplementary Table 2). The subcellular locations of these resistance mediators suggest that imidazolopiperazines might interfere with the transport of parasite biomolecules into the ER or Golgi 115 . Parasite resistance to different chemotypes has also been associated with mutations in pfcarl , suggesting evidence of its role as a multidrug-resistance mediator rather than the actual drug target 114,116 .…”