Defining the Role of PfCRT and PfMDR1 as Pleiotropic Mediators of Plasmodium falciparum Multidrug Resistance

项目概述（见说明）：

PROJECT SUMMARY (See instructions): Antimalarial drug resistance has repeatedly thwarted global efforts to treat and control malaria. Chloroquine, the former gold-standard drug, earlier succumbed to parasite strains harboring mutations in the Plasmodium falciparum chloroquine resistance transporter, PfCRT. Now, artemisinin-based combination therapies have failed in Southeast Asia, driven in part by novel PfCRT variants that confer resistance to the first-line partner drug piperaquine. Resistance to other partner drugs including amodiaquine, lumefantrine, and mefloquine can be conferred by variant forms of PfCRT or the ABC transporter PfMDR1. Our R37 AI050234, funded since 2001, has substantially advanced research in this area including: publishing definitive evidence that PfCRT mutations are the primary determinant of chloroquine and piperaquine resistance; attributing resistance to PfCRT-mediated drug efflux; solving the PfCRT structure; defining fitness costs and physiological effects that impact the spread of resistant alleles; globally mapping pfcrt and pfmdr1 variants across the globe; and characterizing PfMDR 1-mediated multidrug resistance. Since our prior submission in August 2017 we have published 23 articles including in Nature, Nature Microbiol, Lancet Infect Dis, and Cell Chem Biol. Our R37 MERIT Extension application proposes to advance this with four new Specific Aims. Aim 1: Define whether mutant PfCRT can mediate piperaquine resistance in African strains. We will determine whether PfCRT mutations that cause piperaquine resistance in Asian parasites will similarly confer resistance in African strains with regionally distinct PfCRT isoforms. We will also assess the impact of these isoforms on other antimalarials and on parasite fitness. Aim 2: Define genetic determinants that function epistatically with PfCRT to mediate resistance. Using our recent P. falciparum genetic cross, we will positionally map and characterize genes that are coinherited with mutant PfCRT in chloroquine- and/or quinine-resistant progeny and determine whether these determinants augment resistance or have evolved to offset fitness costs. Aim 3: Identify PfCRT-specific inhibitors that can reverse drug resistance. We propose to screen diverse chemical libraries to identify compounds that block the efflux of chloroquine or piperaquine in drug-resistant parasite lines. Assays with pfcrt-conditional knockdown parasites and recombinant PfCRT will test for compounds that can bind this transporter, block efflux, and restore drug potency. Aim 4: Solve the PfMDR1 structure and define its drug efflux properties compared with PfCRT. Following our recent success with PfCRT, we propose to solve the PfMDR1 structure using cryo-electron microscopy. We will also perform binding and transport assays with purified recombinant protein to characterize the resistance properties of PfMDR1 variants. This proposal, which aligns with the NIAID priority of supporting research on antimicrobial drug resistance, will substantially advance our understanding of antimalarial resistance and yield biomarkers and new strategies to counter drug-resistant malaria.