Leveraging PfCRT Structure to Discern Function and Predict Emergence of Drug-Resistant Malaria

Columbia University Health Sciences, New York NY

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Abstract

PROJECT SUMMARY Drug resistance in Plasmodium falciparum (Pf), the deadliest of the malaria parasites, continues to thwart efforts to effectively treat, control, and eradicate malaria worldwide. Genetic variations in pfcrt, coding for the Pf chloroquine resistance transporter (PfCRT) that is situated in the intra-erythrocytic parasite’s digestive vacuole (DV) membrane, are directly responsible for Pf resistance to both previously and currently used first-line 4- aminoquinoline (4-AQ) antimalarials. 4-AQs such as chloroquine (CQ), piperaquine (PPQ), and amodiaquine (ADQ) inhibit the detoxification of heme, produced during the degradation of host hemoglobin inside the Pf DV. During the first funding period, we determined the structure of the full-length CQ-resistant PfCRT 7G8 isoform to 3.2 Å resolution by single-particle cryo-electron microscopy. Leveraging this breakthrough, our team has made critical advances in gaining a deeper understanding of PfCRT-mediated transport of drugs and its natural peptide substrates, using functional assays with PfCRT isoforms in artificial membrane systems and parasite-based assays with pfcrt-modified lines. We now add a computational arm to provide state-of-the-art molecular dynamics and machine-learning elements to our approach that, by combining cutting-edge biochemical/ biophysical and structural methods, gene-editing studies, multiphoton-based imaging, and computational tools, establishes a strong premise for the successful continuation of this project. The long-term goal of our synergistic research program is to close the gap in understanding how PfCRT interacts with drugs, how it works by balancing the extrusion of drugs and the transport of its natural peptide substrates, and how mutations evolve in response to specific therapeutic regiments in the field. These studies are essential to predict and counter multidrug resistance across malaria-endemic regions. Three inter-connected, but not inter-dependent aims guide our line of attack: In Aim 1, we will focus on the structural basis of PfCRT-mediated 4-AQ drug resistance by solving structures of globally variant isoforms bound to 4-AQs in combination with in silico and binding approaches. In Aim 2, we will decipher the interplay between the transport of peptide substrates and 4-AQs in different PfCRT isoforms using functional and real-time imaging approaches. In Aim 3, we will interrogate the extraordinary diversity of pfcrt sequence variants that have evolved in response to local drug pressures in various endemic regions and apply machine-learning algorithms coupled with experimental validation using gene-edited parasites, to accurately predict which mutations can confer resistance to individual 4-AQs. This highly integrated Multi-PI project, led by Drs. Mancia, Quick and Fidock of Columbia University, with expertise in membrane protein structure, bioenergetics, and Pf biology, respectively, and involving Dr. Tajkhorshid as Co-I, a world-renowned expert in computational studies, has tremendous potential to establish a new paradigm in PfCRT research through powerful insights into the molecular mechanisms that affect human health throughout the malaria-endemic world. Our results will aid in the design of combination therapies to effectively treat multidrug-resistant malaria.