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

> **NIH NIH R01** · COLUMBIA UNIVERSITY HEALTH SCIENCES · 2022 · $694,580

## Abstract

Drug resistance in Plasmodium falciparum (Pf), the deadliest of the malaria parasites that threatens almost half
the world’s population, has been associated with genetic changes in specific parasite alleles from field isolates.
The protein responsible for Pf asexual blood stage (ABS) parasite resistance to both previously and currently
used first-line antimalarials, chloroquine (CQ) piperaquine (PPQ) and amodiaquine (ADQ), is the 48-kDa P.
falciparum chloroquine resistance transporter (PfCRT). CQ, PPQ and ADQ, all 4-aminoquinolines, eliminate
drug-sensitive Pf ABS parasites by inhibiting the detoxification of host heme, a product of parasite-mediated
hemoglobin degradation, inside their digestive vacuole (DV). PfCRT, situated in the DV membrane, is thought
to mediate CQ resistance via drug efflux. Our progress in understanding how PfCRT functions, and the
molecular basis of PfCRT-mediated drug resistance, has been seriously hampered by the lack of an atomic
model of this transporter. Using antigen-binding fragment (Fab) technology and single-particle cryo-electron
microscopy, we have determined the structure of the full-length CQ-resistant 7G8 mutant isoform of this 10-
transmembrane protein to 3.2 Å resolution, in an inward-open conformation. These preliminary data are
presented herein, together with functional assays using purified protein in nanodiscs and in liposomes, and
parasite-based assays with pfcrt-modified lines. In this application, we propose to compressively define PfCRT
structure and function and leverage this into experimentally testable predictions of how PfCRT can further
evolve to drive new patterns of multidrug resistance across malaria-endemic regions. In Aim 1, we will solve
the PfCRT structure for globally variant isoforms, including complexes with the antimalarial drugs CQ, PPQ
and ADQ, and physiologic substrates. In Aim 2, we will implement biophysical approaches with recombinant
protein to elucidate the natural function of PfCRT and develop a model of PfCRT-mediated substrate and drug
transport. In Aim 3, we will apply validated gene-editing approaches to predict emerging PfCRT-mediated
resistance and elucidate its functional impact in parasites. Gene-editing studies will focus on PPQ and ADQ in
the context of major global PfCRT variants, as a way to anticipate how mutant PfCRT could evolve new drug
resistance traits, including with isoforms present in high-transmission African settings where drug-resistant
malaria exerts by far its greatest impact. This coordinated research effort combines three Columbia University
groups led by Drs. Mancia, Quick and Fidock, who bring expertise in membrane protein biochemistry and
structure, bioenergetics of membrane transport, and Pf biology including mechanisms of antimalarial drug
resistance, respectively. This highly integrated project has the potential to transform our understanding of how
PfCRT mediates multidrug resistance, by providing powerful advances in deciphering PfCRT str...

## Key facts

- **NIH application ID:** 10443625
- **Project number:** 5R01AI147628-04
- **Recipient organization:** COLUMBIA UNIVERSITY HEALTH SCIENCES
- **Principal Investigator:** David A Fidock
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2022
- **Award amount:** $694,580
- **Award type:** 5
- **Project period:** 2019-07-01 → 2024-06-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10443625

## Citation

> US National Institutes of Health, RePORTER application 10443625, Leveraging PfCRT Structure to Discern Function and Predict Emergence of Drug-Resistant Malaria (5R01AI147628-04). Retrieved via AI Analytics 2026-05-28 from https://api.ai-analytics.org/grant/nih/10443625. Licensed CC0.

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