Additional PfCRT mutations driven by selective pressure for improved fitness can result in the loss of piperaquine resistance and altered Plasmodium falciparum physiology.

IMPORTANCE: Our study leverages gene editing techniques in Plasmodium falciparum asexual blood stage parasites to profile novel mutations in mutant PfCRT, an important mediator of piperaquine resistance, which developed in Southeast Asian field isolates or in parasites cultured for long periods of time. We provide evidence that increased parasite fitness of these lines is the primary driver for the emergence of these PfCRT variants. These mutations differentially impact parasite susceptibility to piperaquine and chloroquine, highlighting the multifaceted effects of single point mutations in this transporter. Molecular features of drug resistance and parasite physiology were examined in depth using proteoliposome-based drug uptake studies and peptidomics, respectively. Energy minimization calculations, showing how these novel mutations might impact the PfCRT structure, suggested a small but significant effect on drug interactions. This study reveals the subtle interplay between antimalarial resistance, parasite fitness, PfCRT structure, and intracellular peptide availability in PfCRT-mediated parasite responses to changing drug selective pressures.

Structures of Plasmodium falciparum Chloroquine Resistance Transporter (PfCRT) Isoforms and Their Interactions with Chloroquine.

Using a recently elucidated atomic-resolution cryogenic electron microscopy (cryo-EM) structure for the Plasmodium falciparum chloroquine resistance transporter (PfCRT) protein 7G8 isoform as template [Kim, J.; Nature 2019, 576, 315-320], we use Monte Carlo molecular dynamics (MC/MD) simulations of PfCRT embedded in a 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membrane to solve energy-minimized structures for 7G8 PfCRT and two additional PfCRT isoforms that harbor 5 or 7 amino acid substitutions relative to 7G8 PfCRT. Guided by drug binding previously defined using chloroquine (CQ) photoaffinity probe labeling, we also use MC/MD energy minimization to elucidate likely CQ binding geometries for the three membrane-embedded isoforms. We inventory salt bridges and hydrogen bonds in these structures and summarize how the limited changes in primary sequence subtly perturb local PfCRT isoform structure. In addition, we use the "AlphaFold" artificial intelligence AlphaFold2 (AF2) algorithm to solve for domain structure that was not resolved in the previously reported 7G8 PfCRT cryo-EM structure, and perform MC/MD energy minimization for the membrane-embedded AF2 structures of all three PfCRT isoforms. We compare energy-minimized structures generated using cryo-EM vs AF2 templates. The results suggest how amino acid substitutions in drug resistance-associated isoforms of PfCRT influence PfCRT structure and CQ transport.

Artemisinin-Based Drugs Target the Plasmodium falciparum Heme Detoxification Pathway.

Artemisinin (ART)-based antimalarial drugs are believed to exert lethal effects on malarial parasites by alkylating a variety of intracellular molecular targets. Recent work with live parasites has shown that one of the alkylated targets is free heme within the parasite digestive vacuole, which is liberated upon hemoglobin catabolism by the intraerythrocytic parasite, and that reduced levels of heme alkylation occur in artemisinin-resistant parasites. One implication of heme alkylation is that these drugs may inhibit parasite detoxification of free heme via inhibition of heme-to-hemozoin crystallization; however, previous reports that have investigated this hypothesis present conflicting data. By controlling reducing conditions and, hence, the availability of ferrous versus ferric forms of free heme, we modify a previously reported hemozoin inhibition assay to quantify the ability of ART-based drugs to target the heme detoxification pathway under reduced versus oxidizing conditions. Contrary to some previous reports, we find that artemisinins are potent inhibitors of hemozoin crystallization, with effective half-maximal concentrations approximately an order of magnitude lower than those for most quinoline-based antimalarial drugs. We also examine hemozoin and in vitro parasite growth inhibition for drug pairs found in the most commonly used ART-based combination therapies (ACTs). All ACTs examined inhibit hemozoin crystallization in an additive fashion, and all but one inhibit parasite growth in an additive fashion.

ß-Strand inspired bifacial π-conjugated polymers.

Access to diverse, relatively high molecular weight soluble linear polymers without pendant solubilizing chains is the key to solution state synthesis of structurally diverse nanoribbons of conjugated materials. However, realizing soluble 1D-π-conjugated polymers without pendant solubilizing chains is a daunting task. Herein, inspired from the polypeptide ß-strand architecture, we have designed and developed novel bifacial π-conjugated polymers (M n: ca. 24 kDa) that are soluble (ca. 70 to >250 mM) despite the absence of pendant solubilizing chains. The impact of varying the bifacial monomer height on polymer solubility, optical properties, and interactions with small molecules is reported.

A practical diastereoselective synthesis of (-)-bestatin.

Diastereoselective addition of nitromethane to Boc-D-Phe-H in the presence of sodium hydride in diethyl ether/hexane containing 15-crown-5 and subsequent N,O-protection with 2,2-dimethoxypropane gave trans-oxazolidine in a diastereomeric ratio of >16:1. The oxazolidine was easily separated by column chromatography, which after Nef reaction was coupled to H-Leu-OtBu. The 8-step synthesis afforded (-)-bestatin in an overall yield of 24.7% after deprotection and ion exchange.

Modified primers for rapid and direct electrochemical analysis of coeliac disease associated HLA alleles.

Direct detection of PCR product via hybridisation assay, would facilitate the development of rapid tools for genetic analysis. Here, a PCR primer designed to generate a PCR amplicon tagged with single stranded DNA tails at each end of the duplex, which can be used for direct hybridisation with a surface immobilised probe and an enzyme labelled reporter probe is presented. Four modified sequence specific primers (SSP) pairs were designed for the selective amplification of coeliac disease associated alleles (DQA1*05, DQB1*02, DQB1*03:02 alleles), and human growth hormone (positive control). Multiplex PCR products were electrochemically detected in less than 5 min at 37 °C via direct hybridisation to short probes immobilised on individual electrodes of a genosensor array, and subsequent hybridisation to an enzyme labelled reporter probe. The developed electrochemical genosensor array exploiting the modified primers for the direct detection of PCR products was applied to the genotyping of real patient samples.