Pan-active imidazolopiperazine antimalarials target the Plasmodium falciparum intracellular secretory pathway.

A promising new compound class for treating human malaria is the imidazolopiperazines (IZP) class. IZP compounds KAF156 (Ganaplacide) and GNF179 are effective against Plasmodium symptomatic asexual blood-stage infections, and are able to prevent transmission and block infection in animal models. But despite the identification of resistance mechanisms in P. falciparum, the mode of action of IZPs remains unknown. To investigate, we here combine in vitro evolution and genome analysis in Saccharomyces cerevisiae with molecular, metabolomic, and chemogenomic methods in P. falciparum. Our findings reveal that IZP-resistant S. cerevisiae clones carry mutations in genes involved in Endoplasmic Reticulum (ER)-based lipid homeostasis and autophagy. In Plasmodium, IZPs inhibit protein trafficking, block the establishment of new permeation pathways, and cause ER expansion. Our data highlight a mechanism for blocking parasite development that is distinct from those of standard compounds used to treat malaria, and demonstrate the potential of IZPs for studying ER-dependent protein processing

PfMFR3: A multidrug-resistant modulator in Plasmodium falciparum

In malaria, chemical genetics is a powerful method for assigning function to uncharacterized genes. MMV085203 and GNF-Pf-3600 are two structurally related napthoquinone phenotypic screening hits that kill both blood- and sexual-stag

The origins of malaria artemisinin resistance defined by a genetic and transcriptomic background

The predisposition of parasites acquiring artemisinin resistance still remains unclear beyond the mutations in Pfk13 gene and modulation of the unfolded protein response pathway. To explore the chain of casualty underlying artemisinin resistance, we reanalyze 773 P. falciparum isolates from TRACI-study integrating TWAS, GWAS, and eQTL analyses. We find the majority of P. falciparum parasites are transcriptomically converged within each geographic site with two broader physiological profiles across the Greater Mekong Subregion (GMS). We report 8720 SNP-expression linkages in the eastern GMS parasites and 4537 in the western. The minimal overlap between them suggests differential gene regulatory networks facilitating parasite adaptations to their unique host environments. Finally, we identify two genetic and physiological backgrounds associating with artemisinin resistance in the GMS, together with a farnesyltransferase protein and a thioredoxin-like protein which may act as vital intermediators linking the Pfk13 C580Y mutation to the prolonged parasite clearance time

Semi-synthetic analogues of cryptolepine as a potential source of sustainable drugs for the treatment of malaria, human African trypanosomiasis, and cancer

The prospect of eradicating malaria continues to be challenging in the face of increasing parasite resistance to antimalarial drugs so that novel antimalarials active against asexual, sexual, and liver-stage malaria parasites are urgently needed. In addition, new antimalarials need to be affordable and available to those most in need and, bearing in mind climate change, should ideally be sustainable. The West African climbing shrub Cryptolepis sanguinolenta is used traditionally for the treatment of malaria; its principal alkaloid, cryptolepine (1), has been shown to have antimalarial properties, and the synthetic analogue 2,7-dibromocryptolepine (2) is of interest as a lead toward new antimalarial agents. Cryptolepine (1) was isolated using a two-step Soxhlet extraction of C. sanguinolenta roots, followed by crystallization (yield 0.8% calculated as a base with respect to the dried roots). Semi-synthetic 7-bromo- (3), 7, 9-dibromo- (4), 7-iodo- (5), and 7, 9-dibromocryptolepine (6) were obtained in excellent yields by reaction of 1 with N-bromo- or N-iodosuccinimide in trifluoroacetic acid as a solvent. All compounds were active against Plasmodia in vitro, but 6 showed the most selective profile with respect to Hep G2 cells: P. falciparum (chloroquine-resistant strain K1), IC50 = 0.25 µM, SI = 113; late stage, gametocytes, IC50 = 2.2 µM, SI = 13; liver stage, P. berghei sporozoites IC50 = 6.13 µM, SI = 4.6. Compounds 3–6 were also active against the emerging zoonotic species P. knowlesi with 5 being the most potent (IC50 = 0.11 µM). In addition, 3–6 potently inhibited T. brucei in vitro at nM concentrations and good selectivity with 6 again being the most selective (IC50 = 59 nM, SI = 478). These compounds were also cytotoxic to wild-type ovarian cancer cells as well as adriamycin-resistant and, except for 5, cisplatin-resistant ovarian cancer cells. In an acute oral toxicity test in mice, 3–6 did not exhibit toxic effects at doses of up to 100 mg/kg/dose × 3 consecutive days. This study demonstrates that C. sanguinolenta may be utilized as a sustainable source of novel compounds that may lead to the development of novel agents for the treatment of malaria, African trypanosomiasis, and cancer.UK Medical Research Council (MRC) and a Medicines for Malaria Venture Grant.http://www.frontiersin.org/Pharmacologyhj2022BiochemistryGeneticsMicrobiology and Plant PathologyUP Centre for Sustainable Malaria Control (UP CSMC

Advances in Malaria Pharmacology and the online Guide to MALARIA PHARMACOLOGY: IUPHAR Review X

Antimalarial drug discovery has until recently been driven by high-throughput phenotypic cellular screening, allowing millions of compounds to be assayed and delivering clinical drug candidates. In this review, we will focus on target-based approaches, describing recent advances in our understanding of druggable targets in the malaria parasite. Targeting multiple stages of the Plasmodium lifecycle, rather than just the clinically symptomatic asexual blood stage, has become a requirement for new antimalarial medicines, and we link pharmacological data clearly to the parasite stages to which it applies. Finally, we highlight the IUPHAR/MMV Guide to MALARIA PHARMACOLOGY, a web resource developed for the malaria research community that provides open and optimized access to published data on malaria pharmacology

Characterization of artemisinin resistance in plasmodium falciparum

Artemisinins are an important class of antimalarials because of their remarkable antimalarial activity and exceptional safety profile. However, the decline in efficacy of artemisinin-based drugs jeopardizes global efforts that aim to control and ultimately eradicate malaria. In order to better understand the Artemisinin resistance phenotype, we developed Artemisinin-resistant parasite lines derived from two isogenic clones (6A and 11C) of the 3D7 strain of Plasmodium falciparum using a selection regimen that mimics how parasites interact with the drug in infected patients. Long term in vitro selection produced two parasite lines that displayed profound stage-specific resistance to artemisinin and its relative compounds. Chemosensitivity and transcriptional profiling of ART-resistant parasites indicate that enhanced adaptive responses against oxidative stress and protein damage are associated with the artemisinin resistance phenotype. Genomic characterization of both parasite lines also identified a spectrum of mutated and copy number-variable genes that could play a role in mediating artemisinin sensitivity. Collectively, this work represents a useful resource for investigating artemisinin response and resistance in P. falciparum.​Doctor of Philosophy (SBS

CNV profiling of <i>in vitro-</i>selected <i>P</i>. <i>falciparum</i>.

<p>Copy number variations in 6A-R and 11C-R were identified using microarray-based comparative genomic hybridization (CGH). Chromosome plots reflect the subtracted log₂ratio of the artemisinin-resistant parasite lines relative to their control counterparts. Copy number variable genes in 6A-R vs. 6A, and 11C-R vs 11C are indicated in the red and green boxes, respectively. Additional data can be found in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s011" target="_blank">S5 Fig</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s012" target="_blank">S6 Table</a></b>.</p

Chemosensitivity profiling of <i>in vitro</i>-selected <i>P</i>. <i>falciparum</i>.

<p>Chemosensitivity phenotypes of our artemisinin-resistant parasites were evaluated. <b>(A)</b>Apart from their ring-stage sensitivity, the artemisinin IC50_4hr for the trophozoite (IC50_20hpi/4hr) and schizont (IC50_30hpi/4hr) stages was also measured in all parasite lines. Differential susceptibility to artemisinin between selected and control parasites, was subsequently validated using a standard <i>in vitro</i> (72-hour) drug assay (IC50) <b>(B)</b>, and the Ring stage Survival Assay (RSA) <b>(C)</b>. Additional data can be found in <b>Figs <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.g002" target="_blank">2A</a> and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s002" target="_blank">S2B</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s008" target="_blank">S2 Table</a></b>. <b>(D)</b> Ring-stage susceptibility (IC50_10hpi/4hr) against a pulse exposure to artemisinin derivatives Dihydroartemisinin (DHA) and Artesunate (ATS) were also monitored, while chemosensitivity against non-artemisinin antimalarials, quinine (QN), chloroquine (CQ), mefloquine (MEF) and pyrimethamine (PYR), were evaluated using a standard drug assay (IC50). Additional data can be found in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s009" target="_blank">S3A and S3B Fig</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s009" target="_blank">S3 Table</a></b>. <b>(E)</b>Apart from antimalarial drugs, the ring-stage sensitivity of all parasite lines was also measured for compounds that are related to the mechanism of action of artemisinin: hydrogen peroxide (H₂O₂), dithiothreitol (DTT), and epoxomicin (EPX). Additional data can be found in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s009" target="_blank">S3C Fig</a></b> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s009" target="_blank">S3 Table</a></b>. All drug assays were performed in biological triplicates; error bars represent the standard deviation. Pairwise comparison of percent survival (RSA), IC50_10hpi/4hr and IC50 values between resistant and sensitive parasites was performed using student’s t-test.</p

<i>In vitro</i> selection of artemisinin resistance in two <i>P</i>. <i>falciparum</i> clones 6A and 11C.

<p>Artemisinin resistance was induced in two subclones (6A and 11C) of the 3D7 strain of <i>P</i>. <i>falciparum</i> through periodic exposure of the parasite to short pulses of a clinically relevant dose of artemisinin. <b>(A)</b>The <i>in vitro</i> artemisinin selection protocol involved repeated 4-hour pulse treatments of synchronized mid-ring stage parasites (6A and 11C) to 900 nM artemisinin. DMSO-treated parasites were grown alongside the artemisinin-treated parasites (renamed as 6A-R and 11C-R) to serve as controls. Both sets of parasites were subjected to the same number of artemisinin and DMSO treatments throughout drug selection, and at the same generations. Stage-specific artemisinin sensitivity was monitored throughout the course of selection using a 4-hour drug pulse assay at the ring (IC50_10hpi/4hr), trophozoite (IC50_20hpi/4hr), and schizont (IC50_30hpi/4hr) stages of the IDC. <b>(B)</b>In order to monitor incremental changes in ring stage artemisinin sensitivity over time, artemisinin IC50_10hpi/4hr was measured throughout increasing cycles of drug selection between artemisinin-treated parasites and their controls. Additional data can be found in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s007" target="_blank">S1 Table</a></b>. <b>(C)</b>At the start of artemisinin selection, parasite viability and morphology after 4-hour treatment was monitored using microscopic evaluation of Giemsa-stained blood smears. The solid gray line depicts the proportion of surviving parasites 24 hours post treatment normalized to the starting parasitemia, while stacked bars depict proportions of ring, trophozoite and schizont stage morphologies observed among the remaining parasites that appeared to be viable. Examples of parasite morphologies after pulse artemisinin treatment are depicted in <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s001" target="_blank">S1 Fig</a></b>.</p

Effect of overexpression of <i>pftrx1</i>, <i>pf6Pgd</i> and <i>pfspp</i> on artemisinin sensitivity.

<p>To validate the role of stress response gene overexpression on artemisinin resistance, transfectant lines overexpressing select candidate genes were generated, and subsequently assayed for artemisinin sensitivity. <b>(A)</b>Differential mRNA expression in the copy number-amplified genes identified in chromosomes in 10, 12, and 14 was evaluated between artemisinin-resistant parasite lines and their corresponding controls. <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s012" target="_blank">S6 Table</a></b> lists down corrected p- and FDR values for each gene in the Chr 10, Chr 12 and Chr 14 CNV clusters identified. Chromosome plots depict the z-score calculated for each gene based on differences in expression levels between resistant and sensitive parasites across the IDC, while the heatmaps represent the fold-difference between resistant and sensitive parasites for each gene at 6 timepoints taken at 8-hour intervals across a single IDC. Also indicated are the Normalized Enrichment Score (NES) values for transcriptional upregulation in each cluster, obtained by GSEA (p-value < 0.05, FDR < 0.25). Marked in red boxes are candidate stress response genes that were found to be significantly upregulated across the IDC (corrected p-value < 0.05, FDR < 0.25) and amplified in 6A-R. These three genes (PF3D7_1454700 (<i>pf6pgd</i>), PF3D7_1457000 (<i>pfspp)</i>, and PF3D7_1457200 (<i>pftrx1</i>)) were subsequently episomally overexpressed and investigated for their capacity to modulate artemisinin sensitivity. Prior to phenotyping, <b>(B)</b>Real-time qPCR was used to determine the relative overexpression of <i>pftrx1</i>, <i>pf6pgd</i> and <i>pfspp</i> from their respective overexpression parasite lines. Mean fold-change values are derived from three biological replicates; error bars represent the standard deviation. <b>(C)</b>Western blot analysis was also carried out on all overexpression and control parasite lines using a monoclonal mouse anti-HA antibody to validate tagged-protein production. Bands denoted by red arrows indicate the tagged proteins at their expected molecular weights. <b>(D)</b>Ring-stage artemisinin sensitivity (IC50_10hpi/4hr) was then measured for all overexpression parasite lines and compared against the vector control. Drug assays were performed in biological triplicates; error bars represent the standard deviation. Pairwise comparison of IC50_10hpi/4hr between each overexpression line and the vector control was performed using student’s t-test. Additional data can be found in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s012" target="_blank"><b>S6</b> Fig</a> and <b><a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006930#ppat.1006930.s013" target="_blank">S7 Table</a></b>.</p