Quinoxaline-Based Anti-Schistosomal Compounds Have Potent Anti-Malarial Activity.

The human pathogens Plasmodium and Schistosoma are each responsible for over 200 million infections annually, being particularly problematic in low- and middle-income countries. There is a pressing need for new drug targets for these diseases, driven by emergence of drug-resistance in Plasmodium and the overall dearth of new drug targets for Schistosoma. Here, we explored the opportunity for pathogen-hopping by evaluating a series of quinoxaline-based anti-schistosomal compounds for activity against P. falciparum. We identified compounds with low nanomolar potency against 3D7 and multidrug-resistant strains. Evolution of resistance using a mutator P. falciparum line revealed a low propensity for resistance. Only one of the series, compound 22, yielded resistance mutations, including point mutations in a non-essential putative hydrolase pfqrp1, as well as copy-number amplification of a phospholipid-translocating ATPase, pfatp2, a potential target. Notably, independently generated CRISPR-edited mutants in pfqrp1 also showed resistance to compound 22 and a related analogue. Moreover, previous lines with pfatp2 copy-number variations were similarly less susceptible to challenge with the new compounds. Finally, we examined whether the predicted hydrolase activity of PfQRP1 underlies its mechanism of resistance, showing that both mutation of the putative catalytic triad and a more severe loss of function mutation elicited resistance. Collectively, we describe a compound series with potent activity against two important pathogens and their potential target in P. falciparum.

tRNA modification reprogramming contributes to artemisinin resistance in Plasmodium falciparum.

Plasmodium falciparum artemisinin (ART) resistance is driven by mutations in kelch-like protein 13 (PfK13). Quiescence, a key aspect of resistance, may also be regulated by a yet unidentified epigenetic pathway. Transfer RNA modification reprogramming and codon bias translation is a conserved epitranscriptomic translational control mechanism that allows cells to rapidly respond to stress. We report a role for this mechanism in ART-resistant parasites by combining tRNA modification, proteomic and codon usage analyses in ring-stage ART-sensitive and ART-resistant parasites in response to drug. Post-drug, ART-resistant parasites differentially hypomodify mcm5s2U on tRNA and possess a subset of proteins, including PfK13, that are regulated by Lys codon-biased translation. Conditional knockdown of the terminal s2U thiouridylase, PfMnmA, in an ART-sensitive parasite background led to increased ART survival, suggesting that hypomodification can alter the parasite ART response. This study describes an epitranscriptomic pathway via tRNA s2U reprogramming that ART-resistant parasites may employ to survive ART-induced stress.

Novel hydrazone compounds with broad-spectrum antiplasmodial activity and synergistic interactions with antimalarial drugs.

The development of novel antiplasmodial compounds with broad-spectrum activity against different stages of Plasmodium parasites is crucial to prevent malaria disease and parasite transmission. This study evaluated the antiplasmodial activity of seven novel hydrazone compounds (referred to as CB compounds: CB-27, CB-41, CB-50, CB-53, CB-58, CB-59, and CB-61) against multiple stages of Plasmodium parasites. All CB compounds inhibited blood stage proliferation of drug-resistant or sensitive strains of Plasmodium falciparum in the low micromolar to nanomolar range. Interestingly, CB-41 exhibited prophylactic activity against hypnozoites and liver schizonts in Plasmodium cynomolgi, a primate model for Plasmodium vivax. Four CB compounds (CB-27, CB-41, CB-53, and CB-61) inhibited P. falciparum oocyst formation in mosquitoes, and five CB compounds (CB-27, CB-41, CB-53, CB-58, and CB-61) hindered the in vitro development of Plasmodium berghei ookinetes. The CB compounds did not inhibit the activation of P. berghei female and male gametocytes in vitro. Isobologram assays demonstrated synergistic interactions between CB-61 and the FDA-approved antimalarial drugs, clindamycin and halofantrine. Testing of six CB compounds showed no inhibition of Plasmodium glutathione S-transferase as a putative target and no cytotoxicity in HepG2 liver cells. CB compounds are promising candidates for further development as antimalarial drugs against multidrug-resistant parasites, which could also prevent malaria transmission.

The emergence of artemisinin partial resistance in Africa: how do we respond?

Malaria remains one of the most important infectious diseases in the world, with the greatest burden in sub-Saharan Africa, primarily from Plasmodium falciparum infection. The treatment and control of malaria is challenged by resistance to most available drugs, but partial resistance to artemisinins (ART-R), the most important class for the treatment of malaria, was until recently confined to southeast Asia. This situation has changed, with the emergence of ART-R in multiple countries in eastern Africa. ART-R is mediated primarily by single point mutations in the P falciparum kelch13 protein, with several mutations present in African parasites that are now validated resistance mediators based on clinical and laboratory criteria. Major priorities at present are the expansion of genomic surveillance for ART-R mutations across the continent, more frequent testing of the efficacies of artemisinin-based regimens against uncomplicated and severe malaria in trials, more regular assessment of ex-vivo antimalarial drug susceptibilities, consideration of changes in treatment policy to deter the spread of ART-R, and accelerated development of new antimalarial regimens to overcome the impacts of ART-R. The emergence of ART-R in Africa is an urgent concern, and it is essential that we increase efforts to characterise its spread and mitigate its impact.

Activity refinement of aryl amino acetamides that target the P. falciparum STAR-related lipid transfer 1 protein.

Malaria is a devastating disease that causes significant morbidity worldwide. The development of new antimalarial chemotypes is urgently needed because of the emergence of resistance to frontline therapies. Independent phenotypic screening campaigns against the Plasmodium asexual parasite, including our own, identified the aryl amino acetamide hit scaffold. In a prior study, we identified the STAR-related lipid transfer protein (PfSTART1) as the molecular target of this antimalarial chemotype. In this study, we combined structural elements from the different aryl acetamide hit subtypes and explored the structure-activity relationship. It was shown that the inclusion of an endocyclic nitrogen, to generate the tool compound WJM-715, improved aqueous solubility and modestly improved metabolic stability in rat hepatocytes. Metabolic stability in human liver microsomes remains a challenge for future development of the aryl acetamide class, which was underscored by modest systemic exposure and a short half-life in mice. The optimized aryl acetamide analogs were cross resistant to parasites with mutations in PfSTART1, but not to other drug-resistant mutations, and showed potent binding to recombinant PfSTART1 by biophysical analysis, further supporting PfSTART1 as the likely molecular target. The optimized aryl acetamide analogue, WJM-715 will be a useful tool for further investigating the druggability of PfSTART1 across the lifecycle of the malaria parasite.

Drug-induced stress mediates Plasmodium falciparum ring-stage growth arrest and reduces in vitro parasite susceptibility to artemisinin.

During blood-stage infection, Plasmodium falciparum parasites are constantly exposed to a range of extracellular stimuli, including host molecules and drugs such as artemisinin derivatives, the mainstay of artemisinin-based combination therapies currently used as first-line treatment worldwide. Partial resistance of P. falciparum to artemisinin has been associated with mutations in the propeller domain of the Pfkelch13 gene, resulting in a fraction of ring stages that are able to survive exposure to artemisinin through a temporary growth arrest. Here, we investigated whether the growth arrest in ring-stage parasites reflects a general response to stress. We mimicked a stressful environment in vitro by exposing parasites to chloroquine or dihydroartemisinin (DHA). We observed that early ring-stage parasites pre-exposed to a stressed culture supernatant exhibited a temporary growth arrest and a reduced susceptibility to DHA, as assessed by the ring-stage survival assay, irrespective of their Pfkelch13 genotype. These data suggest that temporary growth arrest of early ring stages may be a constitutive, Pfkelch13-independent survival mechanism in P. falciparum.IMPORTANCEPlasmodium falciparum ring stages have the ability to sense the extracellular environment, regulate their growth, and enter a temporary growth arrest state in response to adverse conditions such as drug exposure. This temporary growth arrest results in reduced susceptibility to artemisinin in vitro. The signal responsible for this process is thought to be small molecules (less than 3 kDa) released by stressed mature-stage parasites. These data suggest that Pfkelch13-dependent artemisinin resistance and the growth arrest phenotype are two complementary but unrelated mechanisms of ring-stage survival in P. falciparum. This finding provides new insights into the field of P. falciparum antimalarial drug resistance by highlighting the extracellular compartment and cellular communication as an understudied mechanism.

Mixed Alkyl/Aryl Phosphonates Identify Metabolic Serine Hydrolases as Antimalarial Targets.

Malaria, caused by Plasmodium falciparum, remains a significant health burden. A barrier for developing anti-malarial drugs is the ability of the parasite to rapidly generate resistance. We demonstrated that Salinipostin A (SalA), a natural product, kills parasites by inhibiting multiple lipid metabolizing serine hydrolases, a mechanism with a low propensity for resistance. Given the difficulty of employing natural products as therapeutic agents, we synthesized a library of lipidic mixed alkyl/aryl phosphonates as bioisosteres of SalA. Two constitutional isomers exhibited divergent anti-parasitic potencies which enabled identification of therapeutically relevant targets. We also confirm that this compound kills parasites through a mechanism that is distinct from both SalA and the pan-lipase inhibitor, Orlistat. Like SalA, our compound induces only weak resistance, attributable to mutations in a single protein involved in multidrug resistance. These data suggest that mixed alkyl/aryl phosphonates are a promising, synthetically tractable anti-malarials with a low-propensity to induce resistance.

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl-tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure-activity relationship and the selectivity mechanism.

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.

Mapping the genomic landscape of multidrug resistance in Plasmodium falciparum and its impact on parasite fitness.

Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance in vitro and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping using 34 recombinant haplotypes, and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.

Covalent Macrocyclic Proteasome Inhibitors Mitigate Resistance in Plasmodium falciparum.

The Plasmodium proteasome is a promising antimalarial drug target due to its essential role in all parasite lifecycle stages. Furthermore, proteasome inhibitors have synergistic effects when combined with current first-line artemisinin and related analogues. Linear peptides that covalently inhibit the proteasome are effective at killing parasites and have a low propensity for inducing resistance. However, these scaffolds generally suffer from poor pharmacokinetics and bioavailability. Here we describe the development of covalent, irreversible, macrocyclic inhibitors of the Plasmodium falciparum proteasome. We identified compounds with excellent potency and low cytotoxicity; however, the first generation suffered from poor microsomal stability. Further optimization of an existing macrocyclic scaffold resulted in an irreversible covalent inhibitor carrying a vinyl sulfone electrophile that retained high potency and low cytotoxicity and had acceptable metabolic stability. Importantly, unlike the parent reversible inhibitor that selected for multiple mutations in the proteasome, with one resulting in a 5,000-fold loss of potency, the irreversible analogue only showed a 5-fold loss in potency for any single point mutation. Furthermore, an epoxyketone analogue of the same scaffold retained potency against a panel of known proteasome mutants. These results confirm that macrocycles are optimal scaffolds to target the malarial proteasome and that the use of a covalent electrophile can greatly reduce the ability of the parasite to generate drug resistance mutations.

Increasing Prevalence of Artemisinin-Resistant HRP2-Negative Malaria in Eritrea.

BACKGROUND: Although the clinical efficacy of antimalarial artemisinin-based combination therapies in Africa remains high, the recent emergence of partial resistance to artemisinin in Plasmodium falciparum on the continent is troubling, given the lack of alternative treatments. METHODS: In this study, we used data from drug-efficacy studies conducted between 2016 and 2019 that evaluated 3-day courses of artemisinin-based combination therapy (artesunate-amodiaquine or artemether-lumefantrine) for uncomplicated malaria in Eritrea to estimate the percentage of patients with day-3 positivity (i.e., persistent P. falciparum parasitemia 3 days after the initiation of therapy). We also assayed parasites for mutations in Pfkelch13 as predictive markers of partial resistance to artemisinin and screened for deletions in hrp2 and hrp3 that result in variable performance of histidine rich protein 2 (HRP2)-based rapid diagnostic tests for malaria. RESULTS: We noted an increase in the percentage of patients with day-3 positivity from 0.4% (1 of 273) in 2016 to 1.9% (4 of 209) in 2017 and 4.2% (15 of 359) in 2019. An increase was also noted in the prevalence of the Pfkelch13 R622I mutation, which was detected in 109 of 818 isolates before treatment, from 8.6% (24 of 278) in 2016 to 21.0% (69 of 329) in 2019. The odds of day-3 positivity increased by a factor of 6.2 (95% confidence interval, 2.5 to 15.5) among the patients with Pfkelch13 622I variant parasites. Partial resistance to artemisinin, as defined by the World Health Organization, was observed in Eritrea. More than 5% of the patients younger than 15 years of age with day-3 positivity also had parasites that carried Pfkelch13 R622I. In vitro, the R622I mutation conferred a low level of resistance to artemisinin when edited into NF54 and Dd2 parasite lines. Deletions in both hrp2 and hrp3 were identified in 16.9% of the parasites that carried the Pfkelch13 R622I mutation, which made them potentially undetectable by HRP2-based rapid diagnostic tests. CONCLUSIONS: The emergence and spread of P. falciparum lineages with both Pfkelch13-mediated partial resistance to artemisinin and deletions in hrp2 and hrp3 in Eritrea threaten to compromise regional malaria control and elimination campaigns. (Funded by the Bill and Melinda Gates Foundation and others; Australian New Zealand Clinical Trials Registry numbers, ACTRN12618001223224, ACTRN12618000353291, and ACTRN12619000859189.).

Propensity of selecting mutant parasites for the antimalarial drug cabamiquine.

We report an analysis of the propensity of the antimalarial agent cabamiquine, a Plasmodium-specific eukaryotic elongation factor 2 inhibitor, to select for resistant Plasmodium falciparum parasites. Through in vitro studies of laboratory strains and clinical isolates, a humanized mouse model, and volunteer infection studies, we identified resistance-associated mutations at 11 amino acid positions. Of these, six (55%) were present in more than one infection model, indicating translatability across models. Mathematical modelling suggested that resistant mutants were likely pre-existent at the time of drug exposure across studies. Here, we estimated a wide range of frequencies of resistant mutants across the different infection models, much of which can be attributed to stochastic differences resulting from experimental design choices. Structural modelling implicates binding of cabamiquine to a shallow mRNA binding site adjacent to two of the most frequently identified resistance mutations.

Antimalarial drug discovery: progress and approaches.

Recent antimalarial drug discovery has been a race to produce new medicines that overcome emerging drug resistance, whilst considering safety and improving dosing convenience. Discovery efforts have yielded a variety of new molecules, many with novel modes of action, and the most advanced are in late-stage clinical development. These discoveries have led to a deeper understanding of how antimalarial drugs act, the identification of a new generation of drug targets, and multiple structure-based chemistry initiatives. The limited pool of funding means it is vital to prioritize new drug candidates. They should exhibit high potency, a low propensity for resistance, a pharmacokinetic profile that favours infrequent dosing, low cost, preclinical results that demonstrate safety and tolerability in women and infants, and preferably the ability to block Plasmodium transmission to Anopheles mosquito vectors. In this Review, we describe the approaches that have been successful, progress in preclinical and clinical development, and existing challenges. We illustrate how antimalarial drug discovery can serve as a model for drug discovery in diseases of poverty.

Reaction hijacking inhibition of Plasmodium falciparum asparagine tRNA synthetase.

Malaria poses an enormous threat to human health. With ever increasing resistance to currently deployed drugs, breakthrough compounds with novel mechanisms of action are urgently needed. Here, we explore pyrimidine-based sulfonamides as a new low molecular weight inhibitor class with drug-like physical parameters and a synthetically accessible scaffold. We show that the exemplar, OSM-S-106, has potent activity against parasite cultures, low mammalian cell toxicity and low propensity for resistance development. In vitro evolution of resistance using a slow ramp-up approach pointed to the Plasmodium falciparum cytoplasmic asparaginyl tRNA synthetase (PfAsnRS) as the target, consistent with our finding that OSM-S-106 inhibits protein translation and activates the amino acid starvation response. Targeted mass spectrometry confirms that OSM-S-106 is a pro-inhibitor and that inhibition of PfAsnRS occurs via enzyme-mediated production of an Asn-OSM-S-106 adduct. Human AsnRS is much less susceptible to this reaction hijacking mechanism. X-ray crystallographic studies of human AsnRS in complex with inhibitor adducts and docking of pro-inhibitors into a model of Asn-tRNA-bound PfAsnRS provide insights into the structure activity relationship and the selectivity mechanism.

Mapping the genomic landscape of multidrug resistance in Plasmodium falciparum and its impact on parasite fitness.

Drug-resistant Plasmodium falciparum parasites have swept across Southeast Asia and now threaten Africa. By implementing a P. falciparum genetic cross using humanized mice, we report the identification of key determinants of resistance to artemisinin (ART) and piperaquine (PPQ) in the dominant Asian KEL1/PLA1 lineage. We mapped k13 as the central mediator of ART resistance and identified secondary markers. Applying bulk segregant analysis, quantitative trait loci mapping and gene editing, our data reveal an epistatic interaction between mutant PfCRT and multicopy plasmepsins 2/3 in mediating high-grade PPQ resistance. Susceptibility and parasite fitness assays implicate PPQ as a driver of selection for KEL1/PLA1 parasites. Mutant PfCRT enhanced susceptibility to lumefantrine, the first-line partner drug in Africa, highlighting a potential benefit of opposing selective pressures with this drug and PPQ. We also identified that the ABCI3 transporter can operate in concert with PfCRT and plasmepsins 2/3 in mediating multigenic resistance to antimalarial agents.