Covalent Plasmodium falciparum-selective proteasome inhibitors exhibit a low propensity for generating resistance in vitro and synergize with multiple antimalarial agents.

Therapeutics with novel modes of action and a low risk of generating resistance are urgently needed to combat drug-resistant Plasmodium falciparum malaria. Here, we report that the peptide vinyl sulfones WLL-vs (WLL) and WLW-vs (WLW), highly selective covalent inhibitors of the P. falciparum proteasome, potently eliminate genetically diverse parasites, including K13-mutant, artemisinin-resistant lines, and are particularly active against ring-stage parasites. Selection studies reveal that parasites do not readily acquire resistance to WLL or WLW and that mutations in the ß2, ß5 or ß6 subunits of the 20S proteasome core particle or in components of the 19S proteasome regulatory particle yield only hundred-fold decreases in susceptibility. We observed no cross-resistance between WLL and WLW. Moreover, most mutations that conferred a modest loss of parasite susceptibility to one inhibitor significantly increased sensitivity to the other. These inhibitors potently synergized multiple chemically diverse classes of antimalarial agents, implicating a shared disruption of proteostasis in their modes of action. These results underscore the potential of targeting the Plasmodium proteasome with covalent small molecule inhibitors as a means of combating multidrug-resistant malaria.

Chemogenomics identifies acetyl-coenzyme A synthetase as a target for malaria treatment and prevention.

We identify the Plasmodium falciparum acetyl-coenzyme A synthetase (PfAcAS) as a druggable target, using genetic and chemical validation. In vitro evolution of resistance with two antiplasmodial drug-like compounds (MMV019721 and MMV084978) selects for mutations in PfAcAS. Metabolic profiling of compound-treated parasites reveals changes in acetyl-CoA levels for both compounds. Genome editing confirms that mutations in PfAcAS are sufficient to confer resistance. Knockdown studies demonstrate that PfAcAS is essential for asexual growth, and partial knockdown induces hypersensitivity to both compounds. In vitro biochemical assays using recombinantly expressed PfAcAS validates that MMV019721 and MMV084978 directly inhibit the enzyme by preventing CoA and acetate binding, respectively. Immunolocalization studies reveal that PfAcAS is primarily localized to the nucleus. Functional studies demonstrate inhibition of histone acetylation in compound-treated wild-type, but not in resistant parasites. Our findings identify and validate PfAcAS as an essential, druggable target involved in the epigenetic regulation of gene expression.

A G358S mutation in the Plasmodium falciparum Na+ pump PfATP4 confers clinically-relevant resistance to cipargamin.

Diverse compounds target the Plasmodium falciparum Na+ pump PfATP4, with cipargamin and (+)-SJ733 the most clinically-advanced. In a recent clinical trial for cipargamin, recrudescent parasites emerged, with most having a G358S mutation in PfATP4. Here, we show that PfATP4G358S parasites can withstand micromolar concentrations of cipargamin and (+)-SJ733, while remaining susceptible to antimalarials that do not target PfATP4. The G358S mutation in PfATP4, and the equivalent mutation in Toxoplasma gondii ATP4, decrease the sensitivity of ATP4 to inhibition by cipargamin and (+)-SJ733, thereby protecting parasites from disruption of Na+ regulation. The G358S mutation reduces the affinity of PfATP4 for Na+ and is associated with an increase in the parasite's resting cytosolic [Na+]. However, no defect in parasite growth or transmissibility is observed. Our findings suggest that PfATP4 inhibitors in clinical development should be tested against PfATP4G358S parasites, and that their combination with unrelated antimalarials may mitigate against resistance development.

The anticancer human mTOR inhibitor sapanisertib potently inhibits multiple Plasmodium kinases and life cycle stages.

Compounds acting on multiple targets are critical to combating antimalarial drug resistance. Here, we report that the human "mammalian target of rapamycin" (mTOR) inhibitor sapanisertib has potent prophylactic liver stage activity, in vitro and in vivo asexual blood stage (ABS) activity, and transmission-blocking activity against the protozoan parasite Plasmodium spp. Chemoproteomics studies revealed multiple potential Plasmodium kinase targets, and potent inhibition of Plasmodium phosphatidylinositol 4-kinase type III beta (PI4Kß) and cyclic guanosine monophosphate-dependent protein kinase (PKG) was confirmed in vitro. Conditional knockdown of PI4Kß in ABS cultures modulated parasite sensitivity to sapanisertib, and laboratory-generated P. falciparum sapanisertib resistance was mediated by mutations in PI4Kß. Parasite metabolomic perturbation profiles associated with sapanisertib and other known PI4Kß and/or PKG inhibitors revealed similarities and differences between chemotypes, potentially caused by sapanisertib targeting multiple parasite kinases. The multistage activity of sapanisertib and its in vivo antimalarial efficacy, coupled with potent inhibition of at least two promising drug targets, provides an opportunity to reposition this pyrazolopyrimidine for malaria.

The Plasmodium falciparum ABC transporter ABCI3 confers parasite strain-dependent pleiotropic antimalarial drug resistance.

Widespread Plasmodium falciparum resistance to first-line antimalarials underscores the vital need to develop compounds with novel modes of action and identify new druggable targets. Here, we profile five compounds that potently inhibit P. falciparum asexual blood stages. Resistance selection studies with three carboxamide-containing compounds, confirmed by gene editing and conditional knockdowns, identify point mutations in the parasite transporter ABCI3 as the primary mediator of resistance. Selection studies with imidazopyridine or quinoline-carboxamide compounds also yield changes in ABCI3, this time through gene amplification. Imidazopyridine mode of action is attributed to inhibition of heme detoxification, as evidenced by cellular accumulation and heme fractionation assays. For the copy-number variation-selecting imidazopyridine and quinoline-carboxamide compounds, we find that resistance, manifesting as a biphasic concentration-response curve, can independently be mediated by mutations in the chloroquine resistance transporter PfCRT. These studies reveal the interconnectedness of P. falciparum transporters in overcoming drug pressure in different parasite strains.

The antimalarial efficacy and mechanism of resistance of the novel chemotype DDD01034957.

New antimalarial therapeutics are needed to ensure that malaria cases continue to be driven down, as both emerging parasite resistance to frontline chemotherapies and mosquito resistance to current insecticides threaten control programmes. Plasmodium, the apicomplexan parasite responsible for malaria, causes disease pathology through repeated cycles of invasion and replication within host erythrocytes (the asexual cycle). Antimalarial drugs primarily target this cycle, seeking to reduce parasite burden within the host as fast as possible and to supress recrudescence for as long as possible. Intense phenotypic drug screening efforts have identified a number of promising new antimalarial molecules. Particularly important is the identification of compounds with new modes of action within the parasite to combat existing drug resistance and suitable for formulation of efficacious combination therapies. Here we detail the antimalarial properties of DDD01034957-a novel antimalarial molecule which is fast-acting and potent against drug resistant strains in vitro, shows activity in vivo, and possesses a resistance mechanism linked to the membrane transporter PfABCI3. These data support further medicinal chemistry lead-optimization of DDD01034957 as a novel antimalarial chemical class and provide new insights to further reduce in vivo metabolic clearance.

The antimalarial MMV688533 provides potential for single-dose cures with a high barrier to Plasmodium falciparum parasite resistance.

The emergence and spread of Plasmodium falciparum resistance to first-line antimalarials creates an imperative to identify and develop potent preclinical candidates with distinct modes of action. Here, we report the identification of MMV688533, an acylguanidine that was developed following a whole-cell screen with compounds known to hit high-value targets in human cells. MMV688533 displays fast parasite clearance in vitro and is not cross-resistant with known antimalarials. In a P. falciparum NSG mouse model, MMV688533 displays a long-lasting pharmacokinetic profile and excellent safety. Selection studies reveal a low propensity for resistance, with modest loss of potency mediated by point mutations in PfACG1 and PfEHD. These proteins are implicated in intracellular trafficking, lipid utilization, and endocytosis, suggesting interference with these pathways as a potential mode of action. This preclinical candidate may offer the potential for a single low-dose cure for malaria.

Combining Stage Specificity and Metabolomic Profiling to Advance Antimalarial Drug Discovery.

We report detailed susceptibility profiling of asexual blood stages of the malaria parasite Plasmodium falciparum to clinical and experimental antimalarials, combined with metabolomic fingerprinting. Results revealed a variety of stage-specific and metabolic profiles that differentiated the modes of action of clinical antimalarials including chloroquine, piperaquine, lumefantrine, and mefloquine, and identified late trophozoite-specific peak activity and stage-specific biphasic dose-responses for the mitochondrial inhibitors DSM265 and atovaquone. We also identified experimental antimalarials hitting previously unexplored druggable pathways as reflected by their unique stage specificity and/or metabolic profiles. These included several ring-active compounds, ones affecting hemoglobin catabolism through distinct pathways, and mitochondrial inhibitors with lower propensities for resistance than either DSM265 or atovaquone. This approach, also applicable to other microbes that undergo multiple differentiation steps, provides an effective tool to prioritize compounds for further development within the context of combination therapies.

Inhibition of Resistance-Refractory P. falciparum Kinase PKG Delivers Prophylactic, Blood Stage, and Transmission-Blocking Antiplasmodial Activity.

The search for antimalarial chemotypes with modes of action unrelated to existing drugs has intensified with the recent failure of first-line therapies across Southeast Asia. Here, we show that the trisubstituted imidazole MMV030084 potently inhibits hepatocyte invasion by Plasmodium sporozoites, merozoite egress from asexual blood stage schizonts, and male gamete exflagellation. Metabolomic, phosphoproteomic, and chemoproteomic studies, validated with conditional knockdown parasites, molecular docking, and recombinant kinase assays, identified cGMP-dependent protein kinase (PKG) as the primary target of MMV030084. PKG is known to play essential roles in Plasmodium invasion of and egress from host cells, matching MMV030084's activity profile. Resistance selections and gene editing identified tyrosine kinase-like protein 3 as a low-level resistance mediator for PKG inhibitors, while PKG itself never mutated under pressure. These studies highlight PKG as a resistance-refractory antimalarial target throughout the Plasmodium life cycle and promote MMV030084 as a promising Plasmodium PKG-targeting chemotype.

Mapping the malaria parasite druggable genome by using in vitro evolution and chemogenomics.

Chemogenetic characterization through in vitro evolution combined with whole-genome analysis can identify antimalarial drug targets and drug-resistance genes. We performed a genome analysis of 262 Plasmodium falciparum parasites resistant to 37 diverse compounds. We found 159 gene amplifications and 148 nonsynonymous changes in 83 genes associated with drug-resistance acquisition, where gene amplifications contributed to one-third of resistance acquisition events. Beyond confirming previously identified multidrug-resistance mechanisms, we discovered hitherto unrecognized drug target-inhibitor pairs, including thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This exploration of the P. falciparum resistome and druggable genome will likely guide drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms available to the malaria parasite.

Dioxins and related environmental contaminants increase TDP-43 levels.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a debilitating neurodegenerative condition that is characterized by progressive loss of motor neurons and the accumulation of aggregated TAR DNA Binding Protein-43 (TDP-43, gene: TARDBP). Increasing evidence indicates that environmental factors contribute to the risk of ALS. Dioxins, related planar polychlorinated biphenyls (PCBs), and polycyclic aromatic hydrocarbons (PAHs) are environmental contaminants that activate the aryl hydrocarbon receptor (AHR), a ligand-activated, PAS family transcription factor. Recently, exposure to these toxicants was identified as a risk factor for ALS. METHODS: We examined levels of TDP-43 reporter activity, transcript and protein. Quantification was done using cell lines, induced pluripotent stem cells (iPSCs) and mouse brain. The target samples were treated with AHR agonists, including 6-Formylindolo[3,2-b]carbazole (FICZ, a potential endogenous ligand, 2,3,7,8-tetrachlorodibenzo(p)dioxin, and benzo(a)pyrene, an abundant carcinogen in cigarette smoke). The action of the agonists was inhibited by concomitant addition of AHR antagonists or by AHR-specific shRNA. RESULTS: We now report that AHR agonists induce up to a 3-fold increase in TDP-43 protein in human neuronal cell lines (BE-M17 cells), motor neuron differentiated iPSCs, and in murine brain. Chronic treatment with AHR agonists elicits over 2-fold accumulation of soluble and insoluble TDP-43, primarily because of reduced TDP-43 catabolism. AHR antagonists or AHR knockdown inhibits agonist-induced increases in TDP-43 protein and TARDBP transcription demonstrating that the ligands act through the AHR. CONCLUSIONS: These results provide the first evidence that environmental AHR ligands increase TDP-43, which is the principle pathological protein associated with ALS. These results suggest novel molecular mechanisms through which a variety of prevalent environmental factors might directly contribute to ALS. The widespread distribution of dioxins, PCBs and PAHs is considered to be a risk factor for cancer and autoimmune diseases, but could also be a significant public health concern for ALS.

Hexahydroquinolines are antimalarial candidates with potent blood-stage and transmission-blocking activity.

Antimalarial compounds with dual therapeutic and transmission-blocking activity are desired as high-value partners for combination therapies. Here, we report the identification and characterization of hexahydroquinolines (HHQs) that show low nanomolar potency against both pathogenic and transmissible intra-erythrocytic forms of the malaria parasite Plasmodium falciparum. This activity translates into potent transmission-blocking potential, as shown by in vitro male gamete formation assays and reduced oocyst infection and prevalence in Anopheles mosquitoes. In vivo studies illustrated the ability of lead HHQs to suppress Plasmodium berghei blood-stage parasite proliferation. Resistance selection studies, confirmed by CRISPR-Cas9-based gene editing, identified the digestive vacuole membrane-spanning transporter PfMDR1 (P. falciparum multidrug resistance gene-1) as a determinant of parasite resistance to HHQs. Haemoglobin and haem fractionation assays suggest a mode of action that results in reduced haemozoin levels and might involve inhibition of host haemoglobin uptake into intra-erythrocytic parasites. Furthermore, parasites resistant to HHQs displayed increased susceptibility to several first-line antimalarial drugs, including lumefantrine, confirming that HHQs have a different mode of action to other antimalarials drugs for which PfMDR1 is known to confer resistance. This work evokes therapeutic strategies that combine opposing selective pressures on this parasite transporter as an approach to countering the emergence and transmission of multidrug-resistant P. falciparum malaria.