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Summary 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.

Significance There is an urgent need for alternative antimalarials with the emergence of artemisinin-resistant malaria parasites. Blocking sugar uptake in Plasmodium falciparum by selectively inhibiting the hexose transporter P. falciparum hexose transporter 1 (PfHT1) kills the blood-stage parasites without affecting the host cells, making PfHT1 a promising therapeutic target. Here, we report the development of a series of small-molecule inhibitors that simultaneously target the orthosteric and the allosteric binding sites of PfHT1. These inhibitors all exhibit selective potency on the P. falciparum strains over human cell lines. Our findings establish the basis for the rational design of next-generation antimalarial drugs., Artemisinin-resistant malaria parasites have emerged and have been spreading, posing a significant public health challenge. Antimalarial drugs with novel mechanisms of action are therefore urgently needed. In this report, we exploit a “selective starvation” strategy by inhibiting Plasmodium falciparum hexose transporter 1 (PfHT1), the sole hexose transporter in P. falciparum, over human glucose transporter 1 (hGLUT1), providing an alternative approach to fight against multidrug-resistant malaria parasites. The crystal structure of hGLUT3, which shares 80% sequence similarity with hGLUT1, was resolved in complex with C3361, a moderate PfHT1-specific inhibitor, at 2.3-Å resolution. Structural comparison between the present hGLUT3-C3361 and our previously reported PfHT1-C3361 confirmed the unique inhibitor binding-induced pocket in PfHT1. We then designed small molecules to simultaneously block the orthosteric and allosteric pockets of PfHT1. Through extensive structure–activity relationship studies, the TH-PF series was identified to selectively inhibit PfHT1 over hGLUT1 and potent against multiple strains of the blood-stage P. falciparum. Our findings shed light on the next-generation chemotherapeutics with a paradigm-shifting structure-based design strategy to simultaneously target the orthosteric and allosteric sites of a transporter.

Artemisinin-resistant malaria parasites have emerged and been spreading, posing a significant public health challenge. Anti-malarial drugs with novel mechanisms of action are therefore urgently needed. In this report, we exploit a “selective starvation” strategy by selectively inhibiting Plasmodium falciparum hexose transporter 1 (PfHT1), the sole hexose transporter in Plasmodium falciparum, over human glucose transporter 1 (hGLUT1), providing an alternative approach to fight against multidrug-resistant malaria parasites. Comparison of the crystal structures of human GLUT3 and PfHT1 bound to C3361, a PfHT1-specific moderate inhibitor, revealed an inhibitor binding-induced pocket that presented a promising druggable site. We thereby designed small-molecules to simultaneously block the orthosteric and allosteric pockets of PfHT1. Through extensive structure-activity relationship (SAR) studies, the TH-PF series was identified to selectively inhibit PfHT1 over GLUT1 and potent against multiple strains of the blood-stage P. falciparum. Our findings shed light on the next-generation chemotherapeutics with a paradigm-shifting structure-based design strategy to simultaneously targeting the orthosteric and allosteric sites of a transporter.Significance statementBlocking sugar uptake in P. falciparum by selectively inhibiting the hexose transporter PfHT1 kills the blood-stage parasites without affecting the host cells, indicating PfHT1 as a promising therapeutic target. Here, we report the development of novel small-molecule inhibitors that are selectively potent to the malaria parasites over human cell lines by simultaneously targeting the orthosteric and the allosteric binding sites of PfHT1. Our findings established the basis for the rational design of next-generation anti-malarial drugs.

Gli transcription factors within the Hedgehog (Hh) signaling pathway direct key events in mammalian development and promote a number of human cancers. Current therapies for Gli-driven tumors target Smoothened (SMO), a G protein-coupled receptor-like protein that functions upstream in the Hh pathway. Although these drugs can have remarkable clinical efficacy, mutations in SMO and downstream Hh pathway components frequently lead to chemoresistance. In principle, therapies that act at the level of Gli proteins, through direct or indirect mechanisms, would be more efficacious. We therefore conducted a screen of 325,120 compounds for their ability to block the constitutive Gli activity induced by loss of Suppressor of Fused (SUFU), a scaffolding protein that directly inhibits Gli function. Our studies reveal a family of bicyclic imidazolium derivatives that can inhibit Gli-dependent transcription without affecting the ciliary trafficking or proteolytic processing of these transcription factors. We anticipate that these chemical antagonists will be valuable tools for investigating the mechanisms of Gli regulation and developing new strategies for targeting Gli-driven cancers.

The synthesis of a 2-methyl-substituted analogue of the natural product, neopeltolide, is reported in an effort to analyze the importance of molecular conformation and ligand-target interactions in relation to biological activity. The methyl substitution was incorporated via highly diastereoselective ester enolate alkylation of a late-stage intermediate. Coupling of the oxazole sidechain provided 2-methyl-neopeltolide and synthetic neopeltolide via total synthesis. The substitution was shown to maintain the conformational preferences of its biologically active parent compound through computer modeling and NMR studies. Both compounds were shown to be potential antimalarial compounds through the inhibition of mitochondrial respiration in P. falciparum parasites.

Resistance has developed in Plasmodium malaria parasites to every antimalarial drug in clinical use, prompting the need to characterize the pathways mediating resistance. Here, we report a framework for assessing development of resistance of Plasmodium falciparum to new antimalarial therapeutics. We investigated development of resistance by P. falciparum to the dihydroorotate dehydrogenase (DHODH) inhibitors DSM265 and DSM267 in tissue culture and in a mouse model of P. falciparum infection. We found that resistance to these drugs arose rapidly both in vitro and in vivo. We identified 13 point mutations mediating resistance in the parasite DHODH in vitro that overlapped with the DHODH mutations that arose in the mouse infection model. Mutations in DHODH conferred increased resistance (ranging from 2- to ~400-fold) to DHODH inhibitors in P. falciparum in vitro and in vivo. We further demonstrated that the drug-resistant parasites carrying the C276Y mutation had mitochondrial energetics comparable to the wild-type parasite and also retained their fitness in competitive growth experiments. Our data suggest that in vitro selection of drug-resistant P. falciparum can predict development of resistance in a mouse model of malaria infection.

Given that resistance to all drugs in clinical use has arisen, discovery of new antimalarial drug targets is eagerly anticipated. The Plasmodium mitochondrion has been considered a promising drug target largely based on its significant divergence from the host organelle as well as its involvement in ATP production and pyrimidine biosynthesis. However, the functions of Plasmodium mitochondrial protein complexes and associated metabolic pathways are not fully characterized. Here, we report the development of novel and robust bioenergetic assay protocols for Plasmodium falciparum asexual parasites utilizing a Seahorse Bioscience XFe24 Extracellular Flux Analyzer. These protocols allowed us to simultaneously assess the direct effects of metabolites and inhibitors on mitochondrial respiration and glycolytic activity in real-time with the readout of oxygen consumption rate and extracellular acidification rate. Using saponin-freed parasites at the schizont stage, we found that succinate, malate, glycerol-3-phosphate, and glutamate, but not pyruvate, were able to increase the oxygen consumption rate and that glycerol-3-phosphate dehydrogenase had the largest potential as an electron donor among tested mitochondrial dehydrogenases. Furthermore, we revealed the presence of a glucose-regulated metabolic shift between oxidative phosphorylation and glycolysis. We measured proton leak and reserve capacity and found bioenergetic evidence for oxidative phosphorylation in erythrocytic stage parasites but at a level much lower than that observed in mammalian cells. Lastly, we developed an assay platform for target identification and mode of action studies of mitochondria-targeting antimalarials. This study provides new insights into the bioenergetics and metabolomics of the Plasmodium mitochondria.

Microbial resistance to chemotherapy has caused countless deaths where malaria is endemic. Chemotherapy may fail either due to pre-existing resistance or evolution of drug-resistant parasites. Here we use a diverse set of antimalarial compounds to investigate the acquisition of drug resistance and the degree of cross-resistance against common resistance alleles. We assess cross-resistance using a set of 15 parasite lines carrying resistance-conferring alleles in pfatp4, cytochrome bc1, pfcarl, pfdhod, pfcrt, pfmdr, pfdhfr, cytoplasmic prolyl t-RNA synthetase or hsp90. Subsequently, we assess whether resistant parasites can be obtained after several rounds of drug selection. Twenty-three of the 48 in vitro selections result in resistant parasites, with time to resistance onset ranging from 15 to 300 days. Our data indicate that pre-existing resistance may not be a major hurdle for novel-target antimalarial candidates, and focusing our attention on fast-killing compounds may result in a slower onset of clinical resistance., It is unclear whether new antimalarial compounds may rapidly lose effectiveness in the field because of parasite resistance. Here, Corey et al. investigate the acquisition of drug resistance and the extent to which common resistance mechanisms decrease susceptibility to a diverse set of 50 antimalarial compounds.

Antimalarial drugs have thus far been chiefly derived from two sources-natural products and synthetic drug-like compounds. Here we investigate whether antimalarial agents with novel mechanisms of action could be discovered using a diverse collection of synthetic compounds that have three-dimensional features reminiscent of natural products and are underrepresented in typical screening collections. We report the identification of such compounds with both previously reported and undescribed mechanisms of action, including a series of bicyclic azetidines that inhibit a new antimalarial target, phenylalanyl-tRNA synthetase. These molecules are curative in mice at a single, low dose and show activity against all parasite life stages in multiple in vivo efficacy models. Our findings identify bicyclic azetidines with the potential to both cure and prevent transmission of the disease as well as protect at-risk populations with a single oral dose, highlighting the strength of diversity-oriented synthesis in revealing promising therapeutic targets.

Summary Plasmodium species, the causative agent of malaria, rely on glucose for energy supply during blood stage. Inhibition of glucose uptake thus represents a potential strategy for the development of antimalarial drugs. Here, we present the crystal structures of PfHT1, the sole hexose transporter in the genome of Plasmodium species, at resolutions of 2.6 A in complex with D-glucose and 3.7 A with a moderately selective inhibitor, C3361. Although both structures exhibit occluded conformations, binding of C3361 induces marked rearrangements that result in an additional pocket. This inhibitor-binding-induced pocket presents an opportunity for the rational design of PfHT1-specific inhibitors. Among our designed C3361 derivatives, several exhibited improved inhibition of PfHT1 and cellular potency against P. falciparum, with excellent selectivity to human GLUT1. These findings serve as a proof of concept for the development of the next-generation antimalarial chemotherapeutics by simultaneously targeting the orthosteric and allosteric sites of PfHT1.

To discover leads for next-generation chemoprotective antimalarial drugs,we tested more than 500,000 compounds for their ability to inhibit liver-stage development of luciferase-expressing Plasmodium spp. parasites (681 compounds showed a half-maximal inhibitory concentration of less than 1micromolar).Cluster analysis identified potent and previously unreported scaffold families as well as other series previously associated with chemoprophylaxis. Further testing through multiple phenotypic assays that predict stage-specific and multispecies antimalarial activity distinguished compound classes that are likely to provide symptomatic relief by reducing asexual blood-stage parasitemia from those which are likely to only prevent malaria. Target identification by using functional assays, in vitro evolution, or metabolic profiling revealed 58 mitochondrial inhibitors but also many chemotypes possibly with previously unidentified mechanisms of action. INTRODUCTION Malaria remains a devastating disease, affecting 216 million people annually, with 445,000 deaths occurring primarily in children under 5 years old. Malaria treatment relies primarily on drugs that target the diseasecausing asexual blood stages (ABS) of Plasmodium parasites, the organisms responsible for human malaria. Whereas travelers may rely on shortterm daily chemoprotective drugs, those living in endemic regions require long-termmalaria protection such as insecticide-treated nets (ITNs) and vector control. However, ITNs do not fully shield individuals from malaria, may lose potency with time, and can be bulky and difficult to use. Another concern is that mosquitosmay become resistant to the active insecticides that are used in ITNs and vector control. RATIONALE As the possibility of malaria elimination becomesmore tangible, the ideal antimalarial medicine profile should include chemoprotection. Chemoprotectivemedicines typically work against the exoerythrocytic parasite forms that invade and develop in the liver and are responsible for the earliest asymptomatic stage of the infection. Such medicines could be formulated to provide long-acting prophylaxis, safeguarding individuals that are living near or traveling to areas that have been cleared of parasites. Long-acting chemoprotection in endemic regions could also greatly reduce circulating parasite numbersandpotentially replace a vaccine in an elimination campaign. Although millions of compounds have been screened for activity against parasiteABS, and some have been subsequently tested for potential prophylactic activity, large-scale searches that beginwith prophylactic activity have not been performed because of the complexity of the assay: This assay requires the production of infected laboratory-rearedmosquitoes and hand-dissection of the sporozoiteinfected salivary glands frommosquito thoraxes. A Plasmodium vivax liver-stage schizont on a lawn of hepatocytes. The parasite schizont has been stained with antibodies to parasite HSP70 (red) and UIS4 (yellow). Cell (parasite and hepatoma) nuclei are shown in blue. This study identifies compounds that can prevent the development of these liver-stage parasites and may function as chemoprotective drugs for malaria. RESULTS To discover leads for next-generation chemoprotective antimalarial drugs, we used luciferase-expressing Plasmodium spp. parasites, dissected from more than a million mosquitoes over a 2-year period, to test more than 500,000 compounds for their ability to inhibit liver-stage development of malaria (681 compounds showed a half-maximal inhibitory concentration of

Dissecting Plasmodium drug resistance Malaria is a deadly disease with no effective vaccine. Physicians thus depend on antimalarial drugs to save lives, but such compounds are often rendered ineffective when parasites evolve resistance. Cowell et al. systematically studied patterns of Plasmodium falciparum genome evolution by analyzing the sequences of clones that were resistant to diverse antimalarial compounds across the P. falciparum life cycle (see the Perspective by Carlton). The findings identify hitherto unrecognized drug targets and drug-resistance genes, as well as additional alleles in known drug-resistance genes. Science , this issue p. 191 ; see also p. 159

Cytoplasmic dyneins 1 and 2 are related members of the AAA+ superfamily (ATPases associated with diverse cellular activities) that function as the predominant minus-end-directed microtubule motors in eukaryotic cells. Dynein 1 controls mitotic spindle assembly, organelle movement, axonal transport, and other cytosolic, microtubule-guided processes, whereas dynein 2 mediates retrograde trafficking within motile and primary cilia. Small-molecule inhibitors are important tools for investigating motor protein-dependent mechanisms, and ciliobrevins were recently discovered as the first dynein-specific chemical antagonists. Here, we demonstrate that ciliobrevins directly target the heavy chains of both dynein isoforms and explore the structure-activity landscape of these inhibitors in vitro and in cells. In addition to identifying chemical motifs that are essential for dynein blockade, we have discovered analogs with increased potency and dynein 2 selectivity. These antagonists effectively disrupt Hedgehog signaling, intraflagellar transport, and ciliogenesis, making them useful probes of these and other cytoplasmic dynein 2-dependent cellular processes.

Chemogenetic characterization throughin vitroevolution combined with whole genome analysis is a powerful tool to discover novel antimalarial drug targets and identify drug resistance genes. Our comprehensive genome analysis of 262Plasmodium falciparumparasites treated with 37 diverse compounds reveals how the parasite evolves to evade the action of small molecule growth inhibitors. This detailed data set revealed 159 gene amplifications and 148 nonsynonymous changes in 83 genes which developed during resistance acquisition. Using a new algorithm, we show that gene amplifications contribute to 1/3 of drug resistance acquisition events. In addition to confirming known multidrug resistance mechanisms, we discovered novel multidrug resistance genes. Furthermore, we identified promising new drug target-inhibitor pairs to advance the malaria elimination campaign, including: thymidylate synthase and a benzoquinazolinone, farnesyltransferase and a pyrimidinedione, and a dipeptidylpeptidase and an arylurea. This deep exploration of theP. falciparumresistome and drug-able genome will guide future drug discovery and structural biology efforts, while also advancing our understanding of resistance mechanisms of the deadliest malaria parasite.One Sentence SummaryWhole genome sequencing reveals howPlasmodium falciparumevolves resistance to diverse compounds and identifies new antimalarial drug targets.

Inflammation disrupts tissue architecture and function, thereby contributing to the pathogenesis of diverse diseases; the signals that promote or restrict tissue inflammation thus represent potential targets for therapeutic intervention. Here, we report that genetic or pharmacologic Hedgehog pathway inhibition intensifies colon inflammation (colitis) in mice. Conversely, genetic augmentation of Hedgehog response and systemic small-molecule Hedgehog pathway activation potently ameliorate colitis and restrain initiation and progression of colitis-induced adenocarcinoma. Within the colon, the Hedgehog protein signal does not act directly on the epithelium itself, but on underlying stromal cells to induce expression of IL-10, an immune-modulatory cytokine long known to suppress inflammatory intestinal damage. IL-10 function is required for the full protective effect of small-molecule Hedgehog pathway activation in colitis; this pharmacologic augmentation of Hedgehog pathway activity and stromal IL-10 expression are associated with increased presence of CD4+Foxp3+ regulatory T cells. We thus identify stromal cells as cellular coordinators of colon inflammation and suggest their pharmacologic manipulation as a potential means to treat colitis.

Pancreatic ductal adenocarcinoma (PDA) is the most lethal of common human malignancies, with no truly effective therapies for advanced disease. Preclinical studies have suggested a therapeutic benefit of targeting the Hedgehog (Hh) signaling pathway, which is activated throughout the course of PDA progression by expression of Hh ligands in the neoplastic epithelium and paracrine response in the stromal fibroblasts. Clinical trials to test this possibility, however, have yielded disappointing results. To further investigate the role of Hh signaling in the formation of PDA and its precursor lesion, pancreatic intraepithelial neoplasia (PanIN), we examined the effects of genetic or pharmacologic inhibition of Hh pathway activity in three distinct genetically engineered mouse models and found that Hh pathway inhibition accelerates rather than delays progression of oncogenic Kras-driven disease. Notably, pharmacologic inhibition of Hh pathway activity affected the balance between epithelial and stromal elements, suppressing stromal desmoplasia but also causing accelerated growth of the PanIN epithelium. In striking contrast, pathway activation using a small molecule agonist caused stromal hyperplasia and reduced epithelial proliferation. These results indicate that stromal response to Hh signaling is protective against PDA and that pharmacologic activation of pathway response can slow tumorigenesis. Our results provide evidence for a restraining role of stroma in PDA progression, suggesting an explanation for the failure of Hh inhibitors in clinical trials and pointing to the possibility of a novel type of therapeutic intervention.