1-Methyl-1H-pyrazole-5-carboxamide Derivatives Exhibit Unexpected Acute Mammalian Toxicity.

A series of 1-methyl-1H-pyrazole-5-carboxamides were synthesized as potent inhibitors of the parasitic nematode of sheep, Haemonchus contortus. These compounds did not show overt cytotoxicity to a range of mammalian cell lines under standard in vitro culture conditions, had high selectivity indices, and were progressed to an acute toxicity study in a rodent model. Strikingly, acute toxicity was observed in mice. Experiments measuring cellular respiration showed a dose-dependent inhibition of mitochondrial respiration. Under these conditions, potent cytotoxicity was observed for these compounds in rat hepatocytes suggesting that the potent acute mammalian toxicity of this chemotype is most likely associated with respiratory inhibition. In contrast, parasite toxicity was not correlated to acute toxicity or cytotoxicity in respiring cells. This paper highlights the importance of identifying an appropriate in vitro predictor of in vivo toxicity early on in the drug discovery pipeline, in particular assessment for in vitro mitochondrial toxicity.

Human Aurora kinase inhibitor Hesperadin reveals epistatic interaction between Plasmodium falciparum PfArk1 and PfNek1 kinases.

Mitosis has been validated by numerous anti-cancer drugs as being a druggable process, and selective inhibition of parasite proliferation provides an obvious opportunity for therapeutic intervention against malaria. Mitosis is controlled through the interplay between several protein kinases and phosphatases. We show here that inhibitors of human mitotic kinases belonging to the Aurora family inhibit P. falciparum proliferation in vitro with various potencies, and that a genetic selection for mutant parasites resistant to one of the drugs, Hesperadin, identifies a resistance mechanism mediated by a member of a different kinase family, PfNek1 (PF3D7_1228300). Intriguingly, loss of PfNek1 catalytic activity provides protection against drug action. This points to an undescribed functional interaction between Ark and Nek kinases and shows that existing inhibitors can be used to validate additional essential and druggable kinase functions in the parasite.

Interaction of Plasmodium falciparum casein kinase 1 with components of host cell protein trafficking machinery.

A pool of Plasmodium falciparum casein kinase 1 (PfCK1) has been shown to localize to the host red blood cell (RBC) membrane and be secreted to the extracellular medium during trophozoite stage of development. We attempted to identify mechanisms for secretion of PfCK1 and its appearance on the RBC membrane. We found that two host proteins with established functions in membrane trafficking in higher eukaryotes, GTPase-activating protein and Vps9 domain-containing protein 1 (GAPVD1), and Sorting nexin 22, consistently co-purify with PfCK1, suggesting that the parasite utilizes trafficking pathways previously thought to be inactive in RBCs. Furthermore, reciprocal immunoprecipitation experiments with GAPVD1 identified parasite proteins suggestive of a protein recycling pathway hitherto only described in higher eukaryotes. Thus, we have identified components of a trafficking pathway involving parasite proteins that act in concert with host proteins, and which we hypothesize mediates trafficking of PfCK1 to the RBC during infection.

Synthetic Kavalactone Analogues with Increased Potency and Selective Anthelmintic Activity against Larvae of Haemonchus contortus In Vitro.

Kava extract, an aqueous rhizome emulsion of the plant Piper methysticum, has been used for centuries by Pacific Islanders as a ceremonial beverage, and has been sold as an anxiolytic agent for some decades. Kavalactones are a major constituent of kava extract. In a previous investigation, we had identified three kavalactones that inhibit larval development of Haemonchus contortus in an in vitro-bioassay. In the present study, we synthesized two kavalactones, desmethoxyyangonin and yangonin, as well as 17 analogues thereof, and evaluated their anthelmintic activities using the same bioassay as employed previously. Structure activity relationship (SAR) studies showed that a 4-substituent on the pendant aryl ring was required for activity. In particular, compounds with 4-trifluoromethoxy, 4-difluoromethoxy, 4-phenoxy, and 4-N-morpholine substitutions had anthelmintic activities (IC50 values in the range of 1.9 to 8.9 µM) that were greater than either of the parent natural products-desmethoxyyangonin (IC50 of 37.1 µM) and yangonin (IC50 of 15.0 µM). The synthesized analogues did not exhibit toxicity on HepG2 human hepatoma cells in vitro at concentrations of up to 40 µM. These findings confirm the previously-identified kavalactone scaffold as a promising chemotype for new anthelmintics and provide a basis for a detailed SAR investigation focused on developing a novel anthelmintic agent.

Synthesis and structure-activity relationship study of pyrrolidine-oxadiazoles as anthelmintics against Haemonchus contortus.

Parasitic roundworms (nematodes) are significant pathogens of humans and animals and cause substantive socioeconomic losses due to the diseases that they cause. The control of nematodes in livestock animals relies heavily on the use of anthelmintic drugs. However, their extensive use has led to a widespread problem of drug resistance in these worms. Thus, the discovery and development of novel chemical entities for the treatment of parasitic worms of humans and animals is needed. Herein, we describe our medicinal chemistry optimization efforts of a phenotypic hit against Haemonchus contortus based on a pyrrolidine-oxadiazole scaffold. This led to the identification of compounds with potent inhibitory activities (IC50 = 0.78-22.4 µM) on the motility and development of parasitic stages of H. contortus, and which were found to be highly selective in a mammalian cell counter-screen. These compounds could be used as suitable chemical tools for drug target identification or as lead compounds for further optimization.

Identification of Fromiamycalin and Halaminol A from Australian Marine Sponge Extracts with Anthelmintic Activity against Haemonchus contortus.

There is an urgent need to discover and develop new anthelmintics for the treatment of parasitic nematodes of veterinary importance to circumvent challenges linked to drug resistant parasites. Being one of the most diverse natural ecosystems, the marine environment represents a rich resource of novel chemical entities. This study investigated 2000 extracts from marine invertebrates, collected from Australian waters, for anthelmintic activity. Using a well-established in vitro bioassay, these extracts were screened for nematocidal activity against Haemonchus contortus-a socioeconomically important parasitic nematode of livestock animals. Extracts (designated Mu-1, Ha-1 and Ha-2) from two marine sponges (Monanchora unguiculata and Haliclona sp.) each significantly affected larvae of H. contortus. Individual extracts displayed a dose-dependent inhibition of both the motility of exsheathed third-stage larvae (xL3s) and the development of xL3s to fourth-stage larvae (L4s). Active fractions in each of the three extracts were identified using bioassay-guided fractionation. From the active fractions from Monanchora unguiculata, a known pentacyclic guanidine alkaloid, fromiamycalin (1), was purified. This alkaloid was shown to be a moderately potent inhibitor of L4 development (half-maximum inhibitory concentration (IC50) = 26.6 ± 0.74 µM) and L4 motility (IC50 = 39.4 ± 4.83 µM), although it had a relatively low potency at inhibiting of xL3 motility (IC50 ≥ 100 µM). Investigation of the active fractions from the two Haliclona collections led to identification of a mixture of amino alcohol lipids, and, subsequently, a known natural product halaminol A (5). Anthelmintic profiling showed that 5 had limited potency at inhibiting larval development and motility. These data indicate that fromiamycalin, other related pentacyclic guanidine alkaloids and/or halaminols could have potential as anthelmintics following future medicinal chemistry efforts.

Anti-Helicobacter pylori activity of ethoxzolamide.

Ethoxzolamide (EZA), acetazolamide, and methazolamide are clinically used sulphonamide drugs designed to treat non-bacteria-related illnesses (e.g. glaucoma), but they also show antimicrobial activity against the gastric pathogen Helicobacter pylori. EZA showed the highest activity, and was effective against clinical isolates resistant to metronidazole, clarithromycin, and/or amoxicillin, suggesting that EZA kills H. pylori via mechanisms different from that of these antibiotics. The frequency of single-step spontaneous resistance acquisition by H. pylori was less than 5 × 10-9, showing that resistance to EZA does not develop easily. Resistance was associated with mutations in three genes, including the one that encodes undecaprenyl pyrophosphate synthase, a known target of sulphonamides. The data indicate that EZA impacts multiple targets in killing H. pylori. Our findings suggest that developing the approved anti-glaucoma drug EZA into a more effective anti-H. pylori agent may offer a faster and cost-effective route towards new antimicrobials with a novel mechanism of action.

An appraisal of natural products active against parasitic nematodes of animals.

Here, the scientific and patent literature on the activities of purified natural compounds has been reviewed, with the aim of assessing their suitability as anthelmintic drug discovery starting points. Only compounds described as active against parasitic nematodes of animals or against the model nematode Caenorhabditis elegans have been analysed. Scientific articles published since 2010 and patents granted from 2000, both inclusive, have been included in this analysis. The results show a scarcity of novel chemical structures, a limited follow-up of compounds disclosed before 2010 and a bias towards the screening of plant products, almost to the exclusion of other sources, when microbial extracts have, historically, provided most starting points for anti-infective drugs. All plant products published in this period were previously known, alerting to the high re-discovery rates of a limited number of chemical classes from this source. The most promising compounds described in the literature reviewed here, namely the linear nemadectin-derivatives, are novel and of bacterial origin. Patented but otherwise unpublished spiroketal structures also appear as interesting scaffolds for future development. The patent literature confirmed that it is possible to patent derivatives of previously known products, making them valid starting points for translational research.

Novel 1-Methyl-1 H-pyrazole-5-carboxamide Derivatives with Potent Anthelmintic Activity.

A phenotypic screen of two different libraries of small molecules against the motility and development of the parasitic nematode Haemonchus contortus led to the identification of two 1-methyl-1 H-pyrazole-5-carboxamide derivatives. Medicinal chemistry optimization targeted modifications of the left-hand side, middle section, and right-hand side of the hybrid structure of these two hits to elucidate the structure-activity relationship (SAR). Initial SAR around these hits allowed for the iterative and directed assembly of a focused set of 30 analogues of their hybrid structure. Compounds 10, 17, 20, and 22 were identified as the most potent compounds, inhibiting the development of the fourth larval (L4) stage of H. contortus at sub-nanomolar potencies while displaying strong selectivity toward the parasite when tested in vitro against the human MCF10A cell line. In addition, compounds 9 and 27 showed promising activity against a panel of other parasitic nematodes, including hookworms and whipworms.

Selected α-pyrones from the plants Cryptocarya novoguineensis (Lauraceae) and Piper methysticum (Piperaceae) with activity against Haemonchus contortus in vitro.

Due to the widespread occurrence and spread of anthelmintic resistance, there is a need to develop new drugs against resistant parasitic nematodes of livestock animals. The Nobel Prize-winning discovery and development of the anti-parasitic drugs avermectin and artemisinin has renewed the interest in exploring natural products as anthelmintics. In the present study, we screened 7500 plant extracts for in vitro-activity against the barber's pole worm, Haemonchus contortus, a highly significant pathogen of ruminants. The anthelmintic extracts from two plants, Cryptocarya novoguineensis and Piper methysticum, were fractionated by high-performance liquid chromatography (HPLC). Subsequently, compounds were purified from fractions with significant biological activity. Four α-pyrones, namely goniothalamin (GNT), dihydrokavain (DHK), desmethoxyyangonin (DMY) and yangonin (YGN), were purified from fractions from the two plants, GNT from C. novoguineensis, and DHK, DMY and YGN (= kavalactones) from P. methysticum. The three kavalactones induced a lethal, eviscerated (Evi) phenotype in treated exsheathed third-stage larvae (xL3s), and DMY and YGN had moderate potencies (IC50 values of 31.7 ±â€¯0.23 µM and 23.7 ±â€¯2.05 µM, respectively) at inhibiting the development of xL3s to fourth-stage larvae (L4s). Although GNT had limited potency (IC50 of 200-300 µM) at inhibiting L4 development, it was the only compound that reduced L4 motility (IC50 of 6.25-12.50 µM). The compounds purified from each plant affected H. contortus in an irreversible manner. These findings suggest that structure-activity relationship studies of α-pyrones should be pursued to assess their potential as anthelmintics.

Tetrahydroquinoxalines induce a lethal evisceration phenotype in Haemonchus contortus in vitro.

In the present study, the anthelmintic activity of a human tyrosine kinase inhibitor, AG-1295, and 14 related tetrahydroquinoxaline analogues against Haemonchus contortus was explored. These compounds were screened against parasitic larvae - exsheathed third-stage (xL3) and fourth-stage (L4) - using a whole-organism screening assay. All compounds were shown to have inhibitory effects on larval motility, development and growth, and induced evisceration through the excretory pore in xL3s. The estimated IC50 values ranged from 3.5 to 52.0 µM for inhibition of larval motility or development. Cytotoxicity IC50 against human MCF10A cells was generally higher than 50 µM. Microscopic studies revealed that this eviscerated (Evi) phenotype occurs rapidly (<20 min) and relates to a protrusion of internal tissues and organs (evisceration) through the excretory pore in xL3s; severe pathological damage in L4s as well as a suppression of larval growth in both stages were also observed. Using a relatively low concentration (12.5 µM) of compound m10, it was established that the inhibitor has to be present for a relatively short time (between 30 h and 42 h) during in vitro development from xL3 to L4, to induce the Evi phenotype. Increasing external osmotic pressure prevented evisceration and moulting, and xL3s remained unaffected by the test compound. These results point to a mode of action involving a dysregulation of morphogenetic processes during a critical time-frame, in agreement with the expected behaviour of a tyrosine kinase inhibitor, and suggest potential for development of this compound class as nematocidal drugs.

Structure-Activity Relationship Studies of Tolfenpyrad Reveal Subnanomolar Inhibitors of Haemonchus contortus Development.

Recently, we have discovered that the registered pesticide, tolfenpyrad, unexpectedly and potently inhibits the development of the L4 larval stage of the parasitic nematode Haemonchus contortus with an IC50 value of 0.03 µM while displaying good selectivity, with an IC50 of 37.9 µM for cytotoxicity. As a promising molecular template for medicinal chemistry optimization, we undertook anthelmintic structure-activity relationships for this chemical. Modifications of the left-hand side (LHS), right-hand side (RHS), and middle section of the scaffold were explored to produce a set of 57 analogues. Analogues 25, 29, and 33 were shown to be the most potent compounds of the series, with IC50 values at a subnanomolar level of potency against the chemotherapeutically relevant fourth larval (L4) stage of H. contortus. Selected compounds from the series also showed promising activity against a panel of other different parasitic nematodes, such as hookworms and whipworms.

Optimization of Novel 1-Methyl-1 H-Pyrazole-5-carboxamides Leads to High Potency Larval Development Inhibitors of the Barber's Pole Worm.

A phenotypic screen of a diverse library of small molecules for inhibition of the development of larvae of the parasitic nematode Haemonchus contortus led to the identification of a 1-methyl-1 H-pyrazole-5-carboxamide derivative with an IC50 of 0.29 µM. Medicinal chemistry optimization targeted modifications on the left-hand side (LHS), middle section, and right-hand side (RHS) of the scaffold in order to elucidate the structure-activity relationship (SAR). Strong SAR allowed for the iterative and directed assembly of a focus set of 64 analogues, from which compound 60 was identified as the most potent compound, inhibiting the development of the fourth larval (L4) stage with an IC50 of 0.01 µM. In contrast, only 18% inhibition of the mammary epithelial cell line MCF10A viability was observed, even at concentrations as high as 50 µM.

Arylpyrrole and fipronil analogues that inhibit the motility and/or development of Haemonchus contortus in vitro.

Due to widespread drug resistance in parasitic nematodes, there is a need to develop new anthelmintics. Given the cost and time involved in developing a new drug, the repurposing of known chemicals can be a promising, alternative approach. In this context, we tested a library (n = 600) of natural product-inspired pesticide analogues against exsheathed third stage-larvae (xL3s) of Haemonchus contortus (barber's pole worm) using a whole-organism, phenotypic screening technique that measures the inhibition of motility and development in treated larvae. In the primary screen, we identified 32 active analogues derived from chemical scaffolds of arylpyrrole or fipronil. The seven most promising compounds, selected based on their anthelmintic activity and/or limited cytotoxicity, are arylpyrroles that reduced the motility of fourth-stage larvae (L4s) with significant potency (IC50 values ranged from 0.04 ±â€¯0.01 µM to 4.25 ±â€¯0.82 µM, and selectivity indices ranged from 10.6 to 412.5). Since the parent structures of the active compounds are uncouplers of oxidative phosphorylation, we tested the effect of selected analogues on oxygen consumption in xL3s using the Seahorse XF24 flux analyser. Larvae treated with the test compounds showed a significant increase in oxygen consumption compared with the untreated control, demonstrating their uncoupling activity. Overall, the results of the present study have identified natural product-derived molecules that are worth considering for chemical optimisation as anthelmintic drug leads.

Functional screening of selective mitochondrial inhibitors of Plasmodium.

Phenotypic screening has produced most of the new chemical entities currently in clinical development for malaria, plus many lead compounds active against Plasmodium falciparum asexual stages. However, lack of knowledge about the mode of action of these compounds delays and may even hamper their future development. Identifying the mode of action of the inhibitors greatly helps to prioritise compounds for further development as novel antimalarials. Here we describe a whole-cell method to detect inhibitors of the mitochondrial electron transport chain, using oxygen consumption as high throughput readout in 384-well plate format. The usefulness of the method has been confirmed with the Tres Cantos Antimalarial Compound Set (TCAMS). The assay identified 124 respiratory inhibitors in TCAMS, seven of which were novel anti-plasmodial chemical structures never before described as mitochondrial inhibitors.

Parasites under the Spotlight: Applications of Vibrational Spectroscopy to Malaria Research.

New technologies to diagnose malaria at high sensitivity and specificity are urgently needed in the developing world where the disease continues to pose a huge burden on society. Infrared and Raman spectroscopy-based diagnostic methods have a number of advantages compared with other diagnostic tests currently on the market. These include high sensitivity and specificity for detecting low levels of parasitemia along with ease of use and portability. Here, we review the application of vibrational spectroscopic techniques for monitoring and detecting malaria infection. We discuss the role of vibrational (infrared and Raman) spectroscopy in understanding the processes of parasite biology and its application to the study of interactions with antimalarial drugs. The distinct molecular phenotype that characterizes malaria infection and the high sensitivity enabling detection of low parasite densities provides a genuine opportunity for vibrational spectroscopy to become a front-line tool in the elimination of this deadly disease and provide molecular insights into the chemistry of this unique organism.

Multispectral Atomic Force Microscopy-Infrared Nano-Imaging of Malaria Infected Red Blood Cells.

Atomic force microscopy-infrared (AFM-IR) spectroscopy is a powerful new technique that can be applied to study molecular composition of cells and tissues at the nanoscale. AFM-IR maps are acquired using a single wavenumber value: they show either the absorbance plotted against a single wavenumber value or a ratio of two absorbance values. Here, we implement multivariate image analysis to generate multivariate AFM-IR maps and use this approach to resolve subcellular structural information in red blood cells infected with Plasmodium falciparum at different stages of development. This was achieved by converting the discrete spectral points into a multispectral line spectrum prior to multivariate image reconstruction. The approach was used to generate compositional maps of subcellular structures in the parasites, including the food vacuole, lipid inclusions, and the nucleus, on the basis of the intensity of hemozoin, hemoglobin, lipid, and DNA IR marker bands, respectively. Confocal Raman spectroscopy was used to validate the presence of hemozoin in the regions identified by the AFM-IR technique. The high spatial resolution of AFM-IR combined with hyperspectral modeling enables the direct detection of subcellular components, without the need for cell sectioning or immunological/biochemical staining. Multispectral-AFM-IR thus has the capacity to probe the phenotype of the malaria parasite during its intraerythrocytic development. This enables novel approaches to studying the mode of action of antimalarial drugs and the phenotypes of drug-resistant parasites, thus contributing to the development of diagnostic and control measures.

Malaria Parasites in a Hungry Host: Kinases and Host Caloric Restriction Brought Together.

A study recently published in Nature links reduced calorie nutritional intake of host mice with (i) reduced severity of cerebral malaria, (ii) decreased parasitemia, and (iii) activation of a nutrient-sensing pathway that regulates the parasite's proliferation rate. Here, we discuss these findings in the context of human malaria pathology and Plasmodium kinomics.

Antitubercular drugs for an old target: GSK693 as a promising InhA direct inhibitor.

Despite being one of the first antitubercular agents identified, isoniazid (INH) is still the most prescribed drug for prophylaxis and tuberculosis (TB) treatment and, together with rifampicin, the pillars of current chemotherapy. A high percentage of isoniazid resistance is linked to mutations in the pro-drug activating enzyme KatG, so the discovery of direct inhibitors (DI) of the enoyl-ACP reductase (InhA) has been pursued by many groups leading to the identification of different enzyme inhibitors, active against Mycobacterium tuberculosis (Mtb), but with poor physicochemical properties to be considered as preclinical candidates. Here, we present a series of InhA DI active against multidrug (MDR) and extensively (XDR) drug-resistant clinical isolates as well as in TB murine models when orally dosed that can be a promising foundation for a future treatment.

The ins and outs of phosphosignalling in Plasmodium: Parasite regulation and host cell manipulation.

Signal transduction and kinomics have been rapidly expanding areas of investigation within the malaria research field. Here, we provide an overview of phosphosignalling pathways that operate in all stages of the Plasmodium life cycle. We review signalling pathways in the parasite itself, in the cells it invades, and in other cells of the vertebrate host with which it interacts. We also discuss the potential of these pathways as novel targets for antimalarial intervention.