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A series of pleuromutilins modified by introduction of a boron-containing heterocycle on C(14) of the polycyclic core are described. These analogs were found to be potent anti- Wolbachia antibiotics and, as such, may be useful in the treatment of filarial infections caused by Onchocerca volvulus, resulting in Onchocerciasis or river blindness, or Wuchereria bancrofti and Brugia malayi and related parasitic nematodes resulting in lymphatic filariasis. These two important neglected tropical diseases disproportionately impact patients in the developing world. The lead preclinical candidate compound containing 7-fluoro-6-oxybenzoxaborole (15, AN11251) was shown to have good in vitro anti- Wolbachia activity and physicochemical and pharmacokinetic properties providing high exposure in plasma. The lead was effective in reducing the Wolbachia load in filarial worms following oral administration to mice.

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 1 micromolar). 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., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

The chemical diversity and known safety profiles of drugs previously tested in humans make them a valuable set of compounds to explore potential therapeutic utility in indications outside those originally targeted, especially neglected tropical diseases. This practice of "drug repurposing" has become commonplace in academic and other nonprofit drug-discovery efforts, with the appeal that significantly less time and resources are required to advance a candidate into the clinic. Here, we report a comprehensive open-access, drug repositioning screening set of 12,000 compounds (termed ReFRAME; Repurposing, Focused Rescue, and Accelerated Medchem) that was assembled by combining three widely used commercial drug competitive intelligence databases (Clarivate Integrity, GVK Excelra GoStar, and Citeline Pharmaprojects), together with extensive patent mining of small molecules that have been dosed in humans. To date, 12,000 compounds (∼80% of compounds identified from data mining) have been purchased or synthesized and subsequently plated for screening. To exemplify its utility, this collection was screened against Cryptosporidium spp., a major cause of childhood diarrhea in the developing world, and two active compounds previously tested in humans for other therapeutic indications were identified. Both compounds, VB-201 and a structurally related analog of ASP-7962, were subsequently shown to be efficacious in animal models of Cryptosporidium infection at clinically relevant doses, based on available human doses. In addition, an open-access data portal (https://reframedb.org) has been developed to share ReFRAME screen hits to encourage additional follow-up and maximize the impact of the ReFRAME screening collection., Competing Interests: The authors declare no conflict of interest., (Copyright © 2018 the Author(s). Published by PNAS.)

Mycobacterium tuberculosis (Mtb) continues to be a threat to Global Public Health, and its control will require an array of therapeutic strategies. It has been appreciated that high-throughput screens using cell-based assays to identify compounds targeting Mtb within macrophages represent a valuable tool for drug discovery. However, the host immune environment, in the form of lymphocytes and cytokines, is completely absent in a chemical screening platform based on infected macrophages alone. The absence of these players unnecessarily limits the breadth of novel host target pathways to be interrogated. In this study, we detail a new drug screening platform based on dissociated murine TB granulomas, named the Deconstructed Granuloma (DGr), that utilizes fluorescent Mtb reporter strains screened in the host immune environment of the infection site. The platform has been used to screen a collection of known drug candidates. Data from a representative 384-well plate containing known anti-bacterial compounds are shown, illustrating the robustness of the screening platform. The novel deconstructed granuloma platform represents an accessible, sensitive and robust high-throughput screen suitable for the inclusive interrogation of immune targets for Host-Directed Therapeutics.

New drugs are needed to treat gram-negative bacterial infections. These bacteria are protected by an outer membrane which prevents many antibiotics from reaching their cellular targets. The outer leaflet of the outer membrane contains LPS, which is responsible for creating this permeability barrier. Interfering with LPS biogenesis affects bacterial viability. We developed a cell-based screen that identifies inhibitors of LPS biosynthesis and transport by exploiting the nonessentiality of this pathway in Acinetobacter We used this screen to find an inhibitor of MsbA, an ATP-dependent flippase that translocates LPS across the inner membrane. Treatment with the inhibitor caused mislocalization of LPS to the cell interior. The discovery of an MsbA inhibitor, which is universally conserved in all gram-negative bacteria, validates MsbA as an antibacterial target. Because our cell-based screen reports on the function of the entire LPS biogenesis pathway, it could be used to identify compounds that inhibit other targets in the pathway, which can provide insights into vulnerabilities of the gram-negative cell envelope., Competing Interests: The authors declare no conflict of interest.

The original version of this Article contained an error in the spelling of Richard Thomson-Luque, which was incorrectly given as Richard Thomson Luque. This error has now been corrected in both the PDF and HTML versions of the Article.

Malaria liver stages represent an ideal therapeutic target with a bottleneck in parasite load and reduced clinical symptoms; however, current in vitro pre-erythrocytic (PE) models for Plasmodium vivax and P. falciparum lack the efficiency necessary for rapid identification and effective evaluation of new vaccines and drugs, especially targeting late liver-stage development and hypnozoites. Herein we report the development of a 384-well plate culture system using commercially available materials, including cryopreserved primary human hepatocytes. Hepatocyte physiology is maintained for at least 30 days and supports development of P. vivax hypnozoites and complete maturation of P. vivax and P. falciparum schizonts. Our multimodal analysis in antimalarial therapeutic research identifies important PE inhibition mechanisms: immune antibodies against sporozoite surface proteins functionally inhibit liver stage development and ion homeostasis is essential for schizont and hypnozoite viability. This model can be implemented in laboratories in disease-endemic areas to accelerate vaccine and drug discovery research.

A high-content imaging assay was used to screen the fraction collection of the Natural Product Library at The Scripps Research Institute for inhibitors of Cryptosporidium parvum. A chemical investigation of one strain, Streptomyces sp. CB01388, resulted in the isolation of six herbicidins (1-6), one of which is new (herbicidin L, 1). Five of the six herbicidins (1-3, 5, 6) showed moderate inhibitory activity against C. parvum, with 1 and 6 comparable to the FDA-approved drug nitazoxanide, and 2-6 showed no toxicity to the host HCT-8 cells and human HEK293T and HepG2 cells. These findings highlight the herbicidin scaffold for anti- Cryptosporidium drug development.

Cryptosporidiosis causes life-threatening diarrhea in children under the age of 5 years and prolonged diarrhea in immunodeficient people, especially AIDS patients. The standard of care, nitazoxanide, is modestly effective in children and ineffective in immunocompromised individuals. In addition to the need for new drugs, better knowledge of drug properties that drive in vivo efficacy is needed to facilitate drug development. We report the identification of a piperazine-based lead compound for Cryptosporidium drug development, MMV665917, and a new pharmacodynamic method used for its characterization. The identification of MMV665917 from the Medicines for Malaria Venture Malaria Box was followed by dose-response studies, in vitro toxicity studies, and structure-activity relationship studies using commercial analogues. The potency of this compound against Cryptosporidium parvum Iowa and field isolates was comparable to that against Cryptosporidium hominis Furthermore, unlike nitazoxanide, clofazimine, and paromomycin, MMV665917 appeared to be curative in a NOD SCID gamma mouse model of chronic cryptosporidiosis. MMV665917 was also efficacious in a gamma interferon knockout mouse model of acute cryptosporidiosis. To determine if efficacy in this mouse model of chronic infection might relate to whether compounds are parasiticidal or parasitistatic for C. parvum , we developed a novel in vitro parasite persistence assay. This assay suggested that MMV665917 was parasiticidal, unlike nitazoxanide, clofazimine, and paromomycin. The assay also enabled determination of the concentration of the compound required to maximize the rate of parasite elimination. This time-kill assay can be used to prioritize early-stage Cryptosporidium drug leads and may aid in planning in vivo efficacy experiments. Collectively, these results identify MMV665917 as a promising lead and establish a new method for characterizing potential anticryptosporidial agents., (Copyright © 2018 Jumani et al.)

Improvements have been made to the safety and efficacy of bumped kinase inhibitors, and they are advancing toward human and animal use for treatment of cryptosporidiosis. As the understanding of bumped kinase inhibitor pharmacodynamics for cryptosporidiosis therapy has increased, it has become clear that better compounds for efficacy do not necessarily require substantial systemic exposure. We now have a bumped kinase inhibitor with reduced systemic exposure, acceptable safety parameters, and efficacy in both the mouse and newborn calf models of cryptosporidiosis. Potential cardiotoxicity is the limiting safety parameter to monitor for this bumped kinase inhibitor. This compound is a promising pre-clinical lead for cryptosporidiosis therapy in animals and humans., (Copyright © 2017 Australian Society for Parasitology. Published by Elsevier Ltd. All rights reserved.)

Cryptosporidiosis has emerged as a leading cause of non-viral diarrhea in children under five years of age in the developing world, yet the current standard of care to treat Cryptosporidium infections, nitazoxanide, demonstrates limited and immune-dependent efficacy. Given the lack of treatments with universal efficacy, drug discovery efforts against cryptosporidiosis are necessary to find therapeutics more efficacious than the standard of care. To date, cryptosporidiosis drug discovery efforts have been limited to a few targeted mechanisms in the parasite and whole cell phenotypic screens against small, focused collections of compounds. Using a previous screen as a basis, we initiated the largest known drug discovery effort to identify novel anticryptosporidial agents. A high-content imaging assay for inhibitors of Cryptosporidium parvum proliferation within a human intestinal epithelial cell line was miniaturized and automated to enable high-throughput phenotypic screening against a large, diverse library of small molecules. A screen of 78,942 compounds identified 12 anticryptosporidial hits with sub-micromolar activity, including clofazimine, an FDA-approved drug for the treatment of leprosy, which demonstrated potent and selective in vitro activity (EC50 = 15 nM) against C. parvum. Clofazimine also displayed activity against C. hominis-the other most clinically-relevant species of Cryptosporidium. Importantly, clofazimine is known to accumulate within epithelial cells of the small intestine, the primary site of Cryptosporidium infection. In a mouse model of acute cryptosporidiosis, a once daily dosage regimen for three consecutive days or a single high dose resulted in reduction of oocyst shedding below the limit detectable by flow cytometry. Recently, a target product profile (TPP) for an anticryptosporidial compound was proposed by Huston et al. and highlights the need for a short dosing regimen (< 7 days) and formulations for children < 2 years. Clofazimine has a long history of use and has demonstrated a good safety profile for a disease that requires chronic dosing for a period of time ranging 3-36 months. These results, taken with clofazimine's status as an FDA-approved drug with over four decades of use for the treatment of leprosy, support the continued investigation of clofazimine both as a new chemical tool for understanding cryptosporidium biology and a potential new treatment of cryptosporidiosis.

The spiroindolones, a new class of antimalarial medicines discovered in a cellular screen, are rendered less active by mutations in a parasite P-type ATPase, PfATP4. We show here that S. cerevisiae also acquires mutations in a gene encoding a P-type ATPase (ScPMA1) after exposure to spiroindolones and that these mutations are sufficient for resistance. KAE609 resistance mutations in ScPMA1 do not confer resistance to unrelated antimicrobials, but do confer cross sensitivity to the alkyl-lysophospholipid edelfosine, which is known to displace ScPma1p from the plasma membrane. Using an in vitro cell-free assay, we demonstrate that KAE609 directly inhibits ScPma1p ATPase activity. KAE609 also increases cytoplasmic hydrogen ion concentrations in yeast cells. Computer docking into a ScPma1p homology model identifies a binding mode that supports genetic resistance determinants and in vitro experimental structure-activity relationships in both P. falciparum and S. cerevisiae. This model also suggests a shared binding site with the dihydroisoquinolones antimalarials. Our data support a model in which KAE609 exerts its antimalarial activity by directly interfering with P-type ATPase activity.

In order to identify the most attractive starting points for drugs that can be used to prevent malaria, a diverse chemical space comprising tens of thousands to millions of small molecules may need to be examined. Achieving this throughput necessitates the development of efficient ultra-high-throughput screening methods. Here, we report the development and evaluation of a luciferase-based phenotypic screen of malaria exoerythrocytic-stage parasites optimized for a 1536-well format. This assay uses the exoerythrocytic stage of the rodent malaria parasite, Plasmodium berghei , and a human hepatoma cell line. We use this assay to evaluate several biased and unbiased compound libraries, including two small sets of molecules (400 and 89 compounds, respectively) with known activity against malaria erythrocytic-stage parasites and a set of 9886 diversity-oriented synthesis (DOS)-derived compounds. Of the compounds screened, we obtain hit rates of 12-13 and 0.6% in preselected and naïve libraries, respectively, and identify 52 compounds with exoerythrocytic-stage activity less than 1 μM and having minimal host cell toxicity. Our data demonstrate the ability of this method to identify compounds known to have causal prophylactic activity in both human and animal models of malaria, as well as novel compounds, including some exclusively active against parasite exoerythrocytic stages.

Background: Electronic administration of clinician-reported outcomes (eClinROs) has advantages over paper-based methods, but the mode of administration change has the potential to affect the validity of the scale. The literature on migration of patient-reported outcomes (PROs) suggests that there are different levels of modification, which necessitate different approaches to demonstrating mode equivalence. However, little has been written on the migration of ClinROs to electronic administration., Methods: We propose a method of comparing paper and electronic versions of scales that includes a comparison based on content and a comparison based on format. The determination of whether the eClinRO has undergone minor, moderate, or substantial modification will drive the necessary studies required for validation., Results: The unique characteristics of ClinROs suggest 2 additional types of modifications, including functionality adaptation and adaptation of instructions., Conclusions: In many respects, the migration of a ClinRO to electronic administration is similar to that of a PRO. This article has explored the ways in which there might be special considerations for ClinROs that have not been elaborated for PROs.

Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1) in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50) of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic screen with potent activity against Plasmodium falciparum asexual and sexual stage parasites. To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance. Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7&#95;1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell. Similarly to the spiroindolones, GNF-Pf4492 blocks parasite transmission to mosquitoes and disrupts intracellular sodium homeostasis. Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target.

Background: Whole-genome sequencing represents a powerful experimental tool for pathogen research. We present methods for the analysis of small eukaryotic genomes, including a streamlined system (called Platypus) for finding single nucleotide and copy number variants as well as recombination events., Results: We have validated our pipeline using four sets of Plasmodium falciparum drug resistant data containing 26 clones from 3D7 and Dd2 background strains, identifying an average of 11 single nucleotide variants per clone. We also identify 8 copy number variants with contributions to resistance, and report for the first time that all analyzed amplification events are in tandem., Conclusions: The Platypus pipeline provides malaria researchers with a powerful tool to analyze short read sequencing data. It provides an accurate way to detect SNVs using known software packages, and a novel methodology for detection of CNVs, though it does not currently support detection of small indels. We have validated that the pipeline detects known SNVs in a variety of samples while filtering out spurious data. We bundle the methods into a freely available package.

Preventing relapses of Plasmodium vivax malaria through a radical cure depends on use of the 8-aminoquinoline primaquine, which is associated with safety and compliance issues. For future malaria eradication strategies, new, safer radical curative compounds that efficiently kill dormant liver stages (hypnozoites) will be essential. A new compound with potential radical cure activity was identified using a low-throughput assay of in vitro-cultured hypnozoite forms of Plasmodium cynomolgi (an excellent and accessible model for Plasmodium vivax). In this assay, primary rhesus hepatocytes are infected with P. cynomolgi sporozoites, and exoerythrocytic development is monitored in the presence of compounds. Liver stage cultures are fixed after 6 days and stained with anti-Hsp70 antibodies, and the relative proportions of small (hypnozoite) and large (schizont) forms relative to the untreated controls are determined. This assay was used to screen a series of 18 known antimalarials and 14 new non-8-aminoquinolines (preselected for blood and/or liver stage activity) in three-point 10-fold dilutions (0.1, 1, and 10 μM final concentrations). A novel compound, designated KAI407 showed an activity profile similar to that of primaquine (PQ), efficiently killing the earliest stages of the parasites that become either primary hepatic schizonts or hypnozoites (50% inhibitory concentration [IC50] for hypnozoites, KAI407, 0.69 μM, and PQ, 0.84 μM; for developing liver stages, KAI407, 0.64 μM, and PQ, 0.37 μM). When given as causal prophylaxis, a single oral dose of 100 mg/kg of body weight prevented blood stage parasitemia in mice. From these results, we conclude that KAI407 may represent a new compound class for P. vivax malaria prophylaxis and potentially a radical cure.

Achieving the goal of malaria elimination will depend on targeting Plasmodium pathways essential across all life stages. Here we identify a lipid kinase, phosphatidylinositol-4-OH kinase (PI(4)K), as the target of imidazopyrazines, a new antimalarial compound class that inhibits the intracellular development of multiple Plasmodium species at each stage of infection in the vertebrate host. Imidazopyrazines demonstrate potent preventive, therapeutic, and transmission-blocking activity in rodent malaria models, are active against blood-stage field isolates of the major human pathogens P. falciparum and P. vivax, and inhibit liver-stage hypnozoites in the simian parasite P. cynomolgi. We show that imidazopyrazines exert their effect through inhibitory interaction with the ATP-binding pocket of PI(4)K, altering the intracellular distribution of phosphatidylinositol-4-phosphate. Collectively, our data define PI(4)K as a key Plasmodium vulnerability, opening up new avenues of target-based discovery to identify drugs with an ideal activity profile for the prevention, treatment and elimination of malaria.

The malaria parasite Plasmodium falciparum establishes in the host erythrocyte plasma membrane new permeability pathways that mediate nutrient uptake into the infected cell. These pathways simultaneously allow Na(+) influx, causing [Na(+)] in the infected erythrocyte cytosol to increase to high levels. The intraerythrocytic parasite itself maintains a low cytosolic [Na(+)] via unknown mechanisms. Here we present evidence that the intraerythrocytic parasite actively extrudes Na(+) against an inward gradient via PfATP4, a parasite plasma membrane protein with sequence similarities to Na(+)-ATPases of lower eukaryotes. Mutations in PfATP4 confer resistance to a potent class of antimalarials, the spiroindolones. Consistent with this, the spiroindolones cause a profound disruption in parasite Na(+) homeostasis, which is attenuated in parasites bearing resistance-conferring mutations in PfATP4. The mutant parasites also show some impairment of Na(+) regulation. Taken together, our results are consistent with PfATP4 being a Na(+) efflux ATPase and a target of the spiroindolones., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Malaria parasites elude eradication attempts both within the human host and across nations. At the individual level, parasites evade the host immune responses through antigenic variation. At the global level, parasites escape drug pressure through single nucleotide variants and gene copy amplification events conferring drug resistance. Despite their importance to global health, the rates at which these genomic alterations emerge have not been determined. We studied the complete genomes of different Plasmodium falciparum clones that had been propagated asexually over one year in the presence and absence of drug pressure. A combination of whole-genome microarray analysis and next-generation deep resequencing (totaling 14 terabases) revealed a stable core genome with only 38 novel single nucleotide variants appearing in seventeen evolved clones (avg. 5.4 per clone). In clones exposed to atovaquone, we found cytochrome b mutations as well as an amplification event encompassing the P. falciparum multidrug resistance associated protein (mrp1) on chromosome 1. We observed 18 large-scale (>1 kb on average) deletions of telomere-proximal regions encoding multigene families, involved in immune evasion (9.5×10(-6) structural variants per base pair per generation). Six of these deletions were associated with chromosomal crossovers generated during mitosis. We found only minor differences in rates between genetically distinct strains and between parasites cultured in the presence or absence of drug. Using these derived mutation rates for P. falciparum (1.0-9.7×10(-9) mutations per base pair per generation), we can now model the frequency at which drug or immune resistance alleles will emerge under a well-defined set of assumptions. Further, the detection of mitotic recombination events in var gene families illustrates how multigene families can arise and change over time in P. falciparum. These results will help improve our understanding of how P. falciparum evolves to evade control efforts within both the individual hosts and large populations., Competing Interests: The authors have declared that no competing interests exist.

Early secretory and endoplasmic reticulum (ER)-localized proteins that are terminally misfolded or misassembled are degraded by a ubiquitin- and proteasome-mediated process known as ER-associated degradation (ERAD). Protozoan pathogens, including the causative agents of malaria, toxoplasmosis, trypanosomiasis, and leishmaniasis, contain a minimal ERAD network relative to higher eukaryotic cells, and, because of this, we observe that the malaria parasite Plasmodium falciparum is highly sensitive to the inhibition of components of this protein quality control system. Inhibitors that specifically target a putative protease component of ERAD, signal peptide peptidase (SPP), have high selectivity and potency for P. falciparum. By using a variety of methodologies, we validate that SPP inhibitors target P. falciparum SPP in parasites, disrupt the protein's ability to facilitate degradation of unstable proteins, and inhibit its proteolytic activity. These compounds also show low nanomolar activity against liver-stage malaria parasites and are also equipotent against a panel of pathogenic protozoan parasites. Collectively, these data suggest ER quality control as a vulnerability of protozoan parasites, and that SPP inhibition may represent a suitable transmission blocking antimalarial strategy and potential pan-protozoan drug target.

With renewed calls for malaria eradication, next-generation antimalarials need be active against drug-resistant parasites and efficacious against both liver- and blood-stage infections. We screened a natural product library to identify inhibitors of Plasmodium falciparum blood- and liver-stage proliferation. Cladosporin, a fungal secondary metabolite whose target and mechanism of action are not known for any species, was identified as having potent, nanomolar, antiparasitic activity against both blood and liver stages. Using postgenomic methods, including a yeast deletion strains collection, we show that cladosporin specifically inhibits protein synthesis by directly targeting P. falciparum cytosolic lysyl-tRNA synthetase. Further, cladosporin is >100-fold more potent against parasite lysyl-tRNA synthetase relative to the human enzyme, which is conferred by the identity of two amino acids within the enzyme active site. Our data indicate that lysyl-tRNA synthetase is an attractive, druggable, antimalarial target that can be selectively inhibited., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Most malaria drug development focuses on parasite stages detected in red blood cells, even though, to achieve eradication, next-generation drugs active against both erythrocytic and exo-erythrocytic forms would be preferable. We applied a multifactorial approach to a set of >4000 commercially available compounds with previously demonstrated blood-stage activity (median inhibitory concentration < 1 micromolar) and identified chemical scaffolds with potent activity against both forms. From this screen, we identified an imidazolopiperazine scaffold series that was highly enriched among compounds active against Plasmodium liver stages. The orally bioavailable lead imidazolopiperazine confers complete causal prophylactic protection (15 milligrams/kilogram) in rodent models of malaria and shows potent in vivo blood-stage therapeutic activity. The open-source chemical tools resulting from our effort provide starting points for future drug discovery programs, as well as opportunities for researchers to investigate the biology of exo-erythrocytic forms.

Decoquinate has single-digit nanomolar activity against in vitro blood stage Plasmodium falciparum parasites, the causative agent of human malaria. In vitro evolution of decoquinate-resistant parasites and subsequent comparative genomic analysis to the drug-sensitive parental strain revealed resistance was conferred by two nonsynonymous single nucleotide polymorphisms in the gene encoding cytochrome b. The resultant amino acid mutations, A122T and Y126C, reside within helix C in the ubiquinol-binding pocket of cytochrome b, an essential subunit of the cytochrome bc(1) complex. As with other cytochrome bc(1) inhibitors, such as atovaquone, decoquinate has low nanomolar activity against in vitro liver stage P. yoelii and provides partial prophylaxis protection when administered to infected mice at 50 mg kg(-1). In addition, transgenic parasites expressing yeast dihydroorotate dehydrogenase are >200-fold less sensitive to decoquinate, which provides additional evidence that this drug inhibits the parasite's mitochondrial electron transport chain. Importantly, decoquinate exhibits limited cross-resistance to a panel of atovaquone-resistant parasites evolved to harbor various mutations in cytochrome b. The basis for this difference was revealed by molecular docking studies, in which both of these inhibitors were shown to have distinctly different modes of binding within the ubiquinol-binding site of cytochrome b.

M1 protein contributes to Group A Streptococcus (GAS) systemic virulence by interfering with phagocytosis and through proinflammatory activities when released from the cell surface. Here we identify a novel role of M1 protein in the stimulation of neutrophil and mast cell extracellular trap formation, yet also subsequent survival of the pathogen within these DNA-based innate defense structures. Targeted mutagenesis and heterologous expression studies demonstrate M1 protein promotes resistance to the human cathelicidin antimicrobial peptide LL-37, an important effector of bacterial killing within such phagocyte extracellular traps. Studies with purified recombinant protein fragments mapped the inhibition of cathelicidin killing to the M1 hypervariable N-terminal domain. A survey of GAS clinical isolates found that strains from patients with necrotizing fasciitis or toxic shock syndrome were significantly more likely to be resistant to cathelicidin than GAS M types not associated with invasive disease; M1 isolates were uniformly resistant. We conclude increased resistance to host cathelicidin and killing within phagocyte extracellular traps contribute to the propensity of M1 GAS strains to produce invasive infections., (Copyright 2009 S. Karger AG, Basel.)

Unstable self-esteem is thought to reflect fragile and vulnerable feelings of self-worth that are affected by specific positive and negative events. Direct evidence for this contention is lacking, however. To redress this situation, we examined the extent to which level and stability of self-esteem predicted the impact that everyday positive and negative events had on individuals' feelings about themselves. Participants recorded the most positive and most negative event that occurred each day Monday through Thursday for a period of 2 weeks. They then indicated the extent to which each event made them feel better or worse about themselves. As anticipated, negative and positive events had a greater impact on the self-feelings of individuals with unstable as opposed to stable self-esteem (although the effect for positive events was marginal). Additional findings indicated that event qualities (i.e., self-esteem relevance and concerns about social acceptance/rejection) could account for the unstable self-esteem/greater reactivity link for negative events, but not for positive events. Negative, but not positive, events had a greater impact on the self-feelings of individuals with low as compared to high levels of self-esteem. Theoretical implications are discussed.