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© 2015 Khare et al.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.

Safe, effective, and low-cost oral antiviral therapies are needed to treat those at high risk for developing severe COVID-19. To that end, we performed a high-throughput screen to identify non-peptidic, non-covalent inhibitors of the SARS-CoV-2 main protease (Mpro), an essential enzyme in viral replication. NZ-804 was developed from a screening hit through iterative rounds of structure-guided medicinal chemistry. NZ-804 potently inhibits SARS-CoV-2 Mpro (0.009 μM IC 50 ) as well as SARS-CoV-2 replication in human lung cell lines (0.008 μM EC 50 ) and primary human airway epithelial cell cultures. Antiviral activity is maintained against distantly related sarbecoviruses and endemic human CoV OC43. In SARS-CoV-2 mouse and hamster disease models, NZ-804 therapy given once or twice daily significantly diminished SARS-CoV-2 replication and pathogenesis. NZ-804 synthesis is low cost and uncomplicated, simplifying global production and access. These data support the exploration of NZ-804 as a therapy for COVID-19 and future emerging sarbecovirus infections., Competing Interests: Declaration of interests J.C.S., S.T., X.B., I.V.K., J.L.W., N.E.Z., M.K.P., A.A., P.K.J., R.B., A.F., and Z.S. are listed as inventors on a patent for NZ-804. R.S.B. is a member of the advisory boards of VaxArt and Invivyd and has collaborations with Takeda, Pfizer, Moderna, Ridgeback Biosciences, Gilead, and Eli Lily. Y.K. has received unrelated funding support from Daiichi Sankyo Pharmaceutical, Toyama Chemical, Tauns Laboratories, Inc., Shionogi & Co., Ltd., Otsuka Pharmaceutical, KM Biologics, Kyoritsu Seiyaku, Shinya Corporation, and Fuji Rebio., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Toxoplasma gondii causes widespread chronic infections that are not cured by current treatments due to inability to affect semi-dormant bradyzoite stages within tissue cysts. To identify compounds to eliminate chronic infection, we developed a HTS using a recently characterized strain of T. gondii that undergoes efficient conversion to bradyzoites in intro. Stage-specific expression of luciferase was used to selectively monitor growth inhibition of bradyzoites by the Library of Pharmacological Active Compounds, consisting of 1,280 drug-like compounds. We identified 44 compounds with >50% inhibitory effects against bradyzoites, including new highly potent compounds, several of which have precedent for antimicrobial activity. Subsequent characterization of the compound Sanguinarine sulfate revealed potent and rapid killing against in vitro produced bradyzoites and bradyzoites harvested from chronically infected mice. These findings provide a platform for expanded screening and identify promising compounds for further preclinical development against T. gondii bradyzoites responsible for chronic infection.

In efforts towards eliminating malaria, a discovery program was initiated to identify a novel antimalarial using KAF156 as a starting point. Following the most recent TCP/TPP guidelines, we have identified mCMQ069 with a predicted single oral dose for treatment (∼40-106 mg) and one-month chemoprevention (∼96-216 mg). We have improved unbound MPC and predicted human clearance by 18-fold and 10-fold respectively when compared to KAF156.

Recent malaria drug discovery approaches have been extensively focused on the development of oral, smallmolecule inhibitors for disease treatment whereas parenteral routes of administration have been avoided due to limitations in deploying a shelf-stable injectable even though it could be dosed less frequently. However, an updated target candidate profile from Medicines for Malaria Venture (MMV) and stakeholders have advocated for long-acting injectable chemopreventive agents as an important interventive tool to improve malaria prevention. Here, we present strategies for the development of a long-acting, intramuscular, injectable atovaquone prophylactic therapy. We have generated three prodrug approaches that are contrasted by their differential physiochemical properties and pharmacokinetic profiles: mCBK068, a docosahexaenoic acid ester of atovaquone formulated in sesame oil, mCKX352, a heptanoic acid ester of atovaquone formulated as a solution in sesame oil, and mCBE161, an acetic acid ester of atovaquone formulated as an aqueous suspension. As a result, from a single 20 mg/kg intramuscular injection, mCKX352 and mCBE161 maintain blood plasma exposure of atovaquone above the minimal efficacious concentration for >70 days and >30 days, respectively, in cynomolgus monkeys. The differences in plasma exposure are reflective of the prodrug strategy, which imparts altered chemical properties that ultimately influence aqueous solubility and depot release kinetics. On the strength of the pharmacokinetic and safety profiles, mCBE161 is being advanced as a first-in-class clinical candidate for first-in-human trials.

Drug discovery is an intricate and costly process. Repurposing existing drugs and active compounds offers a viable pathway to develop new therapies for various diseases. By leveraging publicly available biomedical information, it is possible to predict compounds' activity and identify their potential targets across diverse organisms. In this study, we aimed to assess the antiplasmodial activity of compounds from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library using in vitro and bioinformatics approaches. We assessed the in vitro antiplasmodial activity of the compounds using blood-stage and liver-stage drug susceptibility assays. We used protein sequences of known targets of the ReFRAME compounds with high antiplasmodial activity (EC 50  < 10 uM) to conduct a protein-pairwise search to identify similar Plasmodium falciparum 3D7 proteins (from PlasmoDB) using NCBI protein BLAST. We further assessed the association between the compounds' in vitro antiplasmodial activity and level of similarity between their known and predicted P. falciparum target proteins using simple linear regression analyses. BLAST analyses revealed 735 P. falciparum proteins that were similar to the 226 known protein targets associated with the ReFRAME compounds. Antiplasmodial activity of the compounds was positively associated with the degree of similarity between the compounds' known targets and predicted P. falciparum protein targets (percentage identity, E value, and bit score), the number of the predicted P. falciparum targets, and their respective mutagenesis index and fitness scores (R 2 between 0.066 and 0.92, P < 0.05). Compounds predicted to target essential P. falciparum proteins or those with a druggability index of 1 showed the highest antiplasmodial activity., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

The apicomplexan parasite Cryptosporidium is a leading cause of childhood diarrhea in developing countries. Current treatment options are inadequate and multiple preclinical compounds are being actively pursued as potential drugs for cryptosporidiosis. Unlike most apicomplexans, Cryptosporidium spp. sequentially replicate asexually and then sexually within a single host to complete their lifecycles. Anti-cryptosporidial compounds are generally identified or tested through in vitro phenotypic assays that only assess the asexual stages. Therefore, compounds that specifically target the sexual stages remain unexplored. In this study, we leveraged the ReFRAME drug repurposing library against a newly devised multi-readout imaging assay to identify small-molecule compounds that modulate macrogamont differentiation and maturation. RNA-seq studies confirmed selective modulation of macrogamont differentiation for 10 identified compounds (9 inhibitors and 1 accelerator). The collective transcriptomic profiles of these compounds indicates that translational repression accompanies Cryptosporidium sexual differentiation, which we validated experimentally. Additionally, cross comparison of the RNA-seq data with promoter sequence analysis for stage-specific genes converged on a key role for an Apetala 2 (AP2) transcription factor (cgd2&#95;3490) in differentiation into macrogamonts. Finally, drug annotation for the ReFRAME hits indicates that an elevated supply of energy equivalence in the host cell is critical for macrogamont formation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Hasan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Leishmaniasis is a group of vector-borne, parasitic diseases caused by over 20 species of the protozoan Leishmania spp. The three major disease classifications, cutaneous, visceral, and mucocutaneous, have a range of clinical manifestations from self-healing skin lesions to hepatosplenomegaly and mucosal membrane damage to fatality. As a neglected tropical disease, leishmaniasis represents a major international health challenge, with nearly 350 million people living at risk of infection a year. The current chemotherapeutics used to treat leishmaniasis have harsh side effects, prolonged and costly treatment regimens, as well as emerging drug resistance, and are predominantly used for the treatment of visceral leishmaniasis. There is an undeniable need for the identification and development of novel chemotherapeutics targeting cutaneous leishmaniasis (CL), largely ignored by concerted drug development efforts. CL is mostly non-lethal and the most common presentation of this disease, with nearly 1 million new cases reported annually. Recognizing this unaddressed need, substantial yet fragmented progress in early drug discovery efforts for CL has occurred in the past 15 years and was outlined in this review. However, further work needs to be carried out to advance early discovery candidates towards the clinic. Importantly, there is a paucity of investment in the translation and development of therapies for CL, limiting the emergence of viable solutions to deal with this serious and complex international health problem.

Malaria parasites like Plasmodium falciparum multiply in red blood cells (RBC), which are cleared from the bloodstream by the spleen when their deformability is altered. Drug-induced stiffening of Plasmodium falciparum-infected RBC should therefore induce their elimination from the bloodstream. Here, based on this original mechanical approach, we identify safe drugs with strong potential to block the malaria transmission. By screening 13 555 compounds with spleen-mimetic microfilters, we identified 82 that target circulating transmissible form of P. falciparum. NITD609, an orally administered PfATPase inhibitor with known effects on P. falciparum, killed and stiffened transmission stages in vitro at nanomolar concentrations. Short exposures to TD-6450, an orally-administered NS5A hepatitis C virus inhibitor, stiffened transmission parasite stages and killed asexual stages in vitro at high nanomolar concentrations. A Phase 1 study in humans with a primary safety outcome and a secondary pharmacokinetics outcome ( https://clinicaltrials.gov , ID: NCT02022306) showed no severe adverse events either with single or multiple doses. Pharmacokinetic modelling showed that these concentrations can be reached in the plasma of subjects receiving short courses of TD-6450. This physiologically relevant screen identified multiple mechanisms of action, and safe drugs with strong potential as malaria transmission-blocking agents which could be rapidly tested in clinical trials., (© 2023. The Author(s).)

The Parkinson's Disease Activities of Daily Living, Interference, and Dependence Instrument© (PD-AID) is a patient-reported outcome (PRO) instrument, recently developed to assess the clinical benefit of Parkinson's Disease (PD) treatment. The PD-AID consists of morning and evening assessments, administered daily. To benefit from the full set of the repeated observations over time, analytic approaches that account for both within- and between-individual variability are required. The current study aimed to employ the advantages of exploratory Multilevel Factor Analysis (MFA) on data collected from 93 participants with moderate to advanced PD, currently using and responding to Levodopa (L-Dopa), who completed the PD-AID twice daily as part of a prospective, non-intervention, observational study for ~28 days. Average daily completion rates were comparable for the Morning and the Evening PD-AID (78% and 74%, respectively). The intraclass correlation coefficients for the Morning and Evening PD-AID items were in the range of 0.70-0.90, with an average of 0.81 for the Morning PD-AID items and 0.83 for the Evening PD-AID items, suggesting that most variability (81%-83%) in responses was due to between-individual variability. For the Morning PD-AID, one factor (including nine out of 10 Morning PD-AID items) emerged at the between-individual level and four factors (core physical actions, basic self-care activities, feeding, and interference & dependence) at the within-individual level. For the Evening PD-AID, there were four between-individual factors (basic activities of daily living ADLs, life interference, impact on planning, and emotional consequences) and five within-individual factors (basic ADLs, toileting, life interference, medication planning, and emotional impact). The factors had high reliability., Competing Interests: CS, BS, and CM are employees of Cronos Clinical Consulting Services, Inc., the entity responsible for licensing of the PD-AID, including commercial license fees for use in industry-sponsored clinical trials. LD is the lead developer of the PD-AID and an employee of Pfizer, Inc., the funding source for the research. No authors receive any licensing fees from the PD-AID., (Copyright © 2022 Sirbu, Saxby, McNamara and Deal.)

A phenotypic screen of the ReFRAME compound library was performed to identify cell-active inhibitors that could be developed as therapeutics for giardiasis. A primary screen against Giardia lamblia GS clone H7 identified 85 cell-active compounds at a hit rate of 0.72%. A cytotoxicity counterscreen against HEK293T cells was carried out to assess hit compound selectivity for further prioritization. Mavelertinib (PF-06747775), a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), was identified as a potential new therapeutic based on indication, activity, and availability after reconfirmation. Mavelertinib has in vitro efficacy against metronidazole-resistant 713-M3 strains. Other EGFR-TKIs screened in follow-up assays exhibited insignificant inhibition of G. lamblia at 5 μM, suggesting that the primary molecular target of mavelertinib may have a different mechanistic binding mode from human EGFR-tyrosine kinase. Mavelertinib, dosed as low as 5 mg/kg of body weight or as high as 50 mg/kg, was efficacious in the acute murine Giardia infection model. These results suggest that mavelertinib merits consideration for repurposing and advancement to giardiasis clinical trials while its analogues are further developed.

In vitro evolution and whole genome analysis were used to comprehensively identify the genetic determinants of chemical resistance in Saccharomyces cerevisiae. Sequence analysis identified many genes contributing to the resistance phenotype as well as numerous amino acids in potential targets that may play a role in compound binding. Our work shows that compound-target pairs can be conserved across multiple species. The set of 25 most frequently mutated genes was enriched for transcription factors, and for almost 25 percent of the compounds, resistance was mediated by one of 100 independently derived, gain-of-function SNVs found in a 170 amino acid domain in the two Zn 2 C 6 transcription factors YRR1 and YRM1 (p < 1 × 10 -100 ). This remarkable enrichment for transcription factors as drug resistance genes highlights their important role in the evolution of antifungal xenobiotic resistance and underscores the challenge to develop antifungal treatments that maintain potency., (© 2022. The Author(s).)

There is a growing appreciation for the idea that bacterial utilization of host-derived lipids, including cholesterol, supports Mycobacterium tuberculosis (Mtb) pathogenesis. This has generated interest in identifying novel antibiotics that can disrupt cholesterol utilization by Mtb in vivo. Here we identify a novel small molecule agonist (V-59) of the Mtb adenylyl cyclase Rv1625c, which stimulates 3', 5'-cyclic adenosine monophosphate (cAMP) synthesis and inhibits cholesterol utilization by Mtb. Similarly, using a complementary genetic approach that induces bacterial cAMP synthesis independent of Rv1625c, we demonstrate that inducing cAMP synthesis is sufficient to inhibit cholesterol utilization in Mtb. Although the physiological roles of individual adenylyl cyclase enzymes in Mtb are largely unknown, here we demonstrate that the transmembrane region of Rv1625c is required during cholesterol metabolism. Finally, the pharmacokinetic properties of Rv1625c agonists have been optimized, producing an orally-available Rv1625c agonist that impairs Mtb pathogenesis in infected mice. Collectively, this work demonstrates a role for Rv1625c and cAMP signaling in controlling cholesterol metabolism in Mtb and establishes that cAMP signaling can be pharmacologically manipulated for the development of new antibiotic strategies., Competing Interests: The authors have declared that no competing interests exist.

Infection with Cryptosporidium spp. can cause severe diarrhea, leading to long-term adverse impacts and even death in malnourished children and immunocompromised patients. The only FDA-approved drug for treating cryptosporidiosis, nitazoxanide, has limited efficacy in the populations impacted the most by the diarrheal disease, and safe, effective treatment options are urgently needed. Initially identified by a large-scale phenotypic screening campaign, the antimycobacterial therapeutic clofazimine demonstrated great promise in both in vitro and in vivo preclinical models of Cryptosporidium infection. Unfortunately, a phase 2a clinical trial in HIV-infected adults with cryptosporidiosis did not identify any clofazimine treatment effect on Cryptosporidium infection burden or clinical outcomes. To explore whether clofazimine's lack of efficacy in the phase 2a trial may have been due to subtherapeutic clofazimine concentrations, a pharmacokinetic/pharmacodynamic modeling approach was undertaken to determine the relationship between clofazimine in vivo concentrations and treatment effects in multiple preclinical infection models. Exposure-response relationships were characterized using E max and logistic models, which allowed predictions of efficacious clofazimine concentrations for the control and reduction of disease burden. After establishing exposure-response relationships for clofazimine treatment of Cryptosporidium infection in our preclinical model studies, it was unmistakable that the clofazimine levels observed in the phase 2a study participants were well below concentrations associated with anti- Cryptosporidium efficacy. Thus, despite a dosing regimen above the highest doses recommended for mycobacterial therapy, it is very likely the lack of treatment effect in the phase 2a trial was at least partially due to clofazimine concentrations below those required for efficacy against cryptosporidiosis. It is unlikely that clofazimine will provide a remedy for the large number of cryptosporidiosis patients currently without a viable treatment option unless alternative, safe clofazimine formulations with improved oral absorption are developed. (This study has been registered in ClinicalTrials.gov under identifier NCT03341767.).

The ongoing pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), necessitates strategies to identify prophylactic and therapeutic drug candidates for rapid clinical deployment. Here, we describe a screening pipeline for the discovery of efficacious SARS-CoV-2 inhibitors. We screen a best-in-class drug repurposing library, ReFRAME, against two high-throughput, high-content imaging infection assays: one using HeLa cells expressing SARS-CoV-2 receptor ACE2 and the other using lung epithelial Calu-3 cells. From nearly 12,000 compounds, we identify 49 (in HeLa-ACE2) and 41 (in Calu-3) compounds capable of selectively inhibiting SARS-CoV-2 replication. Notably, most screen hits are cell-line specific, likely due to different virus entry mechanisms or host cell-specific sensitivities to modulators. Among these promising hits, the antivirals nelfinavir and the parent of prodrug MK-4482 possess desirable in vitro activity, pharmacokinetic and human safety profiles, and both reduce SARS-CoV-2 replication in an orthogonal human differentiated primary cell model. Furthermore, MK-4482 effectively blocks SARS-CoV-2 infection in a hamster model. Overall, we identify direct-acting antivirals as the most promising compounds for drug repurposing, additional compounds that may have value in combination therapies, and tool compounds for identification of viral host cell targets.

Filarial worms cause multiple debilitating diseases in millions of people worldwide, including river blindness. Currently available drugs reduce transmission by killing larvae (microfilariae), but there are no effective cures targeting the adult parasites (macrofilaricides) which survive and reproduce in the host for very long periods. To identify effective macrofilaricides, we carried out phenotypic screening of a library of 2121 approved drugs for clinical use against adult Brugia pahangi and prioritized the hits for further studies by integrating those results with a computational prioritization of drugs and associated targets. This resulted in the identification of 18 hits with anti-macrofilaricidal activity, of which two classes, azoles and aspartic protease inhibitors, were further expanded upon. Follow up screening against Onchocerca spp. (adult Onchocerca ochengi and pre-adult O. volvulus ) confirmed activity for 13 drugs (the majority having IC 50 < 10 μM), and a counter screen of a subset against L. loa microfilariae showed the potential to identify selective drugs that prevent adverse events when co-infected individuals are treated. Stage specific activity was also observed. Many of these drugs are amenable to structural optimization, and also have known canonical targets, making them promising candidates for further optimization that can lead to identifying and characterizing novel anti-macrofilarial drugs.

The quinazolines CBR417 and CBR490 were previously shown to be potent anti-wolbachials that deplete Wolbachia endosymbionts of filarial nematodes and present promising pre-clinical candidates for human filarial diseases such as onchocerciasis. In the present study we tested both candidates in two models of chronic filarial infection, namely the Litomosoides sigmodontis and Brugia pahangi jird model and assessed their long-term effect on Wolbachia depletion, microfilariae counts and filarial embryogenesis 16-18 weeks after treatment initiation (wpt). Once per day (QD) oral treatment with CBR417 (50 mg/kg) for 4 days or twice per day (BID) with CBR490 (25 mg/kg) for 7 days during patent L. sigmodontis infection reduced the Wolbachia load by >99% and completely cleared peripheral microfilaremia from 10-14 wpt. Similarly, 7 days of QD treatments (40 mg/kg) with CBR417 or CBR490 cleared >99% of Wolbachia from B. pahangi and reduced peritoneal microfilariae counts by 93% in the case of CBR417 treatment. Transmission electron microscopy analysis indicated intensive damage to the B. pahangi ovaries following CBR417 treatment and in accordance filarial embryogenesis was inhibited in both models after CBR417 or CBR490 treatment. Suboptimal treatment regimens of CBR417 or CBR490 did not lead to a maintained reduction of the microfilariae and Wolbachia load. In conclusion, CBR417 or CBR490 are pre-clinical candidates for filarial diseases, which achieve long-term clearance of Wolbachia endosymbionts of filarial nematodes, inhibit filarial embryogenesis and clear microfilaremia with treatments as short as 7 days., (Copyright © 2019 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Chagas disease, caused by the kinetoplastid parasite Trypanosoma cruzi , affects between 6 and 7 million people worldwide, with an estimated 300,000 to 1 million of these cases in the United States. In the chronic phase of infection, T. cruzi can cause severe gastrointestinal and cardiac disease, which can be fatal. Currently, only benznidazole is clinically approved by the FDA for pediatric use to treat this infection in the USA. Toxicity associated with this compound has driven the search for new anti-Chagas agents. Drug repurposing is a particularly attractive strategy for neglected diseases, as pharmacological parameters and toxicity are already known for these compounds, reducing costs and saving time in the drug development pipeline. Here, we screened 7680 compounds from the Repurposing, Focused Rescue, and Accelerated Medchem (ReFRAME) library, a collection of drugs or compounds with confirmed clinical safety, against T. cruzi . We identified seven compounds of interest with potent in vitro activity against the parasite with a therapeutic index of 10 or greater, including the previously unreported activity of the antiherpetic compound 348U87. These results provide the framework for further development of new T. cruzi leads that can potentially move quickly to the clinic., Competing Interests: The authors declare no conflicts of interest.

A new cyclic peptide, kakeromamide B ( 1 ), and previously described cytotoxic cyanobacterial natural products ulongamide A ( 2 ), lyngbyabellin A ( 3 ), 18 E -lyngbyaloside C ( 4 ), and lyngbyaloside ( 5 ) were identified from an antimalarial extract of the Fijian marine cyanobacterium Moorea producens . Compounds 1 and 1 exhibited moderate activity against Plasmodium falciparum blood-stages with EC 50 values of 0.89 and 0.99 µM, respectively, whereas 3 was more potent with an EC 50 value of 0.15 nM, respectively. Compounds 1 , 4 , and 5 displayed moderate liver-stage antimalarial activity against P. berghei liver schizonts with EC 50 values of 1.1, 0.71, and 0.45 µM, respectively. The threading-based computational method FINDSITE comb2.0 predicted the binding of 1 and 2 to potentially druggable proteins of Plasmodium falciparum , prompting formulation of hypotheses about possible mechanisms of action. Kakeromamide B ( 1 ) was predicted to bind to several Plasmodium actin-like proteins and a sortilin protein suggesting possible interference with parasite invasion of host cells. When 1 was tested in a mammalian actin polymerization assay, it stimulated actin polymerization in a dose-dependent manner, suggesting that 1 does, in fact, interact with actin., Competing Interests: The authors declare no conflict of interest.

Visceral leishmaniasis (VL) and Chagas disease (CD) are caused by kinetoplastid parasites that affect millions of people worldwide and impart a heavy burden against human health. Due to the partial efficacy and toxicity-related limitations of the existing treatments, there is an urgent need to develop novel therapies with superior efficacy and safety profiles to successfully treat these diseases. Herein we report the application of whole-cell phenotypic assays to screen a set of 150,000 compounds against Leishmania donovani, a causative agent of VL, and Trypanosoma cruzi, the causative agent of CD, with the objective of finding new starting points to develop novel drugs to effectively treat and control these diseases. The screening campaign, conducted with the purpose of global open access, identified twelve novel chemotypes with low to sub-micromolar activity against T. cruzi and/or L. donovani. We disclose these hit structures and associated activity with the goal to contribute to the drug discovery community by providing unique chemical tools to probe kinetoplastid biology and as hit-to-lead candidates for drug discovery., (Copyright © 2019 The Authors. Published by Elsevier Ltd.. All rights reserved.)

The intracellular bacteria now known as Wolbachia were first described in filarial worms in the 1970s, but the idea of Wolbachia being used as a macrofilaricidal target did not gain wide attention until the early 2000s, with research in filariae suggesting the requirement of worms for the endosymbiont. This new-found interest prompted the eventual organization of the Anti- Wolbachia Consortium (A-WOL) at the Liverpool School of Tropical Medicine, who, among others have been active in the field of antiwolbachial drug discovery to treat filarial infections. Clinical proof of concept studies using doxycycline demonstrated the utility of the antiwolbachial therapy, but efficacious treatments were of long duration and not safe for all infected. With the advance of robotics, automation, and high-speed computing, the search for superior antiwolbachials shifted away from smaller studies with a select number of antibiotics to high-throughput screening approaches, centered largely around cell-based phenotypic screens due to the rather limited knowledge about, and tools available to manipulate, this bacterium. A concomitant effort was put towards developing validation approaches and in vivo models supporting drug discovery efforts. In this review, we summarize the strategies behind and outcomes of recent large phenotypic screens published within the last 5 years, hit compound validation approaches and promising candidates with profiles superior to doxycycline, including ones positioned to advance into clinical trials for treatment of filarial worm infections.

Two sulfated diterpene glycosides featuring a highly substituted and sterically encumbered cyclopropane ring have been isolated from the marine red alga Peyssonnelia sp. Combination of a wide array of 2D NMR spectroscopic experiments, in a systematic structure elucidation workflow, revealed that peyssonnosides A-B (1-2) represent a new class of diterpene glycosides with a tetracyclo [7.5.0.0 1,10 .0 5,9 ] tetradecane architecture. A salient feature of this workflow is the unique application of quantitative interproton distances obtained from the rotating frame Overhauser effect spectroscopy (ROESY) NMR experiment, wherein the β-d-glucose moiety of 1 was used as an internal probe to unequivocally determine the absolute configuration, which was also supported by optical rotatory dispersion (ORD). Peyssonnoside A (1) exhibited promising activity against liver stage Plasmodium berghei and moderate antimethicillin-resistant Staphylococcus aureus (MRSA) activity, with no cytotoxicity against human keratinocytes. Additionally, 1 showed strong growth inhibition of the marine fungus Dendryphiella salina indicating an antifungal ecological role in its natural environment. The high natural abundance and novel carbon skeleton of 1 suggests a rare terpene cyclase machinery, exemplifying the chemical diversity in this phylogenetically distinct marine red alga.

Cryptosporidiosis is a leading cause of life-threatening diarrhea in young children and causes chronic diarrhea in AIDS patients, but the only approved treatment is ineffective in malnourished children and immunocompromised people. We here use a drug repositioning strategy and identify a promising anticryptosporidial drug candidate. Screening a library of benzoxaboroles comprised of analogs to four antiprotozoal chemical scaffolds under pre-clinical development for neglected tropical diseases for Cryptosporidium growth inhibitors identifies the 6-carboxamide benzoxaborole AN7973. AN7973 blocks intracellular parasite development, appears to be parasiticidal, and potently inhibits the two Cryptosporidium species most relevant to human health, C. parvum and C. hominis. It is efficacious in murine models of both acute and established infection, and in a neonatal dairy calf model of cryptosporidiosis. AN7973 also possesses favorable safety, stability, and PK parameters, and therefore, is an exciting drug candidate for treating cryptosporidiosis.

A series of oligomeric phenols including the known natural product 3,4,3',4'-tetrahydroxy-1,1'-biphenyl (3), the previously synthesized 2,3,8,9-tetrahydroxybenzo[ c]chromen-6-one (4), and eight new related natural products, cladophorols B-I (5-12), were isolated from the Fijian green alga Cladophora socialis and identified by a combination of NMR spectroscopy, mass spectrometric analysis, and computational modeling using DFT calculations. J-resolved spectroscopy and line width reduction by picric acid addition aided in resolving the heavily overlapped aromatic signals. A panel of Gram-positive and Gram-negative pathogens used to evaluate pharmacological potential led to the determination that cladophorol C (6) exhibits potent antibiotic activity selective toward methicillin-resistant Staphylococcus aureus (MRSA) with an MIC of 1.4 μg/mL. Cladophorols B (5) and D-H (7-11) had more modest but also selective antibiotic potency. Activities of cladophorols A-I (4-12) were also assessed against the asexual blood stages of Plasmodium falciparum and revealed cladophorols A (4) and B (5) to have modest activity with EC 50 values of 0.7 and 1.9 μg/mL, respectively.

The union of two powerful transformations, directed C-H activation and decarboxylative cross-coupling, for the enantioselective synthesis of vicinally functionalized alkyl, carbocyclic, and heterocyclic compounds is described. Starting from simple carboxylic acid building blocks, this modular sequence exploits the residual directing group to access more than 50 scaffolds that would be otherwise extremely difficult to prepare. The tactical use of these two transformations accomplishes a formal vicinal difunctionalization of carbon centers in a way that is modular and thus, amenable to rapid diversity incorporation. A simplification of routes to known preclinical drug candidates is presented along with the rapid diversification of an antimalarial compound series., Competing Interests: Conflict of interest statement: S.L.S. is a member of the Board of Directors of the Genomics Institute of the Novartis Research Foundation (GNF); a shareholder and member of the Board of Directors of Jnana Therapeutics; a shareholder of Forma Therapeutics; a shareholder of and adviser to Decibel Therapeutics; an adviser to Eisai, Inc., the Ono Pharma Foundation, and F-Prime Capital Partners; and a Novartis Faculty Scholar. J.-Q.Y. and P.S.B. are cofounders of Vividion., (Copyright © 2019 the Author(s). Published by PNAS.)

Cryptosporidiosis is a leading cause of life-threatening diarrhea in children, and the only currently approved drug is ineffective in malnourished children and immunocompromised people. Large-scale phenotypic screens are ongoing to identify anticryptosporidial compounds, but optimal approaches to prioritize inhibitors and establish a mechanistically diverse drug development pipeline are unknown. Here, we present a panel of medium-throughput mode of action assays that enable testing of compounds in several stages of the Cryptosporidium life cycle. Phenotypic profiles are given for thirty-nine anticryptosporidials. Using a clustering algorithm, the compounds sort by phenotypic profile into distinct groups of inhibitors that are either chemical analogs (i.e. same molecular mechanism of action (MMOA)) or known to have similar MMOA. Furthermore, compounds belonging to multiple phenotypic clusters are efficacious in a chronic mouse model of cryptosporidiosis. This suite of phenotypic assays should ensure a drug development pipeline with diverse MMOA without the need to identify underlying mechanisms.

Malaria and cryptosporidiosis, caused by apicomplexan parasites, remain major drivers of global child mortality. New drugs for the treatment of malaria and cryptosporidiosis, in particular, are of high priority; however, there are few chemically validated targets. The natural product cladosporin is active against blood- and liver-stage Plasmodium falciparum and Cryptosporidium parvum in cell-culture studies. Target deconvolution in P. falciparum has shown that cladosporin inhibits lysyl-tRNA synthetase ( Pf KRS1). Here, we report the identification of a series of selective inhibitors of apicomplexan KRSs. Following a biochemical screen, a small-molecule hit was identified and then optimized by using a structure-based approach, supported by structures of both Pf KRS1 and C. parvum KRS ( Cp KRS). In vivo proof of concept was established in an SCID mouse model of malaria, after oral administration (ED 90 = 1.5 mg/kg, once a day for 4 d). Furthermore, we successfully identified an opportunity for pathogen hopping based on the structural homology between Pf KRS1 and Cp KRS. This series of compounds inhibit Cp KRS and C. parvum and Cryptosporidium hominis in culture, and our lead compound shows oral efficacy in two cryptosporidiosis mouse models. X-ray crystallography and molecular dynamics simulations have provided a model to rationalize the selectivity of our compounds for Pf KRS1 and Cp KRS vs. (human) Hs KRS. Our work validates apicomplexan KRSs as promising targets for the development of drugs for malaria and cryptosporidiosis., Competing Interests: Conflict of interest statement: A patent relating to this work has been filed (PCT/GB2017/051809). F.-J.G. and L.M.S. are employees of GlaxoSmithKline and own shares of the company. M.B.J.-D. and I.A.-B. have shares in The Art of Discovery. Editor D.E.G. is a recent coauthor with two authors of this paper. He published a research article with M.A. in 2015. With E.A.W. he published two research articles in 2016, one research article in 2018, and coauthored a research article forthcoming in 2019. D.E.G. is a coinvestigator with E.A.W. on a 2012–2019 grant., (Copyright © 2019 the Author(s). Published by PNAS.)

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.

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.)

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.

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.

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.)