Novel inhibitors that target bacterial virulence identified via HTS against intra-macrophage survival of Shigella flexneri.

Shigella flexneri is a facultative intracellular pathogen that causes shigellosis, a human diarrheal disease characterized by the destruction of the colonic epithelium. Novel antimicrobial compounds to treat infections are urgently needed due to the proliferation of bacterial antibiotic resistance and lack of new effective antimicrobials in the market. Our approach to find compounds that block the Shigella virulence pathway has three potential advantages: (i) resistance development should be minimized due to the lack of growth selection pressure, (ii) no resistance due to environmental antibiotic exposure should be developed since the virulence pathways are not activated outside of host infection, and (iii) the normal intestinal microbiota, which do not have the targeted virulence pathways, should be unharmed. We chose to utilize two phenotypic assays, inhibition of Shigella survival in macrophages and Shigella growth inhibition (minimum inhibitory concentration), to interrogate the 1.7 M compound screening collection subset of the GlaxoSmithKline drug discovery chemical library. A number of secondary assays on the hit compounds resulting from the primary screens were conducted, which, in combination with chemical, structural, and physical property analyses, narrowed the final hit list to 44 promising compounds for further drug discovery efforts. The rapid development of antibiotic resistance is a critical problem that has the potential of returning the world to a "pre-antibiotic" type of environment, where millions of people will die from previously treatable infections. One relatively newer approach to minimize the selection pressures for the development of resistance is to target virulence pathways. This is anticipated to eliminate any resistance selection pressure in environmental exposure to virulence-targeted antibiotics and will have the added benefit of not affecting the non-virulent microbiome. This paper describes the development and application of a simple, reproducible, and sensitive assay to interrogate an extensive chemical library in high-throughput screening format for activity against the survival of Shigella flexneri 2457T-nl in THP-1 macrophages. The ability to screen very large numbers of compounds in a reasonable time frame (~1.7 M compounds in ~8 months) distinguishes this assay as a powerful tool in further exploring new compounds with intracellular effect on S. flexneri or other pathogens with similar pathways of pathogenesis. The assay utilizes a luciferase reporter which is extremely rapid, simple, relatively inexpensive, and sensitive and possesses a broad linear range. The assay also utilized THP-1 cells that resemble primary monocytes and macrophages in morphology and differentiation properties. THP-1 cells have advantages over human primary monocytes or macrophages because they are highly plastic and their homogeneous genetic background minimizes the degree of variability in the cell phenotype (1). The intracellular and virulence-targeted selectivity of our methodology, determined via secondary screening, is an enormous advantage. Our main interest focuses on hits that are targeting virulence, and the most promising compounds with adequate physicochemical and drug metabolism and pharmacokinetic (DMPK) properties will be progressed to a suitable in vivo shigellosis model to evaluate the therapeutic potential of this approach. Additionally, compounds that act via a host-directed mechanism could be a promising source for further research given that it would allow a whole new, specific, and controlled approach to the treatment of diseases caused by some pathogenic bacteria.

Gametocytes from K13 Propeller Mutant Plasmodium falciparum Clinical Isolates Demonstrate Reduced Susceptibility to Dihydroartemisinin in the Male Gamete Exflagellation Inhibition Assay.

Mutations in the kelch propeller domain (K13 propeller) of Plasmodium falciparum parasites from Southeast Asia are associated with reduced susceptibility to artemisinin. We exposed in vitro-cultured stage V gametocytes from Cambodian K13 propeller mutant parasites to dihydroartemisinin and evaluated the inhibition of male gamete formation in an in vitro exflagellation inhibition assay (EIA). Gametocytes with the R539T and C580Y K13 propeller alleles were less susceptible to dihydroartemisinin and had significantly higher 50% inhibitory concentrations (IC50s) than did gametocytes with wild-type alleles.

Predicting transmission blocking potential of anti-malarial compounds in the Mosquito Feeding Assay using Plasmodium falciparum Male Gamete Inhibition Assay.

Plasmodium falciparum Standard Membrane Feeding Assay (PfSMFA) is the current gold standard mosquito based confirmatory transmission blocking (TrB) assay for human malaria. However, owing to its complexity only selected gametocytocidal molecules are progressed into SMFA. Predictive tools for evaluation of TrB behavior of compounds in SMFA would be extremely beneficial, but lack of substantially large data sets from many mosquito feeds preempts the ability to perform correlations between outcomes from in vitro assays and SMFA. Here, a total of 44 different anti-malarial compounds were screened for inhibitory effect on male gamete formation in exflagellation inhibition assay (EIA) and the same drug-treated parasites were fed to mosquitoes in SMFA. Regression analysis was performed between outcomes of the two assays and regression models were applied to a randomly selected validation set of four compounds indicating no overfitting and good predictive power. In addition, the pIC50 for 11 different compounds obtained in the EIA was also correlated with pIC50's in SMFA. Resulting regression models provided pIC50 predictions in SMFA with reasonably good accuracy thereby demonstrating the use of a simple in vitro assay to predict TrB of molecules in a complex mosquito based assay.

Hundreds of dual-stage antimalarial molecules discovered by a functional gametocyte screen.

Plasmodium falciparum stage V gametocytes are responsible for parasite transmission, and drugs targeting this stage are needed to support malaria elimination. We here screen the Tres Cantos Antimalarial Set (TCAMS) using the previously developed P. falciparum female gametocyte activation assay (Pf FGAA), which assesses stage V female gametocyte viability and functionality using Pfs25 expression. We identify over 400 compounds with activities <2 µM, chemically classified into 57 clusters and 33 singletons. Up to 68% of the hits are chemotypes described for the first time as late-stage gametocyte-targeting molecules. In addition, the biological profile of 90 compounds representing the chemical diversity is assessed. We confirm in vitro transmission-blocking activity of four of the six selected molecules belonging to three distinct scaffold clusters. Overall, this TCAMS gametocyte screen provides 276 promising antimalarial molecules with dual asexual/sexual activity, representing starting points for target identification and candidate selection.

Swallow Prognosis and Follow-Up Protocol in Infantile Onset Pompe Disease.

Oro-pharyngeal dysphagia commonly occurs in patients with infantile onset Pompe disease (IOPD), which is a rare recessive neuromuscular disorder caused by deficiency of the lysosomal enzyme acid alpha-glucosidase. Without treatment, death occurs by 1 year of age from cardiorespiratory failure. Enzyme replacement therapy (ERT) has been used to increase life expectancy, however emerging developmental and medical morbidities have become apparent. A case file review of the feeding outcomes of 12 patients with IOPD, managed at a single tertiary centre, was undertaken. Two types of assessment had been completed: clinical feeding assessment (CFA) and instrumental videofluoroscopy swallow study (VFSS). A rating of functional oral intake at every Speech and Language Therapy feeding assessment from initial diagnosis to the most recent assessment was applied using the functional oral intake scale (FOIS).Results indicate, initial diagnosis VFSS predicts long-term feeding outcomes. Even if a patient had an improvement in oral feeding after diagnosis, over a period of time their oral intake returned to the initial diagnosis VFSS level or below. All patients (8/8) who required non-oral feeding support under 6 months of age went on to require non-oral feeding support, even if they had periods of full oral feeding. CRIM negative status predicted significant oral feeding difficulties. An evidence-based follow-up protocol was developed. The information is used at diagnosis to counsel families regarding feeding prognosis and consideration of early gastrostomy when cardiac status allows safe anaesthesia. The results reinforce that feeding changes over time and patients require on-going dysphagia monitoring.

A novel validated assay to support the discovery of new anti-malarial gametocytocidal agents.

BACKGROUND: Drugs that kill or inhibit Plasmodium gametocytes in the human host could potentially synergize the impact of other chemotherapeutic interventions by blocking transmission. To develop such agents, reliable methods are needed to study the in vitro activity of compounds against gametocytes. This study describes a novel assay for characterizing the activity of anti-malarial drugs against the later stages of Plasmodium falciparum gametocyte development using real-time PCR (qPCR). METHODS: Genes previously reported to be transcribed at the different sexual stages of the gametocytogenesis were selected for study and their mRNA expression was measured in a gametocytogenesis course by qPCR. Genes mainly expressed in the later stages of gametocyte development were used as a surrogate measurement of drug activity. To distinguish between cidal and static drug effects, two different experiments were performed in parallel, one with constant drug pressure throughout the experiment (144 h), and another in which the gametocyte cultures were exposed to the compound for only 48 h. RESULTS: Four P. falciparum genes coding for proteins Pf77, ROM3, Pfs25, and Pfg377 with transcription specific for late-stage gametocyte development were identified. The in vitro anti-malarial activity of compounds against such gametocytes was assessed by measuring mRNA levels of these genes using qPCR. The assay was validated against standard anti-malarial drugs (epoxomicin, dihydroartemisinin, chloroquine, thiostrepton, and methylene blue) and compounds from the GSK compound library with known anti-gametocyte activity. CONCLUSIONS: This study describes a novel assay for characterizing the activity of anti-malarial drugs against the later stages of P. falciparum gametocyte development using qPCR in genetically unmodified parasites. The method described is a reliable and user-friendly technique with a medium throughput that could be easily implemented in any laboratory.

A New Set of Chemical Starting Points with Plasmodium falciparum Transmission-Blocking Potential for Antimalarial Drug Discovery.

The discovery of new antimalarials with transmission blocking activity remains a key issue in efforts to control malaria and eventually eradicate the disease. Recently, high-throughput screening (HTS) assays have been successfully applied to Plasmodium falciparum asexual stages to screen millions of compounds, with the identification of thousands of new active molecules, some of which are already in clinical phases. The same approach has now been applied to identify compounds that are active against P. falciparum gametocytes, the parasite stage responsible for transmission. This study reports screening results for the Tres Cantos Antimalarial Set (TCAMS), of approximately 13,533 molecules, against P. falciparum stage V gametocytes. Secondary confirmation and cytotoxicity assays led to the identification of 98 selective molecules with dual activity against gametocytes and asexual stages. Hit compounds were chemically clustered and analyzed for appropriate physicochemical properties. The TCAMS chemical space around the prioritized hits was also studied. A selection of hit compounds was assessed ex vivo in the standard membrane feeding assay and demonstrated complete block in transmission. As a result of this effort, new chemical structures not connected to previously described antimalarials have been identified. This new set of compounds may serve as starting points for future drug discovery programs as well as tool compounds for identifying new modes of action involved in malaria transmission.

Fueling open-source drug discovery: 177 small-molecule leads against tuberculosis.

With the aim of fuelling open-source, translational, early-stage drug discovery activities, the results of the recently completed antimycobacterial phenotypic screening campaign against Mycobacterium bovis BCG with hit confirmation in M. tuberculosis H37Rv were made publicly accessible. A set of 177 potent non-cytotoxic H37Rv hits was identified and will be made available to maximize the potential impact of the compounds toward a chemical genetics/proteomics exercise, while at the same time providing a plethora of potential starting points for new synthetic lead-generation activities. Two additional drug-discovery-relevant datasets are included: a) a drug-like property analysis reflecting the latest lead-like guidelines and b) an early lead-generation package of the most promising hits within the clusters identified.

Activity of clinically relevant antimalarial drugs on Plasmodium falciparum mature gametocytes in an ATP bioluminescence "transmission blocking" assay.

BACKGROUND: Current anti-malarial drugs have been selected on the basis of their activity against the symptom-causing asexual blood stage of the parasite. Which of these drugs also target gametocytes, in the sexual stage responsible for disease transmission, remains unknown. Blocking transmission is one of the main strategies in the eradication agenda and requires the identification of new molecules that are active against gametocytes. However, to date, the main limitation for measuring the effect of molecules against mature gametocytes on a large scale is the lack of a standardized and reliable method. Here we provide an efficient method to produce and purify mature gametocytes in vitro. Based on this new procedure, we developed a robust, affordable, and sensitive ATP bioluminescence-based assay. We then assessed the activity of 17 gold-standard anti-malarial drugs on Plasmodium late stage gametocytes. METHODS AND FINDINGS: Difficulties in producing large amounts of gametocytes have limited progress in the development of malaria transmission blocking assays. We improved the method established by Ifediba and Vanderberg to obtain viable, mature gametocytes en masse, whatever the strain used. We designed an assay to determine the activity of antimalarial drugs based on the intracellular ATP content of purified stage IV-V gametocytes after 48 h of drug exposure in 96/384-well microplates. Measurements of drug activity on asexual stages and cytotoxicity on HepG2 cells were also obtained to estimate the specificity of the active drugs. CONCLUSIONS: The work described here represents another significant step towards determination of the activity of new molecules on mature gametocytes of any strain with an automated assay suitable for medium/high-throughput screening. Considering that the biology of the forms involved in the sexual and asexual stages is very different, a screen of our 2 million-compound library may allow us to discover novel anti-malarial drugs to target gametocyte-specific metabolic pathways.

Cyclopropyl carboxamides, a chemically novel class of antimalarial agents identified in a phenotypic screen.

Malaria is one of the deadliest infectious diseases in the world, with the eukaryotic parasite Plasmodium falciparum causing the most severe form of the disease. Discovery of new classes of antimalarial drugs has become an urgent task to counteract the increasing problem of drug resistance. Screening directly for compounds able to inhibit parasite growth in vitro is one of the main approaches the malaria research community is now pursuing for the identification of novel antimalarial drug leads. Very recently, thousands of compounds with potent activity against the parasite P. falciparum have been identified and information about their molecular descriptors, antiplasmodial potency, and cytotoxicity is publicly available. Now the challenges are how to identify the most promising chemotypes for further development and how best to progress these compounds through a lead optimization program to generate antimalarial drug candidates. We report here the first chemical series to be characterized from one of those screenings, a completely novel chemical class with the generic name cyclopropyl carboxamides that has never before been described as having antimalarial or other pharmacological activities. Cyclopropyl carboxamides are potent inhibitors of drug-sensitive and -resistant strains of P. falciparum in vitro and show in vivo oral efficacy in malaria mouse models. In the present work, we describe the biological characterization of this chemical family, showing that inhibition of their still unknown target has very favorable pharmacological consequences but the compounds themselves seem to select for resistance at a high frequency.

Characterization of the phospholipidogenic potential of 4(1H)-pyridone antimalarial derivatives.

Drug-induced phospholipidosis (PLD) is characterized by the excessive accumulation of phospholipids in lysosomes. It is accompanied by intracellular retention of drug that could be associated with increased cytotoxicity. Drug-induced PLD is recognized as a significant challenge for drug development, depending on the severity of the effect it could be reversible or caused cell death. Therefore, the identification at early stages of drug discovery of the potential to induce PLD can be advantageous for selecting improved development candidates. PLD has commonly been associated with cationic amphiphilic drugs (CADs) composed by a hydrophobic ring structure and a hydrophilic side chain with a charged amine group. 4(1H)-pyridone derivatives are a family of antimalarial agents that act as potent selective inhibitors of Plasmodium falciparum mitochondrial function and according to their chemical structure might be considered to be CADs. In the present study, the potential of 4(1H)-pyridone derivatives to induce PLD in vitro and their general cytotoxicity properties were investigated. A cell-based fluorescence assay using the fluorescent phospholipid probe NBD-PE [N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt] was established. Five PLD-inducing reference compounds and six negative reference compounds were evaluated in vitro in HepG2 cell line. The pyridones tested were ranked by using a chloroquine-equivalent scale (chloroquine constituting a well-known antimalarial drug that acts as a potent inducer of lysosomal storage of phospholipids in both cell cultures and in vivo studies). The present findings indicate that these novel chemical antimalarial compounds are not PLD inducers despite to be considered structurally as CADs. Furthermore, none of the compounds tested showed significant cytotoxicity at their maximum solubility.

Importation et destin de la première théorie des germes au Mexique: développement des premières recherches sur la fièvre jaune dans les années 1880 / Importation and destination of the first germ theory in Mexico: development of the first researches into yellow fever in the 1880s

Ce travail s'inscrit dans le cadre de la discussion du transfert de connaissances entre l'Occident et l'Amérique latine, le Mexique en particulier. Nous essayerons de montrer les enjeux internationaux et locaux qui ont encouragé l'importation de la théorie des germes au Mexique pendant les années 1880. Par ailleurs, on montrera quelles ont été les difficultés conceptuelles et matérielles pour incorporer la théorie des germes et les techniques bactériologiques encore en train de se bâtir en Europe. Au moyen de deux exemples, on essayera de mettre en évidence les intentions politiques des médecins mexicains, à l'origine de l'étude des maladies infectieuses au Mexique.

This article aims to contribute to discussions about knowledge transfer between the West and Latin America, especially Mexico. We seek to show the international and local efforts to foster the importation of germ theory during the 1880s. We also highlight the conceptual and material hurdles which stood in the way of the incorporation of this theory and bacteriology techniques at a time when they were still being developed in Europe. Two examples are given as evidence of the political motivations of Mexican physicians in studying infectious diseases in that country.

Félix d´Hérelle. Aportes a la biografía de un pasteuriano en México (1904-1911) / Félix d´Hérelle. Contributions to the biography of a Pasteurian in México (1904-1911)

Presenta una aportación a la biografía de Félix d´Hérelle (1873-1949), así como algunas precisiones sobre su paso por México y las posibles implicaciones que esta estadía de cuatro años pudo haber tenido en la conformación de su pensamiento y de su carrera profesional.(AU)