Jose Garcia-Bustos

Thousands of chemical starting points for antimalarial lead identification

Malaria is a devastating infection caused by protozoa of the genus Plasmodium. Drug resistance is widespread, no new chemical class of antimalarials has been introduced into clinical practice since 1996 and there is a recent rise of parasite strains with reduced sensitivity to the newest drugs. We screened nearly 2 million compounds in GlaxoSmithKline’s chemical library for inhibitors of P. falciparum, of which 13,533 were confirmed to inhibit parasite growth by at least 80% at 2 µM concentration. More than 8,000 also showed potent activity against the multidrug resistant strain Dd2. Most (82%) compounds originate from internal company projects and are new to the malaria community. Analyses using historic assay data suggest several novel mechanisms of antimalarial action, such as inhibition of protein kinases and host–pathogen interaction related targets. Chemical structures and associated data are hereby made public to encourage additional drug lead identification efforts and further research into this disease.

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.

Two-dimensional single-cell patterning with one cell per well driven by surface acoustic waves

In single-cell analysis, cellular activity and parameters are assayed on an individual, rather than population-average basis. Essential to observing the activity of these cells over time is the ability to trap, pattern and retain them, for which previous single-cell-patterning work has principally made use of mechanical methods. While successful as a long-term cell-patterning strategy, these devices remain essentially single use. Here we introduce a new method for the patterning of multiple spatially separated single particles and cells using high-frequency acoustic fields with one cell per acoustic well. We characterize and demonstrate patterning for both a range of particle sizes and the capture and patterning of cells, including human lymphocytes and red blood cells infected by the malarial parasite Plasmodium falciparum. This ability is made possible by a hitherto unexplored regime where the acoustic wavelength is on the same order as the cell dimensions.

Annotated draft genomic sequence from a Streptococcus pneumoniae type 19F clinical isolate.

The public availability of numerous microbial genomes is enabling the analysis of bacterial biology in great detail and with an unprecedented, organism-wide and taxon-wide, broad scope. Streptococcus pneumoniae is one of the most important bacterial pathogens throughout the world. We present here sequences and functional annotations for 2.1-Mbp of pneumococcal DNA, covering more than 90% of the total estimated size of the genome. The sequenced strain is a clinical isolate resistant to macrolides and tetracycline. It carries a type 19F capsular locus, but multilocus sequence typing for several conserved genetic loci suggests that the strain sequenced belongs to a pneumococcal lineage that most often expresses a serotype 15 capsular polysaccharide. A total of 2,046 putative open reading frames (ORFs) longer than 100 amino acids were identified (average of 1,009 bp per ORF), including all described two-component systems and aminoacyl tRNA synthetases. Comparisons to other complete, or nearly complete, bacterial genomes were made and are presented in a graphical form for all the predicted proteins.

P. falciparum in vitro killing rates allow to discriminate between different antimalarial mode-of-action.

Chemotherapy is still the cornerstone for malaria control. Developing drugs against Plasmodium parasites and monitoring their efficacy requires methods to accurately determine the parasite killing rate in response to treatment. Commonly used techniques essentially measure metabolic activity as a proxy for parasite viability. However, these approaches are susceptible to artefacts, as viability and metabolism are two parameters that are coupled during the parasite life cycle but can be differentially affected in response to drug actions. Moreover, traditional techniques do not allow to measure the speed-of-action of compounds on parasite viability, which is an essential efficacy determinant. We present here a comprehensive methodology to measure in vitro the direct effect of antimalarial compounds over the parasite viability, which is based on limiting serial dilution of treated parasites and re-growth monitoring. This methodology allows to precisely determine the killing rate of antimalarial compounds, which can be quantified by the parasite reduction ratio and parasite clearance time, which are key mode-of-action parameters. Importantly, we demonstrate that this technique readily permits to determine compound killing activities that might be otherwise missed by traditional, metabolism-based techniques. The analysis of a large set of antimalarial drugs reveals that this viability-based assay allows to discriminate compounds based on their antimalarial mode-of-action. This approach has been adapted to perform medium throughput screening, facilitating the identification of fast-acting antimalarial compounds, which are crucially needed for the control and possibly the eradication of malaria.

Ribosomal P-protein Stalk Function Is Targeted by Sordarin Antifungals

Sordarin derivatives are remarkably selective inhibitors of fungal protein synthesis. Available evidence points to a binding site for these inhibitors on elongation factor 2, but high affinity binding requires the presence of ribosomes. The gene mutated in one of the two isolated complementation groups ofSaccharomyces cerevisiae mutants resistant to the sordarin derivative GM193663 has now been identified. It is RPP0, encoding the essential protein of the large ribosomal subunit stalk rpP0. Resistant mutants are found to retain most of the binding capacity for the drug, indicating that mutations in rpP0 endow the ribosome with the capacity to perform translation elongation in the presence of the inhibitor. Other proteins of the ribosomal stalk influence the expression of resistance, pointing to a wealth of interactions between stalk components and elongation factors. The involvement of multiple elements of the translation machinery in the mode of action of sordarin antifungals may explain the large selectivity of these compounds, even though the individual target components are highly conserved proteins.

Fast Standardized Therapeutic-Efficacy Assay for Drug Discovery against Tuberculosis

ABSTRACT Murine models of Mycobacteriumtuberculosis infection are essential tools in drug discovery. Here we describe a fast standardized 9-day acute assay intended to measure the efficacy of drugs against M. tuberculosis growing in the lungs of immunocompetent mice. This assay is highly reproducible, allows good throughput, and was validated for drug lead optimization using isoniazid, rifampin, ethambutol, pyrazinamide, linezolid, and moxifloxacin.

A new in vivo screening paradigm to accelerate antimalarial drug discovery.

The emergence of resistance to available antimalarials requires the urgent development of new medicines. The recent disclosure of several thousand compounds active in vitro against the erythrocyte stage of Plasmodium falciparum has been a major breakthrough, though converting these hits into new medicines challenges current strategies. A new in vivo screening concept was evaluated as a strategy to increase the speed and efficiency of drug discovery projects in malaria. The new in vivo screening concept was developed based on human disease parameters, i.e. parasitemia in the peripheral blood of patients on hospital admission and parasite reduction ratio (PRR), which were allometrically down-scaled into P. berghei-infected mice. Mice with an initial parasitemia (P0) of 1.5% were treated orally for two consecutive days and parasitemia measured 24 h after the second dose. The assay was optimized for detection of compounds able to stop parasite replication (PRR = 1) or induce parasite clearance (PRR >1) with statistical power >99% using only two mice per experimental group. In the P. berghei in vivo screening assay, the PRR of a set of eleven antimalarials with different mechanisms of action correlated with human-equivalent data. Subsequently, 590 compounds from the Tres Cantos Antimalarial Set with activity in vitro against P. falciparum were tested at 50 mg/kg (orally) in an assay format that allowed the evaluation of hundreds of compounds per month. The rate of compounds with detectable efficacy was 11.2% and about one third of active compounds showed in vivo efficacy comparable with the most potent antimalarials used clinically. High-throughput, high-content in vivo screening could rapidly select new compounds, dramatically speeding up the discovery of new antimalarial medicines. A global multilateral collaborative project aimed at screening the significant chemical diversity within the antimalarial in vitro hits described in the literature is a feasible task.

Identification and Validation of Tetracyclic Benzothiazepines as Plasmodium falciparum Cytochrome bc1 Inhibitors

Here we report the discovery of tetracyclic benzothiazepines (BTZs) as highly potent and selective antimalarials along with the identification of the Plasmodium falciparum cytochrome bc(1) complex as the primary functional target of this novel compound class. Investigation of the structure activity relationship within this previously unexplored chemical scaffold has yielded inhibitors with low nanomolar activity. A combined approach employing genetically modified parasites, biochemical profiling, and resistance selection validated inhibition of cytochrome bc(1) activity, an essential component of the parasite respiratory chain and target of the widely used antimalarial drug atovaquone, as the mode of action of this novel compound class. Resistance to atovaquone is eroding the efficacy of this widely used antimalarial drug. Intriguingly, BTZ-based inhibitors retain activity against atovaquone resistant parasites, suggesting this chemical class may provide an alternative to atovaquone in combination therapy.

Candida albicans infection enhances immunosuppression induced by cyclophosphamide by selective priming of suppressive myeloid progenitors for NO production

Systemic infections caused by fungi after cytoreductive therapies are especially difficult to deal with in spite of currently available antimicrobials. However, little is known about the effects of fungi on the immune system of immunosuppressed hosts. We have addressed this by studying the in vitro T cell responses after systemic infection with Candida albicans in cyclophosphamide-treated mice. After cyclophosphamide treatment, a massive splenic colonization of the spleens, but not lymph nodes, by immature myeloid progenitor (Ly-6G(+)CD11b(+))cells is observed. These cells are able to suppress proliferation of T lymphocytes via a nitric oxide (NO)-dependent mechanism. Systemic infection with a sublethal dose of C. albicans did not cause immunosuppression per se but strongly increased NO-dependent suppression in cyclophosphamide-treated mice, by selective priming of suppressive myeloid progenitors (Ly-6G(+)CD11b(+)CD31(+)CD40(+)WGA(+)CD117(low/-)CD34(low/-)) for iNOS protein expression. The results indicate that systemic C. albicans infection can augment the effects of immunosuppressive therapies by promoting functional changes in immunosuppressive cells.