Hit and lead criteria in drug discovery for infectious diseases of the developing world.

Reducing the burden of infectious diseases that affect people in the developing world requires sustained collaborative drug discovery efforts. The quality of the chemical starting points for such projects is a key factor in improving the likelihood of clinical success, and so it is important to set clear go/no-go criteria for the progression of hit and lead compounds. With this in mind, the Japanese Global Health Innovative Technology (GHIT) Fund convened with experts from the Medicines for Malaria Venture, the Drugs for Neglected Diseases initiative and the TB Alliance, together with representatives from the Bill &Melinda Gates Foundation, to set disease-specific criteria for hits and leads for malaria, tuberculosis, visceral leishmaniasis and Chagas disease. Here, we present the agreed criteria and discuss the underlying rationale.

Distinct phases of blood gene expression pattern through tuberculosis treatment reflect modulation of the humoral immune response.

BACKGROUND: Accurate assessment of treatment efficacy would facilitate clinical trials of new antituberculosis drugs. We hypothesized that early alterations in peripheral immunity could be measured by gene expression profiling in tuberculosis patients undergoing successful conventional combination treatment. METHODS: Ex vivo blood samples from 27 pulmonary tuberculosis patients were assayed at diagnosis and during treatment. RNA was processed and hybridized to Affymetrix GeneChips, to determine expression of over 47,000 transcripts. RESULTS: There were significant ≥ 2-fold changes in expression of >4000 genes during treatment. Rapid, large-scale changes were detected, with down-regulated expression of 1261 genes within the first week, including inflammatory markers such as complement components C1q and C2. This was followed by slower changes in expression of different networks of genes, including a later increase in expression of B-cell markers, transcription factors, and signaling molecules. CONCLUSIONS: The fast initial down-regulation of expression of inflammatory mediators coincided with rapid killing of actively dividing bacilli, whereas slower delayed changes occurred as drugs acted on dormant bacilli and coincided with lung pathology resolution. Measurement of biosignatures during clinical trials of new drugs could be useful predictors of rapid bactericidal or sterilizing drug activity, and would expedite the licensing of new treatment regimens.

Detection and treatment of subclinical tuberculosis.

Reduction of active disease by preventive therapy has the potential to make an important contribution towards the goal of tuberculosis (TB) elimination. This report summarises discussions amongst a Working Group convened to consider areas of research that will be important in optimising the design and delivery of preventative therapies. The Working Group met in Cape Town on 26th February 2012, following presentation of results from the GC11 Grand Challenges in Global Health project to discover drugs for latent TB.

Confronting the scientific obstacles to global control of tuberculosis.

Tuberculosis (TB) is a major threat to global health, recently exacerbated by the emergence of highly drug-resistant forms of the disease-causing pathogen and synergy with HIV/AIDS. In 2006, the Stop TB Partnership published "The global plan to stop TB: 2006--2015," which set out a vision of halving the prevalence of and mortality caused by the disease by 2015, followed by eliminating the disease as a public health problem by 2050. This vision depends on the development of improved diagnostics, simpler treatment, and more effective vaccination. Recently, active translational research pipelines directed toward each of these goals have been established, but improved understanding of the fundamental biology of this complex disease will prove to be the key to radical advances in TB control.

Current strategies for identifying and validating targets for new treatment-shortening drugs for TB.

There is an urgent need for new drugs to treat tuberculosis. During the last forty years the only drugs to have been developed are variations on existing ones, but new drug candidates must offer improvements over existing agents. In particular, we require new drugs having novel mechanisms of action that are active against drug-resistant strains and also kill persistent bacilli, thus shortening the length of chemotherapy. Recent advances in our understanding of the biology of Mycobacterium tuberculosis, in particularly the availability of the genome sequence coupled with development of new genetic tools, have greatly contributed to the discovery of potential drug targets for new antituberculars. However, although many potential new drug targets have been identified, greater effort is required in target validation to show properly that they are essential for bacterial growth and survival. In this review, the current drug development pipeline and the strategies employed to identify and validate novel tuberculosis drug targets are presented.

Evaluation of N-(phenylmethyl)-4-[5-(phenylmethyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridin-4-yl]benzamide inhibitors of Mycobacterium tuberculosis growth.

The biological evaluation of imidazopiperidines as FAS II inhibitors of Mycobacterium tuberculosis growth has been carried out with a view to assessment of potential as lead compounds for the development of a new TB drug. A summary of the hit evaluation and current challenges is described herein.

Functional complementation of the essential gene fabG1 of Mycobacterium tuberculosis by Mycobacterium smegmatis fabG but not Escherichia coli fabG.

Mycolic acids are a key component of the mycobacterial cell wall, providing structure and forming a major permeability barrier. In Mycobacterium tuberculosis mycolic acids are synthesized by type I and type II fatty acid synthases. One of the enzymes of the type II system is encoded by fabG1. We demonstrate here that this gene can be deleted from the M. tuberculosis chromosome only when another functional copy is provided elsewhere, showing that under normal culture conditions fabG1 is essential. FabG1 activity can be replaced by the corresponding enzyme from the closely related species Mycobacterium smegmatis but not by the enzyme from Escherichia coli. M. tuberculosis carrying FabG from M. smegmatis showed no phenotypic changes, and both the mycolic acids and cell wall permeability were unchanged. Thus, M. tuberculosis and M. smegmatis enzymes are interchangeable and do not control the lengths and types of mycolic acids synthesized.

Systems biology and its impact on anti-infective drug development.

Systems biology offers the potential for more effective selection of novel targets for anti-infective drugs. In contrast to conventional reductionist biology, a systems approach allows targets to be viewed in a wider context of the entire physiology of the cell, with the potential to identify key susceptible nodes and to predict synergistic effects of blocking multiple pathways. In addition to the holistic perspective provided by systems biology, the emphasis on quantitative analysis is likely to add further rigour to the process of target selection. Systems biology also offers the potential to incorporate different levels of information into the selection process. Consideration of data from microbial population biology may be important in the context of predicting future drug-resistance profiles associated with targeting a particular pathway, for example. This chapter provides an overview of major themes in the developing field of systems biology, summarising the core technologies and the strategies used to translate datasets into useful quantitative models capable of predicting complex biological behaviour.

Gene-expression patterns in whole blood identify subjects at risk for recurrent tuberculosis.

BACKGROUND: The majority of patients with tuberculosis who comply with appropriate treatment are cured. However, approximately 5% subsequently have a repeat disease episode, usually within 2 years of successful combination therapy. Presently, there is no way of predicting which patients will experience a relapse. METHODS: We identified 10 subjects who had previously experienced recurrent tuberculosis and carefully matched them to cured subjects who had had only 1 episode of tuberculosis, to patients with active tuberculosis, and to latently infected healthy subjects. We compared their ex vivo whole-blood gene-expression profiles by use of DNA array technology and confirmed the results by use of quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR). RESULTS: The 4 clinical tuberculosis groups exhibited distinct patterns of gene expression. The gene-transcript profiles of the patients with recurrent tuberculosis were more similar to those of the patients with active tuberculosis than to those of the cured or latently infected subjects. Discriminant analysis of a training data set showed that 9 genes were sufficient to classify the subjects. We confirmed that measurement of the expression of these genes by qRT-PCR can accurately discriminate between subjects in a test set of samples. CONCLUSIONS: A simple test based on gene-expression patterns may be used as a biomarker of cure while identifying patients who are at risk for relapse. This would facilitate the introduction of new tuberculosis drugs.

Structure-activity relationships at the 5-position of thiolactomycin: an intact (5R)-isoprene unit is required for activity against the condensing enzymes from Mycobacterium tuberculosis and Escherichia coli.

Thiolactomycin inhibits bacterial cell growth through inhibition of the beta-ketoacyl-ACP synthase activity of type II fatty acid synthases. The effect of modifications of the 5-position isoprenoid side chain on both IC(50) and MIC were determined. Synthesis and screening of a structurally diverse set of 5-position analogues revealed very little tolerance for substitution in purified enzyme assays, but a few analogues retained MIC, presumably through another target. Even subtle modifications such as reducing one or both double bonds of the diene were not tolerated. The only permissible structural modifications were removal of the isoprene methyl group or addition of a methyl group to the terminus. Cocrystallization of these two inhibitors with the condensing enzyme from Escherichia coli revealed that they retained the TLM binding mode at the active site with reduced affinity. These results suggest a strict requirement for a conjugated, planar side chain inserting within the condensing enzyme active site.

HemZ is essential for heme biosynthesis in Mycobacterium tuberculosis.

The complete sequence and subsequent annotation of the Mycobacterium tuberculosis genome has allowed the prediction of many genes and gene functions by homology. HemZ is a predicted ferrochelatase which lies in an apparent operon with two genes involved in mycolic acid biosynthesis, mabA and inhA. We tried to construct hemZ deletion mutants in M. tuberculosis using a two-step recombination strategy, but could only delete the chromosomal copy when we provided a second functional copy on an integrating plasmid. We further confirmed that hemZ is essential under normal culture conditions by demonstrating that the integrated copy of hemZ could not be removed if it was the only wild-type allele in the cell. We were able to obtain hemZ mutants by supplementation with hemin but not with protoporphyrin IX or hemoglobin confirming that this gene does have a role in heme biosynthesis and that M. tuberculosis can transport hemin intracelullarly. The hemin auxotroph required 2 mug/ml hemin for growth and rapid loss of viability occurred after withdrawal of hemin. These data confirm the role of hemZ in heme biosynthesis and indicate that heme is an essential requirement for M. tuberculosis.

Inhibitors of type II NADH:menaquinone oxidoreductase represent a class of antitubercular drugs.

Mycobacterium tuberculosis (Mtb) is an obligate aerobe that is capable of long-term persistence under conditions of low oxygen tension. Analysis of the Mtb genome predicts the existence of a branched aerobic respiratory chain terminating in a cytochrome bd system and a cytochrome aa(3) system. Both chains can be initiated with type II NADH:menaquinone oxidoreductase. We present a detailed biochemical characterization of the aerobic respiratory chains from Mtb and show that phenothiazine analogs specifically inhibit NADH:menaquinone oxidoreductase activity. The emergence of drug-resistant strains of Mtb has prompted a search for antimycobacterial agents. Several phenothiazines analogs are highly tuberculocidal in vitro, suppress Mtb growth in a mouse model of acute infection, and represent lead compounds that may give rise to a class of selective antibiotics.

Prospects for new antitubercular drugs.

The inexorable rise in cases of tuberculosis worldwide, fuelled by the HIV epidemic, highlights the need for new drugs and particularly those that can shorten the duration of treatment. Clinical trials of existing broad-spectrum agents such as the fluoroquinolone moxifloxacin are proceeding, on the basis of efficacy in models of infection and preliminary clinical data. These may provide a stopgap, but the real breakthrough will come when novel agents with potent sterilising activity are discovered. Few such novel pre-clinical drug candidates exist and therefore considerable effort is being exerted to employ new tools to identify drug targets essential for survival of Mycobacterium tuberculosis.

Differential gene expression identifies novel markers of CD4+ and CD8+ T cell activation following stimulation by Mycobacterium tuberculosis.

T cell activation in response to antigenic stimulation is a complex process, involving changes in the expression level of a large number of genes. We have used cDNA array technology to characterize the differences in gene expression between human CD4+ and CD8+ T cells. PBMC from six healthy donors were stimulated with live Mycobacterium tuberculosis, and the gene expression profiles of each donor's CD4+ and CD8+ T cells were analyzed separately. ANOVA revealed 518 genes that were consistently differentially expressed between CD4+ and CD8+ T cells. These differentially expressed genes include a combination of well-known, previously characterized genes with a range of biological functions and unknown in silico predicted hypothetical genes. Where possible, the novel genes have been characterized using bioinformatics, and putative transcription factors, signaling molecules, transmembrane, and secreted factors have been identified. A subset of these differentially expressed genes could be exploited as markers of CD4+ and CD8+ T cell activation for use in vaccine trials. These observed differences in the gene expression profile of CD4+ and CD8+ T cells following activation by a human pathogen contribute to an increased understanding of T cell activation and differentiation and the roles these T cell subsets may play in immunity to infection.

Signature gene expression profiles discriminate between isoniazid-, thiolactomycin-, and triclosan-treated Mycobacterium tuberculosis.

Genomic technologies have the potential to greatly increase the efficiency of the drug development process. As part of our tuberculosis drug discovery program, we used DNA microarray technology to profile drug-induced effects in Mycobacterium tuberculosis. Expression profiles of M. tuberculosis treated with compounds that inhibit key metabolic pathways are required as references for the assessment of novel antimycobacterial agents. We have studied the response of M. tuberculosis to treatment with the mycolic acid biosynthesis inhibitors isoniazid, thiolactomycin, and triclosan. Thiolactomycin targets the beta-ketoacyl-acyl carrier protein (ACP) synthases KasA and KasB, while triclosan inhibits the enoyl-ACP reductase InhA. However, controversy surrounds the precise mode of action of isoniazid, with both InhA and KasA having been proposed as the primary target. We have shown that although the global response profiles of isoniazid and thiolactomycin are more closely related to each other than to that of triclosan, there are differences that distinguish the mode of action of these two drugs. In addition, we have identified two groups of genes, possibly forming efflux and detoxification systems, through which M. tuberculosis may limit the effects of triclosan. We have developed a mathematical model, based on the expression of 21 genes, which is able to perfectly discriminate between isoniazid-, thiolactomycin-, or triclosan-treated M. tuberculosis. This model is likely to prove invaluable as a tool to improve the efficiency of our drug development programs by providing a means to rapidly confirm the mode of action of thiolactomycin analogues or novel InhA inhibitors as well as helping to translate enzyme activity into whole-cell activity.

Progress in TB drug development and what is still needed.

Highly effective drugs for treating TB were introduced over 30 years ago, yet deaths from the disease continue to increase. New tools are needed, including drugs with activity against multi-drug resistant strains of Mycobacterium tuberculosis. Agents that reduce the duration and complexity of the current therapy would have a major impact on compliance and overall cure rate. In recent years, our understanding of the tubercle bacillus and its interaction with the human host has improved dramatically, particularly with the publication in 1998 of the complete genome sequence of M. tuberculosis H37Rv. New genetic tools have been developed and we can now ascertain the function of individual genes. Thus, many potential drug targets have been identified and a number demonstrated to be essential. Several lead compounds have been found, as well as a potential drug candidate, the nitroimidazopyran PA-824. A far greater effort is needed to translate basic research into drug discovery programmes. High throughput screening and rational design must be employed to find lead compounds acting against well-validated targets and a substantial increase in resources devoted to medicinal chemistry is required to take these leads and turn them into drugs. Models of mycobacterial persistence, in which compounds with potent sterilizing activity can be rapidly analysed, must be characterized. Finally, surrogate markers that give an early indication of treatment outcome would facilitate clinical trials.

Associations between toll-like receptors and interleukin-4 in the lungs of patients with tuberculosis.

Toll-like receptors (TLRs) are implicated in the intracellular killing of Mycobacterium tuberculosis, and their expression is modulated by interleukin-4 (IL-4) in vitro. Our aim was to examine the expression of TLRs at the site of pathology in tuberculous lung granulomas and to explore the effect of the immune response on TLR expression. Immunohistochemistry was performed on lung granulomas from nine patients with tuberculosis undergoing lobectomy for haemoptysis. All nine patients expressed all of the TLRs studied (TLRs 1-5 and 9), whereas only five out of the nine patients had any granulomas positive for IL-4. Statistical analysis of TLR and cytokine staining patterns in 183 individual granulomas from the nine patients revealed significant associations between pairs of receptors and IL-4. A positive association between TLR2 and TLR4 (P < 0.0001) and a negative association between TLR2 and IL-4 (P < 0.0001) was observed. The associations between TLRs 1, 5, and 9 were significantly different in IL-4-negative compared with IL-4-positive patients. In conclusion, TLRs are expressed by various cell types in the human tuberculous lung, and their expression patterns are reflected by differences in the immune response.

Gene replacement in mycobacteria by using incompatible plasmids.

A simple and efficient delivery system was developed for making targeted gene knockouts in Mycobacterium smegmatis. This delivery system relies on the use of a pair of replicating plasmids, which are incompatible. Incompatible plasmids share elements of the same replication machinery and so compete with each other during both replication and partitioning into daughter cells. Such plasmids can be maintained together in the presence of antibiotics; however, removal of selection leads to the loss of one or both plasmids. For mutagenesis, two replicating plasmids based on pAL5000 are introduced; one of these plasmids carries a mutated allele of the targeted gene. Homologous recombination is allowed to take place, and either one or both of the vectors are lost through the pressure of incompatibility, allowing the phenotypic effects of the mutant to be studied. Several different plasmid combinations were tested to optimize loss in the absence of antibiotic selection. pAL5000 carries two replication genes (repA and repB), which act in trans, and the use of vectors that each lack one rep gene and complement each other resulted in the loss of both plasmids in M. smegmatis and Mycobacterium bovis BCG. The rate of loss was increased by the incorporation of an additional incompatibility region in one of the plasmids. To facilitate cloning when the system was used, we constructed plasmid vector pairs that allow simple addition of selection and screening genes on flexible gene cassettes. Using this system, we demonstrated that M. smegmatis pyrF mutants could be isolated at high frequency. This method should also be useful in other species in which pAL5000 replicates, including Mycobacterium tuberculosis.