Genetic Approaches to Facilitate Antibacterial Drug Development

Schnappinger, Dirk

doi:10.1101/cshperspect.a021139

Cited by 16 publications

(13 citation statements)

References 123 publications

(127 reference statements)

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“…The development of whole‐cell screenings based on bacterial biosensors sensitized to inhibitors of specific targets represents a novel strategy to identify new antibacterial drugs (Hughes and Karlen, 2014; Schnappinger, 2015). As DprE1 has been shown to be a valid and vulnerable target for M. tuberculosis (Makarov et al ., 2009), we decided to use our set of promoters to develop an assay to identify molecules able to inhibit its activity.…”

Section: Resultsmentioning

confidence: 99%

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Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

Boldrin

Degiacomi

Serafini

et al. 2017

Microbial Biotechnology

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SummaryA range of regulated gene expression systems has been developed for mycobacteria in the last few years to facilitate the study of essential genes, validate novel drug targets and evaluate their vulnerability. Among these, the TetR/Pip‐OFF repressible promoter system was successfully used in several mycobacterial species both in vitro and in vivo. In the first version of the system, the repressible promoter was Pptr, a strong Pip‐repressible promoter of Streptomyces pristinaespiralis, which might hamper effective downregulation of genes with a low basal expression level. Here, we report an enhanced system that allows more effective control of genes expressed at low level. To this end, we subjected Pptr to targeted mutagenesis and produced 16 different promoters with different strength. Three of them, weaker than the wild‐type promoter, were selected and characterized showing that they can indeed improve the performances of TetR/Pip‐OFF repressible system both in vitro and in vivo increasing its stringency. Finally, we used these promoters to construct a series of bacterial biosensors with different sensitivity to DprE1 inhibitors and developed a whole‐cell screening assay to identify inhibitors of this enzyme.

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Section: Resultsmentioning

confidence: 99%

“…Tighty regulated gene expression systems are powerful tools widely used to study essential genes, validate drug targets and evaluate their vulnerability (Schnappinger, 2015). In recent years, several such systems were developed for mycobacteria [reviewed in (Schnappinger and Ehrt, 2014;Choudhary et al, 2016)].…”

Section: Introductionmentioning

confidence: 99%

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

Boldrin

Degiacomi

Serafini

et al. 2017

Microbial Biotechnology

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“…We must refrain from projecting the biology deciphered in one persistence model to other persistence models, for fear that we oversimplify phenotypic tolerance, much as Jacques Monod oversimplified biochemical unity when he declared, “ anything found to be true of E. coli must also be true of elephants ” (290). We need to focus time and resources on developing antibiotics that target mycobacteria in in vitro states that have relevance to persisters found during human tuberculosis, whose structures are accurately understood under the conditions of the assays, that exert bona fide bactericidal activity against nonreplicating mycobacteria, and that have a structure-activity relationship which enables at least one of its derivative compounds to make it through the gauntlet of drug development, or at least, to become an informative tool for chemical biology to guide our understanding of bacterial persistence (291, 292). …”

Section: Future Studies and Conclusionmentioning

confidence: 99%

Targeting Phenotypically TolerantMycobacterium tuberculosis

2017

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While the immune system is credited with averting tuberculosis in billions of individuals exposed to Mycobacterium tuberculosis, the immune system is also culpable for tempering the ability of antibiotics to deliver swift and durable cure of disease. In individuals afflicted with tuberculosis, host immunity produces diverse microenvironmental niches that support suboptimal growth, or complete growth arrest, of M. tuberculosis. The physiological state of nonreplication in bacteria is associated with phenotypic drug tolerance. Many of these host microenvironments, when modeled in vitro by carbon starvation, complete nutrient starvation, stationary phase, acidic pH, reactive nitrogen intermediates, hypoxia, biofilms, and withholding streptomycin from the streptomycin-addicted strain SS18b, render M. tuberculosis profoundly tolerant to many of the antibiotics that are given to tuberculosis patients in a clinical setting. Targeting nonreplicating persisters is anticipated to reduce the duration of antibiotic treatment and rate of post-treatment relapse. Some promising drugs to treat tuberculosis, such as rifampicin and bedaquiline, only kill nonreplicating M. tuberculosis in vitro at concentrations far greater than their minimal inhibitory concentrations against replicating bacilli. There is an urgent demand to identify which of the currently used antibiotics, and which of the molecules in academic and corporate screening collections, have potent bactericidal action on nonreplicating M. tuberculosis. With this goal, we review methods of high throughput screening to target nonreplicating M. tuberculosis and methods to progress candidate molecules. A classification based on structures and putative targets of molecules that have been reported to kill nonreplicating M. tuberculosis revealed a rich diversity in pharmacophores. However, few of these compounds were tested under conditions that would exclude the impact of adsorbed compound acting during the recovery phase of the assay, and few were tested under more than one condition imposing nonreplication. That nonreplicating mycobacteria are metabolically active was corroborated by their susceptibility to several antibiotics and tool compounds that target the synthesis of lipids, RNA, DNA, proteins, and peptidoglycan.

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“…Traditional biochemical methods for example, focus on primary compound-target interaction and use of binding affinity as a surrogate of target identification that, while specific, is not always functional or sufficiently sensitive to yield a specific target. In contrast, transcriptome-based approaches, while biologically broader in scope, report on the response, rather than direct impact, of a given compound, and thus make it hard to identify targets beyond the pathway level, or in comparison to a compendium of previously characterized reference compounds [52]. Whole genome sequencing of drug-resistant clones, though uniquely powerful for their organism-wide scope, is similarly associated with complex isolation procedures and the not infrequent discovery of secondary resistance genes involved in drug-activation, drug efflux and associated DNA transcription, while missing the primary target [53–55].…”

Section: Compound-based Drug Developmentmentioning

confidence: 99%

“…In addition to serving as a specific lens into metabolic drug targets, metabolomics has also enabled biologically unbiased classification of compound MOAs in a way similar to what was achieved with genome-wide RNA profiling [52,60–62]. This application makes specific use of the biological discriminatory power, rather than biological information, of metabolomic profiles as a means of classifying experimental compounds in relation to a compendium of reference profiles of known MOA (Figure 2B) [63].…”

Section: Compound-based Drug Developmentmentioning

confidence: 99%

Emerging Approaches to Tuberculosis Drug Development: At Home in the Metabolome

Jansen

Rhee

2017

Trends in Pharmacological Sciences

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Once considered a crowning achievement of modern drug development, tuberculosis (TB) chemotherapy has proven increasingly unable to keep pace with the spread of the pandemic and rise of drug resistance. Efforts to revive the TB drug development pipeline have, in the meantime, faltered. Closer analysis reveals key experimental deficiencies that have hindered our ability to ‘reverse engineer’ knowledge of antibiotic mechanism into rational drug development. Here, we discuss the emerging potential of metabolomics, the systems level study of small molecule metabolites, to help overcome these gaps and serve as a unique biochemical bridge between the phenotypic properties of chemical compounds and biological targets.

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Genetic Approaches to Facilitate Antibacterial Drug Development

Cited by 16 publications

References 123 publications

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

Promoter mutagenesis for fine‐tuning expression of essential genes in Mycobacterium tuberculosis

Targeting Phenotypically TolerantMycobacterium tuberculosis

Emerging Approaches to Tuberculosis Drug Development: At Home in the Metabolome

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