The emergence of multi- and extensively drug resistant tuberculosis worldwide poses a great threat to human health and highlight the need to discover and develop new, effective and inexpensive ...antituberculosis agents. High-throughput screening assays against well-validated drug targets and structure based drug design have been employed to discover new lead compounds. However, the great majority fail to demonstrate any antimycobacterial activity when tested against
in whole-cell screening assays. This is mainly due to some of the intrinsic properties of the bacilli, such as the extremely low permeability of its cell wall, slow growth, drug resistance, drug tolerance, and persistence. In this sense, understanding the pathways involved in
drug tolerance, persistence, and pathogenesis, may reveal new approaches for drug development. Moreover, the need for compounds presenting a novel mode of action is of utmost importance due to the emergence of resistance not only to the currently used antituberculosis agents, but also to those in the pipeline. Cheminformatics studies have shown that drugs endowed with antituberculosis activity have the peculiarity of being more lipophilic than many other antibacterials, likely because this leads to improved cell penetration through the extremely waxy mycobacterial cell wall. Moreover, the interaction of the lipophilic moiety with the membrane alters its stability and functional integrity due to the disruption of the proton motive force, resulting in cell death. When a ligand-based medicinal chemistry campaign is ongoing, it is always difficult to predict whether a chemical modification or a functional group would be suitable for improving the activity. Nevertheless, in the "instruction manual" of medicinal chemists, certain functional groups or certain physicochemical characteristics (i.e., high lipophilicity) are considered red flags to look out for in order to safeguard drug-likeness and avoid attritions in the drug discovery process. In this review, we describe how antituberculosis compounds challenge established rules such as the Lipinski's "rule of five" and how medicinal chemistry for antituberculosis compounds must be thought beyond such dogmatic schemes.
Strain-specific genomic diversity in the Mycobacterium tuberculosis complex (MTBC) is an important factor in pathogenesis that may affect virulence, transmissibility, host response and emergence of ...drug resistance. Several systems have been proposed to classify MTBC strains into distinct lineages and families. Here, we investigate single-nucleotide polymorphisms (SNPs) as robust (stable) markers of genetic variation for phylogenetic analysis. We identify ~92 k SNP across a global collection of 1,601 genomes. The SNP-based phylogeny is consistent with the gold-standard regions of difference (RD) classification system. Of the ~7 k strain-specific SNPs identified, 62 markers are proposed to discriminate known circulating strains. This SNP-based barcode is the first to cover all main lineages, and classifies a greater number of sublineages than current alternatives. It may be used to classify clinical isolates to evaluate tools to control the disease, including therapeutics and vaccines whose effectiveness may vary by strain type.
In December 2019, the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of coronavirus disease 2019 (COVID-19) was first identified in the province of Wuhan, ...China. Since then, there have been over 400 million confirmed cases and 5.8 million deaths by COVID-19 reported worldwide. The urgent need for therapies against SARS-CoV-2 led researchers to use drug repurposing approaches. This strategy allows the reduction in risks, time, and costs associated with drug development. In many cases, a repurposed drug can enter directly to preclinical testing and clinical trials, thus accelerating the whole drug discovery process. In this work, we will give a general overview of the main developments in COVID-19 treatment, focusing on the contribution of the drug repurposing paradigm to find effective drugs against this disease. Finally, we will present our findings using a new drug repurposing strategy that identified 11 compounds that may be potentially effective against COVID-19. To our knowledge, seven of these drugs have never been tested against SARS-CoV-2 and are potential candidates for in vitro and in vivo studies to evaluate their effectiveness in COVID-19 treatment.
The emergence of infections caused by multi- or pan-resistant bacteria in the hospital or in the community settings is an increasing health concern. Albeit there is no single resistance mechanism ...behind multiresistance, multidrug efflux pumps, proteins that cells use to detoxify from noxious compounds, seem to play a key role in the emergence of these multidrug resistant (MDR) bacteria. During the last decades, experimental data has established their contribution to low level resistance to antimicrobials in bacteria and their potential role in the appearance of MDR phenotypes, by the extrusion of multiple, unrelated compounds. Recent studies suggest that efflux pumps may be used by the cell as a first-line defense mechanism, avoiding the drug to reach lethal concentrations, until a stable, more efficient alteration occurs, that allows survival in the presence of that agent. In this paper we review the current knowledge on MDR efflux pumps and their intricate regulatory network in Staphylococcus aureus, a major pathogen, responsible from mild to life-threatening infections. Particular emphasis will be given to the potential role that S. aureus MDR efflux pumps, either chromosomal or plasmid-encoded, have on resistance towards different antimicrobial agents and on the selection of drug - resistant strains. We will also discuss the many questions that still remain on the role of each specific efflux pump and the need to establish appropriate methodological approaches to address all these questions.
Mycobacterium tuberculosis resistance to anti-tuberculosis drugs is a major threat to global public health. Whole genome sequencing (WGS) is rapidly gaining traction as a diagnostic tool for clinical ...tuberculosis settings. To support this informatically, previous work led to the development of the widely used TBProfiler webtool, which predicts resistance to 14 drugs from WGS data. However, for accurate and rapid high throughput of samples in clinical or epidemiological settings, there is a need for a stand-alone tool and the ability to analyse data across multiple WGS platforms, including Oxford Nanopore MinION.
We present a new command line version of the TBProfiler webserver, which includes hetero-resistance calling and will facilitate the batch processing of samples. The TBProfiler database has been expanded to incorporate 178 new markers across 16 anti-tuberculosis drugs. The predictive performance of the mutation library has been assessed using > 17,000 clinical isolates with WGS and laboratory-based drug susceptibility testing (DST) data. An integrated MinION analysis pipeline was assessed by performing WGS on 34 replicates across 3 multi-drug resistant isolates with known resistance mutations. TBProfiler accuracy varied by individual drug. Assuming DST as the gold standard, sensitivities for detecting multi-drug-resistant TB (MDR-TB) and extensively drug-resistant TB (XDR-TB) were 94% (95%CI 93-95%) and 83% (95%CI 79-87%) with specificities of 98% (95%CI 98-99%) and 96% (95%CI 95-97%) respectively. Using MinION data, only one resistance mutation was missed by TBProfiler, involving an insertion in the tlyA gene coding for capreomycin resistance. When compared to alternative platforms (e.g. Mykrobe predictor TB, the CRyPTIC library), TBProfiler demonstrated superior predictive performance across first- and second-line drugs.
The new version of TBProfiler can rapidly and accurately predict anti-TB drug resistance profiles across large numbers of samples with WGS data. The computing architecture allows for the ability to modify the core bioinformatic pipelines and outputs, including the analysis of WGS data sourced from portable technologies. TBProfiler has the potential to be integrated into the point of care and WGS diagnostic environments, including in resource-poor settings.
Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated ...the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.
This review presents the evidence that supports the use of thioridazine (TZ) for the therapy of a pulmonary tuberculosis infection regardless of its antibiotic resistance status. The evidence ...consists of in vitro and ex vivo assays that demonstrate the activity of TZ against all encountered
(Mtb) regardless of its antibiotic resistance phenotype, as well as in vivo as a therapy for mice infected with multi-drug resistant strains of Mtb, or for human subjects infected with extensively drug resistant (XDR) Mtb. The mechanisms of action by which TZ brings about successful therapeutic outcomes are presented in detail.
Given the ability of M. tuberculosis to survive as an intracellular pathogen and its propensity to develop resistance to the existing antituberculosis drugs, its treatment requires new approaches. ...Here the antimycobacterial properties of verapamil, thioridazine, chlorpromazine, flupenthixol and haloperidol were investigated against a panel of drug resistant M. tuberculosis strains, both in vitro and on human-infected macrophages. These compounds are efflux inhibitors that share among them the characteristic of being ion channel blockers. In vitro, all compounds exhibited synergistic inhibitory activities when combined with isoniazid and rifampicin, and were able to inhibit active efflux, demonstrating their role as efflux inhibitors. Gene expression analysis showed that M. tuberculosis efflux genes were overexpressed in response to antibiotic exposure, in vitro and within macrophages, irrespective of their resistance pattern. These compounds displayed a rapid and high killing activity against M. tuberculosis, associated with a decrease in intracellular ATP levels demonstrating that the bactericidal action of the ion channel blockers against M. tuberculosis clinical strains is associated with their interference with energy metabolism. The compounds led to a decrease in the intracellular mycobacterial load by increasing phagosome acidification and activating lysosomal hydrolases. The results presented in this study enable us to propose the following mechanism of action for these compounds: a) in the bacteria, the compounds generate a cascade of events involving the inhibition of the respiratory chain complexes and energy production for efflux activity. Indirectly, this reduce the resistance level to antituberculosis drugs potentiating their activity; b) on the host cell, the treatment with the ion channel blockers increases phagosome acidification and induces the expression of phagosomal hydrolases, leading to bacterial growth restriction irrespective of their resistance pattern. This work highlights the potential value ion channel blockers as adjuvants of tuberculosis chemotherapy, in particular for the development of new therapeutic strategies, with strong potential for treatment shortening against drug susceptible and resistant forms of tuberculosis. Medicinal chemistry studies are now needed to improve the properties of these compounds, increasing their M. tuberculosis efflux-inhibition and killing-enhancement activity and reduce their toxicity for humans, therefore optimizing their potential for clinical usage.
NorA is one of the main native MDR efflux pumps of
, contributing to reduced susceptibility towards fluoroquinolones and biocides, but little is known about its variability within
or its distribution ...and conservation among other staphylococci. We screened for sequences homologous to
and found it in 61 out of the 63
species described. To the best of our knowledge, this is the first study to report the occurrence of
across the
genus. The
phylogenetic tree follows the evolutionary relations of staphylococci and the closely related
genus. Comparative analyses suggest a conservation of the NorA function in staphylococci. We also analyzed the variability of
within
, for which there are several circulating
alleles, differing up to 10% at the nucleotide level, which may hamper proper
detection. We demonstrate the applicability of a PCR-based algorithm to detect and differentiate
alleles in 52
representing a wider collection of 89 isolates from different hosts. Our results highlight the prevalence of
and
in different settings and the association of
alleles with specific
clonal lineages. Ultimately, it confirms the applicability of our PCR-based algorithm to rapidly detect and assign the different
alleles, a trait that may impact antimicrobial efflux capacity and the search for potential NorA inhibitors.
NorA is the best studied efflux system of
and therefore frequently used as a model for investigating efflux-mediated resistance in this pathogen. NorA activity is associated with resistance to ...fluoroquinolones, several antiseptics and disinfectants and several reports have pointed out the role of efflux systems, including NorA, as a first-line response to antimicrobials in
. Genetic diversity studies of the gene
have described three alleles;
,
and
. However, the epidemiology of these alleles and their impact on NorA activity remains unclear. Additionally, increasing studies do not account for
variability when establishing relations between resistance phenotypes and
presence or reported absence, which actually corresponds, as we now demonstrate, to different
alleles. In the present study we assessed the variability of the
gene present in the genome of over 1,000
isolates, corresponding to 112
strains with whole genome sequences publicly available; 917 MRSA strains sourced from a London-based study and nine MRSA isolates collected in a major Hospital in Lisbon, Portugal. Our analyses show that
is part of the core genome of
It also suggests that occurrence of
variants reflects the population structure of this major pathogen. Overall, this work highlights the ubiquitous nature of
in
which must be taken into account when studying the role played by this important determinant on
resistance to antimicrobials.
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