Evaluation of Different Mycobacterial Species for Drug Discovery and Characterisation of Novel Inhibitors of Mycobacterium Tuberculosis

<p>Tuberculosis (Tb) has plagued mankind for many centuries and is still a leading cause of death worldwide. A worrying development is the emergence of drug-resistant Tb that poses further challenges to the control of the disease. The global Tb burden and high mortality rate indicate that new drugs are needed for Tb treatment. While no new anti-Tb agents have been introduced to the market for about three decades, drugs with novel mechanisms of action can amend the current Tb treatment regimen and may provide an effective solution to drug resistance. The main objectives of this study were to identify an appropriate in vitro model that could be used for anti-Tb drug high-throughput screening (HTS), and to use this model to identify a novel candidate anti-tubercular drug and its cognate cellular target. A sensitive growth inhibition assay was set up with a GFP-labelled Tb vaccine strain, M. bovis BCG, using standard first and second line anti-tubercular drugs. HTS of the drug libraries was performed with various in vitro models to evaluate their efficacy for use in anti-Tb drug discovery. Approximately 50% of the M. tuberculosis inhibitors were not detected in screening with the surrogate species, M. smegmatis; whereas, only 21% of hits were not detected with M. bovis BCG. A comparative genomic study revealed that 97% of M. bovis BCG proteins, compared to 70% in M. smegmatis have conserved orthologues in M. tuberculosis H37Rv. Therefore, M. bovis BCG represented a more sensitive model than M. smegmatis for detecting anti-M. tuberculosis compounds. M. bovis BCG was then used to screen for novel anti-Tb agents by HTS of compound libraries and various plant extracts, followed by validation of new compounds in M. tuberculosis H37Ra. A number of novel M. tuberculosis inhibitors were identified, including sappanone A dimethyl ether from plant sources and compounds NSC112200 and NSC402959 from NIH chemical libraries. The inhibitors that were validated using M. tuberculosis H37Ra were also validated in the virulent Tb strain, M. tuberculosis H37Rv. In addition, their activity was further investigated using a suite of other clinically important human pathogens. One of the key anti-mycobacterial hits identified in this study, NSC402959, has previously been detected in screens for compounds that inhibit ribonuclease H (RNase H), an enzyme that is required for a number of essential cellular processes. NSC402959 inhibited RNase H proteins from HIV as well as from E. coli. Since HIV and Tb are major pandemics, previously-known anti-HIV RNase H compounds were imported and tested for their anti-proliferative activity towards M. tuberculosis H37Ra. HIV RNase H inhibitors, NSC35676, NSC112200, NSC133457 and NSC668394, exhibited good anti-mycobacterial activity in this study. In silico analysis suggested a plausible interaction of these inhibitors with mycobacterial RNase HI. A biochemical assay further confirmed NSC112200 to be specific against RNase HI from M. tuberculosis. These interesting inhibitors were not only structurally different from existing anti-Tb drugs but some of them were also non-toxic to mammalian cells and may have a unique mechanism of action. Thus, these compounds showed good potential for development as dual inhibitors of Tb and HIV; therefore, future studies in animal infection models to determine their dual anti-mycobacterial and anti-HIV activities are warranted.</p>

Evaluation of Different Mycobacterial Species for Drug Discovery and Characterisation of Novel Inhibitors of Mycobacterium Tuberculosis

<p>Tuberculosis (Tb) has plagued mankind for many centuries and is still a leading cause of death worldwide. A worrying development is the emergence of drug-resistant Tb that poses further challenges to the control of the disease. The global Tb burden and high mortality rate indicate that new drugs are needed for Tb treatment. While no new anti-Tb agents have been introduced to the market for about three decades, drugs with novel mechanisms of action can amend the current Tb treatment regimen and may provide an effective solution to drug resistance. The main objectives of this study were to identify an appropriate in vitro model that could be used for anti-Tb drug high-throughput screening (HTS), and to use this model to identify a novel candidate anti-tubercular drug and its cognate cellular target. A sensitive growth inhibition assay was set up with a GFP-labelled Tb vaccine strain, M. bovis BCG, using standard first and second line anti-tubercular drugs. HTS of the drug libraries was performed with various in vitro models to evaluate their efficacy for use in anti-Tb drug discovery. Approximately 50% of the M. tuberculosis inhibitors were not detected in screening with the surrogate species, M. smegmatis; whereas, only 21% of hits were not detected with M. bovis BCG. A comparative genomic study revealed that 97% of M. bovis BCG proteins, compared to 70% in M. smegmatis have conserved orthologues in M. tuberculosis H37Rv. Therefore, M. bovis BCG represented a more sensitive model than M. smegmatis for detecting anti-M. tuberculosis compounds. M. bovis BCG was then used to screen for novel anti-Tb agents by HTS of compound libraries and various plant extracts, followed by validation of new compounds in M. tuberculosis H37Ra. A number of novel M. tuberculosis inhibitors were identified, including sappanone A dimethyl ether from plant sources and compounds NSC112200 and NSC402959 from NIH chemical libraries. The inhibitors that were validated using M. tuberculosis H37Ra were also validated in the virulent Tb strain, M. tuberculosis H37Rv. In addition, their activity was further investigated using a suite of other clinically important human pathogens. One of the key anti-mycobacterial hits identified in this study, NSC402959, has previously been detected in screens for compounds that inhibit ribonuclease H (RNase H), an enzyme that is required for a number of essential cellular processes. NSC402959 inhibited RNase H proteins from HIV as well as from E. coli. Since HIV and Tb are major pandemics, previously-known anti-HIV RNase H compounds were imported and tested for their anti-proliferative activity towards M. tuberculosis H37Ra. HIV RNase H inhibitors, NSC35676, NSC112200, NSC133457 and NSC668394, exhibited good anti-mycobacterial activity in this study. In silico analysis suggested a plausible interaction of these inhibitors with mycobacterial RNase HI. A biochemical assay further confirmed NSC112200 to be specific against RNase HI from M. tuberculosis. These interesting inhibitors were not only structurally different from existing anti-Tb drugs but some of them were also non-toxic to mammalian cells and may have a unique mechanism of action. Thus, these compounds showed good potential for development as dual inhibitors of Tb and HIV; therefore, future studies in animal infection models to determine their dual anti-mycobacterial and anti-HIV activities are warranted.</p>

Ligase A and RNase HI Participate in Completing Replication on the Chromosome in Escherichia coli

In Escherichia coli, several enzymes have been identified that participate in completing replication on the chromosome, including RecG, SbcCD, ExoI, and RecBCD. However, other enzymes are likely to be involved and the precise enzymatic mechanism by which this reaction occurs remains unknown. Two steps predicted to be necessary to complete replication are removal of Okazaki RNA fragments and ligation of the nascent strands at convergent replication forks. E. coli encodes two RNases that remove RNA-DNA hybrids, rnhA and rnhB, as well as two ligases, ligA and ligB. Here, we used replication profiling to show that rnhA and ligA, encoding RNase HI and Ligase A, participate in the completion reaction. Deletion of rnhA impaired the ability to complete replication and resulted in over-replication in the terminus region. It additionally suppressed initiation events from oriC, suggesting a role for the enzyme in oriC-dependent initiation, as has been suggested previously. We also show that a temperature-sensitive mutation in Ligase A led to over-replication at sites where replication completes, and that degradation at these sites occurred upon shifting to the nonpermissive temperature. Deletion of rnhB or ligB did not affect the growth or profile of replication on the genome.

RNase HI depletion strongly potentiates cell killing by rifampicin in mycobacteria

Multidrug resistant (MDR) tuberculosis (TB) is defined by the resistance of Mycobacterium tuberculosis, the causative organism, to the first-line antibiotics rifampicin and isoniazid. Mitigating or reversing resistance to these drugs offers a means of preserving and extending their use in TB treatment. R-loops are RNA/DNA hybrids that are formed in the genome during transcription, and can be lethal to the cell if not resolved. RNase HI is an enzyme that removes R-loops, and this activity is essential in M. tuberculosis: knockouts of rnhC, the gene encoding RNase HI, are non-viable. This essentiality supports it as a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two RNase HI enzymes, RnhA and RnhC. We show that the partial depletion of RNase HI activity in M. smegmatis, by knocking out either of the genes encoding RnhA or RnhC, led to the accumulation of R-loops. The sensitivity of the knockout strains to the antibiotics moxifloxacin, streptomycin and rifampicin was increased, with sensitivity to the transcriptional inhibitor rifampicin strikingly increased by nearly 100-fold. We also show that R-loop accumulation accompanies partial transcriptional inhibition, suggesting a mechanistic basis for the synergy between RNase HI depletion and transcriptional inhibition. A model of how transcriptional inhibition can potentiate R-loop accumulation is presented. Finally, we identified four small molecules that inhibit recombinant RnhC activity and that also potentiated rifampicin activity in whole-cell assays against M. tuberculosis, supporting an on-target mode of action, and providing the first step in developing a new class of anti-mycobacterial drug.

Bacterial type 1A topoisomerases maintain the stability of the genome by preventing and dealing with R-loop-and nucleotide excision repair-dependent topological stress.

E. coli type 1A topoisomerases (topos), topo I (topA) and topo III (topB) have both relaxation and decatenation activities. B. subtilis and E. coli topA topB null cells can survive owing to DNA amplifications allowing overproduction of topo IV, the main cellular decatenase that can also relax supercoiling. We show that overproducing human topo IB, a relaxase but not a decatenase, can substitute for topo IV in allowing E. coli topA null but not topA topB null cells to survive. Deleting topB exacerbates phenotypes of topA null mutants including hypernegative supercoiling, R-loop formation, and RNase HI-sensitive replication, phenotypes that are not corrected by topo IV overproduction. These phenotypes lead to Ter DNA amplification causing a chromosome segregation defect that is corrected by topo IV overproduction. Furthermore, topA topB null mutants not overproducing topo IV acquire uvrB or uvrC mutations, revealing a nucleotide excision repair (NER)-dependent problem with replication fork progression. Thus, type IA topos maintain the stability of the genome by providing essential relaxase and decatenase activities to prevent and solve topological stress related to R-loops and NER. Moreover, excess R-loop formation is well tolerated in cells that have enough topoisomerase activity to support the subsequent replication-related topological stress.

Topoisomerase I Essentiality, DnaA-independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli

Topoisomerase I (Topo I) of Escherichia coli , encoded by topA , acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC -independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including Δ topA , confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued Δ dnaA lethality; (ii) in dnaA + derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by Δ dnaA . We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. coli topA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn lead to transcription-replication conflicts and to cSDR. Importance In all life forms, double helical DNA exists in a topologically supercoiled state. The enzymes DNA gyrase and topoisomerase I act, respectively, to introduce and to relax negative DNA supercoils in Escherichia coli . That gyrase deficiency leads to bacterial death is well established, but the essentiality of topoisomerase I for viability has been less certain. This study confirms that topoisomerase I is essential for E. coli viability, and suggests that in its absence aberrant chromosomal DNA replication and excessive transcription-replication conflicts occur that are responsible for lethality.

Topoisomerase I Essentiality, DnaA-independent Chromosomal Replication, and Transcription-Replication Conflict in Escherichia coli

Topoisomerase I (Topo I) of <Escherichia coli, encoded by topA, acts to relax negative supercoils in DNA. Topo I deficiency results in hypernegative supercoiling, formation of transcription-associated RNA-DNA hybrids (R-loops), and DnaA- and oriC-independent constitutive stable DNA replication (cSDR), but some uncertainty persists as to whether topA is essential for viability in E. coli and related enterobacteria. Here we show that several topA alleles, including ΔtopA>, confer lethality in derivatives of wild-type E. coli strain MG1655. Viability in absence of Topo I was restored with two perturbations, neither of which reversed the hypernegative supercoiling phenotype: (i) in a reduced-genome strain MDS42, or (ii) by an RNA polymerase (RNAP) mutation rpoB*35 that has been reported to alleviate the deleterious consequences of RNAP backtracking and transcription-replication conflicts. Four phenotypes related to cSDR were identified for topA mutants: (i) One of the topA alleles rescued ΔdnaA lethality; (ii) in dnaA+ derivatives, Topo I deficiency generated a characteristic copy number peak in the terminus region of the chromosome; (iii) topA was synthetically lethal with rnhA (encoding RNase HI, whose deficiency also confers cSDR); and (iv) topA rnhA synthetic lethality was itself rescued by ΔdnaA. We propose that the terminal lethal consequence of hypernegative DNA supercoiling in E. colitopA mutants is RNAP backtracking during transcription elongation and associated R-loop formation, which in turn lead to transcription-replication conflicts and to cSDR.

DNA breaks-mediated cost reveals RNase HI as a new target for selectively eliminating antibiotic resistance

Antibiotic resistance often generates a fitness cost to bacteria in drug-free environments. Understanding the causes of the cost is considered the Holy Grail in the antibiotic resistance field, as it is the main determinant of the prevalence of resistances upon reducing antibiotics use. We show that DNA breaks can explain most of the variation in the cost of resistances common in pathogens. Here we demonstrate that targeting the RNase that degrades R-loops, which cause DNA breaks, exacerbates the cost of resistance. Consequently, lack of RNase HI function drives resistant clones to extinction in populations with high initial frequency of resistance, both in laboratory conditions and in a mouse model of gut colonization. Thus, RNase HI provides a target specific against resistant bacteria, which we validate using a repurposed drug. In summary, we revealed key mechanisms underlying the cost of antibiotic resistance that can be exploited to specifically eliminate resistant bacteria.