Modulation of riboflavin biosynthesis and utilization in mycobacteria.

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism and physiology, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins, and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for future studies investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria. IMPORTANCE: The pathway for biosynthesis and utilization of riboflavin, precursor of the essential coenzymes, FMN and FAD, is of particular interest in the flavin-rich pathogen, Mycobacterium tuberculosis (Mtb), for two important reasons: (i) the pathway includes potential tuberculosis (TB) drug targets and (ii) intermediates from the riboflavin biosynthesis pathway provide ligands for mucosal-associated invariant T (MAIT) cells, which have been implicated in TB pathogenesis. However, the riboflavin pathway is poorly understood in mycobacteria, which lack canonical mechanisms to transport this vitamin and to regulate flavin coenzyme homeostasis. By conditionally disrupting each step of the pathway and assessing the impact on mycobacterial viability and on the levels of the pathway proteins as well as riboflavin, our work provides genetic validation of the riboflavin pathway as a target for TB drug discovery and offers a resource for further exploring the association between riboflavin biosynthesis, MAIT cell activation, and TB infection and disease.

Persistent Mycobacterium tuberculosis bioaerosol release in a tuberculosis-endemic setting.

Pioneering studies linking symptomatic disease and cough-mediated release of Mycobacterium tuberculosis (Mtb) established the infectious origin of tuberculosis (TB), simultaneously informing the pervasive notion that pathology is a prerequisite for Mtb transmission. Our prior work has challenged this assumption: by sampling TB clinic attendees, we detected equivalent release of Mtb-containing bioaerosols by confirmed TB patients and individuals not receiving a TB diagnosis, and we demonstrated a time-dependent reduction in Mtb bioaerosol positivity during six-months' follow-up, irrespective of anti-TB chemotherapy. Now, by extending bioaerosol sampling to a randomly selected community cohort, we show that Mtb release is common in a TB-endemic setting: of 89 participants, 79.8% (71/89) produced Mtb bioaerosols independently of QuantiFERON-TB Gold status, a standard test for Mtb infection; moreover, during two-months' longitudinal sampling, only 2% (1/50) were serially Mtb bioaerosol negative. These results necessitate a reframing of the prevailing paradigm of Mtb transmission and infection, and may explain the current inability to elucidate Mtb transmission networks in TB-endemic regions.

Aerosolization of viable Mycobacterium tuberculosis bacilli by tuberculosis clinic attendees independent of sputum-Xpert Ultra status.

Potential Mycobacterium tuberculosis (Mtb) transmission during different pulmonary tuberculosis (TB) disease states is poorly understood. We quantified viable aerosolized Mtb from TB clinic attendees following diagnosis and through six months' follow-up thereafter. Presumptive TB patients (n=102) were classified by laboratory, radiological, and clinical features into Group A: Sputum-Xpert Ultra-positive TB (n=52), Group B: Sputum-Xpert Ultra-negative TB (n=20), or Group C: TB undiagnosed (n=30). All groups were assessed for Mtb bioaerosol release at baseline, and subsequently at 2 wk, 2 mo, and 6 mo. Groups A and B were notified to the national TB program and received standard anti-TB chemotherapy; Mtb was isolated from 92% and 90% at presentation, 87% and 74% at 2 wk, 54% and 44% at 2 mo and 32% and 20% at 6 mo, respectively. Surprisingly, similar numbers were detected in Group C not initiating TB treatment: 93%, 70%, 48% and 22% at the same timepoints. A temporal association was observed between Mtb bioaerosol release and TB symptoms in all three groups. Persistence of Mtb bioaerosol positivity was observed in ~30% of participants irrespective of TB chemotherapy. Captured Mtb bacilli were predominantly acid-fast stain-negative and poorly culturable; however, three bioaerosol samples yielded sufficient biomass following culture for whole-genome sequencing, revealing two different Mtb lineages. Detection of viable aerosolized Mtb in clinic attendees, independent of TB diagnosis, suggests that unidentified Mtb transmitters might contribute a significant attributable proportion of community exposure. Additional longitudinal studies with sputum culture-positive and -negative control participants are required to investigate this possibility.

Synthesis,Antidiabetic and Antitubercular Evaluation of Quinoline-pyrazolopyrimidine hybrids and Quinoline-4-Arylamines.

Two libraries of quinoline-based hybrids 1-(7-chloroquinolin-4-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine and 7-chloro-N-phenylquinolin-4-amine were synthesized and evaluated for their α-glucosidase inhibitory and antioxidant properties. Compounds with 4-methylpiperidine and para-trifluoromethoxy groups, respectively, showed the most promising α-glucosidase inhibition activity with IC50 =46.70 and 40.84 µM, compared to the reference inhibitor, acarbose (IC50 =51.73 µM). Structure-activity relationship analysis suggested that the cyclic secondary amine pendants and para-phenyl substituents account for the variable enzyme inhibition. Antioxidant profiling further revealed that compounds with an N-methylpiperazine and N-ethylpiperazine ring, respectively, have good DPPH scavenging abilities with IC50 =0.18, 0.58 and 0.93 mM, as compared to ascorbic acid (IC50 =0.05 mM), while the best DPPH scavenger is NO2 -substituted compound (IC50 =0.08 mM). Also, compound with N-(2-hydroxyethyl)piperazine moiety emerged as the best NO radical scavenger with IC50 =0.28 mM. Molecular docking studies showed that the present compounds are orthosteric inhibitors with their quinoline, pyrimidine, and 4-amino units as crucial pharmacophores furnishing α-glucosidase binding at the catalytic site. Taken together, these compounds exhibit dual potentials; i. e., potent α-glucosidase inhibitors and excellent free radical scavengers. Hence, they may serve as structural templates in the search for agents to manage Type 2 diabetes mellitus. Finally, in preliminary assays investigating the anti-tubercular potential of these compounds, two pyrazolopyrimidine series compounds and a 7-chloro-N-phenylquinolin-4-amine hybrid showed sub-10 µM whole-cell activities against Mycobacterium tuberculosis.

Classification of early tuberculosis states to guide research for improved care and prevention: an international Delphi consensus exercise.

The current active-latent paradigm of tuberculosis largely neglects the documented spectrum of disease. Inconsistency with regard to definitions, terminology, and diagnostic criteria for different tuberculosis states has limited the progress in research and product development that are needed to achieve tuberculosis elimination. We aimed to develop a new framework of classification for tuberculosis that accommodates key disease states but is sufficiently simple to support pragmatic research and implementation. Through an international Delphi exercise that involved 71 participants representing a wide range of disciplines, sectors, income settings, and geographies, consensus was reached on a set of conceptual states, related terminology, and research gaps. The International Consensus for Early TB (ICE-TB) framework distinguishes disease from infection by the presence of macroscopic pathology and defines two subclinical and two clinical tuberculosis states on the basis of reported symptoms or signs of tuberculosis, further differentiated by likely infectiousness. The presence of viable Mycobacterium tuberculosis and an associated host response are prerequisites for all states of infection and disease. Our framework provides a clear direction for tuberculosis research, which will, in time, improve tuberculosis clinical care and elimination policies.

New azaaurone derivatives as potential multitarget agents in HIV-TB coinfection.

Tuberculosis (TB) disease, caused by Mycobacterium tuberculosis (Mtb) is the leading cause of death among people with human immunodeficiency virus (HIV) infection. No dual-target drug is currently being used to simultaneously treat both infections. This work aimed to obtain new multitarget HIV-TB agents, with the goal of optimizing treatments and preventing this coinfection. These compounds incorporate the structural features of azaaurones as anti-Mtb and zidovudine (AZT) as the antiretroviral moiety. The azaaurone scaffold displayed submicromolar activities against Mtb, and AZT is a potent antiretroviral drug. Six derivatives were synthetically generated, and five were evaluated against both infective agents. Evaluations of anti-HIV activity were carried out in HIV-1-infected MT-4 cells and on endogenous HIV-1 reverse transcriptase (RT) activity. The H37Rv strain was used for anti-Mtb assessments. Most compounds displayed potent antitubercular and moderate anti-HIV activity. (E)-12 exhibited a promising multitarget profile with an MIC90 of 2.82 µM and an IC50 of 1.98 µM in HIV-1-infected T lymphocyte cells, with an 84% inhibition of RT activity. Therefore, (E)-12 could be the first promising compound from a family of multitarget agents used to treat HIV-TB coinfection. In addition, the compound could offer a prototype for the development of new strategies in scientific research to treat this global health issue.

Structural Optimization of Antimycobacterial Azaaurones Towards Improved Solubility and Metabolic Stability.

While N-acetyl azaaurones have already been disclosed for their potential against tuberculosis (TB), their low metabolic stability remains an unaddressed liability. We now report a study designed to improve the metabolic stability and solubility of the azaaurone scaffold and to identify the structural requirements for antimycobacterial activity. Replacing the N-acetyl moiety for a N-carbamoyl group led to analogues with sub- and nanomolar potencies against M. tuberculosis H37Rv, as well as equipotent against drug-susceptible and drug-resistant M. tuberculosis isolates. The new N-carbamoyl azaaurones exhibited improved microsomal stability, compared to their N-acetylated counterparts, with several compounds displaying moderate to high kinetic solubility. The frequency of spontaneous resistance to azaaurones was observed to be in the range of 10-8 , a value that is comparable to current TB drugs in the market. Overall, these results reveal that azaaurones are amenable to structural modifications to improve metabolic and solubility liabilities, and highlight their potential as antimycobacterial agents.

Modulation of riboflavin biosynthesis and utilization in mycobacteria.

Riboflavin (vitamin B2) is the precursor of the flavin coenzymes, FAD and FMN, which play a central role in cellular redox metabolism. While humans must obtain riboflavin from dietary sources, certain microbes, including Mycobacterium tuberculosis (Mtb), can biosynthesize riboflavin de novo. Riboflavin precursors have also been implicated in the activation of mucosal-associated invariant T (MAIT) cells which recognize metabolites derived from the riboflavin biosynthesis pathway complexed to the MHC-I-like molecule, MR1. To investigate the biosynthesis and function of riboflavin and its pathway intermediates in mycobacterial metabolism, physiology and MAIT cell recognition, we constructed conditional knockdowns (hypomorphs) in riboflavin biosynthesis and utilization genes in Mycobacterium smegmatis (Msm) and Mtb by inducible CRISPR interference. Using this comprehensive panel of hypomorphs, we analyzed the impact of gene silencing on viability, on the transcription of (other) riboflavin pathway genes, on the levels of the pathway proteins and on riboflavin itself. Our results revealed that (i) despite lacking a canonical transporter, both Msm and Mtb assimilate exogenous riboflavin when supplied at high concentration; (ii) there is functional redundancy in lumazine synthase activity in Msm; (iii) silencing of ribA2 or ribF is profoundly bactericidal in Mtb; and (iv) in Msm, ribA2 silencing results in concomitant knockdown of other pathway genes coupled with RibA2 and riboflavin depletion and is also bactericidal. In addition to their use in genetic validation of potential drug targets for tuberculosis, this collection of hypomorphs provides a useful resource for investigating the role of pathway intermediates in MAIT cell recognition of mycobacteria.

Investigating the composition and recruitment of the mycobacterial ImuA'-ImuB-DnaE2 mutasome.

A DNA damage-inducible mutagenic gene cassette has been implicated in the emergence of drug resistance in Mycobacterium tuberculosis during anti-tuberculosis (TB) chemotherapy. However, the molecular composition and operation of the encoded 'mycobacterial mutasome' - minimally comprising DnaE2 polymerase and ImuA' and ImuB accessory proteins - remain elusive. Following exposure of mycobacteria to DNA damaging agents, we observe that DnaE2 and ImuB co-localize with the DNA polymerase III ß subunit (ß clamp) in distinct intracellular foci. Notably, genetic inactivation of the mutasome in an imuBAAAAGG mutant containing a disrupted ß clamp-binding motif abolishes ImuB-ß clamp focus formation, a phenotype recapitulated pharmacologically by treating bacilli with griselimycin and in biochemical assays in which this ß clamp-binding antibiotic collapses pre-formed ImuB-ß clamp complexes. These observations establish the essentiality of the ImuB-ß clamp interaction for mutagenic DNA repair in mycobacteria, identifying the mutasome as target for adjunctive therapeutics designed to protect anti-TB drugs against emerging resistance.

High-throughput functional genomics: A (myco)bacterial perspective.

Advances in sequencing technologies have enabled unprecedented insights into bacterial genome composition and dynamics. However, the disconnect between the rapid acquisition of genomic data and the (much slower) confirmation of inferred genetic function threatens to widen unless techniques for fast, high-throughput functional validation can be applied at scale. This applies equally to Mycobacterium tuberculosis, the leading infectious cause of death globally and a pathogen whose genome, despite being among the first to be sequenced two decades ago, still contains many genes of unknown function. Here, we summarize the evolution of bacterial high-throughput functional genomics, focusing primarily on transposon (Tn)-based mutagenesis and the construction of arrayed mutant libraries in diverse bacterial systems. We also consider the contributions of CRISPR interference as a transformative technique for probing bacterial gene function at scale. Throughout, we situate our analysis within the context of functional genomics of mycobacteria, focusing specifically on the potential to yield insights into M. tuberculosis pathogenicity and vulnerabilities for new drug and regimen development. Finally, we offer suggestions for future approaches that might be usefully applied in elucidating the complex cellular biology of this major human pathogen.

Quinolone analogues of benzothiazinone: Synthesis, antitubercular structure-activity relationship and ADME profiling.

Mycobacterium tuberculosis (Mtb) has an impermeable cell wall which gives it an inherent ability to resist many antibiotics. DprE1, an essential enzyme in Mtb cell wall synthesis, has been validated as a target for several TB drug candidates. The most potent and developmentally advanced DprE1 inhibitor, PBTZ169, is still undergoing clinical development. With high attrition rate, there is need to populate the development pipeline. Using a scaffold hopping strategy, we imprinted the benzenoid ring of PBTZ169 onto a quinolone nucleus. Twenty-two compounds were synthesised and screened for activity against Mtb, with six compounds exhibiting sub micromolar activity of MIC90 <0.244 µM. Compound 25 further demonstrated sub-micromolar activity when evaluated against wild-type and fluoroquinolone-resistant Mtb strains. This compound maintained its sub-micromolar activity against a DprE1 P116S mutant strain but showed a significant reduction in activity when tested against the DprE1 C387S mutant.

Discovery and biological evaluation of an adamantyl-amide derivative with likely MmpL3 inhibitory activity.

A series of molecules containing bulky lipophilic scaffolds was screened for activity against Mycobacterium tuberculosis and a number of compounds with antimycobacterial activity were identified. The most active compound, (2E)-N-(adamantan-1-yl)-3-phenylprop-2-enamide (C1), has a low micromolar minimum inhibitory concentration, low cytotoxicity (therapeutic index = 32.26), low mutation frequency and is active against intracellular Mycobacterium tuberculosis. Whole genome sequencing of mutants resistant to C1 showed a mutation in mmpL3 which may point to the involvement of MmpL3 in the antimycobacterial activity of the compound. In silico mutagenesis and molecular modelling studies were performed to better understand the binding of C1 within MmpL3 and the role that the specific mutation may play in the interaction at protein level. These analyses revealed that the mutation increases the energy required for binding of C1 within the protein translocation channel of MmpL3. The mutation also decreases the solvation energy of the protein, suggesting that the mutant protein might be more solvent-accessible, thereby restricting its interaction with other molecules. The results reported here describe a new molecule that may interact with the MmpL3 protein, providing insights into the effect of mutations on protein-ligand interactions and enhancing our understanding of this essential protein as a priority drug target.

Quinolone-3-amidoalkanol: A New Class of Potent and Broad-Spectrum Antimicrobial Agent.

Herein, we describe 39 novel quinolone compounds bearing a hydrophilic amine chain and varied substituted benzyloxy units. These compounds demonstrate broad-spectrum activities against acid-fast bacterium, Gram-positive and -negative bacteria, fungi, and leishmania parasite. Compound 30 maintained antitubercular activity against moxifloxacin-, isoniazid-, and rifampicin-resistant Mycobacterium tuberculosis, while 37 exhibited low micromolar activities (<1 µg/mL) against World Health Organization (WHO) critical pathogens: Cryptococcus neoformans, Acinetobacter baumannii, and Pseudomonas aeruginosa. Compounds in this study are metabolically robust, demonstrating % remnant of >98% after 30 min in the presence of human, rat, and mouse liver microsomes. Several compounds thus reported here are promising leads for the treatment of diseases caused by infectious agents.

RecA-NT homology motif in ImuB is essential for mycobacterial ImuA'-ImuB protein interaction and mutasome function.

The mycobacterial mutasome - minimally comprising ImuA', ImuB, and DnaE2 proteins - has been implicated in DNA damage-induced mutagenesis in Mycobacterium tuberculosis. ImuB, predicted to enable mutasome function via its interaction with the ß clamp, is a catalytically inactive member of the Y-family of DNA polymerases. Like other members of the Y family, ImuB features a recently identified amino acid motif with homology to the RecA-N-terminus (RecA-NT). In RecA, the motif mediates oligomerization of RecA monomers into RecA filaments. Given the role of ImuB in the mycobacterial mutasome, we hypothesized that the ImuB RecA-NT motif might mediate its interaction with ImuA', a RecA homolog of unknown function. To investigate this possibility, we constructed a panel of imuB alleles in which RecA-NT was removed, or mutated. Results from microbiological and biochemical assays indicate that RecA-NT is critical for the interaction of ImuB with ImuA'. A region downstream of RecA-NT (ImuB-C) also appears to stabilize the ImuB-ImuA' interaction, but its removal does not prevent complex formation. In contrast, replacing two key hydrophobic residues of RecA-NT, L378 and V383, is sufficient to disrupt ImuA'-ImuB interaction. To our knowledge, this constitutes the first experimental evidence showing the role of the RecA-NT motif in mediating the interaction between a Y-family member and a RecA homolog.

The Myxobacterial Antibiotic Myxovalargin: Biosynthesis, Structural Revision, Total Synthesis, and Molecular Characterization of Ribosomal Inhibition.

Resistance of bacterial pathogens against antibiotics is declared by WHO as a major global health threat. As novel antibacterial agents are urgently needed, we re-assessed the broad-spectrum myxobacterial antibiotic myxovalargin and found it to be extremely potent against Mycobacterium tuberculosis. To ensure compound supply for further development, we studied myxovalargin biosynthesis in detail enabling production via fermentation of a native producer. Feeding experiments as well as functional genomics analysis suggested a structural revision, which was eventually corroborated by the development of a concise total synthesis. The ribosome was identified as the molecular target based on resistant mutant sequencing, and a cryo-EM structure revealed that myxovalargin binds within and completely occludes the exit tunnel, consistent with a mode of action to arrest translation during a late stage of translation initiation. These studies open avenues for structure-based scaffold improvement toward development as an antibacterial agent.

Hydrazone-Tethered 5-(Pyridin-4-yl)-4H-1,2,4-triazole-3-thiol Hybrids: Synthesis, Characterisation, in silico ADME Studies, and in†vitro Antimycobacterial Evaluation and Cytotoxicity.

Compounds containing arylpyrrole-, 1,2,4-triazole- and hydrazone structural frameworks have been widely studied and demonstrated to exhibit a wide range of pharmacological properties. Herein, an exploratory series of new 1,2,4-triazole derivatives designed by amalgamation of arylpyrrole and 1,2,4-triazole structural units via a hydrazone linkage is reported. The synthesised compounds were tested in vitro for their potential activity against Mycobacterium tuberculosis (MTB) H37 Rv strain. The most promising compound 13 - the derivative without the benzene ring appended to the pyrrole unit displayed acceptable activity (MIC90 =3.99 µM) against MTB H37 Rv, while other compounds from the series exhibited modest to weak antimycobacterial activity with MIC90 values in the range between 7.0 and >125 µM. Furthermore, in silico results, predicated using the SwissADME web tool, show that the prepared compounds display desirable ADME profile with parameters within acceptable range.

Developing synergistic drug combinations to restore antibiotic sensitivity in drug-resistant Mycobacterium tuberculosis.

Tuberculosis (TB) is a leading global cause of mortality owing to an infectious agent, accounting for almost one-third of antimicrobial resistance (AMR) deaths annually. We aimed to identify synergistic anti-TB drug combinations with the capacity to restore therapeutic efficacy against drug-resistant mutants of the causative agent, Mycobacterium tuberculosis We investigated combinations containing the known translational inhibitors, spectinomycin (SPT) and fusidic acid (FA), or the phenothiazine, chlorpromazine (CPZ), which disrupts mycobacterial energy metabolism. Potentiation of whole-cell drug efficacy was observed in SPT-CPZ combinations. This effect was lost against an M. tuberculosis mutant lacking the major facilitator superfamily (MFS) efflux pump, Rv1258c. Notably, the SPT-CPZ combination partially restored SPT efficacy against an SPT-resistant mutant carrying a g1379t point mutation in rrs, encoding the mycobacterial 16S ribosomal RNA. Combinations of SPT with FA, which targets the mycobacterial elongation factor G, exhibited potentiating activity against wild-type M. tuberculosis Moreover, this combination produced a modest potentiating effect against both FA-monoresistant and SPT-monoresistant mutants. Finally, combining SPT with the frontline anti-TB agents, rifampicin (RIF) and isoniazid, resulted in enhanced activity in vitro and ex vivo against both drug-susceptible M. tuberculosis and a RIF-monoresistant rpoB S531L mutant.These results support the utility of novel potentiating drug combinations in restoring antibiotic susceptibility of M. tuberculosis strains carrying genetic resistance to any one of the partner compounds.