High efficacy of the F-ATP synthase inhibitor TBAJ-5307 against nontuberculous mycobacteria in vitro and in vivo.

The F1FO-ATP synthase engine is essential for viability and growth of nontuberculous mycobacteria (NTM) by providing the biological energy ATP and keeping ATP homeostasis under hypoxic stress conditions. Here, we report the discovery of the diarylquinoline TBAJ-5307 as a broad spectrum anti-NTM inhibitor, targeting the FO domain of the engine and preventing rotation and proton translocation. TBAJ-5307 is active at low nanomolar concentrations against fast- and slow-growing NTM as well as clinical isolates by depleting intrabacterial ATP. As demonstrated for the fast grower Mycobacterium abscessus, the compound is potent in vitro and in vivo, without inducing toxicity. Combining TBAJ-5307 with anti-NTM antibiotics or the oral tebipenem-avibactam pair showed attractive potentiation. Furthermore, the TBAJ-5307-tebipenem-avibactam cocktail kills the pathogen, suggesting a novel oral combination for the treatment of NTM lung infections.

An essential protease, FtsH, influences daptomycin resistance acquisition in Enterococcus faecalis.

Daptomycin is a last-line antibiotic commonly used to treat vancomycin-resistant Enterococci, but resistance evolves rapidly and further restricts already limited treatment options. While genetic determinants associated with clinical daptomycin resistance (DAPR) have been described, information on factors affecting the speed of DAPR acquisition is limited. The multiple peptide resistance factor (MprF), a phosphatidylglycerol-modifying enzyme involved in cationic antimicrobial resistance, is linked to DAPR in pathogens such as methicillin-resistant Staphylococcus aureus. Since Enterococcus faecalis encodes two paralogs of mprF and clinical DAPR mutations do not map to mprF, we hypothesized that functional redundancy between the paralogs prevents mprF-mediated resistance and masks other evolutionary pathways to DAPR. Here, we performed in vitro evolution to DAPR in mprF mutant background. We discovered that the absence of mprF results in slowed DAPR evolution and is associated with inactivating mutations in ftsH, resulting in the depletion of the chaperone repressor HrcA. We also report that ftsH is essential in the parental, but not in the ΔmprF, strain where FtsH depletion results in growth impairment in the parental strain, a phenotype associated with reduced extracellular acidification and reduced ability for metabolic reduction. This presents FtsH and HrcA as enticing targets for developing anti-resistance strategies.

An optimized cyclophosphamide-treated mouse model of Mycobacterium abscessus pulmonary infection.

Mycobacterium abscessus pulmonary infections are increasingly problematic, especially for immunocompromised individuals and those with underlying lung conditions. Currently, there is no reliable standardized treatment, underscoring the need for improved preclinical drug testing. We present a simplified immunosuppressed mouse model using only four injections of cyclophosphamide, which allows for sustained M. abscessus lung burden for up to 16 days. This model proved effective for antibiotic efficacy evaluation, as demonstrated with imipenem or amikacin.

HflX is a GTPase that controls hypoxia-induced replication arrest in slow-growing mycobacteria.

GTPase high frequency of lysogenization X (HflX) is highly conserved in prokaryotes and acts as a ribosome-splitting factor as part of the heat shock response in Escherichia coli. Here we report that HflX produced by slow-growing Mycobacterium bovis bacillus Calmette-Guérin (BCG) is a GTPase that plays a critical role in the pathogen's transition to a nonreplicating, drug-tolerant state in response to hypoxia. Indeed, HflX-deficient M. bovis BCG (KO) replicated markedly faster in the microaerophilic phase of a hypoxia model that resulted in premature entry into dormancy. The KO mutant displayed hallmarks of nonreplicating mycobacteria, including phenotypic drug resistance, altered morphology, low intracellular ATP levels, and overexpression of Dormancy (Dos) regulon proteins. Mice nasally infected with HflX KO mutant displayed increased bacterial burden in the lungs, spleen, and lymph nodes during the chronic phase of infection, consistent with the higher replication rate observed in vitro in microaerophilic conditions. Unlike fast growing mycobacteria, M. bovis BCG HlfX was not involved in antibiotic resistance under aerobic growth. Proteomics, pull-down, and ribo-sequencing approaches supported that mycobacterial HflX is a ribosome-binding protein that controls translational activity of the cell. With HflX fully conserved between M. bovis BCG and M. tuberculosis, our work provides further insights into the molecular mechanisms deployed by pathogenic mycobacteria to adapt to their hypoxic microenvironment.

Cryo-Electron Microscopy Structure of the Mycobacterium tuberculosis Cytochrome bcc:aa3 Supercomplex and a Novel Inhibitor Targeting Subunit Cytochrome cI.

The mycobacterial cytochrome bcc:aa3 complex deserves the name "supercomplex" since it combines three cytochrome oxidases-cytochrome bc, cytochrome c, and cytochrome aa3-into one supramolecular machine and performs electron transfer for the reduction of oxygen to water and proton transport to generate the proton motive force for ATP synthesis. Thus, the bcc:aa3 complex represents a valid drug target for Mycobacterium tuberculosis infections. The production and purification of an entire M. tuberculosis cytochrome bcc:aa3 are fundamental for biochemical and structural characterization of this supercomplex, paving the way for new inhibitor targets and molecules. Here, we produced and purified the entire and active M. tuberculosis cyt-bcc:aa3 oxidase, as demonstrated by the different heme spectra and an oxygen consumption assay. The resolved M. tuberculosis cyt-bcc:aa3 cryo-electron microscopy structure reveals a dimer with its functional domains involved in electron, proton, oxygen transfer, and oxygen reduction. The structure shows the two cytochrome cIcII head domains of the dimer, the counterpart of the soluble mitochondrial cytochrome c, in a so-called "closed state," in which electrons are translocated from the bcc to the aa3 domain. The structural and mechanistic insights provided the basis for a virtual screening campaign that identified a potent M. tuberculosis cyt-bcc:aa3 inhibitor, cytMycc1. cytMycc1 targets the mycobacterium-specific α3-helix of cytochrome cI and interferes with oxygen consumption by interrupting electron translocation via the cIcII head. The successful identification of a new cyt-bcc:aa3 inhibitor demonstrates the potential of a structure-mechanism-based approach for novel compound development.

Designer broad-spectrum polyimidazolium antibiotics.

For a myriad of different reasons most antimicrobial peptides (AMPs) have failed to reach clinical application. Different AMPs have different shortcomings including but not limited to toxicity issues, potency, limited spectrum of activity, or reduced activity in situ. We synthesized several cationic peptide mimics, main-chain cationic polyimidazoliums (PIMs), and discovered that, although select PIMs show little acute mammalian cell toxicity, they are potent broad-spectrum antibiotics with activity against even pan-antibiotic-resistant gram-positive and gram-negative bacteria, and mycobacteria. We selected PIM1, a particularly potent PIM, for mechanistic studies. Our experiments indicate PIM1 binds bacterial cell membranes by hydrophobic and electrostatic interactions, enters cells, and ultimately kills bacteria. Unlike cationic AMPs, such as colistin (CST), PIM1 does not permeabilize cell membranes. We show that a membrane electric potential is required for PIM1 activity. In laboratory evolution experiments with the gram-positive Staphylococcus aureus we obtained PIM1-resistant isolates most of which had menaquinone mutations, and we found that a site-directed menaquinone mutation also conferred PIM1 resistance. In similar experiments with the gram-negative pathogen Pseudomonas aeruginosa, PIM1-resistant mutants did not emerge. Although PIM1 was efficacious as a topical agent, intraperitoneal administration of PIM1 in mice showed some toxicity. We synthesized a PIM1 derivative, PIM1D, which is less hydrophobic than PIM1. PIM1D did not show evidence of toxicity but retained antibacterial activity and showed efficacy in murine sepsis infections. Our evidence indicates the PIMs have potential as candidates for development of new drugs for treatment of pan-resistant bacterial infections.

Sensitivity of Mycobacterium leprae to Telacebec.

The treatment of leprosy is long and complex, benefiting from the development of sterilizing, rapidly-acting drugs. Reductive evolution made Mycobacterium leprae exquisitely sensitive to Telacebec, a phase 2 drug candidate for tuberculosis. The unprecedented potency of Telacebec against M. leprae warrants further validation in clinical trials.

New 2-Ethylthio-4-methylaminoquinazoline derivatives inhibiting two subunits of cytochrome bc1 in Mycobacterium tuberculosis.

The emergence of multi-drug (MDR-TB) and extensively-drug resistant tuberculosis (XDR-TB) is a major threat to the global management of tuberculosis (TB) worldwide. New chemical entities are of need to treat drug-resistant TB. In this study, the mode of action of new, potent quinazoline derivatives was investigated against Mycobacterium tuberculosis (M. tb). Four derivatives 11626141, 11626142, 11626252 and 11726148 showed good activity (MIC ranging from 0.02-0.09 µg/mL) and low toxicity (TD50 ≥ 5µg/mL) in vitro against M. tb strain H37Rv and HepG2 cells, respectively. 11626252 was the most selective compound from this series. Quinazoline derivatives were found to target cytochrome bc1 by whole-genome sequencing of mutants selected with 11626142. Two resistant mutants harboured the transversion T943G (Trp312Gly) and the transition G523A (Gly175Ser) in the cytochrome bc1 complex cytochrome b subunit (QcrB). Interestingly, a third mutant QuinR-M1 contained a mutation in the Rieske iron-sulphur protein (QcrA) leading to resistance to quinazoline and other QcrB inhibitors, the first report of cross-resistance involving QcrA. Modelling of both QcrA and QcrB revealed that all three resistance mutations are located in the stigmatellin pocket, as previously observed for other QcrB inhibitors such as Q203, AX-35, and lansoprazole sulfide (LPZs). Further analysis of the mode of action in vitro revealed that 11626252 exposure leads to ATP depletion, a decrease in the oxygen consumption rate and also overexpression of the cytochrome bd oxidase in M. tb. Our findings suggest that quinazoline-derived compounds are a new and attractive chemical entity for M. tb drug development targeting two separate subunits of the cytochrome bc1 complex.

Nontoxic Antimicrobial Cationic Peptide Nanoconstructs with Bacteria-Displaceable Polymeric Counteranions.

Antimicrobial peptides that target the integrity of bacterial envelopes can eradicate pathogens with little development of resistance, but they often inflict nonselective toxicity toward mammalian cells. The prevailing approach to optimize the selectivity of cationic peptides has been to modify their composition. Instead, we invent a new generation of broad-spectrum antibacterial nanoconstructs with negligible mammalian cell toxicity through a competitive displacement of counter polyanions from the complementary polycations. The nanoconstruct, which has a highly cationic Au nanoparticles (NPs) core shielded by polymeric counterions, is inert in nonbacterial environments. When exposed to negatively charged bacterial envelopes, this construct sheds its polyanions, triggering a cationic Au NP/bacterial membrane interaction that rapidly kills Gram-positive and Gram-negative bacteria. The anionic charge and hydrophilicity of the polyanion provides charge neutralization for the peptide-decorated Au NP core, but it is also bacteria-displaceable. These results provide a foundation for the development of other cationic particles and polymeric counterion combinations with potent antimicrobial activity without toxicity.

The QcrB Inhibitors TB47 and Telacebec Do Not Potentiate the Activity of Clofazimine in Mycobacterium abscessus.

The antituberculosis drug telacebec is ineffective against Mycobacterium abscessus. A recent study suggested that TB47, a telacebec analogue, potentiated the efficacy of clofazimine against M. abscessus. Here, we report that TB47 not only is ineffective against M. abscessus in vitro but also does not potentiate the activity of clofazimine.

Inhibitors of energy metabolism interfere with antibiotic-induced death in mycobacteria.

Energy metabolism has recently gained interest as a target space for antibiotic drug development in mycobacteria. Of particular importance is bedaquiline (Sirturo), which kills mycobacteria by inhibiting the F1F0 ATP synthase. Other components of the electron transport chain such as the NADH dehydrogenases (NDH-2 and NdhA) and the terminal respiratory oxidase bc1:aa3 are also susceptible to chemical inhibition. Because antituberculosis drugs are prescribed as part of combination therapies, the interaction between novel drugs targeting energy metabolism and classical first and second line antibiotics must be considered to maximize treatment efficiency. Here, we show that subinhibitory concentration of drugs targeting the F1F0 ATP synthase and the cytochrome bc1:aa3, as well as energy uncouplers, interfere with the bactericidal potency of isoniazid and moxifloxacin. Isoniazid- and moxifloxacin-induced mycobacterial death correlated with a transient increase in intracellular ATP that was dissipated by co-incubation with energy metabolism inhibitors. Although oxidative phosphorylation is a promising target space for drug development, a better understanding of the link between energy metabolism and antibiotic-induced mycobacterial death is essential to develop potent drug combinations for the treatment of tuberculosis.

Toward a Single-Dose Cure for Buruli Ulcer.

A single dose of Q203 (Telacebec), a phase 2 clinical candidate for tuberculosis, eradicates Mycobacterium ulcerans in a mouse model of Buruli ulcer infection without relapse up to 19 weeks posttreatment. Clinical use of Q203 may dramatically simplify the clinical management of Buruli ulcer, a neglected mycobacterial disease.

Exploiting the synthetic lethality between terminal respiratory oxidases to kill Mycobacterium tuberculosis and clear host infection.

The recent discovery of small molecules targeting the cytochrome bc1 :aa3 in Mycobacterium tuberculosis triggered interest in the terminal respiratory oxidases for antituberculosis drug development. The mycobacterial cytochrome bc1 :aa3 consists of a menaquinone:cytochrome c reductase (bc1 ) and a cytochrome aa3 -type oxidase. The clinical-stage drug candidate Q203 interferes with the function of the subunit b of the menaquinone:cytochrome c reductase. Despite the affinity of Q203 for the bc1 :aa3 complex, the drug is only bacteriostatic and does not kill drug-tolerant persisters. This raises the possibility that the alternate terminal bd-type oxidase (cytochrome bd oxidase) is capable of maintaining a membrane potential and menaquinol oxidation in the presence of Q203. Here, we show that the electron flow through the cytochrome bd oxidase is sufficient to maintain respiration and ATP synthesis at a level high enough to protect M. tuberculosis from Q203-induced bacterial death. Upon genetic deletion of the cytochrome bd oxidase-encoding genes cydAB, Q203 inhibited mycobacterial respiration completely, became bactericidal, killed drug-tolerant mycobacterial persisters, and rapidly cleared M. tuberculosis infection in vivo. These results indicate a synthetic lethal interaction between the two terminal respiratory oxidases that can be exploited for anti-TB drug development. Our findings should be considered in the clinical development of drugs targeting the cytochrome bc1 :aa3 , as well as for the development of a drug combination targeting oxidative phosphorylation in M. tuberculosis.

Discovery of a Novel Mycobacterial F-ATP Synthase Inhibitor and its Potency in Combination with Diarylquinolines.

The F1 FO -ATP synthase is required for growth and viability of Mycobacterium tuberculosis and is a validated clinical target. A mycobacterium-specific loop of the enzyme's rotary γ subunit plays a role in the coupling of ATP synthesis within the enzyme complex. We report the discovery of a novel antimycobacterial, termed GaMF1, that targets this γ subunit loop. Biochemical and NMR studies show that GaMF1 inhibits ATP synthase activity by binding to the loop. GaMF1 is bactericidal and is active against multidrug- as well as bedaquiline-resistant strains. Chemistry efforts on the scaffold revealed a dynamic structure activity relationship and delivered analogues with nanomolar potencies. Combining GaMF1 with bedaquiline or novel diarylquinoline analogues showed potentiation without inducing genotoxicity or phenotypic changes in a human embryonic stem cell reporter assay. These results suggest that GaMF1 presents an attractive lead for the discovery of a novel class of anti-tuberculosis F-ATP synthase inhibitors.

A Glycosylated Cationic Block Poly(ß-peptide) Reverses Intrinsic Antibiotic Resistance in All ESKAPE Gram-Negative Bacteria.

Carbapenem-resistant Gram-negative bacteria (GNB) are heading the list of pathogens for which antibiotics are the most critically needed. Many antibiotics are either unable to penetrate the outer-membrane or are excluded by efflux mechanisms. Here, we report a cationic block ß-peptide (PAS8-b-PDM12) that reverses intrinsic antibiotic resistance in GNB by two distinct mechanisms of action. PAS8-b-PDM12 does not only compromise the integrity of the bacterial outer-membrane, it also deactivates efflux pump systems by dissipating the transmembrane electrochemical potential. As a result, PAS8-b-PDM12 sensitizes carbapenem- and colistin-resistant GNB to multiple antibiotics in vitro and in vivo. The ß-peptide allows the perfect alternation of cationic versus hydrophobic side chains, representing a significant improvement over previous antimicrobial α-peptides sensitizing agents. Together, our results indicate that it is technically possible for a single adjuvant to reverse innate antibiotic resistance in all pathogenic GNB of the ESKAPE group, including those resistant to last resort antibiotics.

Target Engagement and Binding Mode of an Antituberculosis Drug to Its Bacterial Target Deciphered in Whole Living Cells by NMR.

Detailed information on hit-target interaction is very valuable for drug discovery efforts and indispensable for rational hit to lead optimization. We developed a new approach combining NMR in whole-cells in-cell NMR) and docking to characterize hit-target interaction at the atomic level. By using in-cell NMR, we validated target engagement of the antituberculosis imidazopyridine amide (IPA) series with the subunit b of the cytochrome bc1:aa3, the major respiratory terminal oxidase in mycobacteria. The most advanced IPA called Q203 is currently in clinical trial. Using its derivative IPA317, we identified the atoms of the drug interacting with the cytochrome b in whole cells. NMR data and the self-organizing map algorithm were used to cluster a large set of drug-target complex models. The selected ensemble revealed IPA317 in a transient cavity of the cytochrome b, interacting directly with the residue T313, which is the site of spontaneous mutation conferring resistance to the IPA series. Our approach constitutes a pipeline to obtain atomic information on hit-target interactions in the cellular context.

Bedaquiline Inhibits the ATP Synthase in Mycobacterium abscessus and Is Effective in Infected Zebrafish.

Pulmonary infections caused by Mycobacterium abscessus are emerging as a global threat, especially in cystic fibrosis patients. Further intensifying the concern of M. abscessus infection is the recent evidence of human-to-human transmission of the infection. M. abscessus is a naturally multidrug-resistant fast-growing pathogen for which pharmacological options are limited. Repurposing antitubercular drugs represents an attractive option for the development of chemotherapeutic alternatives against M. abscessus infections. Bedaquiline (BDQ), an ATP synthase inhibitor, has recently been approved for the treatment of multidrug-resistant tuberculosis. Herein, we show that BDQ has a very low MIC against a vast panel of clinical isolates. Despite being bacteriostatic in vitro, BDQ was highly efficacious in a zebrafish model of M. abscessus infection. Remarkably, a very short period of treatment was sufficient to protect the infected larvae from M. abscessus-induced killing. This was corroborated with reduced numbers of abscesses and cords, considered to be major pathophysiological signs in infected zebrafish. Mode-of-action studies revealed that BDQ triggered a rapid depletion of ATP in M. abscessusin vitro, consistent with the drug targeting the FoF1 ATP synthase. Importantly, despite a failure to select in vitro for spontaneous mutants that are highly resistant to BDQ, the transfer of single nucleotide polymorphisms leading to D29V or A64P substitutions in atpE conferred high resistance, thus resolving the target of BDQ in M. abscessus Overall, this study indicates that BDQ is active against M. abscessusin vitro and in vivo and should be considered for clinical use against the difficult-to-manage M. abscessus pulmonary infections.

Microbiomes in respiratory health and disease: An Asia-Pacific perspective.

There is currently enormous interest in studying the role of the microbiome in health and disease. Microbiome's role is increasingly being applied to respiratory diseases, in particular COPD, asthma, cystic fibrosis and bronchiectasis. The changes in respiratory microbiomes that occur in these diseases and how they are modified by environmental challenges such as cigarette smoke, air pollution and infection are being elucidated. There is also emerging evidence that gut microbiomes play a role in lung diseases through the modulation of systemic immune responses and can be modified by diet and antibiotic treatment. There are issues that are particular to the Asia-Pacific region involving diet and prevalence of specific respiratory diseases. Each of these issues is further complicated by the effects of ageing. The challenges now are to elucidate the cause and effect relationships between changes in microbiomes and respiratory diseases and how to translate these into new treatments and clinical care. Here we review the current understanding and progression in these areas.

The role of acute and chronic respiratory colonization and infections in the pathogenesis of COPD.

COPD is a major global concern, increasingly so in the context of ageing populations. The role of infections in disease pathogenesis and progression is known to be important, yet the mechanisms involved remain to be fully elucidated. While COPD pathogens such as Haemophilus influenzae, Moraxella catarrhalis and Streptococcus pneumoniae are strongly associated with acute exacerbations of COPD (AECOPD), the clinical relevance of these pathogens in stable COPD patients remains unclear. Immune responses in stable and colonized COPD patients are comparable to those detected in AECOPD, supporting a role for chronic colonization in COPD pathogenesis through perpetuation of deleterious immune responses. Advances in molecular diagnostics and metagenomics now allow the assessment of microbe-COPD interactions with unprecedented personalization and precision, revealing changes in microbiota associated with the COPD disease state. As microbial changes associated with AECOPD, disease severity and therapeutic intervention become apparent, a renewed focus has been placed on the microbiology of COPD and the characterization of the lung microbiome in both its acute and chronic states. Characterization of bacterial, viral and fungal microbiota as part of the lung microbiome has the potential to reveal previously unrecognized prognostic markers of COPD that predict disease outcome or infection susceptibility. Addressing such knowledge gaps will ultimately lead to a more complete understanding of the microbe-host interplay in COPD. This will permit clearer distinctions between acute and chronic infections and more granular patient stratification that will enable better management of these features and of COPD.

Isolation and Characterization of a Hybrid Respiratory Supercomplex Consisting of Mycobacterium tuberculosis Cytochrome bcc and Mycobacterium smegmatis Cytochrome aa3.

Recently, energy production pathways have been shown to be viable antitubercular drug targets to combat multidrug-resistant tuberculosis and eliminate pathogen in the dormant state. One family of drugs currently under development, the imidazo[1,2-a]pyridine derivatives, is believed to target the pathogen's homolog of the mitochondrial bc1 complex. This complex, denoted cytochrome bcc, is highly divergent from mitochondrial Complex III both in subunit structure and inhibitor sensitivity, making it a good target for drug development. There is no soluble cytochrome c in mycobacteria to transport electrons from the bcc complex to cytochrome oxidase. Instead, the bcc complex exists in a "supercomplex" with a cytochrome aa3-type cytochrome oxidase, presumably allowing direct electron transfer. We describe here purification and initial characterization of the mycobacterial cytochrome bcc-aa3 supercomplex using a strain of M. smegmatis that has been engineered to express the M. tuberculosis cytochrome bcc. The resulting hybrid supercomplex is stable during extraction and purification in the presence of dodecyl maltoside detergent. It is hoped that this purification procedure will potentiate functional studies of the complex as well as crystallographic studies of drug binding and provide structural insight into a third class of the bc complex superfamily.