CRISPR interference identifies vulnerable cellular pathways with bactericidal phenotypes in Mycobacterium tuberculosis.

Mycobacterium tuberculosis remains a leading cause of death for which new drugs are needed. The identification of drug targets has been advanced by high-throughput and targeted genetic deletion strategies. Each though has limitations including the inability to distinguish between levels of vulnerability, lethality, and scalability as a molecular tool. Using mycobacterial CRISPR interference in combination with phenotypic screening, we have overcome these individual issues to investigate essentiality, vulnerability and lethality for 94 target genes from a diverse array of cellular pathways, many of which are potential antibiotic targets. Essential genes involved in cell wall synthesis and central cellular functions were equally vulnerable and often had bactericidal consequences. Conversely, essential genes involved in metabolism, oxidative phosphorylation, or amino acid synthesis were less vulnerable to inhibition and frequently bacteriostatic. In conclusion, this study provides novel insights into mycobacterial genetics and biology that will help to prioritize potential drug targets.

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 they 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 nonviable. This essentiality makes it a candidate target for the development of new antibiotics. In the model organism Mycolicibacterium smegmatis, RNase HI activity is provided by two 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, the latter by a striking near 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 rifampicin. 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 antimycobacterial drug.

Utilization of CRISPR interference to investigate the contribution of genes to pathogenesis in a macrophage model of Mycobacterium tuberculosis infection.

OBJECTIVES: There is an urgent need for novel drugs that target unique cellular pathways to combat infections caused by Mycobacterium tuberculosis. CRISPR interference (CRISPRi)-mediated transcriptional repression has recently been developed for use in mycobacteria as a genetic tool for identifying and validating essential genes as novel drug targets. Whilst CRISPRi has been applied to extracellular bacteria, no studies to date have determined whether CRISPRi can be used in M. tuberculosis infection models. METHODS: Using the human monocytic macrophage-like THP-1 cell line as a model for M. tuberculosis infection we investigated if CRISPRi can be activated within intracellular M. tuberculosis. RESULTS: The transcriptional repression of two candidate M. tuberculosis genes, i.e. mmpL3 and qcrB, leads to a reduction in viable M. tuberculosis within infected THP-1 cells. The reduction in viable colonies is dependent on both the level of CRISPRi-mediated repression and the duration of repression. CONCLUSIONS: These results highlight the utility of CRISPRi in exploring mycobacterial gene function and essentiality under a variety of conditions pertinent to host infection.

Discovery of 5-methylpyrimidopyridone analogues as selective antimycobacterial agents.

With the emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MDR-TB) and extensive drug-resistant strains (XDR-TB), there is an urgent need to develop novel drugs for the treatment of tuberculosis. Here, we designed and synthesized a series of 5-methylpyrimidopyridone analogues as potential antitubercular agents. The most potent compound 6q exhibited a MIC value of 4 µM in vitro against Mycobacterium tuberculosis. The antitubercular activities of the synthesized compounds were impacted by the amantadine and 2-chlorophenyl groups, and were enhanced by the presence of 3-methyl(4-dimethylamino)piperidinylphenyl. Molecular modeling and binding studies suggest that PknB is the potential molecular target of 5-methylpyrimidopyridone compounds. This study provides insights for the future development of new antimycobacterial agents with novel mechanisms of action.

Total Synthesis and Antimycobacterial Activity of Ohmyungsamycin†A, Deoxyecumicin, and Ecumicin.

The ohmyungsamycin and ecumicin natural product families are structurally related cyclic depsipeptides that display potent antimycobacterial activity. Herein the total syntheses of ohmyungsamycin A, deoxyecumicin, and ecumicin are reported, together with the direct biological comparison of members of these natural product families against Mycobacterium tuberculosis (Mtb), the etiological agent of tuberculosis (TB). The synthesis of each of the natural products employed a solid-phase strategy to assemble the linear peptide precursor, involving a key on-resin esterification and an optional on-resin dimethylation step, before a final solution-phase macrolactamization between the non-proteinogenic N-methyl-4-methoxy-l-tryptophan amino acid and a bulky N-methyl-l-valine residue. The synthetic natural products possessed potent antimycobacterial activity against Mtb with MIC90 's ranging from 110-360 nm and retained activity against Mtb in Mtb-infected macrophages. Deoxyecumicin also exhibited rapid bactericidal killing against Mtb, sterilizing cultures after 21 days.

6-Substituted amiloride derivatives as inhibitors of the urokinase-type plasminogen activator for use in metastatic disease.

The oral K+-sparing diuretic amiloride shows anti-cancer side-activities in multiple rodent models. These effects appear to arise, at least in part, through moderate inhibition of the urokinase-type plasminogen activator (uPA, Ki = 2.4 µM), a pro-metastatic trypsin-like serine protease that is upregulated in many aggressive solid malignancies. In applying the selective optimization of side-activity (SOSA) approach, a focused library of twenty two 6-substituted amiloride derivatives were prepared, with multiple examples displaying uPA inhibitory potencies in the nM range. X-ray co-crystal structures revealed that the potency increases relative to amiloride arise from increased occupancy of uPA's S1ß subsite by the appended 6-substituents. Leading compounds were shown to have high selectivity over related trypsin-like serine proteases and no diuretic or anti-kaliuretic effects in rats. Compound 15 showed anti-metastatic effects in a xenografted mouse model of late-stage lung metastasis.

The synthesis and evaluation of quinolinequinones as anti-mycobacterial agents.

A library of thirty-two quinolinequinones (QQs) with various amine substituents at the 6- and 7-positions were synthesised efficiently and in good yields for evaluation as potential anti-tuberculosis agents. Mycobacterium tuberculosis growth inhibition assays demonstrated that QQs bearing moderate length alkyl chains (i.e. heptylphenylamino- and octylamino-QQs), and aryl groups (i.e. phenylethylamino- and benzylamino-QQs) exhibited encouraging inhibitory activity, while QQ analogue 7-chloro-6-propargylamino-quinoline-5,8-dione (16b) had excellent inhibitory activity (MIC = 8 µM). The cLogP values and redox activities of the QQs were determined, and neither readout correlated with the anti-mycobacterial activities of the compounds. Notwithstanding, mode of action studies of 16b revealed that treatment of M. tuberculosis with this compound led to activation of NADH-dependent oxygen consumption suggesting a redox cycling mechanism. To this end, the promising anti-mycobacterial activity of several QQs and their ability to perturb oxygen management leading to an uncontrolled respiratory burst, as identified in this work and by others, demonstrates the merit of further optimising the anti-mycobacterial activity of this readily synthesised class of compound.

Activation of type II NADH dehydrogenase by quinolinequinones mediates antitubercular cell death.

OBJECTIVES: Quinolinequinones (QQ) have been shown to inhibit the growth of mycobacterial species, but their mode(s) of action and molecular target(s) remain unknown. To facilitate further development of QQ as antimycobacterial drugs, we investigated the molecular mechanism and target of QQ in mycobacteria. METHODS: Cell viability of Mycobacterium tuberculosis and Mycobacterium bovis bacillus Calmette-Guérin was determined in the presence of QQ8c, a representative QQ compound, and isoniazid, a frontline antitubercular drug. The effect of QQ8c on mycobacterial energetics was studied using inverted membrane vesicles. NADH oxidation and formation of reactive oxygen species (ROS) were measured in the presence and absence of KCN. Generation of ROS was measured via oxygen consumption in an oxygen electrode. The effects of QQ8c were compared with the antimycobacterial drug clofazimine in side-by-side experiments. RESULTS: QQ8c challenge resulted in complete sterilization of cultures with no refractory resistant population observed. QQ8c stimulated NADH oxidation by type II NADH dehydrogenase (NDH-2) and oxygen consumption in inverted membrane vesicles. Large quantities of ROS were produced in the presence of QQ8. Even when oxygen consumption was blocked with KCN, activation of NDH-2 by QQ8c occurred suggesting QQ8c was redox cycling. CONCLUSIONS: QQ8c targets NDH-2 of the mycobacterial respiratory chain leading to activation of NADH oxidation and generating bactericidal levels of ROS in a manner similar to, but more effectively than, the antimycobacterial drug clofazimine. Our results validate respiratory-generated ROS as an avenue for antimycobacterial drug development.

Histone deacetylase 6 inhibits influenza A virus release by downregulating the trafficking of viral components to the plasma membrane via its substrate, acetylated microtubules.

UNLABELLED: Mammalian cells produce many proteins, such as IFITM3, ISG15, MxA, and viperin, that inhibit influenza A virus (IAV) infection. Here, we show that a new class of host protein, histone deacetylase 6 (HDAC6), inhibits IAV infection. We found that HDAC6-overexpressing cells release about 3-fold less IAV progeny, whereas HDAC6-depleted cells release about 6-fold more IAV progeny. The deacetylase activity of HDAC6 played a role in its anti-IAV function as tubacin, a specific small-molecule inhibitor of HDAC6, increased the release of IAV progeny in a dose-dependent manner. Further, as visualized by electron microscopy, tubacin-treated cells showed an increase in IAV budding at the plasma membrane, the site of IAV assembly. Tubacin is a domain-specific inhibitor and binds to one of the two HDAC6 catalytic domains possessing tubulin deacetylase activity. This indicated the potential involvement of acetylated microtubules in the trafficking of viral components to the plasma membrane. Indeed, as quantified by flow cytometry, there was about a 2.0- to 2.5-fold increase and about a 2.0-fold decrease in the amount of viral envelope protein hemagglutinin present on the plasma membrane of tubacin-treated/HDAC6-depleted and HDAC6-overexpressing cells, respectively. In addition, the viral ribonucleoprotein complex was colocalized with acetylated microtubule filaments, and viral nucleoprotein coimmunoprecipitated with acetylated tubulin. Together, our findings indicate that HDAC6 is an anti-IAV host factor and exerts its anti-IAV function by negatively regulating the trafficking of viral components to the host cell plasma membrane via its substrate, acetylated microtubules. IMPORTANCE: Host cells produce many proteins that have the natural ability to restrict influenza virus infection. Here, we discovered that another host protein, histone deacetylase 6 (HDAC6), inhibits influenza virus infection. We demonstrate that HDAC6 exerts its anti-influenza virus function by negatively regulating the trafficking of viral components to the site of influenza virus assembly via its substrate, acetylated microtubules. HDAC6 is a multisubstrate enzyme and regulates multiple cellular pathways, including the ones leading to various cancers, neurodegenerative diseases, and inflammatory disorders. Therefore, several drugs targeting HDAC6 are under clinical development for the treatment of a wide range of diseases. Influenza virus continues to be a major global public health problem due to regular emergence of drug-resistant and novel influenza virus strains in humans. As an alternative antiviral strategy, HDAC6 modulators could be employed to stimulate the anti-influenza virus potential of endogenous HDAC6 to inhibit influenza virus infection.

Phenotypic-based Discovery and Exploration of a Resorufin Scaffold with Activity against Mycobacterium Tuberculosis.

Tuberculosis remains a leading cause of death by infectious disease. The long treatment regimen and the spread of drug-resistant strains of the causative agent Mycobacterium tuberculosis (Mtb) necessitates the development of new treatment options. In a phenotypic screen, a nitrofuran-resorufin conjugate 1 was identified as a potent sub-micromolar inhibitor of whole cell Mtb. Complete loss of activity was observed for this compound in Mtb mutants affected in enzyme cofactor F420 biosynthesis (fbiC), suggesting that 1 undergoes prodrug activation in a manner similar to anti-tuberculosis prodrug pretomanid. Exploration of the structure-activity relationship led to the discovery of novel resorufin analogues that do not rely on the deazaflavin-dependent nitroreductase (Ddn) bioactivation pathway for their antimycobacterial activity. These analogues are of interest as they work through an alternative, currently unknown mechanism that may expand our chemical arsenal towards the treatment of this devastating disease.

A dual-targeting succinate dehydrogenase and F1Fo-ATP synthase inhibitor rapidly sterilizes replicating and non-replicating Mycobacterium tuberculosis.

Mycobacterial bioenergetics is a validated target space for antitubercular drug development. Here, we identify BB2-50F, a 6-substituted 5-(N,N-hexamethylene)amiloride derivative as a potent, multi-targeting bioenergetic inhibitor of Mycobacterium tuberculosis. We show that BB2-50F rapidly sterilizes both replicating and non-replicating cultures of M. tuberculosis and synergizes with several tuberculosis drugs. Target identification experiments, supported by docking studies, showed that BB2-50F targets the membrane-embedded c-ring of the F1Fo-ATP synthase and the catalytic subunit (substrate-binding site) of succinate dehydrogenase. Biochemical assays and metabolomic profiling showed that BB2-50F inhibits succinate oxidation, decreases the activity of the tricarboxylic acid (TCA) cycle, and results in succinate secretion from M. tuberculosis. Moreover, we show that the lethality of BB2-50F under aerobic conditions involves the accumulation of reactive oxygen species. Overall, this study identifies BB2-50F as an effective inhibitor of M. tuberculosis and highlights that targeting multiple components of the mycobacterial respiratory chain can produce fast-acting antimicrobials.

Identification of Chemical Scaffolds That Inhibit the Mycobacterium tuberculosis Respiratory Complex Succinate Dehydrogenase.

Drug-resistant Mycobacterium tuberculosis is a significant cause of infectious disease morbidity and mortality for which new antimicrobials are urgently needed. Inhibitors of mycobacterial respiratory energy metabolism have emerged as promising next-generation antimicrobials, but a number of targets remain unexplored. Succinate dehydrogenase (SDH), a focal point in mycobacterial central carbon metabolism and respiratory energy production, is required for growth and survival in M. tuberculosis under a number of conditions, highlighting the potential of inhibitors targeting mycobacterial SDH enzymes. To advance SDH as a novel drug target in M. tuberculosis, we utilized a combination of biochemical screening and in-silico deep learning technologies to identify multiple chemical scaffolds capable of inhibiting mycobacterial SDH activity. Antimicrobial susceptibility assays show that lead inhibitors are bacteriostatic agents with activity against wild-type and drug-resistant strains of M. tuberculosis. Mode of action studies on lead compounds demonstrate that the specific inhibition of SDH activity dysregulates mycobacterial metabolism and respiration and results in the secretion of intracellular succinate. Interaction assays demonstrate that the chemical inhibition of SDH activity potentiates the activity of other bioenergetic inhibitors and prevents the emergence of resistance to a variety of drugs. Overall, this study shows that SDH inhibitors are promising next-generation antimicrobials against M. tuberculosis.

The evolution of antibiotic resistance is associated with collateral drug phenotypes in Mycobacterium tuberculosis.

The increasing incidence of drug resistance in Mycobacterium tuberculosis has diminished the efficacy of almost all available antibiotics, complicating efforts to combat the spread of this global health burden. Alongside the development of new drugs, optimised drug combinations are needed to improve treatment success and prevent the further spread of antibiotic resistance. Typically, antibiotic resistance leads to reduced sensitivity, yet in some cases the evolution of drug resistance can lead to enhanced sensitivity to unrelated drugs. This phenomenon of collateral sensitivity is largely unexplored in M. tuberculosis but has the potential to identify alternative therapeutic strategies to combat drug-resistant strains that are unresponsive to current treatments. Here, by using drug susceptibility profiling, genomics and evolutionary studies we provide evidence for the existence of collateral drug sensitivities in an isogenic collection M. tuberculosis drug-resistant strains. Furthermore, in proof-of-concept studies, we demonstrate how collateral drug phenotypes can be exploited to select against and prevent the emergence of drug-resistant strains. This study highlights that the evolution of drug resistance in M. tuberculosis leads to collateral drug responses that can be exploited to design improved drug regimens.

Discovery of 1-hydroxy-2-methylquinolin-4(1H)-one derivatives as new cytochrome bd oxidase inhibitors for tuberculosis therapy.

The cytochrome bcc-aa3 oxidase (Cyt-bcc) of Mycobacterium tuberculosis (Mtb) is a promising anti-tuberculosis target. However, when Cyt-bcc is inhibited, cytochrome bd terminal oxidase (Cyt-bd) can still maintain the activity of the respiratory chain and drive ATP synthesis. Through virtual screening and biological validation, we discovered two FDA-approved drugs, ivacaftor and roquinimex, exhibited moderate binding affinity to Cyt-bd. Structural modifications of them led to 1-hydroxy-2-methylquinolin-4(1H)-one derivatives as potent new Cyt-bd inhibitors. Compound 8d binds to Cyt-bd with a Kd value of 4.17 µM and inhibits the growth of the Cyt-bcc knock-out strain (ΔqcrCAB, Cyt-bd+) with a MIC value of 6.25 µM. The combination of 8d with the Cyt-bcc inhibitor Q203 completely inhibited oxygen consumption of the wild-type strain and the inverted-membrane vesicles expressing M. tuberculosis Cyt-bd (ΔcydAB::MtbCydAB+). Our study provides a promising starting point for the development of novel dual chemotherapies for tuberculosis.

M. tuberculosis relies on trace oxygen to maintain energy homeostasis and survive in hypoxic environments.

The bioenergetic mechanisms by which Mycobacterium tuberculosis survives hypoxia are poorly understood. Current models assume that the bacterium shifts to an alternate electron acceptor or fermentation to maintain membrane potential and ATP synthesis. Counterintuitively, we find here that oxygen itself is the principal terminal electron acceptor during hypoxic dormancy. M. tuberculosis can metabolize oxygen efficiently at least two orders of magnitude below the concentration predicted to occur in hypoxic lung granulomas. Despite a difference in apparent affinity for oxygen, both the cytochrome bcc:aa3 and cytochrome bd oxidase respiratory branches are required for hypoxic respiration. Simultaneous inhibition of both oxidases blocks oxygen consumption, reduces ATP levels, and kills M. tuberculosis under hypoxia. The capacity of mycobacteria to scavenge trace levels of oxygen, coupled with the absence of complex regulatory mechanisms to achieve hierarchal control of the terminal oxidases, may be a key determinant of long-term M. tuberculosis survival in hypoxic lung granulomas.

Multiplexed transcriptional repression identifies a network of bactericidal interactions between mycobacterial respiratory complexes.

Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Combinations of respiratory inhibitors can have synergistic or synthetic lethal interactions with sterilizing activity, suggesting that regimens with multiple bioenergetic inhibitors could shorten treatment times. However, realizing this potential requires an understanding of which combinations of respiratory complexes, when inhibited, have the strongest consequences on bacterial growth and viability. Here we have used multiplex CRISPR interference (CRISPRi) and Mycobacterium smegmatis as a physiological and molecular model for mycobacterial respiration to identify interactions between respiratory complexes. In this study, we identified synthetic lethal and synergistic interactions between respiratory complexes and demonstrated how the engineering of CRISPRi-guide sequences can be used to further explore networks of interacting gene pairs. These results provide fundamental insights into the functions of and interactions between bioenergetic complexes and the utility of CRISPRi in designing drug combinations.

Synthesis and Biological Evaluation of Aurachin D Analogues as Inhibitors of Mycobacterium tuberculosis Cytochrome bd Oxidase.

A revised total synthesis of aurachin D (1a), an isoprenoid quinolone alkaloid that targets Mycobacterium tuberculosis (Mtb) cytochrome bd (cyt-bd) oxidase, was accomplished using an oxazoline ring-opening reaction. The ring opening enabled access to a range of electron-poor analogues, while electron-rich analogues could be prepared using the Conrad-Limpach reaction. The aryl-substituted and side-chain-modified aurachin D analogues were screened for inhibition of Mtb cyt-bd oxidase and growth inhibition of Mtb. Nanomolar inhibition of Mtb cyt-bd oxidase was observed for the shorter-chain analogue 1d (citronellyl side chain) and the aryl-substituted analogues 1g/1k (fluoro substituent at C6/C7), 1t/1v (hydroxy substituent at C5/C6) and 1u/1w/1x (methoxy substituent at C5/C6/C7). Aurachin D and the analogues did not inhibit growth of nonpathogenic Mycobacterium smegmatis, but the citronellyl (1d) and 6-fluoro-substituted (1g) inhibitors from the Mtb cyt-bd oxidase assay displayed moderate growth inhibition against pathogenic Mtb (MIC = 4-8 µM).

Uncovering interactions between mycobacterial respiratory complexes to target drug-resistant Mycobacterium tuberculosis.

Mycobacterium tuberculosis remains a leading cause of infectious disease morbidity and mortality for which new drug combination therapies are needed. Mycobacterial bioenergetics has emerged as a promising space for the development of novel therapeutics. Further to this, unique combinations of respiratory inhibitors have been shown to have synergistic or synthetic lethal interactions, suggesting that combinations of bioenergetic inhibitors could drastically shorten treatment times. Realizing the full potential of this unique target space requires an understanding of which combinations of respiratory complexes, when inhibited, have the strongest interactions and potential in a clinical setting. In this review, we discuss (i) chemical-interaction, (ii) genetic-interaction and (iii) chemical-genetic interaction studies to explore the consequences of inhibiting multiple mycobacterial respiratory components. We provide potential mechanisms to describe the basis for the strongest interactions. Finally, whilst we place an emphasis on interactions that occur with existing bioenergetic inhibitors, by highlighting interactions that occur with alternative respiratory components we envision that this information will provide a rational to further explore alternative proteins as potential drug targets and as part of unique drug combinations.

Potent Bactericidal Antimycobacterials Targeting the Chaperone ClpC1 Based on the Depsipeptide Natural Products Ecumicin and Ohmyungsamycin A.

Ohmyungsamycin A and ecumicin are structurally related cyclic depsipeptide natural products that possess activity against Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB). Herein, we describe the design and synthesis of a library of analogues of these two natural products using an efficient solid-phase synthesis and late-stage macrolactamization strategy. Lead analogues possessed potent activity against Mtb in vitro (minimum inhibitory concentration 125-500 nM) and were shown to inhibit protein degradation by the mycobacterial ClpC1-ClpP1P2 protease with an associated enhancement of ClpC1 ATPase activity. The most promising analogue from the series exhibited rapid bactericidal killing activity against Mtb, capable of sterilizing cultures after 7 days, and retained bactericidal activity against hypoxic non-replicating Mtb. This natural product analogue was also active in an in vivo zebrafish model of infection.

An amiloride derivative is active against the F1Fo-ATP synthase and cytochrome bd oxidase of Mycobacterium tuberculosis.

Increasing antimicrobial resistance compels the search for next-generation inhibitors with differing or multiple molecular targets. In this regard, energy conservation in Mycobacterium tuberculosis has been clinically validated as a promising new drug target for combatting drug-resistant strains of M. tuberculosis. Here, we show that HM2-16F, a 6-substituted derivative of the FDA-approved drug amiloride, is an anti-tubercular inhibitor with bactericidal properties comparable to the FDA-approved drug bedaquiline (BDQ; Sirturo®) and inhibits the growth of bedaquiline-resistant mutants. We show that HM2-16F weakly inhibits the F1Fo-ATP synthase, depletes ATP, and affects the entry of acetyl-CoA into the Krebs cycle. HM2-16F synergizes with the cytochrome bcc-aa3 oxidase inhibitor Q203 (Telacebec) and co-administration with Q203 sterilizes in vitro cultures in 14 days. Synergy with Q203 occurs via direct inhibition of the cytochrome bd oxidase by HM2-16F. This study shows that amiloride derivatives represent a promising discovery platform for targeting energy generation in drug-resistant tuberculosis.