Towards Selective Mycobacterial ClpP1P2 Inhibitors with Reduced Activity against the Human Proteasome.

Mycobacterium tuberculosis is responsible for the greatest number of deaths worldwide due to a bacterial agent. We recently identified bortezomib (Velcade; compound 1) as a promising antituberculosis (anti-TB) compound. We showed that compound 1 inhibits the mycobacterial caseinolytic proteases P1 and P2 (ClpP1P2) and exhibits bactericidal activity, and we established compound 1 and ClpP1P2 as an attractive lead/target couple. However, compound 1 is a human-proteasome inhibitor currently approved for cancer therapy and, as such, exhibits significant toxicity. Selective inhibition of the bacterial protease over the human proteasome is desirable in order to maintain antibacterial activity while reducing toxicity. We made use of structural data in order to design a series of dipeptidyl-boronate derivatives of compound 1. We tested these derivatives for whole-cell ClpP1P2 and human-proteasome inhibition as well as bacterial-growth inhibition and identified compounds that were up to 100-fold-less active against the human proteasome but that retained ClpP1P2 and mycobacterial-growth inhibition as well as bactericidal potency. The lead compound, compound 58, had low micromolar ClpP1P2 and anti-M. tuberculosis activity, good aqueous solubility, no cytochrome P450 liabilities, moderate plasma protein binding, and low toxicity in two human liver cell lines, and despite high clearance in microsomes, this compound was only moderately cleared when administered intravenously or orally to mice. Higher-dose oral pharmacokinetics indicated good dose linearity. Furthermore, compound 58 was inhibitory to only 11% of a panel of 62 proteases. Our work suggests that selectivity over the human proteasome can be achieved with a drug-like template while retaining potency against ClpP1P2 and, crucially, anti-M. tuberculosis activity.

inPhocus: Current State and Challenges of Phage Research in Singapore.

Bacteriophages and phage-derived proteins are a promising class of antibacterial agents that experience a growing worldwide interest. To map ongoing phage research in Singapore and neighboring countries, Lee Kong Chian School of Medicine, Nanyang Technological University Singapore (NTU) and Yong Loo Lin School of Medicine, National University of Singapore (NUS) recently co-organized a virtual symposium on Bacteriophage and Bacteriophage-Derived Technologies, which was attended by more than 80 participants. Topics were discussed relating to phage life cycles, diversity, the roles of phages in biofilms and the human gut microbiome, engineered phage lysins to combat polymicrobial infections in wounds, and the challenges and prospects of clinical phage therapy. This perspective summarizes major points discussed during the symposium and new perceptions that emerged after the panel discussion.

Hydrogen Sulfide Sensitizes Acinetobacter baumannii to Killing by Antibiotics.

The production of endogenous hydrogen sulfide (H2S) has been shown to confer antibiotic tolerance in all bacteria studied to date. Therefore, this mediator has been speculated to be a universal defense mechanism against antibiotics in bacteria. This is assuming that all bacteria produce endogenous H2S. In this study, we established that the pathogenic bacteria Acinetobacter baumannii does not produce endogenous H2S, giving us the opportunity to test the effect of exogenous H2S on antibiotic tolerance in a bacterium that does not produce it. By using a H2S-releasing compound to modulate the sulfide content in A. baumannii, we demonstrated that instead of conferring antibiotic tolerance, exogenous H2S sensitized A. baumannii to multiple antibiotic classes, and was able to revert acquired resistance to gentamicin. Exogenous H2S triggered a perturbation of redox and energy homeostasis that translated into hypersensitivity to antibiotic killing. We propose that H2S could be used as an antibiotic-potentiator and resistance-reversion agent in bacteria that do not produce it.

A Macromolecule Reversing Antibiotic Resistance Phenotype and Repurposing Drugs as Potent Antibiotics.

In order to mitigate antibiotic resistance, a new strategy to increase antibiotic potency and reverse drug resistance is needed. Herein, the translocation mechanism of an antimicrobial guanidinium-functionalized polycarbonate is leveraged in combination with traditional antibiotics to afford a potent treatment for drug-resistant bacteria. Particularly, this polymer-antibiotic combination approach reverses rifampicin resistance phenotype in Acinetobacter baumannii demonstrating a 2.5 × 105-fold reduction in minimum inhibitory concentration (MIC) and a 4096-fold reduction in minimum bactericidal concentration (MBC). This approach also enables the repurposing of auranofin as an antibiotic against multidrug-resistant (MDR) Gram-negative bacteria with a 512-fold MIC and 128-fold MBC reduction, respectively. Finally, the in vivo efficacy of polymer-rifampicin combination is demonstrated in a MDR bacteremia mouse model. This combination approach lays foundational ground rules for a new class of antibiotic adjuvants capable of reversing drug resistance phenotype and repurposing drugs against MDR Gram-negative bacteria.

The Mycobacterial Membrane: A Novel Target Space for Anti-tubercular Drugs.

Tuberculosis (TB) poses an enduring threat to global health. Consistently ranked among the top 10 causes of death worldwide since 2000, TB has now exceeded HIV-AIDS in terms of deaths inflicted by a single infectious agent. In spite of recently declining TB incident rates, these decreases have been incremental and fall short of threshold levels required to end the global TB epidemic. As in other infectious diseases, the emergence of resistant organisms poses a major impediment to effective TB control. Resistance in mycobacteria may evolve from genetic mutations in target genes which are transmitted during cell multiplication from mother cells to their progeny. A more insidious form of resistance involves sub-populations of non-growing ("dormant") mycobacterial persisters. Quiescent and genetically identical to their susceptible counterparts, persisters exhibit non-inheritable drug tolerance. Their prevalence account for the protracted treatment period that is required for the treatment of TB. In order to improve the efficacy of treatment against mycobacterial persisters and drug-resistant organisms, novel antitubercular agents are urgently required. Selective targeting of bacterial membranes has been proposed as a viable therapeutic strategy against infectious diseases. The underpinning rationale is that a functionally intact cell membrane is vital for both replicating and dormant bacteria. Perturbing the membrane would thus disrupt a multitude of embedded targets with lethal pleiotropic consequences, besides limiting the emergence of resistant strains. There is growing interest in exploring small molecules as selective disruptors of the mycobacterial membrane. In this review, we examined the recent literature on different chemotypes with membrane perturbing properties, the mechanisms by which they induce membrane disruption and their potential as anti-TB agents. Cationic amphiphilicity is a signature motif that is required of membrane targeting agents but adherence to this broad physical requirement does not necessarily translate to conformity in terms of biological outcomes. Nor does it ensure selective targeting of mycobacterial membranes. These are unresolved issues that require further investigation.

Bortezomib Warhead-Switch Confers Dual Activity against Mycobacterial Caseinolytic Protease and Proteasome and Selectivity against Human Proteasome.

Mycobacteria harbor two main degradative proteolytic machineries, the caseinolytic protease ClpP1P2 and a proteasome. We recently showed that Bortezomib inhibits ClpP1P2 and exhibits whole cell activity against Mycobacterium tuberculosis. Bortezomib, a dipeptide with a boronic acid warhead, is a human proteasome inhibitor approved for cancer therapy. The boronic acid warhead of the compound has been shown to drive potency against both the human proteasome and ClpP1P2 protease. Selectivity for the bacterial ClpP1P2 protease over the human proteasome is lacking but needs to be achieved to move this new anti-tuberculosis lead forward. In this study we explored whether an alternative warhead could influence Bortezomib's selectivity. We synthesized an analog containing a chloromethyl ketone instead of the boronic acid warhead and determined potencies against the bacterial and human enzymes. Surprisingly, the analog retained activity against mycobacterial ClpP1P2 and was active against the mycobacterial proteasome, but was devoid of activity against the human proteasome. Interrogation of a set of chloromethyl ketone peptides identified three additional compounds similarly inhibiting both ClpP1P2 and the proteasome in the bacteria while leaving the human proteasome untouched. Finally, we showed that these compounds display bactericidal activity against M. tuberculosis with cytotoxicity ranging from acceptable to undetectable. These results suggest that selectivity over the human proteasome is achievable. Selectivity, together with dual-targeting of mycobacterial ClpP1P2 and proteasome makes this new scaffold an attractive starting point for optimization.

Amphiphilic Indole Derivatives as Antimycobacterial Agents: Structure-Activity Relationships and Membrane Targeting Properties.

Antibacterials that disrupt cell membrane function have the potential to eradicate "persister" organisms and delay the emergence of resistance. Here we report the antimycobacterial activities of 4-fluoro and 6-methoxyindoles bearing a cationic amphiphilic motif represented by a lipophilic n-octyl side chain at position 1 and a positively charged azepanyl or 1,4-dioxa-8-azaspiro[4.5]decane moiety at position 3. These analogues exhibited balanced profiles of potency (Mycobacterium bovis BCG, M tuberculosis H37Rv), selective activity, solubility, and metabolic stability. Bacteriological mechanism of action investigations on a representative analogue revealed cell membrane permeabilization and depolarization in M bovis BCG. These membrane-related changes preceded cell death indicating that the loss in membrane integrity was not an epiphenomenon. Bactericidal activity was observed against both growing and nongrowing mycobacterial cultures. The analogue also upregulated cell envelope stress-inducible promoters piniBAC and pclgR, implicating the involvement of envelope-related targets in its mode of action.

Boromycin Kills Mycobacterial Persisters without Detectable Resistance.

Boromycin is a boron-containing polyether macrolide antibiotic isolated from Streptomyces antibioticus. It was shown to be active against Gram positive bacteria and to act as an ionophore for potassium ions. The antibiotic is ineffective against Gram negative bacteria where the outer membrane appears to block access of the molecule to the cytoplasmic membrane. Here we asked whether boromycin is active against Mycobacterium tuberculosis which, similar to Gram negative bacteria, possesses an outer membrane. The results show that boromycin is a potent inhibitor of mycobacterial growth (MIC50 = 80 nM) with strong bactericidal activity against growing and non-growing drug tolerant persister bacilli. Exposure to boromycin resulted in a rapid loss of membrane potential, reduction of the intracellular ATP level and leakage of cytoplasmic protein. Consistent with boromycin acting as a potassium ionophore, addition of KCl to the medium blocked its antimycobacterial activity. In contrast to the potent antimycobacterial activities of the polyether macrolide, its cytotoxicity and haemolytic activity were low (CC50 = 30 µM, HC50 = 40 µM) with a selectivity index of more than 300. Spontaneous resistant mutants could not be isolated suggesting a mutation frequency of less than 10(-9)/CFU. Taken together, the results suggests that targeting mycobacterial transmembrane ion gradients may be an attractive chemotherapeutic intervention level to kill otherwise drug tolerant persister bacilli, and to slow down the development of genetic antibiotic resistance.

Fragment-Based Whole Cell Screen Delivers Hits against M. tuberculosis and Non-tuberculous Mycobacteria.

Reactive multi-target 'fragment drugs' represent critical components of current tuberculosis regimens. These compounds, such as pyrazinamide, are old synthetic antimycobacterials that are activated inside Mycobacterium tuberculosis bacilli and are smaller than the usual drug-like, single-target molecules. Based on the success of small 'dirty' drugs in the chemotherapy of tuberculosis, we suggested previously that fragment-based whole cell screens should be introduced in our current antimycobacterial drug discovery efforts. Here, we carried out such a screen and characterized bactericidal activity, selectivity and spectrum of hits we obtained. A library of 1725 fragments was tested at a single concentration for growth inhibitory activity against M. bovis BCG as screening strain and 38 of 116 primary hits were confirmed in dose response analyses to be active against virulent M. tuberculosis. Bacterial kill experiments showed that most hits displayed bactericidal activity at their minimal inhibitory concentration. Cytotoxicity assays established that a large proportion of hits displayed a favorable selectivity index for mammalian cells. Importantly, one third of M. tuberculosis active fragments were also active against M. abscessus and M. avium, two emerging non-tuberculous mycobacterial (NTM) pathogens, opening the opportunity to develop broad spectrum antimycobacterials. Activity determination against Gram positive (Staphylococcus aureus) and Gram negative (Escherichia coli, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa) bacteria, as well as fungi (Candida albicans, Cryptococcus neoformans) showed only a small overlap indicating a generally narrow spectrum of these novel antimicrobial hits for mycobacteria. In conclusion, we carried out the first fragment-based whole cell screen against bacteria and identified a substantial number of hits with excellent physicochemical properties and dual activity against M. tuberculosis and NTM pathogens. These hits will now be evaluated in animal models of mycobacterial infection to determine whether any of them can be moved forward as a new antimycobacterial fragment drug candidate.

Target mechanism-based whole-cell screening identifies bortezomib as an inhibitor of caseinolytic protease in mycobacteria.

UNLABELLED: A novel type of antibacterial screening method, a target mechanism-based whole-cell screening method, was developed to combine the advantages of target mechanism- and whole-cell-based approaches. A mycobacterial reporter strain with a synthetic phenotype for caseinolytic protease (ClpP1P2) activity was engineered, allowing the detection of inhibitors of this enzyme inside intact bacilli. A high-throughput screening method identified bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpP1P2 activity and bacterial growth. A battery of secondary assays was employed to demonstrate that bortezomib indeed exerts its antimicrobial activity via inhibition of ClpP1P2: Down- or upmodulation of the intracellular protease level resulted in hyper- or hyposensitivity of the bacteria, the drug showed specific potentiation of translation error-inducing aminoglycosides, ClpP1P2-specific substrate WhiB1 accumulated upon exposure, and growth inhibition potencies of bortezomib derivatives correlated with ClpP1P2 inhibition potencies. Furthermore, molecular modeling showed that the drug can bind to the catalytic sites of ClpP1P2. This work demonstrates the feasibility of target mechanism-based whole-cell screening, provides chemical validation of ClpP1P2 as a target, and identifies a drug in clinical use as a new lead compound for tuberculosis therapy. IMPORTANCE: During the last decade, antibacterial drug discovery relied on biochemical assays, rather than whole-cell approaches, to identify molecules that interact with purified target proteins derived by genomics. This approach failed to deliver antibacterial compounds with whole-cell activity, either because of cell permeability issues that medicinal chemistry cannot easily fix or because genomic data of essentiality insufficiently predicted the vulnerability of the target identified. As a consequence, the field largely moved back to a whole-cell approach whose main limitation is its black-box nature, i.e., that it requires trial-and-error chemistry because the cellular target is unknown. We developed a novel type of antibacterial screening method, target mechanism-based whole-cell screening, to combine the advantages of both approaches. We engineered a mycobacterial reporter strain with a synthetic phenotype allowing us to identify inhibitors of the caseinolytic protease (ClpP1P2) inside the cell. This approach identified bortezomib, an anticancer drug, as a specific inhibitor of ClpP1P2. We further confirmed the specific "on-target" activity of bortezomib by independent approaches including, but not limited to, genetic manipulation of the target level (over- and underexpressing strains) and by establishing a dynamic structure-activity relationship between ClpP1P2 and growth inhibition. Identifying an "on-target" compound is critical to optimize the efficacy of the compound without compromising its specificity. This work demonstrates the feasibility of target mechanism-based whole-cell screening methods, validates ClpP1P2 as a druggable target, and delivers a lead compound for tuberculosis therapy.

The role of reduced pterins in resistance to reactive oxygen and nitrogen intermediates in the protozoan parasite Leishmania.

During its life cycle, the protozoan parasite Leishmania experiences oxidative stress when interacting with macrophages. Reduced pterins are known scavengers of reactive oxygen and nitrogen intermediates. Leishmania has a pteridine reductase, PTR1, whose main function is to provide reduced pterins. We investigated the role of PTR1 in resistance to oxidative and nitrosative stress in Leishmania tarentolae, Leishmania infantum, and Leishmania major PTR1(-/-) mutants. The PTR1(-/-) cells of the three species were more sensitive to H2O2- and NO-induced stress. Using a fluorescent probe allowing ROI quantification, we demonstrated an increase in intracellular oxidant molecules in the PTR1(-/-) mutants. The disruption of PTR1 increased metacyclogenesis in L. infantum and L. major. We purified metacyclic parasites from PTR1(-/-) mutants and control cells and tested their intracellular survival in the J774 mouse cell line and in human monocyte-derived macrophages. Our results showed that PTR1(-/-) null mutants survived less in both macrophage models compared to control cells and this decrease was more pronounced in macrophages activated for oxidant production. This study demonstrates that one physiological role of reduced pterins in Leishmania is to deal with oxidative and nitrosative species, and a decreased ability to provide reduced pterins leads to decreased intracellular survival.