Carolyn Shoen

A chemical genetic screen in Mycobacterium tuberculosis identifies carbon-source-dependent growth inhibitors devoid of in vivo efficacy

Candidate antibacterials are usually identified on the basis of their in vitro activity. However, the apparent inhibitory activity of new leads can be misleading because most culture media do not reproduce an environment relevant to infection in vivo. In this study, while screening for novel anti-tuberculars, we uncovered how carbon metabolism can affect antimicrobial activity. Novel pyrimidine–imidazoles (PIs) were identified in a whole-cell screen against Mycobacterium tuberculosis. Lead optimization generated in vitro potent derivatives with desirable pharmacokinetic properties, yet without in vivo efficacy. Mechanism of action studies linked the PI activity to glycerol metabolism, which is not relevant for M. tuberculosis during infection. PIs induced self-poisoning of M. tuberculosis by promoting the accumulation of glycerol phosphate and rapid ATP depletion. This study underlines the importance of understanding central bacterial metabolism in vivo and of developing predictive in vitro culture conditions as a prerequisite for the rational discovery of new antibiotics. Candidate anti-tuberculosis drugs are often identified in whole-cell screens. Here, Petheet al. show that inappropriate carbon-source selection can lead to the identification of compounds devoid of efficacy in vivo, underlining the importance of developing predictive in vitroscreens.

Antiinfectives targeting enzymes and the proton motive force

Significance Uncoupling agents might be expected to be generally cytotoxic, but many US Food and Drug Administration (FDA)-approved drugs do have activity as uncouplers, in addition to targeting enzymes. There is therefore interest in the discovery of antibiotics that have such multitarget activity. Here, we show that some FDA-approved drugs, such as clofazimine, clomiphene, and bedaquiline, with antiinfective activity act as uncouplers. Using molecular dynamics-based in silico screening, we also discovered that the brain cancer drug lead vacquinol is an uncoupler that inhibits an enzyme involved in the formation of tuberculosis (TB) virulence factors, in addition to killing TB bacteria. Our results indicate strong drug–membrane interactions, and that screening for combined enzyme inhibition plus uncoupler activity will lead to new antibiotic leads. There is a growing need for new antibiotics. Compounds that target the proton motive force (PMF), uncouplers, represent one possible class of compounds that might be developed because they are already used to treat parasitic infections, and there is interest in their use for the treatment of other diseases, such as diabetes. Here, we tested a series of compounds, most with known antiinfective activity, for uncoupler activity. Many cationic amphiphiles tested positive, and some targeted isoprenoid biosynthesis or affected lipid bilayer structure. As an example, we found that clomiphene, a recently discovered undecaprenyl diphosphate synthase inhibitor active against Staphylococcus aureus, is an uncoupler. Using in silico screening, we then found that the anti-glioblastoma multiforme drug lead vacquinol is an inhibitor of Mycobacterium tuberculosis tuberculosinyl adenosine synthase, as well as being an uncoupler. Because vacquinol is also an inhibitor of M. tuberculosis cell growth, we used similarity searches based on the vacquinol structure, finding analogs with potent (∼0.5–2 μg/mL) activity against M. tuberculosis and S. aureus. Our results give a logical explanation of the observation that most new tuberculosis drug leads discovered by phenotypic screens and genome sequencing are highly lipophilic (logP ∼5.7) bases with membrane targets because such species are expected to partition into hydrophobic membranes, inhibiting membrane proteins, in addition to collapsing the PMF. This multiple targeting is expected to be of importance in overcoming the development of drug resistance because targeting membrane physical properties is expected to be less susceptible to the development of resistance.

New Classes of Alanine Racemase Inhibitors Identified by High-Throughput Screening Show Antimicrobial Activity against Mycobacterium tuberculosis

Background In an effort to discover new drugs to treat tuberculosis (TB) we chose alanine racemase as the target of our drug discovery efforts. In Mycobacterium tuberculosis, the causative agent of TB, alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan. Good antimicrobial effects have been achieved by inhibition of this enzyme with suicide substrates, but the clinical utility of this class of inhibitors is limited due to their lack of target specificity and toxicity. Therefore, inhibitors that are not substrate analogs and that act through different mechanisms of enzyme inhibition are necessary for therapeutic development for this drug target. Methodology/Principal Findings To obtain non-substrate alanine racemase inhibitors, we developed a high-throughput screening platform and screened 53,000 small molecule compounds for enzyme-specific inhibitors. We examined the ‘hits’ for structural novelty, antimicrobial activity against M. tuberculosis, general cellular cytotoxicity, and mechanism of enzyme inhibition. We identified seventeen novel non-substrate alanine racemase inhibitors that are structurally different than any currently known enzyme inhibitors. Seven of these are active against M. tuberculosis and minimally cytotoxic against mammalian cells. Conclusions/Significance This study highlights the feasibility of obtaining novel alanine racemase inhibitor lead compounds by high-throughput screening for development of new anti-TB agents.

Peptide deformylase inhibitors of Mycobacterium tuberculosis: synthesis, structural investigations, and biological results.

Bacterial peptide deformylase (PDF) belongs to a subfamily of metalloproteases catalyzing the removal of the N-terminal formyl group from newly synthesized proteins. We report the synthesis and biological activity of highly potent inhibitors of Mycobacterium tuberculosis (Mtb) PDF enzyme as well as the first X-ray crystal structure of Mtb PDF. Structure-activity relationship and crystallographic data clarified the structural requirements for high enzyme potency and cell based potency. Activities against single and multi-drug-resistant Mtb strains are also reported.

Metabolic and Bactericidal Effects of Targeted Suppression of NadD and NadE Enzymes in Mycobacteria

ABSTRACT Mycobacterium tuberculosis remains a major cause of death due to the lack of treatment accessibility, HIV coinfection, and drug resistance. Development of new drugs targeting previously unexplored pathways is essential to shorten treatment time and eliminate persistent M. tuberculosis. A promising biochemical pathway which may be targeted to kill both replicating and nonreplicating M. tuberculosis is the biosynthesis of NAD(H), an essential cofactor in multiple reactions crucial for respiration, redox balance, and biosynthesis of major building blocks. NaMN adenylyltransferase (NadD) and NAD synthetase (NadE), the key enzymes of NAD biosynthesis, were selected as promising candidate drug targets for M. tuberculosis. Here we report for the first time kinetic characterization of the recombinant purified NadD enzyme, setting the stage for its structural analysis and inhibitor development. A protein knockdown approach was applied to validate bothNadD and NadE as target enzymes. Induced degradation of either target enzyme showed a strong bactericidal effect which coincided with anticipated changes in relative levels of NaMN and NaAD intermediates (substrates of NadD and NadE, respectively) and ultimate depletion of the NAD(H) pool. A metabolic catastrophe predicted as a likely result of NAD(H) deprivation of cellular metabolism was confirmed by 13C biosynthetic labeling followed by gas chromatography-mass spectrometry (GC-MS) analysis. A sharp suppression of metabolic flux was observed in multiple NAD(P)(H)-dependent pathways, including synthesis of many amino acids (serine, proline, aromatic amino acids) and fatty acids. Overall, these results provide strong validation of the essential NAD biosynthetic enzymes, NadD and NadE, as antimycobacterial drug targets. IMPORTANCE To address the problems of M. tuberculosis drug resistance and persistence of tuberculosis, new classes of drug targets need to be explored. The biogenesis of NAD cofactors was selected for target validation because of their indispensable role in driving hundreds of biochemical transformations. We hypothesized that the disruption of NAD production in the cell via genetic suppression of the essential enzymes (NadD and NadE) involved in the last two steps of NAD biogenesis would lead to cell death, even under dormancy conditions. In this study, we confirmed the hypothesis using a protein knockdown approach in the model system of Mycobacterium smegmatis. We showed that induced proteolytic degradation of either target enzyme leads to depletion of the NAD cofactor pool, which suppresses metabolic flux through numerous NAD(P)-dependent pathways of central metabolism of carbon and energy production. Remarkably, bactericidal effect was observed even for nondividing bacteria cultivated under carbon starvation conditions. To address the problems of M. tuberculosis drug resistance and persistence of tuberculosis, new classes of drug targets need to be explored. The biogenesis of NAD cofactors was selected for target validation because of their indispensable role in driving hundreds of biochemical transformations. We hypothesized that the disruption of NAD production in the cell via genetic suppression of the essential enzymes (NadD and NadE) involved in the last two steps of NAD biogenesis would lead to cell death, even under dormancy conditions. In this study, we confirmed the hypothesis using a protein knockdown approach in the model system of Mycobacterium smegmatis. We showed that induced proteolytic degradation of either target enzyme leads to depletion of the NAD cofactor pool, which suppresses metabolic flux through numerous NAD(P)-dependent pathways of central metabolism of carbon and energy production. Remarkably, bactericidal effect was observed even for nondividing bacteria cultivated under carbon starvation conditions.

Inhibition of mycobacterial alanine racemase activity and growth by thiadiazolidinones

The genus Mycobacterium includes non-pathogenic species such as M. smegmatis, and pathogenic species such as M. tuberculosis, the causative agent of tuberculosis (TB). Treatment of TB requires a lengthy regimen of several antibiotics, whose effectiveness has been compromised by the emergence of resistant strains. New antibiotics that can shorten the treatment course and those that have not been compromised by bacterial resistance are needed. In this study, we report that thiadiazolidinones, a relatively little-studied heterocyclic class, inhibit the activity of mycobacterial alanine racemase, an essential enzyme that converts l-alanine to d-alanine for peptidoglycan synthesis. Twelve members of the thiadiazolidinone family were evaluated for inhibition of M. tuberculosis and M. smegmatis alanine racemase activity and bacterial growth. Thiadiazolidinones inhibited M. tuberculosis and M. smegmatis alanine racemases to different extents with 50% inhibitory concentrations (IC50) ranging from <0.03 to 28μM and 23 to >150μM, respectively. The compounds also inhibited the growth of these bacteria, including multidrug resistant strains of M. tuberculosis. The minimal inhibitory concentrations (MIC) for drug-susceptible M. tuberculosis and M. smegmatis ranged from 6.25μg/ml to 100μg/ml, and from 1.56 to 6.25μg/ml for drug-resistant M. tuberculosis. The in vitro activities of thiadiazolidinones suggest that this family of compounds might represent starting points for medicinal chemistry efforts aimed at developing novel antimycobacterial agents.

Clinical isolates of Candida albicans, Candida tropicalis, and Candida krusei have different susceptibilities to Co(II) and Cu(II) complexes of 1,10-phenanthroline

The minimal inhibitory concentrations (MICs) of copper and cobalt based dimeric pyrophosphate complexes with capping 1,10-phenanthroline groups on clinical isolates of C. albicans (28 isolates), C. krusei (20 isolates) and C. tropicalis (20 isolates) are reported. C. albicans was inhibited by the cobalt complex better than by the copper complex, while C. krusei demonstrated the opposite results. C. tropicalis showed similar sensitivities to both metals in terms of calculated MIC50 values but was more sensitive to cobalt when MIC90 values were noted. Knockout strains of C. albicans that had the copper efflux protein P-type ATPase (CRP1), the copper binding metallothionein CUP1 or both CRP1/CUP1 removed clearly demonstrate that the origins of copper resistant in C. albicans lies primarily in the P-type ATPase, with the MT playing an important secondary role in the absence of the efflux protein. This study suggests that certain strains of Candida have evolved to protect against particular metal ions and that in the case of C. albicans, a primary invasive fungal species, cobalt may be a good starting-point for new therapeutic development.

In Vitro Activity of JPC 2067 Alone and in Combination with Sulfamethoxazole against Nocardia Species

ABSTRACT JPC 2067 is a novel dihydrotriazine dihydrofolate reductase inhibitor that is being developed as an antimalarial therapeutic. We evaluated the in vitro activity of JPC 2067 alone and in combination with sulfamethoxazole (SMX) against a panel of nocardia isolates. The MIC50s and MIC90s for JPC 2067, SMX, and the combination were 0.125 μg/ml and 4 μg/ml, 16 μg/ml and 32 μg/ml, and 0.03 μg/ml and 2 μg/ml, respectively. JPC 2067 alone and in combination with SMX should be evaluated further to understand its clinical potential.

Propargyl-Linked Antifolates Are Potent Inhibitors of Drug-Sensitive and Drug-Resistant Mycobacterium tuberculosis.

Mycobacterium tuberculosis continues to cause widespread, life-threatening disease. In the last decade, this threat has grown dramatically as multi- and extensively-drug resistant (MDR and XDR) bacteria have spread globally and the number of agents that effectively treat these infections is significantly reduced. We have been developing the propargyl-linked antifolates (PLAs) as potent inhibitors of the essential enzyme dihydrofolate reductase (DHFR) from bacteria and recently found that charged PLAs with partial zwitterionic character showed improved mycobacterial cell permeability. Building on a hypothesis that these PLAs may penetrate the outer membrane of M. tuberculosis and inhibit the essential cytoplasmic DHFR, we screened a group of PLAs for antitubercular activity. In this work, we identified several PLAs as potent inhibitors of the growth of M. tuberculosis with several of the compounds exhibiting minimum inhibition concentrations equal to or less than 1 μg/mL. Furthermore, two of the compounds were very potent inhibitors of MDR and XDR strains. A high resolution crystal structure of one PLA bound to DHFR from M. tuberculosis reveals the interactions of the ligands with the target enzyme.