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.

Characterization of the histone methyltransferase PRDM9 using biochemical, biophysical and chemical biology techniques.

PRDM proteins have emerged as important regulators of disease and developmental processes. To gain insight into the mechanistic actions of the PRDM family, we have performed comprehensive characterization of a prototype member protein, the histone methyltransferase PRDM9, using biochemical, biophysical and chemical biology techniques. In the present paper we report the first known molecular characterization of a PRDM9-methylated recombinant histone octamer and the identification of new histone substrates for the enzyme. A single C321P mutant of the PR/SET domain was demonstrated to significantly weaken PRDM9 activity. Additionally, we have optimized a robust biochemical assay amenable to high-throughput screening to facilitate the generation of small-molecule chemical probes for this protein family. The present study has provided valuable insight into the enzymology of an intrinsically active PRDM protein.

Novel Acetamide Indirectly Targets Mycobacterial Transporter MmpL3 by Proton Motive Force Disruption.

To identify novel inhibitors of Mycobacterium tuberculosis cell envelope biosynthesis, we employed a two-step approach. First, we screened the diverse synthetic small molecule 71,544-compound Enamine library for growth inhibitors using the non-pathogenic surrogate Mycobacterium bovis BCG as screening strain and turbidity as readout. Second, 16 confirmed hits were tested for their ability to induce the cell envelope stress responsive promoter piniBAC controlling expression of red fluorescent protein in an M. bovis BCG reporter strain. Using a fluorescence readout, the acetamide E11 was identified. Resistant mutant selection and whole genome sequencing revealed the mycolic acid transporter Mmpl3 as a candidate target of E11. Biochemical analysis using mycobacterial spheroplasts and various membrane assays suggest that E11 indirectly inhibits MmpL3-facilitated translocation of trehalose monomycolates by proton motive force disruption. E11 showed potent bactericidal activity against growing and non-growing M. tuberculosis, low cytotoxic, and hemolytic activity and a dynamic structure activity relationship. In addition to activity against M. tuberculosis, E11 was active against the non-tuberculous mycobacterium M. abscessus, an emerging opportunistic pathogen. In conclusion, we identified a novel bactericidal anti-mycobacterial lead compound targeting MmpL3 providing an attractive starting point for optimization.

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.

Establishment of a novel whole animal HTS technology platform for melioidosis drug discovery.

Melioidosis is a serious emerging endemic infectious disease caused by Burkholderia pseudomallei, a gram-negative pathogen. Septicemic melioidosis has a mortality rate of 50% even with treatment. Like other gram-negative bacteria, B. pseudomallei is resistant to a number of antibiotics and multi-drug resistant B. pseudomallei is beginning to be encountered in hospitals. There is a clear medical need to develop new treatment options to manage this disease. We used Burkholderia thailandensis (a BSL-2 class organism) to infect Caenorhabditis elegans and set up a surrogate whole animal infection model of melioidosis that we could run in a 384 microtitre plate and establish a whole animal HTS assay. We have optimized and validated this assay in a fluorescence-based format that can be run on our automated screening platforms. This assay has now been used to screen over 300,000 compounds from our small molecule library and we are in the process of characterizing the hits obtained and select compounds for further studies. We have thus established a biologically relevant assay technology platform to screen for antibacterial compounds and used this platform to identify new compounds that may find application in treating melioidosis infections.

Caspase-4 interacts with TNF receptor-associated factor 6 and mediates lipopolysaccharide-induced NF-kappaB-dependent production of IL-8 and CC chemokine ligand 4 (macrophage-inflammatory protein-1 ).

Human caspase-4 does not have a corresponding mouse ortholog. Caspase-4 falls within the class of "inflammatory caspases," being homologous with human caspases 1 and 5 and mouse caspases 1, 11, and 12. To address the function of caspase-4, we generated caspase-4-deficient human THP1 monocytic cell lines which exhibited substantially reduced LPS-induced secretion of several chemokines and cytokines, including IL-8 (CXCL8), CCL4 (macrophage-inflammatory protein-1beta), CCL20 (macrophage-inflammatory protein-3alpha), and IL-1beta. The LPS-induced expression of the mRNAs encoding these cytokines was correspondingly reduced in the caspase-4-deficient clones. Because a specific NF-kappaB inhibitor blocked LPS-induced IL-8 and CCL4 mRNA expression as well as IL-8 and CCL4 secretion in THP1 cells, we investigated the role of caspase-4 in NF-kappaB signaling. LPS-induced NF-kappaB nuclear translocation and activation were inhibited in all caspase-4-deficient clones. LPS stimulation led to the interaction of endogenous caspase-4 and TNFR-associated factor 6 (TRAF6) via a TRAF6-binding motif (PPESGE), which we identified in caspase-4. Mutation of this site in caspase-4 resulted in the loss of the TRAF6-caspase-4 interaction. Similar TRAF6-binding motifs are known to be functionally important for TRAF6 interactions with other molecules including caspase-8, and for mediating NF-kappaB activation in various immune and nonimmune cell types. Our data suggest that the TRAF6-caspase-4 interaction, triggered by LPS, leads to NF-kappaB-dependent transcriptional up-regulation and secretion of important cytokines and chemokines in innate immune signaling in human monocytic cells.

AIF suppresses chemical stress-induced apoptosis and maintains the transformed state of tumor cells.

Apoptosis-inducing factor (AIF) exhibits reactive oxygen species (ROS)-generating NADH oxidase activity of unknown significance, which is dispensable for apoptosis. We knocked out the aif gene in two human colon carcinoma cell lines that displayed lower mitochondrial complex I oxidoreductase activity and produced less ROS, but showed increased sensitivity to peroxide- or drug-induced apoptosis. AIF knockout cells failed to form tumors in athymic mice or grow in soft agar. Only AIF with intact NADH oxidase activity restored complex I activity and anchorage-independent growth of aif knockout cells, and induced aif-transfected mouse NIH3T3 cells to form foci. AIF knockdown in different carcinoma cell types resulted in lower superoxide levels, enhanced apoptosis sensitivity and loss of tumorigenicity. Antioxidants sensitized AIF-expressing cells to apoptosis, but had no effect on tumorigenicity. In summary, AIF-mediated resistance to chemical stress involves ROS and probably also mitochondrial complex I. AIF maintains the transformed state of colon cancer cells through its NADH oxidase activity, by mechanisms that involve complex I function. On both counts, AIF represents a novel type of cancer drug target.