A Suppression Strategy for Antibiotic Discovery

AbstractHigh-throughput phenotype screening and target identification have been combined in an effort to isolate antimicrobial, small-molecule therapeutics [1]. This approach, developed by Brown and colleagues and reported in this issue, is a major technological advance for antimicrobial drug discovery

The Chlamydia Protease CPAF Regulates Host and Bacterial Proteins to Maintain Pathogen Vacuole Integrity and Promote Virulence

The obligate intracellular bacterial pathogen Chlamydia trachomatis injects numerous effector proteins into the epithelial cell cytoplasm to manipulate host functions important for bacterial survival. In addition, the bacterium secretes a serine protease, chlamydial protease-like activity factor (CPAF). Although several CPAF targets are reported, the significance of CPAF-mediated proteolysis is unclear due to the lack of specific CPAF inhibitors and the diversity of host targets. We report that CPAF also targets chlamydial effectors secreted early during the establishment of the pathogen-containing vacuole (“inclusion”). We designed a cell-permeable CPAF-specific inhibitory peptide and used it to determine that CPAF prevents superinfection by degrading early Chlamydia effectors translocated during entry into a pre-infected cell. Prolonged CPAF inhibition leads to loss of inclusion integrity and caspase-1-dependent death of infected epithelial cells. Thus, CPAF functions in niche protection, inclusion integrity and pathogen survival, making the development of CPAF-specific protease inhibitors an attractive anti-chlamydial therapeutic strategy

Discovery of the Class I Antimicrobial Lasso Peptide Arcumycin

Lasso peptides are a structurally diverse superfamily ofconformationally-constrained peptide natural products, of which asubset exhibits broad antimicrobial activity. Although advances inbioinformatics have increased our knowledge of strains harboringthe biosynthetic machinery for lasso peptide production, relatingpeptide sequence to bioactivity remains a continuous challenge.Towards this end, a structure-driven genome mining investigationof Actinobacteria-produced antimicrobial lasso peptides wasperformed to correlate predicted primary structure with antibioticactivity. Bioinformatic evaluation revealed eight putative novelclass I lasso peptide sequences. This subset is predicted topossess antibiotic activity as characterized members of this classhave both broad spectrum and potent activity against Gram positivestrains. Fermentation of one of these hits, StreptomycesNRRL F-5639, resulted in the production of a novel class I lassopeptide, arcumycin, named for the Latin word for bow or arch,arcum. Arcumycin exhibited antibiotic activity against Gram positivebacteria including Bacillus subtilis (4 μg/mL),Staphylococcus aureus (8 μg/mL), and Micrococcus luteus (8μg/mL). Arcumycin treatment of B. subtilis liaI-β-gal promoterfusion reporter strain resulted in upregulation of the system liaRSby the promoter liaI, indicating arcumycin interferes with lipid IIbiosynthesis. Cumulatively, the results illustrate the relationshipbetween phylogenetically related lasso peptides and theirbioactivity as validated through the isolation, structuraldetermination, and evaluation of bioactivity of the novel class Iantimicrobial lasso peptide arcumycin.</p

Histone H3 Lysine 4 Demethylation Is a Target of Nonselective Antidepressive Medications

Demethylation of histone H3 lysine 4 is carried out by BHC110/LSD1, an enzyme with close homology to monoamine oxidases (MAO). Monoamine oxidase A or B are frequent targets of selective and nonselective small molecular inhibitors used for treatment of depression. Here we show that in contrast to selective monoamine oxidase inhibitors such as pargyline, nonselective monoamine oxidase inhibitors potently inhibit nucleosomal demethylation of histone H3 lysine 4. Tranylcypromine (brand name Parnate) displayed the best inhibitory activity with an IC 50 of less than 2 μM. Treatment of P19 embryonal carcinoma cells with tranylcypromine resulted in global increase in H3K4 methylation as well as transcriptional derepression of two BHC110 target genes, Egr1 and the pluripotent stem cell marker Oct4. These results attest to the effectiveness of tranylcypromine as a small molecular inhibitor of histone demethylation

The Chlamydia trachomatis Protease CPAF Contains a Cryptic PDZ-Like Domain with Similarity to Human Cell Polarity and Tight Junction PDZ-Containing Proteins.

The need for more effective anti-chlamydial therapeutics has sparked research efforts geared toward further understanding chlamydial pathogenesis mechanisms. Recent studies have implicated the secreted chlamydial serine protease, chlamydial protease-like activity factor (CPAF) as potentially important for chlamydial pathogenesis. By mechanisms that remain to be elucidated, CPAF is directed to a discrete group of substrates, which are subsequently cleaved or degraded. While inspecting the previously solved CPAF crystal structure, we discovered that CPAF contains a cryptic N-terminal PSD95 Dlg ZO-1 (PDZ) domain spanning residues 106-212 (CPAF106-212). This PDZ domain is unique in that it bears minimal sequence similarity to canonical PDZ-forming sequences and displays little sequence and structural similarity to known chlamydial PDZ domains. We show that the CPAF106-212 sequence is homologous to PDZ domains of human tight junction proteins

Lysine-Specific Histone Demethylase 1 Inhibitors Control Breast Cancer Proliferation in ERα-Dependent and -Independent Manners

Lysine specific demethylase 1 (LSD1, also known as KDM1) is a histone modifying enzyme that regulates the expression of many genes important in cancer progression and proliferation. It is present in various transcriptional complexes including those containing the estrogen receptor (ER). Indeed, inhibition of LSD1 activity and or expression has been shown to attenuate estrogen signaling in breast cancer cells in vitro, implicating this protein in the pathogenesis of cancer. Herein we describe experiments that utilize small molecule inhibitors, phenylcyclopropylamines, along with small interfering RNA to probe the role of LSD1 in breast cancer proliferation and in estrogen-dependent gene transcription. Surprisingly, whereas we have confirmed that inhibition of LSD1 strongly inhibits proliferation of breast cancer cells, we have determined that the cytostatic actions of LSD1 inhibition are not impacted by ER status. These data suggest that LSD1 may be a useful therapeutic target in several types of breast cancer; most notably, inhibitors of LSD1 may have utility in the treatment of ER-negative cancers for which there are minimal therapeutic options