Naoko Kinoshita

Walkmycin B targets WalK (YycG), a histidine kinase essential for bacterial cell growth

The WalK (a histidine kinase)/WalR (a response regulator, aka YycG/YycF) two-component system is indispensable in the signal transduction pathway for the cell-wall metabolism of Bacillus subtilis and Staphylococcus aureus. The inhibitors directed against WalK would be expected to have a bactericidal effect. After we screened 1368 culture broths of Streptomyces sp. by a differential growth assay, walkmycin A, B and C, which were produced by strain MK632-100F11, were purified using silica-gel column chromatography and HPLC. In this paper, the chemical structure of the major product (walkmycin B) was determined to be di-anthracenone (C44H44Cl2O14), which was very similar to BE40665A. MICs of walkmycin B against B. subtilis and S. aureus were 0.39 and 0.20 μg ml−1, and IC50 measurements against WalK were 1.6 and 5.7 μM, respectively. To clarify the affinity between WalK and walkmycin B, surface plasmon resonance was measured to obtain the equilibrium dissociation constant, KD1, of 7.63 μM at the higher affinity site of B. subtilis WalK. These results suggest that walkmycin B inhibits WalK autophosphorylation by binding to the WalK cytoplasmic domain.

Polyoxypeptin isolated from Streptomyces: A bioactive cyclic depsipeptide containing the novel amino acid 3-hydroxy-3-methylproline

Abstract Polyoxypeptin, a potent inducer of apoptosis in human pancreatic carcinoma cells, was isolated from an ethyl acetate extract of a Streptomyces culture broth. Structural determination by 2D-NMR and X-ray crystallographic analysis revealed that it is a novel cyclic hexadepsipeptide containing five hydroxylated amino acids. The unusual and hitherto unreported amino acid 3-hydroxy-3-methylproline was one of them.

Waldiomycin, a novel WalK-histidine kinase inhibitor from Streptomyces sp. MK844-mF10.

WalK, a histidine kinase, and WalR, a response regulator, make up a two-component signal transduction system that is indispensable for the cell-wall metabolism of low GC Gram-positive bacteria. WalK inhibitors are likely to show bactericidal effects against methicillin-resistant Staphylococcus aureus . We discovered a new WalK inhibitor, designated waldiomycin, by screening metabolites from actinomycetes. Waldiomycin belongs to the family of angucycline antibiotics and is structurally related to dioxamycin. Waldiomycin inhibits WalK from S. aureus and Bacillus subtilis at IC50s 8.8 and 10.2 μM, respectively, and shows antibacterial activity with MICs ranging from 4 to 8 μg ml−1 against methicillin-resistant S. aureus and B. subtilis.

Pargamicin A, a Novel Cyclic Peptide Antibiotic from Amycolatopsis sp.

A novel cyclic peptide antibiotic, pargamicin A was isolated from the culture broth of an actinomycete strain. The producing organism, designated ML1-hF4, was identified as a member of the genus Amycolatopsis. Pargamicin A was identified as a novel cyclic hexapeptide antibiotic containing piperazic acid by various spectroscopic analyses. Pargamicin A showed potent antibacterial activity against Staphylococcus aureus strains including MRSA and Enterococcus faecalis/faecium strains including VRE.

Amycolamicin: A Novel Broad‐Spectrum Antibiotic Inhibiting Bacterial Topoisomerase

The abuse of antibacterial drugs imposes a selection pressure on bacteria that has driven the evolution of multidrug resistance in many pathogens. Our efforts to discover novel classes of antibiotics to combat these pathogens resulted in the discovery of amycolamicin (AMM). The absolute structure of AMM was determined by NMR spectroscopy, X-ray analysis, chemical degradation, and modification of its functional groups. AMM consists of trans-decalin, tetramic acid, two unusual sugars (amycolose and amykitanose), and dichloropyrrole carboxylic acid. The pyranose ring named as amykitanose undergoes anomerization in methanol. AMM is a potent and broad-spectrum antibiotic against Gram-positive pathogenic bacteria by inhibiting DNA gyrase and bacterial topoisomerase IV. The target of AMM has been proved to be the DNA gyrase B subunit and its binding mode to DNA gyrase is different from those of novobiocin and coumermycin, the known DNA gyrase inhibitors.

Isolation and structure elucidation of a novel androgen antagonist, arabilin, produced by Streptomyces sp. MK756-CF1

In the course of screening for a new type of androgen receptor (AR) antagonist, we isolated a novel compound, arabilin, with two structural isomers, spectinabilin and SNF4435C, produced by Streptomyces sp. MK756-CF1. Structure elucidation on the basis of the spectroscopic properties showed that arabilin is a novel polypropionate-derived metabolite with a p-nitrophenyl group and a substituted γ-pyrone ring. Arabilin competitively blocked the binding of androgen to the ligand-binding domain of AR in vitro. In addition, arabilin inhibited androgen-induced prostate-specific antigen mRNA expression in prostate cancer LNCaP cells.

Intervenolin, a new antitumor compound with anti-Helicobacter pylori activity, from Nocardia sp. ML96-86F2.

Because stromal cells can regulate the growth and metastasis of tumor cells, a compound that modulates the interaction between the stromal cells and the tumor cells can control the tumor progression. In the course of our screening for such a compound, we have isolated a new compound, intervenolin, from the culture broth of Nocardia sp. ML96-86F2. Intervenolin inhibits the growth of human gastric and colorectal cancer cell lines in the coculture with the respective organ-derived stromal cells more strongly than that of the cancer cells cultured alone. Intervenolin shows antitumor effect against a xenograft model of human colorectal cancer cells in vivo. Furthermore, intervenolin exerts selective anti-Helicobacter pylori effect.

Migracins A and B, new inhibitors of cancer cell migration, produced by Streptomyces sp.

In the course of screening for breast cancer cell migration inhibitors, we isolated two novel compounds, migracins A and B from the culture broth of Streptomyces sp. MI264-NF2. Their structures are related to those of luminacins previously isolated from Streptomyces. Migracins A and B inhibited breast cancer cell migration, monitored by wound healing assay with IC50 values of 1.31 and 1.99 μg ml−1, respectively, in human breast carcinoma MDA-MB-231 cells without showing any cytotoxicity. Migracins also inhibited the migration of human lung adenocarcinoma A549 cells and human fibrosarcoma HT-1080 cells. Therefore, migracins may become new cancer metastasis inhibitors.

A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren

Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multi-drug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904–1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0–7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904–1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904–1. Our method provides a rapid and convenient screen to identify an anti-mycobacterial drug.

Androprostamines A and B, the new anti-prostate cancer agents produced by Streptomyces sp. MK932-CF8

Androgen receptor (AR) is a validated target in all clinical states of prostate cancer. Androprostamines A and B, the new inhibitors of androgen receptor, were isolated from Streptomyces sp. MK932-CF8. Their structures were determined by the spectroscopic analysis, degradation studies and synthesis. Androprostamines showed potent inhibitory effect against androgen-dependent growth of human prostate cancer cells without cytotoxicity and repressed the androgen-induced expression of AR-regulated genes.