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Histone deacetylase (HDAC) inhibitors have been shown to have antitumor activity in vitro and in vivo. Various studies related to their antitumor activity and mechanism of action have been reported for HDAC inhibitors, but the relationship of their antitumor effects to their pharmacodynamic and pharmacokinetic properties in vivo has not ever fully characterized. We report here the discovery of a novel cyclic-peptide-based HDAC inhibitor, YM753. YM753 is a bacteria-derived natural product containing a disulfide bond. It potently inhibited HDAC enzyme with an IC50 of 2.0 nM in the presence of dithiothreitol. YM753 was rapidly converted to a reduced form in tumor cells, and then induced accumulation of acetylated histones, followed by p21WAF1/Cip1 expression, tumor cell growth inhibition and tumor-selective cell death. In an in vitro washout study, YM753 showed prolonged accumulation of acetylated histones in WiDr human colon carcinoma cells. In vivo YM753 dosing of mice harboring WiDr colon tumor xenografts significantly inhibited the tumor growth via sustained accumulation of acetylated histones in the tumor tissue. In a pharmacokinetic study, YM753 rapidly disappeared from the plasma, but its reduced form remained in the tumor tissue. Moreover, the accumulation of acetylated histones induced by YM753 was tumor tissue selective compared to several normal tissues. This study provides evidence that YM753 has antitumor activity that is the result of selective, sustained accumulation of acetylated histones in tumor tissues despite rapid disappearance of the drug from the plasma. These results suggest that the novel HDAC inhibitor, YM753 has attractive pharmacodynamic and pharmacokinetic properties giving it potential as an antitumor agent.

In the course of screening for antitumor antibiotics using 3Y1 rat fibroblasts transformed with adenovirus oncogenes, a new active substance designated thioviridamide was isolated from the culture broth of an actinomycete. The producing organism was identified as Streptomyces olivoviridis on the basis of its culture characteristics and physiological properties. Thioviridamide showed cytotoxicity selectively against Ad12-3Y1 cells (IC50 = 3.9 ng/ml) and E1A-3Y1 cells (IC50 = 32 ng/ml), both of which contain the adenovirus E1A oncogene. Significant numbers of Ad12-3Y1 cells treated with thioviridamide contained condensed chromatin and fragmented nuclei, indicating that thioviridamide induced apoptosis.

A novel apoptosis inducer, thioviridamide, was isolated from an actinomycete identified as Streptomyces olivoviridis. The molecular formula of thioviridamide was determined to be C56H93N14O10S7+ from high-resolution FAB-MS. The structure of thioviridamide was analyzed by NMR spectral analysis including a variety of two-dimensional techniques. COSY and HMBC experiments revealed the presence of a 2-hydroxy-2-methyl-4-oxopentanoyl group and eleven amino acid residues including two novel amino acids, beta-hydroxy-N1,N3-dimethylhistidinium and S-(2-aminovinyl)cysteine. 1H-13C long-range correlations observed in the HMBC, CT-HMBC and HMBC-HOHAHA spectra connected the partial structures with seven amide linkages and five thioamide linkages to establish the planar structure of thioviridamide as a unique cyclic peptide.

The pharmacological properties of YM-254890, a specific G(alpha)q/11 inhibitor, on acute thrombosis and chronic neointima formation after vascular injury have been investigated. FeCl3 was used to induce vascular injury in the carotid artery of mice. For the thrombosis studies, the test drug was either intravenously or orally administered before vascular injury. For the neointima studies, the test drug was orally administered 1 h before and twice daily for 1 week after vascular injury. Histological analysis was then performed 3 weeks later. YM-254890 significantly inhibited ex vivo platelet aggregation 5 min after intravenous bolus injection at 0.03 mg/kg or more, and 1 h after oral administration at 1 mg/kg. YM-254890 significantly inhibited thrombus formation after intravenous bolus injection at 0.03 mg/kg as well as after oral administration at 1 mg/kg, but tail transection bleeding time was significantly prolonged at 0.1 mg/kg for intravenous injection and 3 mg/kg for oral administration. Furthermore, oral administration of YM-254890 dose-dependently inhibited neointima formation 3 weeks after vascular injury with significant effects at 1 mg/kg twice daily for 1 week. Clopidogrel also significantly inhibited neointima formation at its antithrombotic dose, but its inhibitory potency was less than that of YM-254890. However, YM-254890 significantly reduced systemic blood pressure at doses 3 times higher than those that produced significant inhibitory effects on thrombosis and neointima formation. Though the systemic use of YM-254890 may be limited, owing to its narrow therapeutic window, this unique compound is a useful research tool for investigating the physiological roles of G(alpha)q/11 .

YM-216391, a novel cyclic peptide, was isolated from the cultured mycelium of Streptomyces nobilis JCM 4274. It was purified by solvent extraction, silica gel and ODS flash column chromatographies, followed by preparative HPLC. YM-216391 dose-dependently inhibited the growth of human cervical cancer HeLa S3 cells with an IC50 value of 14nM. YM-216391 also showed potent cytotoxic activity against a human cancer cell line panel.

The structure elucidation and biological activity of novel YM-254890 (1) analogues and semi-synthetic derivatives are described. Three natural analogues, YM-254891 (2), YM-254892 (3), and YM-280193 (4), were isolated from the fermentation broth of Chromobacterium sp. QS3666, and two hydrogenated derivatives, YM-385780 (5) and YM-385781 (6), were synthesized from YM-254890. Their structures were determined by one- and two-dimensional NMR studies and mass spectrometry. Among these compounds, two natural analogues 2-3 which possessed acyl groups at beta-HyLeu-1 and one derivative 6 whose conformation was similar to that of 1 showed comparable Galpha(q/11) inhibitory activity to that of 1. This indicates that the acyl beta-HyLeu residue plays an important role in activity and also that the alpha,beta-unsaturated carbonyl group of the N-MeDha residue is not critical to activity. The other hydrogenated derivative 5 had significantly less activity, which could be attributed to conformational differences.

We examined the antithrombotic and thrombolytic effects of the G(q/11) inhibitor YM-254890 in an electrically-induced carotid artery thrombosis model in rats. YM-254890 dose-dependently inhibited ex vivo ADP-induced platelet aggregation after i.v. bolus injection. In the thrombosis study, YM-254890 dosedependently prolonged time to occlusion at doses of 3 and 10 g/kg i.v. and decreased occlusion rate at 10 g/kg i.v. In the thrombolysis study, YM-254890 at 30 micro g/kg i.v. shortened the time to reperfusion and prevented reocclusion after thrombolysis with a modified tissue-type plasminogen activator. YM-254890, at 10 micro g/kg and more, significantly improved carotid patency status after thrombolysis. However, at 30 micro g/kg and more, YM-254890 decreased systemic blood pressure. These results suggest that YM-254890 may be effective for treating G(q)-mediated diseases, and that YM-254890 is a useful tool for investigating the biological roles of G(q/11).

YM-266183 and YM-266184 are new antibacterial substances that have activity against drug-resistant bacteria produced by Bacillus cereus QN03323. These structures were elucidated by MS and NMR spectral analysis. YM-266183 and YM-266184 are the cyclic thiopeptides containing thiazole and pyridine moieties, and several unusual amino acids.

Novel antibiotics, YM-266183 (1) and YM-266184 (2), were found in the culture broth of Bacillus cereus QN03323 which was isolated from the marine sponge Halichondria japonica. The structures of both antibiotics were determined by several spectroscopic experiments as new thiopeptide compounds. They exhibited potent antibacterial activities against staphylococci and enterococci including multiple drug resistant strains, whereas they were inactive against Gram-negative bacteria.