Stereospecific antitumor activity of radicicol oxime derivatives.

PURPOSE: Radicicol is a novel hsp90 antagonist, distinct from the chemically unrelated benzoquinone ansamycin compounds, geldanamycin and herbimycin. Both geldanamycin and radicicol bind in the aminoterminal nucleotide-binding pocket of hsp90, destabilizing the hsp90 client proteins, many of which are essential for tumor cell growth. We describe here antitumor activity of a novel oxime derivative of radicicol, KF58333. We also investigated the mechanism of antitumor activity of KF58333 in comparison with its oxime isomer KF58332. METHODS: Antiproliferative activities were determined in a panel of breast cancer cell lines in vitro. We also examined inhibition of hsp90 function and apoptosis induction in erbB2-overexpressing human breast carcinoma KPL-4 cells in vitro. Direct binding activity to hsp90 was assessed by hsp90-binding assays using geldanamycin or radicicol beads. In animal studies, we investigated plasma concentrations of these compounds after i.v. injection in BALB/c mice and antitumor activity against KPL-4 cells transplanted into nude mice. Inhibition of hsp90 function and induction of apoptosis in vivo were investigated using tumor specimens from drug-treated animals. RESULTS: KF58333 showed potent antiproliferative activity against all breast cancer cell lines tested in vitro, and was more potent than its stereoisomer KF58332. These results are consistent with the ability of KF58333 to deplete hsp90 client proteins and the induction of apoptosis in KPL-4 cells in vitro. Interestingly, KF58333, but not KF58332, showed significant in vivo antitumor activity accompanied by induction of apoptosis in KPL-4 human breast cancer xenografts. Although the plasma concentrations of these compounds were equivalent, KF58333, but not KF58332, depleted hsp90 client proteins such as erbB2, raf-1 and Akt in the tumor specimen recovered from nude mice. CONCLUSIONS: These results suggest that inhibition of hsp90 function, which causes depletion of hsp90 client proteins in tumor, contributes to the antitumor activity of KF58333, and that the stereochemistry of the oxime moiety is important for the biological activity of radicicol oxime derivatives.

Chemoprotective effects of KF41399, a derivative of carbazole compounds, on nimustine-induced thrombocytopenia.

We examined the chemoprotective effects of KF41399, a novel derivative of carbazole compounds, on severe thrombocytopenia induced by nimustine (ACNU, 45 mg/kg administered for 2 consecutive days intravenously) in mice. Administration schedule studies revealed that pretreatment of mice with KF41399 was necessary to improve thrombocytopenia. Oral administration of KF41399 ameliorated thrombocytopenia induced by ACNU and accelerated the rate of platelet recovery in a dose-dependent fashion. In addition, KF41399 pretreatment improved the decrease in body weight and spleen weight and in the colony-forming activity of bone marrow mononuclear cells (MNC). Oral administration of KF41399 to normal mice induced G(0)/G(1)-phase accumulation of MNC as well as hematopoietic progenitor cells (lineage negative cells [Lin(-)]) and reduced the colony-forming activity of MNC. In Lin(-) cells derived from KF41399-treated mice, up-regulation of Bcl-2 and down-regulation of cyclin E and cyclin A proteins were observed. In the same cells, a decrease in the phosphorylated form of Rb protein and an increase in the p130 protein were observed without changes in the protein level of cell cycle-dependent kinase 2 (Cdk2), Cdk4, and Cdk6. More important, KF41399 did not affect the antitumor activity of ACNU against mouse Sarcoma180 and human lung cancer LC-6. However, 25-mg/kg KF41399 treatment reduced the antitumor activity of ACNU against human lung cancer Lu-65, and 5 mg/kg KF41399 caused a slight reduction of the antitumor activity of ACNU without inducing thrombocytopenia. These results suggest that KF41399 might be useful as a chemoprotective agent to improve chemotherapy-induced thrombocytopenia and types of other toxicity. (Blood. 2000;95:3771-3780)

KF25706, a novel oxime derivative of radicicol, exhibits in vivo antitumor activity via selective depletion of Hsp90 binding signaling molecules.

Radicicol, a macrocyclic antifungal antibiotic, has been shown to bind to the heat shock protein 90 (Hsp90) chaperone, interfering with its function. Hsp90 family chaperones have been shown to associate with several signaling molecules and play an essential role in signal transduction, which is important for tumor cell growth. Because radicicol lacks antitumor activity in vivo in experimental animal models, we examined the antitumor activity of a novel radicicol oxime derivative, radicicol 6-oxime (KF25706), on human tumor cell growth both in vitro and in vivo. KF25706 showed potent antiproliferative activities against various human tumor cell lines in vitro and inhibited v-src- and K-ras-activated signaling as well as radicicol. In addition, Hsp90 family chaperone-associated proteins, such as p185erbB2, Raf-1, cyclin-dependent kinase 4, and mutant p53, were depleted by KF25706 at a dose comparable to that required for antiproliferative activity. KF25706 was also shown to compete with geldanamycin for binding to Hsp90. KF29163, which is an inactive derivative of radicicol, was less potent both in p185erbB2 depletion and Hsp90 binding. More importantly, KF25706 showed significant growth-inhibitory activity against human breast carcinoma MX-1 cells transplanted into nude mice at a dose of 100 mg/kg twice daily for five consecutive i.v. injections. KF25706 was also shown to possess antitumor activity against human breast carcinoma MCF-7, colon carcinoma DLD-1, and vulval carcinoma A431 cell lines in vivo in an animal model. Finally, we confirmed the depletion of Hsp90-associated signaling molecules (Raf-1 and cyclin-dependent kinase 4) with ex vivo Western blotting analysis using MX-1 xenografts. In agreement with in vivo antitumor activity, KF25706 depleted Hsp90-associated molecules in vivo, whereas KF29163 and radicicol did not show this activity in vivo. Taken together, these results suggest that antitumor activity of KF25706 may be mediated, at least in part, by binding to Hsp90 family proteins and destabilization of Hsp90-associated signaling molecules.

MS-681a, b, c and d, new inhibitors of myosin light chain kinase from Myrothecium sp. KY6568. I. Characterization of producing strain and production, isolation and biological activities.

Novel compounds MS-681a, b, c and d were isolated from the culture broth of a fungal strain KY6568. The strain was identified as Myrothecium sp. from its morphological characteristics. MS-681a, b, c and d inhibited the activity of purified smooth muscle myosin light chain kinase with IC50 values of 0.11, 0.29, 0.095 and 0.26 microM, respectively. Cyclic AMP-dependent protein kinase, cyclic GMP-dependent protein kinase and protein kinase C were not inhibited at 100 microM by MS-681 compounds.

MS-681a, b, c and d, new inhibitors of myosin light chain kinase from Myrothecium sp. II. Physico-chemical properties and structure elucidation.

MS-681a, b, c and d are new inhibitors of myosin light chain kinase produced by Myrothecium sp. They are novel octapeptides containing alpha-alkyl-alpha-amino acids and a polyamine moiety. The structures were determined by NMR and FAB-MS/MS spectral analyses of the intact peptides. Their absolute configurations were elucidated by GC analysis on a chiral column of the constituent amino acids and by chemical synthesis of the polyamine moieties.

AS-186 compounds, new inhibitors of acyl-CoA: cholesterol acyltransferase from Penicillium asperosporum KY1635.

AS-186a, b, c, d, and g were isolated from the cultured broth of Penicillium asperosporum KY1635 as inhibitors of acyl-CoA: cholesterol acyltransferase (ACAT). IC50 values for the effect of AS-186a, b, c, d, and g against ACAT activity of the microsomes from cholesterol-fed rabbit liver were calculated to be 22.9, 8.2, 11.5, 12.4, and 13.9 microM, respectively. Although AS-186a, and b were identical to penicillide and purpactin A, respectively, AS-186c, d, and g were found to be new compounds.