The synthesis and evaluation of the antiproliferative activity of deacidified GEX1A analogues.

GEX1A/herboxidiene (1) is a natural product isolated from Streptomyces sp. and has been reported to target the pre-mRNA splicing process. Although 1 was shown to have antitumor activity in vivo, weight loss was observed in mice when 1 was consecutively administered. We assumed that the carboxylic acid moiety was one of the causes of this toxicity. In this study, a series of amide, carbamate and urea analogues of 1 were synthesized and their antiproliferative activity was evaluated in vitro. The synthesis of urea analogues featured Curtius rearrangement following amine treatment with the one-pot procedure from 1. Furthermore, a structure-activity relationship study of the urea analogues revealed that the pharmacologically preferable basic side chains were acceptable and that compound 9g was equipotent to parent 1. These basic urea analogues would be promising leads for the development of novel antitumor agents.

Hsp90 inhibitors as anti-cancer agents, from basic discoveries to clinical development.

Heat shock protein (Hsp) 90 is an ATP-dependent molecular chaperone which stabilizes various oncogenic kinases, including HER2, EGFR, BCR-ABL, B-Raf and EML4-ALK, which are essential for tumor growth. Several monoclonal antibodies and small molecule kinase inhibitors which target these kinases have been identified as potential new molecular target therapeutics. Previous reports have shown that many oncogenic proteins essential for cancer transformation are chaperoned by the Hsp90 complex, and some of these client proteins have been discovered by using Hsp90 inhibitors, such as geldanamycin (GA) and radicicol (RD).Thus far more than 200 client proteins have been identified. In past derivatives of these natural products have been evaluated in clinical trials, but none of the 1st generation of Hsp90 inhibitors has been approved yet because of their limitations in physico-chemical properties and/or safety profiles. However, recent reports have indicated that more than 10 new agents, 2nd generation of Hsp90 inhibitors with different chemotypes from GA and RD, have entered clinical trials and some of them showed clinical efficacy. In this review article, we describe the discoveries of major Hsp90 client proteins in the cancer field by RD derivatives, the history of KW-2478 discovery and development by Kyowa Hakko Kirin, and gave an update on the current status of new Hsp90 inhibitors in clinical trials.

New molecular and biological mechanism of antitumor activities of KW-2478, a novel nonansamycin heat shock protein 90 inhibitor, in multiple myeloma cells.

PURPOSE: The heat shock protein 90 (Hsp90) plays an important role in chaperoning oncogenic client proteins in multiple myeloma (MM) cells, and several Hsp90 inhibitors have shown antitumor activities both in vitro and in vivo. However the precise mechanism of action of Hsp90 inhibitor in MM has not been fully elucidated. EXPERIMENTAL DESIGN: We evaluated the antitumor activities of KW-2478, a nonansamycin Hsp90 inhibitor, in MM cells with various chromosomal translocations of immunoglobulin heavy chain (IgH) loci both in vitro and in vivo. RESULTS: Our studies revealed that exposure of KW-2478 to MM cells resulted in growth inhibition and apoptosis, which were associated with degradation of well-known client proteins as well as a decrease in IgH translocation products (FGFR3, c-Maf, and cyclin D1), and FGFR3 was shown to be a new client protein of Hsp90 chaperon complex. In addition, KW-2478 depleted the Hsp90 client Cdk9, a transcriptional kinase, and the phosphorylated 4E-BP1, a translational inhibitor. Both inhibitory effects of KW-2478 on such transcriptional and translational pathways were shown to reduce c-Maf and cyclin D1 expression. In NCI-H929 s.c. inoculated model, KW-2478 showed a significant suppression of tumor growth and induced the degradation of client proteins in tumors. Furthermore, in a novel orthotopic MM model of i.v. inoculated OPM-2/green fluorescent protein, KW-2478 showed a significant reduction of both serum M protein and MM tumor burden in the bone marrow. CONCLUSIONS: These results suggest that targeting such diverse pathways by KW-2478 could be a promising strategy for the treatment of MM with various cytogenetic abnormalities.

Inhibition of Hsp90 activates osteoclast c-Src signaling and promotes growth of prostate carcinoma cells in bone.

Hsp90 inhibitors are being evaluated extensively in patients with advanced cancers. However, the impact of Hsp90 inhibition on signaling pathways in normal tissues and the effect that this may have on the antitumor activity of these molecularly targeted drugs have not been rigorously examined. Breast and prostate carcinomas are among those cancers that respond to Hsp90 inhibitors in animal xenograft models and in early studies in patients. Because these cancers frequently metastasize to bone, it is important to determine the impact of Hsp90 inhibitors in the bone environment. In the current study, we show that, in contrast to its activity against prostate cancer cells in vitro and its inhibition of s.c. prostate cancer xenografts, the Hsp90 inhibitor 17-AAG stimulates the intraosseous growth of PC-3M prostate carcinoma cells. This activity is mediated not by a direct effect on the tumor but by Hsp90-dependent stimulation of osteoclast maturation. Hsp90 inhibition transiently activates osteoclast Src kinase and promotes Src-dependent Akt activation. Both kinases are key drivers of osteoclast maturation, and three agents that block osteoclastogenesis, the Src inhibitor dasatinib, the bisphosphonate alendronate, and the osteoclast-specific apoptosis-inducer reveromycin A, markedly reduced 17-AAG-stimulated tumor growth in bone. These data emphasize the importance of understanding the complex role played by Hsp90 in regulating signal transduction pathways in normal tissues as well as in cancer cells, and they demonstrate that drug-dependent modulation of the local tumor environment may profoundly affect the antitumor efficacy of Hsp90-directed therapy.

Conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin, for Hsp90 inhibitory activity.

Hsp90 is an attractive chemotherapeutic target because it is essential to maturation of multiple oncogenes. We describe the conformational significance of EH21A1-A4, phenolic derivatives of geldanamycin isolated from Streptomyces sp. Their native free structures are similar to the active form of geldanamycin bound to Hsp90 protein. Their conformational character is a probable reason for their high-affinity binding. Lack of toxic benzoquinone in EH21A1-A4 also adds to their potential as lead compounds for anti-tumor drugs.

Design and synthesis of piperidine farnesyltransferase inhibitors with reduced glucuronidation potential.

The design and synthesis of a novel piperidine series of farnesyltransferase (FTase) inhibitors with reduced potential for metabolic glucuronidation are described. The various substitution and exchange of the phenyl group at the C-2 position of the previously described 2-(4-hydroxy)phenyl-3-nitropiperidine 1a (FTase IC(50)=5.4nM) resulted in metabolically stable compounds with potent FTase inhibition (14a IC(50)=4.3nM, 20a IC(50)=3.0nM, and 50a IC(50)=16nM). Molecular modeling studies of these compounds complexed with FTase and farnesyl pyrophosphate are also described.

Development of radicicol analogues.

Radicicol, a macrocyclic antibiotic produced by fungi, was originally isolated many years ago, and was described as tyrosine kinase inhibitor. We also rediscovered radicicol as an inhibitor of signal transduction of oncogene products, such as K-ras and v-Src, using yeast and mammalian cell-based assays. In a study of mechanisms of action, it was revealed that radicicol depletes the Hsp90 client signaling molecules in cells, and thus inhibit the signal transduction pathway. In addition, direct binding of radicicol to the N-terminal ATP/ADP binding site of Hsp90 was shown, and thus radicicol has been recognized as a structurally unique antibiotic that binds and inhibits the molecular chaperone Hsp90. Although radicicol itself has little or no activity in animals because of instability in animals, its oxime derivatives showed potent antitumor activities against human tumor xenograft models. Hsp90 client proteins were depleted and apoptosis was induced in the tumor specimen treated with radicicol oxime derivatives. Taken together, these results suggest that the antitumor activity of radicicol oxime derivatives is mediated by binding to Hsp90 and destabilization of Hsp90 client proteins in the tumor. Among Hsp90 clients, we focused on ErbB2 and Bcr-Abl as examples of important targets of Hsp90 inhibitors. Radicicol oxime showed potent antitumor activity against ER negative/ErbB2 overexpressing breast cancer and Bcr-Abl expressing CML. Putative mechanisms of action and future directions of radicicol oxime against these kinds of tumor are discussed.

Improvement of biological activity and proteolytic stability of peptides by coupling with a cyclic peptide.

The cyclic moiety of an endothelin antagonist peptide RES-701-1, composed of 10 amino acids with an amide bond between alpha-NH(2) of Gly1 and beta-COOH of Asp9, was coupled to some biologically active peptides aiming to improve their activities and stabilities against proteolytic degradation. Coupling of the cyclic peptide to the N-terminal of RGD-peptides, maximally 4-fold improvement of in vitro activity compared to the original peptide has been achieved. Coupling of it to protein farnesyltransferase inhibiting peptides resulted to improve in vitro activity maximally 3-fold. These peptides coupled with the cyclic peptide also showed enhanced stability against some typical proteases. These results indicate that this cyclic peptide can stabilize the conformations of the peptides coupled to its C-terminus. Coupling of our cyclic peptide is anticipated to be a novel conformational stabilizing method for biologically active peptides, results to improve their activity and stability.