Tsutomu Agatsuma

Targeting of the protein chaperone, HSP90, by the transformation suppressing agent, radicicol

Radicicol, a macrocyclic anti-fungal antibiotic, has the ability to suppress transformation by diverse oncogenes such as Src, Ras and Mos. Despite this useful property, the mechanism by which radicicol exerts its anti-transformation effects is currently unknown. To understand the transformation-suppressing effects of radicicol, a biotinylated derivative of radicicol was chemically synthesized and used as a probe in a Western-blot format to visualize cellular proteins that interact with radicicol. In transformed and untransformed mouse fibroblasts, the most prominent cellular protein that bound to radicicol had a molecular weight of approximately 90 kDa. Further analysis revealed that this protein was the mouse homologue of the 90 kDa heat shock protein (HSP90). This was confirmed by demonstrating the ability of radicicol to specifically bind purified human HSP90. Specificity of binding was demonstrated by the inhibition of binding of biotinylated radicicol by the native drug. Taken together with other studies the present observations suggest that the anti-transformation effects of radicicol may be mediated, at least in part, by the association of radicicol with HSP90 and the consequent dissociation of the Raf/HSP90 complex leading to the attenuation of the Ras/MAP kinase signal transduction pathway.

Halohydrin and oxime derivatives of radicicol: synthesis and antitumor activities.

Novel halohydrin and oxime derivatives of radicicol (1) were prepared and evaluated for their v-src tyrosine kinase inhibitory, antiproliferative, and antitumor activities. Some of the resulting derivatives showed significantly improved antitumor activities than those of 1 in vitro as tested in a cell proliferation assay and in vivo using sc-inoculated human breast carcinoma and epidermoid tumor models. Design and synthesis of radicicol-based novel affinity probes are also described.

Synthetic Inhibitors of Proline-Rich Ligand-Mediated Protein-Protein Interaction: Potent Analogs of UCS15A

The proline-rich motif in proteins is known to function as a ligand sequence that binds to protein modules such as SH3, WW, and several other protein interaction domains. These proline-rich ligand-mediated protein-protein interactions (abbreviated PLPI) are important in many signaling pathways that are involved in various diseases. Our previous studies showed that UCS15A, produced by Streptomyces species, inhibited PLPI. Here we report on synthetic analogs of UCS15A that show more potent activity than UCS15A in inhibiting PLPI. A synthetic analog, compound 2c, blocked in vitro PLPI of Sam68-Fyn-SH3 as well as in vivo PLPI of Grb2-Sam68 and Grb2-Sos1. Activation of MEK was also inhibited by compound 2c. Unlike UCS15A, compound 2c was an order of magnitude less cytotoxic and did not cause morphological changes in treated cells.