Conformational significance of EH21A1–A4, phenolic derivatives of geldanamycin, for Hsp90 inhibitory activity Hideyuki Onodera, a Masami Kaneko, a Yuichi Takahashi, b Yumiko Uochi, a Jun Funahashi, b Takayuki Nakashima, b Shiro Soga, b Makoto Suzuki, a Shunichi Ikeda, a Yoshinori Yamashita, b Endang S. Rahayu, c Yutaka Kanda b and Michio Ichimura a, * a BioFrontier Laboratories, Kyowa Hakko Kogyo Co., Ltd., 3-6-6 Asahi-machi, Machida-shi, Tokyo 194-8533, Japan b Pharmaceutical Research Center, Kyowa Hakko Kogyo Co., Ltd., 1188 Shimotogari, Nagaizumi, Sunto, Shizuoka, Japan c Faculty of Agricultural Technology, Gadjah Mada University, Bulaksumur Yogyakarta 55281, Indonesia Received 14 December 2007; accepted 17 January 2008 Available online 24 January 2008 Abstract—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. Ó 2008 Elsevier Ltd. All rights reserved. Heat Shock Protein 90 (Hsp90) is an essential molecular chaperone required for conformational stability and function of multiple growth regulatory signaling pro- teins. Inhibiting the function of Hsp90 leads to subse- quent simultaneous degradation of the client proteins via the ubiquitination-proteosome system. 1 Since the cli- ent proteins cover the six hallmarks of cancer, 2 Hsp90 inhibitors are attractive as chemotherapeutic agents. 3 A first-in-class drug, 17-allylamino-17-demethoxygel- danamycin (17-AAG, 2), a derivative of geldanamycin (1), has provided proof of concept for Hsp90 inhibition in clinical trials. However, 2 showed significant hepato- toxicity probably due to the redox-active benzoquinone moiety, and was difficult to administer because of its poor solubility profile. 4 Our aim, therefore, was to find more potent inhibitors with less toxicity and higher solubility. For screening, we established a dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA) using biotin- ylated radicicol 5 as a probe because radicicol (8), another natural Hsp90 inhibitor, shares the common N-terminal ATP binding pocket in Hsp90 with a higher affinity than 1. In the course of screening, we found that an actinomy- cete Streptomyces sp., isolated from Indonesian soil (see Supplementary data), produced polar inhibitors together with 1. Jar fermentation culture of the strain (62.7 L), followed by repetitive activity-guided column chroma- tography, provided four inhibitors; tentatively named EH21A1–A4 (3–6) with the following yields: 128.9, 34.9, 29.8, and 8.4 mg, respectively (see Supplementary data). The structures of 3–6 were spectroscopically revealed as analogous to 1 with a unique phenol structure rather than the benzoquinone moiety of 1. The distinctive reduction in C-4/C-5 was also a common feature of 3– 6. NMR signals for a methyl group adjacent to C-2 were not observed in 6. Collectively, 3 was identical to reblastatin, 6 and the structures of 4–6 were elucidated as 17-demethoxy, 17-O-demethyl, 6 and 17-demethoxy- 2-demethyl derivatives of 3, respectively (Fig. 1). Although production of 4 and 6 has been reported as KOSN1806 and KOSN1559, respectively, in the actino- mycetes genetically engineered, 7 the present report would be their first isolation as natural products. In vitro affinities for a purified Hsp90 protein were com- pared by compound concentration sufficient to inhibit 50% binding of the probe (EC 50 ) in DELFIA assay 0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.bmcl.2008.01.072 Keywords: Hsp90; Inhibitor; Ansamycin; Geldanamycin; Conformation. * Corresponding author. E-mail: michio.ichimura@kyowa.co.jp Available online at www.sciencedirect.com Bioorganic & Medicinal Chemistry Letters 18 (2008) 1577–1580