Gambogic acid identifies an isoform-specific druggable pocket in the middle domain of Hsp90β
Tuesday, 2016/08/23 | 08:02:32
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Kendrick H. Yim, Thomas L. Prince, Shiwei Qu, Fang Bai, Patricia A. Jennings, José N. Onuchic, Emmanuel A. Theodorakis, and Leonard Neckers CELL BIOLOGY SignificanceThe molecular chaperone heat-shock protein 90 (Hsp90) is a key member of the cellular proteostasis network, and as such helps to protect cells against proteotoxic stress. Cancer cells have up-regulated members of this network, including Hsp90, to promote their survival and growth. Several Hsp90 inhibitors have undergone clinical trials, but these drugs, which bind to a shared nucleotide pocket in the N-terminal domain, do not differentiate between the four Hsp90 family members [Hsp90α, Hsp90β, GRP94 (glucose-regulated protein 94 kDa), and TRAP1 (tumor necrosis receptor-associated protein 1)]. In this report, we identify a pharmacophore contained within the natural product gambogic acid that binds uniquely to a site in Hsp90β, thus identifying this compound as a prototype of a new class of isoform-specific Hsp90 inhibitors. AbstractBecause of their importance in maintaining protein homeostasis, molecular chaperones, including heat-shock protein 90 (Hsp90), represent attractive drug targets. Although a number of Hsp90 inhibitors are in preclinical/clinical development, none strongly differentiate between constitutively expressed Hsp90β and stress-induced Hsp90α, the two cytosolic paralogs of this molecular chaperone. Thus, the importance of inhibiting one or the other paralog in different disease states remains unknown. We show that the natural product, gambogic acid (GBA), binds selectively to a site in the middle domain of Hsp90β, identifying GBA as an Hsp90β-specific Hsp90 inhibitor. Furthermore, using computational and medicinal chemistry, we identified a GBA analog, referred to as DAP-19, which binds potently and selectively to Hsp90β. Because of its unprecedented selectivity for Hsp90β among all Hsp90 paralogs, GBA thus provides a new chemical tool to study the unique biological role of this abundantly expressed molecular chaperone in health and disease.
See: http://www.pnas.org/content/113/33/E4801.full PNAS August 16 2016; vol.113; no.33: E4801–E4809
Fig. 1. Gambogic acid preferentially binds to Hsp90β. (A) Chemical structures of gambogic acid and GBA-biotin. (B–E) Hsp90 from cell lysate or purified protein was isolated with biotinylated GBA and streptavidin beads (abbreviated as Strep beads in all figures). STA-7346, the biotinylated version of the NTD-targeted inhibitor ganetespib, was used for comparison. AP, affinity purification; WB, Western blot. (B) FLAG-Hsp90α, FLAG-Hsp90β, and FLAG-TRAP1 were transfected into HEK293 cells. Only Hsp90β bound to Bio-GBA, whereas STA-7346 interacted strongly with all three isoforms. (C) Purified proteins (10 μg; Hsp90α, Hsp90β, and TRAP1) were incubated with Bio-GBA and STA-7346. Bio-GBA bound strongly to Hsp90β, only weakly to Hsp90α, and not at all to TRAP1. (D and E) Binding preferences of endogenous Hsp90α, Hsp90β, and GRP94 were evaluated in lysate from HEK293 (D) and SKBR3 (E) cells. Bio-GBA bound only to Hsp90β, whereas STA-7346 bound to all isoforms tested. (F) HEK293 cells were transfected with 3×F(LAG)-Hsp90α, 3×F-Hsp90β, HA-Hsp90α, or HA-Hsp90β. The following day, cells were lysed and lysates were treated with increasing concentrations of unlabeled GBA. After incubation for 30 min, Hsp90 protein was isolated with Bio-GBA and streptavidin beads. Compared with 3×F-Hsp90α and HA-Hsp90α, 3×F-Hsp90β and HA-Hsp90β bound strongly to Bio-GBA and binding was competitively and dose-dependently inhibited by pretreatment of cell lysate with unlabeled GBA. (G) LUMIER analysis confirmed that Bio-GBA binds to 3×F-Hsp90β at 10 times the binding strength of 3×F-Hsp90α [using lysates of HEK293 cells transfected with Hsp90 and control (EYFP) vector expression plasmids]. Error bars represent SDs. *P < 0.05. |
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