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. 2016 Aug 16;113(33):E4801-9.
doi: 10.1073/pnas.1606655113. Epub 2016 Jul 27.

Gambogic acid identifies an isoform-specific druggable pocket in the middle domain of Hsp90β

Kendrick H Yim  1 , Thomas L Prince  1 , Shiwei Qu  2 , Fang Bai  3 , Patricia A Jennings  2 , José N Onuchic  4 , Emmanuel A Theodorakis  2 , Leonard Neckers  5
Affiliations

Gambogic acid identifies an isoform-specific druggable pocket in the middle domain of Hsp90β

Kendrick H Yim et al. Proc Natl Acad Sci U S A. .

Abstract

Because 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.

Keywords: caged xanthone; heat-shock protein 90; isoform-specific inhibitor; molecular chaperone; molecular docking.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
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.
Fig. 2.
Fig. 2.
Gambogic acid promotes degradation of Hsp90-dependent clients and demonstrates a unique client and cochaperone binding profile. (A) SKBR3 cells were treated with GBA for 6 h at increasing concentrations. GBA induced potent, dose-dependent degradation of several endogenous Hsp90 client proteins, although not significantly impacting expression of either of the cytoprotective chaperones Hsp70 and Hsp27. (B and C) Hsp90β–client and Hsp90β–cochaperone complexes were isolated from HEK293, SKBR3, and 22Rv1 cell lysates with Bio-GBA and streptavidin beads. (B) Bio-GBA is able to pull down Hsp90 complexed to several endogenous client proteins, unlike most classic NTD-targeted Hsp90 inhibitors. (C) Bio-GBA recognizes Hsp90 in complex with multiple cochaperones recognizing distinct Hsp90 conformational states.
Fig. S1.
Fig. S1.
GBA does not bind directly to Hsp90 clients and cochaperones. HEK293 cells were transfected with control siRNA or Hsp90β siRNA (20 nM). Cell lysates were prepared after 72 h and incubated with biotinylated GBA to isolate Hsp90–client/cochaperone complexes. “Input” lanes reflect lysate concentrations of Hsp90β and each client or cochaperone. Biotinylated GBA pulldown lanes are shown (Left). Although cochaperone and client expression was not affected by silencing Hsp90β, copulldown of these proteins was reduced, consistent with the degree of reduction in expression of Hsp90β following siRNA silencing.
Fig. 3.
Fig. 3.
Gambogic acid binds at a site distinct from NTD and CTD Hsp90 inhibitors. (A) HEK293 cells were lysed and lysates were treated with various Hsp90 inhibitors (10 μM). Bio-GBA and streptavidin beads were then added to isolate Hsp90β. Only unlabeled GBA was able to compete with Bio-GBA for binding. Conversely, HEK293 lysate was also incubated with free GBA and pulled down with STA-7346– or SNX-2112–conjugated drug beads. GBA did not block binding of these NTD-targeted inhibitors. As a positive control, STA-9090 (ganetespib) was added to lysate followed by Hsp90 pulldown with SNX-2112–conjugated beads and competitive binding inhibition was observed. Similar competitive binding inhibition was seen when SNX-2112 was added to lysate followed by Hsp90 pulldown with STA-7346. (B) LUMIER analysis of drug competition confirmed that no NTD- or CTD-targeted inhibitors compete with GBA for binding to Hsp90β. Error bars represent SDs. *P < 0.05.
Fig. 4.
Fig. 4.
Domain dissection of Hsp90β reveals a druggable site in the MD. Various 3×F-Hsp90β truncation mutants were made and transfected into HEK293 cells. STA-7346 was used as a representative NTD-targeted inhibitor and bound to all fragments that included the NTD. Bio-GBA was only able to bind to Hsp90β fragments that contained at least the first 432 residues. In contrast to STA-7346, GBA binding did not require the NTD. See Fig. S2 for the raw data supporting this figure.
Fig. S2.
Fig. S2.
Biotinylated GBA and STA-7346 (biotinylated N-terminal domain inhibitor) binding to Hsp90β truncation mutants. HEK293 cells were transfected with various 3×F-Hsp90β truncation mutants. After cell lysis, biotinylated GBA and STA-7346 were added to isolate 3×F-Hsp90β. (A–C) C-terminal truncations extending into the MD beyond amino acid 432 in Hsp90β are not recognized by biotinylated GBA, whereas STA-7346 bound all C-terminal truncation mutants retaining the NTD. (D) Densitometric quantification of selected data obtained in C, normalized for input. IP, immunoprecipitation. (E) N-terminally truncated Hsp90β (C268–648) retains binding to GBA but not to STA-7346.
Fig. 5.
Fig. 5.
Molecular docking model of GBA bound to Hsp90β. (A) Molecular modeling suggests that GBA (shown in blue stick representation) binds to a pocket within a region composed of residues 350 to 436 in Hsp90β (shown in pale cyan). Hydrogen bonds formed between GBA and the pocket are shown as red dashed lines. Oxygens are shown in red and nitrogens are shown in blue. (B and C) Surface electrostatic potential map of Hsp90β (residues 350 to 436) and Hsp90α (residues 359 to 444). Red, blue, and white colors correspond to negatively charged, positively charged, and neutral areas, respectively. (D) LUMIER analysis showed decreased GBA binding to 3×F-Hsp90β S365N and 3×F-Hsp90β 369SSA (the triple mutant L369S, I370S, E372A) compared with wild-type 3×F-Hsp90β. Error bars represent SDs. *P < 0.05 relative to wild-type. (E) HEK293 cells were transfected with HA-Hsp90β, HA-S365N, or HA-369SSA. The next day, cells were lysed and increasing amounts of Bio-GBA were used to pull down Hsp90β from cells expressing equivalent amounts of wild-type HA-Hsp90β or the mutants HA-S365N and HA-369SSA. The binding-pocket mutants displayed reduced ability to bind to Bio-GBA, consistent with the data obtained by LUMIER.
Fig. S3.
Fig. S3.
Surface electrostatic potential map of the GBA-binding site (residues 350 to 436) in Hsp90β and the impact of binding-site mutation on the interaction of cochaperones. (A) Wild-type human Hsp90β. (B) S375N mutant. (C) Triple mutant L369S, I370S, E372A. Red color corresponds to negatively charged areas, blue color corresponds to positively charged areas, and white color corresponds to neutral areas. The negatively charged area around L369 is because of the D367 (not shown). (D) Plasmids expressing HA-Hsp90β or HA-Hsp90β 369SSA (triple mutant) were transfected into HEK293 cells and then isolated with anti-HA beads. (E) HA-Hsp90β and HA-Hsp90β 369SSA were transfected into HEK293 cells (4 μg plasmid per 10-cm dish) and allowed to express overnight. After 24 h, transfected proteins were immunoprecipitated with HA beads (D) or affinity-purified with STA-7346 and streptavidin beads (E). In D, three cochaperones associated to an equivalent degree with wild-type and mutated Hsp90β. In E, the biotinylated N-terminal inhibitor STA-7346 affinity-purified both wild-type and mutant Hsp90β with equal efficiency. Taken together, the data in D and E confirm that Hsp90β 369SSA is structurally intact.
Fig. 6.
Fig. 6.
Model-based structural modifications of GBA increase or decrease binding to Hsp90β. (A) Drug schematic indicates key structural differences between GBA, MAD28, and DAP-19. Hydrophobic side chains at the periphery of the A ring of GBA are marked with green dashed circles. Substituents at the C29 center are marked with a yellow dashed circle. (B) Predicted binding mode of DAP-19 (shown in blue stick representation) against Hsp90β (shown in pale cyan). The morpholine amide group at C29 is marked with a yellow dashed circle. (C) HEK293 cells were lysed and treated with unlabeled GBA, DAP-19, or MAD28 at various concentrations. Endogenous Hsp90β was then isolated from cell lysates with Bio-GBA and streptavidin beads. Compared with unlabeled GBA, DAP-19 more effectively blocked Hsp90β binding to Bio-GBA, whereas MAD28 was less effective. (D) Data obtained from LUMIER analysis are consistent with the results obtained by Western blot (C). Error bars represent SDs. *P < 0.05 relative to DMSO.
Fig. S4.
Fig. S4.
Structure of the Hsp90β dimer showing the N-terminal, middle, and C-terminal domains and the GBA-binding site in the MD.
Fig. S5.
Fig. S5.
Route of synthesis of DAP-19 from GBA.
Fig. S6.
Fig. S6.
1H NMR spectrum of DAP-19.
Fig. S7.
Fig. S7.
13C NMR spectrum of DAP-19.
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