Gambogic acid, a natural product inhibitor of Hsp90

Abstract

A high-throughput screening of natural product libraries identified (-)-gambogic acid (1), a component of the exudate of Garcinia harburyi, as a potential Hsp90 inhibitor, in addition to the known Hsp90 inhibitor celastrol (2). Subsequent testing established that 1 inhibited cell proliferation, brought about the degradation of Hsp90 client proteins in cultured cells, and induced the expression of Hsp70 and Hsp90, which are hallmarks of Hsp90 inhibition. Gambogic acid also disrupted the interaction of Hsp90, Hsp70, and Cdc37 with the heme-regulated eIF2α kinase (HRI, an Hsp90-dependent client) and blocked the maturation of HRI in vitro. Surface plasmon resonance spectroscopy indicated that 1 bound to the N-terminal domain of Hsp90 with a low micromolar Kd, in a manner that was not competitive with the Hsp90 inhibitor geldanamycin (3). Molecular docking experiments supported the posit that 1 binds Hsp90 at a site distinct from Hsp90s ATP binding pocket. The data obtained have firmly established 1 as a novel Hsp90 inhibitor and have provided evidence of a new site that can be targeted for the development of improved Hsp90 inhibitors.

Figure 1

Effect of gambogic acid (1) and celastrol (2) on Hsp90-dependent luciferase refolding in reticulocyte lysate (A), and effect of 1 on cell proliferation of HeLa cells, and MCF7 and SkBr3 breast cancer cells. Experiments were carried out as described in the Experimental Section.

Figure 2

Gambogic acid (1)-induced degradation of Hsp90 client proteins. Compound 1 was incubated with (A) MCF7 and (B) SkBr3 breast cancer cells at concentrations (µM) indicated in the figure. Gambogic acid (1) was evaluated for its ability to downregulate several client proteins as described in the Experimental Section. Geldanamycin (3) (500 nM) and DMSO were used as positive and negative controls, respectively. Cell extracts were prepared and equivalent amounts of protein were separated by SDS-PAGE and subsequently Western blotted for the indicated proteins as described in the Experimental Section.

Figure 3

Effect of Hsp90 inhibitors on the interaction of Hsp90 and its co-chaperones with HRI (A), on HRI’s Hsp90-dependent maturation (B), and on HRI stability (C). A. [³⁵S]His-tagged HRI/K199R was synthesized by TnT in reticulocyte lysate as described in the Experimental Section. After 10 min, DMSO (4% v/v, lanes 1 & 2), geldanamycin (3, 80 µM, lane 3), sodium molybdate (20 mM, lane 4), celastrol (2, 100 µM, lane 5), novobiocin (4, 4.0 mM, lane 6), coumermycin A1 (5, 400 µM, lane 7), or gambogic acid (1, 50 µM, lane 8) were added, followed by an additional 40 min of incubation. [³⁵S]His-tagged HRI/K199R was then immunoadsorbed with anti-His antibodies and samples were analyzed for co-adsorbing Hsp90, Hsp70, and Cdc37 by SDS-PAGE and Western blotting. Lane 1: TnT lysate containing no plasmid as the control for non-specific binding. Top panel: autoradiogram of immunoadsorbed [³⁵S]HRI/K199R. B. [³⁵S]His-tagged HRI was synthesized in reticulocyte lysate as described in the Experimental Section. After 20 min, DMSO (4% v/v, lanes 1, 2 and 3), geldanamycin (3, 80 µM, lane 4), sodium molybdate (20 mM, lane 5), celastrol (2, 100 µM, lane 6), novobiocin (4, 4.0 mM, lane 7), coumermycin A1 (5, 400 µM, lane 8), or 1 (50 µM, lane 9) were added, followed by an additional 10 min of incubation. An aliquot of the TnT lysate was then transferred to hemin supplemented (20 µM, lane 2) or heme-deficient (lanes 1 and 3–12) lysate containing and equivalent concentration of each addition, followed by an additional 45 min of incubation. The samples were then analyzed for HRI maturation by SDS-PAGE and autoradiography as described in the Experimental Section. Lane 1: TnT lysate containing no plasmid. [³⁵S]HRI*: mature, active HRI. C. [³⁵S]His-tagged HRI was synthesized in reticulocyte lysate and treated with DMSO or Hsp90-inhibitors as described above. Aliquots of each reaction were taken prior to (upper panel, 0 min) and 45 min after (lower panel, 45 min) dilution into and incubation in heme-supplemented (lane1) or heme-deficient (lanes 2–8) lysate. The band intensities in the lower panel were quantified by scanning densitometry and values below the lane numbers are given as %O.D./mm² of minus heme control.

Figure 4

SPR analysis of the interaction of gambogic acid (1) with (A) full length Hsp90 and (B) the N-terminal domain of Hsp90. A. Injection of 1.0, 10, 25 and 50 µM 1 over a SPR chip containing bound full length Hsp90. B. Injection of 0.5, 5, 15 and 25 µM 1 over a SPR chip containing bound Hsp90NT. Black line: sensorgram of binding and dissociation; gray line with dots: curve fit.

Figure 5

Models of gambogic acid (1) docked to geldanamycin-bound Hsp90NT. A. Ribbon diagram of Hsp90NT with the carbons of 1 and geldanamycin shown in yellow and green, respectively. B. Electrostatic surface potential of Hsp90NT shown in the same orientation as in A. C. Close-up showing the salt bridge and H-bond formed between K208 and K204 with 1.