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. 2014 Feb 10;3(1):101-38.
doi: 10.3390/biology3010101.

High-throughput screen of natural product libraries for hsp90 inhibitors

Jason Davenport  1 , Maurie Balch  2 , Lakshmi Galam  3 , Antwan Girgis  4 , Jessica Hall  5 , Brian S J Blagg  6 , Robert L Matts  7
Affiliations

High-throughput screen of natural product libraries for hsp90 inhibitors

Jason Davenport et al. Biology (Basel). .

Abstract

Hsp90 has become the target of intensive investigation, as inhibition of its function has the ability to simultaneously incapacitate proteins that function in pathways that represent the six hallmarks of cancer. While a number of Hsp90 inhibitors have made it into clinical trials, a number of short-comings have been noted, such that the search continues for novel Hsp90 inhibitors with superior pharmacological properties. To identify new potential Hsp90 inhibitors, we have utilized a high-throughput assay based on measuring Hsp90-dependent refolding of thermally denatured luciferase to screen natural compound libraries. Over 4,000 compounds were screen with over 100 hits. Data mining of the literature indicated that 51 compounds had physiological effects that Hsp90 inhibitors also exhibit, and/or the ability to downregulate the expression levels of Hsp90-dependent proteins. Of these 51 compounds, seven were previously characterized as Hsp90 inhibitors. Four compounds, anthothecol, garcinol, piplartine, and rottlerin, were further characterized, and the ability of these compounds to inhibit the refolding of luciferase, and reduce the rate of growth of MCF7 breast cancer cells, correlated with their ability to suppress the Hsp90-dependent maturation of the heme-regulated eIF2α kinase, and deplete cultured cells of Hsp90-dependent client proteins. Thus, this screen has identified an additional 44 compounds with known beneficial pharmacological properties, but with unknown mechanisms of action as possible new inhibitors of the Hsp90 chaperone machine.

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Figures

Figure 1
Figure 1
Scatter plot showing the activities of 3,859 of the compounds screened.
Figure 2
Figure 2
Sesquiterpine lactones, tetracyclic sestorpenoids and sesterpines.
Figure 3
Figure 3
Polyphenols and related compounds.
Figure 4
Figure 4
Flavonoids and related compounds.
Figure 5
Figure 5
Chalones and related compounds.
Figure 6
Figure 6
Pentacyclic triterpenoids.
Figure 7
Figure 7
Alkaloid compounds.
Figure 8
Figure 8
Benzylisoquinoline Alkaloids.
Figure 9
Figure 9
Cyclic peptides.
Figure 10
Figure 10
Quinones.
Figure 11
Figure 11
Other miscellaneous compounds.
Figure 12
Figure 12
Structure of anthothecol, rottlerin garcinol and piplartine/piperlongumine.
Figure 13
Figure 13
Effect of compounds on HRI’s Hsp90-dependent maturation. [35S]Labeled-HRI was synthesized by TnT in RRL and transferred to heme-deficient lysate for maturation. Translated protein was separated by SDS PAGE, transferred to PVDF membrane, and visualized by X-ray film exposure. The phosphorylated active form of the kinase is indicated with an asterisk. Lanes were treated as follows: heme, no heme, 20 µM geldanamycin (GA), 20 mM sodium molybdate, 20 mM novobiocin, and 100 µM each of anthothecol, garcinol, rottlerin, and piplartine/piperlongumine
Figure 14
Figure 14
Western blot for Hsp90-dependent client proteins (Cdk2, pAkt and Her2), and Hsp90 and Hsp70 present in extracts prepared from MCF7 cells treated for 24 h with DMSO (vehicle control), 0.5 µM geldanamycin, or the indicated concentrations of anthothecol, rottlerin, garcinol, or piplartine. Actin was used as the loading control.
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