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. 2010 Jan 1;1(1):4-8.
doi: 10.1021/ml900003t.

Mechanistic studies of Sansalvamide A-amide: an allosteric modulator of Hsp90

Robert C Vasko  1 , Rodrigo A Rodriguez, Christian N Cunningham, Veronica C Ardi, David A Agard, Shelli R McAlpine
Affiliations

Mechanistic studies of Sansalvamide A-amide: an allosteric modulator of Hsp90

Robert C Vasko et al. ACS Med Chem Lett. .

Abstract

Herein we show that San A-amide, a structurally unique molecule, influences a subset of cancer-related pathways involving Hsp90. We show that San A-amide specifically binds to the N-middle domain of Hsp90 allosterically disrupts the binding of proteins thought to interact with the Hsp90 C-terminal domain, while having no effect on an N-terminal domain client protein. This unique mechanism suggests that San A-amide is a potential tool for studying C-terminal binding proteins of Hsp90 as well as a promising lead in the development of new cancer therapeutics.

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Figures

Figure 1
Figure 1
(a) Natural product San A, the derivative San A-amide compound (1), and biotinylated San A-amide (San A-amide-Biotin). (b) Schematic diagram of the pull-down assay.
Figure 2
Figure 2
Bands isolated in the pull-down assay using HCT-116 colon cancer cell lyate. Lanes: 1 and 5, MW marker (kDa); 2, San A-amide-Biotin; 3, negative control (PEGlayted biotin linker); 4, DMSO control; and 6−8, protein input for lanes 2−4, respectively. Major proteins are indicated by asterisks with the following order from top to bottom: Hsp90, keratin, α- and β-tubulin, and actin.
Figure 3
Figure 3
In vitro pull-down binding assay: (a) San A-amide inhibits binding of both IP6K2 (EC50 = ∼1.4 μM) and the FKBP (EC50 = 1.0 μM) to Hsp90. Her2 binding to Hsp90 is not affected. (b) 17AAG inhibits Her2 binding to Hsp90 (EC50 = ∼0.66 μM), while FKBP and IP6K2 binding to Hsp90 are not affected (IP6K2 is not inhibited at 10 μM). %Hsp90 bound to client protein was quantified by densitometric scanning of Hsp90 protein on Western blot with normalization to client protein loading using Image J.
Figure 4
Figure 4
Cytotoxicity of San A-amide in IP6K2 overexpressed cell line (K2-O/E, gray bar) as compared to wild-type control (Con, white bar). *p < 0.05; ***p < 0.001.
Figure 5
Figure 5
San A-amide pull down of Hsc82 (yeast variant of Hsp90) full length and domains. Lanes: 1, MW marker (kDa); 2, N-terminal domain; 3, middle domain; 4, C-terminal domain; 5, N-terminal and middle domain combined; and 6, full length.
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