An Hsp90 modulator that exhibits a unique mechanistic profile

Abstract

Described is the synthesis of two biotinylated derivatives of a cytotoxic macrocycle. Pull-down assays indicate that this macrocycle targets the N-middle domain of Hsp90. Untagged compound can effectively compete away tagged compound-Hsp90 protein complexes, confirming the binding specificity of the macrocycle for Hsp90. The macrocycle is similar in potency to other structurally-related analogs of Sansalvamide A (San A) and induces apoptosis via a caspase 3 mechanism. Unlike other San A derivatives, we show that the macrocycle does not inhibit binding between C-terminal client proteins and co-chaperones and Hsp90, suggesting that it has a unique mechanism of action.

Figure 1

San A-amide (compound 1)⁴ Hsp90 inhibitors (compounds 2⁵ and 3⁶, and compound of interest (compound 4).

Figure 2

Biotin-tagged derivatives of Compound 4.

Figure 3

Western blot of proteins isolated in pull down assay.

Figure 4

Visualization of Hsp90 in HCT 116 cells treated with DMSO (72 hours; 1%), 17-AAG (24 hours; 200 nM), compound 3 (72 hours; 5 µM), or compound 4 (72 hours; 5 µM).

Figure 5

a) Compound 4-T-III selectively pulls down the N-middle domain of Hsp90. [11.5 fold above background] b) Compound 4 and 3 compete off the tagged analog 4-T-III (IC₅₀ 6.2 µM and IC₅₀ 1.9 µM respectively).