Abstract
The attachment of biotin to a small molecule provides a powerful tool in biology. Here, we present a systematic approach to identify biotinylated analogues of the Hsp90 inhibitor PU-H71 that are capable of permeating cell membranes so as to enable the investigation of Hsp90 complexes in live cells. The identified derivative 2g can isolate Hsp90 through affinity purification and, as we show, represents a unique and useful tool to probe tumor Hsp90 biology in live cells by affinity capture, flow cytometry and confocal microscopy. To our knowledge, 2g is the only reported biotinylated Hsp90 probe to have such combined characteristics.
Highlights
Heat shock protein 90 (Hsp90) is a molecular chaperone that functions to properly fold proteins to their active conformation through its ATPase activity [1]
It has played a paramount role as a probe molecule to investigate Hsp90 biology, and the attachment of GM to solid support enabled the identification of Hsp90 as the target of its anticancer activity through affinity purification [16]
We set out here to design a series of biotinylated analogues derived from the purine scaffold Hsp90 inhibitor PU-H71 (1a) with the purpose of identifying compounds capable of permeating cancer-cell membranes, binding selectively to intracellular oncogenic Hsp90 in live cancer cells, and able to trap and isolate Hsp90 bound to tumor-specific oncoclient proteins
Summary
Heat shock protein 90 (Hsp90) is a molecular chaperone that functions to properly fold proteins to their active conformation through its ATPase activity [1]. These client proteins include many that are involved in malignant cell transformations (i.e., HER2, EGFR, mutant ER, HIF1α, Raf-1, AKT, mutant p53). As a result of this, as well as the ability to block multiple signaling pathways through inhibition of a single target, Hsp has become one of the most pursued molecular targets for anticancer therapy [2,3]. As a testament to this, there are numerous ongoing clinical trials evaluating Hsp inhibitors from a Beilstein J.
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