Crystallization of heat-shock protein 90 bound with bruceantin and NSC145366

Abstract

Hsp90 (heat shock protein 90) is part of a molecular chaperone machine that is important for activating, stabilizing, and regulating hundreds of proteins, including oncogenic proteins. Hsp90 is often overexpressed in cancer cells and, because it stabilizes oncogenic proteins, it helps the cancer grow. This makes Hsp90 a promising target for cancer treatment. Drugs that inhibit Hsp90 by binding the N-terminal domain have been tested in clinical trials, but the drugs trigger the heat-shock response which further stimulates Hsp90 expression. Therefore, drugs that inhibit Hsp90 by binding the middle domain or C-terminal domain are being explored. The drugs bruceantin (BCT) and NSC145366 (NSC) are both promising Hsp90 inhibitors. It has been shown that NSC interacts with Hsp90 at the C-terminal domain and inhibits chaperone activity without eliciting a detectable heat-shock response. It is not known exactly where bruceantin interacts with Hsp90, but it has also been shown to inhibit chaperone activity. The objective of this study is to crystallize Hsp90 linked separately with BCT and NSC so that the structure can be further analyzed to determine if and how the inhibitors are binding to Hsp90. In this study, crystals were obtained with sitting drops. Hsp90 was mixed separately with BCT and NSC and tested in several conditions. Once the structure is found from the crystals, the structure can be analyzed to determine if the inhibitors are binding Hsp90 and where. This will give insight into the mechanisms of the inhibitors and the possible effects on Hsp90 function.