Hsp90 C-terminal domain binding site for novobiocin and related coumarins

Heat shock protein 90 (Hsp90) is a highly conserved, eukaryotic, molecular chaperone which stabilizes an assortment of oncogenic proteins. Currently, novobiocin and related coumarins are being developed into higher affinity analogs for the treatment of various cancers based on their inhibition of Hsp90 chaperone function. However, direct binding to Hsp90 has yet to be demonstrated; the existence of an Hsp90 binding site for these compounds has, to date, been based on a number of indirect assays. In order to address this gap in our knowledge, we used the technique of surface plasmon resonance (SPR) to assay the affinities of novobiocin derivatives for full length Hsp90 (Hsp90FL) and a C-terminal Hsp90 truncation (Hsp90CT). We also examined the effect of bound N-terminal ligands, ATP, ADP, and geldanamycin (GA), on the affinities for our compounds of interest. Results demonstrate that novobiocin and related coumarins bind to apo Hsp90FL and apo Hsp90CT with comparable affinities. Additionally, novobiocin and related compounds did not bind to the N terminal nucleotide binding pocket on Hsp90FL. Moreover, coumarin derivatives bound to Hsp90: ADP complexes with enhanced affinity. Our results demonstrate that a binding site for novobiocin related compounds resides on the C-terminus of Hsp90. Our results also support the hypothesis that the N-terminal and C-terminal domains of Hsp90 interact to moderate Hsp90 chaperone activity. Our results provide new insights into the mode of action by which novobiocin related compounds interact with Hsp90 in vitro and may suggest novel approaches to the development of higher affinity novobiocin derivatives in the treatment of cancer and related diseases.