Design, Synthesis, and Evaluation of Site-Specific Hsp90 Inhibitors

Abstract

The heat shock protein 90 (Hsp90) family of molecular chaperones is responsible for the conformational maturation of nascent polypeptides and refolding of denatured proteins. Hsp90 is known to play an important role in the regulation of cell signaling, survival and proliferation, transforming it into a promising target for the treatment of several diseases, including cancer. Proteins associated with all six hallmarks of cancer, as described by Weinberg, are Hsp90 clients. Since these proteins depend upon Hsp90, its inhibition has the potential to simultaneously disrupt all six and halt the malignant progression using a single small molecule. Hsp90 is abundantly expressed in the cell and accounts for 1-2% of the total cellular protein, making itone of the most prevalent proteins in eukaryotic cells. The Hsp90 isoforms are responsible for the conformational maintenance of greater than 150 proteins. There are four distinct isoforms, including Hsp90á, Hsp90â, GRP94 and TRAP1. Hsp90á and â are located in the cytoplasm; Hsp90á is the major inducible form, while Hsp90â is the constitutively active form. In contrast, GRP94 is found in the endoplasmic reticulum and TRAP1 is located in the mitochondrial matrix. Hsp90 exists as a homodimer consisting of an N- and C-terminal domain, connected by a middle domain. The Hsp90 N-terminus contains an ATP binding site, responsible for the ATPase activity of the chaperone associated with folding of clients. Small molecules that preferentially bind to this nucleotide binding site inhibit the ability of Hsp90 to properly fold polypeptides, ultimately tagging them for degradation through the ubiquitin-proteasome pathway. Natural product inhibitors of the N-terminal ATP binding site include the ansamycin antibiotic geldanamycin (GDA) and the macrocyclic lactone radicicol (RDC). Blagg and co-workers have previously reported the chimeric N-terminal inhibitor radamide, which combines the resorcinol portion of RDC and the quinone portion of GDA through a flexible linker. Radamide manifests a slightly greater binding affinity for GRP94 (Kd = 0.52 ìM) versus cytosolic Hsp90 (Kd = 0.87 ìM). This phenomenon is elucidated by examining the unique binding conformations adopted by radamide when crystal structures of were obtained using each isoform. When bound to Hsp90, radamide exhibits a linear conformation, while when bound to GRP94 the quinone portion is bent towards a pocket that is not accessible in Hsp90á or â. By employing conformational constraint, it was proposed that the quinone portion of radamide could exhibit the bent conformation seen in the natural products and result in increased affinity for Hsp90 as well as selective binding to the cytosolic isoforms. In contrast to radamide, a recent paper by Gasiewicz and co-workers examined the effects of flavones and (-)-Epigallocatechin-3-Gallate (EGCG), the major polyphenolic catechin found in green tea, on the aryl hydrocarbon receptor (AHR). Due to its diverse medicinal properties, EGCG has been proposed as a potential treatment for several diseases. The study by Gasiewicz revealed that EGCG functions through a different mechanism of action than the known flavone antagonists, interacting with the Hsp90 C-terminus rather than the AHR. It was confirmed that EGCG binds specifically to the C-terminal nucleotide binding site, either within or proximal to the site where the natural product novobiocin is thought to bind. Previous studies have determined that EGCG manifests an IC50 of ~150 ìM against MCF-7 cells. Upon examination of an overlay of EGCG and novobiocin, the structural similarities of the coumarin and the catechin cores become apparent. It was proposed that design of molecules based on the structure-activity relationships established for novobiocin may result in Hsp90-specific inhibitors based upon the EGCG scaffold