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.Department of Biochemistry and Molecular Biolog

Molecular Mechanisms Underlying Fast-Acting Antidepressant Efficacy

Major Depressive Disorder is a devastating mental illness with a profound disease burden, particularly in the United States. Major Depressive Disorder is a heterogeneous disorder that is characterized by dysregulated mood and/or anhedonia with intense feelings of despair and sadness, agitation, self-deprecation, and suicidal ideation. Antidepressants, such as selective serotonin reuptake inhibitors, are the most common form of treatment for Major Depressive Disorder, however the precise mechanism by which these drugs work is largely unknown. Moreover, the time they take to reach clinical effect can take weeks to months, and some patients never truly respond, leaving a critical need for more rapidly acting antidepressants with sustained efficacy. In the laboratory, we have explored aspects of the neurotrophic hypothesis of depression and have made progress toward understanding the role of brain-derived neurotrophic factor in animal models. We have also made progress in understanding the role of brain-derived neurotrophic factor in cellular and molecular mechanisms which underlie fast-acting antidepressant efficacy. First, we examined whether ketamine, a novel fast acting antidepressant, functioned in a dose dependent manner to elicit its antidepressant effects. We found that only low, nonpsychomimetic doses of ketamine produce antidepressant effects, whereas high, psychomimetic doses did not produce antidepressant responses. We also demonstrated that only low dose ketamine triggered robust increases in BDNF translation, which our lab has previously shown to be required for ketamine's fast acting antidepressant effects. Next we examined the role of calcineurin in relation to our model of ketamine action, and we uncovered a parallel L-type calcium channel mediated calcium signaling pathway that dephosphorylates eukaryotic elongation factor 2 and competes with the previously identified n-methyl-d-aspartate receptor dependent signaling that activates eukaryotic elongation factor 2 kinase function. The balance between these two calcium signaling pathways determines the degree of eukaryotic elongation factor 2 phosphorylation and the extent of BDNF protein translation, which in turn gauges the efficacy of ketamine-mediated rapid antidepressant responses in preclinical mouse models. Finally, we investigated the molecular mechanisms underlying scopolamine's fast acting antidepressant effect and discovered that scopolamine mediated antidepressant effects require brain-derived neurotrophic factor transcription