Investigation of the Hsp90 C-terminal Binding Site, Novel Inhibitors and Isoform-Dependent Client Proteins

The heat shock proteins represent an important class of pro-survival proteins that are intimately involved in cell survival, adaptation to cellular stress, and protein management. Heat shock protein 90 kDa (Hsp90) is a molecular chaperone responsible for the post-translational maturation of nascent polypeptides. Many of the Hsp90-dependent client proteins are involved in oncogenic processes, and accordingly, Hsp90 has emerged as a promising target for anti-cancer therapies. Unfortunately, the clinical evaluation of Hsp90 inhibitors has been met with dosing, scheduling, and toxicity issues. The Hsp90 inhibitors that have reached clinical trials bind to the Hsp90 N-terminal ATP-binding site and demonstrate pan-Hsp90 inhibition, as they bind to and inhibit all four human Hsp90 isoforms. This characteristic may rationalize the undesired toxicities related to Hsp90 inhibition. Interestingly, the identification and characterization of isoform specific client proteins has not been extensively explored. In addition, N-terminal Hsp90 inhibition results in the induction of the heat shock response, whereby the expression of Hsp90 and other heat shock proteins is induced. This attribute of N-terminal inhibitors results in the clinically observed dosing and scheduling detriments as levels of Hsp90 increase with administration of the drug. Described herein is the design, synthesis and biological evaluation of novel Hsp90 inhibitors that avoid the above mentioned therapeutic liabilities of currently known Hsp90 inhibitors. The identification and characterization of an Hsp90-isoform dependent client protein and an Hsp90-isoform selective inhibitor is also presented

Click Chemistry to Probe Hsp90: Synthesis and Evaluation of a Series of Triazole Containing Novobiocin Analogues

A series of triazole-containing novobiocin analogues has been designed, synthesized and their inhibitory activity determined. These compounds contain a triazole ring in lieu of the amide moiety present in the natural product. The anti-proliferative effects of these compounds were evaluated against two breast cancer cell lines (SKBr-3 and MCF-7), and manifested activities similar to their amide-containing counterparts. In addition, Hsp90-dependent client protein degradation was observed via western blot analysis, further supporting a common mode of Hsp90 inhibition for both structural classes

Hook3 is a scaffold for the opposite-polarity microtubule-based motors cytoplasmic dynein-1 and KIF1C.

The unidirectional and opposite-polarity microtubule-based motors, dynein and kinesin, drive long-distance intracellular cargo transport. Cellular observations suggest that opposite-polarity motors may be coupled. We recently identified an interaction between the cytoplasmic dynein-1 activating adaptor Hook3 and the kinesin-3 KIF1C. Here, using in vitro reconstitutions with purified components, we show that KIF1C and dynein/dynactin can exist in a complex scaffolded by Hook3. Full-length Hook3 binds to and activates dynein/dynactin motility. Hook3 also binds to a short region in the "tail" of KIF1C, but unlike dynein/dynactin, this interaction does not activate KIF1C. Hook3 scaffolding allows dynein to transport KIF1C toward the microtubule minus end, and KIF1C to transport dynein toward the microtubule plus end. In cells, KIF1C can recruit Hook3 to the cell periphery, although the cellular role of the complex containing both motors remains unknown. We propose that Hook3's ability to scaffold dynein/dynactin and KIF1C may regulate bidirectional motility, promote motor recycling, or sequester the pool of available dynein/dynactin activating adaptors

Finding character strengths through loss: an extension of Peterson and Seligman (2003)

People can experience positive changes even in the midst of adversity and loss. We investigated character strengths following three recent shooting tragedies. Using an Internet database of respondents to the Values in Action Inventory of Strengths (VIA-IS), we compared responses from three groups of participants (N = 31,429) within close proximity of each event: those who completed it eight months prior to the event, and one month and two months after. Results suggested that for one of the events, participants who completed the VIA-IS after the event showed slightly different levels of self-reported character strengths compared to participants who completed the VIA-IS before the event, with some mean levels higher and others lower. The observed differences in character strengths were inconsistent across follow-up periods, and effect sizes were small (d values from –0.13 to 0.15). These findings raise questions about whether and how tragedies might catalyze differences in character strengths

Gambogic Acid, a Natural Product Inhibitor of Hsp90

A high-throughput screening of natural product libraries identified (−)-gambogic acid (1), a component of the exudate of Garcinia harburyi, as a potential Hsp90 inhibitor, in addition to the known Hsp90 inhibitor celastrol (2). Subsequent testing established that 1 inhibited cell proliferation, brought about the degradation of Hsp90 client proteins in cultured cells, and induced the expression of Hsp70 and Hsp90, which are hallmarks of Hsp90 inhibition. Gambogic acid also disrupted the interaction of Hsp90, Hsp70, and Cdc37 with the heme-regulated eIF2α kinase (HRI, an Hsp90-dependent client) and blocked the maturation of HRI in vitro. Surface plasmon resonance spectroscopy indicated that 1 bound to the N-terminal domain of Hsp90 with a low micromolar Kd, in a manner that was not competitive with the Hsp90 inhibitor geldanamycin (3). Molecular docking experiments supported the posit that 1 binds Hsp90 at a site distinct from Hsp90s ATP binding pocket. The data obtained have firmly established 1 as a novel Hsp90 inhibitor and have provided evidence of a new site that can be targeted for the development of improved Hsp90 inhibitors

Synthesis and Evaluation of Novologues as C-Terminal Hsp90 Inhibitors with Cytoprotective Activity against Sensory Neuron Glucotoxicity

Compound 2 (KU-32) is a first-generation novologue (a novobiocin-based, C-terminal, heat shock protein 90 (Hsp90) inhibitor), that decreases glucose-induced death of primary sensory neurons and reverses numerous clinical indices of diabetic peripheral neuropathy in mice. The current study sought to exploit the C-terminal binding site of Hsp90 to determine whether the optimization of hydrogen bonding and hydrophobic interactions of second generation novologues could enhance neuroprotective activity. Using a series of substituted phenylboronic acids to replace the coumarin lactone of 2, we identified electronegative atoms placed at the meta-position of the B-ring exhibit improved cytoprotective activity, which is believed to result from favorable interactions with Lys539 in the Hsp90 C-terminal binding pocket. Consistent with these results, a meta-3-fluorophenyl substituted novologue (13b) exhibited a 14-fold lower ED50 compared to 2 for protection against glucose-induced toxicity of primary sensory neurons

Targeting the Heat Shock Protein 90 Dimer with Dimeric Inhibitors

The design, synthesis and biological evaluation of conformationally constrained coumermycin A1 analogues are reported. Compounds were evaluated against both breast cancer (SKBr3 and MCF7) and prostate cancer (PC3mm2, A549 and HT29) cell lines. Non-noviosylated coumermycin A1 analogues that manifest potent anti-proliferative activity resulting from Hsp90 inhibition are provided, wherein replacement of the stereochemically complex noviose sugar with readily available piperidine rings resulted in ~100 fold increase in anti-proliferative activities as compared to coumermycin A1, producing small molecule Hsp90 inhibitors that exhibit nanomolar activities

Stability of methylation markers in head and neck squamous cell carcinoma

BackgroundAs cancer progresses, methylation patterns change to promote the tumorigenic phenotype. However, stability of methylation markers over time and the extent that biopsy samples are representative of larger tumor specimens are unknown. This information is critical for clinical use of such biomarkers.MethodsNinety‐eight patients with tumor specimens from 2 timepoints were measured for DNA methylation in the promoter regions across 4 genes.ResultsThere were no significant differences in overall methylation of CCNA1 (cyclin A1), NDN (necdin), deleted in colorectal carcinoma (DCC), and cluster of differentiation 1a (CD1A) within paired specimens (p values = .56, .17, .66, and .58, respectively). All genes showed strong correlations between paired specimens across time. Methylation was most consistent for CCNA1 and NDN over time.ConclusionThis report provides the first evidence that methylation markers measured in biopsy samples are representative of gene methylation in later specimens and suggests that biopsy markers could be representative biomarkers for use in defining personalized treatment utilizing epigenetic changes. © 2015 Wiley Periodicals, Head Neck 38: E1325–E1331, 2016Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/137576/1/hed24223.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/137576/2/hed24223_am.pd

Chagas Disease in a Domestic Transmission Cycle in Southern Texas, USA

After three dogs died from acute Chagas cardiomyopathy at one location, an investigation was conducted of the home, garage, and grounds of the owner. A serologic study was conducted on stray dogs, and an ecologic niche model was developed to predict areas where the vector Trypanosoma gerstaeckeri might be expected

Elucidation of the Hsp90 C-terminal Inhibitor Binding Site

The Hsp90 chaperone machine is required for the folding, activation and/or stabilization of more than 50 proteins directly related to malignant progression. Hsp90 contains small molecule binding sites at both its N- and C-terminal domains, however, limited structural and biochemical data regarding the C-terminal binding site is available. In this report, the small molecule binding site in the Hsp90 C-terminal domain was revealed by protease fingerprinting and photoaffinity labeling utilizing LC-MS/MS. The identified site was characterized by generation of a homology model for hHsp90α using the SAXS open structure of HtpG and docking the bioactive conformation of NB into the generated model. The resulting model for the bioactive conformation of NB bound to Hsp90α is presented herein