Quantification of protein kinase enzymatic activity in unfractionated cell lysates using CSox-based sensors.

Defining perturbations in protein kinase activity within biological samples can provide insight into disease mechanisms as well as potential targets for drug development. In this article, we present a method that utilizes a phosphorylation-sensitive amino acid, termed CSox, to afford kinase-selective biosensors capable of reporting on enzymatic activity directly in biological samples. These sensors produce an increase in fluorescence in response to phosphorylation of an amino acid residue adjacent to CSox.

Selective mitogen activated protein kinase activity sensors through the application of directionally programmable D domain motifs.

Accurate and quantitative methods for measuring the dynamic fluctuations of protein kinase activities are critically needed as diagnostic tools and for the evaluation of kinase-targeted inhibitors, which represent a major therapeutic development area in the treatment of cancer and other diseases. In particular, rapid and economical methods that utilize simple instrumentation and provide quantitative data in a high throughput format will have the most impact on basic research in systems biology and medicine. There are over 500 protein kinases in the human kinome.

Gedunin inactivates the co-chaperone p23 protein causing cancer cell death by apoptosis.

Pharmacological inhibition of Hsp90 is an exciting option for cancer therapy. The clinical efficacy of Hsp90 inhibitors is, however, less than expected. Binding of the co-chaperone p23 to Hsp90 and induced overexpression of anti-apoptotic proteins Hsp70 and Hsp27 are thought to contribute to this outcome.

3-Arylcoumarin derivatives manifest anti-proliferative activity through Hsp90 inhibition.

The potential therapeutic benefits associated with Hsp90 modulation for the treatment of cancer and neurodegenerative diseases highlight the importance of identifying novel Hsp90 scaffolds. KU-398, a novobiocin analogue, and silybin were recently identified as new Hsp90 inhibitors. Consequently, a library of 3-arylcoumarin derivatives that incorporated the structural features of KU-398 and silybin was designed, synthesized and evaluated against two breast cancer cell lines.

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 that 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.

Development of a Grp94 inhibitor.

Heat shock protein 90 (Hsp90) represents a promising therapeutic target for the treatment of cancer and other diseases. Unfortunately, results from clinical trials have been disappointing as off-target effects and toxicities have been observed. These detriments may be a consequence of pan-Hsp90 inhibition, as all clinically evaluated Hsp90 inhibitors simultaneously disrupt all four human Hsp90 isoforms.

The hERG channel is dependent upon the Hsp90α isoform for maturation and trafficking.

Heat shock protein 90 (Hsp90) has emerged as a promising therapeutic target for the treatment of cancer. Several Hsp90 inhibitors have entered clinical trials. However, some toxicological detriments have arisen, such as cardiotoxicity resulting from hERG inhibition following the administration of Hsp90 inhibitors.

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 (PC3 mm2, A549, and HT29) cell lines. Non-noviosylated coumermycin A1 analogues that manifest potent antiproliferative 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 antiproliferative activities as compared to coumermycin A1, producing small molecule Hsp90 inhibitors that exhibit nanomolar activities.

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.

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.

To fold or not to fold: modulation and consequences of Hsp90 inhibition.

Background: The 90-kDa heat-shock proteins (Hsp90) have rapidly evolved into promising therapeutic targets for the treatment of several diseases, including cancer and neurodegenerative diseases. Hsp90 is a molecular chaperone that aids in the conformational maturation of nascent polypeptides, as well as the rematuration of denatured proteins.

Discussion: Many of the Hsp90-dependent client proteins are associated with cellular growth and survival and, consequently, inhibition of Hsp90 represents a promising approach for the treatment of cancer.

Synthesis and evaluation of electron-rich curcumin analogues.

The natural product curcumin has long been recognized for its medicinal properties and is utilized for the treatment of many diseases. However, it remains unknown whether this activity is based on its presumably promiscuous scaffold, or if it results from the Michael acceptor properties of the alpha,beta-unsaturated 1,3-diketone moiety central to its structure. To probe this issue, electron-rich pyrazole and isoxazole analogues were prepared and evaluated against two breast cancer cell lines, which resulted in the identification of several compounds that exhibit low micromolar to mid nanomolar anti-proliferative activity.