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. Probes can be designed for either serine/threonine or tyrosine kinases, and analysis can be performed using standard fluorescence equipment. The procedures provided herein represent our optimized protocols for the design, validation, and application of CSox-based protein kinase activity sensors.

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. Among these, the mitogen activated protein (MAP) kinases are recognized to be central players in key cellular signaling events and are associated with essential processes including growth, proliferation, differentiation, migration, and apoptosis. The major challenge with MAP kinase sensor development is achieving high selectivity since these kinases rely acutely on secondary interactions distal to the phosphorylation site to impart substrate specificity. Herein we describe the development and application of selective sensors for three MAP kinase subfamilies, ERK1/2, p38α/ß, and JNK1/2/3. The new sensors are based on a modular design, which includes a sensing element that exploits a sulfonamido-oxine (Sox) fluorophore for reporting phosphorylation, a recognition and specificity element based on reported docking domain motifs and a variable linker, which can be engineered to optimize the intermodule distance and relative orientation. Following rigorous validation, the capabilities of the new sensors are exemplified through the quantitative analysis of the target MAP kinases in breast cancer progression in a cell culture model, which reveals a strong correlation between p38α/ß activity and increased tumorgenicity.

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. Herein, we report that the natural product gedunin may provide a new alternative to inactivate the Hsp90 machine. We show that gedunin directly binds to p23 and inactivates it, without overexpression of Hsp27 and relatively modest induction of Hsp70. Using molecular docking and mutational analysis, we mapped the gedunin-binding site on p23. Functional analysis shows that gedunin inhibits the p23 chaperoning activity, blocks its cellular interaction with Hsp90, and interferes with p23-mediated gene regulation. Cell treatment with gedunin leads to cancer cell death by apoptosis through inactivation of p23 and activation of caspase 7, which cleaves p23 at the C terminus. These results provide important insight into the molecular mechanism of action of this promising lead compound.

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. Consistent with these results, a meta-3-fluorophenyl substituted novologue (13b) exhibited a 14-fold lower ED(50) for protection against glucose-induced toxicity of primary sensory neurons compared to 2.

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. Using a structure-based approach, we designed an inhibitor of Grp94, the ER-resident Hsp90. The effect manifested by compound 2 on several Grp94 and Hsp90α/ß (cytosolic isoforms) clients were investigated. Compound 2 prevented intracellular trafficking of the Toll receptor, inhibited the secretion of IGF-II, affected the conformation of Grp94, and suppressed Drosophila larval growth, all Grp94-dependent processes. In contrast, compound 2 had no effect on cell viability or cytosolic Hsp90α/ß client proteins at similar concentrations. The design, synthesis, and evaluation of 2 are described herein.

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. We sought to investigate this toxicity as hERG has been previously reported as a client protein that depends upon Hsp90 for its maturation and functional trafficking. In this study we show that hERG depends upon a single Hsp90 isoform. hERG preferentially co-immunoprecipitated with Hsp90α, and genetic knockdown of Hsp90α, but not Hsp90ß, resulted in a trafficking-defective hERG channel. This study demonstrates the importance of delineating the isoform dependence of Hsp90 client proteins and provides rationale for the design of isoform-selective Hsp90 inhibitors that avoid detrimental effects.

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. Western blot analysis confirmed that the resulting 3-arylcoumarin hybrids target the Hsp90 protein folding machinery.

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. 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.

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 analyses, supporting a common mode of Hsp90 inhibition for both structural classes.

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. A conjugate addition study was also performed to compare the relative electrophilicity of the diketone, pyrazole and isoxazole analogues.

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. Conversely, stimulation of heat-shock protein levels has potential therapeutic applications for the treatment of neurodegenerative diseases that result from misfolded and aggregated proteins. CONCLUSION: Hsp90 modulation exhibits the potential to treat unrelated disease states, from cancer to neurodegenerative diseases, and, thus, to fold or not to fold, becomes a question of great value.