ABSTRACT
The 90 kDa heat-shock protein (Hsp90) and other cochaperones allow for proper folding of nascent or misfolded polypeptides. Cancer cells exploit these chaperones by maintaining the stability of mutated and misfolded oncoproteins and allowing them to evade proteosomal degradation. Inhibiting Hsp90 is an attractive strategy for cancer therapy, as the concomitant degradation of multiple oncoproteins may lead to effective anti-neoplastic agents. Unfortunately, early clinical trials have been disappointing with N-terminal Hsp90 inhibitors, as it is unclear whether the problems that plague current Hsp90 inhibitors in clinical trials are related to on-target or off-target activity. One approach to overcome these pitfalls is to identify structurally diverse scaffolds that improve Hsp90 inhibitory activity in the cancer cell milieu. Utilizing a panel of cancer cell lines that express luciferase, we have designed an in-cell Hsp90-dependent luciferase refolding assay. The assay was optimized using previously identified Hsp90 inhibitors and experimental novobiocin analogues against prostate, colon, and lung cancer cell lines. This assay exhibits good interplate precision (% CV), a signal-to-noise ratio (S/N) of ≥7, and an approximate Z-factor ranging from 0.5 to 0.7. Novobiocin analogues that revealed activity in this assay were examined via western blot experiments for client protein degradation, a hallmark of Hsp90 inhibition. Subsequently, a pilot screen was conducted using the Prestwick library, and two compounds, biperiden and ethoxyquin, revealed significant activity. Here, we report the development of an in-cell Hsp90-dependent luciferase refolding assay that is amenable across cancer cell lines for the screening of inhibitors in their specific milieu.
Subject(s)
Antineoplastic Agents/pharmacology , HSP90 Heat-Shock Proteins/antagonists & inhibitors , High-Throughput Screening Assays/methods , Luciferases/chemistry , Protein Refolding/drug effects , Animals , Biological Assay , Blotting, Western , Cell Line, Tumor , Coloring Agents , Dose-Response Relationship, Drug , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/pharmacology , Humans , Luciferases/antagonists & inhibitors , Novobiocin/analogs & derivatives , Novobiocin/pharmacology , Protein Denaturation , Rabbits , Reticulocytes/drug effects , Reticulocytes/metabolism , Rhodamines , Small Molecule LibrariesABSTRACT
BACKGROUND: The molecular chaperone, heat shock protein 90 (Hsp90) has been shown to be overexpressed in a number of cancers, including prostate cancer, making it an important target for drug discovery. Unfortunately, results with N-terminal inhibitors from initial clinical trials have been disappointing, as toxicity and resistance resulting from induction of the heat shock response (HSR) has led to both scheduling and administration concerns. Therefore, Hsp90 inhibitors that do not induce the heat shock response represent a promising new direction for the treatment of prostate cancer. Herein, the development of a C-terminal Hsp90 inhibitor, KU174, is described, which demonstrates anti-cancer activity in prostate cancer cells in the absence of a HSR and describe a novel approach to characterize Hsp90 inhibition in cancer cells. METHODS: PC3-MM2 and LNCaP-LN3 cells were used in both direct and indirect in vitro Hsp90 inhibition assays (DARTS, Surface Plasmon Resonance, co-immunoprecipitation, luciferase, Western blot, anti-proliferative, cytotoxicity and size exclusion chromatography) to characterize the effects of KU174 in prostate cancer cells. Pilot in vivo efficacy studies were also conducted with KU174 in PC3-MM2 xenograft studies. RESULTS: KU174 exhibits robust anti-proliferative and cytotoxic activity along with client protein degradation and disruption of Hsp90 native complexes without induction of a HSR. Furthermore, KU174 demonstrates direct binding to the Hsp90 protein and Hsp90 complexes in cancer cells. In addition, in pilot in-vivo proof-of-concept studies KU174 demonstrates efficacy at 75 mg/kg in a PC3-MM2 rat tumor model. CONCLUSIONS: Overall, these findings suggest C-terminal Hsp90 inhibitors have potential as therapeutic agents for the treatment of prostate cancer.
Subject(s)
Antineoplastic Agents/therapeutic use , HSP90 Heat-Shock Proteins/antagonists & inhibitors , Neoplasm Proteins/antagonists & inhibitors , Prostatic Neoplasms/drug therapy , Prostatic Neoplasms/metabolism , Animals , Blotting, Western , Cell Line, Tumor , Cell Proliferation/drug effects , Growth Inhibitors/pharmacology , HSP90 Heat-Shock Proteins/metabolism , Humans , Male , Neoplasm Proteins/metabolism , Novobiocin/pharmacology , Protein Binding/drug effects , RatsABSTRACT
Over the last half century, major theoretical and experimental advances have been made in understanding the molecular architecture (e.g., sarcomeric organization) and biophysics (e.g., excitation-contraction coupling) of striated muscle. Studies of how the contractile apparatus is assembled have a shorter history, but our understanding has deepened considerably over the last decade. This review focuses on spontaneous intracellular calcium (Ca2+) signals and their role in skeletal muscle myofibrillogenesis. In embryonic skeletal muscle, several classes of spontaneous Ca2+ signal occur both in vivo and in culture, and blocking their production prevents de novo sarcomere assembly. This review includes a brief overview of myofibrillogenesis, discussion of spontaneous Ca2+ signals produced in embryonic skeletal muscle, the Xenopus model system, the role of Ca2+ signals in regulating assembly of the three major filament systems (actin, titin, and myosin), integration of physiological and biochemical approaches to the problem, and the clinical relevance of basic research in this area. Interspersed throughout are suggestions for future directions and citations for reviews in closely related areas not covered herein.
