High-throughput assay for the identification of Hsp90 inhibitors based on Hsp90-dependent refolding of firefly luciferase

Galam, Lakshmi; Hadden, M. Kyle; Ma, Zeqiang; Ye, Qi Zhuang; Yun, Bo Geon; Blagg, Brian S. J.; Matts, Robert L.

doi:10.1016/j.bmc.2007.01.004

Cited by 77 publications

(84 citation statements)

References 44 publications

(20 reference statements)

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“…In the past, RRL systems have been widely used to obtain mechanistic details about the role of various chaperones in refolding of firefly luciferase (Nimmesgern and Hartl, 1993;Schumacher et al, 1996) and to identify small molecules that modify protein folding (Cassel et al, 2012;Galam et al, 2007;Wisen and Gestwicki, 2008). We suggest that using a RRL based Fluc assay, it should be possible in the future to obtain detailed information about the interactions of various chaperones during the folding of differentially stabilized Fluc proteins.…”

Section: Vi2 Assessment Of Folding Capacity Of Cells Using Fluc-basmentioning

confidence: 99%

Firefly luciferase mutants as sensors of proteome stress

et al. 2011

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Section: Vi2 Assessment Of Folding Capacity Of Cells Using Fluc-basmentioning

confidence: 99%

Firefly luciferase mutants as sensors of proteome stress

et al. 2011

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“…In contrast to traditional cancer therapuetics directed proteins, resulting in improperly folded proteins that are targeted for the ubiquitin-proteasomal degradation pathway 9 . The ansamycin antibiotic novobiocin has been demonstrated to bind to the C-terminal site of the Hsp90 molecular chaperone 10,11 . Clinically, novobiocin has been used for its antimicrobial activity with acceptable toxicity and bioavailability.…”

Section: Introductionmentioning

confidence: 99%

“…Clinically, novobiocin has been used for its antimicrobial activity with acceptable toxicity and bioavailability. Unfortunately, it exhibits low Hsp90 affinity with an IC 50 of ~400 µM and would require high concentrations for maximal effects 10,11 . Thus, we hypothesize novobiocin analogues with improved affinity for Hsp90 may represent effective therapy for prostate cancer.…”

Section: Introductionmentioning

confidence: 99%

Development of the C-Terminal Inhibitors of Heat Shock Protein 90 in the Treatment of Prostate Cancer

Holzbeierlein¹

2008

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Public reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing this collection of information. Send comments regarding this burden estimate or any other aspect of this collection of information, including suggestions for reducing this burden to Department of Defense, Washington Headquarters Services, Directorate for Information Operations and Reports (0704-0188), 1215 Jefferson Davis Highway, Suite 1204, Arlington, VA 22202-4302. Respondents should be aware that notwithstanding any other provision of law, no person shall be subject to any penalty for failing to comply with a collection of information if it does not display a currently valid OMB control number. PLEASE DO NOT RETURN YOUR FORM TO THE ABOVE ADDRESS. These results demonstrate an effect of KU-1 at approximately the 10uM range. This is significantly more potent than its parent analogue Novobiocin. Similar results were then obtained when KU-1 was applied to LAPC-4 and PC-3 (graph not shown). An MTT assay was then conducted on LNCaP cell using KU-1 (also called A-4) at various concentrations and for different time periods. Results are seen in Figure These results show that KU-1 does not appear toxic to LNCaP cells even at concentrations of 100uM. Interestingly, this has applications for other disease states such as Alzheimers but toxicity through inhibition is something desirable for cancer studies. Therefore, we believe that this compound has the ability to inhibit Hsp90 at relatively lower concentrations but in and of itself is not toxic to cancer cells. This research was completed in the stated time period of 4 months. REPORT DATEBecause of the time, work and expense of the synthesis of this compound and its analogues it was decided that more potent analogues of KU-1 would be developed prior to their testing in an in vivo model. This is the aim of Task 3 and will be described in detail.The compound which was initially selected based on its MTT assay results and Western blot results was KU-36 (F-4 in manuscript). A manuscript which has been previously submitted to Cancer Research but was not accepted, and has now been revised and will be sent to the Journal of Urology is attached which details the results of the in vitro studies with KU-36. Appendix 1. Specific Aim #2:To test the effectiveness of KU-1 in an LNCaP nude mouse xenograft model and determine the toxicity of the compound.As mentioned previously due to the expense and time commitment required to scale up these compounds to sufficient quantities to be used in an in vivo model, we chose not to test KU-1 in vivo as we had identified KU-36 as having increased potency in vitro. Furthermore, we chose to evaluate this compound in an orthotopic mouse model using PC-3 cells which more closely resembles the human condition of hormone refractory prostate cancer, a condition where a compound of this nature would ...

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“…24 Rabbit reticulocyte lysate (1:2, lysis of one volume of packed cells in two volumes of deionized water) was purchased from Green Hectares (Oregon, WI). Firefly luciferase (L-9506), luciferin, molecular biology grade acetylated bovine serum albumin, ATP, Coenzyme A, and novobiocin were purchased from Sigma-Aldrich.…”

Section: Rabbit Reticulocyte Lysate Luciferase Refolding Assaymentioning

confidence: 99%

“…[21][22][23] Assays based on rabbit reticulocyte lysates have been successfully used to biochemically characterize the refolding kinetics of the Hsp70/Hsp90 system as well as a screening tool to identify compounds that inhibit Hsp90 activity. 24 While the rabbit reticulocyte assay is quite sensitive and robust, questions remain as to the physiological relevance of the active chaperone complexes in this system, as it represents a species more related to normal tissue rather than disease. Over the last decade, there has been considerable effort put forth to develop specific Hsp90 inhibitors toward various cancers.…”

Section: Introductionmentioning

confidence: 99%

Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors

Sadikot

Swink

Eskew

et al. 2013

ASSAY and Drug Development Technologies

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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.

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High-throughput assay for the identification of Hsp90 inhibitors based on Hsp90-dependent refolding of firefly luciferase

Cited by 77 publications

References 44 publications

Firefly luciferase mutants as sensors of proteome stress

Firefly luciferase mutants as sensors of proteome stress

Development of the C-Terminal Inhibitors of Heat Shock Protein 90 in the Treatment of Prostate Cancer

Development of a High-Throughput Screening Cancer Cell-Based Luciferase Refolding Assay for Identifying Hsp90 Inhibitors

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