Regulation of IκBα expression involves both NF-κB and the MAP kinase signaling pathways

IκBα is an inhibitor of the nuclear transcription factor NF-κB. Binding of IκBα to NF-κB inactivates the transcriptional activity of NF-κB. Expression of IκBα itself is regulated by NF-κB, which provides auto-regulation of this signaling pathway. Here we present a mouse model for monitoring in vivo IκBα expression by imaging IκBα-luc transgenic mice for IκBα promoter driven luciferase activity. We demonstrated a rapid and systemic induction of IκBα expression in the transgenic mice following treatment with LPS. The induction was high in liver, spleen, lung and intestine and lower in the kidney, heart and brain. The luciferase induction in the liver correlated with increased IκBα mRNA level. Pre-treatment with proteasome inhibitor bortezomib dramatically suppressed LPS-induced luciferase activity. The p38 kinase inhibitor SB203580 also showed moderate inhibition of LPS-induced luciferase activity. Analysis of IκBα mRNA in the liver tissue showed a surprising increase of the IκBα mRNA after bortezomib and SB203580 treatments, which could be due to increased IκBα mRNA stability. Our data demonstrate that regulation of IκBα expression involves both the NF-κB and the p38 signaling pathways. The IκBα-luc transgenic mice are useful for analyzing IκBα expression and the NF-κB transcriptional activity in vivo

Pharmacodynamic Assays to Facilitate Preclinical and Clinical Development of Pre-mRNA Splicing Modulatory Drug Candidates

The spliceosome has recently emerged as a new target for cancer chemotherapy and novel antitumor spliceosome targeted agents are under development. Here, we describe two types of novel pharmacodynamic assays that facilitate drug discovery and development of this intriguing class of innovative therapeutics; the first assay is useful for preclinical optimization of small-molecule agents that target the SF3B1 spliceosomal protein in animals, the second assay is an ex vivo validated, gel-based assay for the measurement of drug exposure in human leukocytes. The first assay utilizes a highly specific bioluminescent splicing reporter, based on the skipping of exons 4-11 of a Luc-MDM2 construct, which specifically yields active luciferase when treated with small-molecule spliceosome modulators. We demonstrate that this reporter can be used to monitor alternative splicing in whole cells in vitro. We describe here that cell lines carrying the reporter can be used in vivo for the efficient pharmacodynamic analysis of agents during drug optimization and development. We also demonstrate dose- and time-dependent on-target activity of sudemycin D6 (SD6), which leads to dramatic tumor regression. The second assay relies on the treatment of freshly drawn human blood with SD6 ex vivo treatment. Changes in alternative splicing are determined by RT-PCR using genes previously identified in in vitro experiments. The Luc-MDM2 alternative splicing bioluminescent reporter and the splicing changes observed in human leukocytes should allow for the more facile translation of novel splicing modulators into clinical application

The metal-responsive transcription factor-1 protein is elevated in human tumors

We previously identified metal-responsive transcription factor-1 (MTF-1) as a positive contributor to mouse fibrosarcoma growth through effects on cell survival, proliferation, tumor angiogenesis and extracellular matrix remodeling. In the present study, we investigated MTF-1 protein expression in human tissues by specific immunostaining of both normal and tumor tissue samples. Immunohistochemical (IHC) staining of a human tissue microarray (TMA), using a unique anti-human MTF-1 antibody, indicated constitutive MTF-1 expression in most normal tissues, with liver and testis displaying comparatively high levels of expression. Nevertheless, MTF-1 protein levels were found to be significantly elevated in diverse human tumor types, including breast, lung and cervical carcinomas. IHC analysis of a separate panel of full-size tissue sections of human breast cancers, including tumor and normal adjacent, surrounding tissue, confirmed and extended the results of the TMA analysis. Taken with our previous findings, this new study suggests a role for MTF-1 in human tumor development, growth or spread. Moreover, the study suggests that MTF-1 could be a novel therapeutic target that offers the opportunity to manipulate metal or redox homeostasis in tumor cells

Structural insights into the function of the catalytically active human Taspase1

19 pags., 7 figs., 2 tabs.Taspase1 is an Ntn-hydrolase overexpressed in primary human cancers, coordinating cancer cell proliferation, invasion, and metastasis. Loss of Taspase1 activity disrupts proliferation of human cancer cells in vitro and in mouse models of glioblastoma. Taspase1 is synthesized as an inactive proenzyme, becoming active upon intramolecular cleavage. The activation process changes the conformation of a long fragment at the C-terminus of the α subunit, for which no full-length structural information exists and whose function is poorly understood. We present a cloning strategy to generate a circularly permuted form of Taspase1 to determine the crystallographic structure of active Taspase1. We discovered that this region forms a long helix and is indispensable for the catalytic activity of Taspase1. Our study highlights the importance of this element for the enzymatic activity of Ntn-hydrolases, suggesting that it could be a potential target for the design of inhibitors with potential to be developed into anticancer therapeutics.This project has been funded in whole with Federal funds from the National Cancer Institute (NCI), National Institutes of Health (NIH), under Chemical Biology Consortium contract no. HHSN261200800001E

Quantitative Comparison of the Sensitivity of Detection of Fluorescent and Bioluminescent Reporters in Animal Models

Bioluminescent and fluorescent reporters are finding increased use in optical molecular imaging in small animals. In the work presented here, issues related to the sensitivity of in vivo detection are examined for standard reporters. A high-sensitivity imaging system that can detect steady-state emission from both bioluminescent and fluorescent reporters is described. The instrument is absolutely calibrated so that animal images can be analyzed in physical units of radiance allowing more quantitative comparisons to be performed. Background emission from mouse tissue, called autoluminescence and autofluorescence, is measured and found to be an important limitation to detection sensitivity of reporters. Measurements of dual-labeled (bioluminescent/fluorescent) reporter systems, including PC-3M-luc/DsRed2-1 and HeLa-luc/PKH26, are shown. The results indicate that although fluorescent signals are generally brighter than bioluminescent signals, the very low autoluminescent levels usually results in superior signal to background ratios for bioluminescent imaging, particularly compared with fluorescent imaging in the green to red part of the spectrum. Fluorescence detection sensitivity improves in the far-red to near-infrared, provided the animals are fed a low-chlorophyll diet to reduce autofluorescence in the intestinal region. The use of blue-shifted excitation filters is explored as a method to subtract out tissue autofluorescence and improve the sensitivity of fluorescent imaging

Development of functional assays for p97/VCP inhibition

p97 (also called VCP in metazoans and CDC48 in yeast) is a highly conserved, ubiquitously expressed, and essential AAA ATPase. p97 plays a key role in endoplasmic reticulum-associated degradation of misfolded secretory and membrane proteins as well as ubiquitin-dependent turnover of a subset of cytoplasmic substrates of the ubiquitin-proteasome system

An exon skipping screen identifies antitumor drugs that are potent modulators of pre-mRNA splicing, suggesting new therapeutic applications.

Agents that modulate pre-mRNA splicing are of interest in multiple therapeutic areas, including cancer. We report our recent screening results with the application of a cell-based Triple Exon Skipping Luciferase Reporter (TESLR) using a library that is composed of FDA approved drugs, clinical compounds, and mechanistically characterized tool compounds. Confirmatory assays showed that three clinical antitumor therapeutic candidates (milciclib, PF-3758309 and PF-562271) are potent splicing modulators and that these drugs are, in fact, nanomolar inhibitors of multiple kinases involved in the regulation the spliceosome. We also report the identification of new SF3B1 antagonists (sudemycinol C and E) and show that these antagonists can be used to develop a displacement assay for SF3B1 small molecule ligands. These results further support the broad potential for the development of agents that target the spliceosome for the treatment of cancer and other diseases, as well as new avenues for the discovery of new chemotherapeutic agents for a range of diseases

A Mega-High-Throughput Screening Platform for the Discovery of Biologically Relevant Sequence-Defined Non-Natural Polymers

Combinatorial methods enable the synthesis of chemical libraries on scales of millions to billions of compounds, but the ability to efficiently screen and sequence such large libraries has remained a major bottleneck for molecular discovery. We developed a novel technology for screening and sequencing libraries of synthetic molecules of up to a billion compounds in size. This platform utilizes the Fiber-optic Array Scanning Technology (FAST) to screen bead-based libraries of synthetic compounds at a rate of 5 million compounds per minute (~83,000 Hz). This ultra-high-throughput screening platform has been used to screen libraries of synthetic “self-readable” non-natural polymers that can be sequenced at femtomole scale by chemical fragmentation and high-resolution mass spectrometry. The versatility and throughput of the platform was demonstrated by screening two libraries of non-natural polyamide polymers with sizes of 1.77M and 1B compounds against the protein targets K-Ras, asialoglycoprotein receptor 1 (ASGPR), IL-6, IL 6 receptor (IL-6R) and TNFα. Hits with low nanomolar binding affinities were found against all targets, including competitive inhibitors of K-Ras binding to Raf and functionally active uptake ligands for ASGPR facilitating intracellular delivery of a non-glycan ligand