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Yehuda-Shnaidman E.,Hamner Institutes for Health Sciences | Buehrer B.,Zen-Bio, Inc. | Pi J.,Hamner Institutes for Health Sciences | Kumar N.,Scripps Research Institute | And 2 more authors.
Diabetes | Year: 2010

OBJECTIVE - We examined the effect of β-adrenergic receptor (βAR) activation and cAMP-elevating agents on respiration and mitochondrial uncoupling in human adipocytes and probed the underlying molecular mechanisms. RESEARCH DESIGN AND METHODS - Oxygen consumption rate (OCR, aerobic respiration) and extracellular acidification rate (ECAR, anaerobic respiration) were examined in response to isoproterenol (ISO), forskolin (FSK), and dibutyryl-cAMP (DB), coupled with measurements of mitochondrial depolarization, lipolysis, kinase activities, and gene targeting or knockdown approaches. RESULTS - ISO, FSK, or DB rapidly increased oxidative and glycolytic respiration together with mitochondrial depolarization in human and mouse white adipocytes. The increase in OCR was oligomycin-insensitive and contingent on cAMP-dependent protein kinase A (PKA)-induced lipolysis. This increased respiration and the uncoupling were blocked by inhibiting the mitochondrial permeability transition pore (PTP) and its regulator, BAX. Interestingly, compared with lean individuals, adipocytes from obese subjects exhibited reduced OCR and uncoupling capacity in response to ISO. CONCLUSIONS - Lipolysis stimulated by βAR activation or other maneuvers that increase cAMP levels in white adipocytes acutely induces mitochondrial uncoupling and cellular energetics, which are amplified in the absence of scavenging BSA. The increase in OCR is dependent on PKA-induced lipolysis and is mediated by the PTP and BAX. Because this effect is reduced with obesity, further exploration of this uncoupling mechanism will be needed to determine its cause and consequences. © 2010 by the American Diabetes Association. Source


Grant
Agency: Department of Health and Human Services | Branch: | Program: STTR | Phase: Phase I | Award Amount: 289.93K | Year: 2013

DESCRIPTION (provided by applicant): Characterization of human tumor samples and cell lines in combination with inhibitor studies in animal models has established a central role for the Hh pathway in a vast array of cancer types, including small-cell lung, pancreatic, oesophageal, prostate, breast, colon, liver and ovarian cancers. Hh signaling is now implicated in approximately 20-25% of all cancers. GLI1 is the downstream effector in the Hh signaling pathway, and has emerged as a valid therapeutic target. It has been suggested that due to the complexity of signaling inputs in the Hh pathway, targeting GLI1 may provide a more comprehensive strategy for treating both canonical and noncanonical Hh-pathway dependent cancers. This is especially true in light of the acquired resistance to inhibitors of SMO in patients. Screens utilizing GLI1-dependent transcriptional reporter cell-based assays have yielded inhibitors targeting the Hh pathway downstream of SMO including small molecule and natural product compounds that act by targeting GLI1 through differing mechanisms of action. Targeting at the level of GLI1 with small molecules has been effective in a number of cancer model systems including colon, CLL and breast cancer. However, these compounds have only micromolar potencies in vitro, and a subset (GANT61 and ATO) has shown only modest efficacy in in vivo cancer models. These studies suggest that GLI1 is pharmacologically targetable, and that the use of GLI1 inhibitors is a valid and promising approach for targeting GLI1-dependent cancers. Unfortunately, current GLI1 inhibitors are hampered by low potency and a lack of in vivo efficacy and hence are not viable clinical candidates. There are no GLI1-targeted inhibitors in the clinic. Using a novel high throughput, high content cell-based imaging platform, we will screen a diverse collection of small molecules to identify potent inhibitors of GLI1 (Aim 1). Compounds that show inhibitory activity will be further validated in a focused panel of cell-based assays which will incorporate cells derived from primary human breast tumors. These assays will also examine selectivity of the compounds as well as their effects on motility/invasiveness (Aim 2). Promising compounds discovered in Aims 1 and 2 will be subjected to extensive structure-activity relationship to optimize the novel GLI1 inhibitors (Aim 3). PUBLIC HEALTH RELEVANCE PUBLIC HEALTH RELEVANCE: A central role for the Hedgehog (Hh) pathway has been established in a vast array of cancer types and it hasbeen implicated in approximately 20-25% of all cancers. GLI1 is a downstream mediator of Hh signaling, making GLI1 a very attractive therapeutic target. A limited number of small molecule inhibitors of GLI1 have been identified; however none of them are clinically viable. In this application we describe a highly innovative cell-based drug screen that will be applied to identifying novel, potent and selective inhibitors o GLI1 as potential cancer therapeutics.


Basu J.,Tengion | Ludlow J.W.,Zen-Bio, Inc.
Regenerative Medicine | Year: 2014

Potency is a critical quality attribute of biological products, defined by the US FDA as the specific ability or capacity of the product, as indicated by appropriate laboratory tests or by adequately controlled clinical data obtained through the administration of the product in the manner intended, to effect a given result. Ideally, a potency assay will leverage the product's mechanism of action. Alternatively, the assay may focus on a therapeutically relevant biological activity. The absence of rigorous mechanistic data for the majority of cell-based therapeutics currently in the process research pipeline has impeded efforts to design and validate indices of product potency. Development of a systematic battery of parallel functional assays that, taken together, can address all potential mechanisms of action believed to be relevant for the product platform is recommended. Such an approach is especially important during preclinical development. Here, we summarize the principal and unique challenges facing the development of functionally relevant and rigorous potency assays for cell-based therapeutics. We present perspectives regarding potency assay development for these products as illustrated by our experiences in process R&D of cryopreserved hepatocytes (Incara Pharmaceuticals) and selected renal cells (Tengion). © 2014 Future Medicine Ltd. Source


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.32K | Year: 2015

DESCRIPTION provided by applicant The activation and recruitment of brown adipose tissue has become an exciting target in the fight against obesity and its related metabolic diseases Brown adipose tissue differs from white adipose tissue in its critical ability to burn energy as heat through UCP driven adaptive thermogenesis Expression of mitochondrial UCP uncouples oxidative phosphorylation resulting in accelerated substrate oxidation but low ATP production The resulting dissipation of energy is associated with plasma triglyceride clearance a key factor in weight loss The brown adipose depot has long been known to exist in rodents as well as in human infants However recent evidence has demonstrated the existence of active brown adipose tissue in human adults These findings prompted a resurgence of research focusing on both the activation of brown adipose tissue and the andquot browningandquot of white adipose tissue a phenomenon wherein UCP mediated mitochondrial uncoupling is induced by stimuli Studies using rodent models have demonstrated that brown fat activation or induction of browning can promote beneficial metabolic effects and promising therapeutic targets have been identified However the therapeutic potential of these targets has not been confirmed due to the lack of a widely available human brown adipocyte model Therefore there is a pronounced need to validate existing therapeutic targets in the human system ZenBio will address this need by providing both a primary and immortalized human brown adipocyte cell culture system to the research community and will utilize these tools to establish a screen to identify activators of human brown adipogenesis The principal goal of this project is to generate and characterize a human adult brown adipocyte cell system We will achieve this in a stepwise fashion beginning with an initial feasibility study focused on human fetal brown adipocytes Aim I will use functional and genomic analyses to generate a widely available fetal brown adipocyte model which will be used as a tool to establish the adult cell system The fetal model will be developed first due to the comparative ease of detecting fetal brown adipose tissue and its ready availability through existing procurement agencies The second aim is to generate and characterize an immortalized fetal brown adipocyte system for high throughput high content screening These cells will circumvent the limited material and lifespan of primary cells and allow for the development of a high throughput screening platform to identify novel targets of brown adipocyte activation Phase I will be expanded in Phase II with the goal of characterizing and commercializing the human adult brown adipocyte system from donors of differing BMI age gender and diabetic state These studies will generate both primary and immortalized human adult adipocyte cells which will be used to identify novel targets of brown adipocyte energy expenditure for the treatment of obesity PUBLIC HEALTH RELEVANCE The worldwide prevalence of obesity and its related diseases is at epidemic proportions necessitating novel therapeutic approaches The recent identification of human adult brown fat has led researchers to investigate new ways to increase this beneficial adipose tissue to deter obesity related diseases We will generate human brown adipocyte systems that can be used to discover and validate novel therapeutic modalities


Grant
Agency: Department of Health and Human Services | Branch: National Institutes of Health | Program: SBIR | Phase: Phase I | Award Amount: 224.90K | Year: 2015

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