Measurement of Receptor Signaling Bias.
Authors: Kenakin T
Abstract
G protein-coupled receptors (GPCRs) are often pleiotropically linked to numerous cellular signaling mechanisms in cells, and it is now known that many agonists differentially activate some signaling pathways at the expense of others. The mechanism for this effect is the stabilization of different active receptor states by different agonists, and it leads to varying qualities of efficacy for different agonists. Agonist bias is a powerful mechanism to amplify beneficial signals and diminish harmful signals, and thus improve the overall profile of agonist ligands. This unit describ...
Source: Current Protocols in Pharmacology - September 18, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
A Mouse Model of Furosemide-Induced Overactive Bladder.
Authors: Saporito MS, Zuvich E, DiCamillo A
Abstract
Detailed in this unit is a mouse model of overactive bladder and urinary incontinence based on diuretic stress-induced urination. The procedure involves the use of a unique, highly sensitive, and automated urine capturing method to measure urinary latency, frequency, and void volume. Although this method was first described and validated using an anti-muscarinic drug used for treating overactive bladder, subsequent work has shown that effective non-cholinergic agents can be detected. These findings indicate good predictive value for this model regarding ...
Source: Current Protocols in Pharmacology - September 18, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
In Vitro Drug Metabolism Using Liver Microsomes.
Authors: Knights KM, Stresser DM, Miners JO, Crespi CL
Abstract
Knowledge of the metabolic stability of newly discovered drug candidates eliminated by metabolism is essential for predicting the pharmacokinetic (PK) parameters that underpin dosing and dosage frequency. Further, characterization of the enzyme(s) responsible for metabolism (reaction phenotyping) allows prediction, at least at the qualitative level, of factors (including metabolic drug-drug interactions) likely to alter the clearance of both new chemical entities (NCEs) and established drugs. Microsomes are typically used as the enzyme source ...
Source: Current Protocols in Pharmacology - September 18, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Overview of Critical Parameters for the Design and Execution of a High-Throughput Screen for Allosteric Ligands.
Authors: Alt A
Abstract
Allosteric ligands modulate the activity of receptor targets by binding to sites that are distinct from the orthosteric (native agonist) binding site. Allosteric modulators have potential therapeutic advantages over orthosteric agonists and antagonists, including improved selectivity, and maintenance of the spatial and temporal fidelity of native signaling patterns. The identification of allosteric ligands presents unique challenges because of the requirement for screening in the presence of an orthosteric agonist, the small signal window that is produced by many allosteric modulato...
Source: Current Protocols in Pharmacology - September 18, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Mammalian Cell Tissue Culture Techniques.
Authors: Phelan K, May KM
Abstract
Cultured tissues and cells are used extensively in physiological and pharmacological studies. In vitro cultures provide a means of examining cells and tissues without the complex interactions that would be present if the whole organism were studied. A number of special skills are required in order to preserve the structure, function, behavior, and biology of cells in culture. This unit describes the basic skills required to maintain and preserve cell cultures: maintaining aseptic technique, preparing media with the appropriate characteristics, passaging, freezing and stor...
Source: Current Protocols in Pharmacology - June 3, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Genetically Engineered Mouse Models of Pancreatic Cancer: The KPC Model (LSL-Kras(G12D/+) ;LSL-Trp53(R172H/+) ;Pdx-1-Cre), Its Variants, and Their Application in Immuno-oncology Drug Discovery.
Authors: Lee JW, Komar CA, Bengsch F, Graham K, Beatty GL
Abstract
Pancreatic ductal adenocarcinoma (PDAC) ranks fourth among cancer-related deaths in the United States. For patients with unresectable disease, treatment options are limited and lack curative potential. Preclinical mouse models of PDAC that recapitulate the biology of human pancreatic cancer offer an opportunity for the rational development of novel treatment approaches that may improve patient outcomes. With the recent success of immunotherapy for subsets of patients with solid malignancies, interest is mounting in the possible use of immun...
Source: Current Protocols in Pharmacology - June 3, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Assays for Determination of Protein Concentration.
Authors: Olson BJ
Abstract
Biochemical analysis of proteins relies on accurate quantification of protein concentration. Detailed in this appendix are some commonly used methods for protein analysis, e.g., Lowry, Bradford, bicinchoninic acid (BCA), UV spectroscopic, and 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde (CBQCA) assays. The primary focus of this report is assay selection, emphasizing sample and buffer compatibility. The fundamentals of generating protein assay standard curves and of data processing are considered, as are high-throughput adaptations of the more commonly used protein assays. Also ...
Source: Current Protocols in Pharmacology - June 3, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Overview of Transgenic Glioblastoma and Oligoastrocytoma CNS Models and Their Utility in Drug Discovery.
Authors: Chen F, Becker A, LoTurco J
Abstract
Many animal models have been developed to investigate the sources of central nervous system (CNS) tumor heterogeneity. Reviewed in this unit is a recently developed CNS tumor model using the piggyBac transposon system delivered by in utero electroporation, in which sources of tumor heterogeneity can be conveniently studied. Their applications for studying CNS tumors and drug discovery are also reviewed. © 2016 by John Wiley & Sons, Inc.
PMID: 26995546 [PubMed - as supplied by publisher] (Source: Current Protocols in Pharmacology)
Source: Current Protocols in Pharmacology - March 21, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Overview of Genetically Engineered Mouse Models of Distinct Breast Cancer Subtypes.
Authors: Usary J, Darr DB, Pfefferle AD, Perou CM
Abstract
Advances in the screening of new therapeutic options have significantly reduced the breast cancer death rate over the last decade. Despite these advances, breast cancer remains the second leading cause of cancer death among women. This is due in part to the complexity of the disease, which is characterized by multiple subtypes that are driven by different genetic mechanisms and that likely arise from different cell types of origin. Because these differences often drive treatment options and outcomes, it is important to select relevant preclinical m...
Source: Current Protocols in Pharmacology - March 21, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Methods of Inducing Inflammatory Bowel Disease in Mice.
Authors: Bang B, Lichtenberger LM
Abstract
Animal models of experimentally induced inflammatory bowel disease (IBD) are useful for understanding more about the mechanistic basis of the disease, identifying new targets for therapeutic intervention, and testing novel therapeutics. This unit provides detailed protocols for five widely used mouse models of experimentally induced intestinal inflammation: chemical induction of colitis by dextran sodium sulfate (DSS), hapten-induced colitis via 2,4,6-trinitrobenzene sulfonic acid (TNBS), Helicobacter-induced colitis in mdr1a(-/-) mice, the CD4(+) CD45RB(hi) SCID ...
Source: Current Protocols in Pharmacology - March 21, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Models of Inflammation: Carrageenan Air Pouch.
Authors: Duarte DB, Vasko MR, Fehrenbacher JC
Abstract
The subcutaneous air pouch is an in vivo model that can be used to study the components of acute and chronic inflammation, the resolution of the inflammatory response, the oxidative stress response, and potential therapeutic targets for treating inflammation. Injection of irritants into an air pouch in rats or mice induces an inflammatory response that can be quantified by the volume of exudate produced, the infiltration of cells, and the release of inflammatory mediators. The model presented in this unit has been extensively used to identify potential...
Source: Current Protocols in Pharmacology - March 21, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Drug Combinations: Tests and Analysis with Isoboles.
Authors: Tallarida RJ
Abstract
Described in this unit are experimental and computational methods to detect and classify drug interactions. In most cases, this relates to two drugs or compounds with overtly similar effects, e.g., two analgesics or two anti-hypertensives. From the dose-response data of the individual drugs, it is possible to generate a curve, the isobole, which defines all dose combinations that are expected to yield a specified effect. The theory underlying the isobole involves the calculation of doses of drug A that are effectively equivalent to doses of drug B with that equivalence determ...
Source: Current Protocols in Pharmacology - March 21, 2016 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Sigma Receptor Binding Assays.
Authors: Chu UB, Ruoho AE
Abstract
Sigma receptors, both Sigma-1(S1R) and Sigma-2 (S2R), are small molecule-regulated, primarily endoplasmic reticulum (ER) membrane-associated sites. A number of drugs bind to sigma receptors, including the antipsychotic haloperidol and (+)-pentazocine, an opioid analgesic. Sigma receptors are implicated in many central nervous system disorders, in particular Alzheimer's disease and conditions associated with motor control, such as Amyotrophic Lateral Sclerosis (ALS). Described in this unit are radioligand binding assays used for the pharmacological characterization of S1R ...
Source: Current Protocols in Pharmacology - December 12, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
In Vitro Binding of [(3) H]PSB-0413 to P2Y12 Receptors.
Authors: Dupuis A, Heim V, Ohlmann P, Gachet C
Abstract
The P2Y12 /ADP receptor plays a central role in platelet activation. Characterization of this receptor is mandatory for studying disorders associated with a P2Y12 receptor defect and for evaluating P2Y12 receptor agonists and antagonists. In the absence of suitable anti-P2Y12 antibodies, radioligand binding assays are the only way to conduct such studies. While various radioligands were employed in the past for this purpose, none were found to be suitable for routine use. Described in this unit are protocols for quantitatively and qualitatively assess...
Source: Current Protocols in Pharmacology - December 12, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
A Human Induced Pluripotent Stem Cell-Derived Cardiomyocyte (hiPSC-CM) Multielectrode Array Assay for Preclinical Cardiac Electrophysiology Safety Screening.
Authors: Harris K
Abstract
Cardiotoxicity is a leading cause of compound attrition during drug development. Preclinical models used to assess the risk for compound-induced effects on cardiac electrophysiology largely rely on animals that can differ in terms of sensitivity and specificity to the targeted clinical response. There is currently no in vitro human cardiomyocyte model for routine preclinical compound screening, as adult human cardiac tissue is unsuitable for such screening. The commercial availability of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) makes possible the dev...
Source: Current Protocols in Pharmacology - December 12, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
