Measurement of Glucose Uptake in Cultured Cells.
Authors: Yamamoto N, Ueda-Wakagi M, Sato T, Kawasaki K, Sawada K, Kawabata K, Akagawa M, Ashida H
Abstract
Facilitative glucose uptake transport systems are ubiquitous in animal cells and are responsible for transporting glucose across cell surface membranes. Evaluation of glucose uptake is crucial in the study of numerous diseases and metabolic disorders such as myocardial ischemia, diabetes mellitus, and cancer. Detailed in this unit are laboratory methods for assessing glucose uptake into mammalian cells. The unit is divided into five sections: (1) a brief overview of glucose uptake assays in cultured c...
Source: Current Protocols in Pharmacology - December 12, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
In vitro isolated tissue functional muscarinic receptor assays.
Authors: Pulido-Rios MT, Steinfeld T, Armstrong S, Watson N, Choppin A, Eglen R, Hegde SS
Abstract
Muscarinic receptor (mAChRs) subtypes are viable targets for the design of novel agents for use in a number of central and peripheral disorders. In vitro isolated tissue functional assays for muscarinic receptor subtypes have played an invaluable role in basic research and drug discovery. The availability of biological assays for generation of quantitative estimates of affinity and potency of ligands allows evaluation of the contribution of a given mAChR to the functional end organ response and also enables d...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Mouse models of human bladder cancer as a tool for drug discovery.
Authors: Seager C, Puzio-Kuter AM, Cordon-Cardo C, McKiernan J, Abate-Shen C
Abstract
Muscle-invasive bladder cancer is a deadly condition in dire need of effective new treatments. This unit contains a description of mouse models suitable for the evaluation of potential new therapies. Included is a genetically engineered mouse model of bladder cancer generated by the delivery of an adenovirus expressing Cre recombinase into the bladder lumen. Also described is an orthotopic mouse model created by the instillation of human bladder tumor cells into the bladder lumen of immune deficient mice. Protocols are al...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Cyclic nucleotide phosphodiesterase assay technology.
Authors: MacKenzie SJ, Hastings SF, Wells C
Abstract
Because of their critical role in modulating cellular cyclic nucleotide levels, phosphodiesterases (PDEs) are involved in many disease-related signaling pathways. The PDE family is large and diverse, with members having different tissue distribution, sub-cellular localizations, and substrate specificities. Because of these characteristics, the PDEs represent a broad group of potential drug targets. Described in the present unit are the assay development and validation procedures needed to establish a high-throughput screening system for these important e...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Novel object recognition in the rat: a facile assay for cognitive function.
Authors: Mathiasen JR, DiCamillo A
Abstract
The rat novel object recognition (NOR) assay is a relatively high-throughput, robust, and sensitive procedure for evaluating compounds for cognition-enhancing activity. For the test, rats are given the opportunity to explore two identical objects for a predetermined period of time. After a delay, the animals are then presented with two objects to explore, one of which is the same as in the first exploration trial, the other a new object. Depending on the length of the delay between the two trials, the rats will either explore the novel object for a greater time p...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Rodent models of depression: forced swim and tail suspension behavioral despair tests in rats and mice.
Authors: Castagné V, Moser P, Roux S, Porsolt RD
Abstract
The development of antidepressants requires simple rodent behavioral tests for initial screening before undertaking more complex preclinical tests and clinical evaluation. Presented in the unit are two widely used screening tests used for antidepressants, the forced swim (also termed behavioral despair) test in the rat and mouse, and the tail suspension test in the mouse. These tests have good predictive validity and allow rapid and economical detection of substances with potential antidepressant-like activity. The behavioral despair and the tail s...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Pharmacological applications of baculovirus-mediated protein expression in mammalian cells.
Authors: Condreay JP, Watson CA
Abstract
The development of cell-based assays for cellular receptors, ion channels, and transporters requires the delivery and expression of transgenes. Viral-mediated gene delivery is a particularly attractive approach for this purpose because of its efficiency and potential to deliver genes to a wide variety of cell types. Recombinant baculoviruses, long used to deliver genes to insect cells for overexpression, also effectively transfer genes to mammalian cells. The only required modification to the virus for this purpose is the addition of transgene expression cassettes c...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Overview on the Rule of Five.
Authors: Pollastri MP
Abstract
In the mid- to late 1990 s, because of the drug discovery paradigm shift from phenotypic screens to combinatorial chemistry and high-throughput screening, the physicochemical properties of exploratory drug molecules displayed a dramatic shift toward higher molecular weight and lipophilicity. In response, Lipinski and coworkers reported an analysis of compounds that successfully navigated Phase I and entered into Phase II clinical studies, and correlated the computed physicochemical properties of these molecules to their aqueous solubility, permeability, and oral bioavailabili...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Xenograft and Transgenic Mouse Models of Epithelial Ovarian Cancer and Non Invasive Imaging Modalities to Monitor Ovarian Tumor Growth In situ -Applications in Evaluating Novel Therapeutic Agents.
Authors: Connolly DC, Hensley HH
Abstract
Epithelial ovarian cancer (EOC) is the most commonly fatal gynecologic malignancy in developed countries. Most EOC patients are diagnosed at advanced stage when disease has spread beyond the ovary. While many patients initially respond to surgery and chemotherapy, the long term prognosis is generally unfavorable, with recurrence and development of drug resistant disease. There is a critical need to identify new therapeutic agents that prolong disease-free intervals and effectively manage recurrent disease. Murine models of ovarian carcinoma are excellent models to ...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Static biofilm cultures of Gram-positive pathogens grown in a microtiter format used for anti-biofilm drug discovery.
Authors: Kwasny SM, Opperman TJ
Abstract
An in vitro assay is presented for culturing staphylococcal biofilms and biofilms of nonmotile Gram-positive bacteria under static conditions in microtiter assay plates, and for the quantification of biofilm growth, using a simple staining procedure that measures amounts of bacterial cells and extracellular matrix. This basic assay can be adapted readily to study several aspects of biofilm formation, for high-throughput screening to identify small molecule inhibitors of biofilm formation or biofilm-defective mutants, and for quantifying the anti-biofilm activity of ...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Evaluation of compound activity against hepatitis C virus in replicon systems.
Authors: Yang G, Huang M
Abstract
Described in this unit are protocols for evaluation of new chemical entities for activity against hepatitis C virus (HCV) using HCV replicon systems. While agents designed to target NS3 protease, NS3 helicase, and NS5B RNA polymerase can be evaluated in biochemical assays, it is important to confirm their inhibitory effect on HCV RNA replication using HCV replicon systems, especially since replication involves many components besides these enzymes. Screening of compound libraries using replicon systems has led to the discovery of replication inhibitors, which act through d...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Studies on hepatitis C virus resistance to inhibitors in replicon systems.
Authors: Zhao Y, Huang M
Abstract
Viruses evolve under selection pressure from a particular antiviral agent, resulting in the emergence of organisms that are not susceptible to the drug. This process is referred to as "virus resistance induction." While conventional in vitro resistance studies are conducted using infectious viruses, the lack of a robust hepatitis C virus (HCV) infection system in cell culture makes such an approach impossible in this case. Instead, cell lines harboring a self-replicating HCV RNA (or HCV replicon) are used for this purpose. The protocols detailed in this unit describe metho...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Label-free imaging and temporal signature in phenotypic cellular assays: a new approach to high-content screening.
Authors: Martin J
Abstract
Some drug targets are not amenable to screening because of the lack of a practical or validated biological assay. Likewise, some screening assays may not be predictive of compound activity in a more disease-relevant scenario, or assay development may demand excessive allocation of resources (i.e., time, money or personnel) with limited knowledge of the actual tractability of the target. Label-free methodologies, implemented in microtiter plate format, may help address these issues and complement, simplify, or facilitate assays. Label-free biosensors, based on grating resonance or...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Measuring receptor target coverage: a radioligand competition binding protocol for assessing the association and dissociation rates of unlabeled compounds.
Authors: Sykes DA, Dowling MR, Charlton SJ
Abstract
The kinetics of the ligand-receptor interaction is an important feature in lead optimization for new drug candidates. The protocol described in this unit is a kinetic radioligand competition binding assay that makes possible the determination of both the association and dissociation rates of unlabeled receptor ligands.
PMID: 22294377 [PubMed - indexed for MEDLINE] (Source: Current Protocols in Pharmacology)
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Pre-clinical mouse models of human prostate cancer and their utility in drug discovery.
Authors: Park SI, Kim SJ, McCauley LK, Gallick GE
Abstract
In vivo animal experiments are essential to current prostate cancer research, and are particularly critical to studying interactions between tumor cells and their microenvironment. Numerous pre-clinical animal models of prostate cancer are currently available, including transgenic mouse models and human prostate cancer xenograft mouse models. In contrast to transgenic mouse models producing more heterogeneous cohorts of tumors, xenograft mouse models provide more controlled approaches. This unit describes the detailed procedures necessary to establ...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
