Establishment, maintenance and in vitro and in vivo applications of primary human glioblastoma multiforme (GBM) xenograft models for translational biology studies and drug discovery.
Authors: Carlson BL, Pokorny JL, Schroeder MA, Sarkaria JN
Abstract
Development of clinically relevant tumor model systems for glioblastoma multiforme (GBM) is important for advancement of basic and translational biology. One model that has gained wide acceptance in the neuro-oncology community is the primary xenograft model. This model entails the engraftment of patient tumor specimens into the flank of nude mice and subsequent serial passage of these tumors in the flank of mice. These tumors are then used to establish short-term explant cultures or intracranial xenografts. This unit describes detailed pr...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
The fentanyl/etomidate-anesthetized beagle (FEAB) model in safety pharmacology assessment.
Authors: van der Linde HJ, Deuren BV, Somers Y, Teisman A, Gallacher DJ
Abstract
This unit describes a procedure for performing safety studies in the anesthetized beagle dog. Detailed are the anesthetic regime, the surgical procedure, and all materials needed to perform cardiovascular, central nervous system, and respiratory safety studies in these animals. An overview of all parameters that can be measured and calculated is provided, as are experimental protocols. Endpoints discussed include hemodynamic, electrocardiological, respiratory, arterial blood, and electroencephalogical parameters. Also presente...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Preparation of HCV NS3 and NS5B proteins to support small-molecule drug discovery.
Authors: Hung M, Wang R, Liu X
Abstract
Production of high-quality, well-characterized recombinant proteins facilitates screening of compound libraries. The protocols detailed in this unit are used to purify three recombinant enzymes that are widely used in HCV research: the HCV NS3 protease domain, the helicase domain as an NS3+NS4A complex, and the NS5B RNA-dependent RNA polymerase. The active enzymes are purified to homogeneity by two-column chromatography to support a screening program for HCV inhibitors.
PMID: 21898331 [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
Biochemical evaluation of HCV NS3 protease inhibitors.
Authors: Schultz B, Yang H, Delaney WE
Abstract
This unit describes assays for characterizing the potency and mechanism of action of NS3 protease inhibitors. Determination of IC(50) values is described using in vitro expressed and purified NS3 protease. This assay can also be used for the rapid exploration of structure-activity relationships. Another protocol describes using the full-length NS3/4A complexes expressed in HCV replicon cell lines for a rapid alternative method for assessing protease activity without requiring conventional protein expression and purification. A method is then provided for dete...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Orthotopic models of esophageal carcinoma and their use in drug discovery.
Authors: Gros SJ
Abstract
The protocol detailed in this unit is for the establishment of an orthotopic model of human esophageal adenocarcinoma in NMRI/nu mice. The resultant tumor has high metastatic potential, spreading readily to liver, lungs, and lymph nodes. This model is useful for studying primary esophageal carcinoma, tumor biology, pathogenesis, tumor progression, metastatic homing, and the efficacy of therapeutic approaches for treating this condition. The practical use of this preclinical model for drug discovery is illustrated with data from a study on the chemotherapeutic effects of HER2-targe...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Pre-clinical mouse models of primary and metastatic pleural cancers of the lung and breast and the use of bioluminescent imaging to monitor pleural tumor burden.
Authors: Servais EL, Colovos C, Kachala SS, Adusumilli PS
Abstract
Malignant pleural disease (MPD) results in an estimated 150,000 cases of malignant pleural effusions (MPE) annually. The most common malignancies associated with MPD are primary malignant pleural mesothelioma (MPM) and metastatic lung cancer, breast cancer, and lymphoma. MPM is a rare, regionally aggressive malignancy whose incidence is increasing secondarily to the latency of disease progression. MPD is characteristic of advanced-stage pleural disease and portends a grave clinical prognosis with a median survival between 3 and 12 months. P...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Overview of receptor allosterism.
Authors: Gregory KJ, Sexton PM, Christopoulos A
Abstract
In addition to the orthosteric site, which recognizes endogenous ligands, most G protein-coupled receptors (GPCRs) possess topographically distinct allosteric sites that can be recognized by small molecules and accessory cellular proteins. Ligand binding to allosteric sites promotes a conformational change in the GPCR that can alter orthosteric ligand affinity and/or efficacy. Moreover, there has been an increase in recent years in the identification of allosteric agonists that can directly activate the receptor in the absence of orthosteric ligand. ...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Evaluation of neurotoxicity potential in rats: the functional observational battery.
Authors: Boucard A, Bétat AM, Forster R, Simonnard A, Froget G
Abstract
This unit describes the functional observational battery (FOB), a behavioral screening procedure commonly used in safety pharmacology and toxicology studies to assess potentially adverse effects of test agents on the central nervous system. The battery includes general observations and the determination of reactivity to various stimuli. Also presented is the severity score index for analyzing individual measurements and evaluations over a range of endpoints. The severity score index can be used to identify, quantify, and describe the ...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Generation and quantitation of infectious hepatitis C virus derived from cell culture (HCVcc).
Authors: Paulson MS
Abstract
The development of robust genotype 1b and genotype 1a hepatitis C virus (HCV) replicon systems has enabled the convenient in vitro study of part of the virus life cycle. This unit describes detailed protocols for generating and measuring infectious HCV, or cell-culture-derived infectious HCV (HCVcc). The HCVcc infectious system has two essential components: (1) cells that are permissive to de novo infection and allow effective replication of the full virus life cycle; and (2) a virus genome that has robust and efficient replication in tissue culture. The assays in this unit are...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
High-throughput screening of viral entry inhibitors using pseudotyped virus.
Authors: Basu A, Mills DM, Bowlin TL
Abstract
Virus entry into a host cell is an attractive target for therapy because propagation of virus can be blocked at an early stage, minimizing chances for the virus to acquire drug resistance. Anti-infective drug discovery for BSL-4 viruses like Ebola or Lassa hemorrhagic fever virus presents challenges due to the requirement for a BSL-4 laboratory containment facility. Pseudotyped viruses provide a surrogate model in which the native envelope glycoprotein of a BSL-2 level virus (e.g., vesicular stomatitis virus) is replaced with envelope glycoprotein of a foreign ...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
In vitro anti-hepatitis C virus (HCV) resistance selection and characterization.
Authors: Mo H
Abstract
Understanding the resistance profile for an antiviral drug is essential in the drug discovery process. The selection of drug-resistant mutant viruses is critical in characterizing the resistance of hepatitis C virus (HCV) to anti-HCV agents, and can be examined in HCV replicon systems. Three basic methods are employed in this unit to select HCV resistance replicons: (i) cells containing HCV replicons are cultured at low density in the presence of G418 and a fixed concentration of the investigational drug; (ii) cells containing HCV replicons are passaged in the presence of a fixed con...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Orthotopic xenograft model of cervical cancer for studying microenvironmental effects on metastasis formation and response to drug treatment.
Authors: Chaudary N, Hedley DW, Hill RP
Abstract
Cancers arising in the uterine cervix are usually squamous cell carcinomas that develop from preneoplastic lesions. They invade locally, and then typically metastasize to the regional lymph nodes and eventually to distant sites. Orthotopically grown xenografts are technically challenging to perform, but recapitulate the clinical situation to a greater extent than xenografts grown at subcutaneous or intramuscular sites. Thus, orthotopic xenografts develop lymphovascular invasion and metastasize to the para-aortic lymphatic chain in a pattern similar to that s...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Animal models of systemic lupus erythematosus (SLE) and ex vivo assay design for drug discovery.
Authors: Seavey MM, Lu LD, Stump KL
Abstract
Systemic Lupus Erythematosus (SLE) is a debilitating and often fatal autoimmune disease that involves multiple organ systems. It can develop for years before being diagnosed. Current treatments for SLE usually involve the use of cytotoxic or immunosuppressive agents that can lead to infection or cancer. The design of appropriate models and assays will determine the efficiency and speed with which an investigator can test a new chemical entity (NCE) or expect results to move a drug discovery program forward. This unit describes a series of preclinical assays for ...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Quantification of allosteric interactions at G protein-coupled receptors using radioligand binding assays.
Authors: Leach K, Sexton PM, Christopoulos A
Abstract
Allosteric interactions involve the simultaneous binding of two ligands to the same receptor. An allosteric modulator causes a conformational change in the receptor protein that yields a change in the binding or signaling of an orthosteric agent, i.e., an agonist or competitive antagonist that binds to the endogenous agonist binding site. Because of the complex nature of allosteric phenomena, the detection and quantification of their effects on orthosteric ligand binding relies on the use of both equilibrium and non-equilibrium assays to ensure proper i...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Preclinical models of pediatric solid tumors (neuroblastoma) and their use in drug discovery.
Authors: Patterson DM, Shohet JM, Kim ES
Abstract
Neuroblastoma is the most common pediatric abdominal solid tumor. This aggressive embryonal malignancy of neural crest origin has a peak age of onset of 22 months, and accounts for ~11% of all pediatric cancers and 15% of all pediatric cancer deaths. With current treatment protocols, including high-dose chemotherapy with autologous stem cell transplantation, radiation, and surgery, ~80% of high-risk patients go into remission, although the majority relapse and succumb to therapy-resistant tumors. Long-term survival rates (>5 years) are <50%. Mouse mod...
Source: Current Protocols in Pharmacology - November 18, 2015 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
