Targeted Protein Degradation Phenotypic Studies Using HaloTag CRISPR/Cas9 Endogenous Tagging Coupled with HaloPROTAC3.
Authors: Caine EA, Mahan SD, Johnson RL, Nieman AN, Lam N, Warren CR, Riching KM, Urh M, Daniels DL
Abstract
To assess the role of a protein, protein loss phenotypic studies can be used, most commonly through mutagenesis RNAi or CRISPR knockout. Such studies have been critical for the understanding of protein function and the identification of putative therapeutic targets for numerous human disease states. However, these methodological approaches present challenges because they are not easily reversible, and if an essential gene is targeted, an associated loss of cell viability can potentially hinder furth...
Source: Current Protocols in Pharmacology - December 19, 2020 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Preclinical Models for Studying the Impact of Macrophages on Cancer Cachexia.
Authors: Markov SD, Gonzalez D, Mehla K
Abstract
Cancer-associated cachexia is defined by loss of weight and muscle mass, and by the potential loss of adipose tissue accompanied by insulin resistance and increased resting energy expenditure. Cachexia is most prevalent in pancreatic cancer, the third leading cause of cancer-related deaths. While various factors interact to induce cachexia, the precise mechanisms underlying this clinical condition are not fully understood. Clinically relevant animal models of cachexia are needed given the lack of standard diagnostic methods or treatments for this condition. ...
Source: Current Protocols in Pharmacology - December 3, 2020 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
In Vitro Permeation Test (IVPT) for Pharmacokinetic Assessment of Topical Dermatological Formulations.
Authors: Santos LL, Swofford NJ, Santiago BG
Abstract
In vitro assessment of topical (dermal) pharmacokinetics is a critical aspect of the drug development process for semi-solid products (e.g., solutions, foams, sprays, creams, gels, lotions, ointments), allowing for informed selection of new chemical entities, optimization of prototype formulations during the nonclinical stage, and determination of bioequivalence of generics. It can also serve as a tool to further understand the impact of different excipients on drug delivery, product quality, and formulation microstructure when used in parallel with oth...
Source: Current Protocols in Pharmacology - October 1, 2020 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
A Localized Aldara (5% Imiquimod)-Induced Psoriasiform Dermatitis Model in Mice Using Finn Chambers.
This article also details functional measurements performed during assays, including skin thickness, blood perfusion, semiquantitative histopathological evaluation, determination of scaling score to monitor psoriatic symptoms, and collection of spleen and body weight data to identify systemic effects. © 2020 The Authors. Basic Protocol: Use of Finn chambers to induce psoriasiform skin reactions with imiquimod Support Protocol 1: Measurement of double-fold dorsal skin thickness Support Protocol 2: Measurement of blood perfusion Support Protocol 3: Determination of scaling score Support Protocol 4: Semiquantitative histopat...
Source: Current Protocols in Pharmacology - August 15, 2020 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Autologously Humanized Mice for Immune-Oncologic Studies.
Authors: Fu J, Kim YJ
Abstract
With the rapid approval of immune checkpoint inhibitors for lung, melanoma, breast, genitourinary, and hematological malignancies, the hematopoietic cells in the tumor microenvironment (TME) are now considered an important, if not essential, consideration for cancer scientists. In many instances, syngeneic murine models have not been highly predictive for responsiveness in clinical trials. Our limited understanding of the human TME have, therefore, precluded a rational translation of immunotherapeutic combinations. This has led to the adoption of hematopoietic humanized murin...
Source: Current Protocols in Pharmacology - May 30, 2020 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Establishment of Humanized Mice from Peripheral Blood Mononuclear Cells or Cord Blood CD34+ Hematopoietic Stem Cells for Immune-Oncology Studies Evaluating New Therapeutic Agents.
Authors: Verma B, Wesa A
Abstract
The clinical success of immune checkpoint modulators and the development of next-generation immune-oncology (IO) agents underscore the need for robust preclinical models to evaluate novel IO therapeutics. Human immune system (HIS) mouse models enable in vivo studies in the context of the HIS via a human tumor. The immunodeficient mouse strains NOG (Prkdcscid Il2rgtm1Sug ) and triple-transgenic NOG-EXL [Prkdcscid Il2rgtm1Sug Tg (SV40/HTLV-IL3, CSF2)], which expresses human IL-3 and GM-CSF, allow for human CD34+ hematopoietic stem cell (huCD34+ HSC) engraftment and multiline...
Source: Current Protocols in Pharmacology - May 27, 2020 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Screening Assay Protocols Targeting the Nav1.7 Channel Using Qube High-Throughput Automated Patch-Clamp System.
This article describes the basic procedure for setting up the screening protocol and recording data for Nav1.7 on a Qube automated patch-clamp system. Three protocols along with step-by-step details are provided. First, we describe a protocol to estimate Vhalf , the voltage at which half of the channels are inactivated, using traditional steady-state inactivation measurement as well as a new adaptive online estimation. Second, we establish a state-dependent protocol using adaptive online Vhalf measurement to obtain a concentration response curve (CRC) on known reference blockers. Last, we introduce a use-dependent protocol...
Source: Current Protocols in Pharmacology - April 14, 2020 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Electrophysiological Studies of GABAA Receptors Using QPatch II, the Next Generation of Automated Patch-Clamp Instruments.
Authors: Schupp M, Park SH, Qian B, Yu W
Abstract
Ligand-gated ion channels (LGICs) are a group of diverse ion channels that are gated by ligands and play important roles in normal physiological and pathological conditions. Many of them are drug targets that have been pursued, are being pursued, and will likely be pursued in the future by pharmaceutical companies and academic groups for a variety of diseases. One of those LGICs is the GABAA receptor, a heterooligomeric chloride channel that can be blocked and modulated at various sites. In order to study the receptor's functional response to compounds, the...
Source: Current Protocols in Pharmacology - April 10, 2020 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Studying Nicotinic Acetylcholine Receptors Using the IonFlux ™ Microfluidic-Based Automated Patch-Clamp System with Continuous Perfusion and Fast Solution Exchange.
We present protocols to study agonist, antagonist, and PAM activities on nAChRs, particularly the rapidly desensitizing nAChR α7 receptors. The data demonstrate that the IonFlux™ system is a fast, robust, and reliable platform for the study of nAChRs and other ligand-gated ion channels, generating data that closely mimic those from manual patch-clamp conditions. © 2020 by John Wiley & Sons, Inc. Basic Protocol 1: Measuring agonist concentration-dependent response Basic Protocol 2: Measuring antagonist concentration-dependent response Basic Protocol 3: Measuring positive allosteric modulator (PAM) concentration-depe...
Source: Current Protocols in Pharmacology - February 20, 2020 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Fluorescent Western Blotting: High Sensitivity Detection of Multiple Targets.
Authors: Berkelman T
Abstract
Western blotting with fluorescence detection offers the possibility of detecting multiple targets simultaneously on a single blot. Primary antibodies are increasingly available from multiple hosts, and there are now a wide variety of dye labels to exploit multiple imaging channels. If primary and secondary antibodies are selected so that individual targets can be discriminated, multiple antigens can be detected and quantified in a single experiment. Current fluorescence imaging instrumentation offers multiple detection channels and gives sensitivity comparable to other methods...
Source: Current Protocols in Pharmacology - January 19, 2020 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Automated Dynamic Clamp for Simulation of IK1 in Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes in Real Time Using Patchliner Dynamite8.
We describe protocols for optimized cell handling and harvesting for use on the Patchliner and the steps required for automated execution of experiments and data analysis in dynamic clamp mode. © 2019 by John Wiley & Sons, Inc. Basic Protocol: Recording action potential pharmacology from human induced pluripotent stem cell-derived cardiomyocytes in automated patch clamp combined with dynamic clamp to introduce simulated IK1 and compensate seal resistance Support Protocol 1: Cardiomyocyte plating and culture Support Protocol 2: Cell harvesting and dissociation Alternate Protocol: Recording action potential pharmacology...
Source: Current Protocols in Pharmacology - December 25, 2019 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Assays for Modulators of Ryanodine Receptor (RyR)/Ca2+ Release Channel Activity for Drug Discovery for Skeletal Muscle and Heart Diseases.
This article describes assays to evaluate RyR channel activity, including an ER Ca2+ measurement assay that is compatible with high-throughput screening and a [3 H]-ryanodine binding assay that provides a quantitative measure of RyR channel activity as a second screen for compound hits. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: [Ca2+ ]ER assay for ryanodine receptor (RyR) channel activity Support Protocol 1: Determination of dose dependence and isoform selectivity of RyR inhibitors using [Ca2+ ]ER assay Basic Protocol 2: [3 H]-Ryanodine binding assay for RyR channel activity Support Protocol 2: Isolation of ...
Source: Current Protocols in Pharmacology - December 15, 2019 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Role of High-Throughput Electrophysiology in Drug Discovery.
Authors: Liu C, Li T, Chen J
Abstract
Due to their important physiological functions, ion channels are key therapeutic targets for a variety of disorders. However, electrophysiological assessment of ion channel activity is technically challenging and has been a bottleneck in the discovery of drugs that modulate channel function. To address this issue, automated patch clamp platforms have been developed with improved throughput and broader applications. An overview of the current status of high-throughput electrophysiology and its applications in drug discovery is provided. © 2019 The Authors.
PMID...
Source: Current Protocols in Pharmacology - December 7, 2019 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
A Simple Assay to Evaluate the Function of Human Connexin Hemichannels Expressed in Escherichia coli that Can Be Used for Drug Discovery and Mutant Analysis.
Authors: Fiori MC, Cuello LG, Altenberg GA
Abstract
Abnormally increased activity of connexin hemichannels contributes to cell damage in many disorders, including deafness, stroke, and cardiac infarct, and therefore hemichannels constitute a potentially important therapeutic target. Unfortunately, the available hemichannel inhibitors are not specific and most are toxic. The absence of a simple and cost-effective screening assay has made the discovery of hemichannel inhibitors difficult. Here, we present an optimized assay where human connexins are expressed in genetically modified Escherichia coli cells de...
Source: Current Protocols in Pharmacology - November 24, 2019 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
Spontaneous Model of Sj ögren's Syndrome in NOD Mice.
Spontaneous Model of Sjögren's Syndrome in NOD Mice.
Curr Protoc Pharmacol. 2019 Sep;86(1):e65
Authors: Scuron MD, Fay B, Oliver J, Smith P
Abstract
The non-obese diabetic (NOD) mouse model is the most widely described and validated method for investigating human primary Sjögren's syndrome (SS) and represents a useful model for translational studies. However, the systemic disease manifestation in NOD mice is sensitive to the housing environment, as stress modulates the immune system, so it is essential to confirm that readouts are robust, reproducible, and sensitive to known cli...
Source: Current Protocols in Pharmacology - September 22, 2019 Category: Drugs & Pharmacology Tags: Curr Protoc Pharmacol Source Type: research
