Discovery Services

Biology

 

Discovery Services - Biology

 

Overview

With a broad range of capabilities across the spectrum of drug discovery activities- from target identification to lead generation and beyond- the Icagen Discovery Biology group offers a wide range of standalone services, as well as fully integrated discovery programs.


 

Assay Development

Our highly experienced team of scientists employs an extensive array of state-of-the-art technologies to tackle projects across a diverse spectrum of assay types and target classes. Beyond the experienced personnel and state-of-the-art equipment, the team is highly engaged and dedicated to delivering quality at every step. 

Capabilities

Target classes

  • Ion Channels
  • GPCR
  • Receptor-ligand
  • PPI
  • Transporter
  • Anti-infective
  • Kinase
  • Protease
  • Phosphatase
  • Phenotypic

Assay types

  • Biochemical
  • Biochemical enzymatic
  • Cellular (primary)
  • Cellular (patient derived)
  • Cellular (clonal line)

Detection technologies

  • Fluorescence
  • Time-resolved fluorescence
  • Fluorescence polarization
  • Luminescence
  • Flipr
  • High content imaging
  • Alphascreen
  • Colorimetric
  • Radiometric
  • Flow cytometry


 

Stem Cells (Human Disease Models)

Icagen leverages the power of stem cells (iPSCs and primary cells) to create terminally differentiated cells to build in vitro cellular human models of disease. These models are employed to create novel and pathophysiological relevant drug discovery platforms. We can create cell-based assays for rare monogenic diseases and employ these models for drug discovery and profiling. These models can be built from patient cells or generated by CRISPR/CAS9 approaches.

Our Icagen discovery biology team has developed core competencies in development of cell-based assays that use muscle cells derived from human iPSCs and myoblasts. The team is able to employ patient cells to assess the impact of therapeutics ( including cells) on contractility of skeletal and cardiovascular muscle tissues.

We have specifically developed robust transcriptional and protein expression assays using these cells and various types of cells, including iPSC, NSC, neurons, and oligodendrocytes. These bioassay platforms have been employed to screen tens of thousands of compounds as part of phenotypic high content screens. Our ability to employ human iPSC and our experience in development of bioassays with primary cells can quickly establish a novel screening system using physiologically relevant cellular models. All assays are in 384-well plate format and can be optimized for 1536 well assays.


 

HTS – High Throughput Screening

Icagen supports the identification of novel modulators of targets by designing, optimizing, and running high throughput assays that deliver strategic starting points for drug discovery. We have a seasoned team of scientists with over 8 decades of combined experience in drug discovery research and high throughput screening, with expertise in assay development, assay optimization, assay miniaturization, HTS, and hit-to-lead optimization support. Our experienced, motivated, and responsive team employs multiple robotic, liquid handling, and detection platforms, along with robust processes, to deliver the highest quality results.

HTS capabilities
  • Compound and sample management
  • State-of-the-art screening system anchored by 2 HighRes Biosolutions robotic platforms
  • Comprehensive suite of instrumentation for liquid handling, environmental control, and detection 

 

in vitro ADME

Icagen provides the scientific and regulatory expertise to streamline and reduce the costs of the discovery and development of new drugs. We support large or small partners so that they can bring their therapeutic to the market.

in vitro Permeability Assays

Most discovery projects focus on the development of orally administered drugs which are primarily absorbed across the intestinal mucosa. Thus assays that predictably and reliably correlate in vitro permeability to in vivo absorption are essential to development. PAMPA is used for measuring passive diffusion and cell-based assay MDCK assay remain the most valuable assay for lead optimization. The permeability data obtained from cell based assays are composed of many factors including aqueous solubility, cell partitioning, influx/efflux transport and tight-junction module ability making it useful not just for estimating oral bioavailability but total biological distribution as well; in particular estimating permeation into the CNS or BBB. Icagen can provide both PAMPA and MDCK cell based assays for different purposes.

PAMPA

PAMPA is a non-cell based assay designed to predict passive transcellular permeability of compounds in early drug discovery. This method provides the most simplistic approach to examining permeability as it only examines a single mechanism, and does not utilize an actual biological barrier.

MDCK Cell-based Model

Icagen's MDCK Cell-based Model assesses both passive and active transporter processes. The cells are derived from Madin Darby canine kidney cells. The assay is a unidirectional/ bidirectional permeability assay. 

in vitro Metabolic Stability Assays

Hepatic metabolism is a major factor of pharmacokinetic behavior, and rapid first–pass metabolism is a primary cause of low bioavailability. There are a variety of in metabolic stability assays which mainly include pooled liver microsomes, pooled liver S9 fraction, hepatocytes, human recombinant cytochrome P450s, UGTs, blood, and plasma are used for the metabolic assessment of hits, leads, and new chemical entity. In vitro drug metabolism kinetic parameters can provide an estimate of in vivo clearance via “scaling factor”. Metabolism in liver microsomes can serve as good predictive model for in vivo PK in terms of plasma clearance and bioavailability when NCE was metabolized by Cytochrome P450; metabolism in hepatocytes also can be used when Phase II enzymes are involved. CLint, in vitro values were calculated from the substrate disappearance rate in microsomes and hepatocytes as follows. If substrate disappearance can be assumed to follow first-order reaction, then the first-order rate equation can be used to calculate half life (t1/2) and intrinsic clearance (CLint): where ke is the first-order disappearance rate constant of unchanged drug (min-1). Icagen can provide microsomal and hepatocyte metabolic stability assays in multiple species and varying tissue for different purposes.

Phase I Microsomal Stability

Microsomal stability assay is the most popularly used in vitro model to characterize the metabolic conversion by phase I enzymes, such as cytochrome P450s (CYPs). This assay is commonly used as the primary ADME screening because of the time and cost advantage. Liver microsomes that provide a rich source of CYPs, FMOs, and UGTs, are available from human and various animal species, relatively cheap to prepare, simple to use, and can be stored frozen for relatively long period. Icagen uses high throughput 96-well format microsomal stability assay (human, rat and mouse etc. species; 0.5 uM of NCE; 0.5 mg/mL microsomes; with and without NADPH; 0 and 20 min of incubation time) to generate % metabolized results.

Phase II Hepatocyte Stability

In general, the microsomes are usually used to determine P450-mediated metabolism such as phase I enzymes study, however, hepatocytes have intact cell membrane and physiological concentrations of phase I, Phase II enzymes and cofactor and close to whole liver for drug metabolism. NCE can be screened and rank-ordered according to metabolic half-life estimates or in vitro intrinsic clearance values obtained from metabolic stability studies. Cryopreserved hepatocytes retain enzymatic activities similar to those of fresh hepatocytes and offer convenience to the end user. Icagen can use automated HTS 96-format hepatocyte metabolic stability assay (human, rat, mouse etc. specie; 0.5 uM of NCE; 0, 20, 40 and 60 min of incubation) to generate Clint and t1/2 results.

 

Ex-hepatic Stability

Plasma and blood stability

Plasma and blood stability assays have many applications in drug discovery, to determine the stability of new chemical entities in plasma/blood. Icagen provides this service to prioritize compounds for in vivo studies if compounds degrade in plasma/blood in generally show poor in vivo efficacy, and to screen pro-drugs and ante-drugs. 

in vitro DDI Assays

Cytochrome P450 (CYP) enzymes are responsible 75% of metabolism. Clinical drug-drug interactions (DDIs) are a major source of adverse drug reactions and are becoming a growing problem with multiple-drug therapy. The most common DDIs are caused by inhibition of enzymes responsible for drug clearance and result in increased bioavailability, systemic exposure, and half-life of a victim drug. The resulting increased exposure to the affected drug can lead to enhancement of the pharmacodynamic effects or serious side effects in humans. DDIs not only affect patient safety but also add to the cost of drug development because of the high costs of failure in clinical development. These issues have prompted the search for approaches to predict the potential for DDIs at the earliest possible stages of drug development. Information about the inhibition of drug-metabolizing enzymes obtained using in vitro systems can provide a basis for predicting DDIs in humans and thereby facilitate decisions about advancement of drug candidates and aid in planning of clinical DDI studies.

Cytochrome P4503A4 and 2D6 Inhibition

Human 3A4 and 2D6 are the most abundant cytochrome P450 (P450) isozymes and responsible for the metabolism of approximately 75 % of all drugs. The substrates of CYP3A4 are chemically diverse, and this broad substrate specificity renders CYP3A4 susceptible to reversible or irreversible inhibition by a variety of drugs. Icagen provides high throughput screening (HTS) CYP3A4 and 2D6 (CYP3A4 substrate: midazolam and testosterone) assays to generate % inhibition results.

Cytochrome P4503A4, 2B6 and 1A2 Induction

Induction of CYP3A4 is also important. Unlike P450 inhibition, CYP3A4 induction is not as frequent a problem and is not normally thought to be a safety concern. Induction can lead to inadequate efficacy of co-administered drugs, and it is therefore an undesirable property. Induction studies have generally been more difficult to conduct experimentally than inhibition studies because induction is an indirect and slow process of gene up-regulation and increased protein expression; however, the advent of newer technologies such as mRNA analyses has partially overcome this problem. Icagen provides CYP3A4, 2B6 and 1A2 induction assay (human hepatocytes; 1 or 10 uM of NCEs, and bDNA assay) to generate % of positive control results.

in vitro Protein Binding Assay

The free fraction of the drug drives pharmacodynamics effect. Plasma protein binding (PPB) is being increasingly used to explain pharmacokinetics and pharmacodynamics (PK/PD) relationships. After steady state equilibrium has been achieved, free drug concentration in plasma is equal to one in the tissues and only the free drug in the tissue is available for the target receptor binding and pharmacologic activity. Hence, unbound drug in plasma should reflect the pharmacologically relevant concentration of unbound drug at the target site in the tissue. Total drug concentration in plasma may be significantly different than total drug concentration in tissues. To accurate correlate in vitro potency and initial PK parameters, assessment of PPB has been routinely carried out in discovery stages, and RED PPB assay is routinely used for assessing PPB binding. 

Rapid Equilibrium Dialysis Protein Binding

Equilibrium dialysis is a system consisting of two fluid-phase compartments separated by a size-controlled, porous membrane. NCE is diluted into plasma and added to a dialysis chamber. This is then dialyzed against blank buffer in the assay chamber. The protein cannot diffuse through the membrane though the drug can do so freely allowing it to set up equilibrium. At equilibrium, the drug will maintain a ratio of bound/unbound dictated by the affinities and capacities of the various binding sites on the protein. Additionally by laws of diffusion, the unbound concentration of drug should be the same on each side. Thus by measuring the total amount of drug in the assay compartment, the whole system can be defined. Icagen provides RED assay to generate free fraction Fu result.

in vitro Toxicity Assay

Mitochondria Toxicity Assay

Drug induced mitochondrial dysfunction underlies many organ toxicities including hepatotoxicity, cardiotoxicity and acute kidney injury, which leads to safety-related compound attrition and post-market drug withdraws. A simple higher-throughput screen assay can identify mitochondrial and non-mitochondrial toxicants during the early stages of drug discovery. Icagen provides a 96-well plate based, dual parameter assay capable of measuring cell membrane integrity and cellular ATP levels simultaneously in primary human hepatocytes.