Chelsea Clark Profile

High Throughput Screening (HTS) is a drug discovery process that allows automated detection of large numbers of chemical and/or biological compounds for a specific biological target. It speed up target analysis, as large scale compound libraries can be quickly screened in a cost-effective way. HTS should be seen as a fast scan of biological processes by which compounds with poor or no effects can be rapidly excluded from the analysis pipeline.

Creative Bioarray is a full-time, professionally managed facility with highly trained and experienced staff, advanced equipment, and a substantial library of compounds. These features, combined with a centralized knowledge base, increase cost-effectiveness and productivity in the ongoing fight against disease. Our platform allows convenient screening of compounds in 96, 384 and 1536 well formats for both enzyme/protein and cell-based assays using all available technologies (luminescence, fluorescence intensity, fluorescence polarization, FRET, TR-FRET, HTRF, absorbance). We have developed and optimized in vitro enzymatic assays, protein binding assays, protein-protein, protein-nucleic acids binding assays, cell viability, reporter gene and image-based phenotypic screening assays.

Transporters are the largest family of membrane proteins in human organism including members of solute carrier transporter and ATP-binding cassette transporter families. They play pivotal roles in the absorption, distribution and excretion of exogenous and endogenous molecules. Transporters are widely expressed in a variety of human tissues and are routinely evaluated during the process of drug development and approval. Currently, transporters are used as the therapeutic targets for the treatment of various diseases such as diabetes, major depression, hypertension, and constipation. Despite the steady growth of the field of transporter biology, we know very little about the endogenous substrates or physiological functions of more than half of the members in transporter superfamily. Therefore, understanding the interaction between the potential compounds and transporters in the early stage of drug development is critical for selecting candidates with desirable drug metabolism and safety profiles.

Protein kinases are involved in many cellular signaling pathways and comprise one of the largest families of homologous proteins. They play an extensive role in signal transduction and regulation of the complex cellular processes necessary to maintain life. Overexpression and/or dysregulation of protein kinases lead to a variety of diseases, thus providing numerous targets for drug development. Currently, approximately 24% of all research spending on drug discovery and development is focused on kinases. In view of the huge demand for small molecules that target this class of proteins, many technologies and platforms have been developed for the discovery of kinase inhibitors, including biochemical-based functional assays and compound-binding competition assays.

Label-free technologies with the potential to substantially change some aspects of whole-cell assays, including GPCR screening, have emerged in recent years. These technologies detect changes in cellular characteristics including adhesion, proliferation, migration, and cell death, which are regulated by many different receptors, ion channels and signal transduction pathways. The advent of label-free technologies provides a different strategy for signal transduction measurement by using integrated or cumulative responses rather than the resolution of individual events.

Label-free whole cell assays typically employ a biosensor to convert the summation of ligand-induced changes in living cells into optical, electrical, magnetic, acoustic, calorimetric, or other quantifiable signals. In addition, these label-free assays do not require the addition of detection reagents, nor do they involve the expression strategies of forcing G-protein coupling or promiscuous G proteins, thus offering the potential for studying a more physiological state. Some label-free instruments have gained prominence in detecting GPCR function due to their ability to detect the activation of Gs, Gi and Gq signal transduction pathways. These systems are also used in ligand selectivity studies, endogenous receptor profiling, systemic cell biology studies, and many other aspects of GPCR research.

β-Arrestins bind to GPCRs in a conformationally sensitive manner and are known to regulate: 1) GPCR desensitization by inhibition of GPCR coupling to heterotrimeric G proteins, 2) GPCR endocytosis by promoting association of GPCR/β-arrestin complexes, and (3) arrestin-promoted signaling via the extensive adaptor functions of the β-arrestins. The β-arrestin-mediated signaling constitutes an important component of GPCR signaling in addition to the G protein-mediated signaling. Monitoring of β-arrestin recruitment to activated receptors has become a popular way to measure GPCR activity and is described as a ‘universal’ GPCR platform owing to the fact that it is G-protein independent, such that GPCRs will recruit β-arrestins regardless of the nature of the G protein to which they couple.

GPCR internalization is a key regulatory step in determining receptor activity. It is essential to prevent cells from being subjected to excessive receptor stimulation or prolonged inactivity. If GPCR internalization is inhibited by a ligand, protein mutation, or an abnormal signaling, adverse effects may occur, often resulting in drug tolerance, unwanted side effects, and disease.

The broad applicability of GPCR internalization assays is based on the common phenomenon of GPCR "desensitization" and has been demonstrated by numerous GPCRs. During desensitization, GPCR kinases (GRKs) phosphorylate agonist-activated GPCRs on serine and threonine residues. Arrestin, a cytoplasmic protein, is recruited to the plasma membrane by GRK-phosphorylated GPCRs. The arrestin then uncouples the GPCR from the cognate G protein and targets the desensitized receptors to clathrin-coated pits for endocytosis.

GPCRs transmit information within cells via two major signaling pathways: regulation of cAMP levels and increases in intracellular Ca2+ triggered by inositol 1, 4, 5- triphosphate (IP3). These signaling pathways are activated by specific G protein associated with the receptor. Activation of Gs or Gi coupled receptor results in an increase or decrease in cAMP levels, respectively. While activation of Gq coupled receptor activates phospholipase C (PLC), which converts PIP2 into DAG and IP3. The metabolic pathway of IP3 is very complicated, as IP3 can either be directly hydrolyzed or be first phosphorylated to generate D-myo-inositol 1, 3, 4, 5-tetrakisphosphate (IP4) before being degraded. These processes involve several phosphatases, leading to the formation of D-myo-inositol bisphosphates (IP2) and D-myo-inositol monophosphates (IP1), which can be phosphorylated at different positions on the inositol ring. Finally, inositol monophosphatase (IMPase) degrades IP1 into myo-inositol.