Michael P. Nova

A Scintillating Microplate Assay for the Assessment of Protein Kinase Activity

Protein kinases, a class of enzymes that phosphorylate certain tyrosine, serine, and threonine residues, play an important role in cellular functions and are important targets in drug discovery research. Thus, it is of interest to develop a simple assay that can be used to measure protein kinase activity toward specific substrates and is suitable for the high throughput screening (HTS) of potential kinase inhibitors. The scintillation proximity concept has been successfully applied for measuring specific kinase activity using surfaces passively coated with a peptide substrate. In this study, we evaluated kinase assay performance on three ScintiStrip platforms: unmodified surface, streptavidin-coated surface, and streptavidin covalently attached to surface. The high affinity of streptavidin toward biotin-linked peptide substrates makes it a unique platform for measuring specific incorporation of radiolabeled phosphate into selected substrates of specific enzymes in the presence of others. Therefore, this assay may be used with cell extracts containing impure kinases as well as with purified enzymes. The scope of this assay was demonstrated with purified tyrosine kinases (e.g., p60c-src kinase) and A431 cell extracts. This scintillation proximity assay is universal, simple, rapid, accurate, and can be adapted for use with robotics for HTS.

A fluorometric assay for the measurement of endothelial cell density in vitro

SummaryA fluorometric assay for determining endothelial cell numbers based on the endogenous enzyme acid phosphatase is described. In preliminary studies, three substrates—p-nitrophenyl phosphate, 4-methylumbelliferyl phosphate, and 2′-[2-benzthiazoyl]-6′-hydroxy-benthiazole phosphate (AttoPhos™)—were compared with respect to their kinetic, optimum assay conditions, sensitivity, and detection limits. Only AttoPhos™ was found to have a high degree of sensitivity, reliability, and reproducibility for measuring both high and low cell numbers in the same plate. In subsequent experiments, assay conditions were validated for measuring endothelial cell density in response to basic fibroblast growth factor and fumagillin. Furthermore, the AttoPhos™ assay revealed a linear correlation between acid phosphatase activity and cell number in many cell types, including BALB/3T3, CHO-K1, A431, MCF7, 2008, SK-OV-3, T47-D, and OVCAR-3. This assay is potentially valuable for use in many in vitro systems in which the quantitation of cell density and proliferation is necessary. The practical advantages of AttoPhos™ assay for measuring endothelial cell numbers include (1) nonradioactivity, (2) simplicity, (3) economy, (4) speed of assessment of proliferation of large number of samples, and (5) amenability to high-throughput drug screening.

Synthesis Coupled to Scintillation Proximity Affinity Screening

The rapidly changing developments in genomics and combinatorial chemistry, generating new drug targets and large numbers of compounds, are beginning to push the limits of screening efficiently. Thus, there is a need for novel tools and strategies to improve high throughput screening. A novel approach is to couple synthesis and screening on a common platform, rather than to increase the rate at which traditional screening methods can be implemented. We have developed a proprietary grafted polymer with special fluorescence characteristics referred to as Electronically Encoded Fluorescence matriX (EFX™), which has the sturdiness and required functionality for direct chemical synthesis as well as suitable surface characteristics for measuring interactions in aqueous solution. This matrix is fabricated into a MicroTube reactor, and each tube is associated with an electronically encoded tag. The system follows a homogenous assay protocol and is based on the scintillation proximity principle. Using solid-phase chemistry, a variety of small molecules may be synthesized onto the EFX. A simple binding assay can be conducted by combining a collection of MicroTubes with any radiolabeled acceptor molecule. The MicroTubes that carry active compounds are selected based on the photon mission or fluorescence characteristics. We validated this approach by evaluating the interactions of biotin with radiolabeled streptavidin.