Label-Free High-Throughput Functional Lytic Assays

Abstract

Refractive index-sensitive resonant waveguide grating biosensors are used to assay the label-free enzymatic degradation of biomolecules. These assays provide a robust means of screening for functional lytic modulators. The biomolecular substrates in this study were covalently immobilized through amine groups. Using the Corning ® Epic™ System, the digestion signatures for multiple protein substrates on the biosensors are measured. Label-free digestion profiles for these proteins were substrate specific. Similarly, the authors find that the label-free digestion is protease specific. Enzyme-substrate pairs were used to evaluate highthroughput biosensors as tools for screening functional modulators. The lytic inhibitor properties for several proteases and dextranase are determined. The authors find that the IC 50 values for the protease inhibitors agree with the reported values for several known inhibitors. The Z´ values, using biosensor-based functional lytic screens, were routinely greater than 0.5, making this label-free application feasible for high-throughput screening. (Journal of Biomolecular Screening 2007:117-125)

Novel N-Phenyl–Substituted Thiazolidinediones Protect Neural Cells against Glutamate- and tBid-Induced Toxicity

“…The binding of the presented thiazolidinediones to recombinant Bid was measured using the EnSpire label-free platform (Fig. 6, C-E) (O'Malley et al, 2007). Using this biochemical assay, the target Bid was initially immobilized at a concentration of 200 mg/ml onto the aminocoupling surface of the EnSpire label-free biochemical microplate biosensor.…”

“…Biochemical assays were performed using the Corning Epic label-free technology on the EnSpire Multimode Plate Reader (PerkinElmer, Rodgau, Germany) (O'Malley et al, 2007). Bid immobilization on the optical biosensors was accomplished by adding 200 mg/ml protein in 20 mM sodium acetate, pH 4.0, using a 12-channel Thermo Scientific matrix multichannel equalizer pipette followed by overnight incubation at 4°C.…”

Structural insights into the molecular basis responsible for the effects of immobilization on the kinetic parameters of glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi and human

“…Particularly, immobilized enzymes can be implemented as an alternative high-throughput screening (HTS) method aimed at identifying novel drug candidates for the treatment of several life-threatening diseases. [6][7][8] Neglected tropical diseases are widely distributed throughout the tropical and subtropical regions of the world, with significant social and economic impact. 9,10 Chagas' disease, caused by the parasite Trypanosoma cruzi, is a parasitic infection widespread in the Americas, from the great lakes of North America to Southern Patagonia, with devastating consequences in terms of human morbidity and mortality.…”

High-Throughput Affinity-Based Technologies for Small-Molecule Drug Discovery

“…8,9 Based upon data obtained by SPR, kinetic parameters and equilibrium constants of binding (i.e., on-rate, off-rate, and apparent dissociation constants) can be determined. 7,10,11 A recent study of carbonic anhydrase II that compared SPR with the traditional measurement of stopped-flow fluorescence showed that the kinetic parameters determined by SPR are in agreement with those determined by stopped-flow fluorescence.…”

“…8,9 Target protein is immobilized onto a solid surface via covalent or noncovalent interaction. Binding of small-molecule compounds changes the refractive index of the immobilized target, which can be detected by optical sensing.…”