Split and Mix Techniques
Assays are being developed in order to tackle other challenging problems. Michael Organ, Ph.D., of the department of chemistry, York University, Toronto, pointed out that screening mixtures of compounds has presented difficulties in the past because of an averaging effect of assay results which makes it hard to single out those compounds with true potential.
"This has produced a tendency to shy away from mixtures," said Dr. Organ. Yet not rising to this challenge may mean that companies are missing out on valuable combinatorial chemistry collections synthesized by "split and mix" techniques and natural product extracts.
Dr. Organ's team has been developing a technique called Frontal Affinity Chromatography (FAC), in which the target is trapped within a matrix, bypassing problems posed by chemical binding of the protein to the column. Then the mixture of compounds to be screened against the target is infused onto the column.
The most weakly bound compound comes off first, and the mixture is analyzed on a mass spectrometer (MS) which allows real-time analysis. FAC/MS has shown promise with a mixture of 100 tripeptides binding onto thrombin, and the team has also looked at Factor Xa, a clotting protein that is deactivated by conventional affinity chromatography.
Another new technology, with many potential screening applications, is Respirometric Screening Technology (RST) from Luxcel Biosciences (www.luxcel.com), which uses fluorescence quenching of a probe to give a measure of oxygen uptake by cells, organisms, oxygen-dependent enzymes, and metabolites.
"RST is amenable for the development of very sensitive cell-based assays," said Richard Fernandes, Ph.D., founder and CEO of Luxcel.
Oxygen uptake is an important biomarker, giving a very direct assessment of mitochondrial function. It is used as an early indication of apoptosis, metabolic activity, and can also give a good indication of the mechanism of toxicology. Luxcel is working with many Big Pharma companies, applying RST in toxicological screening and ADME studies.