No abstract

High-throughput screening (HTS) efforts to discover "hits" typically rely on the large-scale parallel screening of individual compounds with attempts to screen mixtures of compounds typically and, unfortunately, giving rise to false positives and false negatives due to the nature of the HTS readout (% inhibition/activation above a defined threshold) that makes deconvolution virtually intractable. Bioaffinity screening methods have emerged as an alternative or orthogonal method to classic HTS. One of these methods, frontal affinity chromatography coupled to mass spectrometry detection (FAC-MS), although still a relatively new technique, is turning out to be a viable screening tool. However, to push FAC-MS more to the forefront as a moderate primary HTS system (or a secondary screening assay), automation needs to be addressed. An automated FAC-MS system is described using 2 columns containing immobilized hERbeta, whereby while 1 column is being regenerated, the other is being used. The authors are extrapolating that in a continuous 24-h operation, the number of ligands screened could potentially approach 10,000. In addition, preliminary structure-activity relationship binding information (typically not seen in early primary HTS) can be obtained by observing the rank order of the library members in the various mixtures.

Methotrexate has been a clinical agent used in cancer, immunosuppression, rheumatoid arthritis, and other highly proliferative diseases for many years, yet its underlying molecular mechanism of action in these therapeutic areas is still unclear. We have previously reported using a chemical proteomics technique on several other potential pharmacodynamic targets of methotrexate. Here, using a frontal affinity chromatography with mass spectrometry detection, we confirm one of these targets, hypoxanthine-guanine amidophosphoribosyltransferase, as a true binder of methotrexate with a K d of 4.2 M. These results complement and confirm our recent study, but more importantly, shed light into the mechanism of action of methotrexate in oncology and other highly proliferative diseases and may help explain some unaccounted for effects of this drug. For example, despite the fact that DNA salvage pathway enzymes are highly active, methotrexate can be effective if it only targets enzymes of the de novo pathway.