227 Molecular Targets New Targets for an Old Drug II. Hypoxanthine-Guanine Amidophosphoribosyltransferase As a New Pharmacodynamic Target of Methotrexate Jacek J. Slon-Usakiewicz,* Andrew Pasternak, Neil Reid, and Leticia M.Toledo-Sherman † Protana Inc., OptiMol Drug Discovery Division,Toronto, ON, Canada Clinical Proteomics Journal Copyright © Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN 1542-6416/04/01:227–234/$25.00 *Author to whom all correspondence and reprint requests should be addressed: Jacek J. Slon-Usakiewicz, Protana Inc., OptiMol Drug Discovery Division, 251 Attwell Drive, Toronto, ON, M9W 7H4 Canada. E-mail: jslon@protana.com † Present address: Lymphosign Inc., Markham, ON, Canada Abstract 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 methotrex- ate. Here, using a frontal affinity chromatography with mass spectrometry detection, we confirm one of these targets, hypoxanthine-guanine ami- dophosphoribosyltransferase, 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 ex- plain 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.