A New Mechanism of Methotrexate Action Revealed by Target Screening with Affinity Beads

Abstract

Methotrexate (MTX) is the anticancer and antirheumatoid drug that is believed to block nucleotide synthesis and cell cycle by inhibiting dihydrofolate reductase activity. We have developed novel affinity matrices, termed SG beads, that are easy to manipulate and are compatible with surface functionalization. Using the matrices, here we present evidence that deoxycytidine kinase (dCK), an enzyme that acts in the salvage pathway of nucleotide biosynthesis, is another target of MTX. MTX modulates dCK activity differentially depending on substrate concentrations. 1-␤-D-Arabinofuranosylcytosine (ara-C), a chemotherapy agent often used in combination with MTX, is a nucleoside analog whose incorporation into chromosome requires prior phosphorylation by dCK. We show that, remarkably, MTX enhances incorporation and cytotoxicity of ara-C through regulation of dCK activity in Burkitt's lymphoma cells. Thus, this study provides new insight into the mechanisms underlying MTX actions and demonstrates the usefulness of the SG beads.

Novel Applications of Affinity Beads

“…To identify novel MTX-binding proteins, we prepared two types of SG beads to which MTX was immobilized through different functional groups, either using the amino group of an MTX amino derivative or using the two carboxyl groups of MTX itself ( Fig. 3-10) [12]. An MTX amino derivative bearing an amino group instead of the γ-carboxyl group was synthesized and fixed to SGNE-COOH beads, the SG beads carrying carboxyl groups, through the amino group by amidation ( Fig.…”

Salicylic Acid Induces Mitochondrial Injury by Inhibiting Ferrochelatase Heme Biosynthesis Activity

“…To better understand the mechanism of action of drugs such as salicylic acid, we previously developed high-performance affinity beads that allow one-step affinity purification of drug target proteins from crude cell extracts (Sakamoto et al, 2009). By using this methodology, we successfully identified new molecular targets of pharmacological drugs and elucidated novel cellular mechanisms responsible for their actions (Shimizu et al, 2000;Uga et al, 2006;Sakamoto et al, 2009;Ito et al, 2010). In the present work, this technology was used to identify ferrochelatase (FECH) (EC 4.99.1.1) as a novel target of salicylic acid that is involved in its mitochondrial actions.…”

A New Mechanism of 6-((2-(Dimethylamino)ethyl)amino)-3-hydroxy-7H-indeno(2,1-c)quinolin-7-one Dihydrochloride (TAS-103) Action Discovered by Target Screening with Drug-Immobilized Affinity Beads

“…They enable the rapid and efficient purification of ligand-or drug-binding proteins (Ohtsu et al, 2005). These high-performance affinity beads have been used successfully for purification of various proteins, including transcription factors and drug receptors (Shimizu et al, 2000;Uga et al, 2006) and cisplatin-damaged DNA binding proteins (Tomohiro et al, 2002).…”