Interactors and allosteric effectors of serine racemase

Abstract

Serine racemase is a pyridoxal phosphate-dependent enzyme responsible for the synthesis of D-serine in the central nervous system. D-serine is a co-agonist of the glutamate NMDA receptors, thus playing an important role in glutamatergic neurotransmission. Serine racemase has been recently recognized as a drug target for the control of D-serine levels, recently associated to a number of pathological conditions. This thesis work was focused on the characterization of the interaction of serine racemase with allosteric effectors and interactors, particularly divalent cations, NADH, DISC1 and glyceraldehyde 3-phosphate dehydrogenase (GADH). Divalent cations, Mg2+ and Ca2+, stabilize the enzyme and increase the activity of serine racemase, but with different affinity. Together with ATP, they modulate the quaternary equilibrium of the protein. NADH partially inhibits serine racemase through the N-substituted 1,4-dihydronicotinic ring, which was predicted to bind at a site adjacent to the ATP binding site. NADH binds with a binding constant much higher than its intracellular concentrations, but some of its derivatives exhibited a much higher affinity, thus suggesting a novel class of inhibitors. DISC1, a recently discovered interactor of serine racemase, was expressed in insect cells using the baculovirus expression system, at the Institute of biomedical research of Strasbourg, (Institut Génétique Biologie Moléculaire Cellulaire ). A preliminary characterization confirmed that it binds and stabilizes serine racemase. Human GAPDH, contrary to what was suggested in a recent publication, did not seem to interact with human serine racemase and an alternative explanation for the observed effect was suggested. GAPDH was also used to characterize novel inhibitors to be used as antiprotozoal or anti-cancer drugs: 3-bromo isoxazolines and 2-phenoxy-1,4-naphthoquinone derivatives.