2′(3′)-O-(N-methylanthraniloyl)-substituted GTP analogs: A novel class of potent competitive adenylyl cyclase inhibitors

Abstract

2′(3′)-O-(N-Methylanthraniloyl)-(MANT)-substituted nucleotides are fluorescent and widely used for the kinetic analysis of enzymes and signaling proteins. We studied the effects of MANT-guanosine 5′-[γ-thio]triphosphate (MANT-GTPγS) and MANT-guanosine 5′-[β,γimido]triphosphate (MANT-GppNHp) on Gαs- and Gαiprotein-mediated signaling. MANT-GTPγS/MANT-GppNHp had lower affinities for Gαs and Gαi than GTPγS/GppNHp as assessed by inhibition of GTP hydrolysis of receptor-Gα fusion proteins. MANT-GTPγS was much less effective than GTPγS at disrupting the ternary complex between the formyl peptide receptor and Gαi2. MANT-GTPγS/MANT-GppNHp non-competitively inhibited GTPγS/GppNHp-, AlF4-, β2-adrenoceptor plus GTP-, cholera toxin plus GTP-, and forskolin-stimulated adenylyl cyclase (AC) in Gαs-expressing Sf9 insect cell membranes and S49 wild-type lymphoma cell membranes. AC inhibition by MANT-GTPγS/MANT-GppNHp was not due to Gαs inhibition because it was also observed in Gαs-deficient S49 cyc- lymphoma cell membranes. Mn2+ blocked AC inhibition by GTPγS/GppNHp in S49 cyc- membranes but enhanced the potency of MANT-GTPγS/MANT-GppNHp at inhibiting AC by ∼4-8-fold. MANT-GTPγS and MANT-GppNHp competitively inhibited forskolin/Mn2+-stimulated AC in S49 cyc- membranes with Ki values of 53 and 160 nM, respectively. The Ki value for MANT-GppNHp at insect cell AC was 155 nM. Collectively, MANT-GTPγS/MANT-GppNHp bind to Gαs- and Gαi-proteins with low affinity and are ineffective at activating Gα. Instead, MANT-GTPγS/MANT-GppNHp constitute a novel class of potent competitive AC inhibitors.