Structural Basis for the High-Affinity Inhibition of Mammalian Membranous Adenylyl Cyclase by 2′,3′-O-(N-Methylanthraniloyl)-Inosine 5′-Triphosphate

Abstract

2Ј,3Ј-O-(N-Methylanthraniloyl)-ITP (MANT-ITP)is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, K i , 1 nM) yet discovered and surpasses the potency of MANT-GTP by 55-fold (J Pharmacol Exp Ther 329: 1156 -1165, 2009). AC5 inhibitors may be valuable drugs for treatment of heart failure. The aim of this study was to elucidate the structural basis for the high-affinity inhibition of mAC by MANT-ITP. MANT-ITP was a considerably more potent inhibitor of the purified catalytic domains VC1 and IIC2 of mAC than MANT-GTP (K i , 0.7 versus 18 nM). Moreover, there was considerably more efficient fluorescence resonance energy transfer between Trp1020 of IIC2 and the MANT group of MANT-ITP compared with MANT-GTP, indicating optimal interaction of the MANT group of MANT-ITP with the hydrophobic pocket.The crystal structure of MANT-ITP in complex with the G s ␣-and forskolin-activated catalytic domains VC1:IIC2 compared with the existing MANT-GTP crystal structure revealed only subtle differences in binding mode. The higher affinity of MANT-ITP to mAC compared with MANT-GTP is probably due to fewer stereochemical constraints upon the nucleotide base in the purine binding pocket, allowing a stronger interaction with the hydrophobic regions of IIC2 domain, as assessed by fluorescence spectroscopy. Stronger interaction is also achieved in the phosphate-binding site. The triphosphate group of MANT-ITP exhibits better metal coordination than the triphosphate group of MANT-GTP, as confirmed by molecular dynamics simulations. Collectively, the subtle differences in ligand structure have profound effects on affinity for mAC.

Structure/Activity Relationships of (M)ANT- and TNP-Nucleotides for Inhibition of Rat Soluble Guanylyl Cyclase α1β1

“…When bound to mAC VC1:IIC5, the (M)ANT group acts as a wedge between b1-a1-a2 of VC1 and b79-b89 of IIC2, preventing the formation of the "fully closed", catalytically active conformation (Mou et al, 2005). It is very reasonable to suggest similar binding modes of (M)ANT nucleotides to mAC and sGC by the following reasons: 1) Without the hypoxanthine derivatives with explicit causes of mAC selectivity [hydrogen bonds of the base with Lys938 and Asp1018 (Hübner et al, 2011), replaced by Glu473 and Cys541, respectively, in sGCb], the correlations of sGC and mAC pK i values (Fig. 2) substantially increase (sGC versus mAC1: r, 0.62, versus mAC2: r, 0.68, mAC5: r, 0.67).…”

“…4C). Since the docking poses of the triphosphate groups were derived from the mAC templates (Mou et al, 2005(Mou et al, , 2006Hübner et al, 2011), the given conformations are only exemplary. The great flexibility of the triphosphate chain, of the surrounding residues, and of the Mn 21 positions enable different fits with, however, conserved interactions.…”

“…The recently published (Allerston et al, 2013) crystal structure of an sGCacat/sGCbcat heterodimer [Protein Data Bank (PDB) ID 3UVJ] represents a catalytically inactive, "open" conformation and could therefore not be used directly for docking studies. However, several structures of mAC VC1:IIC2 in complex with (M)ANT-and TNP-nucleotides (PDB IDs 1TL7, 1U0H, 2GVZ, 3G82, and 2GVD) (Mou et al, 2005(Mou et al, , 2006Hübner et al, 2011) may serve as templates for the generation of a "partially closed" sGC conformation that binds this class of inhibitors. mAC VC1:IIC2 in complex with 39-MANT-GTP (PDB ID 1TL7) was selected because of the highest resolution (2.8 Å).…”

“…In contrast, the "fully closed" active state of mACs is well known from several structures of the heterodimeric hybrid VC1:IIC2 (Tesmer et al, 1997(Tesmer et al, , 2000. A major technical advance in the resolution of crystal structures of VC1:IIC2 was achieved by the use of high-affinity competitive mAC inhibitors, i.e., TNP [29,39-O-(2,4,6-trinitrophenyl)]-and (M) ANT [29,39-O-(N-methylanthraniloyl)]-substituted NTPs as stabilizing ligands (Mou et al, 2005(Mou et al, , 2006Hübner et al, 2011). These nucleotides bind to the catalytic site of VC1:IIC2, and the TNP or (M)ANT group projects into a pocket adjacent to the catalytic site, conferring high affinity of the ligands for mACs and stabilizing inactive mAC conformations between the open and closed states (Mou et al, 2005(Mou et al, , 2006Hübner et al, 2011;.…”

“…A major technical advance in the resolution of crystal structures of VC1:IIC2 was achieved by the use of high-affinity competitive mAC inhibitors, i.e., TNP [29,39-O-(2,4,6-trinitrophenyl)]-and (M) ANT [29,39-O-(N-methylanthraniloyl)]-substituted NTPs as stabilizing ligands (Mou et al, 2005(Mou et al, , 2006Hübner et al, 2011). These nucleotides bind to the catalytic site of VC1:IIC2, and the TNP or (M)ANT group projects into a pocket adjacent to the catalytic site, conferring high affinity of the ligands for mACs and stabilizing inactive mAC conformations between the open and closed states (Mou et al, 2005(Mou et al, , 2006Hübner et al, 2011;. Hydrophobic interactions of the TNP-or (M)ANT-nucleotides were also explored to monitor conformational changes in mACs by fluorescence spectroscopy, providing major new insights into the dynamics of enzyme regulation (Mou et al, 2005(Mou et al, , 2006Pinto et al, 2009Pinto et al, , 2011Suryanarayana et al, 2009;Hübner et al, 2011).…”