Differential interactions of the catalytic subunits of adenylyl cyclase with forskolin analogs

Abstract

The diterpene forskolin (FS) binds to, and activates, mammalian membranous adenylyl cyclase (AC) isoforms I–VIII. Diterpenes without C1-OH group do not activate ACs. The C1-OH group forms a hydrogen bond with the backbone oxygen of Val506 of the C1 catalytic subunit of AC (isoform V numbering). To better understand the mechanism of AC activation we examined the interactions of FS and eight FS analogs with purified catalytic AC subunits C1 (AC V) and C2 (AC II) by fluorescence spectroscopy, using 2′,3′-O-(N-methylanthraniloyl)-guanosine 5′-triphosphate (MANT-GTP) as fluorescent reporter probe, and by enzymatic activity. FS analogs induced C1/C2 assembly as assessed by fluorescence resonance energy transfer from Trp1020 of C2 to MANT-GTP and by increased direct MANT-GTP fluorescence in the order of efficacy FS ~ 7-deacetyl-FS ~ 6-acetyl-7-deacetyl-FS ~ 9-deoxy-FS > 7-deacetyl-7-(N-methylpiperazino-γ-butyryloxy)-FS > 1-deoxy-FS ~ 1,9-dideoxy-FS ~ 7-deacetyl-1-deoxy-FS ~ 7-deacetyl-1,9-dideoxy-FS. In contrast, FS analogs activated catalysis in the order of efficacy FS > 7-deacety-FS ~ 6-acetyl-7-deacetyl-FS ~ 9-deoxy-FS > 7-deacetyl-7-(N-methylpiperazino-γ-butyryloxy)-FS ≫ 1-deoxy-FS, 1,9-dideoxy-FS, 7-deacetyl-1-deoxy-FS and 7-deacetyl-1,9-dideoxy-FS (all ineffective). 1-Deoxy-FS analogs inhibited FS-stimulated catalysis by an apparently non-competitive mechanism. Our data suggest a two-step mechanism of AC activation by diterpenes. In the first step, diterpenes, regardless of their substitution pattern, promote C1/C2 assembly. In the second and yet poorly understood step, diterpenes that form a hydrogen bond between C1-OH and Val506 promote a conformational switch that results in activation of catalysis. The apparent non-competitive interaction of FS with 1-deoxy-FS analogs is explained by impaired ligand exchange due to strong hydrophobic interactions with C1/C2.

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

“…These compounds may be useful for future crystallographic and fluorescence spectroscopy studies with sGCa 1 b 1 . By analogy to previous studies with mAC VC1:IIC2 (Mou et al, 2005(Mou et al, , 2006Pinto et al, 2009Pinto et al, , 2011Suryanarayana et al, 2009), such biophysical studies with sGCa 1 b 1 are expected to provide major insights into the molecular mechanisms underlying sGC regulation by NO, sGC activators, and sGC stimulators (Daiber et al, 2010;Derbyshire and Marletta, 2012;Follmann et al, 2013;Stasch and Evegenov, 2013). In fact, Busker et al (2014) has recently reported on the application of (M)ANT-nucleotides for monitoring conformational changes in sGC upon activation.…”

“…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).…”

Adenylate Cyclase/cAMP/Protein Kinase A Signaling Pathway Inhibits Endothelin Type A Receptor-Operated Ca2+ Entry Mediated via Transient Receptor Potential Canonical 6 Channels

“…It is noteworthy that we have found that 1,9-dideoxyforskolin, an inactive analog of forskolin (Pinto et al, 2008(Pinto et al, , 2009, also inhibited ET A R-operated Ca 2ϩ influx via TRPC6 (Supplemental Fig. S3).…”

“…S3). Because it is well known that forskolin can exhibit pleiotropic effects in an AC-independent manner (Laurenza et al, 1989), we examined the effect of 1,9-dideoxyforskolin, an inactive analog of forskolin as a negative control (Pinto et al, 2008(Pinto et al, , 2009, on the ET A R-operated Ca 2ϩ influx via TRPC6. We were surprised to find that 10 M 1,9-dideoxyforskolin as well as 10 M forskolin inhibited the TRPC6-mediated Ca 2ϩ entry (Supplemental Fig.…”

Isoform Selectivity of Adenylyl Cyclase Inhibitors: Characterization of Known and Novel Compounds

“…Although forskolin is an AC activator, inhibitors that target the forskolin binding site have been identified (Pinto et al, 2009;Erdorf et al, 2011). Since our virtual screening focused only on the ATP site, we wanted to rule out that SQ22,536 and the other molecules that inhibited AC activity do not exert their actions through interactions with the forskolin pocket.…”