Abstract
2′,3′-O-(N-Methylanthraniloyl)-ITP (MANT-ITP) is the most potent inhibitor of mammalian membranous adenylyl cyclase (mAC) 5 (AC5, Ki, 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 (Ki, 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 Gsα- 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.
Footnotes
↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
This work was supported by the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases [Grant DK46371] (to S.R.S); the Deutsche Forschungsgemeinschaft [Grant Se 529/5–2] (to R.S.); and the Elite Network of Bavaria (graduate research scholarship to M.H.).
Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
doi:10.1124/mol.111.071894.
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ABBREVIATIONS:
- AC
- adenylyl cyclase
- MANT
- 2′,3′-O-(N-methylanthraniloyl)
- FS
- forskolin
- GTPγS
- guanosine 5′-[γ-thio]triphosphate
- MES
- 2-(N-morpholino)ethanesulfonic acid
- PDB
- Protein Data Bank
- GBSA
- Generalized Born, augmented by solvent-accessible surface
- FRET
- fluorescence resonance energy transfer
- λex
- excitation wavelength
- λem
- emission wavelength
- mAC
- mammalian membranous adenylyl cyclase
- VCI and IIC2
- the N- and C-terminal catalytic domains, respectively, from canine AC5 and rat AC2 expressed as soluble proteins.
- Received February 20, 2011.
- Accepted April 14, 2011.
- Copyright © 2011 The American Society for Pharmacology and Experimental Therapeutics
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