Abstract
Phosphodiesterases (PDEs) regulate the intracellular levels of cAMP and cGMP. The great clinical success of the PDE5 inhibitors, Sildenafil (Viagra), Vardenafil (Levitra) and Tadalafil (Cialis) has led to an increasing interest for this class of enzymes. Recent studies have shown a correlation between tumor growth and PDE5 overexpression, making PDE5-selective inhibitors promising candidates for cancer treatment. The search for such inhibitors rests today on radioactive assays. In this work, we exploit the conserved catalytic domain of the enzyme and propose a faster and safer method for detecting the binding of ligands and evaluate their affinities. The new approach takes advantage of Förster Resonance Energy Transfer (FRET) between, as the donor, a fluorescein-like diarsenical probe able to covalently bind a tetracysteine motif fused to the recombinant PDE5 catalytic domain and, as the acceptor, a rhodamine probe covalently bound to the pseudosubstrate cGMPS. The FRET efficiency decreases when a competitive ligand binds the PDE5 catalytic site and displaces the cGMPS-rhodamine conjugate. We have structurally investigated the PDE5/cGMPS-rhodamine complex by molecular modelling and have used the FRET signal to quantitatively characterize its binding equilibrium. Competitive displacement experiments were carried out with tadalafil and cGMPS. An adaptation of the competitive-displacement equilibrium model yielded the affinities for PDE5 of the incoming ligands, nano- and micromolar, respectively.
Highlights
Phosphodiesterase enzymes (PDEs) are phosphohydrolases that selectively hydrolyse ubiquitous second messengers thereby regulating their signaling pathways and downstream biological effects [1,2,3]
The new approach takes advantage of Förster Resonance Energy Transfer (FRET) between, as the donor, a fluorescein-like diarsenical probe able to covalently bind a tetracysteine motif fused to the recombinant Phosphodiesterase 5 (PDE5) catalytic domain and, as the acceptor, a rhodamine probe covalently bound to the pseudosubstrate cGMPS
The sequence corresponding to the C-terminal catalytic domain of PDE5A2 was cloned in two different forms: the wt (PDE5C) and the tetracysteine-tagged catalytic domain (PDE5CTC)
Summary
Phosphodiesterase enzymes (PDEs) are phosphohydrolases that selectively hydrolyse ubiquitous second messengers (cGMP and cAMP) thereby regulating their signaling pathways and downstream biological effects [1,2,3]. Di Rocco et al / Journal of Pharmaceutical and Biomedical Analysis 149 (2018) 335–342 we demonstrate that the tetracysteine-tag technology applied to the PDE5 catalytic domain (PDE5C, coding region Glu536-Gln860) is a promising approach in this respect. This method exploits the binding of a fluorescent probe, the fluorescein diarsenical hairpin binder (FlAsH), to a short peptide sequence which includes four cysteine residues, namely Cys-Cys-Xaa-Yaa-Cys-Cys (CCXYCC, in which X and Y denote any amino acid) to be engineered on the target protein [8,9]. Because the presently available X-ray diffraction structures for PDEs are limited to the catalytic domains, the homology model of the related human PDE5A2 isoform created by us was limited to this portion of the protein
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