Fluorometric detection of protein-ligand engagement: The case of phosphodiesterase5.
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.
['Di Rocco|Giulia|G|', 'Martinelli|Ilaria|I|', 'Pacifico|Salvatore|S|', 'Guerrini|Remo|R|', 'Cichero|Elena|E|', 'Fossa|Paola|P|', 'Franchini|Silvia|S|', 'Cardarelli|Silvia|S|', 'Giorgi|Mauro|M|', 'Sola|Marco|M|', 'Ponterini|Glauco|G|']
D020134:Catalytic Domain D002626:Chemistry, Pharmaceutical D006152:Cyclic GMP D054706:Cyclic Nucleotide Phosphodiesterases, Type 5 D031541:Fluorescence Resonance Energy Transfer D005456:Fluorescent Dyes D006801:Humans D007527:Isoenzymes D008024:Ligands D008956:Models, Chemical D062105:Molecular Docking Simulation D058986:Phosphodiesterase 5 Inhibitors D011485:Protein Binding D011994:Recombinant Proteins D012235:Rhodamines D000068581:Tadalafil
Förster resonance energy transfer (FRET) cGMPS-rhodamine Affinity constant (K(d)) Competitive displacement analysis Fluorometric ligand binding detection Phosphodiesterase 5