Unveiling the binding mode of adenosine deaminase inhibitors to the active site of the enzyme: implication for rational drug design.

Limongelli and colleagues describe a novel class of potent adenosine deaminase (ADA) inhibitors, developed as drug candidate for the treatment of inflammatory disorders [1]. Since a close correlation has been found between the severity of inflammation and a local increase in both expression and activity of ADA [2], the pharmacological inhibition of this enzyme has recently being regarded as a novel therapeutic approach to counteract inflammation in several pathological conditions. Actually, blockade of the irreversible deamination of adenosine to inosine, normally catalyzed by the enzyme, leads to an increased availability of the biologically active purine at the site of inflammation. Adenosine, in turn, may modulate purinergic responses to these pathological events. Pursuing their interest in this research field [3–6], and exploiting their synthetic expertise in the synthesis of heterocyclic compounds, authors propose a number of pirazolo[ 1,5-a]pyrimidin-7-one derivatives bearing suitable alkyl and arylalkyl substituents in the position 4 of the heterocyclic core, which have been shown to inhibit ADA with Ki values in the nanomolar range. Although successful in providing novel ADA inhibitors, authors recognize the difficulty of unveiling their mechanism of binding to the active site of the enzyme by means of docking calculations. Actually, both the high flexibility of the protein and the role played by the solvent in accommodating the novel compounds into the ADA site increase the complexity of the interaction between ligands and the target enzyme, thus preventing docking algorithms to clearly describe the binding event at a molecular level. In the PNAS paper, Limongelli and colleagues brightly overcame this drawback by exploiting well-tempered metadynamics, an emerging technique which allows dealing with protein motion and solvation during ligand binding. With this in hand, they clarified the binding mode of the most active compound, 4-decyl-5-methylpyrazolo[1,5-a]pyrimidin- 7(4H)-one, thus reconstructing the free-energy profile of the ligand–enzyme interaction and highlighting the preferred binding mode of the ligand inside the protein. Besides representing a novel class of ADA inhibitors, the pyrazolopyrimidine here described is an excellent case study of sampling protein motion and solvent effect during ligand binding.