RESUMO
We have developed a dual enzymatic system assay involving liquid chromatography-mass spectrometry (LC-MS) to screen AChE and BACE1 ligands. A fused silica capillary (30 cm × 0.1 mm i.d. × 0.362 mm e.d.) was used as solid support. The co-immobilization procedure encompassed two steps and random immobilization. The resulting huAChE+BACE1-ICER/MS was characterized by using acetylcholine (ACh) and JMV2236 as substrates. The best conditions for the dual enzymatic system assay were evaluated and compared to the conditions of the individual enzymatic system assays. Analysis was performed in series for each enzyme. The kinetic parameters (KMapp) and inhibition assays were evaluated. To validate the system, galantamine and a ß-secretase inhibitor were employed as standard inhibitors, which confirmed that the developed screening assay was able to identify reference ligands and to provide quantitative parameters. The combination of these two enzymes in a single on-line system allowed possible multi-target inhibitors to be screened and identified. The innovative huAChE+BACE1-ICER/MS dual enzymatic system reported herein proved to be a reliable tool to identify and to characterize hit ligands for AChE and BACE1 in an enzymatic competitive environment. This innovative system assay involved lower costs; measured the product from enzymatic hydrolysis directly by MS; enabled immediate recovery of the enzymatic activity; showed specificity, selectivity, and sensitivity; and mimicked the cellular process.
RESUMO
BACKGROUND: Virtual methodologies have become essential components of the drug discovery pipeline. Specifically, structure-based drug design methodologies exploit the 3D structure of molecular targets to discover new drug candidates through molecular docking. Recently, dual target ligands of the Adenosine A2A Receptor and Monoamine Oxidase B enzyme have been proposed as effective therapies for the treatment of Parkinson's disease. METHODS: In this paper we propose a structure-based methodology, which is extensively validated, for the discovery of dual Adenosine A2A Receptor/Monoamine Oxidase B ligands. This methodology involves molecular docking studies against both receptors and the evaluation of different scoring functions fusion strategies for maximizing the initial virtual screening enrichment of known dual ligands. RESULTS: The developed methodology provides high values of enrichment of known ligands, which outperform that of the individual scoring functions. At the same time, the obtained ensemble can be translated in a sequence of steps that should be followed to maximize the enrichment of dual target Adenosine A2A Receptor antagonists and Monoamine Oxidase B inhibitors. CONCLUSION: Information relative to docking scores to both targets have to be combined for achieving high dual ligands enrichment. Combining the rankings derived from different scoring functions proved to be a valuable strategy for improving the enrichment relative to single scoring function in virtual screening experiments.