'Enzymes' immobilization on monolithic matrices: applications in drug discovery'

During the last decade new combinatorial techniques have been developed that allow for synthesis of vast quantities of potential therapeutic compounds. At the same time, in parallel with the synthetic process, analytical techniques must be capable of screening a large number of compounds in a high-throughput manner with precision and accuracy. In the last few years, immobilised enzyme reactors (IMERs) on monolithic matrices have been developed by numerous groups and have proven to be a useful and economic alternative to conventional in-solution methods, when automation and fast data output are required. In particular, considering the high cost and difficulty in over-expression, isolation and purification of recombinant enzymes, this analytical technique represents an extremely useful approach to preserve the activity of the small amount of enzyme available. In the IMER format, enzymes are ready to be reused and can be coupled to chromatographic systems and appropriate detectors. This coupling generally increases automation, reproducibility and analyses accuracy and reduces sample handling and operator time consumption. Monoliths are considered a novel generation of stationary phases whose special feature is the fast separation and enzymatic conversion due to lack of diffusion resistance during mass transfer. The large pores of monolithic materials allow high-speed analysis and low back pressure. Preliminary experiments carried out with enzymes immobilized onto compact porous supports have shown that such enzyme reactors have much higher rates of conversion than the reactors in which enzymes have been immobilized onto bulk porous supports. In particular, monolithic disks based on a new polymeric macroporous material are chosen because promising for analytical application due to reduced time analysis and high enzyme efficiency. Monolithic disks are characterized by millimetric dimensions and consist of a monolithic stationary phase as chromatographic support for immobilization of enzymes and ligands In the field of drug discovery, monolithic IMERs can be reliably applied to rapidly screen for potential drugs candidates to lead selection, to perform potent inhibitor’s kinetic studies, and for ADMET parameters determination, an interesting application being for the early estimates of the drug metabolism, toxicity and possible drug-drug interactions. In this chapter, a few IMERs applications will be presented, including cholinesterase and BACE-1 (beta secretase) immobilized reactors, which were validated for the determination of the mechanism of action and inhibitory constants of new leads for the treatment of Alzheimer’s disease in a highly reliable and automated mode. Moreover, the preparation of monolithic disk-shaped mini-columns (2 mm x 6 mm I.D.) containing immobilized cytochrome P450 as tools for drug metabolim studies, will be described.