9-Benzoyl 9-deazaguanines as potent xanthine oxidase inhibitors.

A novel potent xanthine oxidase inhibitor, 3-nitrobenzoyl 9-deazaguanine (LSPN451), was selected from a series of 10 synthetic derivatives. The enzymatic assays were carried out using an on-flow bidimensional liquid chromatography (2D LC) system, which allowed the screening¸ the measurement of the kinetic inhibition constant and the characterization of the inhibition mode. This compound showed a non-competitive inhibition mechanism with more affinity for the enzyme-substrate complex than for the free enzyme, and inhibition constant of 55.1±9.80 nM, about thirty times more potent than allopurinol. Further details of synthesis and enzymatic studies are presented herein.

Angiotensin converting enzyme immobilized on magnetic beads as a tool for ligand fishing.

Angiotensin converting enzyme (ACE) presents an important role in blood pressure regulation, since that converts angiotensin I to the vasoconstrictor angiotensin II. Some commercially available ACE inhibitors are captopril, lisinopril and enalapril; due to their side effects, naturally occurring inhibitors have been prospected. In order to endorse this research field we have developed a new tool for ACE ligand screening. To this end, ACE was extracted from bovine lung, purified and chemically immobilized in modified ferrite magnetic beads (ACE-MBs). The ACE-MBs have shown a Michaelian kinetic behavior towards hippuryl-histidyl-leucine. Moreover, as proof of concept, the ACE-MBs was inhibited by lisinopril with a half maximal inhibitory concentration (IC50) of 10nM. At the fishing assay, ACE-MBs were able not only to fish out the reference inhibitor, but also one peptide from a pool of tryptic digested BSA. In conclusion, ACE-MBs emerge as new straightforward tool for ACE kinetics determination, inhibition and binder screening.

New trends in LC protein ligand screening.

The diversity of small molecules available to produce truly innovative drugs associated with the wealth of known biological targets calls for key strategies in protein ligand screening. This review encompasses the recently developed bioaffinity-based strategies. A critical view of the use of zonal, frontal, and nonlinear chromatography with immobilized proteins is given. The association of these elution modes with the ligand fishing method, which uses nanomagnetics particles, is also addressed. A series of applications and how these new screening strategies can be used to determine the function, affinity, and activity parameters of proteins is discussed.

Acetylcholinesterase immobilized capillary reactors-tandem mass spectrometry: an on-flow tool for ligand screening.

The use of immobilized capillary enzyme reactors (ICERs) for online ligand screening has been adopted as a new technique for high-throughput screening (HTS). In this work, the selected target was the enzyme acetylcholinesterase (AChE), and the AChE-ICERs produced were used in a liquid chromatograph-tandem ion-trap mass spectrometer. The activity and kinetic parameters were evaluated by monitoring the choline's precursor ion (M + H)(+)m/z 104.0 and its ion fragment (C2H3OH) - (M + H)(+)m/z 60.0. The assay method was validated using the reference AChE inhibitors tacrine and galanthamine. Two new ligands, out of a library of 17 coumarin derivatives, were identified, and the half-maximal inhibitory concentration (IC50), inhibition constant (K(i)), and the inhibition mechanism were determined. A coumarin derivative with IC50 similar to tacrine was highlighted.