Mass spectrometry in structure-property relationships studies: allosteric inhibitors of monoacylglycerol lipase and prodrugs of palmitoylethanolamide

Mass spectrometry is a highly versatile and multi faceted analytical technique, which can be successfully applied to the study of structure-property relationships of small molecules, as new drug candidates, and to better characterize macromolecular targets. During the three years of Ph.D. project, mass spectrometry was employed as analytical tool to solve different tasks related to two main research themes: prodrugs of palmitoylethanolamide (PEA) and allosteric inhibitors of the serine hydrolase monoacylglycerol lipase (MGL). Palmitoylethanolamide (PEA), belonging to the family of fatty acid amide lipid mediators, has antinflammatory and antinociceptive properties widely exploited in veterinary and human medicine, despite its poor pharmacokinetics. Looking for prodrugs that could progressively release PEA to maintain effective plasma concentrations, carbonates, esters and carbamates at the hydroxyl group of PEA were tested for their chemical stability (pH=7.4) and stability in rat plasma and liver homogenate by in vitro assays and high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Carbonates and carbamates resulted too labile and too resistant in plasma, respectively. With ester derivatives, prepared by conjugating PEA with various amino acids, it was possible to modulate the kinetics of PEA release in plasma and their stability in liver homogenate. L-Val-PEA, with suitable PEA release in plasma, and D-Val-PEA, with high resistance to hepatic degradation, were orally administered to rats and plasma levels of prodrugs and PEA were measured at different time points. Both prodrugs showed significant release of PEA, but lower than that obtained with equimolar doses of PEA. Amino-acid esters of PEA are a promising class to develop prodrugs, even if they need further chemical optimization. The second research project involved the characterization of the chemical reactivity for a series of benzisothiazolone-based compounds, designed to allosterically inhibit the serine hydrolase MGL, a chief enzyme in the metabolism of the main endocannabinoid, 2-arachidonoylglycerol, by targeting selected cysteine residues known to regulate MGL enzymatic activity. Starting from a lead compound, a series of chemical modifications were made by introducing at position 5 and 6 of the benzisothiazolone ring a set of substituents endowed with opposite electronic properties. The reactivity of the newly synthesized compounds was tested in vitro versus glutathione setting up HPLC-MS/MS analytical methods to detect all reaction species in order to have a crucial information about the oxidative potential of each benzisothiazolinone. The role of these regulatory cysteine residues in the fine tuning of MGL activity, their sensitivity towards the redox state of the surrounding environment and their capacity to respond to changes in the redox potential were further explored in a series of pharmacological assays on purified human MGL. In these experiments, hMGL was pre-incubated for a certain period of time in buffer with different oxidizing or reducing agents, subsequently with a probe (dimedone) for identifying an hypothesized oxidized species of cysteine, sulfenic acid. Employing MALDI-TOF/TOF mass spectrometry on oxidized hMGL after trypsin digestion, it was possible to identify which cysteine residue was oxidized to sulfenic acid and which cysteine-containing peptide had been covalently modified by dimedone. Finally, during a three months period spent in the laboratory of Prof. Kasper Rand, at the university of Copenhagen, the conformational changes in hMGL following the interaction with a benzisothiazolone inhibitor were investigated, employing the hydrogen/deuterium exchange mass spectrometry technique (H/DX-MS).