Mass spectrometry (MS) has recently emerged as an efficient analytical tool in early drug discovery, thanks to its ability to obtain detailed information on covalent and non-covalent binding between small and large molecules with protein or nucleic acid targets. Non-covalent protein binding by native (not denaturating conditions) MS is mainly focused on identifying ligands by fishing libraries or using fragment based approaches. Top-down and bottom-up MS approaches are both used to obtain information on the covalent modifications of proteins induced by damaging or bioactive, endogenous or xenobiotic compounds and are particularly useful for identifying the adducted protein/s in complex matrices, the stoichiometry of reaction, the aa site undergoing biotransformation, the reaction products and the mechanism of the covalent modification. Covalent binding of proteins has been extensively used in our lab in parallel to native MS, for different applications in drug discovery and development, including drug target identification, biological activity screening, pre-ADMET, and elucidation of the mechanism of action of bioactive compounds. In more detail, by using MS we recently found that protein covalently modified by reactive carbonyl species (RCS) which are generated by lipid-oxidation and metabolism, as well as by non-enzymatic glycation, are potential drug targets for the development of bioactive compounds effective in oxidative based diseases, including atherosclerosis, diabetes related diseases and neurological disorders [1-3]. By considering AGEs and ALEs as drug targets, a high resolution MS application has been set-up to screen compounds effective as inhibitors of such damaging oxidation products. The method consists of measuring the extent of protein modification induced by the most damaging RCS, including glyoxal, methylglyoxal, acrolein and 4-hydroxynonenal as well as by glycating reducing sugars, in the presence and in the absence of the potential inhibitors. Ubiquitin was chosen as model protein target and orbitrap as MS analyzer. The method is suitable for measuring non deconvoluted libraries as well as natural and complex mixtures. High resolution MS has been successfully applied to studying the ability of drug or metabolite to covalently bind protein with a view to predicting potential idiosyncratic reactions as well as to fully detail the protein binding. In this context a MS strategy based on precursor ion scanning has recently been set-up, in order to fully elucidate the protein haptenation of amoxicillin and in particular the main serum protein target, the adducted aminoacids, and the mechanism of haptenation , thus providing a novel insight for the study of protein haptenation and the mechanisms involved in penicillin-elicited allergic reactions. Further applications of protein covalent binding studied by MS are represented by the detection and quantification of protein oxidation products as reliable biomarker of oxidative damage, such as hydroxynonenal albumin adducts, glutationylated haemoglobin and cysteinylated albumin. References  Aldini G et al. J Mass Spectrom., 2008, 43, 1470-81.  Aldini G et al. Chem Res Toxicol. 2008,21, 824-35.  Aldini G et al. Biochemistry, 2007, 46, 2707-18.  Ariza A et al. J Proteomics, 2012, 21, 504-20.
|Titolo:||Mass spectrometry of covalent protein binding: applications in drug discovery|
|Data di pubblicazione:||1-lug-2013|
|Settore Scientifico Disciplinare:||Settore CHIM/08 - Chimica Farmaceutica|
|Citazione:||Mass spectrometry of covalent protein binding: applications in drug discovery / G. Aldini. ((Intervento presentato al 24. convegno International symposium on pharmaceutical and biomedical analysis (PBA) tenutosi a Bologna nel 2013.|
|Appare nelle tipologie:||03 - Contributo in volume|