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Innovative high resolution mass spectrometry applications in drug discovery
No full textIn the past, the main applications of mass spectrometry in the pharmaceutical field
were mainly oriented towards structure elucidation and quantitative analysis of drug
and metabolites in biological matrices. Since the advent in the 90’s of soft ion sources
such as ESI and MALDI, allowing the analysis of biomacromolecules (biopolymers
such as DNA, proteins, peptides and sugars), MS has found growing applications in the
different steps of drug discovery and development processes, including target
identification, hit identification and lead identification and optimization.
In particular, ESI source is the workhorse for the mass spectrometric analysis of
noncovalent bound protein complexes since it allows intact weakly bound complexes to
be detected reflecting the nature of the interaction found in the condensed phase.
Several applications of such techniques have been reported, the most popular being
the identification of selective and high affinity ligands by fishing libraries or using
fragment based approaches. In particular, ligands are easily identified on the basis of
the MW of the protein adduct and their affinity established on the basis of their relative
abundance. The strength of this technique lies in its inherent sensitivity (picomolar
quantities of protein and ligand required), its speed (as low as 30 s for each analysis),
and its ability to simultaneously determine binding stoichiometry and dissociation
constants. A further improvement in studying non covalent interactions came about
more recently with the advent of nano-ESI source and high resolution MS analyzers
such as orbitrap. Several applications have been reported and we have successfully
applied this technique to screen targeted libraries against proteins [1,2] and nucleic
acid [3].
Moreover, the effect of ligand interaction on protein conformation can be evaluated by
studying the distribution of multicharged ions recorded in non denaturating conditions
as well as the protein aa involved in the ligand recognition by H/D exchange
experiments.
The most recent investigation indicates that MS is also an excellent method for
determining binding strength of protein-protein interactions within noncovalent
complexes. The substantial advantages of MS over other techniques (e.g., ITC) are
sensitivity and the ability to provide information on composition, stoichiometry and
subunit interactions of protein complexes.
References
[1] L. Regazzoni, L. Bertoletti, G. Vistoli et al., ChemMedChem, 2010, 5, 1015-1025
[2] L. Regazzoni, R. Colombo, L. Bertoletti et al., Anal Chim Acta, 2011
[3] F. Riccardi Sirtori, G. Aldini, M. Colombo et al., submitte
Mass spectrometry of covalent protein binding: applications in drug discovery
No full textMass 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 [4], 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
[1] Aldini G et al. J Mass Spectrom., 2008, 43, 1470-81.
[2] Aldini G et al. Chem Res Toxicol. 2008,21, 824-35.
[3] Aldini G et al. Biochemistry, 2007, 46, 2707-18.
[4] Ariza A et al. J Proteomics, 2012, 21, 504-20
Carnosine derivatives as novel RCS sequestering agents in preventing protein carbonylation and related cellular dysfunction
No full textSeveral evidences strongly support a pathogenic role for Reactive Carbonyl Species (RCS), such as in the case of diabetic-related diseases, age-dependent tissue dysfunction, and metabolic distress syndrome. Hence, RCS can be considered a potential biological target for drug discovery. The most promising pharmacological strategy to neutralize/reduce RCS is based on nucleophilic compounds capable to form covalent and unreactive adducts with RCS (RCS sequestering agents) such as pyridoxamine (PYR), hydralazine (HY), dihydralazine (di-HY), and aminoguanidine (AG). However these compounds are characterized by a lack of selectivity since they also react with physiological aldehydes, such as pyridoxal. We recently found that the endogenous dipeptide carnosine (beta-alanyl-L-histidine, CAR) is a specific quencher of alfa,beta-unsaturated aldehydes due to its peculiar mechanism involving the Schiff base formation between the beta-alanine amino group and the RCS aldehyde followed by the Michael adduction between the C3 of the aldehyde and the Ntau of the histidine group. We also found that CAR exogenously given to Zucker obese rats (30 mg/Kg die for 24 weeks) greatly reduces dyslipidemia, hypertension, albuminuria and protein carbonylation. However, the therapeutic use of CAR is limited since it is unstable in human plasma due to serum carnosinase. Hence, our interest was to derive carnosine analogues characterized by (i) carnosinase stability and (ii) a grater reactivity towards RCS, even maintaining the same selectivity. The stability was reached by the isomerization of L- to D-histidine, leading to β-alanyl-D-histidine (D-CAR), which is not recognized by carnosinase, but maintains the same quenching activity of L-CAR. The increase of reactivity was reached by modulating the conformational profile of the Schiff’s base intermediate, in order to favour a close conformation in which the imidazole ring approaches enough the C3 of the Schiff’s base to form the corresponding Michael adduct. A series of D-CAR derivatives was analyzed by in silico approaches to find out those characterized by a favorable folded conformational profile. The most promising were synthesized and the stability and quenching ability evaluated. By this way a set of phenyl derivatives was identified, characterized by high stability in human plasma, and by a three fold HNE-quenching ability increase compared to D-CAR
Covalent modifications of albumin CYS34 as biomarker of oxidative stress
No full textProtein carbonylation is an irreversible process, induced by oxidants and reactive carbonyl species (RCS), the latter deriving from lipid peroxidation and oxidation of reducing sugars. Protein carbonylation is a cause/effect factor in oxidative based diseases and also an established biomarker of oxidative stress. From an analytical point of view, protein carbonylation is largely determined by immunological and spectroscopic techniques, which suffer from the intrinsic limitation of providing a very general and unspecific index of carbonyl damage. Hence, it is of great importance to set-up an analytical strategy aimed to identify, characterize and quantitate protein carbonyls in order to have a specific and reliable biomarker of oxidative stress.
Cys34 residue of human serum albumin (HSA) represents the main target of oxidation and carbonylation in human plasma and this is explained by considering the high plasma content of HSA (≈ 0.6 mM), as well as the high reactivity and accessibility of the free Cys residue.[1] Different mass spectrometric approaches have been set-up in our laboratory to map the covalently oxidative modifications of Cys34, in order to identify suitable markers of early systemic oxidative stress. By using a top-down MS approach we found that Cys34 undergoes the following main oxidative modifications: cysteinylation, oxidation to sulfenic, sulfinic and sulfonic acid derivatives and carbonylation by reacting with RCS. Cysteinylated HSA was then monitored in different physio-pathological conditions involving oxidative stress, such as aging, diabetes, metabolic syndrome and nephropathy. Furthermore, we recently described an MS strategy based on the precursor-ion scanning technique which is able to specifically detect unknown covalent modifications of the Cys34 residue.[2] We are now on a further MS approach based on nanoLC coupled to the Orbitrap as the mass analyzer. Cys34 covalent adducts and oxidized forms are searched in a database of predicted variable modifications on Cys residues. We recently employed this approach to rapidly identify the covalent modifications of Cys34 in albumin exposed to a whole-phase cigarette smoke extract. The covalent Michael adducts of Cys34 with ACR and crotonaldehyde were identified, as well as the sulfinic and sulfonic acid derivatives.[3]
[1] Aldini G et al., Chem Res Toxicol. 2008. [2] Aldini G. et al., J. Mass Spec. 2008. [3] Colombo G et al., Antioxid Redox Signal. 2010
High resolution mass spectrometric strategies for studying AGEs inhibitors and RAGE antagonists
No full textAGEs are involved in the onset and progression of different oxidative based diseases and their inhibition, together with the blockade of the AGEs-RAGE interaction, represents a promising drug target. In order to exploit AGEs and RAGE as drug targets, validated analytical methods able to screen compounds acting as AGEs inhibitors or as RAGE antagonists are required. An analytical platform based on high-resolution mass spectrometry (MS) which permits the evaluation of the ability of the tested compounds to inhibit AGEs-ALEs formation and their interaction with RAGE is here reported.
Testing AGEs/ALEs inhibitors: the method we set-up offers the unique advantage of evaluating the efficacy of pure compound, mixture and raw extract on inhibiting AGEs and ALEs generated by incubating ubiquitin as protein target with reactive carbonyl species (RCS) such as glyoxal, methylglyoxal, 2-hydroxynonenal, acrolein, or reducing sugars (glucose and fructose). The method is based on automated injection and quantitative analysis of ubiquitin in native and adducted forms, using an Orbitrap mass spectrometer. The method was validated by investigating the effect of known inhibitors of AGEs and ALEs formation such as carnosine, hydralazine, aminoguanidine and pyridoxamine as well as of some natural extracts.
Testing RAGE antagonists: a MS method was firstly set up to study the non-covalent interactions between ligands and recombinant sRAGEs (V1-C1), representing the ligand binding domain of RAGE. The V1-C1 protein target was expressed in E. coli and the ligand-protein binding properties (stoichiometry and Kd values) were determined by a high-resolution mass spectrometric (orbitrap) approach carried out in not-denaturating conditions and using a static nano-ESI source. The method was then validated by using well known low molecular weight sRAGE ligands and the Kd values were in line with those previously reported by fluorescence titration experiments
REACTIVE CARBONYL SPECIES AS POTENTIAL DRUG TARGETS IN PREVENTING PROTEIN CARBONYLATION AND RELATED CELLULAR DYSFUNCTION
No full textProtein carbonylation induced by reactive carbonyl species (RCS) generated by peroxidation of polyunsaturated fatty acids plays a significant role in the etiology and/or progression of several human diseases, such as cardiovascular (e.g., atherosclerosis, long-term complications of diabetes) and neurodegenerative diseases (e.g., Alzheimer's disease, Parkinson's disease, and cerebral ischemia)(1). Most of the biological effects of intermediate RCS, mainly alpha,beta-unsaturated aldehydes, di-aldehydes, and keto-aldehydes, are due to their capacity to react with the nucleophilic sites of proteins, forming advanced lipoxidation end-products (ALEs). Because of the emerging deleterious role of RCS/protein adducts in several human diseases, different potential therapeutic strategies have been developed in the last few years. The talk will focus on the fundamental studies on lipid-derived RCS generation, their biological effects, and their reactivity with proteins, with particular emphasis to 4-hydroxy-trans-2-nonenal (HNE)-, acrolein (ACR)-, malondialdehyde (MDA)-, and glyoxal (GO)-modified proteins (2). It will also consider the recently developed pharmacological approaches for the management of chronic diseases in which oxidative stress and RCS formation are massively involved. The most promising strategy to neutralize/reduce these pathogenetic factors is based on nucleophilic compounds capable to form covalent and unreactive adducts with RCS (RCS sequestering agents) such as pyridoxamine (PYR), hydralazine (HY), dihydralazine (di-HY), aminoguanidine (AG), and metformin (MF)(2). However these compounds are characterized by a severe aspecificty since they react also with physiological aldehydes such as pyridoxal (3). The talk will also describe a new class of RCS-sequestering agents developed in our laboratory, derived form the endogenous peptide Carnosine (β-alanyl-L-hisitidine), characterized by a significant quenching activity towards electrophilic aldehydes, and high selectivity. The efficacy of this new class of compounds in preventing dyslipidemia, hypertension and kidney damage in Zucker obese rats will be also presented
BIOANALYSIS IN DRUG DISCOVERY: AN INTRODUCTION
No full textFor several years, bioanalytical techniques and in particular mass spectrometry (MS), have found several applications in the different stages of drug development, including identification of new compounds (organic, inorganic and metallo-organic compounds), their structure elucidation and confirmation, identification of drugs and their metabolites in body-fluids, and assessment of compound purity. With the advent of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI) techniques, the use of MS has also extended to all the discovery stages and in particular: 1) target identification and characterization, 2) screening and hit evaluation, 3) lead identification, and optimization.
Drug target identification attempts to identify new targets, normally proteins, whose modulation might inhibit or reverse disease progression. Not too long ago, scientists searched for new targets employing a long and costly process of trial and error. Today, the collective contribution of “omics” approaches allow for the much more rapid and precise discovery of those genes and/or proteins involved in the etiology of certain diseases. In particular, proteomics represents a promising tool for drug target discovery, and this thank to the development of bioanalytical approaches for protein separation and identification/characterization leading to the analysis of protein expression on a broad scale and in complex mixtures. Examples of proteomics application in drug target identification and validation will be given during the seminar.
Regarding screening and hit evaluation, ESI-MS has been widely recognized as a suitable technique in the measurement of the effects of compounds on the biological activity of a target molecule (function-based screening). The MS approach (mainly LC-ESI-MS) permits to quantitate analytes by selected ion monitoring (SIM) or multiple reaction monitoring (MRM) and to evaluate the inhibition of a target enzyme at a single compound concentration (% inhibition) or by serial concentrations to obtain IC50. MS is also useful to determine the affinities of compounds for target macromolecules such as proteins, RNA, DNA (affinity-based screening). The screening is based on a MS approach able to study non-covalent complexes between the target and ligands, which can be characterized either by direct detection of the complexes by MS (gas-phase drug screening) or by detection of bound compound after the compound is dissociated from the complex (condensed-phase drug screening). Several condensed-phase ESI-MS approaches have been up to now set-up and applied, among them: frontal affinity chromatography, affinity ultrafiltration, pulsed ultrafiltration, GPC-spin column
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