Screening and Characterization of Reactive Compounds with In Vitro Peptide-Trapping and Liquid Chromatography/High-Resolution Accurate Mass Spectrometry
10
Citation
21
Reference
10
Related Paper
Citation Trend
Abstract:
The present study describes a novel methodology for the detection of reactive compounds using in vitro peptide-trapping and liquid chromatography-high-resolution accurate mass spectrometry (LC-HRMS). Compounds that contain electrophilic groups can covalently bind to nucleophilic moieties in proteins and form adducts. Such adducts are thought to be associated with drug-mediated toxicity and therefore represent potential liabilities in drug discovery programs. In addition, reactive compounds identified in biological screening can be associated with data that can be misinterpreted if the reactive nature of the compound is not appreciated. In this work, to facilitate the triage of hits from high-throughput screening (HTS), a novel assay was developed to monitor the formation of covalent peptide adducts by compounds suspected to be chemically reactive. The assay consists of in vitro incubations of test compounds (under conditions of physiological pH) with synthetically prepared peptides presenting a variety of nucleophilic moieties such as cysteine, lysine, histidine, arginine, serine, and tyrosine. Reaction mixtures were analyzed using full-scan LC-HRMS, the data were interrogated using postacquisition data mining, and modified amino acids were identified by subsequent LC-HRMS/mass spectrometry. The study demonstrated that in vitro nucleophilic peptide trapping followed by LC-HRMS analysis is a useful approach for screening of intrinsically reactive compounds identified from HTS exercises, which are then removed from follow-up processes, thus obviating the generation of data from biochemical activity assays.Keywords:
Reactive intermediate
Contrary to other studies, here we describe cysteine (Cys) pseudoproline-containing peptides with short deprotection times in TFA. The deprotection times fell in the same range as other protecting groups commonly used in SPPS (e.g., 1-3 h). Moreover, when using Cys pseudoprolines as peptide macrocyclization-enhancing moieties a considerable reduction in reaction time was observed compared to a peptide containing trityl protected Cys.
Thiol
Peptide Synthesis
Solid-Phase Synthesis
Cite
Citations (22)
Sample Preparation
Metribuzin
Cite
Citations (11)
Summary This work aimed to investigate the reactivity of hydroxymethylfurfural ( HMF ) with selected amino acids, to identify the produced adducts and to clarify whether or not the adducts release HMF after their digestion under gastric conditions. Results showed that cysteine, glycine and lysine can deplete the added HMF , and their reactivity increased with increasing pH and temperature. Cysteine (25 μmol mL −1 ) depleted 91.0% of the added HMF (315.3 μg mL −1 ) at 40 °C in 15 min, lysine did not eliminate HMF until 80 °C, and glycine started to eliminate HMF at 100 °C. Four adducts for cysteine, three adducts for lysine and only one adduct for glycine were identified through HPLC – MS – MS after they reacted with HMF . The adducts formed from the reaction mixture of cysteine, lysine and glycine with HMF only released 1.7%, 2.6% and 10.5% of eliminated HMF , respectively, after their digestion in simulated gastric conditions.
Reactivity
5-hydroxymethylfurfural
Digestion
Cite
Citations (17)
Synthetic methodologies to chemically modify peptide molecules have long been investigated for their impact in the field of chemical biology. They allow the introduction of biochemical probes useful for studying protein functions, for manipulating peptides with therapeutic potential, and for structure-activity relationship investigations. The commonly used approach was the derivatization of an amino acid side chain. In this regard, the cysteine, for its unique reactivity, has been widely employed as the substrate for such modifications. Herein, we report on methodologies developed to modify the cysteine thiol group through the S-alkylation reaction. Some procedures perform the alkylation of cysteine derivatives, in order to prepare building blocks to be used during the peptide synthesis, whilst some others selectively modify peptide sequences containing a cysteine residue with a free thiol group, both in solution and in the solid phase.
Cite
Citations (32)
Abstract We report the genetic incorporation of caged cysteine and caged homocysteine into proteins in bacterial and mammalian cells. The genetic code of these cells was expanded with an engineered pyrrolysine tRNA/tRNA synthetase pair that accepts both light‐activatable amino acids as substrates. Incorporation was validated by reporter assays, western blots, and mass spectrometry, and differences in incorporation efficiency were explained by molecular modeling of synthetase–amino acid interactions. As a proof‐of‐principle application, the genetic replacement of an active‐site cysteine residue with a caged cysteine residue in Renilla luciferase led to a complete loss of enzyme activity; however, upon brief exposure to UV light, a >150‐fold increase in enzymatic activity was observed, thus showcasing the applicability of the caged cysteine in live human cells. A simultaneously conducted genetic replacement with homocysteine yielded an enzyme with greatly reduced activity, thereby demonstrating the precise probing of a protein active site. These discoveries provide a new tool for the optochemical control of protein function in mammalian cells and expand the set of genetically encoded unnatural amino acids.
Genetic Code
Amino Acyl-tRNA Synthetases
Cite
Citations (64)
The toxicity of the ubiquitous pollutant and endogenous metabolite, acrolein, is due in part to covalent protein modifications. Acrolein reacts readily with protein nucleophiles via Michael addition and Schiff base formation. Potential acrolein targets in protein include the nucleophilic side chains of cysteine, histidine, and lysine residues as well as the free amino terminus of proteins. Although cysteine is the most acrolein-reactive residue, cysteine−acrolein adducts are difficult to identify in vitro and in vivo. In this study, model peptides with cysteine, lysine, and histidine residues were used to examine the reactivity of acrolein. Results from these experiments show that acrolein reacts rapidly with cysteine residues through Michael addition to form M+56 Da adducts. These M+56 adducts are, however, not stable, even though spontaneous dissociation of the adduct is slow. Further studies demonstrated that when acrolein and model peptides are incubated at physiological pH and temperature, the M+56 adducts decreased gradually accompanied by the increase of M+38 adducts, which are formed from intramolecular Schiff base formation. Adduct formation with the side chains of other amino acid residues (lysine and histidine) was much slower than cysteine and required higher acrolein concentration. When cysteine residues were blocked by reaction with iodoacetamide and higher concentrations of acrolein were used, adducts of the N-terminal amino group or histidyl residues were formed, but lysine adducts were not detected. Collectively, these data demonstrate that acrolein reacts avidly with protein cysteine residues and that the apparent loss of protein−acrolein Michael adducts over time may be related to the appearance of a novel (M+38) adduct. These findings may be important in identification of in vivo adducts of acrolein with protein cysteine residues.
Acrolein
Residue (chemistry)
Cite
Citations (167)
Cite
Citations (1)
Summary Human platelets are known to contain various protein components. Among them are fibrinogen and other soluble proteins. The origin of such proteins has not been clear. Studies were designed to demonstrate the ability of the platelet to incorporate amino acids and subsequently utilise these for de novo protein synthesis. Seven different 14C labelled amino acids were used, cysteine, lysine, serine, glutamic acid, proline, valine und leucine. Active incorporation into platelets of all these amino acids was demonstrated with some evidence for incorporation of cysteine, lysine and serine into the fibrinogen and soluble protein.
Glutamic acid
Cite
Citations (6)
The Spectrophotometric direct peptide reactivity assay (Spectro-DPRA) is now in process for the development and validation of evaluation on skin-sensitizing chemicals. The cysteine peptides (Ac-RFAACAACOOH) and lysine peptides (Ac-RFAAKAA-COOH) is used for Spectro-DPRA.
The objective of this study is to confirm the stability of cysteine and lysine peptides which are used for Spectro-DPRA through the storage conditions. We analyzed the stability of peptide using HPLC according to the conditions, such as storage temperature and storage period.
The peptide powders of cysteine and lysine were stable on storage -80℃, for 6 and 12 month. And 10 mM stock peptides of cysteine and lysine were also stable on storage -70℃, for 1, 3 and 6 month. But 10 mM stock peptides of cysteine were denatured and not stable on storage -20℃, for 1 month. Taken together, peptides powder for Spectro-DPRA should be stored under -80℃, less than 12 month. And, 10 mM stock peptides should be also stored under -70℃, less than 6 month.
Cite
Citations (0)
Glutamic acid
Amino acid synthesis
Aspartic acid
Complete protein
Alanine
Essential amino acid
Cite
Citations (12)