Determining site occupancy of acetaminophen covalent binding to target proteins in vitro

Allergic reactions to antibiotics are a major concern in the clinic. ß-lactam antibiotics are the class most frequently reported to cause hypersensitivity reactions. One of the mechanisms involved in this outcome is the modification of proteins by covalent binding of the drug (haptenation). Hence, interest in identifying the corresponding serum and cellular protein targets arises. Importantly, haptenation susceptibility and extent can be modulated by the context, including factors affecting protein conformation or the occurrence of other posttranslational modifications. We previously identified the glycolytic enzyme α-enolase as a target for haptenation by amoxicillin, both in cells and in the extracellular milieu. Here, we performed an in vitro study to analyze amoxicillin haptenation of α-enolase using gel-based and activity assays. Moreover, the possible interplay or interference between amoxicillin haptenation and acetylation of α-enolase was studied in 1D- and 2D-gels that showed decreased haptenation and displacement of the haptenation signal to lower pI spots after chemical acetylation of the protein, respectively. In addition, the peptide containing lysine 239 was identified by mass spectrometry as the amoxicillin target sequence on α-enolase, thus suggesting a selective haptenation under our conditions. The putative amoxicillin binding site and the surrounding interactions were investigated using the α-enolase crystal structure and molecular docking. Altogether, the results obtained provide the basis for the design of novel diagnostic tools or approaches in the study of amoxicillin-induced allergic reactions.

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Annexin a2 as a target protein for chlorogenic acid in human lung cancer A549 cells

10.1101/2020.06.11.146027 ◽

2020 ◽

Author(s):

Lei Wang ◽

Hongwu Du ◽

Peng Chen

Keyword(s):

Chlorogenic Acid ◽

Covalent Binding ◽

Animal Experiments ◽

A549 Cells ◽

Annexin A2 ◽

Human Lung Cancer ◽

Target Proteins ◽

Apoptotic Genes

AbstractChlorogenic acid, an important active component of coffee with anti-tumor activities, has been found for many years. However, the lack of understanding about its target proteins greatly limits the exploration of its anti-tumor molecular mechanism and clinical application. Here, in vitro and animal experiments showed that chlorogenic acid had a significant inhibitory effect on the proliferation of A549 cells. Using the spontaneous fluorescence characteristic of chlorogenic acid to screen the target proteins cleverly to avoid the problem of chemical modification increasing false positive, we identify and verify annexin A2 (ANXA2) as a covalent binding target of chlorogenic acid in A549 cells. Then, we discover that chlorogenic acid as an inhibitor of the binding of ANXA2 to p50 subunit inhibited the expression of downstream anti-apoptotic genes cIAP1 and cIAP2 of NF-κB signaling pathway in A549 cells in vitro and vivo. Moreover, we find chlorogenic acid hindered the binding of ANXA2 and actin maybe involved in the impediment of tumor cell cycle and migration. Thus, this work demonstrates that chlorogenic acid, as a binding ligand of ANXA2, decrease the expression of NF-κB downstream anti-apoptotic genes, inhibiting the proliferation of A549 cells in vivo and vitro.

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Properties and Biotechnological Application of mutant Derivatives of Mini-intein PRP8 from Penicillium chrysogenum with Improved Control of C-terminal Processing

Biotekhnologiya ◽

10.21519/0234-2758-2019-35-6-91-101 ◽

2019 ◽

Vol 35 (6) ◽

pp. 91-101

Author(s):

F.A. Klebanov ◽

S.E. Cheperegin ◽

D.G. Kozlov

Keyword(s):

Penicillium Chrysogenum ◽

Protein Denaturation ◽

Biologically Active ◽

Cultivation Temperature ◽

Target Proteins ◽

E Coli ◽

Complete Inactivation ◽

Target Molecules ◽

Proteins And Peptides

Mutant variants of mini-intein PRP8 from Penicillium chrysogenum (Int4b) with improved control of C-terminal processing were characterized. The presented variants can serve as a basis for self-removed polypeptide tags capable of carrying an affine label and allowing to optimize the process of obtaining target proteins and peptides in E. coli cells. They allow to synthesize target molecules in the composition of soluble and insoluble hybrid proteins (fusions), provide their afnne purification, autocatalytic processing and obtaining mature target products. The presented variants have a number of features in comparison with the known prototypes. In particular the mutant mini-intein Int4bPRO, containing the L93P mutation, has temperature-dependent properties. At cultivation temperature below 30 °C it allows the production of target molecules as part of soluble fusions, but after increasing of cultivation temperature to 37 °C it directs the most of synthesized fusions into insoluble intracellular aggregates. The transition of Int4bPRO into insoluble form is accompanied by complete inactivation of C-terminal processing. Further application of standard protein denaturation-renaturation procedures enable efficiently reactivate Int4bPRO and to carry out processing of its fusions in vitro. Two other variants, Int4b56 and Int4b36, containing a point mutation T62N or combination of mutations D144N and L146T respectively, have a reduced rate of C-terminal processing. Their use in E. coli cells allows to optimize the biosynthesis of biologically active target proteins and peptides in the composition of soluble fusions, suitable for afnne purification and subsequent intein-dependent processing without the use of protein denaturation-renaturation procedures. intein, fusion, processing, processing rate, gelonin The work was supported within the framework of the State Assignment no. 595-00003-19 PR.

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Novel Iodinated Hydrazide-hydrazones and their Analogues as Acetyl- and Butyrylcholinesterase Inhibitors

Current Topics in Medicinal Chemistry ◽

10.2174/1568026620666200819155503 ◽

2020 ◽

Vol 20 (23) ◽

pp. 2106-2117

Author(s):

Martin Krátký ◽

Šárka Štěpánková ◽

Michaela Brablíková ◽

Katarína Svrčková ◽

Markéta Švarcová ◽

...

Keyword(s):

Biological Activities ◽

Structural Parameters ◽

Covalent Binding ◽

Docking Studies ◽

Potent Inhibition ◽

Brain Barrier Permeability ◽

Electric Eel ◽

Ic50 Values ◽

Ellman’S Method

Background: Hydrazide-hydrazones have been known as scaffold with various biological activities including inhibition of acetyl- (AChE) and butyrylcholinesterase (BuChE). Cholinesterase inhibitors are mainstays of dementias’ treatment. Objective: Twenty-five iodinated hydrazide-hydrazones and their analogues were designed as potential central AChE and BuChE inhibitors. Methods: Hydrazide-hydrazones were synthesized from 4-substituted benzohydrazides and 2-/4- hydroxy-3,5-diiodobenzaldehydes. The compounds were investigated in vitro for their potency to inhibit AChE from electric eel and BuChE from equine serum using Ellman’s method. We calculated also physicochemical and structural parameters for CNS delivery. Results: The derivatives exhibited a moderate dual inhibition with IC50 values ranging from 15.1-140.5 and 35.5 to 170.5 μmol.L-1 for AChE and BuChE, respectively. Generally, the compounds produced a balanced or more potent inhibition of AChE. N'-[(E)-(4-Hydroxy-3,5-diiodophenyl)methylidene]-4- nitrobenzohydrazide 2k and 4-fluoro-N'-(2-hydroxy-3,5-diiodobenzyl)benzohydrazide 3a were the most potent inhibitors of AChE and BuChE, respectively. Structure-activity relationships were established, and molecular docking studies confirmed interaction with enzymes. Conclusion: Many novel hydrazide-hydrazones showed lower IC50 values than rivastigmine against AChE and some of them were comparable for BuChE to this drug used for the treatment of dementia. They interact with cholinesterases via non-covalent binding into the active site. Based on the BOILEDEgg approach, the majority of the derivatives met the criteria for blood-brain-barrier permeability.

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Role of Phase II Enzymes in the Bioactivation of 1,4-Dichlorobenzene and 1,4-Dibromobenzene

Alternatives to Laboratory Animals ◽

10.1177/026119299602400422 ◽

1996 ◽

Vol 24 (4) ◽

pp. 603-608

Author(s):

Moreno Paolini ◽

Laura Pozzetti ◽

Renata Mesirca ◽

Andrea Sapone ◽

Paola Silingardi ◽

...

Keyword(s):

Dna Binding ◽

Phase I ◽

Phase Ii ◽

Covalent Binding ◽

Fold Increase ◽

Test System ◽

Oxidative Enzymes ◽

Transferase Activity ◽

Genetic Toxicology

The use of sodium phenobarbital (PB, CYP2B1 inducer) combined with β-naphthoflavone (β-NF, 1A1) to induce certain Phase I reactions in S9 liver fractions is a standard method for conducting short-term bioassays for genotoxicity. However, because post-oxidative enzymes are also able to activate many precarcinogens, we tested the possibility of adapting S9 liver fractions derived from Phase II-induced rodents to the field of genetic toxicology. In this study, S9 liver fractions derived from Swiss albino CD1 mice fed 7.5g/kg 2-(3)-tert-butyl-4-hydroxyanisole (BHA; a monofunctional Phase II-inducer) for 3 weeks, show a clear pattern of induction with an approximately 3.5–9.5-fold increase in glutathione S-transferase activity. In vitro DNA binding of the promutagenic agents, [14C]-l,4-dichlorobenzene (DCB) and [14C]-1,4-dibromobenzene (DBB), is mediated by such metabolic liver preparations and showed a significant increase in covalent binding capability. In some instances, enzyme activity was more elevated when compared to that obtained with traditional (Phase I-induced) S9. Together with DNA binding, the genetic response of these chemicals in the diploid D7 strain of Saccharomyces cerevisiae used as a biological test system, revealed the ability of the BHA-derived preparations to activate the promutagenic agents, as exemplified by the significant enhancement of mitotic gene-conversion (up to 5.2-fold for DCB and 3.4-fold for DBB) and reverse point mutation (up to 3.6-fold for DCB and 2.5-fold for DBB) at a 4mM concentration. This novel metabolising biosystem, with enhanced Phase II activity, is recommended together with a traditional S9, for detecting unknown promutagens in genotoxicity studies. The routine use of either oxidative or post-oxidative S9 increases the responsiveness of the test and can contribute to the identification of promutagens not detected when using traditional protocols.

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Investigating the Mechanism of Trimethoprim-Induced Skin Rash and Liver Injury

Toxicological Sciences ◽

10.1093/toxsci/kfaa182 ◽

2021 ◽

Author(s):

Yanshan Cao ◽

Ahsan Bairam ◽

Alison Jee ◽

Ming Liu ◽

Jack Uetrecht

Keyword(s):

Liver Injury ◽

Skin Rash ◽

Covalent Binding ◽

Reactive Metabolites ◽

Important Data ◽

Drug Induced ◽

Interindividual Differences ◽

Immune Mediated ◽

Sulfate Conjugate

Abstract Trimethoprim (TMP)-induced skin rash and liver injury are likely to involve the formation of reactive metabolites. Analogous to nevirapine-induced skin rash, one possible reactive metabolite is the sulfate conjugate of α-hydroxyTMP, a metabolite of TMP. We synthesized this sulfate and found that it reacts with proteins in vitro. We produced a TMP-antiserum and found covalent binding of TMP in the liver of TMP-treated rats. However, we found that α-hydroxyTMP is not a substrate for human sulfotransferases, and we did not detect covalent binding in the skin of TMP-treated rats. Although less reactive than the sulfate, α-hydroxyTMP was found to covalently bind to liver and skin proteins in vitro. Even though there was covalent binding to liver proteins, TMP did not cause liver injury in rats or in our impaired immune tolerance mouse model that has been able to unmask the ability of other drugs to cause immune-mediated liver injury. This is likely because there was much less covalent binding of TMP in the livers of TMP-treated mice than TMP-treated rats. It is possible that some patients have a sulfotransferase that can produce the reactive benzylic sulfate; however, α-hydroxyTMP, itself, has sufficient reactivity to covalently bind to proteins in the skin and may be responsible for TMP-induced skin rash. Interspecies and interindividual differences in TMP metabolism may be one factor that determines the risk of TMP-induced skin rash. This study provides important data required to understand the mechanism of TMP-induced skin rash and drug-induced skin rash in general.

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Synthesis, in Vitro Covalent Binding Evaluation, and Metabolism of14C-Labeled Inhibitors of 11β-HSD1

ACS Medicinal Chemistry Letters ◽

10.1021/ml500331y ◽

2014 ◽

Vol 5 (11) ◽

pp. 1245-1250 ◽

Cited By ~ 1

Author(s):

Daqing Sun ◽

Qiuping Ye ◽

Xuelei Yan ◽

Yosup Rew ◽

Peter Fan ◽

...

Keyword(s):

Covalent Binding

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Monitoring Drug-Target Interactions through Target Engagement-Mediated Amplification on Arrays and in situ

10.21203/rs.3.rs-1109577/v1 ◽

2021 ◽

Author(s):

Rasel Al-Amin ◽

Lars Johansson ◽

Eldar Abdurakhmanov ◽

Nils Landegren ◽

Liza Löf ◽

...

Keyword(s):

Drug Target ◽

Kinase Inhibitors ◽

Expression Profiles ◽

Clinical Samples ◽

Target Engagement ◽

Rolling Circle Replication ◽

Target Proteins ◽

Dna Conjugates

Abstract Drugs are designed to bind their target proteins in physiologically relevant tissues and organs to modulate biological functions and elicit desirable clinical outcomes. Information about target engagement at cellular and subcellular resolution is therefore critical for guiding compound optimization in drug discovery, and for probing resistance mechanisms to targeted therapies in clinical samples. We describe a target engagement-mediated amplification (TEMA) technology, where oligonucleotide-conjugated drugs are used to visualize and measure target engagement in situ, amplified via rolling-circle replication of circularized oligonucleotide probes. We illustrate the TEMA technique using dasatinib and gefitinib, two kinase inhibitors with distinct selectivity profiles. In vitro binding by dasatinib probe to arrays of displayed proteins accurately reproduced known selectivity profiles, while their differential binding to a panel of fixed adherent cells agreed with expectations from expression profiles of the cells. These findings were corroborated by competition experiments using kinase inhibitors with overlapping and non-overlapping target specificities, and translated to pathology tissue sections. We also introduce a proximity ligation variant of TEMA in which these drug-DNA conjugates are combined with antibody-DNA conjugates to selectively investigate binding to specific target proteins of interest. This form of the assay serves to improve resolution of binding to on- and off-target proteins. In conclusion, TEMA has the potential to aid in drug development and clinical routine by conferring valuable insights in drug-target interactions at spatial resolution in protein arrays, cells and tissues.

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