scholarly journals Amoxicillin Haptenation of α-Enolase is Modulated by Active Site Occupancy and Acetylation

2022 ◽  
Vol 12 ◽  
Author(s):  
Juan M. González-Morena ◽  
Francisco J. Sánchez-Gómez ◽  
Yolanda Vida ◽  
Ezequiel Pérez-Inestrosa ◽  
María Salas ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Covalent Binding ◽  
Site Occupancy ◽  
Cellular Protein ◽  
Glycolytic Enzyme ◽  
Diagnostic Tools ◽  
Target Sequence ◽  
Allergic Reactions ◽  
Extracellular Milieu

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.

scholarly journals Gene-Based Antibody Strategies for Prion Diseases

2013 ◽  
Vol 2013 ◽  
pp. 1-6 ◽  
Author(s):  
Alessio Cardinale ◽  
Silvia Biocca
Keyword(s):  
Therapeutic Potential ◽  
Prion Diseases ◽  
Recombinant Antibody ◽  
Cellular Protein ◽  
Transmissible Spongiform Encephalopathies ◽  
Diagnostic Tools ◽  
Spongiform Encephalopathies ◽  
Interaction Sites

Prion diseases or transmissible spongiform encephalopathies (TSE) are a group of neurodegenerative and infectious disorders characterized by the conversion of a normal cellular protein PrPCinto a pathological abnormally folded form, termed PrPSc. There are neither available therapies nor diagnostic tools for an early identification of individuals affected by these diseases. New gene-based antibody strategies are emerging as valuable therapeutic tools. Among these, intrabodies are chimeric molecules composed by recombinant antibody fragments fused to intracellular trafficking sequences, aimed at inhibiting,in vivo, the function of specific therapeutic targets. The advantage of intrabodies is that they can be selected against a precise epitope of target proteins, including protein-protein interaction sites and cytotoxic conformers (i.e., oligomeric and fibrillar assemblies). Herein, we address and discussin vitroandin vivoapplications of intrabodies in prion diseases, focussing on their therapeutic potential.

scholarly journals Quinolone Allergy

Pharmacy ◽  
2019 ◽  
Vol 7 (3) ◽  
pp. 97 ◽  
Author(s):  
Edoabasi U. McGee ◽  
Essie Samuel ◽  
Bernadett Boronea ◽  
Nakoasha Dillard ◽  
Madison N. Milby ◽  
...  
Keyword(s):  
Clinical Manifestations ◽  
Provocation Test ◽  
Cross Reactivity ◽  
Skin Tests ◽  
Diagnostic Tools ◽  
In Vitro Tests ◽  
Allergic Reactions ◽  
Drug Induced ◽  
Delayed Reaction

Quinolones are the second most common antibiotic class associated with drug-induced allergic reactions, but data on quinolone allergy are scarce. This review article discusses the available evidence on quinolone allergy, including prevalence, risk factors, diagnosis, clinical manifestations, cross-reactivity, and management of allergic reactions. Although the incidence of quinolone allergy is still lower than beta-lactams, it has been increasingly reported in recent decades, most likely from its expanded use and the introduction of moxifloxacin. Thorough patient history remains essential in the evaluation of quinolone allergy. Many diagnostic tools have been investigated, but skin tests can yield false-positive results and in vitro tests have not been validated. The drug provocation test is considered the test of choice to confirm a quinolone allergy but is not without risk. Evidence regarding cross-reactivity among the quinolones is limited and conflicting. Quinolone allergy can be manifested either as an immediate or delayed reaction, but is not uniform across the class, with moxifloxacin posing the highest risk of anaphylaxis. Quinolone should be discontinued when an allergic reaction occurs and avoided in future scenarios, but desensitization may be warranted if no alternatives are available.

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

2021 ◽  
Author(s):  
Timon Geib ◽  
Cristina Lento ◽  
Vanessa Marensi ◽  
Madhuranayaki Thulasingam ◽  
Jesper Z. Haeggström ◽  
...  
Keyword(s):  
Covalent Binding ◽  
Site Occupancy ◽  
Target Proteins

scholarly journals Deamidated Human Triosephosphate Isomerase is a Promising Druggable Target

Biomolecules ◽  
2020 ◽  
Vol 10 (7) ◽  
pp. 1050
Author(s):  
Sergio Enríquez-Flores ◽  
Luis Antonio Flores-López ◽  
Itzhel García-Torres ◽  
Ignacio de la Mora-de la Mora ◽  
Nallely Cabrera ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Triosephosphate Isomerase ◽  
Noncovalent Interactions ◽  
Severe Disease ◽  
Selective Inhibition ◽  
Therapeutic Strategies ◽  
Glycolytic Enzyme ◽  
Enzyme Inactivation ◽  
Isoaspartic Acid

Therapeutic strategies for the treatment of any severe disease are based on the discovery and validation of druggable targets. The human genome encodes only 600–1500 targets for small-molecule drugs, but posttranslational modifications lead to a considerably larger druggable proteome. The spontaneous conversion of asparagine (Asn) residues to aspartic acid or isoaspartic acid is a frequent modification in proteins as part of the process called deamidation. Triosephosphate isomerase (TIM) is a glycolytic enzyme whose deamidation has been thoroughly studied, but the prospects of exploiting this phenomenon for drug design remain poorly understood. The purpose of this study is to demonstrate the properties of deamidated human TIM (HsTIM) as a selective molecular target. Using in silico prediction, in vitro analyses, and a bacterial model lacking the tim gene, this study analyzed the structural and functional differences between deamidated and nondeamidated HsTIM, which account for the efficacy of this protein as a druggable target. The highly increased permeability and loss of noncovalent interactions of deamidated TIM were found to play a central role in the process of selective enzyme inactivation and methylglyoxal production. This study elucidates the properties of deamidated HsTIM regarding its selective inhibition by thiol-reactive drugs and how these drugs can contribute to the development of cell-specific therapeutic strategies for a variety of diseases, such as COVID-19 and cancer.

scholarly journals Identification of Host Cellular Protein Substrates of SARS-COV-2 Main Protease

2020 ◽  
Vol 21 (24) ◽  
pp. 9523
Author(s):  
Márió Miczi ◽  
Mária Golda ◽  
Balázs Kunkli ◽  
Tibor Nagy ◽  
József Tőzsér ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Severe Pneumonia ◽  
Cellular Protein ◽  
Target Sequence ◽  
Cleavage Sites ◽  
Cellular Targets ◽  
Main Protease ◽  
Human Proteins ◽  
Successful Replication

The novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the causative agent of coronavirus disease-19 (COVID-19) being associated with severe pneumonia. Like with other viruses, the interaction of SARS-CoV-2 with host cell proteins is necessary for successful replication, and cleavage of cellular targets by the viral protease also may contribute to the pathogenesis, but knowledge about the human proteins that are processed by the main protease (3CLpro) of SARS-CoV-2 is still limited. We tested the prediction potentials of two different in silico methods for the identification of SARS-CoV-2 3CLpro cleavage sites in human proteins. Short stretches of homologous host-pathogen protein sequences (SSHHPS) that are present in SARS-CoV-2 polyprotein and human proteins were identified using BLAST analysis, and the NetCorona 1.0 webserver was used to successfully predict cleavage sites, although this method was primarily developed for SARS-CoV. Human C-terminal-binding protein 1 (CTBP1) was found to be cleaved in vitro by SARS-CoV-2 3CLpro, the existence of the cleavage site was proved experimentally by using a His6-MBP-mEYFP recombinant substrate containing the predicted target sequence. Our results highlight both potentials and limitations of the tested algorithms. The identification of candidate host substrates of 3CLpro may help better develop an understanding of the molecular mechanisms behind the replication and pathogenesis of SARS-CoV-2.

scholarly journals Nitrosative stress inhibits aminoacylation and editing activities of mitochondrial threonyl-tRNA synthetase by S-nitrosation

2020 ◽  
Vol 48 (12) ◽  
pp. 6799-6810
Author(s):  
Wen-Qiang Zheng ◽  
Yuying Zhang ◽  
Qin Yao ◽  
Yuzhe Chen ◽  
Xinhua Qiao ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Active Sites ◽  
Nitrosative Stress ◽  
Ex Vivo ◽  
Inhibitory Effect ◽  
Cellular Protein ◽  
Trna Synthetase ◽  
Mouse Tissues

Abstract Structure and/or function of proteins are frequently affected by oxidative/nitrosative stress via posttranslational modifications. Aminoacyl-tRNA synthetases (aaRSs) constitute a class of ubiquitously expressed enzymes that control cellular protein homeostasis. Here, we found the activity of human mitochondrial (mt) threonyl-tRNA synthetase (hmtThrRS) is resistant to oxidative stress (H2O2) but profoundly sensitive to nitrosative stress (S-nitrosoglutathione, GSNO). Further study showed four Cys residues in hmtThrRS were modified by S-nitrosation upon GSNO treatment, and one residue was one of synthetic active sites. We analyzed the effect of modification at individual Cys residue on aminoacylation and editing activities of hmtThrRS in vitro and found that both activities were decreased. We further confirmed that S-nitrosation of mtThrRS could be readily detected in vivo in both human cells and various mouse tissues, and we systematically identified dozens of S-nitrosation-modified sites in most aaRSs, thus establishing both mitochondrial and cytoplasmic aaRS species with S-nitrosation ex vivo and in vivo, respectively. Interestingly, a decrease in the S-nitrosation modification level of mtThrRS was observed in a Huntington disease mouse model. Overall, our results establish, for the first time, a comprehensive S-nitrosation-modified aaRS network and a previously unknown mechanism on the basis of the inhibitory effect of S-nitrosation on hmtThrRS.

Neurofilaments and Paired Helical Filaments

1985 ◽  
Vol 43 ◽  
pp. 740-743
Author(s):  
J. Metuzals
Keyword(s):  
Animal Model ◽  
Posttranslational Modifications ◽  
Posttranslational Modification ◽  
Giant Axon ◽  
Paired Helical Filaments ◽  
Squid Giant Axon ◽  
In Vitro Experiments ◽  
Thin Sections ◽  
Structural Relationships

It has been demonstrated that the neurofibrillary tangles in biopsies of Alzheimer patients, composed of typical paired helical filaments (PHF), consist also of typical neurofilaments (NF) and 15nm wide filaments. Close structural relationships, and even continuity between NF and PHF, have been observed. In this paper, such relationships are investigated from the standpoint that the PHF are formed through posttranslational modifications of NF. To investigate the validity of the posttranslational modification hypothesis of PHF formation, we have identified in thin sections from frontal lobe biopsies of Alzheimer patients all existing conformations of NF and PHF and ordered these conformations in a hypothetical sequence. However, only experiments with animal model preparations will prove or disprove the validity of the interpretations of static structural observations made on patients. For this purpose, the results of in vitro experiments with the squid giant axon preparations are compared with those obtained from human patients. This approach is essential in discovering etiological factors of Alzheimer's disease and its early diagnosis.

Effect of ornidazole on fertility of male rats: inhibition of a glycolysis-related motility pattern and zona binding required for fertilization in vitro

Reproduction ◽  
2000 ◽  
pp. 127-135 ◽  
Author(s):  
W Bone ◽  
NG Jones ◽  
G Kamp ◽  
CH Yeung ◽  
TG Cooper
Keyword(s):  
Zona Pellucida ◽  
Glucose Utilization ◽  
Cumulus Cells ◽  
Glycolytic Enzyme ◽  
Male Rats ◽  
Fertilization In Vitro ◽  
Swimming Pattern ◽  
Principal Piece ◽  
Free Media

The effects of the male antifertility agent ornidazole on glycolysis as a prerequisite for fertilization were investigated in rats. Antifertility doses of ornidazole inhibited glycolysis within mature spermatozoa as determined from the lack of glucose utilization, reduced acidosis under anaerobic conditions and reduced glycolytic enzyme activity. As a consequence, cauda epididymidal spermatozoa from ornidazole-fed rats were unable to fertilize rat oocytes in vitro, with or without cumulus cells, which was not due to transfer of an inhibitor in epididymal fluid with the spermatozoa. Under IVF conditions, binding to the zona pellucida was reduced in spermatozoa from ornidazole-fed males and the spermatozoa did not undergo a change in swimming pattern, which was observed in controls. The block to fertilization could be explained by the disruption of glycolysis-dependent events, since reduced binding to the zona pellucida and a lack of kinematic changes were demonstrated by control spermatozoa in glucose-free media in the presence of respiratory substrates. The importance of glycolysis for binding to, and penetration of, the zona pellucida, and hyperactivation in rats is discussed in relation to the glycolytic production of ATP in the principal piece in which local deprivation of energy may explain the reduced force of spermatozoa from ornidazole-fed males.

Peculiarities of the course of type I allergic reactions in patients with blood diseases

2020 ◽  
pp. 40-50
Author(s):  
A. Nikitina
Keyword(s):  
Search Engines ◽  
Anaphylactic Shock ◽  
Type I ◽  
Allergic Reactions ◽  
Common Cause ◽  
Blood Diseases ◽  
Treatment Regimens ◽  
Modern Methods

Analysis of literature data presented in search engines — Elibrary, PubMed, Cochrane — concerning the risk of developing type I allergic reactions in patients with blood diseases is presented. It is shown that the most common cause of type I allergic reactions is drugs included in the treatment regimens of this category of patients. The article presents statistics on the increase in the number of drug allergies leading to cases of anaphylactic shock in patients with blood diseases. Modern methods for the diagnosis of type I allergic reactions in vivo and in vitro are considered.

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

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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