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Jun'ichi Oda - One of the best experts on this subject based on the ideXlab platform.

  • Recognition of a Cysteine Substrate by E. coli γ-Glutamylcysteine Synthetase Probed by Sulfoximine-based Transition-state Analogue Inhibitors
    Bioscience biotechnology and biochemistry, 2002
    Co-Authors: Jun Hiratake, Takayuki Irie, Nobuya Tokutake, Jun'ichi Oda
    Abstract:

    A series of sulfoximine-based transition-state analogue inhibitors with a varying alkyl side chain was synthesized to probe the recognition of a Cys substrate by E. coliγ-glutamylcysteine synthetase (γ-GCS). The sulfoximines with a small alkyl group (H, methyl, ethyl, propyl, butyl and CH2OH) each served as a slow-binding inhibitor, the sulfoximine with an ethyl being by far the most potent inhibitor to cause facile and irreversible Enzyme inhibition. As the size of the side chain changed from an ethyl, the inhibition potency markedly decreased to reduce the overall affinity with concomitant loss in the inactivation rate and with facile Enzyme Reactivation by dilution. The sulfoximine without a side chain inhibited the Enzyme with almost the same potency as that of L-buthionine-(SR)-sulfoximine (BSO). The free energy difference calculated from the inhibition constants indicates that the side chain of Cys was recognized by its size through hydrophobic interaction and contributed almost equally or even more...

  • Mechanism-based inactivation of E. coli γ-glutamylcysteine synthetase by phosphinic acid- and sulfoximine-based transition-state analogues
    Bioorganic & Medicinal Chemistry Letters, 1996
    Co-Authors: Makoto Katoh, Jun Hiratake, Hiroaki Kato, Jun'ichi Oda
    Abstract:

    Abstract Phosphinic acid- and sulfoximine-based transition state analogues having a carboxyl group at the β-carbon to the hetero atom exhibited significantly higher potency as mechanism-based inhibitors of E. coli γ-glutamylcysteine synthetase as compared with l -buthionine -SR- sulfoximine . The enhanced inhibition potency is evidenced by both tight binding of the inhibitor and slow Enzyme Reactivation.

Changwen Jin - One of the best experts on this subject based on the ideXlab platform.

  • A Hybrid Mechanism for the Synechocystis Arsenate Reductase Revealed by Structural Snapshots during Arsenate Reduction.
    The Journal of biological chemistry, 2015
    Co-Authors: Yanhua Liu, Xianhui Hou, Xiaoyun Liu, Changwen Jin
    Abstract:

    Evolution of Enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification Enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its Reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the "hybrid" mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the Enzyme Reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family.

Yunfei Hu - One of the best experts on this subject based on the ideXlab platform.

  • A Hybrid Mechanism for the Synechocystis Arsenate Reductase Revealed by Structural Snapshots during Arsenate Reduction.
    Journal of Biological Chemistry, 2015
    Co-Authors: Cuiyun Hu, Caifang Yu, Yunfei Hu
    Abstract:

    Abstract Evolution of Enzymes plays a crucial role in obtaining new biological functions for all life forms. Arsenate reductases (ArsC) are several families of arsenic detoxification Enzymes that reduce arsenate to arsenite, which can subsequently be extruded from cells by specific transporters. Among these, the Synechocystis ArsC (SynArsC) is structurally homologous to the well characterized thioredoxin (Trx)-coupled ArsC family but requires the glutaredoxin (Grx) system for its Reactivation, therefore classified as a unique Trx/Grx-hybrid family. The detailed catalytic mechanism of SynArsC is unclear and how the “hybrid” mechanism evolved remains enigmatic. Herein, we report the molecular mechanism of SynArsC by biochemical and structural studies. Our work demonstrates that arsenate reduction is carried out via an intramolecular thiol-disulfide cascade similar to the Trx-coupled family, whereas the Enzyme Reactivation step is diverted to the coupling of the glutathione-Grx pathway due to the local structural difference. The current results support the hypothesis that SynArsC is likely a molecular fossil representing an intermediate stage during the evolution of the Trx-coupled ArsC family from the low molecular weight protein phosphotyrosine phosphatase (LMW-PTPase) family.

Peter C. Searson - One of the best experts on this subject based on the ideXlab platform.

  • In vitro characterization of pralidoxime transport and acetylcholinesterase Reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs).
    Fluids and barriers of the CNS, 2016
    Co-Authors: Erin Gallagher, Il Minn, Janice E. Chambers, Peter C. Searson
    Abstract:

    Current therapies for organophosphate poisoning involve administration of oximes, such as pralidoxime (2-PAM), that reactivate the Enzyme acetylcholinesterase. Studies in animal models have shown a low concentration in the brain following systemic injection. To assess 2-PAM transport, we studied transwell permeability in three Madin-Darby canine kidney (MDCKII) cell lines and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs). To determine whether 2-PAM is a substrate for common brain efflux pumps, experiments were performed in the MDCKII-MDR1 cell line, transfected to overexpress the P-gp efflux pump, and the MDCKII-FLuc-ABCG2 cell line, transfected to overexpress the BCRP efflux pump. To determine how transcellular transport influences Enzyme Reactivation, we developed a modified transwell assay where the inhibited acetylcholinesterase Enzyme, substrate, and reporter are introduced into the basolateral chamber. Enzymatic activity was inhibited using paraoxon and parathion. The permeability of 2-PAM is about 2 × 10−6 cm s−1 in MDCK cells and about 1 × 10−6 cm s−1 in BC1-hBMECs. Permeability is not influenced by pre-treatment with atropine. In addition, 2-PAM is not a substrate for the P-gp or BCRP efflux pumps. The low permeability explains poor brain penetration of 2-PAM and therefore the slow Enzyme Reactivation. This elucidates one of the reasons for the necessity of sustained intravascular (IV) infusion in response to organophosphate poisoning.

  • In vitro characterization of pralidoxime transport and acetylcholinesterase Reactivation across MDCK cells and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs)
    Fluids and Barriers of the CNS, 2016
    Co-Authors: Erin Gallagher, Il Minn, Janice E. Chambers, Peter C. Searson
    Abstract:

    Background Current therapies for organophosphate poisoning involve administration of oximes, such as pralidoxime (2-PAM), that reactivate the Enzyme acetylcholinesterase. Studies in animal models have shown a low concentration in the brain following systemic injection. Methods To assess 2-PAM transport, we studied transwell permeability in three Madin-Darby canine kidney (MDCKII) cell lines and stem cell-derived human brain microvascular endothelial cells (BC1-hBMECs). To determine whether 2-PAM is a substrate for common brain efflux pumps, experiments were performed in the MDCKII-MDR1 cell line, transfected to overexpress the P-gp efflux pump, and the MDCKII-FLuc-ABCG2 cell line, transfected to overexpress the BCRP efflux pump. To determine how transcellular transport influences Enzyme Reactivation, we developed a modified transwell assay where the inhibited acetylcholinesterase Enzyme, substrate, and reporter are introduced into the basolateral chamber. Enzymatic activity was inhibited using paraoxon and parathion. Results The permeability of 2-PAM is about 2 × 10^−6 cm s^−1 in MDCK cells and about 1 × 10^−6 cm s^−1 in BC1-hBMECs. Permeability is not influenced by pre-treatment with atropine. In addition, 2-PAM is not a substrate for the P-gp or BCRP efflux pumps. Conclusions The low permeability explains poor brain penetration of 2-PAM and therefore the slow Enzyme Reactivation. This elucidates one of the reasons for the necessity of sustained intravascular (IV) infusion in response to organophosphate poisoning.

Kamil Kuca - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis and in vitro evaluation of neutral aryloximes as reactivators of Electrophorus eel acetylcholinesterase inhibited by NEMP, a VX surrogate.
    Chemico-biological interactions, 2019
    Co-Authors: Samir F. De A. Cavalcante, Daniel A. S. Kitagawa, Rafael B. Rodrigues, Leandro B. Bernardo, Thiago N. Da Silva, Wellington V. Dos Santos, Joyce S. F. D. De Almeida, Ana Beatriz De A. Correa, Tanos C. C. França, Kamil Kuca
    Abstract:

    Casualties caused by nerve agents, potent acetylcholinesterase inhibitors, have attracted attention from media recently. Poisoning with these chemicals may be fatal if not correctly addressed. Therefore, research on novel antidotes is clearly warranted. Pyridinium oximes are the only clinically available compounds, but poor penetration into the blood-brain barrier hampers efficient Enzyme Reactivation at the central nervous system. In searching for structural factors that may be explored in SAR studies, we synthesized and evaluated neutral aryloximes as reactivators for acetylcholinesterase inhibited by NEMP, a VX surrogate. Although few tested compounds reached comparable Reactivation results with clinical standards, they may be considered as leads for further optimization.

  • A comparison of tabun-inhibited rat brain acetylcholinesterase Reactivation by three oximes (HI-6, obidoxime, and K048) in vivo detected by biochemical and histochemical techniques
    Journal of enzyme inhibition and medicinal chemistry, 2010
    Co-Authors: Jiri Bajgar, Kamil Kuca, Petr Hájek, Jana Karasova Zdarova, Jiri Kassa, Antonin Paseka, Dasa Slizova, Otakar Krs, Daniel Jun, Josef Fusek
    Abstract:

    Tabun belongs to the most toxic nerve agents. Its mechanism of action is based on acetylcholinesterase (AChE) inhibition at the peripheral and central nervous systems. Therapeutic countermeasures comprise administration of atropine with cholinesterase reactivators able to reactivate the inhibited Enzyme. Reactivation of AChE is determined mostly biochemically without specification of different brain structures. Histochemical determination allows a fine search for different structures but is performed mostly without quantitative evaluation. In rats intoxicated with tabun and treated with a combination of atropine and HI-6, obidoxime, or new oxime K048, AChE activities in different brain structures were determined using biochemical and quantitative histochemical methods. Inhibition of AChE following untreated tabun intoxication was different in the various brain structures, having the highest degree in the frontal cortex and reticular formation and lowest in the basal ganglia and substantia nigra. Treatment...

  • Reactivation of VX-inhibited AChE by novel oximes having two oxygen atoms in the linker.
    Environmental toxicology and pharmacology, 2010
    Co-Authors: Kamil Kuca, Daniel Jun, Jiri Cabal, Kamil Musilek, Ondrej Soukup, Miroslav Pohanka, Jaroslav Pejchal, Garp Yeol Yang, Young-sik Jung
    Abstract:

    Abstract Two newly developed AChE reactivators possessing two oxime groups in 4-position of the pyridinium rings with linkers CH 2 O(CH 2 ) 2 OCH 2 and CH 2 O(CH 2 ) 4 OCH 2 were tested for their potency to reactivate VX-inhibited AChE. Their Reactivation potency was compared with currently available oximes such as pralidoxime, obidoxime and HI-6. Appropriate constants (affinity towards the intact and inhibited Enzyme, Reactivation rate) characterizing the Reactivation process were determined. According to the data obtained, a new oxime with CH 2 O(CH 2 ) 2 OCH 2 linker reached as high Reactivation potency as HI-6. The percentage of Reactivation of the oxime with CH 2 O(CH 2 ) 2 OCH 2 linker was comparable to that of obidoxime at a concentration 10 −3  M. Hence, these oximes may be worthy of future development for the treatment of nerve agent intoxications, especially, with lipophilic agents such as soman and cyclosarin.