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Статті в журналах з теми "Inhibition covalente":
1
Aljoundi, Aimen, Ahmed El Rashedy, Patrick Appiah-Kubi та Mahmoud E. S. Soliman. "Coupling of HSP72 α-Helix Subdomains by the Unexpected Irreversible Targeting of Lysine-56 over Cysteine-17; Coevolution of Covalent Bonding". Molecules 25, № 18 (16 вересня 2020): 4239. http://dx.doi.org/10.3390/molecules25184239.
Анотація:
Covalent inhibition has recently gained a resurgence of interest in several drug discovery areas. The expansion of this approach is based on evidence elucidating the selectivity and potency of covalent inhibitors when bound to particular amino acids of a biological target. The unexpected covalent inhibition of heat shock protein 72 (HSP72) by covalently targeting Lys-56 instead of Cys-17 was an interesting observation. However, the structural basis and conformational changes associated with this preferential coupling to Lys-56 over Cys-17 remain unclear. To resolve this mystery, we employed structural and dynamic analyses to investigate the structural basis and conformational dynamics associated with the unexpected covalent inhibition. Our analyses reveal that the coupling of the irreversible inhibitor to Lys-56 is intrinsically less dynamic than Cys-17. Conformational dynamics analyses further reveal that the coupling of the inhibitor to Lys-56 induced a closed conformation of the nucleotide-binding subdomain (NBD) α-helices, in contrast, an open conformation was observed in the case of Cys-17. The closed conformation maintained the crucial salt-bridge between Glu-268 and Lys-56 residues, which strengthens the interaction affinity of the inhibitor nearly identical to adenosine triphosphate (ADP/Pi) bound to the HSP72-NBD. The outcome of this report provides a substantial shift in the conventional direction for the design of more potent covalent inhibitors.
2
Liu, S. Q., and P. A. Knauf. "Lys-430, site of irreversible inhibition of band 3 Cl- flux by eosin-5-maleimide, is not at the transport site." American Journal of Physiology-Cell Physiology 264, no. 5 (May 1, 1993): C1155—C1164. http://dx.doi.org/10.1152/ajpcell.1993.264.5.c1155.
Анотація:
Although eosin-5-maleimide (EM) covalently labels band 3 and has been thought to react at the external-facing anion transport site, EM reversibly inhibits Cl- exchange at 0 degrees C in a noncompetitive fashion, indicating that under these conditions it does not bind to the transport site [Knauf, P.A., N.M. Strong, J. Penikas, R.B. Wheeler, Jr., and S.J. Liu. Am. J. Physiol. 264 (Cell Physiol. 33): C1144-C1154 1993]. To see whether or not the covalent labeling by EM takes place at the same noncompetitive site as the reversible binding, we examined the dependence of reaction rate on EM concentration. The reaction rate saturates with increasing EM concentration, indicating that reversible binding precedes covalent reaction and that EM therefore acts as an affinity label. A more complex model in which reversible binding prevents a bimolecular reaction at a different site cannot, however, be ruled out. Cl- gradients across the membrane affect EM reversible binding in a manner suggesting that EM binds preferentially to the Eo form of band 3, with the transport site unloaded and facing outward. Thus EM binds to and probably reacts covalently with a site that is different from the transport site, but whose conformation is affected by the orientation of the transport site. Lysine-430, the amino acid residue which is covalently labeled by EM (4), may be near the transport site but does not seem to be directly involved in the binding of transported substrates such as chloride. EM binding to one band 3 monomer decreases the reactivity of the adjacent monomer but does not decrease the affinity constant of the reversible binding step that precedes covalent reaction. Although a small fraction (approximately 1%) of band 3 monomers fail to react with EM, EM nearly completely inhibits transport in those monomers with which it reacts.
3
Yang, Jianhong, Yong Li, Wei Yan, Weimin Li, Qiang Qiu, Haoyu Ye та Lijuan Chen. "Covalent modification of Cys-239 in β-tubulin by small molecules as a strategy to promote tubulin heterodimer degradation". Journal of Biological Chemistry 294, № 20 (2 квітня 2019): 8161–70. http://dx.doi.org/10.1074/jbc.ra118.006325.
Анотація:
Clinical microtubule-targeting drugs are functionally divided into microtubule-destabilizing and microtubule-stabilizing agents. Drugs from both classes achieve microtubule inhibition by binding different sites on tubulin and inhibiting or promoting polymerization with no concomitant effects on the protein levels of tubulin heterodimers. Here, we have identified a series of small molecules with diverse structures potentially representing a third class of novel tubulin inhibitors that promote degradation by covalent binding to Cys-239 of β-tubulin. The small molecules highlighted in this study include T0070907 (a peroxisome proliferator-activated receptor γ inhibitor), T007-1 (a T0070907 derivative), T138067, N,N′-ethylene-bis(iodoacetamide) (EBI), and allyl isothiocyanate (AITC). Label-free quantitative proteomic analysis revealed that T007-1 promotes tubulin degradation with high selectivity. Mass spectrometry findings showed covalent binding of both T0070907 and T007-01 to Cys-239 of β-tubulin. Furthermore, T007-1 exerted a degradative effect on tubulin isoforms possessing Cys-239 (β2, β4, and β5(β)) but not those containing Ser-239 (β3, β6) or mutant β-tubulin with a C239S substitution. Three small molecules (T138067, EBI, and AITC) also reported to bind covalently to Cys-239 of β-tubulin similarly induced tubulin degradation. Our results strongly suggest that covalent modification of Cys-239 of β-tubulin by small molecules could serve as a novel strategy to promote tubulin heterodimer degradation. We propose that these small molecules represent a third novel class of tubulin inhibitor agents that exert their effects through degradation activity.
4
Maksimenko, A. V., and R. Sh Beabealashvili. "Theoretical Grounding and Formation of Experimental Approaches to Hyaluronidase Structure Consolidation due to Its Computational Interactions with Shortchain Glycosaminoglycan Ligands." Биоорганическая химия 49, no. 4 (July 1, 2023): 369–83. http://dx.doi.org/10.31857/s0132342323020161.
Анотація:
The computational study of 3D model hyaluronidase interaction with shortchain glycosaminoglycan ligands demonstrated the diversity and significance of their reaction on enzyme structure. It has been realized due to electrostatic noncovalent interactions (without specific coupling with active site) inducing the perceptible conformational alterations of biocatalyst molecule. As a result of this the inactivation and stabilization of enzyme globule are observed, change of inhibition of biocatalyst by heparin. The binding of chondroitin trimers (on centers cn6, cn3, cn1) to hyaluronidase molecular surface increased the enzyme stability, binding of chondroitin sulfate trimers (on centers cs2, cs4, cs7, cs8 or cs1, cs2, cs4, cs7, cs8) decreased the inhibition of enzyme by tetramer heparin. It should be noted the importance of ligand binding for regulation of enzyme functioning and existence of multiform and multicomponent microenvironment of enzyme. The sequence of preferable coupling of ligands with hyaluronidase is elicited in our study and with its help was evaluate reality of experimental selective modification of enzyme (possibly no covalently or covalently, for instance, with chondroitin sulfate trimers on centers cs7, cs1, cs5) for experimental obtaining of stabilized enzyme forms of medical destination. The perspective approaches for this aim may be the no covalent reaction on hyaluronidase by chondroitin or chondroitin sulfate trimers as well covalent modification of biocatalyst by chondroitin sulfate trimers.
5
Bhatia, Sumeena, Steven C. Almo, Stanley G. Nathenson, and Richard J. Hodes. "Dynamic equilibrium of B7-1 dimers and monomers is important for regulation of TCR/CD28 – mediated T cell activation (33.28)." Journal of Immunology 182, no. 1_Supplement (April 1, 2009): 33.28. http://dx.doi.org/10.4049/jimmunol.182.supp.33.28.
Анотація:
Abstract Under steady state conditions, B7-1 is present as a mixed population of non-covalent dimers and monomers on the cell surface. Here we have examined the physiological significance of this unique dimer-monomer equilibrium state of B7-1. We demonstrate that altering B7-1 to uniformly covalent dimeric state results in increased frequency of CD28 mediated T cell-APC conjugates. This augmented T cell-APC conjugate formation correlates with persistent concentration of signaling molecules, PKC-θ and lck, at the immunological synapse (IS) and with a higher calcium flux in T cells. In contrast, B7-1 acquisition by T cells, an event that occurs as a consequence of CD28 engagement with B7-1/B7-2, is highly reduced when B7-1 is present in covalently dimeric state. The ability of covalently dimeric and wild type B7-1 to costimulate antigen-specific T cell proliferation was also assessed. In contrast to the enhanced ability of dimeric B7-1 to support proximal T cell activation events, sensitivity to competitive inhibition by soluble CTLA-4:Ig indicated that the covalent dimeric form of B7-1 is in fact less efficient in costimulating T cell proliferation. These findings suggest a novel model in which optimal T cell costimulatory function of B7-1 requires CD28 engagement by B7-1 in a non-covalent dimeric state, followed by dissociation of these B7-1 dimers, facilitating downregulation of CD28 and internalization of B7-1.
6
Kuznetsova, Anastasiya, Philipp Klein, and Till Opatz. "Halogenated 2,1,3-benzoxadiazoles as Potential Fluorescent Warheads for Covalent Protease Inhibitors." Proceedings 9, no. 1 (November 14, 2018): 54. http://dx.doi.org/10.3390/ecsoc-22-05670.
Анотація:
Recently there has been a growing interest in covalent protease inhibitors in both industry and academia, caused by their longer residence times, their higher potency and their high ligand efficiency. Covalently reactive moieties which interact with activated amino acid residues such as serine or cysteine in enzymes like proteases or esterases mostly act through nucleophilic addition, substitution or ring opening. In contrast, nucleophilic aromatic substitution (SNAr) is rarely employed. In our previous work, we prepared and investigated electrophilic “warheads”, which contain aromatic, heteroaromatic or quinoid fragments. Some of them show potent inhibition constants for cathepsin L, cathepsin B, rhodesain or dengue-protease, and depending on the exact nature of the electrophile they exhibit reversible covalent or irreversible inhibition modes. In the present work, we demonstrate the synthesis of fluorescent “warhead” candidates based on 2,1,3-benzoxadiazoles and the investigation of their physicochemical and photophysical properties. These molecules shall serve as probes for the detailed analysis of association/dissociation mechanism and of the kinetic parameters of the bond forming event.
7
Hognon, Cécilia, Marco Marazzi, and Cristina García-Iriepa. "Atomistic-Level Description of the Covalent Inhibition of SARS-CoV-2 Papain-like Protease." International Journal of Molecular Sciences 23, no. 10 (May 23, 2022): 5855. http://dx.doi.org/10.3390/ijms23105855.
Анотація:
Inhibition of the papain-like protease (PLpro) of SARS-CoV-2 has been demonstrated to be a successful target to prevent the spreading of the coronavirus in the infected body. In this regard, covalent inhibitors, such as the recently proposed VIR251 ligand, can irreversibly inactivate PLpro by forming a covalent bond with a specific residue of the catalytic site (Cys111), through a Michael addition reaction. An inhibition mechanism can therefore be proposed, including four steps: (i) ligand entry into the protease pocket; (ii) Cys111 deprotonation of the thiol group by a Brønsted–Lowry base; (iii) Cys111-S− addition to the ligand; and (iv) proton transfer from the protonated base to the covalently bound ligand. Evaluating the energetics and PLpro conformational changes at each of these steps could aid the design of more efficient and selective covalent inhibitors. For this aim, we have studied by means of MD simulations and QM/MM calculations the whole mechanism. Regarding the first step, we show that the inhibitor entry in the PLpro pocket is thermodynamically favorable only when considering the neutral Cys111, that is, prior to the Cys111 deprotonation. For the second step, MD simulations revealed that His272 would deprotonate Cys111 after overcoming an energy barrier of ca. 32 kcal/mol (at the QM/MM level), but implying a decrease of the inhibitor stability inside the protease pocket. This information points to a reversible Cys111 deprotonation, whose equilibrium is largely shifted toward the neutral Cys111 form. Although thermodynamically disfavored, if Cys111 is deprotonated in close proximity to the vinylic carbon of the ligand, then covalent binding takes place in an irreversible way (third step) to form the enolate intermediate. Finally, due to Cys111-S− negative charge redistribution over the bound ligand, proton transfer from the initially protonated His272 is favored, finally leading to an irreversibly modified Cys111 and a restored His272. These results elucidate the selectivity of Cys111 to enable formation of a covalent bond, even if a weak proton acceptor is available, as His272.
8
Bisconte, Angelina, Ronald Hill, Michael Bradshaw, Erik Verner, David Finkle, Ken Brameld, Jens Funk, David Goldstein, and Phil Nunn. "Efficacy in collagen induced arthritis models with a selective, reversible covalent Bruton’s tyrosine kinase inhibitor PRN473 is driven by durable target occupancy rather than extended plasma exposure (THER5P.904)." Journal of Immunology 194, no. 1_Supplement (May 1, 2015): 139.6. http://dx.doi.org/10.4049/jimmunol.194.supp.139.6.
Анотація:
Abstract Bruton’s Tyrosine Kinase (BTK) is an essential signaling element downstream of the B-cell receptor (BCR). Inhibition of BTK activity in B cells produces phenotypic changes consistent with blockade of the BCR, including inhibition of cell proliferation, differentiation, maturation, and survival. A selective BTK inhibitor has the potential to treat diseases involving inflammation and autoimmunity. Using Principia Biopharma’s proprietary Tailored Covalency™ technology, we discovered PRN473, a reversible covalent BTK inhibitor that selectively binds BTK with a slow off-rate as assessed in biochemical and cell based assays. A slow off-rate molecule with rapid systemic clearance may lead to a long duration of action and high efficacy, while reducing the potential for off target toxicities. The duration of binding of PRN473 was measured in vivo, with prolonged BTK occupancy confirmed after drug was cleared from plasma. In mouse and rat collagen-induced arthritis (CIA) models, PRN473 when dosed therapeutically demonstrated dose-dependent inhibition, reversal of arthritis, with almost complete abrogation of clinical scores and preservation of joint integrity and histology. BTK occupancy in splenocytes was found to be a robust measure of BTK inhibition and correlated with efficacy in the CIA models. These data in combination with GLP toxicology studies, where no organ specific toxicities were observed, validate using a reversible covalent BTK inhibitor to treat autoimmune diseases.
9
Beck, Philipp, Christian Dubiella, and Michael Groll. "Covalent and non-covalent reversible proteasome inhibition." Biological Chemistry 393, no. 10 (October 1, 2012): 1101–20. http://dx.doi.org/10.1515/hsz-2012-0212.
Анотація:
Abstract The 20S proteasome core particle (CP) is the proteolytically active key element of the ubiquitin proteasome system that directs the majority of intracellular protein degradation in eukaryotic cells. Over the past decade, the CP has emerged as an anticancer therapy target after approval of the first-in-class drug bortezomib (Velcade®) by the US Food and Drug Administration. However, bortezomib and all second-generation CP inhibitors that are currently explored in clinical phase studies react covalently and most often irreversibly with the proteolytic sites of the CP, hereby causing permanent CP blockage. Furthermore, reactive head groups result in unspecific binding to proteasomal active centers and in substantial enzymatic off-target activities that translate to severe side effects. Thus, reversible proteasome inhibitors might be a promising alternative, overcoming these drawbacks, but are challenging with respect to their urge for thorough enthalpic and entropic optimization. This review describes developments in the hitherto neglected field of reversible proteasome inhibitors focusing on insights gained from crystal structures, which provide valuable knowledge and strategies for future directions in drug development.
10
Piestrzeniewicz, Mariola K., Dorota Wilmańska, Janusz Szemraj, Kazimierz Studzian, and Marek Gniazdowski. "Interactions of Novel Morpholine and Hexamethylene Derivatives of Anthracycline Antibiotics with DNA." Zeitschrift für Naturforschung C 59, no. 9-10 (October 1, 2004): 739–48. http://dx.doi.org/10.1515/znc-2004-9-1020.
Анотація:
Abstract Doxorubicin (DOX), daunorubicin (DRB), epidoxorubicin (EDOX) and their analogues with a 3′-NH2 group in daunosamine form a covalent bond with a 2-NH2 group of guanine via a methylene group from formaldehyde (CH2O). It is assumed that a Schiff base type intermediate is formed between CH2O and the 3′-NH2 group in the reaction. This reaction is supposed to occur in the cell. New analogues of anthracyclines with formamidine functionality bound to C-3′ of daunosamine and containing the bulky morpholine (DRBM, DOXM and EDOXM) or hexamethyleneimine rings attached are studied in our laboratory. These substituents decrease the association of the drugs to DNA and potentially hinder the formation of Schiff base-intermediates. Our experiments indicate that the formation of the covalent complexes by DRB, DOX and EDOX under these conditions is confirmed by a high enhancement (17-40x) of the inhibition of overall RNA synthesis by E. coli RNA polymerase on T7 DNA. DRBM and DOXM exhibit a lower enhancement of the inhibition by CH2O (7-13x). The other analogues show a 1.6-3x increase of inhibition. Hence, their covalent binding is lower than that of the parent compounds. These conclusions are confirmed by spectrophotometric estimations following removal of non-covalently associated drugs. Electrophoretic analysis of drug-DNA complexes formed in the presence of CH2O indicates that DRBM and DOXM as their parent compounds induce labile cross-links in DNA. Comparison of the results obtained at the subcellular level with cytotoxicity estimations indicates that there is a correlation between cytotoxicity of the anthracyclines on L1210 cells and transcriptional template activity of drug-DNA complexes formed in the presence of CH2O (r = 0.64; n = 9). These data confirm a notion that covalent attachment of anthracyclines to DNA is an essential event leading to cytotoxicity.
Дисертації з теми "Inhibition covalente":
1
Fındık, Volkan. "Simulations atomistiques de la réaction d’acétylation d’amines et de l’inhibition covalente de l’enzyme Phosphoinositide 3-kinase (PI3K)." Electronic Thesis or Diss., Université de Lorraine, 2022. http://www.theses.fr/2022LORR0266.
Анотація:
Les inhibiteurs covalents ciblés (TCI) sont très prometteurs pour la recherche de nouveaux médicaments. Ils offrent un certain nombre d’avantages par rapport aux inhibiteurs réversibles traditionnels, comme un temps de séjour prolongé, une puissance accrue et la possibilité d’apporter des modifications pour une conception efficace. Les inhibiteurs de kinases sont les exemples les plus courants d’ITC. Les enzymes phosphoinositide 3-kinase (PI3K) sont des cibles médicamenteuses importantes en oncologie car elles sont impliquées dans la voie de signalisation de nombreuses fonctions cellulaires telles que le contrôle de la croissance, le métabolisme et l’initiation de la traduction. Les résidus lysine (Lys) ont suscité un intérêt croissant comme alternative pour l’inhibition covalente ciblée. Récemment, les premiers inhibiteurs sélectifs et irréversibles avec des groupes esters comme tête électrophile ciblant le résidu Lys779 et inactivant de manière covalente l’enzyme PI3Kδ ont été rapportés. L’objectif principal de cette thèse est d’élucider le mécanisme de l’inhibition covalente de PI3Kδ par ces inhibiteurs ester afin d’aider à la conception future de nouveaux inhibiteurs avec des activités supérieures. Avant les études mécanistiques sur l’enzyme, nous avons d’abord effectué des calculs ab initio et DFT sur la réaction modèle entre la méthylamine et les acétates de méthyle, phényle et p-NO2 phényle en solution aqueuse. Les mêmes systèmes modèles ont ensuite été étudiés par l’approche de dynamique moléculaire QM/MM "à double niveau". Pour l’option "bas niveau", les simulations QM/MM ont été conduites au niveau PM3/TIP3P, et elles ont permis d’obtenir l’échantillonnage du système dans le cadre de la technique « umbrella sampling ». Les structures obtenues ont ensuite été utilisées pour obtenir des corrections perturbatives à l’énergie libre avec une région QM de "haut niveau" décrite par la méthode M06-2X/6-311+G(d,p). Les résultats montrent que la première étape implique la formation d’un intermédiaire tétraédrique zwittérionique. Ensuite, pour des esters suffisamment électrophiles, tels que le dérivé p-NO2, la réaction se déroule par dissociation du zwittérion sous forme de paire d’ions, suivie d’un transfert de protons conduisant à la formation des produits attendus. Nous avons utilisé des outils théoriques similaires pour étudier les mécanismes d’inhibition dans le cas de l’enzyme. Tout d’abord, un modèle de site actif de l’enzyme a été construit par des simulations classiques de dynamique moléculaire. Ensuite, l’approche ONIOM QM:QM au niveau M06-2X/6-31+G(d,p):PM6 a été appliquée pour obtenir les mécanismes de réaction envisageables dans ce site actif. Ces calculs nous ont permis d’affiner les mécanismes de réaction dans l’environnement enzymatique qui confirment globalement les étapes obtenues à partir du petit système modèle. Nous avons finalement utilisé ces informations pour aborder une étude QM/MM dynamique sur l’enzyme en utilisant le même protocole "double niveau" établi pour le petit système modèle, ce qui nous a permis d’obtenir le profil d’énergie libre du mécanisme d’inhibition de PI3Kδ pour le dérivé p-NO2 de l’inhibiteur ester. La barrière calculée est en bon accord avec les données cinétiques expérimentales disponibles, ce qui valide l’approche théorique proposée et les mécanismes obtenus. Grâce à l’élucidation du mécanisme d’inhibition de composés précédemment testés expérimentalement, notre étude ouvre la voie à la découverte de nouveaux inhibiteurs à activité améliorée à l’aide des outils de la chimie théoriqueTargeted Covalent Inhibitors (TCIs) hold great promise for search of new drugs. They offer a number of potential advantages over traditional reversible inhibitors, such as extended residence time, increased potency, and the ability to make modifications for effective design. Kinase inhibitors are the most common examples of TCIs. Phosphoinositide 3-kinase (PI3K) enzymes are important drug targets in oncology as they are involved in the signaling pathway for many cellular functions such as growth control, metabolism and translation initiation. Lysine (Lys) residues have gained increasing interest as an alternative for targeted covalent inhibition. Recently, the first selective and irreversible inhibitors with ester groups as electrophilic head targeting the Lys779 residue and covalently inactivating the PI3Kδ enzyme were reported. The main objective of this thesis is to elucidate the mechanism of the covalent inhibition of PI3Kδ by these ester inhibitors in order to assist future design of new inhibitors with superior activities. Prior to the mechanistic studies on the enzyme, initially, we performed ab initio and DFT calculations on the model reaction between methylamine and methyl, phenyl and p-NO2 phenyl acetates in aqueous solution. The same model systems were then studied by the "dual-level" QM/MM molecular dynamics approach. For the “low-level” option, PM3/TIP3P umbrella sampling QM/MM simulations were applied for the sampling. The obtained structures were then used to obtain perturbative corrections to the free energy with a “high-level” QM region at the M06-2X/6-311+G(d,p) level. The results show that thefirst step involves the formation of the zwitterionic tetrahedral intermediate. Then, for sufficiently electrophilic esters, such as the p-NO2 derivative, the reaction proceeds by dissociation of the zwitterion as an ion pair, followed by proton transfer leading to the formation of the expected products. We, then, employed similar computational tools to shed light on the mechanistic aspects of the enzyme. First, an active site model of the enzyme was built through classical molecular dynamics simulations. Then, ONIOM QM:QM approach at the M06-2X/6 -31+G(d,p):PM6 level was applied to get possible reaction mechanisms in this active site. These calculations guided us to refine the reaction mechanisms in enzyme environment which globally confirm the steps obtained from the small model system. We finally used this information to approach a dynamic QM/MM study on the enzyme using the same“dual-level” protocol established for the small model system, which allowed us to obtain the free energy profile of the inhibition mechanism of PI3Kδ for p-NO2 derivative of the ester inhibitor. The calculated barrier is in good agreement with the available experimental kinetic data, which validates the proposed theoretical approach and the obtained mechanisms. Through the elucidation of the inhibition mechanism of previously experimentally tested compounds, our study paves the way for the discovery of new inhibitors with improved activity with the help of theoretical chemistry tools
2
Akbar, Abdullah. "Design, Synthesis and Evaluation of Covalent Inhibitors for Tissue Transglutaminase and Factor XIIIa." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39645.
Анотація:
Transglutaminases are a family of enzymes expressed in various tissues of our body. Some are expressed ubiquitously while others are specific to a tissue. Their primary catalytic activity is to crosslink substrates via an isopeptidic bond. The work described in this thesis focuses on two of these transglutaminases; human tissue transglutaminase (hTG2) and human factor XIIIa (FXIIIa). Divided into two projects for each enzyme, the main objective of this thesis was directed towards the discovery of potent and selective covalent inhibitors for each isozyme, namely hTG2 and hFXIIIa. The first project was concentrated on the inhibition of hTG2 activity. Ubiquitously expressed in tissues, hTG2 is a multifunctional enzyme. Its primary activity is the formation of isopeptide bonds between glutamine and lysine residues found on the surface of proteins or substrates. In addition to its catalytic activity, hTG2 is also a G-protein, distinguishing it from other members of the transglutaminase family. Much evidence illustrates that hTG2’s multifunctional abilities are conformationally regulated between its “open” and “closed” forms. Overexpression and unregulated hTG2 activity has been associated with numerous human diseases; however, most evidence has been collected for its association with fibrosis and celiac sprue. More recently, elevated hTG2 expression has been linked to cancer stem cell survival and metastatic phenotype in certain cancer cells. These findings call for the development of suitable and potent inhibitors that selectivity inactivate human hTG2 as a potential therapeutic target. Starting with previously designed acrylamide based peptidomimetic irreversible inhibitors, a structure-activity relationship (SAR) study was conducted. In this work, >20 novel irreversible inhibitors were prepared and kinetically evaluated. Our lead inhibitors allosterically inhibited GTP binding by locking the enzyme in its open conformation, as demonstrated both in vitro and in cells. Furthermore, our most potent and efficient irreversible inhibitors revealed selectivity for hTG2 over other relevant members of the transglutaminase family (hTG1, hTG3, hTG6 and hFXIIIa), providing higher confidence towards our goal of developing an ideal drug candidate. The second project was concentrated on the inhibition of hFXIIIa activity. In the blood, coagulation factor XIII (FXIII) is a tetrameric protein consisting of two catalytic A subunits (FXIII-A2) and two carrier/inhibitory B (FXIII-B2) subunits. It is a zymogen, which is converted into active transglutaminase (FXIIIa) in the final phase of coagulation cascade by thrombin proteolytic activity and Ca2+ binding. hFXIII is essential for hemostasis and thus its deficiency results in severe bleeding conditions. Further, hFXIIIa mechanically stabilizes fibrin and protects it from fibrinolysis. Due to the enzyme’s involvement in the stability of blood clots, inhibition of hFXIIIa activity has been linked to thrombotic diseases. Furthermore, inhibitors of the enzyme have the therapeutic potential to be used as anticoagulant agents. The current number of selective and potent inhibitors of hFXIIIa are few, mainly due to the similarity between its catalytic pockets and hTG2. Inspired by a poorly reactive hTG2 inhibitor discovered in this work’s hTG2 SAR study, we synthesized a small library of covalent inhibitors for hFXIIIa. Our kinetic results from this pioneering SAR study will pave the way for future hFXIIIa inhibitor SAR studies.
3
Belghazi, Maya. "Etude de modifications covalentes de protéines par spectrométrie de masse Maldi-Tof et Esi-Tof." Palaiseau, Ecole polytechnique, 2001. http://www.theses.fr/2001EPXX0030.
Анотація:
La spectrométrie de masse MALDI-TOF (matrix-assisted laser desorption/ionization- time of flight) et ESI-TOF (electrospray ionization- time of flight) permettent d'accéder à des méthodologies performantes pour l'étude de la structure primaire des protéines. Ce travail a mis en oeuvre ces deux modes d'ionisation pour étudier les modifications covalentes de trois protéines impliquées dans des contextes biologiques différents. En premier lieu, les modifications post-traductionnelles de deux nitrile hydratases (NHases), métalloenzymes bactériennes catalysant l'hydrolyse de nitriles en amides, ont été caractérisées. Des études sur les protéines intactes ont mis en évidence deux modifications post-traductionnelles--un acide sulfénique et un acide sulfinique--sur les cystéines du centre actif de deux NHases de bactéries différentes. Ensuite, nous avons caractérisé les modifications post-traductionnelles d'une protéine intervenant dans la coagulation sanguine, le facteur IX (FIX), dans le but d'établir une référence structurale du FIX plasmatique. Cette protéine comporte un ensemble de modifications complexes incluant en particulier N-glycosylations, O-glycosylations, phosphorylation et sulfatation, localisées sur une région de la protéine, le peptide d'activation. En dernier lieu, nous avons étudié les mécanismes d'inhibition suicide de deux cytochromes P450. Pour cela, nous avons caractérisé, grâce à des techniques de marquage par des isotopes stables, les métabolites produits lors de l'hydroxylation de l'acide tiénilique par le P450 2C9, réaction conduisant également à l'inactivation de l'enzyme. Ces expériences ont conduit à proposer deux mécanismes réactionnels différents pour ce composé. Le mécanisme de l'inhibition suicide d'un P450 d'origine végétale a été abordé en cherchant à identifier les peptides marqués pendant la réaction. Cette identification n'a pas été obtenue, vraisemblablement en raison de l'instabilité du marquage dans nos conditions d'analyse.
4
Erdmann, Alexandre. "Conception, synthèse et caractérisation de nouveaux inhibiteurs de méthyltranférases d'ADN à visée anticancéreuse." Thesis, Toulouse 3, 2015. http://www.theses.fr/2015TOU30270.
Анотація:
Le domaine de l'épigénétique couvre l'ensemble des phénomènes héritables et transmissibles qui interviennent dans l'expression du génome sans modifier la séquence nucléotidique. L'information épigénétique est régulée par les modifications de la chromatine impliquant les histones et l'ADN. La méthylation de l'ADN est un phénomène réversible jouant un rôle crucial dans l'expression des gènes puisque la méthylation des promoteurs de gènes empêche leur transcription. La modulation aberrante de cette marque épigénétique est associée à diverses pathologies telles que le cancer. Cette méthylation étant réversible, elle peut être ciblée afin de reprogrammer la cellule cancéreuse. Les méthyltransferases d'ADN (DNMT), étant les enzymes responsables de la méthylation, représentent la cible principale de notre stratégie de recherche. Leur inhibition par des petites molécules est au centre de nos recherches de thérapies anticancéreuses dont les bases sont représentées par deux catégories d'inhibiteurs de DNMT existant. Les premiers sont des analogues de cytosine qui est la cible de la méthylation. Ils sont connus pour s'intégrer dans l'ADN et former un complexe covalent irréversible avec l’enzyme (complexe suicide) mais ils souffrent d'un manque de stabilité et de certains effets indésirables dus à leur incorporation dans l’ADN. Les seconds sont les inhibiteurs non nucléosidiques qui sont divers et parfois connus pour cibler d’autres enzymes. Ils ont l’avantage de pouvoir être utilisés comme sondes pour comprendre plus précisément le mécanisme d'inhibition mais ils manquent de spécificité et de sélectivité. Au cours de cette thèse, une banque de molécules a été criblée à partir de la combinaison d'un test enzymatique et d'un test cellulaire visant à inhiber ces enzymes. Les synthèses de trois familles de molécules potentiellement inhibitrices de DNMT issus de ce criblage sont décrites en expliquant le chemin de drug design emprunté pour obtenir des informations mécanistiques d’inhibition de la méthylation d’ADN, notamment de réactivité avec la cible. Les découvertes ont été inspirées par des études de modélisation permettant de mettre en évidence une sélectivité de certains inhibiteurs. La synthèse chimique a également abouti à une nouvelle voie de synthèse d’accès aux diaminopyrimidines dont l’impact permet de faciliter les études chimiques de dérivés quinazolines comme inhibiteur non nucléosidiques utiles pour les thérapies anticancéreusesEpigenetic is defined as the study of heritable changes in the genes expression without altering the DNA sequence. Two main processes are implicated in this field, the histones modifications and the DNA methylation. By introducing an acetyl or a methyl group on the histone tails or by methylation of DNA, the chromatin state is modified and the gene expression is controlled. Aberrant epigenetic modifications are associated with several diseases, in particular with cancer. In cancer cells, the whole DNA is hypomethylated, thus promoting genome instability, while the promoter region is hypermethylated, inducing silencing of these genes. Overall, these observations indicate that DNA methylation is a central epigenetic process in cancerogenesis. Since DNA methylation is reversible, it is possible to target the methylation process in order to reactivate tumor suppressor genes. The DNA methyltransferases (DNMTs), the enzymes responsible for DNA methylation, use the SAM co-factor at specific CpG sites to product 5-methylcytosine. Three main isoforms (DNMT1, DNMT3A and DNMT3B) are described to ensure efficient methylation process during replication. Two families of DNMT inhibitors already exist, the nucleosidiques analogues are cytidine derivatives and are toxic molecules because of their incorporation into DNA, and the non-nucleosidic analogues are less toxic but also less potent. Our strategy of drug design is based on docking study and high throughput screening (HTS) information. First, novel potent derivatives of reference inhibitors are designed from molecular modelling. Then, three different families of compounds from HTS are described with appropriate structure-activity relationship studies. Mechanistic information on DNA methylation process are described through the discovery of a reactive inhibitor of DNMT3A. The study on a family of hydrazone derivatives of gallic acid is depicted and shows its selectivity for DNMT3A, compared to DNMT1, based on docking study. An alternative chemical pathway to diaminopyrimidines is described and extended to the synthesis of quinazolone in order to synthesize new quinazoline derivatives as potent inhibitors of DNMT. Promising informations are described in this thesis to enrich epigenetic knowledge of tumor genesis and to provide new molecules for anticancer therapy
5
Ueda, Tsuyoshi. "Development of Covalent Inhibitors and Drug Screening using Ligand-Directed NASA Chemistry." Doctoral thesis, Kyoto University, 2020. http://hdl.handle.net/2433/253248.
Анотація:
京都大学0048
新制・課程博士
博士(工学)
甲第22412号
工博第4673号
新制||工||1729(附属図書館)
京都大学大学院工学研究科合成・生物化学専攻
(主査)教授 浜地 格, 教授 森 泰生, 教授 生越 友樹
学位規則第4条第1項該当
Doctor of Philosophy (Engineering)
Kyoto University
DGAM
6
Vermeer, Lydia Maria Mexas. "Covalent modification and inhibition of tyrosine hydroxylase by 3,4-dihydroxyphenylacetaldehyde, an endogenously produced neurotoxin relevant to Parkinson's disease." Diss., University of Iowa, 2012. https://ir.uiowa.edu/etd/1923.
Анотація:
Parkinson's disease (PD) is a prevalent neurodegenerative disorder which affects over a million people in the United States. This disease is marked by the selective loss of dopaminergic neurons in the substantia nigra, leading to a decrease in the important neurotransmitter dopamine (DA), which is essential for the initiation and execution of coordinated movement. Currently, the pathogenesis behind PD is unknown, but there is evidence that both exogenous causes, such as pesticides and metals, as well as endogenous causes, such as reactive oxygen species or reactive metabolism intermediates, may play a role in the onset and progression of the disease. DA is catabolized by monoamine oxidase to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is further metabolized by aldehyde dehydrogenase and aldehyde reductase to the acid and alcohol products, respectively. Studies have demonstrated the reactivity of DOPAL with peptides and proteins, leading to covalent modification which may be detrimental to protein action. Furthermore, studies have shown that DOPAL is toxic, leading to a decrease in cell viability. Due to this, it was of interest to further study DOPAL and how it may play a role in the onset and progression of PD.
It was of particular interest to determine protein targets of DOPAL modification. Until recently, no protein targets were identified and the cellular consequence of elevated DOPAL had not been fully studied. It has been previously shown that the important enzyme, tyrosine hydroxylase (TH) is inhibited by other catechols, including DA. This enzyme catalyzes the rate-limiting step in DA synthesis, oxidizing tyrosine to L-DOPA which is further metabolized to DA. Therefore, it was of interest to determine the effect of DOPAL on TH activity. It was hypothesized that DOPAL modifies and inhibits TH, leading to a decrease in the production of L-DOPA and DA. This work employed the use of a dopaminergic cell model (PC6-3 cells), to positively identify TH as a protein target of DOPAL modification. It also used both cell lysate as well as PC6-3 cell studies to investigate the effect of DOPAL modification on TH activity. Mass spectrometry was also utilized to determine sites of protein modification on TH.
Results show that TH is potently inhibited by DOPAL modification, leading to a significant decrease in both L-DOPA and DA. Furthermore, DOPAL inhibition appears to be slowly-irreversible, with enzyme activity showing a time- and concentration dependent in recovery after preincubation with DOPAL. A novel cloning and purification procedure was used to clone human recombinant TH, which was used in mass spectrometry studies in which five sites of DOPAL modification were discovered. Furthermore, a real-time assay for TH activity was developed using a plate reader to spectrophotometrically observe the formation of L-DOPA over time. These data demonstrate the toxicity and potent enzyme inhibition by DOPAL and implicate DOPAL as a neurotoxin relevant in the pathogenesis of PD.
7
Tay, Sew Wah. "Factors affecting the thrombin inhibiting activity of heparin when immobilised to hydrogels by covalent bonding." Thesis, Massachusetts Institute of Technology, 1986. http://hdl.handle.net/1721.1/16490.
Анотація:
Thesis (Sc. D.)--Massachusetts Institute of Technology, Dept. of Applied Biological Sciences, 1986.MICROFICHE COPY AVAILABLE IN ARCHIVES AND SCIENCE.
Bibliography: leaves 157-166.
by Sew-Wah Tay.
Sc.D.
8
Bourgeois, Karine. "Towards in vitro Pharmacokinetic Assessment of Novel Targeted Covalent Inhibitors for Human Tissue Transglutaminase." Thesis, Université d'Ottawa / University of Ottawa, 2019. http://hdl.handle.net/10393/39472.
Анотація:
Human tissue transglutaminase (TG2) is a calcium-dependent multifunctional enzyme that natively catalyzes the post-translational modification of proteins, namely by the formation of isopeptide bonds between protein- or peptide-bound glutamine and lysine residues. This ubiquitously expressed enzyme plays important roles in cellular differentiation, extracellular matrix stabilization, and apoptosis, to name a few. However, its unregulated activity has been associated with many pathologies such as fibrosis, cancer, neurodegenerative disorders and celiac disease. Most of these disorders are associated with unregulated acyl-transferase activity. As such, the Keillor group has directed its efforts towards the development of TG2 inhibitors.
Over the years, the Keillor group has synthesized large libraries of targeted covalent inhibitors against TG2. These compounds have undergone pharmacodynamic testing in order to examine their kinetic parameters of inhibition. Having gained knowledge of their enzyme kinetics, the logical next step was to consider their pharmacokinetic profiles. In the context of this thesis, we considered two important pharmacokinetic properties: membrane permeability and off-target reactivity.
Firstly, we aimed to evaluate our inhibitors for their ability to permeate the cell membrane. In efforts to do so, we were able to adapt, optimize, and validate a parallel artificial membrane permeability assay (PAMPA) utilizing hexadecane as our artificial membrane. We were able to test a few of our own inhibitors and found that compounds NC9, VA4 and AA9 possess Log Pe values of -5.26 ± 0.01, -4.66 ± 0.04 and -6.5 ± 0.5 respectively.
Secondly, we sought to investigate the susceptibility of our inhibitors to glutathione addition reactions under physiological conditions. We adapted and optimized a colorimetric assay using Ellman’s reagent (DTNB) and found that our inhibitors are minimally reactive with glutathione.
The methods developed over the course of this work provide protocols that can be adopted for the characterization of future inhibitors in the Keillor group, along the process of developing TG2 inhibitors into drug candidates.
9
CAMPANER, ELENA. "A new covalent PIN1 inhibitor selectively targets cancer cells by a dual mechanism of action." Doctoral thesis, Università degli Studi di Trieste, 2017. http://hdl.handle.net/11368/2908180.
Анотація:
In the last decades targeted drugs have improved cancer treatment, but revealed to be ineffective mainly in the treatment of solid tumors, largely because of tumor heterogeneity, activation of redundant pathways, and drug resistance. A common and central signal transduction mechanism in many oncogenic pathways is the phosphorylation of proteins at serine or threonine residues followed by proline (S/T-P). Importantly, the phospho-S/T-P motifs of these proteins are recognized by the peptidyl-prolyl cis/trans isomerase (PPIase) PIN1, which catalyzes the cis-trans or trans-cis conformational change around the S-P or T-P bond. Among PPIases, PIN1 is the only enzyme able to efficiently bind proteins containing phosphorylated S/T-P motifs. As a consequence, the phosphorylation dependent prolyl-isomerase PIN1 acts as a critical modifier of multiple signaling pathways. It is overexpressed in the majority of cancers and its activity strongly contributes to tumor initiation and progression. Conversely, inactivation of PIN1 function curbs tumor growth and cancer stem cell expansion, restores chemosensitivity and blocks metastatic spread, thus providing the rationale for a therapeutic strategy based on PIN1 inhibition. Notwithstanding, potent PIN1 inhibitors are still missing from the arsenal of anti-cancer drugs. By a mechanism-based screening we have identified a novel covalent PIN1 inhibitor, KPT-6566, able to selectively inhibit PIN1 among other prolyl-isomerases, and target it for degradation. We demonstrate that KPT-6566 covalently binds to the catalytic site of PIN1. This interaction results in the release of a quinine-mimicking drug that generates reactive oxygen species and DNA damage inducing cell death specifically in cancer cells. Accordingly, KPT-6566 treatment impairs PIN1-dependent cancer phenotypes in vitro and growth of lung metastasis in vivo.
10
Serrano, Aparicio Natalia. "Inhibition studies on the human 20S proteasome: molecular insights from a computational approach." Doctoral thesis, Universitat Jaume I, 2022. http://dx.doi.org/10.6035/14122.2022.684242.
Анотація:
The human 20S proteasome activity and malfunction has been related to numerous diseases and validated as a protein target for inhibition in the treatment of cancer, with three proteasome inhibitors approved as a drug. But these compounds could be improved, since usually the molecular mechanism of action is unknown. Thus, computational studies can clarify the mode of action of proteasome inhibitors, helping to understand the system and improve the inhibition process The present thesis is devoted to understand the mode of action of two classes of covalent inhibitors of the 20S proteasome, α,β-epoxyketones and γ-lactam-β-lactones. Molecular dynamics simulations with hybrid QM/MM potentials have been used to characterize the free energy landscape for the inhibition mechanism of these compounds and to provide the structures necessary to analyze and understand the inhibition process in the β5 active site of the proteasome, providing valuable knowledge to optimize the compounds into more efficient inhibitors.Programa de Doctorat en Química Teòrica i Modelització Computacional
Частини книг з теми "Inhibition covalente":
1
de Bruin, Gerjan, and Tjeerd Barf. "CHAPTER 4. Covalent Inhibition of Kinases." In Drug Discovery, 61–96. Cambridge: Royal Society of Chemistry, 2018. http://dx.doi.org/10.1039/9781788013093-00061.
2
Mehdi, Shujaath. "COVALENT ENZYME INHIBITION IN DRUG DISCOVERY AND DEVELOPMENT." In Enzyme Technologies, 81–129. Hoboken, NJ: John Wiley & Sons, Inc, 2013. http://dx.doi.org/10.1002/9781118739907.ch3.
3
Alexander, Patrick, and Andrew G. Stephen. "Affinity Measurement of Non-covalent Interactions of the Covalent KRAS G12C GDP Inhibitor MRTX849 to RAS Isoforms Using Surface Plasmon Resonance." In Methods in Molecular Biology, 103–14. New York, NY: Springer US, 2024. http://dx.doi.org/10.1007/978-1-0716-3822-4_8.
4
Potier, Noelle, Patrick Barth, Denis Tritsch, Jean-François Biellmann, and Alain Van Dorsselaer. "Study of Non-Covalent Enzyme-Inhibitor Complexes of Aldose Reductase by Electrospray Mass Spectrometry." In Advances in Experimental Medicine and Biology, 453–54. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4615-5871-2_51.
5
Crawford, James J., and Haiming Zhang. "Discovery and Development of Non-Covalent, Reversible Bruton’s Tyrosine Kinase Inhibitor Fenebrutinib (GDC-0853)." In ACS Symposium Series, 239–66. Washington, DC: American Chemical Society, 2019. http://dx.doi.org/10.1021/bk-2019-1332.ch009.
6
Liedtke, Harald, and Günter Legler. "Splenic Glucocerebrosidase and Its Cytosolic Activator Protein: Effects on Substrate Hydrolysis and Covalent Inhibition by Conduritol B Epoxides." In Lipid Storage Disorders, 353–58. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4613-1029-7_43.
7
Surh, Y. J. "Chemopreventative Activity of Chlorophyllin: Inhibition of Mutagenicity and Covalent DNA Binding of Benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide." In Advances in Experimental Medicine and Biology, 228. Boston, MA: Springer US, 1994. http://dx.doi.org/10.1007/978-1-4899-0939-8_28.
8
Kalgutkar, Amit S., Brenda C. Crews, Scott W. Rowlinson, Carlos Garner, and Lawrence J. Marnett. "Discovery of a New Class of Selective Cyclooxygenase-2 (COX-2) Inhibitor that Covalently Modifies the Isozyme." In Advances in Experimental Medicine and Biology, 139–43. Boston, MA: Springer US, 1999. http://dx.doi.org/10.1007/978-1-4615-4793-8_21.
9
Yingsung, Wannarat, Lisheng Zhuo, Masahiko Yoneda, Naoki Ishiguro, Hisashi Iwata, and Koji Kimata. "The covalent complex formation of hyaluronan with heavy chains of inter-α-trypsin inhibitor family is important for its functions." In The Many Faces of Osteoarthritis, 207–12. Basel: Birkhäuser Basel, 2002. http://dx.doi.org/10.1007/978-3-0348-8133-3_20.
10
Zeller, Hans-Dieter, and Sandro Ghisla. "Inactivation of general acyl-CoA dehydrogenase from pig k i dney by the suicide substrate methylenecyclopropylacetyl - CoA. Stucture of one of the covalent flavin-inhibitor adducts." In Flavins and Flavoproteins 1987, edited by D. E. Edmondson and D. B. McCormick, 161–64. Berlin, Boston: De Gruyter, 1987. http://dx.doi.org/10.1515/9783110884715-027.
Тези доповідей конференцій з теми "Inhibition covalente":
1
Santi, Claudio, Luca Sancineto, Francesca Mangiavacchi, Cecilia Scimmi, and Sougat Misra. "ELECTROPHILIC ORGANOSELENIUM COMPOUNDS AND SARS-COV-2: PRO-OXIDANT ACTIVITY AS A MORE PROMISING WAY TOWARDS THE DRUGGABILITY." In 1st INTERNATIONAL Conference on Chemo and BioInformatics. Institute for Information Technologies, University of Kragujevac,, 2021. http://dx.doi.org/10.46793/iccbi21.020s.
Анотація:
Ebselent has been recently reported as the most efficient hinibitors of Sars-Cov-2 main protease (Mpro) thought the electrophilic covalent pro-oxidation of the reactive Cysteine 145. According to similar evidences in literature we can propose a general mechanism to explore a novel and promising application of mild organoselenium centered electrophiles in medicinal chemistry. New insights in the field of covalent and non-covalent inhibition of Mpro as well as the antiviral SARS-Cov2 activity of novel organoselenium compounds will be here discussed
2
Venetsanakos, Eleni, Yan Xing, Natalie Loewenstein, J. Michael Bradshaw, Dane Karr, Jacob LaStant, Philip Nunn, et al. "Abstract 2091: PRN1371, an irreversible, covalent inhibitor of FGFR1-4 exhibits sustained pathway inhibition in cancer cell lines." In Proceedings: AACR Annual Meeting 2017; April 1-5, 2017; Washington, DC. American Association for Cancer Research, 2017. http://dx.doi.org/10.1158/1538-7445.am2017-2091.
3
Koneti Rao, A., and Maria A. Kowalska. "ADP-INDUCED CYTOPLASMIC CALCIUM MOBILIZATION AND SHAPE CHANGE IN PLATELETS ARE MEDIATED BY DIFFERENT BINDING SITES." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644466.
Анотація:
Platelet stimulation with ADP results in a number of responses including increase in cytoplasmic ionized calcium concentration [Ca2+]i, shape change, aggregation, secretion, and inhibition of cAMP accumulation caused by PGI2.5'-Fluorosulphonylbenzoyladenosine (FSBA), which covalently labels ADP binding site on platelets, blocks platelet shape change but not inhibition of cyclic AMP levels by ADP, while p-chloromercuribenzenesulfonate (pCMBS), a non-penetrating thiol reagent, blocks ADP-induced inhibition of adenylate cyclase but not shape change. We examined the effect of FSBA and pCMBS on ADP-induced increase in [Ca2+]i to determine whether it is linked to the binding site mediating shape change or that for inhibition of adenylate cyclase. In platelets loaded with Ca2+ indicators, quin 2 or fura 2, and in presence of adenosine deaminase (AD), FSBA (50-200 μM) induced a dose-dependent, rapid rise in [Ca2+]i. from basal levels of 70-90 nM to peak levels of 300-500 nM in the presence of 1 mM external Ca2+ providing direct evidence that FSBA is a platelet agonist. The [Ca2+ ]i. returned to near basal levels over 30 min. The effect of FSBA on [Ca2+]i. was inhibited by ZK 36,374 (40 nM), a stable PGI2 analog. AdP concentrations eliciting similar responses were about 10-fold less than those for FSBA. Platelet incubation with FSBA (50-100 μM) in the presence of AD for 30 min (to ensure optimal covalent labelling of the ADP binding sites) abolished shape change but jjid not inhibit ADP (5, 25 μM)-induced increase in [Ca2+]i. or block the inhibitory effect of ADP on cAMP accumulation in1platelets exposed to ZK 36,374 (50 nM) in.presence of theophylline (7 mM). Incubation with pCMBS (5-100 pM, 2 min) abolished the effect of ADP on [Ca2+]. and on the inhibition of cAMP levels; shape change was not 1 inhabited even at 1 mM. pCMBS (0.5-1 mM) inhibited the rise in [Ca2+ ]. by FSBA alone. These observations suggest that ADP-induced Ca mobilization is mediated by platelet binding sites which are distinct from those mediating shape change but probably the same as those modulating adenylate cyclase.
4
Yue, Zhiwei, Qiang Fu, Nancy Lang, and Chunfang Fan. "Liquid Scale Inhibitors for Metallic-Crosslinked Gel Fracturing Systems." In SPE International Oilfield Scale Conference and Exhibition. SPE, 2014. http://dx.doi.org/10.2118/spe-169806-ms.
Анотація:
Abstract Scale inhibitors are important additives in fracturing fluids to help prevent mineral scale depositions during hydraulic fracturing, shut-in, and flowback stages. The inhibition mechanisms rely heavily on the interactions of certain functional groups from the inhibitor molecules and the lattice metals on the scale crystal surface. In stimulation using crosslinked gel fluids based on metallic crosslinkers, such as zirconium (Zr), titanium (Ti), or aluminum (Al), these metals form strong covalent bonds with guar and guar derivatives and therefore significantly increase overall gel stability. When anionic liquid scale inhibitors are present, the crosslinker metals are susceptible to attack from scale inhibitors because of similar interactions, often resulting in lower viscosity and compromised fluid performance. For this reason, liquid scale inhibitors are usually pumped in prepads during many fracturing treatments. During these treatments, fracturing fluids from other stages and reservoirs that contact these fluids are not protected from scale deposition. Although this incompatibility issue is an important factor in designing fracturing fluid systems, the authors have not found sufficient prior literature providing valid solutions other than applications of time-released solid scale inhibitors. This paper investigates the interferences of various types of liquid scale inhibitors on Zr-based crosslinkers. Their chemical compatibilities are evaluated in terms of crosslinked viscosity stability and scale inhibition efficiencies. Data presented in this paper indicate that two polymer-based liquid scale inhibitors can be added to selected Zr-crosslinked fluids with minimal impact on crosslinking performance. Scale inhibition efficiency can be maintained by adjusting the pumping flow rate to compensate for the effect of Zr-based crosslinkers.
5
Niederst, P. N., M. Asbach, M. Ott, and R. E. Zimmermann. "IN VITRO REACTION MODELS OF THROMBIN AND ITS PHYSIOLOGICAL INHIBITOR ANTITHROMBIN III IN THE PRESENCE OF HEPARIN." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644356.
Анотація:
Antithrombin III (AT III) neutralizes thrombin and other serine proteases of plasma coagulation system by forming a stable 1:1 covalent complex. The inhibition rates are greatly increased by the potent catalyst heparin. The catalytic mechanism of heparin was studied in the presence of dextran sulfate (DS), a thrombin-binding sulfated Polysaccharid. DS did not influence the reaction of AT III with heparin and the amidolytic activity of thrombin, but preincubation with thrombin could inhibit the catalytic activity of heparin in the reaction of thrombin with AT III. We conclude that the reaction of heparin with enzyme and inhibitor, thus forming a ternary complex, is necessary for its catalytic activity.It is known that heparin also converts AT III from an inhibitor to a substrate for thrombin in a dose dependent manner. By cleavage of the reaction site bound Arg(385)-Ser(386) an AT III-fragment (MG 50000 d) occurs, which has a decreased affinity to heparin and does not inhibit F I la. At physiological ionic strength we have only measured a small percentage of AT 111-proteolysis (4%, 1 U/ml Hep). The extent of AT III-fragment formation could be enhanced by lowering the ionic strength (max 44%, 1 U/ml Hep., 1=0,02).
6
Poddutoori, Ramulu, Leena K. Satyam, Girish Daginakatte, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Raghuveer Ramachandra, et al. "Abstract C190: Potent and selective inhibition of CDK7 by novel covalent inhibitors." In Abstracts: AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; November 5-9, 2015; Boston, MA. American Association for Cancer Research, 2015. http://dx.doi.org/10.1158/1535-7163.targ-15-c190.
7
Satyam, Leena Khare, Ramulu Poddutoori, Subhendu Mukherjee, Sivapriya Marappan, Sreevalsam Gopinath, Raghuveer Ramachandra, Manoj Kumar Pothuganti, et al. "Abstract 3070: Potent and selective inhibition of CDK7 by novel covalent inhibitors." In Proceedings: AACR 107th Annual Meeting 2016; April 16-20, 2016; New Orleans, LA. American Association for Cancer Research, 2016. http://dx.doi.org/10.1158/1538-7445.am2016-3070.
8
Simone, E. R., T. A. Davies, N. A. Zabe, S. M. Greenberg-seperaky, and N. E. Larsen. "EARLY PLATELET-THROMBIN RECEPTORS AND THEIR FUNCTIONS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643730.
Анотація:
Human platelets possess less than 1000 high affinity [Kd=10-9]and 50-100,000 receptors of lower [Kd=10-7] affinity for o(α-thrombin. The selective derivatization of thrombin with the bifunctional crosslinking agent, DNCO, has enabled us to identify these receptorsvia covalent binding of either active siteinhibited tosyllyslmethylketothrombin (TLCK-T) or active Ctf-thrombin (T).Kinetic studies of the inhibition of the platelet-thrombin response by covalently and noncovalently bound TLCK-T have helped to elucidate the roles of the high and low affinity thrombin receptors. The activation parameters examined were initial membrane depolarization, cytoplasmic alkalinization,dense granule secretion of serotonin and lysosomal secretion of β-glucuronidase.Isolation and characterization of the thrombin receptors after covalent photocoupling of the TLCK-T or active T- were performed after solubilization by gel filtration. The intact, high affinity receptor moiety, a glycoprotein, has an approximate molecular weight of∽lSO.OOO daltons; occasionally this protein is found as a dimer of ∽360,000 daltons. When exposed to o(α-T the receptor undergoes proteolysis, leaving a protein of∽80,000 daltons and releasing the remaining glycoprotein into the medium.Higher doses of active T have been shown to bind with lower affinity to a larger protein of approximate molecular weight 600,000 daltons anda smaller protein of 46,000 daltons. Both proteins are nonsusceptible to thrombin proteolysis. Reduction and alkylation of the600,000 dalton complex yielded two and possibly three high molecular weight components (200,000, 160,000, and possibly 145,000daltons) which may correspond to previously suggested GP-Ia and GP-Ib of the GP-I complex. Under different solubilization conditions, two other membrane proteins have been found to be part of the GP-I complex; one which is not a glycoprotein, GP-Ic, while the other is associated with the glycocalyx and is called glycocalicin. Glycocalicin and GP-Icdo inhibit thrombin binding,implying that the low affinity receptor is indeed the previously suggested GP-I complex and does not appear to be directly involved withplatelet activation.Examination of the effect of dose and duration of incubation with non-covalently binding TLCK-T on subsequent α-thrombin response suggests the existence of positive cooperativity among thrombin receptors.Although TLCK-T has the same affinity for platelets (Kd) as T , the rateof binding and therefore that of dissociation are lower. Thus for incubation times of 1 minute or less with up to a 2x saturating TLCK-T dose, the subsequent depolarization response to a saturating T dose was enhanced. Exposure to higher TLCK-T (5x saturating)doses led to significant inhibition.Verification of the potentiation observed in noncovalent TLCK-T studies was performed using TLCK-T covalently bound to the platelet receptor with DNCO. Several hundred thrombin molecules were bound to the platelet when a subsaturating dose of TLCK-T(0.0025 U/ml) was used to crosslink, whileseveral thousand resulted with a saturating (0.05 U/ml) TLCK-T dose. Positive cooperativity was observed with low αT doses (0.005 U/ml) when several hundred high affinity receptors are blocked. The parameters studied which exhibited this positive cooperativity were depolarization, pH change and serotonin secretion, α-Glucuronidase secretion was normal. The presence and degreeof enhancement were donor-variableand suggest different threshhold thrombin dose requirements. The enhancement observed can be attributed to either an increased rate of binding (increased affinity) or to an increased number of exposed binding sites. Since little difference was found between the number of TLCK-T molecules bound after30 versus 60 seconds, we conclude that thepotentiation is more likely due to an increased number of exposed binding sites. Results from covalent crosslinks using a fluorescein and rhodamine labeled-TLCK-T and the fluorescence activated cellsorter support this hypothesis. The sensitization of the high affinity binding sitesby partial occupancy implies these bindingsites are responsible for depolarization, pH change and dense granule secretion (the rapid initial activation response), while βglucuronidase secretion, a secondary response, is otherwise controlled.
9
Kosmachevskaya, Olga, Elvira Nasybullina, Konstantin Shumaev, and Alexey Topunov. "HEMOGLOBIN-BOUND DYNITROSIL IRON COMPLEXES PROTECT IT FROM OXIDATIVE MODIFICATION." In NEW TECHNOLOGIES IN MEDICINE, BIOLOGY, PHARMACOLOGY AND ECOLOGY. Institute of information technology, 2021. http://dx.doi.org/10.47501/978-5-6044060-1-4.51.
Анотація:
Under the action of peroxynitrite, DNICs associated with hemoglobin are dose-dependently destroyed, while inhibiting the oxidation of tryptophan and tyrosine residues, the formation of carbonyl derivatives, preventing the formation of covalent cross-links between subunits, and preventing the degradation of the heme group.
10
Guo, Zipeng, Ruizhe Yang, Jun Liu, Jason Armstrong, Ruogang Zhao, and Chi Zhou. "Continuous Stereolithography 3D Printing of Multi-Network Hydrogels in Triply Periodic Minimal Structures (TPMS) With Tunable Mechanical Strength for Energy Absorption." In ASME 2022 International Mechanical Engineering Congress and Exposition. American Society of Mechanical Engineers, 2022. http://dx.doi.org/10.1115/imece2022-95806.
Анотація:
Abstract A fast additive manufacturing (AM) protocol to fabricate multi-network hydrogels is reported in this work. The gas-permeable PDMS film creates a polymerization-inhibition zone, facilitating the continuous stereolithography (SLA) 3D printing of hydrogels. The fabricated multi-bonding network integrates the rigid covalent bonding and the tough ionic bonding. The elastic modulus and strength could be effectively tuned by varying the ratio between the covalent and ionic bonding networks to fulfill various loading conditions. The printed triply periodic minimal structures (TPMS) hydrogels demonstrated high compressibility for up to 80% recoverable strain. Moreover, the dried TPMS hydrogels show novel energy absorption properties. We fabricated uniform and gradient hydrogels and compared their energy absorption capability. The anisotropy and quasi-isotropy behavior of TPMS structures were analyzed using simulation studies, providing insights into designing and controlling the TPMS structures for energy absorption. The results showed that the gradient TPMS hydrogels are preferable energy absorbers and have potential applications in impact resistance and absorption.