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Journal articles on the topic "Non-covalent inhibitor"
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Bjij, Imane, Pritika Ramharack, Shama Khan, Driss Cherqaoui, and Mahmoud Soliman. "Tracing Potential Covalent Inhibitors of an E3 Ubiquitin Ligase Through Target-Focused Modelling." Proceedings 22, no. 1 (November 14, 2019): 103. http://dx.doi.org/10.3390/proceedings2019022103.
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The Nedd4-1 E3 Ubiquitin ligase has been implicated in multiple disease conditions due its overexpression. Although the Nedd4-1 E3 Ubiquitin ligase is an enzyme that may be targeted either covalently, or non-covalently, there are few studies that demonstrate effective inhibitors of the enzyme. In this work, we aimed to identify covalent inhibitors of Nedd4-1. This task however, proved to be challenging due to the limited available electrophilic moieties in virtual libraries. We therefore opted to divide an existing covalent Nedd4-1 inhibitor in two parts: A non-covalent binding part and a pre-selected α, β-unsaturated ester that forms the covalent linkage with the protein. A non-covalent pharmacophore model was built based on the active site binding investigations followed by validating the covalent conjugation. Thirty compounds were selected and covalently docked into the catalytic site of the Nedd4-1. Multiple filtrations were effected before selecting 5 hits that were later analysed by molecular dynamic simulations to check their stability and explore their binding landscape in complex with the protein. All in all, two inhibitors with optimum overall stability and more stabilising interactions were kept for eventual biological evaluation. Our improved pharmacophore model approach serves as a robust method that will illuminate the screening for novel covalent inhibitor in drug discovery.
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Lockbaum, Gordon J., Archie C. Reyes, Jeong Min Lee, Ronak Tilvawala, Ellen A. Nalivaika, Akbar Ali, Nese Kurt Yilmaz, Paul R. Thompson, and Celia A. Schiffer. "Crystal Structure of SARS-CoV-2 Main Protease in Complex with the Non-Covalent Inhibitor ML188." Viruses 13, no. 2 (January 25, 2021): 174. http://dx.doi.org/10.3390/v13020174.
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Viral proteases are critical enzymes for the maturation of many human pathogenic viruses and thus are key targets for direct acting antivirals (DAAs). The current viral pandemic caused by SARS-CoV-2 is in dire need of DAAs. The Main protease (Mpro) is the focus of extensive structure-based drug design efforts which are mostly covalent inhibitors targeting the catalytic cysteine. ML188 is a non-covalent inhibitor designed to target SARS-CoV-1 Mpro, and provides an initial scaffold for the creation of effective pan-coronavirus inhibitors. In the current study, we found that ML188 inhibits SARS-CoV-2 Mpro at 2.5 µM, which is more potent than against SAR-CoV-1 Mpro. We determined the crystal structure of ML188 in complex with SARS-CoV-2 Mpro to 2.39 Å resolution. Sharing 96% sequence identity, structural comparison of the two complexes only shows subtle differences. Non-covalent protease inhibitors complement the design of covalent inhibitors against SARS-CoV-2 main protease and are critical initial steps in the design of DAAs to treat CoVID 19.
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Gomez, Eliana B., Lippincott Isabel, Mary S. Rosendahal, Stephen M. Rothenberg, Steven W. Andrews, and Barb J. Brandhuber. "Loxo-305, a Highly Selective and Non-Covalent Next Generation BTK Inhibitor, Inhibits Diverse BTK C481 Substitution Mutations." Blood 134, Supplement_1 (November 13, 2019): 4644. http://dx.doi.org/10.1182/blood-2019-126114.
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Introduction: Bruton's Tyrosine Kinase (BTK) is an essential component of normal and malignant B-cell receptor signaling. Covalent BTK inhibitors have transformed the treatment of B-cell malignancies but are limited by off-target toxicity and acquired resistance, leading to eventual treatment discontinuation and disease progression. Emerging evidence suggests that acquired resistance is mediated predominantly by BTK C481 substitution mutations at the covalent BTK inhibitors' binding site. There is significant unmet clinical need for new treatment approaches that overcome acquired resistance and minimize toxicity. LOXO-305 is a highly selective, non-covalent, next generation BTK inhibitor. We previously showed that LOXO-305 potently inhibited both wild-type (WT) BTK and BTK C481S -mediated kinase activity in enzyme and cell-based assays with nanomolar potency, caused regression of BTK-dependent lymphoma mouse xenograft models, and was more than 300-fold selective for BTK over 98% of 370 other kinases tested and showed no significant inhibition of non-kinase off targets at 1 mM (Brandhuber et al. SOHO 2018). In addition, ADME and pharmacokinetic experiments in two preclinical species predicted that LOXO-305 will have high human exposure and sustained BTK C481S target coverage in patients at clinically achievable doses. Here we describe the activity of LOXO-305 against additional BTK C481 substitution mutations, including mutations identified in patients with acquired resistance to covalent BTK inhibitors. We further determine equilibrium-binding affinities for LOXO-305 for diverse mutant BTK enzymes in comparison to other clinically available BTK inhibitors. Methods: To assess cellular BTK inhibitor potency, HEK293T cell lines transiently expressing wild-type BTK and BTK C481 substitution mutations were serum starved and incubated with LOXO-305 overnight. Cells were next incubated with serum and orthovanadate for 5 min and the phosphorylated Y223 BTK was analyzed by immunoblot. Bands were quantified and the IC50 values calculated with GraphPad Prism. The equilibrium-binding affinities for targeted BTK inhibitors to BTK enzyme variants were determined by surface plasmon resonance (SPR) using the Biacore T200. Biotinylated BTK variants were immobilized on a docked streptavidin coated sensor chip. Five concentrations of each inhibitor plus blank controls were analyzed. Association/dissociation rate constants were calculated by global fitting of the data to a 1:1 binding interaction model. Results: While BTK C481S possessed similar levels of basal Y223 autophosphorylation as wild-type BTK in cells, BTK C481T autophosphorylation was reduced by ~50%, C481R by ~90%, and mutants C481F, and C481Y were inactive in HEK293T cells. LOXO-305 inhibited Y223 phosphorylation of all active mutants with similar nanomolar potency. In contrast, autophosphorylation of all BTK C481 mutants were resistant to both Ibrutinib and acalabrutinib. Equilibrium-binding affinities of LOXO-305 for select BTK C481 substitution mutations confirmed LOXO-305's superior potency versus commercially available BTK inhibitors (ibrutinib and acalabrutinib). Conclusions: The next generation, non-covalent, highly selective BTK inhibitor LOXO-305 potently inhibited the cellular activity of BTK C481S, T and R mutations and displayed strong equilibrium binding to WT BTK and several BTK C481 substitution mutations. Together with high selectivity and significant BTK target coverage in vivo, these results indicate that LOXO-305 may overcome acquired resistance to covalent BTK inhibitors in patients without significant off-target toxicity. A phase 1 clinical trial of LOXO-305 is currently underway. Disclosures Gomez: LOXO Oncology Inc.: Employment, Equity Ownership. Isabel:Loxo Oncology: Employment. Rosendahal:Loxo Oncology: Employment. Rothenberg:LOXO Oncology Inc.: Employment. Andrews:Loxo Oncology: Employment. Brandhuber:LOXO Oncology Inc.: Employment, Equity Ownership.
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Buneeva, O. A., L. N. Aksenova, and A. E. Medvedev. "A Simple Approach for Pilot Analysis of Time-dependent Enzyme Inhibition: Discrimination Between Mechanism-based Inactivation and Tight Binding Inhibitor Behavior." Biomedical Chemistry: Research and Methods 3, no. 1 (2020): e00115. http://dx.doi.org/10.18097/bmcrm00115.
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The increase in enzyme inhibition developed during prolonged incubation of an enzyme preparation with a chemical substance may be associated with both the non-covalent and also with covalent enzyme-inhibitor complex formation. The latter case involves catalytic conversion of a mechanism-based irreversible inhibitor (a poor substrate) into a reactive species forming covalent adduct(s) with the enzyme and thus irreversibly inactivating the enzyme molecule. Using a simple approach, based on comparison of enzyme inhibition after preincubation with a potential inhibitor at 4ºC or 37ºC we have analyzed inhibition of monoamine oxidase A (MAO A) by known MAO inhibitors pargyline and pirlindole (pyrazidol). MAO A inhibitory activity of pirlindole (reversible tight binding inhibitor of MAO A) assayed after mitochondrial wash was basically the same for the incubation at both 4ºC and 37ºC. In contrast to pirlindole, the effect of pargyline (mechanism based irreversible MAO inhibitor) strongly depended on the temperature of the incubation medium. At 37ºC the residual activity MAO A in the mitochondrial fraction after washing was significantly lower than in the mitochondrial samples incubated with pargyline at 4ºC. Results of this study suggest that using analysis of both time- and temperature-dependence of inhibition it is possible to discriminate mechanism-based irreversible inhibition and reversible tight binding inhibition of target enzym
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Zhang, Datong, He Gong, and Fancui Meng. "Recent Advances in BTK Inhibitors for the Treatment of Inflammatory and Autoimmune Diseases." Molecules 26, no. 16 (August 13, 2021): 4907. http://dx.doi.org/10.3390/molecules26164907.
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Bruton’s tyrosine kinase (BTK) plays a crucial role in B-cell receptor and Fc receptor signaling pathways. BTK is also involved in the regulation of Toll-like receptors and chemokine receptors. Given the central role of BTK in immunity, BTK inhibition represents a promising therapeutic approach for the treatment of inflammatory and autoimmune diseases. Great efforts have been made in developing BTK inhibitors for potential clinical applications in inflammatory and autoimmune diseases. This review covers the recent development of BTK inhibitors at preclinical and clinical stages in treating these diseases. Individual examples of three types of inhibitors, namely covalent irreversible inhibitors, covalent reversible inhibitors, and non-covalent reversible inhibitors, are discussed with a focus on their structure, bioactivity and selectivity. Contrary to expectations, reversible BTK inhibitors have not yielded a significant breakthrough so far. The development of covalent, irreversible BTK inhibitors has progressed more rapidly. Many candidates entered different stages of clinical trials; tolebrutinib and evobrutinib are undergoing phase 3 clinical evaluation. Rilzabrutinib, a covalent reversible BTK inhibitor, is now in phase 3 clinical trials and also offers a promising future. An analysis of the protein–inhibitor interactions based on published co-crystal structures provides useful clues for the rational design of safe and effective small-molecule BTK inhibitors.
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Ehmann, D. E., H. Jahic, P. L. Ross, R. F. Gu, J. Hu, G. Kern, G. K. Walkup, and S. L. Fisher. "Avibactam is a covalent, reversible, non- -lactam -lactamase inhibitor." Proceedings of the National Academy of Sciences 109, no. 29 (July 2, 2012): 11663–68. http://dx.doi.org/10.1073/pnas.1205073109.
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Bjij, Imane, Pritika Ramharack, Shama Khan, Driss Cherqaoui, and Mahmoud E. S. Soliman. "Tracing Potential Covalent Inhibitors of an E3 Ubiquitin Ligase through Target-Focused Modelling." Molecules 24, no. 17 (August 28, 2019): 3125. http://dx.doi.org/10.3390/molecules24173125.
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The Nedd4-1 E3 Ubiquitin ligase has been implicated in multiple disease conditions due its overexpression. Although the enzyme may be targeted both covalently and non-covalently, minimal studies provide effective inhibitors against it. Recently, research has focused on covalent inhibitors based on their characteristic, highly-selective warheads and ability to prevent drug resistance. This prompted us to screen for new covalent inhibitors of Nedd4-1 using a combination of computational approaches. However, this task proved challenging due to the limited number of electrophilic moieties available in virtual libraries. Therefore, we opted to divide an existing covalent Nedd4-1 inhibitor into two parts: a non-covalent binding group and a pre-selected α, β-unsaturated ester that forms the covalent linkage with the protein. A non-covalent pharmacophore model was built based on molecular interactions at the binding site. The pharmacophore was then subjected to virtual screening to identify structurally similar hit compounds. Multiple filtrations were implemented prior to selecting four hits, which were validated with a covalent conjugation and later assessed by molecular dynamic simulations. The results showed that, of the four hit molecules, Zinc00937975 exhibited advantageous molecular groups, allowing for favourable interactions with one of the characteristic cysteine residues. Predictive pharmacokinetic analysis further justified the compound as a potential lead molecule, prompting its recommendation for confirmatory biological evaluation. Our inhouse, refined, pharmacophore model approach serves as a robust method that will encourage screening for novel covalent inhibitors in drug discovery.
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Sugiyama, Katsumi, Zhou Chen, Yong S. Lee, and Peter F. Kador. "Isolation of a non-covalent aldose reductase–nucleotide–inhibitor complex." Biochemical Pharmacology 59, no. 4 (February 2000): 329–36. http://dx.doi.org/10.1016/s0006-2952(99)00332-9.
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Yang, Zhimin, Hui Liu, Botao Pan, Fengli He, and Zhengying Pan. "Design and synthesis of (aza)indolyl maleimide-based covalent inhibitors of glycogen synthase kinase 3β." Organic & Biomolecular Chemistry 16, no. 22 (2018): 4127–40. http://dx.doi.org/10.1039/c8ob00642c.
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Sivakumar, Dakshinamurthy, and Matthias Stein. "Binding of SARS-CoV Covalent Non-Covalent Inhibitors to the SARS-CoV-2 Papain-Like Protease and Ovarian Tumor Domain Deubiquitinases." Biomolecules 11, no. 6 (May 28, 2021): 802. http://dx.doi.org/10.3390/biom11060802.
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The urgent need for novel and effective drugs against the SARS-CoV-2 coronavirus pandemic has stimulated research worldwide. The Papain-like protease (PLpro), which is essential for viral replication, shares a similar active site structural architecture to other cysteine proteases. Here, we have used representatives of the Ovarian Tumor Domain deubiquitinase family OTUB1 and OTUB2 along with the PLpro of SARS-CoV-2 to validate and rationalize the binding of inhibitors from previous SARS-CoV candidate compounds. By forming a new chemical bond with the cysteine residue of the catalytic triad, covalent inhibitors irreversibly suppress the protein’s activity. Modeling covalent inhibitor binding requires detailed knowledge about the compounds’ reactivities and binding. Molecular Dynamics refinement simulations of top poses reveal detailed ligand-protein interactions and show their stability over time. The recently discovered selective OTUB2 covalent inhibitors were used to establish and validate the computational protocol. Structural parameters and ligand dynamics are in excellent agreement with the ligand-bound OTUB2 crystal structures. For SARS-CoV-2 PLpro, recent covalent peptidomimetic inhibitors were simulated and reveal that the ligand-protein interaction is very dynamic. The covalent and non-covalent docking plus subsequent MD refinement of known SARS-CoV inhibitors into DUBs and the SARS-CoV-2 PLpro point out a possible approach to target the PLpro cysteine protease from SARS-CoV-2. The results show that such an approach gives insight into ligand-protein interactions, their dynamic character, and indicates a path for selective ligand design.
Dissertations / Theses on the topic "Non-covalent inhibitor"
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Bordessa, Andrea. "Design, synthesis and structural evaluation of peptidomimetics towards foldamers, PNAs and non covalent inhibitors of the 20S proteasome." kostenfrei, 2008. http://www.opus-bayern.de/uni-regensburg/volltexte/2009/1112/.
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Engdahl, Cecilia. "Selective inhibition of acetylcholinesterase 1 from disease-transmitting mosquitoes : design and development of new insecticides for vector control." Doctoral thesis, Umeå universitet, Kemiska institutionen, 2017. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-134625.
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Acetylcholinesterase (AChE) is an essential enzyme with an evolutionary conserved function: to terminate nerve signaling by rapid hydrolysis of the neurotransmitter acetylcholine. AChE is an important target for insecticides. Vector control by the use of insecticide-based interventions is today the main strategy for controlling mosquito-borne diseases that affect millions of people each year. However, the efficiency of many insecticides is challenged by resistant mosquito populations, lack of selectivity and off-target toxicity of currently used compounds. New selective and resistance-breaking insecticides are needed for an efficient vector control also in the future. In the work presented in this thesis, we have combined structural biology, biochemistry and medicinal chemistry to characterize mosquito AChEs and to develop selective and resistance-breaking inhibitors of this essential enzyme from two disease-transmitting mosquitoes.We have identified small but important structural and functional differences between AChE from mosquitoes and AChE from vertebrates. The significance of these differences was emphasized by a high throughput screening campaign, which made it evident that the evolutionary distant AChEs display significant differences in their molecular recognition. These findings were exploited in the design of new inhibitors. Rationally designed and developed thiourea- and phenoxyacetamide-based non-covalent inhibitors displayed high potency on both wild type and insecticide insensitive AChE from mosquitoes. The best inhibitors showed over 100-fold stronger inhibition of mosquito than human AChE, and proved insecticide potential as they killed both adult and larvae mosquitoes.We show that mosquito and human AChE have different molecular recognition and that non-covalent selective inhibition of AChE from mosquitoes is possible. We also demonstrate that inhibitors can combine selectivity with sub-micromolar potency for insecticide resistant AChE.
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Le, Thien Anh [Verfasser], Bernd [Gutachter] Engels, and Volker [Gutachter] Engel. "Theoretical investigations of proton transfer and interactions or reactions of covalent and non-covalent inhibitors in different proteins / Thien Anh Le ; Gutachter: Bernd Engels, Volker Engel." Würzburg : Universität Würzburg, 2020. http://d-nb.info/1219429864/34.
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Mehrtens, (nee Nikkel) Janna Marie. "The Design, Synthesis and Biological Assay of Cysteine Protease Specific Inhibitors." Thesis, University of Canterbury. Chemistry, 2007. http://hdl.handle.net/10092/3271.
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This thesis investigates the design, synthesis and biological assay of cysteine protease inhibitors within the papain superfamily of cysteine proteases. This is achieved by examining the effect of inhibitor design, especially warheads, on IC₅₀ values and structureactivity relationships between cysteine protease inhibitors of the papain superfamily. The representative proteases used are m-calpain, μ-calpain, cathepsin B and papain. Chapter One is an introductory chapter; Chapters Two-Four describe the design and synthesis of cysteine protease inhibitors; Chapter Five discusses assay protocol; and Chapter Six contains the assay results and structure-activity relationships of the synthesised inhibitors. Chapter One introduces cysteine proteases of the papain family and examines the structure, physiology and role in disease of papain, cathepsin B, m-calpain and μ-calpain. The close structural homology that exists between these members of the papain superfamily is identified, as well characteristics unique to each protease. Covalent reversible, covalent irreversible and non-covalent warheads are defined. The generic inhibitor scaffold of address region, recognition and warhead, upon which the inhibitors synthesised in this thesis are based, is also introduced. Chapter Two introduces reversible cysteine protease inhibitors found in the literature and that little is known about the effect of inhibitor warhead on selectivity within the papain superfamily. Oxidation of the dipeptidyl alcohols 2.6, 2.26, 2.29, 2.30, 2.35 and 2.36 utilising the sulfur trioxide-pyridine complex gave the aldehydes 2.3, 2.27, 2.19, 2.2, 2.21 and 2.22. Semicarbazones 2.37-2.40 were synthesised by a condensation reaction between the alcohol 2.3 and four available semicarbazides. The amidoximes 2.48 and 2.49 separately underwent thermal intramolecular cyclodehydration to give the 3-methyl-1,2,4- oxadiazoles 2.41 and 2.50. The aldehydes 2.3 and 2.27 were reacted with potassium cyanide to give the cyanohydrins 2.51 and 2.52. The cyanohydrins 2.51 and 2.52 were separately reacted to give 1) the α-ketotetrazoles 2.43 and 2.55; 2) the α-ketooxazolines 2.42 and 2.58; 3) the esterified cyanohydrins 2.60 and 2.61. A two step SN2 displacement reaction of the alcohol 2.6 to give the azide 2.62, an example of a non-covalent cysteine protease inhibitor. Chapter Three introduces inhibitors with irreversible warheads. The well-known examples of epoxysuccinic acids 3.1 and 3.5 are discussed in detail, highlighting the lack of irreversible cysteine protease specific inhibitors. The aldehydes 2.3 and 2.27 were reacted under Wittig conditions to give the α,β-unsaturated carbonyls 3.14-3.18. Horner- Emmons-Wadsworth methodology was utilised for the synthesis of the vinyl sulfones 3.20- 3.23. The dipeptidyl acids 2.24 and 2.28 were separately reacted with diazomethane to give the diazoketones 3.25 and 3.26. The diazoketones 3.25 and 3.26 were separately reacted with hydrogen bromide in acetic acid (33%) to give the α-bromomethyl ketones 3.27 and 3.28, which were subsequently reduced to give the α-bromomethyl alcohols 3.29-3.32. Under basic conditions the α-bromomethyl alcohols 3.29-3.32 ring-closed to form the peptidyl epoxides 3.33-3.36. Chapter Four introduces the disadvantages of peptide-based inhibitors. A discussion is given on the benefits of constraining inhibitors into the extended bioactive conformation known as a β-strand. Ring closing metathesis is utilised in the synthesis of the macrocyclic aldehyde 4.4, macrocyclic semicarbazone 4.15, the macrocyclic cyanohydrin 4.16, the macrocyclic α-ketotetrazole 4.18 and the macrocyclic azide 4.19. Chapter Five introduces enzyme inhibition studies. The BODIPY-casein fluorogenic assay used for establishing inhibitor potency against m-calpain and μ-calpain is validated. Assay protocols are also established and validated for cathepsin B, papain, pepsin and α- chymotrypsin. A discussion of the effect of solvent on enzyme activity is also included as part of this study. Chapter Six presents the assay results for all the inhibitors synthesised throughout this thesis and an extensive structure-activity relationship study between inhibitors is included. The alcohols 2.26 and 2.30 are unprecedented examples of non-covalent, potent, cathepsin B inhibitors (IC₅₀ = 0.075 μM selectivity 80-fold and 1.1 μM, selectivity 18-fold). The macrocyclic semicarbazone 4.15 is an unprecedented example of a potent macrocyclic cysteine protease inhibitor (m-calpain: IC₅₀ = 0.16 μM, selectivity 8-fold). The cyanohydrin 2.51 contains an unprecedented cysteine protease warhead and is a potent and selective inhibitor of papain (IC₅₀ = 0.030 μM, selectivity 3-fold). The O-protected cyanohydrin 2.61 is a potent and selective inhibitor of pepsin (IC₅₀ = 1.6 μM, selectivity 1.5-fold). The top ten warheads for potent, selective cathepsin B inhibition are: carboxylic acid, methyl ester, diazoketone, esterified cyanohydrin, α-bromomethyl ketone, α,β- unsaturated aldehyde, vinyl sulfones, α-bromomethyl-C₃-S,R-alcohol, alcohol and α,β- unsaturated ethyl ester. The selectivity of these warheads was between 5- and 130-fold for cathepsin B. The best inhibitors for cathepsin B were the α-bromomethyl ketone 3.26 (IC₅₀ = 0.075 μM, selectivity 16-fold), the α,β-unsaturated aldehyde 3.18 (IC₅₀ = 0.13 μM, selectivity 13-fold) and the esterified cyanohydrin 3.59 (IC₅₀ = 0.35 μM, selectivity 22- fold). Chapter Seven outlines the experimental details and synthesis of the compounds prepared in this thesis.
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Nichols, Derek Allen. "Structure-Based Design of Novel Inhibitors and Ultra High Resolution Analysis of CTX-M Beta-Lactamase." Scholar Commons, 2014. https://scholarcommons.usf.edu/etd/5284.
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The emergence of CTX-M class-A extended-spectrum β-lactamases, which confer resistance to second and third-generation cephalosporins, poses a serious health threat to the public. CTX-M β-lactamases use a catalytic serine to hydrolyze the β-lactam ring. Specifically, the hydrolysis reaction catalyzed by CTX-M β-lactamase proceeds through a pre-covalent complex, a high-energy tetrahedral acylation intermediate, a low-energy acyl-enzyme complex, a high-energy tetrahedral deacylation intermediate after attack via a catalytic water, and lastly, the hydrolyzed β-lactam ring product which is released from the enzyme complex. The crystallographic structure of CTX-M at sub-angstrom resolution has enabled us to study enzyme catalysis as well as perform computational molecular docking in our efforts to develop new inhibitors against CTX-M. The goal of this project was to determine the hydrogen bonding network and proton transfer process at different stages of the reaction pathway as well as develop novel inhibitors against CTX-M β-lactamases. The results I have obtained from the project have elucidated the catalytic mechanism of CTX-M β-lactamase in unprecedented detail and facilitated the development of novel inhibitors for antibiotic drug discovery.
The first aim of the project focused on developing high affinity inhibitors against class A β-lactamase using a structure-based drug discovery approach, which ultimately led to the identification of CTX-M9 inhibitors with nanomolar affinity. Compound design was based on the initial use of computational molecular docking results along with x-ray crystal structures with known inhibitors bound in the active site. In addition, chemical synthesis was used to build and extend the existing inhibitor scaffold to improve affinity to CTX-M9 and related serine β-lactamases. Through a fragment-based screening approach, we recently identified a novel non-covalent tetrazole-containing inhibitor of CTX-M. Structure-based design was used to improve the potency of the original tetrazole lead compound more than 200-fold with the use of small, targeted structural modifications. A series of compounds were used to probe specific binding hotspots present in CTX-M. The designed compounds represent the first nM-affinity non-covalent inhibitors of a class A β-lactamase. The complex structures of these potent compounds have been solved using high resolution x-ray crystallography at ~ 1.2-1.4 Å, which provides valuable insight about ligand binding and future inhibitor design against class A β-lactamases.
Specifically, the first aim of the project was to use ultra-high resolution x-ray crystallography to study β-lactamase catalysis. Through the use of ultra-high resolution x-ray crystallography with non-covalent and covalent inhibitors, I was able to structurally characterize the critical stages of the enzyme mechanism. Here we report a series of ultra-high resolution x-ray crystallographic structures that reveal the proton transfer process for the early stages of the class A β-lactamase catalytic mechanism. The structures obtained include an a 0.89 Å crystal structure of CTX-M β-lactamase in complex with a recently-developed 89 nM non-covalent inhibitor, and a 0.80 Å structure in complex with an acylation transition state boronic acid inhibitor. Nearly all the hydrogen atoms in the active site, including those on the ligand, polar protein side chains and catalytic water, can be identified in the unbiased difference electron density map. Most surprisingly, compared with a previously determined 0.88 Å apo structure determined under the same conditions, the hydrogen-bonding network has undergone a series of reshuffling upon the binding of the non-covalent ligand. Two key catalytic residues, Lys73 and Glu166, appear to have both changed from a charged state to being neutral. Interestingly, structural evidence suggests the presence of a low barrier hydrogen bond (LBHB) shared between Lys73 and Ser70. These unprecedented detailed snapshots offer direct evidence that ligand binding can alter the pKa's of polar protein side chains and their affinities for protons. Such effects can be a common mechanism utilized by enzymes to facilitate the proton transfer process of a reaction pathway. They also have important implications for computational modeling of protein-ligand interactions. Ultra-high resolution x-ray crystallography allowed us to determine the hydrogen atom positions for key active site residues involved in catalysis. As a result, the ability to characterize the hydrogen bonding network led to the determination of the specific proton transfer process that occurs during the reaction stages of the CTX-M β-lactamase mechanism. Overall, the results from this project demonstrate the effectiveness of using ultra high resolution x-ray crystallography as a useful tool to study enzyme catalysis as well as develop and discover novel inhibitors.
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Scholtes, Jan Felix [Verfasser], and Oliver [Akademischer Betreuer] Trapp. "Chiral induction in stereodynamic catalysts by non-covalent interactions : ligand design, supramolecular self-recognition, deracemization and enantioselective self-inhibition / Jan Felix Scholtes ; Betreuer: Oliver Trapp." München : Universitätsbibliothek der Ludwig-Maximilians-Universität, 2019. http://d-nb.info/1188564129/34.
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Keita, Massaba. "Conception, synthèse et évaluation biologique d'inhibiteurs fluorés non covalents du protéasome." Phd thesis, Université Paris Sud - Paris XI, 2012. http://tel.archives-ouvertes.fr/tel-01059792.
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Le protéasome 26S est une macromolécule impliquée dans la dégradation de la majorité des protéines cellulaires. Parmi ces protéines, il y a les différents régulateurs de processus cruciaux tels que les protéines responsables de la progression du cycle cellulaire, de l'apoptose, des réponses inflammatoires, de l'activation de NF-B, de la présentation antigénique et de la différenciation cellulaire. Par conséquent, les inhibiteurs du protéasome sont des agents thérapeutiques dans des pathologies tels que le cancer, l'inflammation et les maladies auto-immunes. En effet, les inhibiteurs du protéasome sont connus pour induire la mort sélective des cellules cancéreuses tout en les rendant plus sensibles aux autres traitements anticancéreux existants (chimiothérapie, radiothérapie...). L'objectif de notre laboratoire est de développer des inhibiteurs non covalents du protéasome de structures peptidomimétiques fluorés ou non fluorés, et de montrer l'intérêt du fluor en chimie médicinale. Mon projet de thèse s'inscrit dans ce cadre. Dans un premier temps nous avons mis en évidence la grande diversité et la quantité des inhibiteurs du protéasome montrant ainsi l'importance de cette macromolécule comme cible dans le traitement du cancer. D'ailleurs, deux de ces inhibiteurs sont utilisés dans le traitement du myélome multiple et du lymphome du manteau et, plusieurs composés sont en études cliniques pour différents cancers. Nous avons aussi mis en évidence le bénéfice apporté par l'incorporation de groupement fluoré sur une molécule bioactive en particulier dans les structures peptidomimétiques. En revanche, ce rappel bibliographique a aussi montré que les peptidomimétiques contraints et fluorés sont peu décrits dans la littérature et le seul exemple à notre connaissance est l'analogue contraint et fluoré de la substance P contenant le motif (Z)-fluoroalcène.La deuxième partie de ces travaux de thèse s'est focalisée sur la conception, la synthèse et l'évaluation biologique d'inhibiteurs originaux du protéasome. Nous avons mis au point une synthèse facile et efficace de pseudopeptides possédant les motifs α et β-hydrazino acides et le motif β-hydrazino acide trifluorométhyle (schéma 1). Ces molécules inhibent de manière efficace le site CT-L du protéasome du lapin avec une IC50 de l'ordre du submicromolaire. Nous avons ainsi démontré que l'activité biologique est maintenue en remplaçant un α-amino acide par un scaffold α ou β-hydrazino acide. La pharmacomodulation effectuée autour de ces motifs nous a permis d'établir des relations structure-activité. Nous avons aussi mis au point un modèle de docking assez fiable qui va nous permettre de prédire le potentiel inhibiteur de nos futures molécules.Enfin, nous avons déterminé l'IC50 de nos molécules en utilisant la technique du FABS en RMN du 19F. Schéma1: voies d'accès aux peptidomimétiques contenant les motifs α et β-hydrazino acide et le motif β-hydrazino acide trifluorométhyl.Ces travaux de thèses ont été complétés par une méthodologie de synthèse portant sur le développement de nouveaux synthons contraints fluorés dans le but de les incorporer dans nos inhibiteurs de protéasome. Les cyclopropanes trifluorométhyles ont été obtenus en utilisant la réaction tandem de Michael, addition nucléophile suivie de cyclisation avec une excellente diastéréosélectivité pour certaines réactions. Les cyclopropanes obtenus ont été fonctionnalisés en amino acides ce qui faciliterait leur incorporation dans nos pseudopeptides. Les N-aminoaziridines fluorés ont été synthétisés à partir d'oléfines fluorés et de précurseurs de nitrène en présence de diacétate d'iodobenzène (PhI(OAc)2. L'incorporation de ces nouveaux scaffolds dans la structure de nos inhibiteurs de protéasome est en cours de réalisation dans le laboratoire.
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Kuo, Chin-Jung, and 郭瑾融. "A non-covalent small inhibitor blocking β-tubulin:CCT-β complex induces apoptosis and suppresses migration and invasionin CL1-5 cells." Thesis, 2019. http://ndltd.ncl.edu.tw/handle/yj45b7.
Full textAbstract:
碩士國立臺灣大學
生化科學研究所
107
Previously, we reported the protein-protein interaction (PPI) between β-tubulin and CCT-β complex as a potential anti-cancer chemotherapeutic target. Through virtual screening, a compound 3112210 from Sigma-Aldrich compound bank was identified to be a reversible inhibitor of the PPI by docking into hot spots on this PPI interface of β- tubulin. In this study, 3112210 was tested on a highly metastatic non-small cell lung cancer (NSCLC) cell line, CL1-5. The co-IP experiments showed that, in 3112210-treated cancer cells, β-tubulin and CCT-β complex was disrupted. Furthermore, 3112210 caused CL1-5 cell death through ER stress and apoptosis. In addition to verifying its toxicity toward CL1-5, we performed migration and invasion assays using dosage at about IC20. The results indicated that 3112210 also inhibited cancer cell migration and invasion, and MMP-2, -9 were also inhibited. These anti-metastatic effects were endowed via integrin- related pathways and EMT transcriptional factors, as demonstrated by western blot experiments. To sum, 3112210 is a novel non-covalent inhibitor for β-tubulin:CCT-β complex in CL1-5 lung adenocarcinoma cells to induce cancer cell death and impeded cell metastasis.
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Le, Thien Anh. "Theoretical investigations of proton transfer and interactions or reactions of covalent and non-covalent inhibitors in different proteins." Doctoral thesis, 2020. https://doi.org/10.25972/OPUS-17051.
Full textAbstract:
Nowadays, computational-aided investigations become an essential part in the chemical, biochemical or pharmaceutical research. With increasing computing power, the calculation of larger biological systems becomes feasible. In this work molecular mechanical (MM) and quantum mechanical approaches (QM) and the combination of both (QM/MM) have been applied to study several questions which arose from different working groups. Thus, this work comprises eight different subjects which deals with chemical reactions or proton transfer in enzymes, conformational changes of ligands or proteins and verification of experimental data. This work firstly deals with reaction mechanisms of aromatic inhibitors of cysteine proteases which can be found in many organisms. These enzymes are responsible for various cancer or diseases as for example Human African Trypanosomiasis (HAT) or the Chagas disease. Aromatic SNAr-type electrophiles might offer a new possibility to covalently modify these proteases. Quantum mechanical calculations have been performed to gain insights into the energetics and possible mechanisms. The next chapter also deals with Trypanosomiasis but the focus was set on a different enzyme. The particularity of Trypanosomiasis is the thiol metabolism which can also be modified by covalent inhibitors. In this context, the wild type and point mutations of the enzyme tryparedoxin have been investigated via molecular dynamic (MD) simulations to examine the influence of specific amino acids in regard to the inhibitor. Experimental data showed that a dimerization of the enzyme occurs if the inhibitor is present. Simulations revealed that the stability of the dimer decreases in absence of the inhibitor and thus confirms these experiments. Further investigations concerning cysteine proteases such as cruzain and rhodesain have been conducted with respect to experimental kinetic data of covalent vinylsulfone inhibitors. Several approaches such as QM or QM/MM calculations and docking, MD or MMPBSA/MMGBSA simulations have been applied to reproduce these data. The utilization of force field approaches resulted in a qualitatively accurate prediction. The kinase AKT is involved in a range of diseases and plays an important role in the formation of cancer. Novel covalent-allosteric inhibitors have been developed and crystallized in complex with AKT. It was shown that depending on the inhibitor a different cysteine residue is modified. To investigate these differences in covalent modification computational simulations have been applied. Enoyl-(acyl carrier) (ENR) proteins are essential in the last step of the fatty acid biosynthesis II (FAS) and represent a good target for inhibition. The diphenylether inhibitor SKTS1 which was originally designed to target the ENR’s of Staphylococcus aureus was also crystallized in InhA, the ENR of Mycobacterium tuberculosis (TB). Crystal structures indicate a change of the inhibitor's tautomeric form. This subject was investigated via MD simulations. Results of these simulations confirmed the tautomerization of the inhibitor. This work also deals with the development of a covalent inhibitor originating from a non-covalent ligand. The target FadA5 is an essential enzyme for the degradation of steroids in TB and is responsible for chronic tuberculosis. This enzyme was crystallized in complex with a non-covalent ligand which served as starting point for this study. Computations on QM or QM/MM level and docking and MD simulations have been applied to evaluate potential candidates. The next chapter focuses on the modification of the product spectrum of Bacillus megaterium levansucrase, a polymerase which catalyzes the biosynthesis of fructans. The covalent modification of the wild type or mutants of the enzyme lead to an accumulation of oligosaccharides but also to polymers with higher polymerization degree. To understand these changes in product spectra MD simulations have been performed. Finally, the proton transfer in catalytic cysteine histidine dyads was investigated. The focus was set on the influence of the relaxation of the protein environment to the reaction. Calculations of the enzymes FadA5 and rhodesain revealed that the preferred protonation state of the dyade depends on the protein environment and has an impact on the reaction barrier. Furthermore, the adaptation of the environment to a fixed protonation state was analyzed via MD simulationsHeutzutage sind computergestützte Untersuchungen ein essentieller Teil in der chemischen, biochemischen oder pharmazeutischen Forschung. Durch die in den Jahren gestiegene Rechenleistung ist die Berechnung biologischer Systeme möglich. Im Rahmen dieser Arbeit wurden molekularmechanische (MM) und quantenmechanische (QM) Methoden sowie die Kombination beider (QM/MM) für verschiedene Studien eingesetzt, die teilweise aus Fragestellungen verschiedener Arbeitsgruppen hervorgegangen sind. Dadurch umfasst diese Arbeit acht verschiedene Themenkomplexe, bei denen chemische Reaktionen, aber auch der Protonentransfer in Enzymen, Konformationsänderungen von Liganden oder Proteinen und die Verifizierung experimenteller Daten im Fokus standen. Die Arbeit befasst sich anfangs mit Reaktionsmechansimen aromatischer Inhibitoren für Cysteinproteasen, Enzyme, welche in vielen Organismen enthalten sind. Diese Enzyme sind für verschiedene Karzinome oder Krankheiten wie der Afrikanischen Trypanosomiasis oder der Chagas-Krankheit verantwortlich. Aromatische SNAr-Elektrophile bieten hierbei eine neue Möglichkeit der kovalenten Modifikation dieser Proteasen. Quantenmechanische wurden durchgeführt, um Einblicke in die Energetik und mögliche Mechanismen zu erhalten. Das nächste Kapitel befasst sich ebenfalls mit Trypanosomiasis, setzt aber den Fokus auf ein anderes Enzym. Die Besonderheit von Trypanosomiasis ist der Thiol Metabolismus, welcher durch kovalente Inhibitoren modifiziert werden kann. In diesem Kontext wurden der Wildtyp und Punktmutationen des Enzyms Tryparedoxin mittels Molekulardynamik Simulationen untersucht, um Interaktionen einzelner Aminosäuren mit dem kovalenten Inhibitor zu evaluieren. Experimentelle Daten zeigten, dass eine Dimerisierung des Enzyms in Anwesenheit des Inhibitors stattfindet. Durch MD-Simulationen konnte gezeigt werden, dass die Stabilität des Dimers in Abwesenheit des Inhibitors sinkt, wodurch experimentellen Daten bestätigt wurden. Weitere Untersuchungen zu Cysteinproteasen wie Cruzain und Rhodeasin wurden durchgeführt, um experimentelle kinetische Daten von kovalenten Vinylsulfon Inhibitoren zu reproduzieren. Hierbei wurden Methoden wie QM oder QM/MM Rechnungen aber auch Docking, MD und MMPBSA/MMGBSA Simulationen angewandt, um diese Daten zu reproduzieren. In den Untersuchungen zeigte sich, dass die Verwendung der Kraftfeld-basierten Methoden zu qualitativ richtigen Vorhersagen führte. Die Kinase AKT ist in einer Reihe von Krankheiten involviert und spielt eine wichtige Rolle bei der Entstehung von Krebs. Neue kovalent-allosterische Inhibitoren wurden entwickelt und im kovalenten Komplex mit AKT kristallisiert. Die Kristallstrukturen zeigten, dass je nach Inhibitor ein anderes Cystein adressiert wurde. Um diese Unterschiede zu untersuchen, wurden computergestützte Simulationen verwendet. Enoyl-(acyl carrier) (ENR) Proteine sind essentiell für den letzten Schritt in der Fettsäurebiosynthese II (FAS) und bilden ein gutes Target zur Inhibition. Der Diphenylether Inhibitor SKTS1, welchen man ursprünglich als Target für den ENR von Staphylococcus aureus entwarf, wurde auch in InhA, dem ENR von Mycobacterium Tuberculosis (TB), kristallisiert. Die Kristallstrukturen weisen je nach Protein auf einen Wechsel der tautomeren Form des Inhibitors hin. Dieser Sachverhalt wurde mittels MD Simulationen untersucht. Hierbei zeigten die Ergebnisse eine Übereinstimmung mit den experimentellen Daten. Diese Arbeit befasst sich ebenfalls mit der Entwicklung eines kovalenten Inhibitors ausgehend von einem nicht-kovalenten Liganden. Das Target FadA5 ist ein integrales Enzym zur Degradation von Steroiden in TB und ist für die chronische Tuberkulose verantwortlich. Dieses Enzym wurde im Komplex mit einem nicht-kovalenten Liganden kristallisiert, welches als Startpunkt dieser Untersuchungen diente. QM, QM/MM, Docking und MD Simulationen wurden hierbei verwandt, um potentielle Kandidaten zu evaluieren. Das nächste Kapitel befasst sich mit der Modifikation des Produktspektrums von Bacillus megaterium Levansucrase, eine Polymerase, welche die Biosynthese von Fruktanen katalysiert. Durch kovalente Modifikatoren im Wildtyp oder bei Mutanten des Enzyms konnte sowohl eine Anreicherung von Oligosacchariden, aber auch von Polymeren mit höherem Polymerisationsgrad erzielt werden. Um diese Änderungen im Produktspektrum zu verstehen, wurden MD Simulationen durchgeführt. Schließlich wurde die Untersuchung des Protonentransfers in katalytischen Cystein Histidin Dyaden durchgeführt. Hierbei stand der Einfluss der Relaxation der Proteinumgebung auf diese Reaktion im Fokus. Berechnungen in den Enzymen FadA5 und Rhodesain zeigten, dass der präferierte Protonierungszustand der Diade von der Proteinumgebung abhängt und einen großen Einfluss auf die Reaktionsbarriere hat. Um dynamische Effekte einzubeziehen, wurde die Adaption der Umgebung auf einen fixierten Protonierungszustand mittels MD Simulationen analysiert
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Tseng, Guo-Hsing, and 曾國興. "Identification and Characterization of Optimal Inhibitors and Non-covalent Interaction with Enzyme Using Mass Spectrometry." Thesis, 2005. http://ndltd.ncl.edu.tw/handle/71966133273175971286.
Full textAbstract:
碩士國立彰化師範大學
化學系
93
Revolutional progress in combinatorial chemistry in recent years necessitated the development of high-throughput screening tools capable of purification/isolation, structural characterization with maximum sensitivity and speed. The intrinsic properties for the structural and analytical characterization render mass spectrometry an essential tool in combinatorial technologies. In this study, we attempted to use electrospray ionization mass spectrometry (ESI-MS) to investigate the non-covalent interaction between enzyme and its inhibitor. The ��-L-fucosidase from corynebacterium sp was chosen as a model for non-covalent interaction. Enzyme-inhibitor (E-I) complexes were investigated by nanoflow ESI-MS under non-denaturing conditions. Several methods were investigated to characterize the non-covalent interaction of enzyme-inhibitor. In the first part of the thesis, the non-covalent nature and the specificity of complexes are studied in detail with a number of control experiments. Mass spectrometry can be a tool to determine the inhibition constant (KI) of ��-L-fucosidase inhibitors. The ratio of the free enzyme and E-I complex in mass spectrum could be a measure to reflect their concentrations in solution to evaluate KI. Under the current nanoflow ESI-MS sensitivity, we found that the method was valid for KI ranging from micromolar to nanomolar. However, direct MS-based evaluation of more potent inhibitors can not obtain accurate values and still present a challenge by mass spectrometry. Alternatively, we tried to determine the most potent inhibitor in an inhibitor mixture used tandem mass spectrometry. In the collisional-induced dissociation (CID) mass spectrum, the intensity of fragment ion for the precursor of each enzyme-inhibitor can be used to identify the most potent inhibitor. The potency of the inhibitor in the range of nanomolar obtained from the tandem mass spectrometry was in agreement to the literature. In addition, we found that the proton affinity of inhibitor can be obtained from CID experiment, and the tendency correlated well with the inhibition capability reported in the literature. Whether the proton affinity of inhibitor can be used as a probe to select the more potent inhibitors from CID mass spectrum remains further study. On the other hand, in source CID experiment monitored by mass spectrometry permitted us to evaluate rapidly the relative gas phase stabilities of non-covalent fucosidase-inhibitor complexes. In this experiment, dissociation in the gas phase was provoked by increasing the accelerating voltage of the ion (DP2) in the source-analyzer interface region. The DP2 value needed to dissociate 50% of the non-covalent complex initially present was taken as a gas phase stability parameter of the enzyme-inhibitor complex. The DP2 values that needed to dissociate 50% of the complexes initially present were may correlate the energy of the electrostatic and H-bond interactions between ��-L-fucosidase and inhibitor. Therefore, we could use the method to probe the binding force of fucosidase-inhibitor.
Book chapters on the topic "Non-covalent inhibitor"
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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.
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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.
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Legler, Günter. "β-Glucocerebrosidase: Mechanistic Studies With Covalent and Non-Covalent Inhibitors." In Lipid Storage Disorders, 63–72. Boston, MA: Springer US, 1988. http://dx.doi.org/10.1007/978-1-4613-1029-7_7.
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Watterson, Scott H., and Joseph A. Tino. "Advances in the Discovery and Development of Non-Covalent and Covalent BTK Inhibitors Targeting Autoimmune Diseases." In 2020 Medicinal Chemistry Reviews, 195–226. Medicinal Chemistry Division of the American Chemical Society, 2020. http://dx.doi.org/10.29200/acsmedchemrev-v55.ch8.
Full textConference papers on the topic "Non-covalent inhibitor"
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Asami, Tokiko, Wataru Kawahata, Shigeki Kashimoto, and Masaaki Sawa. "Abstract B152: CB1763, a highly selective, novel non-covalent BTK inhibitor, targeting ibrutinib-resistant BTK C481S mutant." In Abstracts: AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; October 26-30, 2017; Philadelphia, PA. American Association for Cancer Research, 2018. http://dx.doi.org/10.1158/1535-7163.targ-17-b152.
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Quereda, V., S. Bayle, V. Francesca, M. Andrii, R. William, and D. Derek. "Abstract P1-06-03: A selective Cdk12/13 non-covalent inhibitor with potent anti-breast cancer activity." In Abstracts: 2018 San Antonio Breast Cancer Symposium; December 4-8, 2018; San Antonio, Texas. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1538-7445.sabcs18-p1-06-03.
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Sebti, Said M., Aslamuzzaman Kazi, Sevil Ozcan, Awet G. Tecleab, Ying Sun, and Harshani Lawrence. "Abstract 1810: PI-1840, a novel non-covalent and rapidly reversible proteasome inhibitor with anti-tumor activity." In Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA. American Association for Cancer Research, 2014. http://dx.doi.org/10.1158/1538-7445.am2014-1810.
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Johannessen, Liv H., Shanhu Hu, Nan Ke, Anthony D'Ippolito, Nisha Rajagopal, Jason Marineau, Anneli Savinainen, William Zamboni, and Graeme Hodgson. "Abstract C091: Preclinical evaluation of PK, PD, and antitumor activity of the oral, non-covalent, potent and highly selective CDK7 inhibitor, SY-5609, provides rationale for clinical development in multiple solid tumor indications." In Abstracts: AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; October 26-30, 2019; Boston, MA. American Association for Cancer Research, 2019. http://dx.doi.org/10.1158/1535-7163.targ-19-c091.
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Beltran, Pedro J., Jinghui Zhan, Petia Mitchell, Ryan P. Wurz, Liping Pettus, Tian Wu, Mary Chaves, et al. "Abstract 2587: A novel covalent inhibitor of mutant but not wild-type (WT) epidermal growth factor receptor (EGFR) has activity in vitro and in vivo in non-small cell lung cancer (NSCLC) models." In Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA. American Association for Cancer Research, 2015. http://dx.doi.org/10.1158/1538-7445.am2015-2587.
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Freund, M., J.-P. Cazenave, M.-L. Wiesel, C. Roitsch, N. Riehl-Bellon, G. Loison, Y. E. Lemoine, S. Brown, and M. Courtney. "RECOMBINANT HIRUDIN INHIBITS EXPERIMENTAL VENOUS THROMBOSIS INDUCED BY INJECTION OF TISSUE FACTOR AND STASIS." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643917.
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Hirudin (HIR), a polypeptide of 65 aminoacids, is the most potent natural inhibitor of coagulation by forming rapidly a very stable and specific non covalent 1:1 complex with α-thrombin, independent of antithrombin III. Although natural HIR has in vivo anticoagulant and antithrombotic properties, its limited availability for large scale purification has prevented further clinical testing and potential use; this can now be solved by recombinant DNA technology. We have previously reported the cloning and expression of a cDNA encoding one variant (called HV-2) of Hirudo medicinalis HIR (Proc. Natl. Acad. Sci. USA. 1986, 83, 1084-1088). The main factors responsible for venous thrombosis are stasis and thrombin generation secondary to tissue factor liberation from vascular cells and monocytes by injury, endotoxin, interleukin-1 or cachectin and the subsequent activation and circulation of activated clotting factors. We have studied the antithrombotic properties of recombinant HIR, HV-2, in a rat experiemental model of venous thrombosis. HV-2 was expressed in yeast, extracted from culture supernatant and purified by HPLC. Pure HV-2 had an isoleucine NH2-terminus and a specific activity of 13000 ATU/mg.30 male Wistar rats (225-300g) were anesthetized with pentobarbital. At time t (0 min) an i.v. (penis) injection of 0.4 ml of saline or HV-2 (2000 to 8000 ATU/kg) was given, followed at t (5min) by 25 mg/kg tissue factor (Thromboplastin C, Dade) i.v. ; 10 s later stasis of the exposed vena cava between 2 sutures 0.7 cm apart and at t (15 min) removal, blotting, fixation and weighing of the thrombus. Linear regression analysis showed a correlation (r=0.99) between the dose of HV-2 and thrombus weight and a calculated IC50 = 3000 ATU/kg. Total inhibition of thrombus formation was seen after injection of 6000 ATU/kg HV-2 and lasted up to 15 min of circulation, HV-2 being completely eliminated from blood in 60 min and accumulated in the kidneys as shown by gamma imaging with 131I-HV-2. In conclusion, the recombinant HIR HV-2 is a potent immediate antithrombin which inhibits venous thrombosis induced by tissue factor and stasis.
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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.
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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.
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Kodama, K., O. Larm, P. Olsson, B. Pasehe, J. Risenfeldt, and J. Swedenborg. "ANTITHROMBIN III BINDING TO IMMOBILIZED HEPARIN FRAGMENTS AND ITS RELATION TO F XA INHIBITION." In XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643091.
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Covalent end-point attachment of heparin fragments (8 000 daltons) to artitifical materials results as published before in a highly thromboresistant surface. Approximately one out of six bonded fragments carry the antithrombin III (AT) binding sequence. i.e. the high affinity site. With regard to thrombin inhibition the heparin surface resembles the endothelium. The present work deals with the uptake on the immobilized heparin and its significance for F Xa inhibition. The uptake of AT was studied at 0.15 M and 0.35 M NaCl concentration (TRIS buffer, pH 7.4) respectively and determined as disappearance of AT activity in the exposed solutions. Large amounts were adsorbed at 0.15 M and the uptake was both concentration and time dependent. At 0.35 M the uptake was the same at all the tested AT concentrations: 5 pico-moles/square cm. It was deduced that mainly high affinity sites had taken up AT at 0.35 M and that both high and low affinity sites had taken up AT at 0.15 M.The non-AT-adsorbed heparin surface did not induce inhibition of F Xa (in TRIS buffer solution) after exposure. The surface AT-adsorbed at 0.15 M induced F Xa inhibition with the same rate in several consecutive exposed aliquots. The surface AT-adsorbed at 0.35 M had a lower inhibitory capacity and only the first F Xa aliquot was inhibited at the same rate as on the surface AT-adsorbed at 0.15 M. The inhibition rate for a second aliquot was slowlier due to the facts that AT had been consumed and that the density of AT on high affinity sites had decreased. It is concluded that AT on high affinity sites determines the rate at which F Xa is inhibited whereas the amount of AT on low affinity sites determines the inhibitory capacity by continously providing the high affinity sites with AT. The migration of AT must take place horizontally in the 100-200 A thick surface layer and may mimic events happening on a cellular membrane with binding sites of different classes for a substance.
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Kazi, Aslamuzzaman, Sevil Ozcan, Harshani Lawrence, and Said M. Sebti. "Abstract 2778: Development of non-covalent reversible proteasome inhibitors." In Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL. American Association for Cancer Research, 2012. http://dx.doi.org/10.1158/1538-7445.am2012-2778.
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Lee, Ho-June, Gabriele Schaefer, Tim Heffron, Shiva Malek, Mark Merchant, Robert L. Yauch, Valentina Pirazzoli, Katerina Politi, and Jeff Settleman. "Abstract LB-309: Non-covalent wild-type-sparing inhibitors of EGFR T790M ." In Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC. American Association for Cancer Research, 2013. http://dx.doi.org/10.1158/1538-7445.am2013-lb-309.
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