Cover Picture: Angew. Chem. Int. Ed. 6/2002

Lewis, Warren G.; Green, Luke G.; Grynszpan, Flavio; Radić, Zoran; Carlier, Paul R.; Taylor, Palmer; Finn, M. G.; Sharpless, K. Barry

doi:10.1002/1521-3773(20020315)41:6<875::aid-anie875>3.0.co;2-y

Cited by 138 publications

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“…Monomeric mAChE expressed from human embryonic kidney-293 cells (9) was purified by affinity chromatography by using propidium elution (10). The anti1-and syn1-mAChE complexes were prepared by using a 2-fold molar excess of the inhibitors and concentrations well above their K d s (Ϸ135 M i.e., 15 10 6 ϫ K i(anti) and 330 10 6 ϫ K i(syn) ; Table 2) (7,10). Titration of the isomer stock solutions ( 490nm ϭ 6,000 M Ϫ1 ⅐cm Ϫ1 ) and analysis of the complex solutions were carried out spectrophotometrically (10).…”

Section: Methodsmentioning

confidence: 99%

“…The TZ2PA6 anti1 and syn1 isomers were synthesized as described (7). Monomeric mAChE expressed from human embryonic kidney-293 cells (9) was purified by affinity chromatography by using propidium elution (10).…”

Section: Methodsmentioning

confidence: 99%

“…In contrast, chemical synthesis by thermal reaction in the absence of enzyme proceeds very slowly and provides an Ϸ1:1 mixture of syn1 and anti1 regioisomers, which differ in the nitrogen substitution positions on the 1,2,3-triazole. Although both are high-affinity inhibitors, the syn1 isomer, with a 100-fold greater affinity and a subpicomolar dissociation constant for certain AChEs (7), has a potency greater than all known noncovalent organic AChE inhibitors and high selectivity for individual cholinesterases.…”

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confidence: 99%

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Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

Bourne

Kolb

Radić

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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311

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The 1,3-dipolar cycloaddition reaction between unactivated azides and acetylenes proceeds exceedingly slowly at room temperature. However, considerable rate acceleration is observed when this reaction occurs inside the active center gorge of acetylcholinesterase (AChE) between certain azide and acetylene reactants, attached via methylene chains to specific inhibitor moieties selective for the active center and peripheral site of the enzyme. AChE catalyzes the formation of its own inhibitor in a highly selective fashion: only a single syn1-triazole regioisomer with defined substitution positions and linker distances is generated from a series of reagent combinations. Inhibition measurements revealed this syn1-triazole isomer to be the highest affinity reversible organic inhibitor of AChE with association rate constants near the diffusion limit. The corresponding anti1 isomer, not formed by the enzyme, proved to be a respectable but weaker inhibitor. The crystal structures of the syn1-and anti1-mouse AChE complexes at 2.45-to 2.65-Å resolution reveal not only substantial binding contributions from the triazole moieties, but also that binding of the syn1 isomer induces large and unprecedented enzyme conformational changes not observed in the anti1 complex nor predicted from structures of the apoenzyme and complexes with the precursor reactants. Hence, the freeze-frame reaction offers both a strategically original approach for drug discovery and a means for kinetically controlled capture, as a high-affinity complex between the enzyme and its self-created inhibitor, of a highly reactive minor abundance conformer of a fluctuating protein template.A cetylcholinesterase (AChE) rapidly terminates cholinergic neurotransmission by catalyzing the hydrolysis of the neurotransmitter, acetylcholine, and inhibitors of AChE have been used for over a century in various therapeutic regimens (1, 2). The structure of the target enzyme reveals a narrow gorge Ϸ20 Å in depth with the catalytic triad of the active center at its base (3). Distinctive inhibitors bind to the active center or to a peripheral anionic site (PAS) located at the rim of the gorge near the enzyme surface (4-6). Previously, we generated a library of active site and PAS inhibitors with respective tacrine and phenanthridinium nuclei, each equipped with an azide or acetylene group at the end of a flexible methylene chain, to enable the reporting 1,3-dipolar cycloaddition to occur (Scheme 1) (7). AChE itself served as the reaction vessel, synthesizing its own inhibitor from these building blocks, in effect, by equilibriumcontrolled sampling of various possible pairs of reactants in its active center gorge until irreversible cycloaddition between azide and acetylene ensued at an intersecting point within the gorge, between the two anchoring positions. From 49 building block combinations, the enzyme selected the TZ2͞PA6 pair to form, with an enhanced reaction rate, a highly regioselective syn1 triazole as the sole product (Scheme 1). In contrast, chemical synthesis by t...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

Bourne

Kolb

Radić

et al. 2004

Proc. Natl. Acad. Sci. U.S.A.

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311

262

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“…In situ click chemistry screening of binary mixtures of tacrine/phenylphenanthridinium-based building blocks for the discovery of bivalent inhibitors to AChE. 91,98 curbituril, a macrocycle made of glycouril, 89 Lewis et al were the first to investigate the potential of Huisgen's cycloaddition for application to target-guided synthesis. 91 In their proof-of-concept study, they selected enzyme acetylcholinesterase (AChE; EC 3.1.1.7) which plays a vital role in neuro-transmission in central and peripheral nervous system.…”

Section: In Situ Click Chemistry Using Acetylcholinesterase As a Tmentioning

confidence: 99%

The Lock is the Key: Development of Novel Drugs through Receptor Based Combinatorial Chemistry

Maraković

Šinko

2017

ACSi

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Modern drug discovery is mainly based on the de novo synthesis of a large number of compounds with a diversity of chemical functionalities. Though the introduction of combinatorial chemistry enabled the preparation of large libraries of compounds from so-called building blocks, the problem of successfully identifying leads remains. The introduction of a dynamic combinatorial chemistry method served as a step forward due to the involvement of biological macromolecular targets (receptors) in the synthesis of high affinity products. The major breakthrough was a synthetic method in which building blocks are irreversibly combined due to the presence of a receptor. Here we present various receptor-based combinatorial chemistry approaches. Huisgen's cycloaddition (1,3-dipolar cycloaddition of azides and alkynes) forms stabile 1,2,3-triazoles with very high receptor affinity that can reach femtomolar levels, as the case with acetylcholinesterase inhibitors shows. Huisgen's cycloaddition can be applied to various receptors including acetylcholinesterase, acetylcholine binding protein, carbonic anhydrase-II, serine/threonine-protein kinase and minor groove of DNA.

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“…The imaginative chemistry of Barry Sharpless and his colleagues leads to that end. 13 Acetylcholinesterase contains two discrete binding sites for inhibitors: one exists at the active center at the base of the gorge some 20 Å in depth; the other resides at the rim of the gorge. By extending aliphatic chains from the two binding molecules terminating in either an acetylene or azide moiety, a cycloaddition coupling reaction occurs leading to the formation of a syn or anti substituted 1,2,3-triazole.…”

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confidence: 99%

Contemporary paradigms for cholinergic ligand design guided by biological structure

Taylor

Hansen

Talley

et al. 2004

Bioorganic & Medicinal Chemistry Letters

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The identification of the various nicotinic receptor subtypes, when coupled with the recent development of three-dimensional structures of surrogate extracellular receptor domains, offers new opportunities to design nicotinic ligands. Conformation and fluctuations in receptor structure are critical to ligand selectivity, and we present here how a flexible receptor template can be used in the development of selective ligands affecting cholinergic neurotransmission.Close to a century ago Langley and his student Elliot proposed that cellular communication within the nervous system occurred through chemical neurotransmission. A necessary corollary of their proposal is the existence of receptors, termed by them as 'receptive substances'. 1 For the first 70 years, receptors were studied by inference through structureactivity relationships, enantiomeric selectivity, agonist-antagonist relationships and, somewhat later, covalent modifications to influence responsiveness. Only in the last 30 years, with the discovery of α-bungarotoxin to identify and characterize the nicotinic acetylcholine receptor, 2 have we witnessed the treatment of the receptor as a discrete chemical entity. A century following Langley's studies in this genome-based era a new paradigm has emerged, where a structural template or surrogate structure does or will soon exist for all receptors and biological targets.Sequencing the genome yielded a potential primary structure for all of the targets of drug action, and technical advances in crystallography and NMR spectrometry have produced structures of the gene products or individual domains of gene products at atomic level resolution. In some cases the actual receptor target has been characterized structurally; 3 in others, a homologous surrogate target has a resolved structure. 4-6 Accordingly, these structures, similar to lead compounds in small molecule design, provide templates for drug discovery. However, sequence differences between members of homologous proteins, structural constraints imposed by the crystallization process and the recognition that the ligand itself may induce changes in structure are considerations that require continuous refinement of the template.An appropriate example comes from considerations of the nicotinic acetylcholine receptor (nAChR), where a recently crystallized homopentameric acetylcholine binding protein (AChBP) isolated from the fresh water snail Lymnaea, was shown to be homologous to the N-terminal 210 amino acids in the nAChR. 5,7 As a soluble entity homologous to the extracellular domain of the receptor, but lacking in its transmembrane spans, the protein can be purified and crystallized. We have synthesized the encoding gene and expressed this protein from Lymnaea as well as from the saltwater species, Aplysia. 8,9 The sequences and

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Cover Picture: Angew. Chem. Int. Ed. 6/2002

Cited by 138 publications

References 0 publications

Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

Freeze-frame inhibitor captures acetylcholinesterase in a unique conformation

The Lock is the Key: Development of Novel Drugs through Receptor Based Combinatorial Chemistry

Contemporary paradigms for cholinergic ligand design guided by biological structure

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