scholarly journals Identification of a critical binding site for local anesthetics in the side pockets of Kv1 channels

2021 ◽  
Author(s):  
Aytug K. Kiper ◽  
Mauricio Bedoya ◽  
Sarah Stalke ◽  
Stefanie Marzian ◽  
David Ramírez ◽  
...  
Keyword(s):  
Binding Site ◽  
Local Anesthetics ◽  
Critical Binding

Is there a second external lidocaine binding site on mammalian cardiac cells?

1989 ◽  
Vol 257 (1) ◽  
pp. H79-H84 ◽  
Author(s):  
L. A. Alpert ◽  
H. A. Fozzard ◽  
D. A. Hanck ◽  
J. C. Makielski
Keyword(s):  
Binding Site ◽  
Cardiac Arrhythmias ◽  
Local Anesthetics ◽  
Sodium Current ◽  
Cardiac Cells ◽  
Na Channel ◽  
Functional Difference ◽  
Na Channels ◽  
Internal Perfusion ◽  
Cardiac Purkinje Cells

Lidocaine and its permanently charged analogue QX-314 block sodium current (INa) in nerve, and by this mechanism, lidocaine produces local anesthesia. When administered clinically, lidocaine prevents cardiac arrhythmias. Nerve and skeletal muscle are much more sensitive to local anesthetics when the drugs are applied inside the cell, indicating that the binding site for local anesthetics is located on the inside of those Na channels. Using a large suction pipette for voltage clamp and internal perfusion of single cardiac Purkinje cells, we demonstrate that a charged lidocaine analogue blocks INa not only when applied from the inside but also from the outside, unlike noncardiac tissue. This functional difference in heart predicts that a second local anesthetic binding site exists outside or near the outside of cardiac Na channels and emphasizes that the cardiac Na channel is different from that in nerve.

scholarly journals Discovery and Design of Novel Small Molecule GSK-3 Inhibitors Targeting the Substrate Binding Site

2020 ◽  
Vol 21 (22) ◽  
pp. 8709
Author(s):  
Ido Rippin ◽  
Netaly Khazanov ◽  
Shirley Ben Joseph ◽  
Tania Kudinov ◽  
Eva Berent ◽  
...  
Keyword(s):  
Binding Site ◽  
Small Molecule ◽  
Substrate Binding ◽  
Pharmacophore Model ◽  
Drug Induced ◽  
Substrate Binding Site ◽  
Zinc Database ◽  
Competitive Inhibitors ◽  
Ic50 Values ◽  
Critical Binding

The serine/threonine kinase, GSK-3, is a promising drug discovery target for treating multiple pathological disorders. Most GSK-3 inhibitors that were developed function as ATP competitive inhibitors, with typical limitations in specificity, safety and drug-induced resistance. In contrast, substrate competitive inhibitors (SCIs), are considered highly selective, and more suitable for clinical practice. The development of SCIs has been largely neglected in the past because the ambiguous, undefined nature of the substrate-binding site makes them difficult to design. In this study, we used our previously described structural models of GSK-3 bound to SCI peptides, to design a pharmacophore model and to virtually screen the “drug-like” Zinc database (~6.3 million compounds). We identified leading hits that interact with critical binding elements in the GSK-3 substrate binding site and are chemically distinct from known GSK-3 inhibitors. Accordingly, novel GSK-3 SCI compounds were designed and synthesized with IC50 values of~1–4 μM. Biological activity of the SCI compound was confirmed in cells and in primary neurons that showed increased β-catenin levels and reduced tau phosphorylation in response to compound treatment. We have generated a new type of small molecule GSK-3 inhibitors and propose to use this strategy to further develop SCIs for other protein kinases.

A Common Binding Site for Channel-Permeant Cations and Local Anesthetics on the Nicotinic Acetylcholine Receptor

1991 ◽  
Vol 625 (1 Molecular and) ◽  
pp. 645-648
Author(s):  
JEFFREY M. HERZ ◽  
STEPHEN KOLB ◽  
THOMAS ERLINGER
Keyword(s):  
Binding Site ◽  
Acetylcholine Receptor ◽  
Nicotinic Acetylcholine Receptor ◽  
Local Anesthetics ◽  
Nicotinic Acetylcholine

scholarly journals Identification of an essential binding site for local anesthetics in the ‘side pockets´ of Kv1 channels

Authorea ◽  
2020 ◽  
Author(s):  
Aytug Kiper ◽  
Sarah Stalke ◽  
Stefanie Marzian ◽  
Mauricio Bedoya ◽  
David Ram rez ◽  
...  
Keyword(s):  
Binding Site ◽  
Local Anesthetics

Detailed Mapping of the Binding Site within Platelet Glycoprotein Ibα for Leukocyte Integrin Mac-1 (αMβ2).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1547-1547
Author(s):  
Qiang Han ◽  
Yuandong Peng ◽  
Zhiping Chen ◽  
Valentin A. Ustinov ◽  
Tatiana P. Ugarova ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Platelet Aggregation ◽  
Binding Site ◽  
Vessel Wall ◽  
Vascular Inflammation ◽  
Peptide Binding ◽  
Targeted Mutagenesis ◽  
Platelet Glycoprotein ◽  
Human Sequence ◽  
Critical Binding

Abstract We have shown that the platelet counter-receptor for integrin Mac-1 (αMβ2, CD11b/CD18) is glycoprotein (GP) Ibα, the largest component of the GP Ib-IX-V complex, better known for its role in mediating platelet attachment to sites of vessel wall injury. The GP Ibα-Mac-1 interaction mediates the firm adhesion of leukocytes on platelet thrombi, enabling their subsequent transmigration into the vessel wall. Our previous studies showed that the I-domain of Mac-1 binds the C-terminal flanking sequences (Phe201–Gly268) of GP Ibα, as demonstrated by the fact that binding is almost completely inhibited by the I domain ligands fibrinogen and heparin, the I-domain monoclonal antibody LPM19c, and the GP Ibα-specific monoclonal antibody AP1. The latter antibody is of interest because it also inhibits the binding of von Willebrand factor and thrombin. We have mapped the AP1 epitope to a 10-amino acid sequence spanning Arg218 to Tyr228. In the current investigation, we constructed a series of cell lines expressing mutants of human GP Ibα, either by replacement of the human sequence with the corresponding dog sequence (dog GP Ibα does not bind human Mac-1) or by targeted mutagenesis, and tested their ability to bind the recombinant Mac-1 I domain. The GP Ibα region Phe201-Asn223 was crucial for Mac-1 binding, with residues Arg218, Asp222 and Asn223 playing vital roles. In addition, a peptide containing the AP1 epitope (Leu214-Val229) bound Mac-1 I-domain specifically and saturably. Peptide binding was blocked by LPM19c and soluble GP Ibα, and by the M2 peptide, which corresponds to the GP Ibα-binding site in the Mac-1 I domain (Phe201-Lys217). Peptide binding was also blocked by an antibody against the M2 sequence. The AP1 peptide inhibited the attachment of GP Ib-IX-V complex-expressing CHO cells to immobilized Mac-1 I domain, and the adhesion of THP-1 cells—a monocytoid line expressing Mac-1—to immobilized GP Ibα. This peptide did not inhibit attachment of the cells VWF surfaces, or thrombin-induced platelet aggregation. In summary, we have defined the GP Ibα sequence Arg214 to Val229 as a critical binding site for Mac-1. Because a peptide corresponding to this region inhibits GP Ibα binding to Mac-1 but blocks neither platelet adhesion to immobilized VWF nor thrombin-induced platelet aggregation, it has potential to guide the development of agents that will specifically inhibit leukocyte-platelet complexes that promote vascular inflammation.

scholarly journals Rational Design of Resveratrol O-methyltransferase for the Production of Pinostilbene

2021 ◽  
Vol 22 (9) ◽  
pp. 4345
Author(s):  
Daniela P. Herrera ◽  
Andrea M. Chánique ◽  
Ascensión Martínez-Márquez ◽  
Roque Bru-Martínez ◽  
Robert Kourist ◽  
...  
Keyword(s):  
Binding Site ◽  
Rational Design ◽  
Three Dimensional ◽  
Point Mutations ◽  
Catalytic Efficiency ◽  
Monomethyl Ether ◽  
Protein Model ◽  
Health Promoting ◽  
The Stability ◽  
Critical Binding

Pinostilbene is a monomethyl ether analog of the well-known nutraceutical resveratrol. Both compounds have health-promoting properties, but the latter undergoes rapid metabolization and has low bioavailability. O-methylation improves the stability and bioavailability of resveratrol. In plants, these reactions are performed by O-methyltransferases (OMTs). Few efficient OMTs that monomethylate resveratrol to yield pinostilbene have been described so far. Here, we report the engineering of a resveratrol OMT from Vitis vinifera (VvROMT), which has the highest catalytic efficiency in di-methylating resveratrol to yield pterostilbene. In the absence of a crystal structure, we constructed a three-dimensional protein model of VvROMT and identified four critical binding site residues by applying different in silico approaches. We performed point mutations in these positions generating W20A, F24A, F311A, and F318A variants, which greatly reduced resveratrol’s enzymatic conversion. Then, we rationally designed eight variants through comparison of the binding site residues with other stilbene OMTs. We successfully modified the native substrate selectivity of VvROMT. Variant L117F/F311W showed the highest conversion to pinostilbene, and variant L117F presented an overall increase in enzymatic activity. Our results suggest that VvROMT has potential for the tailor-made production of stilbenes.

scholarly journals Cocaine-induced closures of single batrachotoxin-activated Na+ channels in planar lipid bilayers.

1988 ◽  
Vol 92 (6) ◽  
pp. 747-765 ◽  
Author(s):  
G K Wang
Keyword(s):  
Binding Site ◽  
Binding Affinity ◽  
Lipid Bilayers ◽  
Local Anesthetics ◽  
Direct Interaction ◽  
Na Channel ◽  
Planar Lipid Bilayers ◽  
Na Channels ◽  
Single Class ◽  
Na Ions

Batrachotoxin (BTX)-activated Na+ channels from rabbit skeletal muscle were incorporated into planar lipid bilayers. These channels appear to open most of the time at voltages greater than -60 mV. Local anesthetics, including QX-314, bupivacaine, and cocaine when applied internally, induce different durations of channel closures and can be characterized as "fast" (mean closed duration less than 10 ms at +50 mV), "intermediate" (approximately 80 ms), and "slow" (approximately 400 ms) blockers, respectively. The action of these local anesthetics on the Na+ channel is voltage dependent; larger depolarizations give rise to stronger binding interactions. Both the dose-response curve and the kinetics of the cocaine-induced closures indicate that there is a single class of cocaine-binding site. QX-314, though a quaternary-amine local anesthetic, apparently competes with the same binding site. External cocaine or bupivacaine application is almost as effective as internal application, whereas external QX-314 is ineffective. Interestingly, external Na+ ions reduce the cocaine binding affinity drastically, whereas internal Na+ ions have little effect. Both the cocaine association and dissociation rate constants are altered when external Na+ ion concentrations are raised. We conclude that (a) one cocaine molecule closes one BTX-activated Na+ channel in an all-or-none manner, (b) the binding affinity of cocaine is voltage sensitive, (c) this cocaine binding site can be reached by a hydrophilic pathway through internal surface and by a hydrophobic pathway through bilayer membrane, and (d) that this binding site interacts indirectly with the Na+ ions. A direct interaction between the receptor and Na+ ions seems minimal.

scholarly journals Identification of an active disaccharide unit of a glycoconjugate receptor for pneumococci attaching to human pharyngeal epithelial cells.

1983 ◽  
Vol 158 (2) ◽  
pp. 559-570 ◽  
Author(s):  
B Andersson ◽  
J Dahmén ◽  
T Frejd ◽  
H Leffler ◽  
G Magnusson ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Binding Site ◽  
Inhibitory Activity ◽  
Receptor Activity ◽  
Target Cells ◽  
Soluble Receptor ◽  
Synthetic Oligosaccharides ◽  
Critical Binding

Glycoconjugates containing the disaccharide unit GlcNAc beta 1 leads to 3Gal beta were suggested as receptors for pneumococci adhering to human pharyngeal epithelial cells. The receptor activity was detected both by inhibition of adhesion by an excess of free oligosaccharide and by induction or increase of adhesion after coating of target cells with glycolipid. Studies with free natural and synthetic oligosaccharides identified the disaccharide GlcNAc beta 1 leads to 3Gal beta as one critical binding site. The specificity of recognition was shown inter alia by the lack of inhibitory activity of GlcNAc beta 1 leads to 4Gal beta, which differs only in the linkage of the two sugars. Specific interference with pneumococcal adhesion by administration of soluble receptor sugar may improve our understanding of the role of adhesion in vivo.

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