ATP-binding site
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The Na+/K+-ATPase plays a key role in ion transport across the plasma membrane of all animal cells. The voltage-sensitive styrylpyrimidium dye RH421 has been used in several laboratories for monitoring of Na+/K+-ATPase kinetics. It is known, that RH421 can interact with the enzyme and it can influence its activity at micromolar concentrations, but structural details of this interaction are only poorly understood. Experiments with isolated large cytoplasmic loop (C45) of Na+/K+-ATPase revealed that RH421 can interact with this part of the protein with dissociation constant 1μM. The Trp-to-RH421 FRET performed on six single-tryptophan mutants revealed that RH421 binds directly into the ATP-binding site. This conclusion was further supported by results from molecular docking, site-directed mutagenesis and by competitive experiments using ATP. Experiments with C45/DPPC mixture revealed that RH421 can bind to both C45 and lipids, but only the former interaction was influenced by the presence of ATP.
- MeSH
- adenosintrifosfát metabolismus MeSH
- buněčná membrána metabolismus MeSH
- cytoplazma metabolismus MeSH
- fluorescenční barviva metabolismus MeSH
- kinetika MeSH
- mutageneze cílená metody MeSH
- simulace molekulového dockingu MeSH
- sodíko-draslíková ATPasa metabolismus MeSH
- tryptofan metabolismus MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Transient receptor potential channel vanilloid receptor subunit 1 (TRPV1) is a thermosensitive cation channel activated by noxious heat as well as a wide range of chemical stimuli. Although ATP by itself does not directly activate TRPV1, it was shown that intracellular ATP increases its activity by directly interacting with the Walker A motif residing on the C-terminus of TRPV1. In order to identify the amino acid residues that are essential for the binding of ATP to the TRPV1 channel, we performed the following point mutations of the Walker A motif: P732A, D733A, G734A, K735A, D736A, and D737A. Employing bulk fluorescence measurements, namely a TNP-ATP competition assay and FITC labelling and quenching experiments, we identified the key role of the K735 residue in the binding of the nucleotide. Experimental data was interpreted according to our molecular modelling simulations.
Název „ATP binding cassette“ představuje velkou rodinu transmembránových proteinů, které vážou ATP a energii z tohoto zdroje využívají k aktivnímu řízenému transportu chemicky různorodých látek z vnitřního prostředí buňky do extracelulárního prostoru. Mezi endogenní látky, na jejichž transportu se zástupci této transportní rodiny podílejí, patří lipidy, steroidy, hormony či bilirubin; významnou úlohu však mají i v transportu xenobiotik.
Over the years, Mycobacterium tuberculosis has been one of the major causes of death worldwide. As several clinical isolates of the bacteria have developed drug resistance against the target sites of the current therapeutic agents, the development of a novel drug is the pressing priority. According to recent studies on Mycobacterium tuberculosis, ATP binding sites of Mycobacterium tuberculosis serine/threonine protein kinases (MTPKs) have been identified as the new promising drug target. Among the several other protein kinases (PKs), Protein kinase G (PknG) was selected for the study because of its crucial role in modulating bacterium's metabolism to survive in host macrophages. In this work, we have focused on the H37Rv strain of Mycobacterium tuberculosis. A list of 477 flavanones obtained from the PubChem database was docked one by one against the crystallized and refined structure of PknG by in-silico techniques. Initially, potential inhibitors were narrowed down by preliminary docking. Flavanones were then selected using binding energies ranging from -7.9 kcal.mol-1 to -10.8 kcal.mol-1. This was followed by drug-likeness prediction, redocking analysis, and molecular dynamics simulations. Here, we have used experimentally confirmed drug AX20017 as a reference to determine candidate compounds that can act as potential inhibitors for PknG. PubChem165506, PubChem242065, PubChem688859, PubChem101367767, PubChem3534982, and PubChem42607933 were identified as possible target site inhibitors for PknG with a desirable negative binding energy of -8.1, -8.3, -8.4, -8.8, -8.6 and -7.9 kcal.mol-1 respectively. Communicated by Ramaswamy H. Sarma.
- MeSH
- adenosintrifosfát metabolismus MeSH
- bakteriální proteiny chemie MeSH
- Mycobacterium tuberculosis * metabolismus MeSH
- proteinkinasy závislé na cyklickém GMP chemie metabolismus MeSH
- simulace molekulární dynamiky MeSH
- simulace molekulového dockingu MeSH
- vazebná místa MeSH
- Publikační typ
- časopisecké články MeSH
Autor podává krátký přehled nedávné historie vývoje prvních farmakologických inhibitorů působících kompetitivně a selektivně v ATP-vazebných místech proteinkináz. Vzhledem k tomu, že enzymové domény, které obsahují vazebná místa pro ATP, jsou navzájem vysoce homologní, byly nálezy inhibitorů tohoto typu překvapivé. V době, kdy se tyto inhibitory objevily, tj. v první polovině 90. let minulého století, zcela převládalo přesvědčení, že selektivní kompetitivní inhibice v ATP-vazebném místě není pravděpodobná. Tento názor byl překonán zejména odhalením selektivních syntetických inhibitorů kinázy c-Abl, tyrosinkinázy receptoru epidermálního růstového faktoru, a konečně purinových inhibitorů cyklin-dependentních kináz (CDK). K objevu a vývoji inhibitorů CDK významně přispěla spolupráce mezi českými a francouzskými autory.
A brief survey on the development of the first synthetic selective inibitors that competitively inhibit proteinkinases in their ATP-binding pocket is presented. Until 1990s, selective inhibition of this kind was considered improbable. The reason for such a belief was the high degree of homology existing between proteinkinase ATP-binding domains. The small synthetic molecules inhibiting c-Abl kinase, epidermal growth factor receptor kinase, and cyclin-dependent kinases (CDKs) were the first selective ATP-competitive inhibitors to have been described about ten years ago. French and Czech scientists made an important contribution in this field by discovering lead purine inhibitor olomoucine that competitively targets the CDK catalytic domain.
- MeSH
- cyklin-dependentní kinasy antagonisté a inhibitory MeSH
- epidermální růstový faktor MeSH
- inhibitory enzymů dějiny farmakologie chemie MeSH
- inhibitory proteinkinas MeSH
- proteinkinasy MeSH
- puriny antagonisté a inhibitory chemická syntéza MeSH
- tyrosinkinasy MeSH
- vazebná místa MeSH
- Publikační typ
- přehledy MeSH
- MeSH
- karcinogeny MeSH
- krysa rodu rattus MeSH
- pyreny MeSH
- vazebná místa MeSH
- Check Tag
- krysa rodu rattus MeSH
