scholarly journals The molecular basis for an allosteric inhibition of K+-flux gating in K2P channels

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Susanne Rinné ◽  
Aytug K Kiper ◽  
Kirsty S Vowinkel ◽  
David Ramírez ◽  
Marcus Schewe ◽  
...  
Keyword(s):  
Binding Site ◽  
Drug Targets ◽  
Drug Binding ◽  
Inhibition Mechanism ◽  
Central Cavity ◽  
Allosteric Inhibition ◽  
Mechanism Of Inhibition ◽  
Gating Mechanism ◽  
Channel Inhibition ◽  
K2p Channels

Two-pore-domain potassium (K2P) channels are key regulators of many physiological and pathophysiological processes and thus emerged as promising drug targets. As for other potassium channels, there is a lack of selective blockers, since drugs preferentially bind to a conserved binding site located in the central cavity. Thus, there is a high medical need to identify novel drug-binding sites outside the conserved lipophilic central cavity and to identify new allosteric mechanisms of channel inhibition. Here, we identified a novel binding site and allosteric inhibition mechanism, disrupting the recently proposed K+-flux gating mechanism of K2P channels, which results in an unusual voltage-dependent block of leak channels belonging to the TASK subfamily. The new binding site and allosteric mechanism of inhibition provide structural and mechanistic insights into the gating of TASK channels and the basis for the drug design of a new class of potent blockers targeting specific types of K2P channels.

scholarly journals Molecular Pharmacology of K2P Potassium Channels

2021 ◽  
Vol 55 (S3) ◽  
pp. 87-107
Keyword(s):  
Potassium Channels ◽  
Binding Sites ◽  
Drug Targets ◽  
Therapeutic Potential ◽  
Drug Binding ◽  
Channel Blockers ◽  
Molecular Pharmacology ◽  
K2p Channel ◽  
Channel Inhibition ◽  
K2p Channels

Potassium channels of the tandem of two-pore-domain (K2P) family were among the last potassium channels cloned. However, recent progress in understanding their physiological relevance and molecular pharmacology revealed their therapeutic potential and thus these channels evolved as major drug targets against a large variety of diseases. However, after the initial cloning of the fifteen family members there was a lack of potent and/or selective modulators. By now a large variety of K2P channel modulators (activators and blockers) have been described, especially for TASK-1, TASK-3, TREK-1, TREK2, TRAAK and TRESK channels. Recently obtained crystal structures of K2P channels, alanine scanning approaches to map drug binding sites, in silico experiments with molecular dynamics simulations (MDs) combined with electrophysiological studies to reveal the mechanism of channel inhibition/activation, yielded a good understanding of the molecular pharmacology of these channels. Besides summarizing drugs that were identified to modulate K2P channels, the main focus of this article is on describing the differential binding sites and mechanisms of channel modulation that are utilized by the different K2P channel blockers and activators.

scholarly journals Ligand Docking Methods to Recognize Allosteric Inhibitors for G-Protein-Coupled Receptors

2021 ◽  
Vol 15 ◽  
pp. 117793222110377
Author(s):  
K Harini ◽  
S Jayashree ◽  
Vikas Tiwari ◽  
Sneha Vishwanath ◽  
Ramanathan Sowdhamini
Keyword(s):  
Binding Site ◽  
G Protein ◽  
Drug Targets ◽  
Drug Binding ◽  
G Protein Coupled Receptors ◽  
Ligand Docking ◽  
Amino Acid Residues ◽  
Computational Docking ◽  
Cellular Processes ◽  
G Protein Coupled

G-protein-coupled receptors (GPCRs) are membrane proteins which play an important role in many cellular processes and are excellent drug targets. Despite the existence of several US Food and Drug Administration (FDA)-approved GPCR-targeting drugs, there is a continuing challenge of side effects owing to the nonspecific nature of drug binding. We have investigated the diversity of the ligand binding site for this class of proteins against their cognate ligands using computational docking, even if their structures are known already in the ligand-complexed form. The cognate ligand of some of these receptors dock at allosteric binding site with better score than the binding at the conservative site. Interestingly, amino acid residues at such allosteric binding site are not conserved across GPCR subfamilies. Such a computational approach can assist in the prediction of specific allosteric binders for GPCRs.

scholarly journals Dynamics of human protein kinases linked to drug selectivity

2018 ◽  
Author(s):  
Warintra Pitsawong ◽  
Vanessa Buosi ◽  
Renee Otten ◽  
Roman V. Agafonov ◽  
Adelajda Zorba ◽  
...  
Keyword(s):  
Protein Dynamics ◽  
Protein Kinases ◽  
Conformational Changes ◽  
Drug Targets ◽  
Active Sites ◽  
Kinase Inhibitors ◽  
Drug Binding ◽  
Inhibition Mechanism ◽  
Aurora A ◽  
Drug Selectivity

AbstractProtein kinases are major drug targets, but the development of highly-selective inhibitors has been challenging due to the similarity of their active sites. The observation of distinct structural states of the fully-conserved Asp-Phe-Gly (DFG) loop has put the concept of conformational selection for the DFG-state at the center of kinase drug discovery. Recently, it was shown that Gleevec selectivity for the Tyr-kinases Abl was instead rooted in conformational changes after drug binding. Here, we investigate whether protein dynamics after binding is a more general paradigm for drug selectivity by characterizing the binding of several approved drugs to the Ser/Thr-kinase Aurora A. Using a combination of biophysical techniques, we propose a universal drug-binding mechanism, that rationalizes selectivity, affinity and long on-target residence time for kinase inhibitors. These new concepts, where protein dynamics in the drug-bound state plays the crucial role, can be applied to inhibitor design of targets outside the kinome.eLife digestThe Ser/Thr kinase Aurora A is an important target for the development of new anticancer therapies. A longstanding question is how to specifically and effectively inhibit only this kinase in a background of over 550 protein kinases with very similar structures. To this end, understanding the inhibition mechanism of Aurora A by different drugs is essential. Here, we characterize the kinetic mechanism of three distinct kinase drugs, Gleevec (Imatinib), Danusertib (PHA739358) and AT9283 (Pyrazol-4-yl Urea) for Aurora A. We show that inhibitor affinities do not rely exclusively on the recognition of a specific conformation of the Asp-Phe-Gly loop of the kinase. Our quantitative kinetics data put forward an opposing mechanism in which a slow conformational change after drug binding (i.e., induced-fit step) dictates drug affinity.

scholarly journals A Periplasmic Drug-Binding Site of the AcrB Multidrug Efflux Pump: a Crystallographic and Site-Directed Mutagenesis Study

2005 ◽  
Vol 187 (19) ◽  
pp. 6804-6815 ◽  
Author(s):  
Edward W. Yu ◽  
Julio R. Aires ◽  
Gerry McDermott ◽  
Hiroshi Nikaido
Keyword(s):  
Binding Site ◽  
Transmembrane Domain ◽  
Efflux Pump ◽  
Drug Binding ◽  
Site Directed Mutagenesis ◽  
Multidrug Efflux ◽  
Multidrug Efflux Pump ◽  
Central Cavity ◽  
Intact Cells ◽  
Drug Binding Site

ABSTRACT The Escherichia coli AcrB multidrug efflux pump is a membrane protein that recognizes many structurally dissimilar toxic compounds. We previously reported the X-ray structures of four AcrB-ligand complexes in which the ligands were bound to the wall of the extremely large central cavity in the transmembrane domain of the pump. Genetic studies, however, suggested that discrimination between the substrates occurs mainly in the periplasmic domain rather than the transmembrane domain of the pump. We here describe the crystal structures of the AcrB mutant in which Asn109 was replaced by Ala, with five structurally diverse ligands, ethidium, rhodamine 6G, ciprofloxacin, nafcillin, and Phe-Arg-β-naphthylamide. The ligands bind not only to the wall of central cavity but also to a new periplasmic site within the deep external depression formed by the C-terminal periplasmic loop. This depression also includes residues identified earlier as being important in the specificity. We show here that conversion into alanine of the Phe664, Phe666, or Glu673 residue in the periplasmic binding site produced significant decreases in the MIC of most agents in the N109A background. Furthermore, decreased MICs were also observed when these residues were mutated in the wild-type AcrB background, although the effects were more modest. The MIC data were also confirmed by assays of ethidium influx rates in intact cells, and our results suggest that the periplasmic binding site plays a role in the physiological process of drug efflux.

scholarly journals DNA G-quadruplexes for native mass spectrometry in potassium: a database of validated structures in electrospray-compatible conditions

2021 ◽  
Author(s):  
Anirban Ghosh ◽  
Eric Largy ◽  
Valérie Gabelica
Keyword(s):  
Mass Spectrometry ◽  
Drug Targets ◽  
Native Mass Spectrometry ◽  
Drug Binding ◽  
Quadruplex Dna ◽  
Dna Structures ◽  
Nmr Structures ◽  
G Quadruplex ◽  
Screening Assays

Abstract G-quadruplex DNA structures have become attractive drug targets, and native mass spectrometry can provide detailed characterization of drug binding stoichiometry and affinity, potentially at high throughput. However, the G-quadruplex DNA polymorphism poses problems for interpreting ligand screening assays. In order to establish standardized MS-based screening assays, we studied 28 sequences with documented NMR structures in (usually ∼100 mM) potassium, and report here their circular dichroism (CD), melting temperature (Tm), NMR spectra and electrospray mass spectra in 1 mM KCl/100 mM trimethylammonium acetate. Based on these results, we make a short-list of sequences that adopt the same structure in the MS assay as reported by NMR, and provide recommendations on using them for MS-based assays. We also built an R-based open-source application to build and consult a database, wherein further sequences can be incorporated in the future. The application handles automatically most of the data processing, and allows generating custom figures and reports. The database is included in the g4dbr package (https://github.com/EricLarG4/g4dbr) and can be explored online (https://ericlarg4.github.io/G4_database.html).

scholarly journals Function Trumps Form in Two Sugar Symporters, LacY and vSGLT

2021 ◽  
Vol 22 (7) ◽  
pp. 3572
Author(s):  
Jeff Abramson ◽  
Ernest M. Wright
Keyword(s):  
Binding Site ◽  
Sugar Transport ◽  
Electrochemical Potential ◽  
Inverted Repeats ◽  
Side Chains ◽  
Transmembrane Helices ◽  
Central Cavity ◽  
Potential Gradients ◽  
Sodium Binding ◽  
Major Facilitator

Active transport of sugars into bacteria occurs through symporters driven by ion gradients. LacY is the most well-studied proton sugar symporter, whereas vSGLT is the most characterized sodium sugar symporter. These are members of the major facilitator (MFS) and the amino acid-Polyamine organocation (APS) transporter superfamilies. While there is no structural homology between these transporters, they operate by a similar mechanism. They are nano-machines driven by their respective ion electrochemical potential gradients across the membrane. LacY has 12 transmembrane helices (TMs) organized in two 6-TM bundles, each containing two 3-helix TM repeats. vSGLT has a core structure of 10 TM helices organized in two inverted repeats (TM 1–5 and TM 6–10). In each case, a single sugar is bound in a central cavity and sugar selectivity is determined by hydrogen- and hydrophobic- bonding with side chains in the binding site. In vSGLT, the sodium-binding site is formed through coordination with carbonyl- and hydroxyl-oxygens from neighboring side chains, whereas in LacY the proton (H3O+) site is thought to be a single glutamate residue (Glu325). The remaining challenge for both transporters is to determine how ion electrochemical potential gradients drive uphill sugar transport.

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