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1.
Proc Natl Acad Sci U S A ; 119(13): e2109431119, 2022 03 29.
Article in English | MEDLINE | ID: mdl-35333652

ABSTRACT

SignificanceCholesterol is one of the main components found in plasma membranes and is involved in lipid-dependent signaling enabled by integral membrane proteins such as inwardly rectifying potassium (Kir) channels. Similar to other ion channels, most of the Kir channels are down-regulated by cholesterol. One of the very few notable exceptions is Kir3.4, which is up-regulated by this important lipid. Here, we discovered and characterized a molecular switch that controls the impact (up-regulation vs. down-regulation) of cholesterol on Kir3.4. Our results provide a detailed molecular mechanism of tunable cholesterol regulation of a potassium channel.


Subject(s)
Cholesterol , G Protein-Coupled Inwardly-Rectifying Potassium Channels , Cell Membrane/metabolism , Cholesterol/metabolism , G Protein-Coupled Inwardly-Rectifying Potassium Channels/genetics , G Protein-Coupled Inwardly-Rectifying Potassium Channels/metabolism , Potassium/metabolism , Signal Transduction
2.
Circ Res ; 126(8): 947-964, 2020 04 10.
Article in English | MEDLINE | ID: mdl-32091972

ABSTRACT

RATIONALE: Drug-induced proarrhythmia is so tightly associated with prolongation of the QT interval that QT prolongation is an accepted surrogate marker for arrhythmia. But QT interval is too sensitive a marker and not selective, resulting in many useful drugs eliminated in drug discovery. OBJECTIVE: To predict the impact of a drug from the drug chemistry on the cardiac rhythm. METHODS AND RESULTS: In a new linkage, we connected atomistic scale information to protein, cell, and tissue scales by predicting drug-binding affinities and rates from simulation of ion channel and drug structure interactions and then used these values to model drug effects on the hERG channel. Model components were integrated into predictive models at the cell and tissue scales to expose fundamental arrhythmia vulnerability mechanisms and complex interactions underlying emergent behaviors. Human clinical data were used for model framework validation and showed excellent agreement, demonstrating feasibility of a new approach for cardiotoxicity prediction. CONCLUSIONS: We present a multiscale model framework to predict electrotoxicity in the heart from the atom to the rhythm. Novel mechanistic insights emerged at all scales of the system, from the specific nature of proarrhythmic drug interaction with the hERG channel, to the fundamental cellular and tissue-level arrhythmia mechanisms. Applications of machine learning indicate necessary and sufficient parameters that predict arrhythmia vulnerability. We expect that the model framework may be expanded to make an impact in drug discovery, drug safety screening for a variety of compounds and targets, and in a variety of regulatory processes.


Subject(s)
Anti-Arrhythmia Agents/chemistry , Arrhythmias, Cardiac/drug therapy , Cardiotoxins/chemistry , Computer Simulation , Drug Discovery/methods , ERG1 Potassium Channel/chemistry , Anti-Arrhythmia Agents/metabolism , Anti-Arrhythmia Agents/therapeutic use , Arrhythmias, Cardiac/metabolism , Cardiotoxicity/metabolism , Cardiotoxicity/prevention & control , Cardiotoxins/adverse effects , Cardiotoxins/metabolism , Drug Discovery/trends , ERG1 Potassium Channel/metabolism , Female , Humans , Long QT Syndrome/drug therapy , Long QT Syndrome/metabolism , Machine Learning , Male , Moxifloxacin/chemistry , Moxifloxacin/metabolism , Moxifloxacin/therapeutic use , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiology , Phenethylamines/chemistry , Phenethylamines/metabolism , Phenethylamines/therapeutic use , Protein Structure, Secondary , Sulfonamides/chemistry , Sulfonamides/metabolism , Sulfonamides/therapeutic use , Topoisomerase II Inhibitors/chemistry , Topoisomerase II Inhibitors/metabolism , Topoisomerase II Inhibitors/therapeutic use
3.
Biophys J ; 120(4): 738-748, 2021 02 16.
Article in English | MEDLINE | ID: mdl-33476597

ABSTRACT

The human-ether-a-go-go-related gene (hERG) encodes the voltage-gated potassium channel (KCNH2 or Kv11.1, commonly known as hERG). This channel plays a pivotal role in the stability of phase 3 repolarization of the cardiac action potential. Although a high-resolution cryo-EM structure is available for its depolarized (open) state, the structure surprisingly did not feature many functionally important interactions established by previous biochemical and electrophysiology experiments. Using molecular dynamics flexible fitting (MDFF), we refined the structure and recovered the missing functionally relevant salt bridges in hERG in its depolarized state. We also performed electrophysiology experiments to confirm the functional relevance of a novel salt bridge predicted by our refinement protocol. Our work shows how refinement of a high-resolution cryo-EM structure helps to bridge the existing gap between the structure and function in the voltage-sensing domain (VSD) of hERG.


Subject(s)
Ether-A-Go-Go Potassium Channels , Molecular Dynamics Simulation , Action Potentials , Cryoelectron Microscopy , ERG1 Potassium Channel/metabolism , Ether-A-Go-Go Potassium Channels/metabolism , Humans
4.
J Mol Cell Cardiol ; 158: 163-177, 2021 09.
Article in English | MEDLINE | ID: mdl-34062207

ABSTRACT

Drug isomers may differ in their proarrhythmia risk. An interesting example is the drug sotalol, an antiarrhythmic drug comprising d- and l- enantiomers that both block the hERG cardiac potassium channel and confer differing degrees of proarrhythmic risk. We developed a multi-scale in silico pipeline focusing on hERG channel - drug interactions and used it to probe and predict the mechanisms of pro-arrhythmia risks of the two enantiomers of sotalol. Molecular dynamics (MD) simulations predicted comparable hERG channel binding affinities for d- and l-sotalol, which were validated with electrophysiology experiments. MD derived thermodynamic and kinetic parameters were used to build multi-scale functional computational models of cardiac electrophysiology at the cell and tissue scales. Functional models were used to predict inactivated state binding affinities to recapitulate electrocardiogram (ECG) QT interval prolongation observed in clinical data. Our study demonstrates how modeling and simulation can be applied to predict drug effects from the atom to the rhythm for dl-sotalol and also increased proarrhythmia proclivity of d- vs. l-sotalol when accounting for stereospecific beta-adrenergic receptor blocking.


Subject(s)
Adrenergic beta-Antagonists/chemistry , Adrenergic beta-Antagonists/metabolism , Anti-Arrhythmia Agents/chemistry , Anti-Arrhythmia Agents/metabolism , Ether-A-Go-Go Potassium Channels/metabolism , Long QT Syndrome/metabolism , Potassium Channel Blockers/chemistry , Potassium Channel Blockers/metabolism , Signal Transduction/drug effects , Sotalol/chemistry , Sotalol/metabolism , Adrenergic beta-Antagonists/pharmacology , Anti-Arrhythmia Agents/pharmacology , Cryoelectron Microscopy/methods , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Ether-A-Go-Go Potassium Channels/chemistry , HEK293 Cells , Humans , Molecular Dynamics Simulation , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/metabolism , Potassium Channel Blockers/pharmacology , Protein Binding/drug effects , Sotalol/pharmacology , Stereoisomerism
5.
J Chem Inf Model ; 61(9): 4266-4279, 2021 09 27.
Article in English | MEDLINE | ID: mdl-34420294

ABSTRACT

Psychostimulant drugs, such as cocaine, inhibit dopamine reuptake via blockading the dopamine transporter (DAT), which is the primary mechanism underpinning their abuse. Atypical DAT inhibitors are dissimilar to cocaine and can block cocaine- or methamphetamine-induced behaviors, supporting their development as part of a treatment regimen for psychostimulant use disorders. When developing these atypical DAT inhibitors as medications, it is necessary to avoid off-target binding that can produce unwanted side effects or toxicities. In particular, the blockade of a potassium channel, human ether-a-go-go (hERG), can lead to potentially lethal ventricular tachycardia. In this study, we established a counter screening platform for DAT and against hERG binding by combining machine learning-based quantitative structure-activity relationship (QSAR) modeling, experimental validation, and molecular modeling and simulations. Our results show that the available data are adequate to establish robust QSAR models, as validated by chemical synthesis and pharmacological evaluation of a validation set of DAT inhibitors. Furthermore, the QSAR models based on subsets of the data according to experimental approaches used have predictive power as well, which opens the door to target specific functional states of a protein. Complementarily, our molecular modeling and simulations identified the structural elements responsible for a pair of DAT inhibitors having opposite binding affinity trends at DAT and hERG, which can be leveraged for rational optimization of lead atypical DAT inhibitors with desired pharmacological properties.


Subject(s)
Cocaine , Dopamine Plasma Membrane Transport Proteins , Ether , Humans , Machine Learning , Models, Molecular
6.
Proc Natl Acad Sci U S A ; 114(18): E3622-E3631, 2017 05 02.
Article in English | MEDLINE | ID: mdl-28420794

ABSTRACT

Dimeric tubulin, an abundant water-soluble cytosolic protein known primarily for its role in the cytoskeleton, is routinely found to be associated with mitochondrial outer membranes, although the structure and physiological role of mitochondria-bound tubulin are still unknown. There is also no consensus on whether tubulin is a peripheral membrane protein or is integrated into the outer mitochondrial membrane. Here the results of five independent techniques-surface plasmon resonance, electrochemical impedance spectroscopy, bilayer overtone analysis, neutron reflectometry, and molecular dynamics simulations-suggest that α-tubulin's amphipathic helix H10 is responsible for peripheral binding of dimeric tubulin to biomimetic "mitochondrial" membranes in a manner that differentiates between the two primary lipid headgroups found in mitochondrial membranes, phosphatidylethanolamine and phosphatidylcholine. The identification of the tubulin dimer orientation and membrane-binding domain represents an essential step toward our understanding of the complex mechanisms by which tubulin interacts with integral proteins of the mitochondrial outer membrane and is important for the structure-inspired design of tubulin-targeting agents.


Subject(s)
Biomimetic Materials/chemistry , Mitochondrial Membranes/chemistry , Tubulin/chemistry , Animals , Biomimetic Materials/metabolism , Cattle , Mitochondrial Membranes/metabolism , Protein Binding , Protein Domains , Tubulin/metabolism
7.
Proc Natl Acad Sci U S A ; 113(40): E5962-E5971, 2016 10 04.
Article in English | MEDLINE | ID: mdl-27647906

ABSTRACT

Voltage-gated proton (Hv1) channels are involved in many physiological processes, such as pH homeostasis and the innate immune response. Zn2+ is an important physiological inhibitor of Hv1. Sperm cells are quiescent in the male reproductive system due to Zn2+ inhibition of Hv1 channels, but become active once introduced into the low-Zn2+-concentration environment of the female reproductive tract. How Zn2+ inhibits Hv1 is not completely understood. In this study, we use the voltage clamp fluorometry technique to identify the molecular mechanism of Zn2+ inhibition of Hv1. We find that Zn2+ binds to both the activated closed and resting closed states of the Hv1 channel, thereby inhibiting both voltage sensor motion and gate opening. Mutations of some Hv1 residues affect only Zn2+ inhibition of the voltage sensor motion, whereas mutations of other residues also affect Zn2+ inhibition of gate opening. These effects are similar in monomeric and dimeric Hv1 channels, suggesting that the Zn2+-binding sites are localized within each subunit of the dimeric Hv1. We propose that Zn2+ binding has two major effects on Hv1: (i) at low concentrations, Zn2+ binds to one site and prevents the opening conformational change of the pore of Hv1, thereby inhibiting proton conduction; and (ii) at high concentrations, Zn2+, in addition, binds to a second site and inhibits the outward movement of the voltage sensor of Hv1. Elucidating the molecular mechanism of how Zn2+ inhibits Hv1 will further our understanding of Hv1 function and might provide valuable information for future drug development for Hv1 channels.


Subject(s)
Ion Channel Gating/genetics , Ion Channels/genetics , Zinc/metabolism , Animals , Binding Sites , Female , Fluorometry/methods , Humans , Hydrogen-Ion Concentration , Immunity, Innate/genetics , Ion Channels/metabolism , Mutation , Patch-Clamp Techniques/methods , Protons , Xenopus laevis/metabolism , Zinc/chemistry
8.
Biophys J ; 112(8): 1645-1653, 2017 Apr 25.
Article in English | MEDLINE | ID: mdl-28445755

ABSTRACT

Ryanodine (Ryd) irreversibly targets ryanodine receptors (RyRs), a family of intracellular calcium release channels essential for many cellular processes ranging from muscle contraction to learning and memory. Little is known of the atomistic details about how Ryd binds to RyRs. In this study, we used all-atom molecular dynamics simulations with both enhanced and bidirectional sampling to gain direct insights into how Ryd interacts with major residues in RyRs that were experimentally determined to be critical for its binding. We found that the pyrrolic ring of Ryd displays preference for the R4892AGGG-F4921 residues in the cavity of RyR1, which explain the effects of the corresponding mutations in RyR2 in experiments. Particularly, the mutant Q4933A (or Q4863A in RyR2) critical for both the gating and Ryd binding not only has significantly less interaction with Ryd than the wild-type, but also yields more space for Ryd and water molecules in the cavity. These results describe clear binding modes of Ryd in the RyR cavity and offer structural mechanisms explaining functional data collected on RyR blockade.


Subject(s)
Ryanodine Receptor Calcium Release Channel/metabolism , Ryanodine/metabolism , Animals , Binding Sites , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Protein Structure, Secondary , Ryanodine/chemistry , Ryanodine Receptor Calcium Release Channel/chemistry , Ryanodine Receptor Calcium Release Channel/genetics , Thermodynamics , Water/chemistry
9.
J Physiol ; 595(14): 4695-4723, 2017 07 15.
Article in English | MEDLINE | ID: mdl-28516454

ABSTRACT

KEY POINTS: This study represents a first step toward predicting mechanisms of sex-based arrhythmias that may lead to important developments in risk stratification and may inform future drug design and screening. We undertook simulations to reveal the conditions (i.e. pacing, drugs, sympathetic stimulation) required for triggering and sustaining reentrant arrhythmias. Using the recently solved cryo-EM structure for the Eag-family channel as a template, we revealed potential interactions of oestrogen with the pore loop hERG mutation (G604S). Molecular models suggest that oestrogen and dofetilide blockade can concur simultaneously in the hERG channel pore. ABSTRACT: Female sex is a risk factor for inherited and acquired long-QT associated torsade de pointes (TdP) arrhythmias, and sympathetic discharge is a major factor in triggering TdP in female long-QT syndrome patients. We used a combined experimental and computational approach to predict 'the perfect storm' of hormone concentration, IKr block and sympathetic stimulation that induces arrhythmia in females with inherited and acquired long-QT. More specifically, we developed mathematical models of acquired and inherited long-QT syndrome in male and female ventricular human myocytes by combining effects of a hormone and a hERG blocker, dofetilide, or hERG mutations. These 'male' and 'female' model myocytes and tissues then were used to predict how various sex-based differences underlie arrhythmia risk in the setting of acute sympathetic nervous system discharge. The model predicted increased risk for arrhythmia in females when acute sympathetic nervous system discharge was applied in the settings of both inherited and acquired long-QT syndrome. Females were predicted to have protection from arrhythmia induction when progesterone is high. Males were protected by the presence of testosterone. Structural modelling points towards two plausible and distinct mechanisms of oestrogen action enhancing torsadogenic effects: oestradiol interaction with hERG mutations in the pore loop containing G604 or with common TdP-related blockers in the intra-cavity binding site. Our study presents findings that constitute the first evidence linking structure to function mechanisms underlying female dominance of arousal-induced arrhythmias.


Subject(s)
Arousal/physiology , Arrhythmias, Cardiac/physiopathology , Models, Biological , Adrenergic beta-Agonists/pharmacology , Animals , Anti-Arrhythmia Agents/pharmacology , Estradiol/pharmacology , Ether-A-Go-Go Potassium Channels/physiology , Female , Guinea Pigs , Isoproterenol/pharmacology , Male , Molecular Docking Simulation , Myocytes, Cardiac/physiology , Phenethylamines/pharmacology , Sex Characteristics , Sulfonamides/pharmacology
10.
PLoS Comput Biol ; 12(1): e1004482, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26727271

ABSTRACT

Crystal structures of several bacterial Na(v) channels have been recently published and molecular dynamics simulations of ion permeation through these channels are consistent with many electrophysiological properties of eukaryotic channels. Bacterial Na(v) channels have been characterized as functionally asymmetric, and the mechanism of this asymmetry has not been clearly understood. To address this question, we combined non-equilibrium simulation data with two-dimensional equilibrium unperturbed landscapes generated by umbrella sampling and Weighted Histogram Analysis Methods for multiple ions traversing the selectivity filter of bacterial Na(v)Ab channel. This approach provided new insight into the mechanism of selective ion permeation in bacterial Na(v) channels. The non-equilibrium simulations indicate that two or three extracellular K+ ions can block the entrance to the selectivity filter of Na(v)Ab in the presence of applied forces in the inward direction, but not in the outward direction. The block state occurs in an unstable local minimum of the equilibrium unperturbed free-energy landscape of two K+ ions that can be 'locked' in place by modest applied forces. In contrast to K+, three Na+ ions move favorably through the selectivity filter together as a unit in a loose "knock-on" mechanism of permeation in both inward and outward directions, and there is no similar local minimum in the two-dimensional free-energy landscape of two Na+ ions for a block state. The useful work predicted by the non-equilibrium simulations that is required to break the K+ block is equivalent to large applied potentials experimentally measured for two bacterial Na(v) channels to induce inward currents of K+ ions. These results illustrate how inclusion of non-equilibrium factors in the simulations can provide detailed information about mechanisms of ion selectivity that is missing from mechanisms derived from either crystal structures or equilibrium unperturbed free-energy landscapes.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Potassium/chemistry , Potassium/metabolism , Voltage-Gated Sodium Channels/chemistry , Voltage-Gated Sodium Channels/metabolism , Computational Biology , Computer Simulation , Models, Molecular , Thermodynamics
11.
Proc Natl Acad Sci U S A ; 111(2): E273-82, 2014 Jan 14.
Article in English | MEDLINE | ID: mdl-24379371

ABSTRACT

Voltage-gated proton (Hv1) channels play important roles in the respiratory burst, in pH regulation, in spermatozoa, in apoptosis, and in cancer metastasis. Unlike other voltage-gated cation channels, the Hv1 channel lacks a centrally located pore formed by the assembly of subunits. Instead, the proton permeation pathway in the Hv1 channel is within the voltage-sensing domain of each subunit. The gating mechanism of this pathway is still unclear. Mutagenic and fluorescence studies suggest that the fourth transmembrane (TM) segment (S4) functions as a voltage sensor and that there is an outward movement of S4 during channel activation. Using thermodynamic mutant cycle analysis, we find that the conserved positively charged residues in S4 are stabilized by countercharges in the other TM segments both in the closed and open states. We constructed models of both the closed and open states of Hv1 channels that are consistent with the mutant cycle analysis. These structural models suggest that electrostatic interactions between TM segments in the closed state pull hydrophobic residues together to form a hydrophobic plug in the center of the voltage-sensing domain. Outward S4 movement during channel activation induces conformational changes that remove this hydrophobic plug and instead insert protonatable residues in the center of the channel that, together with water molecules, can form a hydrogen bond chain across the channel for proton permeation. This suggests that salt bridge networks and the hydrophobic plug function as the gate in Hv1 channels and that outward movement of S4 leads to the opening of this gate.


Subject(s)
Ion Channel Gating/physiology , Ion Channels/chemistry , Models, Molecular , Protein Conformation , Base Sequence , Hydrophobic and Hydrophilic Interactions , Ion Channels/metabolism , Molecular Dynamics Simulation , Molecular Sequence Data , Mutagenesis, Site-Directed , Patch-Clamp Techniques , Protein Binding , Sequence Analysis, DNA , Thermodynamics
12.
J Physiol ; 594(3): 567-93, 2016 Feb 01.
Article in English | MEDLINE | ID: mdl-26515697

ABSTRACT

KEY POINTS: The mechanism of therapeutic efficacy of flecainide for catecholaminergic polymorphic ventricular tachycardia (CPVT) is unclear. Model predictions suggest that Na(+) channel effects are insufficient to explain flecainide efficacy in CPVT. This study represents a first step toward predicting therapeutic mechanisms of drug efficacy in the setting of CPVT and then using these mechanisms to guide modelling and simulation to predict alternative drug therapies. Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmia syndrome characterized by fatal ventricular arrhythmias in structurally normal hearts during ß-adrenergic stimulation. Current treatment strategies include ß-blockade, flecainide and ICD implementation--none of which is fully effective and each comes with associated risk. Recently, flecainide has gained considerable interest in CPVT treatment, but its mechanism of action for therapeutic efficacy is unclear. In this study, we performed in silico mutagenesis to construct a CPVT model and then used a computational modelling and simulation approach to make predictions of drug mechanisms and efficacy in the setting of CPVT. Experiments were carried out to validate model results. Our simulations revealed that Na(+) channel effects are insufficient to explain flecainide efficacy in CPVT. The pure Na(+) channel blocker lidocaine and the antianginal ranolazine were additionally tested and also found to be ineffective. When we tested lower dose combination therapy with flecainide, ß-blockade and CaMKII inhibition, our model predicted superior therapeutic efficacy than with flecainide monotherapy. Simulations indicate a polytherapeutic approach may mitigate side-effects and proarrhythmic potential plaguing CPVT pharmacological management today. Importantly, our prediction of a novel polytherapy for CPVT was confirmed experimentally. Our simulations suggest that flecainide therapeutic efficacy in CPVT is unlikely to derive from primary interactions with the Na(+) channel, and benefit may be gained from an alternative multi-drug regimen.


Subject(s)
Anti-Arrhythmia Agents/pharmacology , Flecainide/pharmacology , Models, Cardiovascular , Tachycardia, Ventricular/physiopathology , Animals , Animals, Genetically Modified , Anti-Arrhythmia Agents/therapeutic use , Electrocardiography , Flecainide/therapeutic use , Mice , Rabbits , Ryanodine Receptor Calcium Release Channel/physiology , Sodium Channels/physiology , Tachycardia, Ventricular/drug therapy
13.
Mol Cell ; 30(4): 472-85, 2008 May 23.
Article in English | MEDLINE | ID: mdl-18424209

ABSTRACT

Group II introns are self-splicing ribozymes believed to be the ancestors of spliceosomal introns. Many group II introns encode reverse transcriptases that promote both RNA splicing and intron mobility to new genomic sites. Here we used a circular permutation and crosslinking method to establish 16 intramolecular distance relationships within the mobile Lactococcus lactis Ll.LtrB-DeltaORF intron. Using these new constraints together with 13 established tertiary interactions and eight published crosslinks, we modeled a complete three-dimensional structure of the intron. We also used the circular permutation strategy to map RNA-protein interaction sites through fluorescence quenching and crosslinking assays. Our model provides a comprehensive structural framework for understanding the function of group II ribozymes, their natural structural variations, and the mechanisms by which the intron-encoded protein promotes RNA splicing and intron mobility. The model also suggests an arrangement of active site elements that may be conserved in the spliceosome.


Subject(s)
Bacterial Proteins , Introns/genetics , Models, Molecular , Nucleic Acid Conformation , RNA-Directed DNA Polymerase , RNA , Amino Acid Sequence , Bacterial Proteins/chemistry , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Binding Sites , Cross-Linking Reagents/metabolism , Molecular Sequence Data , Phylogeny , Protein Conformation , RNA/chemistry , RNA/genetics , RNA Splicing , RNA, Catalytic/chemistry , RNA, Catalytic/genetics , RNA, Catalytic/metabolism , RNA-Directed DNA Polymerase/chemistry , RNA-Directed DNA Polymerase/genetics , RNA-Directed DNA Polymerase/metabolism
14.
J Enzyme Inhib Med Chem ; 31(1): 112-20, 2016.
Article in English | MEDLINE | ID: mdl-26083304

ABSTRACT

The rational design of high-affinity inhibitors of poly-ADP-ribose polymerase-1 (PARP-1) is at the heart of modern anti-cancer drug design. While relevance of enzyme to DNA repair processes in cellular environment is firmly established, the structural and functional understanding of the main determinants for high-affinity ligands controlling PARP-1 activity is still lacking. The conserved active site of PARP-1 represents an ideal target for inhibitors and may offer a novel target at the treatment of breast cancer. To fill the gap in the structural knowledge, we report on the combination of molecular dynamics (MD) simulations, principal component analysis (PCA), and conformational analysis that analyzes in great details novel binding mode for a number of inhibitors at the PARP-1. While optimization of the binding affinity for original target is an important goal in the drug design, many of the promising molecules for treatment of the breast cancer are plagued by significant cardiotoxicity. One of the most common side-effects reported for a number of polymerase inhibitors is its off-target interactions with cardiac ion channels and hERG1 channel, in particular. Thus, selected candidate PARP-1 inhibitors were also screened in silico at the central cavities of hERG1 potassium ion channel.


Subject(s)
Apoenzymes/chemistry , Catalytic Domain , Drug Design , Holoenzymes/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/chemistry , Poly(ADP-ribose) Polymerase Inhibitors/pharmacology , Poly(ADP-ribose) Polymerases/chemistry , Poly(ADP-ribose) Polymerases/metabolism , Apoenzymes/metabolism , Biocatalysis , Computer Simulation , Dose-Response Relationship, Drug , Holoenzymes/metabolism , Humans , Molecular Dynamics Simulation , Molecular Structure , Poly (ADP-Ribose) Polymerase-1 , Poly(ADP-ribose) Polymerase Inhibitors/chemical synthesis , Structure-Activity Relationship
15.
Biophys J ; 109(6): 1163-8, 2015 Sep 15.
Article in English | MEDLINE | ID: mdl-26340817

ABSTRACT

PDEδ is a small protein that binds and controls the trafficking of RAS subfamily proteins. Its inhibition protects initiation of RAS signaling, and it is one of the common targets considered for oncological drug development. In this study, we used solved x-ray structures of inhibitor-bound PDEδ targets to investigate mechanisms of action of six independent all-atom MD simulations. An analysis of atomic simulations combined with the molecular mechanic-Poisson-Boltzmann solvent accessible surface area/generalized Born solvent accessible surface area calculations led to the identification of action mechanisms for a panel of novel PDEδ inhibitors. To the best of our knowledge, this study is one of the first in silico investigations on co-crystallized PDEδ protein. A detailed atomic-scale understanding of the molecular mechanism of PDEδ inhibition may assist in the design of novel PDEδ inhibitors. One of the most common side effects for diverse small molecules/kinase inhibitors is their off-target interactions with cardiac ion channels and human-ether-a-go-go channel specifically. Thus, all of the studied PDEδ inhibitors are also screened in silico at the central cavities of hERG1 potassium channels.


Subject(s)
Cyclic Nucleotide Phosphodiesterases, Type 6/antagonists & inhibitors , Phosphodiesterase Inhibitors/pharmacology , Binding Sites , Cyclic Nucleotide Phosphodiesterases, Type 6/chemistry , Cyclic Nucleotide Phosphodiesterases, Type 6/metabolism , Hydrogen Bonding , Molecular Docking Simulation , Molecular Dynamics Simulation , Molecular Structure , Phosphodiesterase Inhibitors/adverse effects , Phosphodiesterase Inhibitors/chemistry , Water/chemistry
16.
Biophys J ; 108(6): 1414-1424, 2015 Mar 24.
Article in English | MEDLINE | ID: mdl-25809254

ABSTRACT

Congenital and acquired (drug-induced) forms of the human long-QT syndrome are associated with alterations in Kv11.1 (hERG) channel-controlled repolarizing IKr currents of cardiac action potentials. A mandatory drug screen implemented by many countries led to a discovery of a large group of small molecules that can activate hERG currents and thus may act as potent antiarrhythmic agents. Despite significant progress in identification of channel activators, little is known about their mechanism of action. A combination of electrophysiological studies with molecular and kinetic modeling was used to examine the mechanism of a model activator (NS1643) action on the hERG channel and its L529I mutant. The L529I mutant has gating dynamics similar to that of wild-type while its response to application of NS1643 is markedly different. We propose a mechanism compatible with experiments in which the model activator binds to the closed (C3) and open states (O). We suggest that NS1643 is affecting early gating transitions, probably during movements of the voltage sensor that precede the opening of the activation gate.


Subject(s)
Cresols/pharmacology , Ether-A-Go-Go Potassium Channels/metabolism , Membrane Transport Modulators/pharmacology , Models, Molecular , Phenylurea Compounds/pharmacology , Cell Line , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/genetics , Humans , Kinetics , Membrane Potentials/drug effects , Membrane Potentials/physiology , Mutation , Patch-Clamp Techniques , Transfection
17.
Biophys J ; 108(6): 1400-1413, 2015 Mar 24.
Article in English | MEDLINE | ID: mdl-25809253

ABSTRACT

Activators of hERG1 such as NS1643 are being developed for congenital/acquired long QT syndrome. Previous studies identify the neighborhood of L529 around the voltage-sensor as a putative interacting site for NS1643. With NS1643, the V1/2 of activation of L529I (-34 ± 4 mV) is similar to wild-type (WT) (-37 ± 3 mV; P > 0.05). WT and L529I showed no difference in the slope factor in the absence of NS1643 (8 ± 0 vs. 9 ± 0) but showed a difference in the presence of NS1643 (9 ± 0.3 vs. 22 ± 1; P < 0.01). Voltage-clamp-fluorimetry studies also indicated that in L529I, NS1643 reduces the voltage-sensitivity of S4 movement. To further assess mechanism of NS1643 action, mutations were made in this neighborhood. NS1643 shifts the V1/2 of activation of both K525C and K525C/L529I to hyperpolarized potentials (-131 ± 4 mV for K525C and -120 ± 21 mV for K525C/L529I). Both K525C and K525C/K529I had similar slope factors in the absence of NS1643 (18 ± 2 vs. 34 ± 5, respectively) but with NS1643, the slope factor of K525C/L529I increased from 34 ± 5 to 71 ± 10 (P < 0.01) whereas for K525C the slope factor did not change (18 ± 2 at baseline and 16 ± 2 for NS1643). At baseline, K525R had a slope factor similar to WT (9 vs. 8) but in the presence of NS1643, the slope factor of K525R was increased to 24 ± 4 vs. 9 ± 0 mV for WT (P < 0.01). Molecular modeling indicates that L529I induces a kink in the S4 voltage-sensor helix, altering a salt-bridge involving K525. Moreover, docking studies indicate that NS1643 binds to the kinked structure induced by the mutation with a higher affinity. Combining biophysical, computational, and electrophysiological evidence, a mechanistic principle governing the action of some activators of hERG1 channels is proposed.


Subject(s)
Cresols/metabolism , Cresols/pharmacology , Ether-A-Go-Go Potassium Channels/metabolism , Phenylurea Compounds/metabolism , Phenylurea Compounds/pharmacology , Animals , Binding Sites , Cell Line , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/genetics , Fluorometry , Humans , Membrane Potentials/physiology , Molecular Docking Simulation , Molecular Dynamics Simulation , Mutation , Oocytes , Patch-Clamp Techniques , Protein Structure, Secondary , Transfection , Xenopus laevis
18.
J Mol Cell Cardiol ; 85: 71-8, 2015 Aug.
Article in English | MEDLINE | ID: mdl-25986146

ABSTRACT

In Europe, ivabradine has recently been approved to treat patients with angina who have intolerance to beta blockers and/or heart failure. Ivabradine is considered to act specifically on the sinoatrial node by inhibiting the If current (the funny current) to slow automaticity. However, in vitro studies show that ivabradine prolongs phase 3 repolarization in ventricular tissue. No episodes of Torsades de Pointes have been reported in randomized clinical studies. The objective of this study is to assess whether ivabradine blocked the hERG1 current. In the present study we discovered that ivabradine prolongs action potential and blocks the hERG current over a range of concentrations overlapping with those required to block HCN4. Ivabradine produced tonic, rather than use-dependent block. The mutation Y652A significantly suppressed pharmacologic block of hERG by ivabradine. Disruption of C-type inactivation also suppressed block of hERG1 by ivabradine. Molecular docking and molecular dynamics simulations indicate that ivabradine may access the inner cavity of the hERG1 via a lipophilic route and has a well-defined binding site in the closed state of the channel. Structural organization of the binding pockets for ivabradine is discussed. Ivabradine blocks hERG and prolongs action potential duration. Our study is potentially important because it indicates the need for active post marketing surveillance of ivabradine. Importantly, proarrhythmia of a number of other drugs has only been discovered during post marketing surveillance.


Subject(s)
Benzazepines/pharmacology , Ether-A-Go-Go Potassium Channels/antagonists & inhibitors , Hyperpolarization-Activated Cyclic Nucleotide-Gated Channels/antagonists & inhibitors , Myocytes, Cardiac/physiology , Potassium Channel Blockers/pharmacology , Action Potentials , Animals , Benzazepines/chemistry , Binding Sites , Dose-Response Relationship, Drug , ERG1 Potassium Channel , Ether-A-Go-Go Potassium Channels/chemistry , Humans , Inhibitory Concentration 50 , Ivabradine , Lipid Bilayers/chemistry , Mice , Molecular Docking Simulation , Myocytes, Cardiac/drug effects , Potassium Channel Blockers/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary
19.
Biochemistry ; 54(4): 994-1005, 2015 Feb 03.
Article in English | MEDLINE | ID: mdl-25545271

ABSTRACT

The N-terminus of the voltage-dependent anion channel (VDAC) has been proposed to contain the mechanistically important gating helices that modulate channel opening and closing. In this study, we utilize magic angle spinning nuclear magnetic resonance (MAS NMR) to determine the location and structure of the N-terminus for functional channels in lipid bilayers by measuring long-range (13)C-(13)C distances between residues in the N-terminus and other domains of VDAC reconstituted into DMPC lipid bilayers. Our structural studies show that the distance between A14 Cß in the N-terminal helix and S193 Cß is ∼4-6 Å. Furthermore, VDAC phosphorylation by a mitochondrial kinase at residue S193 has been claimed to delay mitochondrial cell death by causing a conformational change that closes the channel, and a VDAC-Ser193Glu mutant has been reported to show properties very similar to those of phosphorylated VDAC in a cellular context. We expressed VDAC-S193E and reconstituted it into DMPC lipid bilayers. Two-dimensional (13)C-(13)C correlation experiments showed chemical shift perturbations for residues located in the N-terminus, indicating possible structural perturbations to that region. However, electrophysiological data recorded on VDAC-S193E showed that channel characteristics were identical to those of wild type samples, indicating that phosphorylation of S193 does not directly affect channel gating. The combination of NMR and electrophysiological results allows us to discuss the validity of proposed gating models.


Subject(s)
Lipid Bilayers/chemistry , Lipid Bilayers/metabolism , Magnetic Resonance Spectroscopy/methods , Voltage-Dependent Anion Channel 1/chemistry , Voltage-Dependent Anion Channel 1/metabolism , Crystallization , Humans , Ion Channel Gating/physiology , Protein Structure, Secondary , Protein Structure, Tertiary
20.
J Biol Chem ; 289(34): 23670-82, 2014 Aug 22.
Article in English | MEDLINE | ID: mdl-24962576

ABSTRACT

The voltage-dependent anion channel (VDAC) is the major pathway for ATP, ADP, and other respiratory substrates through the mitochondrial outer membrane, constituting a crucial point of mitochondrial metabolism regulation. VDAC is characterized by its ability to "gate" between an open and several "closed" states under applied voltage. In the early stages of tumorigenesis or during ischemia, partial or total absence of oxygen supply to cells results in cytosolic acidification. Motivated by these facts, we investigated the effects of pH variations on VDAC gating properties. We reconstituted VDAC into planar lipid membranes and found that acidification reversibly increases its voltage-dependent gating. Furthermore, both VDAC anion selectivity and single channel conductance increased with acidification, in agreement with the titration of the negatively charged VDAC residues at low pH values. Analysis of the pH dependences of the gating and open channel parameters yielded similar pKa values close to 4.0. We also found that the response of VDAC gating to acidification was highly asymmetric. The presumably cytosolic (cis) side of the channel was the most sensitive to acidification, whereas the mitochondrial intermembrane space (trans) side barely responded to pH changes. Molecular dynamic simulations suggested that stable salt bridges at the cis side, which are susceptible to disruption upon acidification, contribute to this asymmetry. The pronounced sensitivity of the cis side to pH variations found here in vitro might provide helpful insights into the regulatory role of VDAC in the protective effect of cytosolic acidification during ischemia in vivo.


Subject(s)
Acids/chemistry , Voltage-Dependent Anion Channels/physiology , Animals , Cytosol/metabolism , Hydrogen-Ion Concentration , Ion Channel Gating , Mitochondrial Membranes/metabolism , Molecular Dynamics Simulation , Rats
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