A molecular switch controls the impact of cholesterol on a Kir channel.

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.

Toward Reducing hERG Affinities for DAT Inhibitors with a Combined Machine Learning and Molecular Modeling Approach.

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.

Molecular determinants of pro-arrhythmia proclivity of d- and l-sotalol via a multi-scale modeling pipeline.

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.

Refinement of a cryo-EM structure of hERG: Bridging structure and function.

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.

Inter-species variation in monovalent anion substrate selectivity and inhibitor sensitivity in the sodium iodide symporter (NIS).

The sodium iodide symporter (NIS) transports iodide, which is necessary for thyroid hormone production. NIS also transports other monovalent anions such as tetrafluoroborate (BF4-), pertechnetate (TcO4-), and thiocyanate (SCN-), and is competitively inhibited by perchlorate (ClO4-). However, the mechanisms of substrate selectivity and inhibitor sensitivity are poorly understood. Here, a comparative approach was taken to determine whether naturally evolved NIS proteins exhibit variability in their substrate transport properties. The NIS proteins of thirteen animal species were initially assessed, and three species from environments with differing iodide availability, freshwater species Danio rerio (zebrafish), saltwater species Balaenoptera acutorostrata scammoni (minke whale), and non-aquatic mammalian species Homo sapiens (human) were studied in detail. NIS genes from each of these species were lentivirally transduced into HeLa cells, which were then characterized using radioisotope uptake assays, 125I- competitive substrate uptake assays, and kinetic assays. Homology models of human, minke whale and zebrafish NIS were used to evaluate sequence-dependent impact on the organization of Na+ and I- binding pockets. Whereas each of the three proteins that were analyzed in detail concentrated iodide to a similar degree, their sensitivity to perchlorate inhibition varied significantly: minke whale NIS was the least impacted by perchlorate inhibition (IC50 = 4.599 µM), zebrafish NIS was highly sensitive (IC50 = 0.081 µM), and human NIS showed intermediate sensitivity (IC50 = 1.566 µM). Further studies with fifteen additional substrates and inhibitors revealed similar patterns of iodide uptake inhibition, though the degree of 125I- uptake inhibition varied with each compound. Kinetic analysis revealed whale NIS had the lowest Km-I and the highest Vmax-I. Conversely, zebrafish NIS had the highest Km and lowest Vmax. Again, human NIS was intermediate. Molecular modeling revealed a high degree of conservation in the putative ion binding pockets of NIS proteins from different species, which suggests the residues responsible for the observed differences in substrate selectivity lie elsewhere in the protein. Ongoing studies are focusing on residues in the extracellular loops of NIS as determinants of anion specificity. These data demonstrate significant transport differences between the NIS proteins of different species, which may be influenced by the unique physiological needs of each organism. Our results also identify naturally-existing NIS proteins with significant variability in substrate transport kinetics and inhibitor sensitivity, which suggest that the affinity and selectivity of NIS for certain substrates can be altered for biotechnological and clinical applications. Further examination of interspecies differences may improve understanding of the substrate transport mechanism.

A Computational Pipeline to Predict Cardiotoxicity: From the Atom to the Rhythm.

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.

Lipid roles in hERG function and interactions with drugs.

Human-ether-a-go-go-related channel (hERG) is a voltage gated potassium channel (Kv11.1) abundantly expressed in heart and brain tissues. In addition to playing an important role in mediation of repolarizing K+ currents (IKr) in Action Potential (AP), hERG is notorious for its propensity to interact with various medications. The drug-induced block of K+ currents across hERG channel are strongly associated with dysrhythmic conditions collectively known as drug-induced long-QT-syndrome. The recent availability of the high-resolution Cryo-EM structures for the hERG channel has provided unique opportunity to resolve structural mechanisms involved into the process of voltage-gating of hERG channels, map various roles played by components of ventricular and neuronal membranes and then to connect it to cellular pathways through which diverse chemical compounds might be affecting function of the channel. Specifically, lipids and lipid derivatives such as polyunsaturated fatty acids (PUFAs), ceramides and steroids have been shown to directly interact with the lipid facing amino acids in various Kv channels including hERG. In this review, possible lipophilic pathways of hERG activators and blockers, together with the existence of fenestration windows and effects of PUFAs, ceramides and steroids are explored throughout different sections. Finally, the interplay between long QT inducing drugs and phospholipidosis is briefly discussed.

Determinants of Isoform-Specific Gating Kinetics of hERG1 Channel: Combined Experimental and Simulation Study.

IKr is the rapidly activating component of the delayed rectifier potassium current, the ion current largely responsible for the repolarization of the cardiac action potential. Inherited forms of long QT syndrome (LQTS) (Lees-Miller et al., 1997) in humans are linked to functional modifications in the Kv11.1 (hERG) ion channel and potentially life threatening arrhythmias. There is little doubt now that hERG-related component of IKr in the heart depends on the tetrameric (homo- or hetero-) channels formed by two alternatively processed isoforms of hERG, termed hERG1a and hERG1b. Isoform composition (hERG1a- vs. the b-isoform) has recently been reported to alter pharmacologic responses to some hERG blockers and was proposed to be an essential factor pre-disposing patients for drug-induced QT prolongation. Very little is known about the gating and pharmacological properties of two isoforms in heart membranes. For example, how gating mechanisms of the hERG1a channels differ from that of hERG1b is still unknown. The mechanisms by which hERG 1a/1b hetero-tetramers contribute to function in the heart, or what role hERG1b might play in disease are all questions to be answered. Structurally, the two isoforms differ only in the N-terminal region located in the cytoplasm: hERG1b is 340 residues shorter than hERG1a and the initial 36 residues of hERG1b are unique to this isoform. In this study, we combined electrophysiological measurements for HEK cells, kinetics and structural modeling to tease out the individual contributions of each isoform to Action Potential formation and then make predictions about the effects of having various mixture ratios of the two isoforms. By coupling electrophysiological data with computational kinetic modeling, two proposed mechanisms of hERG gating in two homo-tetramers were examined. Sets of data from various experimental stimulation protocols (HEK cells) were analyzed simultaneously and fitted to Markov-chain models (M-models). The minimization procedure presented here, allowed assessment of suitability of different Markov model topologies and the corresponding parameters that describe the channel kinetics. The kinetics modeling pointed to key differences in the gating kinetics that were linked to the full channel structure. Interactions between soluble domains and the transmembrane part of the channel appeared to be critical determinants of the gating kinetics. The structures of the full channel in the open and closed states were compared for the first time using the recent Cryo-EM resolved structure for full open hERG channel and an homology model for the closed state, based on the highly homolog EAG1 channel. Key potential interactions which emphasize the importance of electrostatic interactions between N-PAS cap, S4-S5, and C-linker are suggested based on the structural analysis. The derived kinetic parameters were later used in higher order models of cells and tissue to track down the effect of varying the ratios of hERG1a and hERG1b on cardiac action potentials and computed electrocardiograms. Simulations suggest that the recovery from inactivation of hERG1b may contribute to its physiologic role of this isoform in the action potential. Finally, the results presented here contribute to the growing body of evidence that hERG1b significantly affects the generation of the cardiac Ikr and plays an important role in cardiac electrophysiology. We highlight the importance of carefully revisiting the Markov models previously proposed in order to properly account for the relative abundance of the hERG1 a- and b- isoforms.

Digging into Lipid Membrane Permeation for Cardiac Ion Channel Blocker d-Sotalol with All-Atom Simulations.

Interactions of drug molecules with lipid membranes play crucial role in their accessibility of cellular targets and can be an important predictor of their therapeutic and safety profiles. Very little is known about spatial localization of various drugs in the lipid bilayers, their active form (ionization state) or translocation rates and therefore potency to bind to different sites in membrane proteins. All-atom molecular simulations may help to map drug partitioning kinetics and thermodynamics, thus providing in-depth assessment of drug lipophilicity. As a proof of principle, we evaluated extensively lipid membrane partitioning of d-sotalol, well-known blocker of a cardiac potassium channel Kv11.1 encoded by the hERG gene, with reported substantial proclivity for arrhythmogenesis. We developed the positively charged (cationic) and neutral d-sotalol models, compatible with the biomolecular CHARMM force field, and subjected them to all-atom molecular dynamics (MD) simulations of drug partitioning through hydrated lipid membranes, aiming to elucidate thermodynamics and kinetics of their translocation and thus putative propensities for hydrophobic and aqueous hERG access. We found that only a neutral form of d-sotalol accumulates in the membrane interior and can move across the bilayer within millisecond time scale, and can be relevant to a lipophilic channel access. The computed water-membrane partitioning coefficient for this form is in good agreement with experiment. There is a large energetic barrier for a cationic form of the drug, dominant in water, to cross the membrane, resulting in slow membrane translocation kinetics. However, this form of the drug can be important for an aqueous access pathway through the intracellular gate of hERG. This route will likely occur after a neutral form of a drug crosses the membrane and subsequently re-protonates. Our study serves to demonstrate a first step toward a framework for multi-scale in silico safety pharmacology, and identifies some of the challenges that lie therein.

First universal pharmacophore model for hERG1 K+ channel activators: acthER.

The intra-cavitary drug blockade of hERG1 channel has been extensively studied, both experimentally and theoretically. Structurally diverse ligands inadvertently block the hERG1 K+ channel currents lead to drug induced Long QT Syndrome (LQTS). Accordingly, designing either hERG1 channel openers or current activators, with the potential to target other binding pockets of the channel, has been introduced as a viable approach in modern anti-arrhythmia drug development. However, reports and investigations on the molecular mechanisms underlying activators binding to the hERG1 channel remain sparse and the overall molecular design principles are largely unknown. Most of the hERG1 activators were discovered during mandatory screening for hERG1 blockade. To fill this apparent deficit, the first universal pharmacophore model for hERG1 K+ channel activators was developed using PHASE. 3D structures of 18 hERG1 K+ channel activators and their corresponding measured binding affinity values were used in the development of pharmacophore models. These compounds spanned a range of structurally different chemotypes with moderate variation in binding affinity. A five sites AAHRR (A, hydrogen-bond accepting, H, hydrophobic, R, aromatic) pharmacophore model has shown reasonable high statistical results compared to the other developed more than 1000 hypotheses. This model was used to construct steric and electrostatic contour maps. The predictive power of the model was tested with 3 external test set compounds as true unknowns. Finally, the pharmacophore model was combined with the previously developed receptor-based model of hERG1 K+ channel to develop and screen novel activators. The results are quite striking and it suggests a greater future role for pharmacophore modeling and virtual drug screening simulations in deciphering complex patterns of molecular mechanisms of hERG1 channel openers at the target sites. The developed model is available upon request and it may serve as basis for the synthesis of novel therapeutic hERG1 activators.

A multiscale computational modelling approach predicts mechanisms of female sex risk in the setting of arousal-induced arrhythmias.

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.

Mapping Ryanodine Binding Sites in the Pore Cavity of Ryanodine Receptors.

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.

Structural features and lipid binding domain of tubulin on biomimetic mitochondrial membranes.

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.

Molecular mechanism of Zn2+ inhibition of a voltage-gated proton channel.

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.

K+ Block Is the Mechanism of Functional Asymmetry in Bacterial Na(v) Channels.

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.

Estimation of Potentials of Mean Force from Nonequilibrium Pulling Simulations Using Both Minh-Adib Estimator and Weighted Histogram Analysis Method.

Nonequilibrium pulling simulations have been a useful approach for investigating a variety of physical and biological problems. The major target in the simulations is to reconstruct reliable potentials of mean force (PMFs) or unperturbed free-energy profiles for quantitatively addressing both equilibrium mechanistic properties and contributions from nonequilibrium processes. While several current nonequilibrium methods were shown to be accurate in computing free-energy profiles in systems with relatively simple dynamics, they have proved to be unsuitable in more complicated systems. To extend the applicability of nonequilibrium sampling, we demonstrate a novel method that combines Minh-Adib's bidirectional estimator with nonlinear WHAM equations to reconstruct and assess PMFs from relatively fast pulling trajectories. We test the method in a one-dimensional model system and in a system of an antibiotic gramicidin-A (gA) channel, which is considered a significant challenge for nonequilibrium sampling. We identify key parameters for efficiently performing pulling simulations to improve and ensure the convergence and accuracy of estimated PMFs. We show that a few pulling trajectories of a relatively fast pulling speed v = 10 Å/ns can return a fair estimate of the PMF of a single potassium ion in gA.

Two-Dimensional Potentials of Mean Force of Nile Red in Intact and Damaged Model Bilayers. Application to Calculations of Fluorescence Spectra.

Fluorescent dyes revolutionized and expanded our understanding of biological membranes. The interpretation of experimental fluorescence data in terms of membrane structure, however, requires detailed information about the molecular environment of the dyes. Nile red is a fluorescent molecule whose excitation and emission maxima depend on the polarity of the solvent. It is mainly used as a probe to study lipid microenvironments, for example in imaging the progression of damage to the myelin sheath in multiple sclerosis. In this study, we determine the position and orientation of Nile red in lipid bilayers by calculating two-dimensional Potential of Mean Force (2D-PMF) profiles in a defect-free 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) bilayer and in damaged bilayers containing two mixtures of the oxidized lipid 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine and POPC. From 2D-PMF simulations we obtain positions and orientations of Nile Red corresponding to the minimum on the binding free energy surface in three different membrane environments with increasing amounts of water, mimicking damage in biological tissue. Using representative snapshots from the simulations, we use combined quantum mechanical/molecular mechanical (QM/MM) models to calculate the emission spectrum of Nile red as a function of its local solvation environment. The results of QM and QM/MM computations are in qualitative agreement with the experimentally observed shift in fluorescence for the dye moving from aqueous solution to the more hydrophobic environment of the lipid interiors. The range of the conformation dependent values of the computed absorption-emission spectra and the lack of solvent relaxation effects in the QM/MM calculations made it challenging to delineate specific differences between the intact and damaged bilayers.

In silico prediction of drug therapy in catecholaminergic polymorphic ventricular tachycardia.

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.