Modeling micropatterned antigen-antibody binding kinetics in a microfluidic chip

The reaction kinetics of antigen-antibody binding in the electrokinetically controlled microfluidic heterogeneous immunoassays has been investigated by numerical simulations. A two-dimensional computational model was employed to include the mass transport (convection and diffusion) and binding reaction between the antigen in the bulk flow and the immobilized antibody at the channel surface. The influence of the bulk velocity, the concentrations of the antibody and antigen, and the geometry of the microchips was studied for a variation of conditions and the guidance for designing of microfluidic immunoassay was provided. The model also shows that electrokinetically driven immunoassays have better reaction kinetics than pressure-driven ones, resulting from the plug-like velocity profile. Finally, a multi-patch immunoassay chip was analyzed and the reaction kinetics was optimized by rearranging the reaction patches at the channel surfaces.     

Microfluidic bead-based enzymatic primer extension for single-nucleotide discrimination using quantum dots as labels

This study reports the development of an on-chip enzyme-mediated primer extension process based on a microfluidic device with microbeads array for single-nucleotide discrimination using quantum dots as labels. The functionalized microbeads were independently introduced into the arrayed chambers using the loading chip slab. A single channel was used to generate weir structures to confine the microbeads and make the beads array accessible by microfluidics. The applied allele-specific primer extension method employed a nucleotide-degrading enzyme (apyrase) to achieve specific single-nucleotide detection. Based on the apyrase-mediated allele-specific primer extension with quantum dots as labels, on-chip single-nucleotide discrimination was demonstrated with high discrimination specificity and sensitivity (0.5 pM, signal/noise > 3) using synthesized target DNA. The chip-based signal enhancement for single-nucleotide discrimination resulted in 200 times higher sensitivity than that of an off-chip test. This microfluidic device successfully achieved simultaneous detection of two disease-associated single-nucleotide polymorphism sites using polymerase chain reaction products as target. This apyrase-mediated microfluidic primer extension approach combines the rapid binding kinetics of homogeneous assays of suspended microbeads array, the liquid handling capability of microfluidics, and the fluorescence detection sensitivity of quantum dots to provide a platform for single-base analysis with small reagent consumption, short assay time, and parallel detection.     

RNA–protein binding kinetics in an automated microfluidic reactor

Microfluidic chips can automate biochemical assays on the nanoliter scale, which is of considerable utility for RNA–protein binding reactions that would otherwise require large quantities of proteins. Unfortunately, complex reactions involving multiple reactants cannot be prepared in current microfluidic mixer designs, nor is investigation of long-time scale reactions possible. Here, a microfluidic ‘Riboreactor’ has been designed and constructed to facilitate the study of kinetics of RNA–protein complex formation over long time scales. With computer automation, the reactor can prepare binding reactions from any combination of eight reagents, and is optimized to monitor long reaction times. By integrating a two-photon microscope into the microfluidic platform, 5-nl reactions can be observed for longer than 1000 s with single-molecule sensitivity and negligible photobleaching. Using the Riboreactor, RNA–protein binding reactions with a fragment of the bacterial 30S ribosome were prepared in a fully automated fashion and binding rates were consistent with rates obtained from conventional assays. The microfluidic chip successfully combines automation, low sample consumption, ultra-sensitive fluorescence detection and a high degree of reproducibility. The chip should be able to probe complex reaction networks describing the assembly of large multicomponent RNPs such as the ribosome.     

Insulin receptor binding kinetics: modeling and simulation studies

Biological actions of insulin regulate glucose metabolism and other essential physiological functions. Binding of insulin to its cell surface receptor initiates signal transduction pathways that mediate cellular responses. Thus, it is of great interest to understand the mechanisms underlying insulin receptor binding kinetics. Interestingly, negative cooperative interactions are observed at high insulin concentrations while positive cooperativity may be present at low insulin concentrations. Clearly, insulin receptor binding kinetics cannot be simply explained by a classical bimolecular reaction. Mature insulin receptors have a dimeric structure capable of binding two molecules of insulin. The binding affinity of the receptor for the second insulin molecule is significantly lower than for the first bound insulin molecule. In addition, insulin receptor aggregation occurs in response to ligand binding and aggregation may also influence binding kinetics. In this study, we develop a mathematical model for insulin receptor binding kinetics that explicitly represents the divalent nature of the insulin receptor and incorporates receptor aggregation into the kinetic model. Model parameters are based upon published data where available. Computer simulations with our model are capable of reproducing both negative and positive cooperativity at the appropriate insulin concentrations. This model may be a useful tool for helping to understand the mechanisms underlying insulin receptor binding and the coupling of receptor binding to downstream signaling events.     

Direct monitoring of molecular recognition processes using fluorescence enhancement at colloid-coated microplates

Direct monitoring of recognition processes at the molecular level is a valuable tool for studying reaction kinetics to assess affinity constants (e.g. drugs to receptors) and for designing rapid single step immunoassays. Methods currently used to gain information about binding processes predominantly depend on surface plasmon resonance. These systems use excitation with coherent light in attenuated total reflection geometry to obtain discrimination between surface-bound and free molecules in solution. Therefore labeling of the compounds is not necessary, but due to the complexity of the measuring setup the method is rather costly. In this contribution we present a simple method for performing kinetic single step biorecognition assays with fluorophore labeled compounds using the fluorescence enhancement properties of surface bound silver colloids. Silver colloids are bound to standard microplates via silanization of the plastic surface. Fluorophores close to this colloid coated surface show a significant gain in fluorescence compared to fluorophores farther away in the bulk solution. Therefore discrimination between surface bound and free fluorophores is possible and the binding of, for example, fluorophore labeled antibodies to antigens immobilized on the colloid surface results in increasing fluorescence intensity. Utilization of standard microplates makes this method fully compatible with conventional microplate processing and reading devices. Neither excitation with coherent laser light nor ATR geometry is required, the measurement is performed in a standard fluorescence microplate reader in front face geometry with a xenon flash lamp as excitation source. Methods for the preparation of colloid-coated microplates and fluorescence-enhanced biorecognition assays are presented. Additionally the dependence of the system performance on the structure and properties of the metal colloid coated surface is described. A two-component biorecognition model system shows a detection limit in the subnanomolar range. The ease of colloid-surface preparation and the high sensitivity makes fluorescence enhancement at colloid-coated microplates a valuable tool for studying reaction kinetics and performing rapid single-step immunoassays.     

Theoretical assessments of errors in rapid immunoassays-how critical is the exact timing and reagent concentrations?

The reliability of rapid immunoassay is a concern due to an incomplete incubation to a non-equilibrium state and is susceptible to different error factors causing variance. The most critical point in the process should be found in order to improve the accuracy, and reproducibility of immunoassays, and enhance the system robustness. In this paper, the behavior of rapid assays is predicted by simulations using mechanistic assay model, based on antibody-analyte binding reaction kinetics. This antibody-analyte binding reaction kinetics model was constructed for a generic three-component (immunometric) assay and the parameters were chosen to be those of a known surface binding assay. The effects of the exact incubation timing and the initial reagent concentrations were studied focusing on the early phase of incubation, the non-equilibrium state. The magnitudes of errors in the input parameters were estimated using knowledge from practical immunoassays. According to simulations, inaccurate incubation timing adds error in the results at very short incubation times, especially in low analyte concentrations. The inaccurate reagent concentrations increase variance at short incubation times, as well. The error decreases rapidly after the first few minutes of incubation.     

On the binding determinants of the glutamate agonist with the glutamate receptor ligand binding domain

Ionotropic glutamate receptors (GluRs) are ligand-gated membrane channel proteins found in the central neural system that mediate a fast excitatory response of neurons. In this paper, we report theoretical analysis of the ligand-protein interactions in the binding pocket of the S1S2 (ligand binding) domain of the GluR2 receptor in the closed conformation. By utilizing several theoretical methods ranging from continuum electrostatics to all-atom molecular dynamics simulations and quantum chemical calculations, we were able to characterize in detail glutamate agonist binding to the wild-type and E705D mutant proteins. A theoretical model of the protein-ligand interactions is validated via direct comparison of theoretical and Fourier transform infrared spectroscopy (FTIR) measured frequency shifts of the ligand's carboxylate group vibrations [Jayaraman et al. (2000) Biochemistry 39, 8693-8697; Cheng et al. (2002) Biochemistry 41, 1602-1608]. A detailed picture of the interactions in the binding site is inferred by analyzing contributions to vibrational frequencies produced by protein residues forming the ligand-binding pocket. The role of mobility and hydrogen-bonding network of water in the ligand-binding pocket and the contribution of protein residues exposed in the binding pocket to the binding and selectivity of the ligand are discussed. It is demonstrated that the molecular surface of the protein in the ligand-free state has mainly positive electrostatic potential attractive to the negatively charged ligand, and the potential produced by the protein in the ligand-binding pocket in the closed state is complementary to the distribution of the electrostatic potential produced by the ligand itself. Such charge complementarity ensures specificity to the unique charge distribution of the ligand.     

Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody

Surface plasmon resonance biosensor analysis was used to evaluate the thermodynamics and binding kinetics of naturally occurring and synthetic cobalamins interacting with vitamin B(12) binding proteins. Cyanocobalamin-b-(5-aminopentylamide) was immobilized on a biosensor chip surface to determine the affinity of different cobalamins for transcobalamin, intrinsic factor, and nonintrinsic factor. A solution competition binding assay, in which a surface immobilized cobalamin analog competes with analyte cobalamin for B(12) protein binding, shows that only recombinant human transcobalamin is sensitive to modification of the corrin ring b-propionamide of cyanocobalamin. A direct binding assay, where recombinant human transcobalamin is conjugated to a biosensor chip, allows kinetic analysis of cobalamin binding. Response data for cyanocobalamin binding to the transcobalamin protein surface were globally fitted to a bimolecular interaction model that includes a term for mass transport. This model yields association and dissociation rate constants of k(a) = 3 x 10(7) M(-1) s(-1) and k(d) = 6 x 10(-4) s(-1), respectively, with an overall dissociation constant of K(D) = 20 pM at 30 degrees C. Transcobalamin binds cyanocobalamin-b-(5-aminopentylamide) with association and dissociation rates that are twofold slower and threefold faster, respectively, than transcobalamin binding to cyanocobalamin. The affinities determined for protein-ligand interaction, using the solution competition and direct binding assays, are comparable, demonstrating that surface plasmon resonance provides a versatile way to study the molecular recognition properties of vitamin B(12) binding proteins.     

Structural basis for μ-opioid receptor binding and activation

Opioids that stimulate the μ-opioid receptor (MOR1) are the most frequently prescribed and effective analgesics. Here we present a structural model of MOR1. Molecular dynamics simulations show a ligand-dependent increase in the conformational flexibility of the third intracellular loop that couples with the G protein complex. These simulations likewise identified residues that form frequent contacts with ligands. We validated the binding residues using site-directed mutagenesis coupled with radioligand binding and functional assays. The model was used to blindly screen a library of ～1.2 million compounds. From the 34 compounds predicted to be strong binders, the top three candidates were examined using biochemical assays. One compound showed high efficacy and potency. Post hoc testing revealed this compound to be nalmefene, a potent clinically used antagonist, thus further validating the model. In summary, the MOR1 model provides a tool for elucidating the structural mechanism of ligand-initiated cell signaling and for screening novel analgesics.     

Mapping of Enzyme Kinetics on a Microfluidic Device

A microfluidic platform or “microfluidic mapper” is demonstrated, which in a single experiment performs 36 parallel biochemical reactions with 36 different combinations of two reagents in stepwise concentration gradients. The volume used in each individual reaction was 36 nl. With the microfluidic mapper, we obtained a 3D enzyme reaction plot of horseradish peroxidase (HRP) with Amplex Red (AR) and hydrogen peroxide (H₂O₂), for concentration ranges of 11.7 μM to 100.0 μM and 11.1 μM to 66.7 μM for AR and H₂O₂, respectively. This system and methodology could be used as a fast analytical tool to evaluate various chemical and biochemical reactions especially where two or more reagents interact with each other. The generation of dual concentration gradients in the present format has many advantages such as parallelization of reactions in a nanoliter-scale volume and the real-time monitoring of processes leading to quick concentration gradients. The microfluidic mapper could be applied to various problems in analytical chemistry such as revealing of binding kinetics, and optimization of reaction kinetics.     

The Microfluidic Probe: Operation and Use for Localized Surface Processing

Microfluidic devices allow assays to be performed using minute amounts of sample and have recently been used to control the microenvironment of cells. Microfluidics is commonly associated with closed microchannels which limit their use to samples that can be introduced, and cultured in the case of cells, within a confined volume. On the other hand, micropipetting system have been used to locally perfuse cells and surfaces, notably using push-pull setups where one pipette acts as source and the other one as sink, but the confinement of the flow is difficult in three dimensions. Furthermore, pipettes are fragile and difficult to position and hence are used in static configuration only.The microfluidic probe (MFP) circumvents the constraints imposed by the construction of closed microfluidic channels and instead of enclosing the sample into the microfluidic system, the microfluidic flow can be directly delivered onto the sample, and scanned across the sample, using the MFP. . The injection and aspiration openings are located within a few tens of micrometers of one another so that a microjet injected into the gap is confined by the hydrodynamic forces of the surrounding liquid and entirely aspirated back into the other opening. The microjet can be flushed across the substrate surface and provides a precise tool for localized deposition/delivery of reagents which can be used over large areas by scanning the probe across the surface. In this video we present the microfluidic probe¹ (MFP). We explain in detail how to assemble the MFP, mount it atop an inverted microscope, and align it relative to the substrate surface, and finally show how to use it to process a substrate surface immersed in a buffer.Open in a separate windowClick here to view.^{(47M, flv)}     

Fluidics-resolved estimation of protein adsorption kinetics in a biomicrofluidic system

Protein adsorption on surfaces is a complex phenomenon that is described by the balance of convective/diffusive transport of the protein species to the surface and its adsorption/desorption at the surface. The extent of binding depends on a variety of factors such as protein/surface interactions, availability of binding sites, localized concentrations of protein near biomaterial surfaces and flow characteristics of the protein in that region. Factors such as time-varying flows, complex device geometries, presence of multiple competitive species, or possible denaturing of proteins when they attach to the surface make it extremely difficult to quantitatively analyze protein interactions with surfaces. Adsorption/desorption rate constants are often inferred using simplistic models which neglect mass transport and have limited use across different microfluidic systems and flow protocols. In this work, we have developed and demonstrated a fluidics-resolved model that evaluates protein adsorption, accounting for both the fluidic transport and the biochemical kinetics in complex biomicrofluidic devices. The model is valid for both flow and static conditions. An automated procedure was also developed to extract the "intrinsic" mass-transport-independent adsorption kinetic rate constants from experimental data using a least squares optimization method. The automated data extraction methodology is applied to two proteins (alkaline phosphatase and glucose oxidase) that have been brought into contact with poly(etheretherketone) and Teflon capillaries. The applicability of the procedure in analyzing flow and adsorption in complex microfluidic structures is also demonstrated.     

Combined affinity and rate constant distributions of ligand populations from experimental surface binding kinetics and equilibria

The present article considers the influence of heterogeneity in a mobile analyte or in an immobilized ligand population on the surface binding kinetics and equilibrium isotherms. We describe strategies for solving the inverse problem of calculating two-dimensional distributions of rate and affinity constants from experimental data on surface binding kinetics, such as obtained from optical biosensors. Although the characterization of a heterogeneous population of analytes binding to uniform surface sites may be possible under suitable experimental conditions, computational difficulties currently limit this approach. In contrast, the case of uniform analytes binding to heterogeneous populations of surface sites is computationally feasible, and can be combined with Tikhonov-Phillips and maximum entropy regularization techniques that provide the simplest distribution that is consistent with the data. The properties of this ligand distribution analysis are explored with several experimental and simulated data sets. The resulting two-dimensional rate and affinity constant distributions can describe well experimental kinetic traces measured with optical biosensors. The use of kinetic surface binding data can give significantly higher resolution than affinity distributions from the binding isotherms alone. The shape and the level of detail of the calculated distributions depend on the experimental conditions, such as contact times and the concentration range of the analyte. Despite the flexibility introduced by considering surface site distributions, the impostor application of this model to surface binding data from transport limited binding processes or from analyte distributions can be identified by large residuals, if a sufficient range of analyte concentrations and contact times are used. The distribution analysis can provide a rational interpretation of complex experimental surface binding kinetics, and provides an analytical tool for probing the homogeneity of the populations of immobilized protein.     

A method of binding kinetics of a ligand to micropatterned proteins on a microfluidic chip

A combination of microfluidic protein patterning and quantitative microfluidic handling has been used to analyze the binding kinetics of protein-ligand interactions on the nanoliter scale. The microfluidic handling method employing hydrophobic valving and pneumatic control allowed us to control nanoliter volumes of ligand or protein on a microfluidic chip. A hydrophobic and inert fluorocarbon thin film was patterned on a silicon nitride substrate to prevent non-specific binding on the background. Selectively patterned protein patterns of various sizes were used for quantitative analysis of the kinetic parameters of immobilized proteins on the circular patterns. As a model system, a streptavidin-patterned array of the same-sized pattern, i.e. 150 microm diameter, was used to capture FITC-BSA-biotin present in solution. The fluorescence intensity was well matched with the Langmuir isotherm model results, showing a dissociation constant of 2.43x10(-8)M. Similar streptavidin arrays with different-sized spots, ranging from 50 to 200 microm, showed a consistent dissociation constant of FITC-BSA-biotin with streptavidin pattern. Therefore, the reduction of pattern size of an immobilized protein did not change the dissociation rate of the ligand.     

Theoretical model and experimental study of red blood cell (RBC) deformation in microchannels

The motion and deformation of red blood cells (RBCs) flowing in a microchannel were studied using a theoretical model and a novel automated rheoscope. The theoretical model was developed to predict the cells deformation under shear as a function of the cells geometry and mechanical properties. Fluid dynamics and membrane mechanics are incorporated, calculating the traction and deformation in an iterative manner. The model was utilized to evaluate the effect of different biophysical parameters, such as: inner cell viscosity, membrane shear modulus and surface to volume ratio on deformation measurements. The experimental system enables the measurement of individual RBCs velocity and their deformation at defined planes within the microchannel. Good agreement was observed between the simulation results, the rheoscope measurements and published ektacytometry results. The theoretical model results imply that such deformability measuring techniques are weakly influenced by changes in the inner viscosity of the cell or the ambient fluid viscosity. However, these measurements are highly sensitive to RBC shear modulus. The shear modulus, estimated by the model and the rheoscope measurements, falls between the values obtained by micropipette aspiration and laser trapping. The study demonstrates the integration of a theoretical model with a microfabricated device in order to achieve a better understanding of RBC mechanics and their measurement using microfluidic shear assays. The system and the model have the potential of serving as quantitative clinical tools for diagnosing deformability disorders in RBCs.     

Great interactions: How binding incorrect partners can teach us about protein recognition and function

Protein–protein interactions play a key part in most biological processes and understanding their mechanism is a fundamental problem leading to numerous practical applications. The prediction of protein binding sites in particular is of paramount importance since proteins now represent a major class of therapeutic targets. Amongst others methods, docking simulations between two proteins known to interact can be a useful tool for the prediction of likely binding patches on a protein surface. From the analysis of the protein interfaces generated by a massive cross‐docking experiment using the 168 proteins of the Docking Benchmark 2.0, where all possible protein pairs, and not only experimental ones, have been docked together, we show that it is also possible to predict a protein's binding residues without having any prior knowledge regarding its potential interaction partners. Evaluating the performance of cross‐docking predictions using the area under the specificity‐sensitivity ROC curve (AUC) leads to an AUC value of 0.77 for the complete benchmark (compared to the 0.5 AUC value obtained for random predictions). Furthermore, a new clustering analysis performed on the binding patches that are scattered on the protein surface show that their distribution and growth will depend on the protein's functional group. Finally, in several cases, the binding‐site predictions resulting from the cross‐docking simulations will lead to the identification of an alternate interface, which corresponds to the interaction with a biomolecular partner that is not included in the original benchmark. Proteins 2016; 84:1408–1421. © 2016 The Authors Proteins: Structure, Function, and Bioinformatics Published by Wiley Periodicals, Inc.     

Relationship among types of nerve growth factor receptors on PC12 cells

We analyzed the kinetics and thermodynamics of 125I-nerve growth factor (125I-NGF) binding to NGF-receptor on PC12 cells. We used conditions of pseudo-first order kinetics and techniques to quantitate internalized complexes, "slow" or high affinity binding complexes, and cell surface "fast" or low affinity complexes. Two possible models were examined: binding to two independent receptors at the cell surface (i.e. high and low affinity forms of NGF-receptor) and a model for consecutive formation of fast, low affinity binding followed by slow, high affinity binding or internalization. Our data are consistent with the consecutive model only. The rates of association and dissociation of NGF with slow, high affinity sites and internalized, acid wash-resistant sites are indistinguishable from each other. We also analyzed, in detail, the two assays primarily used to distinguish slow binding complexes from internalized complexes. Scatchard analysis of total binding and dissociation of pre-equilibrated 125I-NGF in the presence of unlabeled NGF at high concentration (cold wash). Neither of these assays shows any evidence that the slow, high affinity binding step is different from internalization of the 125I-NGF-receptor complex. Based on this analysis, there are only two detectable forms of NGF-receptor on PC12 cells: complexes on the surface of the cells with a binding affinity of 0.5 nM at 37 degrees C and complexes internalized by the cells. Furthermore, the data are consistent with a model in which NGF-receptor is internalized constitutively and independently of occupancy by NGF. We also examined the fate of internalized 125I-NGF. In the first 60 min after contact with PC12 cells, no degradation of 125I-NGF was observed. Moreover, a significant amount of 125I-NGF recirculates to the cell surface and is released as intact, Mr = 13,000 NGF. The cells were also stimulated by NGF in a primary neurite outgrowth assay with an ED50 of 2-16 pM under conditions of low initial cell numbers in a large extracellular volume of NGF-containing medium. Thus, low level occupancy of the cell surface receptors, Kd = 0.5 nM, for several days is sufficient to stimulate neurite outgrowth. This indicates the presence of spare NGF-receptors on the surface PC12 cells.     

An experimental and theoretical study of the morphine binding capacity and kinetics of an engineered opioid receptor

Electrochemical real-time monitoring of ligand binding to an engineered opioid receptor specific for morphine is reported. In the particular systems studied, 90% of the binding was found to be completed after only 85-120 s. Thus, the binding kinetics has proven to be more rapid than previously believed. The observed association rate constant for the morphine binding reaction was calculated to be 215 M(-1)s(-1). A theoretical analysis of the experimental binding data suggested that the binding sites of the engineered opioid receptor could best be described by a model having two populations of binding sites: K(D)=40 microM (13 micromol/g) and K(D)=205 microM (29 micromol/g). Furthermore, a theoretical model was developed in order to explain the observed binding of the engineered opioid receptor. This model suggested that the binding sites on the polymer surface are up to 5.1A deep and they allow 100% of the ligand (morphine) to anchor itself into the site. The predicted theoretical maximum binding capacity for the reported receptor is calculated to be approximately 2 mmol/g polymer (based on an increase of cavity density).     

Finite Element Model of Oxygen Transport for the Design of Geometrically Complex Microfluidic Devices Used in Biological Studies

Red blood cells play a crucial role in the local regulation of oxygen supply in the microcirculation through the oxygen dependent release of ATP. Since red blood cells serve as an oxygen sensor for the circulatory system, the dynamics of ATP release determine the effectiveness of red blood cells to relate the oxygen levels to the vessels. Previous work has focused on the feasibility of developing a microfluidic system to measure the dynamics of ATP release. The objective was to determine if a steep oxygen gradient could be developed in the channel to cause a rapid decrease in hemoglobin oxygen saturation in order to measure the corresponding levels of ATP released from the red blood cells. In the present study, oxygen transport simulations were used to optimize the geometric design parameters for a similar system which is easier to fabricate. The system is composed of a microfluidic device stacked on top of a large, gas impermeable flow channel with a hole to allow gas exchange. The microfluidic device is fabricated using soft lithography in polydimethyl-siloxane, an oxygen permeable material. Our objective is twofold: (1) optimize the parameters of our system and (2) develop a method to assess the oxygen distribution in complex 3D microfluidic device geometries. 3D simulations of oxygen transport were performed to simulate oxygen distribution throughout the device. The simulations demonstrate that microfluidic device geometry plays a critical role in molecule exchange, for instance, changing the orientation of the short wide microfluidic channel results in a 97.17% increase in oxygen exchange. Since microfluidic devices have become a more prominent tool in biological studies, understanding the transport of oxygen and other biological molecules in microfluidic devices is critical for maintaining a physiologically relevant environment. We have also demonstrated a method to assess oxygen levels in geometrically complex microfluidic devices.     

Binding free energies for nicotine analogs inhibiting cytochrome P450 2A6 by a combined use of molecular dynamics simulations and QM/MM-PBSA calculations

Molecular dynamics (MD) simulations and hybrid quantum mechanical/molecular mechanical (QM/MM) calculations have been performed to explore the dynamic behaviors of cytochrome P450 2A6 (CYP2A6) binding with nicotine analogs (that are typical inhibitors) and to calculate their binding free energies in combination with Poisson–Boltzmann surface area (PBSA) calculations. The combined MD simulations and QM/MM-PBSA calculations reveal that the most important structural parameters affecting the CYP2A6-inhibitor binding affinity are two crucial internuclear distances, that is, the distance between the heme iron atom of CYP2A6 and the coordinating atom of the inhibitor, and the hydrogen-bonding distance between the N297 side chain of CYP2A6 and the pyridine nitrogen of the inhibitor. The combined MD simulations and QM/MM-PBSA calculations have led to dynamic CYP2A6-inhibitor binding structures that are consistent with the observed dynamic behaviors and structural features of CYP2A6-inhibitor binding, and led to the binding free energies that are in good agreement with the experimentally-derived binding free energies. The agreement between the calculated binding free energies and the experimentally-derived binding free energies suggests that the combined MD and QM/MM-PBSA approach may be used as a valuable tool to accurately predict the CYP2A6-inhibitor binding affinities in future computational design of new, potent and selective CYP2A6 inhibitors.