Binh Khanh Mai

Top leads for swine influenza A/H1N1 virus revealed by steered molecular dynamics approach.

Since March 2009, the rapid spread of infection during the recent A/H1N1 swine flu pandemic has raised concerns of a far more dangerous outcome should this virus become resistant to current drug therapies. Currently oseltamivir (tamiflu) is intensively used for the treatment of influenza and is reported effective for 2009 A/H1N1 virus. However, as this virus is evolving fast, some drug-resistant strains are emerging. Therefore, it is critical to seek alternative treatments and identify roots of the drug resistance. In this paper, we use the steered molecular dynamics (SMD) approach to estimate the binding affinity of ligands to the glycoprotein neuraminidase. Our idea is based on the hypothesis that the larger is the force needed to unbind a ligand from a receptor the higher its binding affinity. Using all-atom models with Gromos force field 43a1 and explicit water, we have studied the binding ability of 32 ligands to glycoprotein neuraminidase from swine flu virus A/H1N1. The electrostatic interaction is shown to play a more important role in binding affinity than the van der Waals one. We have found that four ligands 141562, 5069, 46080, and 117079 from the NSC set are the most promising candidates to cope with this virus, while peramivir, oseltamivir, and zanamivir are ranked 8, 11, and 20. The observation that these four ligands are better than existing commercial drugs has been also confirmed by our results on the binding free energies obtained by the molecular mechanics-Poisson-Boltzmann surface area (MM-PBSA) method. Our prediction may be useful for the therapeutic application.

Neuraminidase inhibitor R-125489 - A promising drug for treating influenza virus: Steered molecular dynamics approach

Two neuraminidase inhibitors, oseltamivir and zanamivir, are important drug treatments for influenza. Oseltamivir-resistant mutants of the influenza virus A/H1N1 and A/H5N1 have emerged, necessitating the development of new long-acting antiviral agents. One such agent is a new neuraminidase inhibitor R-125489 and its prodrug CS-8958. An atomic level understanding of the nature of this antiviral agents binding is still missing. We address this gap in our knowledge by applying steered molecular dynamics (SMD) simulations to different subtypes of seasonal and highly pathogenic influenza viruses. We show that, in agreement with experiments, R-125489 binds to neuraminidase more tightly than CS-8958. Based on results obtained by SMD and the molecular mechanics-Poisson-Boltzmann surface area method, we predict that R-125489 can be used to treat not only wild-type but also tamiflu-resistant N294S, H274Y variants of A/H5N1 virus as its binding affinity does not vary much across these systems. The high correlation level between theoretically determined rupture forces and experimental data on binding energies for the large number of systems studied here implies that SMD is a promising tool for drug design.

Study of Tamiflu Sensitivity to Variants of A/H5N1 Virus Using Different Force Fields

An accurate estimation of binding free energy of a ligand to receptor ΔG(bind) is one of the most important problems in drug design. The success of solution of this problem is expected to depend on force fields used for modeling a ligand-receptor complex. In this paper, we consider the impact of four main force fields, AMBER99SB, CHARMM27, GROMOS96 43a1, and OPLS-AA/L, on the binding affinity of Oseltamivir carboxylate to the wild-type and Y252H, N294S, and H274Y mutants of glycoprotein neuraminidase from the pandemic A/H5N1 virus. Having used the molecular mechanic-Poisson-Boltzmann surface area method, we have shown that ΔG(bind), obtained by AMBER99SB, OPLS-AA/L, and CHARMM27, shows the high correlation with the available experimental data. They correctly capture the binding ranking Y252H → WT → N294S → H274Y observed in experiments (Collins, P. J. et al. Nature 2008, 453, 1258). In terms of absolute values of binding scores, results obtained by AMBER99SB are in the nearest range with experiments, while OPLS-AA/L, which is applied to study binding of Oseltamivir to the influenza virus for the first time, gives rather big negative values for ΔG(bind). GROMOS96 43a1 provides a lower correlation as it supports Oseltamivir to be more resistant to N294S than H274Y. Our study suggests that force fields have pronounced influence on theoretical estimations of binding free energy of a ligand to receptor. The effect of all-atom models on dynamics of the binding pocket as well as on the hydrogen-bond network between Oseltamivir and receptors is studied in detail. The hydrogen network, obtained by GROMOS, is weakest among four studied force fields.

Mechanical Unfolding of Acylphosphatase Studied by Single-Molecule Force Spectroscopy and MD Simulations

Single-molecule manipulation methods provide a powerful means to study protein transitions. Here we combined single-molecule force spectroscopy and steered molecular-dynamics simulations to study the mechanical properties and unfolding behavior of the small enzyme acylphosphatase (AcP). We find that mechanical unfolding of AcP occurs at relatively low forces in an all-or-none fashion and is decelerated in the presence of a ligand, as observed in solution measurements. The prominent energy barrier for the transition is separated from the native state by a distance that is unusually long for alpha/beta proteins. Unfolding is initiated at the C-terminal strand (beta(T)) that lies at one edge of the beta-sheet of AcP, followed by unraveling of the strand located at the other. The central strand of the sheet and the two helices in the protein unfold last. Ligand binding counteracts unfolding by stabilizing contacts between an arginine residue (Arg-23) and the catalytic loop, as well as with beta(T) of AcP, which renders the force-bearing units of the protein resistant to force. This stabilizing effect may also account for the decelerated unfolding of ligand-bound AcP in the absence of force.

Estimation of the Binding Free Energy of AC1NX476 to HIV-1 Protease Wild Type and Mutations Using Free Energy Perturbation Method

The binding mechanism of AC1NX476 to HIV‐1 protease wild type and mutations was studied by the docking and molecular dynamics simulations. The binding free energy was calculated using the double‐annihilation binding free energy method. It is shown that the binding affinity of AC1NX476 to wild type is higher than not only ritonavir but also darunavir, making AC1NX476 become attractive candidate for HIV treatment. Our theoretical results are in excellent agreement with the experimental data as the correlation coefficient between calculated and experimentally measured binding free energies R = 0.993. Residues Asp25‐A, Asp29‐A, Asp30‐A, Ile47‐A, Gly48‐A, and Val50‐A from chain A, and Asp25‐B from chain B play a crucial role in the ligand binding. The mutations were found to reduce the receptor–ligand interaction by widening the binding cavity, and the binding propensity is mainly driven by the van der Waals interaction. Our finding may be useful for designing potential drugs to combat with HIV.

Development and Characterization of Monomeric N‑End Rule Inhibitors through In Vitro Model Substrates

In the N-end rule pathway, a set of N-terminal amino acids, called N-degrons, are recognized and ubiquitinated by the UBR proteins. Here we examined various N-end rule inhibitors to identify essential structural components of the system. Our study using in vitro biochemical assay indicated that the l-conformation and protonated α-amino group of the first residue were critical for N-degrons to properly interact with the UBR proteins. The monomeric molecules with minimum interacting motifs showed endopeptidase resistance and better inhibitory activities than traditional dipeptide inhibitors. Collectively, our study identifies a pharmacophore of N-end rule inhibitors, which provides a structural platform to improve the efficiency and druggable properties of inhibitors. Considering that the N-end rule has been implicated in many pathophysiological processes in cells, inhibitors of this pathway, such as p-chloroamphetamine, are potentially of clinical interest in a novel aspect of action mechanisms.

Characterization of mammalian N-degrons and development of heterovalent inhibitors of the N-end rule pathway

The N-end rule pathway relates the in vivo half-life of a protein with its N-terminal residue. Recent understanding of the molecular mechanism underlying N-degron recognition implies that the yeast N-degrons may not be identical to those of mammals. Here we re-evaluate the role of N-terminal amino acids as degradation determinants through an in vitro degradation assay and computational docking analysis. To take advantage of the distinct binding modes of type 1 and type 2 destabilizing residues, we developed and optimized heterovalent inhibitors of the N-end rule pathway. These small-molecules effectively delayed the degradation of the physiological N-end rule substrates in vitro and in living cells, including cardiomyocytes, suggesting that the heterovalent inhibitors could be applied to various cardiac diseases that originate from abnormal N-end rule regulation.

Theoretical Studies for Large Tunneling and the Hydrogen‐Transfer Mechanism in the CH Activation of CH3CN by a Di(μ‐oxo)diiron(IV) Complex: A Model for Intermediate Q in Soluble Methane Monooxygenase

Methane monooxygenase (MMO) is an enzyme that catalyzes the oxidation of methane to methanol when using dioxygen as the oxidant. This enzyme has been found in two forms, soluble MMO (sMMO) and particulate MMO (pMMO). The mechanism of hydrocarbon hydroxylation by sMMO has attracted much attention in recent years. The sMMO enzyme contains three protein components: a hydroxylase (MMOH), a cofactorless coupling protein (MMOB), and a reductase (MMOR). The MMOH component has a pair of carboxylate-bridged diiron centers, which are involved in O2 activation and the hydroxylation of C H bonds. The diACHTUNGTRENNUNG(m-oxo)diiron(IV) intermediate Q generated in the catalytic cycle has been identified as the primary component for the hydroxylation of methane and related alkanes. The details of the hydroxylation step are not fully understood, although extensive experimental studies have provided much insight. By using a chiral ethane substrate, CH3CHDT, Valentine et al. [6] and Priestley et al. suggested that the hydroxylation of methane by intermediate Q occurs by means of two steps, whereby hydrogen abstraction occurs to form a bound radical species followed by OH rebound to yield an alcohol. Various substrate probes have been used to reveal the nature of the C H bond-activating step with intermediate Q. The most striking observation during the hydrogen transfer that triggers the hydroxylation of CH4 and its derivatives is the unusually large H/D kinetic isotope effects (KIEs). The measured KIE values were 23.1 1 and 46.4 2.3 for CH4 and CH3CN at 20 8C, respectively, 62 4 for CH3CN at 4 8C in Methylococcus capsulatus (Bath), [9] and 42 for CH4 in Methylosinus trichosporium OB3b at 4 8C. The hydrogen-atom tunneling is believed to play an important role. Gherman et al. used the quantum mechanics (QM)/molecular mechanics (MM) method to calculate rates and KIEs and included tunneling by using the Skodje–Truhlar approximation. However, they could not estimate the KIE value for CH3CN. A simple one-dimensional tunneling approximation greatly underestimates the tunneling probabilities and/ or misrepresents the physical meaning of the tunneling dynamics. To interpret these KIE values correctly, a global potential-energy surfaces (PESs) and a dynamics theory with realistic description of tunneling are required. In the present study, the variational transition-state theory including multidimensional tunneling (VTST/MT) was used to calculate rate constants and KIEs on the basis of the state-of-the-art quantum mechanical calculations of the intrinsic reaction coordinate (IRC). The potential and vibrationally adiabatic energy (sum of potential and zero-point energies) curves were constructed by using the IVTST-M algorithm with 21 Hessian with 250 gradient points, which is described briefly in the Supporting Information. This method is more realistic because the hydrogen-transfer kinetics can be more precisely described on well-defined multidimensional potential-energy landscapes. Recently, the VTST/MT was applied to study the C H activation by the model of type III dicopper enzymes in which the experimental KIEs and Arrhenius parameters were successfully reproduced. The M06 level of theory was used because this functional produced good benchmark results for various metal complexes. The “small” Morokuma–Basch model of intermediate Q was used to mimic the hydroxylation in sMMO, and a full range of QM calculations were carried out to determine the IRC of hydrogen transfer with CH3CN. Intermediate Q was shown to be diamagnetic and contain two antiferromagnetically coupled high-spin Fe atoms. Multi-determinant QM methods should be used to calculate such systems, which is practically impossible for large systems. The broken-symmetry (BS) approach developed by Yamaguchi et al. and Noodleman et al. is also impractical for frequency and IRC calculations. Basch et al. suggested that it is computationally more practical to ignore the antiferromagnetic nature of these systems when the magnitude of the spin coupling between two Fe atoms is not strong and to perform spin-unrestricted open-shell single-determinant calculations. We also assumed that the dynamics and mechanism of hydrogen transfer would not be significantly influenced by the antiferromagnetic nature of the complexes and performed spin-unrestricted open-shell single-determinant DFT calculations for the ferromagnetically coupled highspin states of A to obtain the potential and adiabatic energies along the IRC. The optimized structure of model Q at [a] B. Khanh Mai, Prof. Y. Kim Department of Applied Chemistry Kyung Hee University 1 Seochun-Dong, Giheung-Gu, Yongin-Si Gyeonggi-Do, 446-701 (Korea) Fax: (+82) 31-203-5773 E-mail : yhkim@khu.ac.kr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/chem.201204488.

The Kinetic Isotope Effect as a Probe of Spin Crossover in the CH Activation of Methane by the FeO+ Cation†

Two-state reactivity (TSR) is often used to explain the reaction of transition-metal-oxo reagents in the bare form or in the complex form. The evidence of the TSR model typically comes from quantum-mechanical calculations for energy profiles with a spin crossover in the rate-limiting step. To prove the TSR concept, kinetic profiles for CH activation by the FeO(+) cation were explored. A direct dynamics approach was used to generate potential energy surfaces of the sextet and quartet H-transfers and rate constants and kinetic isotope effects (KIEs) were calculated using variational transition-state theory including multidimensional tunneling. The minimum energy crossing point with very large spin-orbit coupling matrix element was very close to the intrinsic reaction paths of both sextet and quartet H-transfers. Excellent agreement with experiments were obtained when the sextet reactant and quartet transition state were used with a spin crossover, which strongly support the TSR model.

Substrate‐Dependent H/D Kinetic Isotope Effects and the Role of the Di(μ‐oxo)diiron(IV) Core in Soluble Methane Monooxygenase: A Theoretical Study

Soluble methane monooxygenase (sMMO) is an enzyme that converts alkanes to alcohols using a di(μ-oxo)diiron(IV) intermediate Q at the active site. Very large kinetic isotope effects (KIEs) indicative of significant tunneling are observed for the hydrogen transfer (H-transfer) of CH4 and CH3 CN; however, a relatively small KIE is observed for CH3NO2. The detailed mechanism of the enzymatic H-transfer responsible for the diverse range of KIEs is not yet fully understood. In this study, variational transition-state theory including the multidimensional tunneling approximation is used to calculate rate constants to predict KIEs based on the quantum-mechanically generated intrinsic reaction coordinates of the H-transfer by the di(μ-oxo)diiron(IV) complex. The results of our study reveal that the role of the di(μ-oxo)diiron(IV) core and the H-transfer mechanism are dependent on the substrate. For CH4 , substrate binding induces an electron transfer from the oxygen to one Fe(IV) center, which in turn makes the μ-O ligand more electrophilic and assists the H-transfer by abstracting an electron from the C-H σ orbital. For CH3CN, the reduction of Fe(IV) to Fe(III) occurs gradually with substrate binding and H-transfer. The charge density and electrophilicity of the μ-O ligand hardly change upon substrate binding; however, for CH3NO2, there seems to be no electron movement from μ-O to Fe(IV) during the H-transfer. Thus, the μ-O ligand appears to abstract a proton without an electron from the C-H σ orbital. The calculated KIEs for CH4, CH3CN, and CH3NO2 are 24.4, 49.0, and 8.27, respectively, at 293 K, in remarkably good agreement with the experimental values. This study reveals that diverse KIE values originate mainly from tunneling to the same di(μ-oxo)diiron(IV) core for all substrates, and demonstrate that the reaction dynamics are essential for reproducing experimental results and understanding the role of the diiron core for methane oxidation in sMMO.