EPR study of melanin-protein interaction: photoinduced free radicals and progressive microwave power saturation.

The photo-induction of free radicals in synthetic L-dihydroxyphenylalanine (L-DOPA) melanin in the presence of bovine serum albumin (BSA) was studied by electron paramagnetic resonance (EPR) spectroscopy. By monitoring the signal intensities and progressive microwave power saturation it was shown that L-DOPA melanin in solution behaves as a single macromolecule, interacting with BSA and molecular oxygen. In the absence of oxygen, the EPR signal of L-DOPA melanin was homogeneously broadened; the magnetic interaction with oxygen induced inhomogeneous broadening. In aqueous solution, the presence of BSA decreased the accessibility of oxygen to paramagnetic centres in the melanin. On UV-visible illumination, the presence of BSA modified the rates of formation and decay of photoinduced free radicals, resulting in a net enhancement of the EPR signal compared with that observed in pure L-DOPA melanin.     

Theoretical studies on water–tetracaine interaction

The action of local anesthetics (LA) is controversial. There is experimental evidence that the unprotonated form of LA penetrates the axon, while the charged form acts in the intracellular phase. To obtain some insight on the structure of the local anesthetics tetracaine and its pharmacological action, we made calculations using the density functional theory (DFT) method. After those calculations, we performed molecular dynamics (MD) simulations in a p, N, T ensemble, in an aqueous environment, on both unprotonated and protonated forms of the molecule. The radial distribution function was used to study water solvent effects, through the characterization of the affinity of tetracaine to water. The results indicate that the molecule has regions with different degree of hydrophobicity, and the N‐terminal of the anesthetic was primarily affected by changes in the protonation state of the anesthetic. The pH‐dependent activity of TTC should then be analyzed in view of local changes in different regions of the molecule, rather than in terms of general effects on the hydrophobicity of the molecule as a whole. © 2005 Wiley Periodicals, Inc. Int J Quantum Chem, 2006     

Nonspecific interaction forces at water-membrane interface by forced molecular dynamics simulations

Nonspecific interactions are the main driving forces for the behavior of molecules with great affinity for biologic membranes. To investigate not only the molecular details of these interactions but to estimate their magnitude as well, the theoretical method of Forced Molecular Dynamics Simulations, based on the Atomic Force Spectroscopy experimental technique, was applied. In this approach, an additional one-dimensional elastic force, representing the cantilever probe, was incorporated to the force field of a Molecular Dynamics computational program. This force represents a spring fixed on one end to a selected atom of the molecule; the other end of the spring is displaced at constant velocity to pull the molecule out of the membrane. The force experimented by the molecule due to the spring, is proportional to the spring elongation relative to its equilibrium position. This value is registered during the entire simulation, and its maximum value will determine the molecule-membrane interaction force. Nonexplicit medium simulations were carried out. Polar and apolar media were considered according to their polarizability degree and a specific dielectric constant value was assigned. In this approach, the membrane was considered as the apolar region limited by two flat surfaces with a polar aqueous medium. The potential energy discontinuity at the interfaces was smoothed by considering the polarization-induced effects using the image method. The results of this methodology are presented using a small system, a single Alanine amino acid model, which enables extended simulations in a microsecond time scale. The confinement of this amino acid at the interface reduces its degrees of freedom and forces it to adopt one of the six defined conformations. A correlation between these stable structures at the water-membrane interface and the interaction force value was determined.     

New parameterization approaches of the LIE method to improve free energy calculations of PlmII‐Inhibitors complexes

The standard parameterization of the Linear Interaction Energy (LIE) method has been applied with quite good results to reproduce the experimental absolute binding free energies for several protein–ligand systems. However, we found that this parameterization failed to reproduce the experimental binding free energy of Plasmepsin II (PlmII) in complexes with inhibitors belonging to four dissimilar scaffolds. To overcome this fact, we developed three approaches of LIE, which combine systematic approaches to predict the inhibitor‐specific values of α, β, and γ parameters, to gauge their ability to calculate the absolute binding free energies for these PlmII‐Inhibitor complexes. Specifically: (i) we modified the linear relationship between the weighted nonpolar desolvation ratio (WNDR) and the α parameter, by introducing two models of the β parameter determined by the free energy perturbation (FEP) method in the absence of the constant term γ, and (ii) we developed a new parameterization model to investigate the linear correlation between WNDR and the correction term γ. Using these parameterizations, we were able to reproduce the experimental binding free energy from these systems with mean absolute errors lower than 1.5 kcal/mol. © 2010 Wiley Periodicals, Inc. J Comput Chem 2010     

Polarization effects on peptide conformations at water–membrane interface by molecular dynamics simulation

The electrostatic image method was applied to investigate the conformation of peptides characterized by different hydrophobicities in a water–membrane interface model. The interface was represented by a surface of discontinuity between two media with different dielectric constants, taking into account the difference between the polarizabilities of the aqueous medium and the hydrocarbon one. The method consists of a substitution of the real problem, which involves the charges and the induced polarization at the surface of discontinuity, by a simpler problem formed with charges and their images. The electric field due to the polarization induced at the surface by charge q was calculated using a hypothetical charge q′ (image of q), symmetrically located on the opposite side of the surface. The value of q′ was determined using the appropriate electrostatic boundary conditions at the surface. By means of this procedure, the effect of the interface can be introduced easily in the usual force field. We included this extension in the computational package that we are developing for molecular dynamics simulations (Thor ). The peptides studied included hydrophilic tetraaspartic acid (Asp–Asp–Asp–Asp), tetralysine (Lys–Lys–Lys–Lys), hydrophobic tetrapeptide (His–Phe–Arg–Trp), an amphiphilic fragment of β-endorphin, and the signal sequence of the E. coli λ-receptor. The simulation results are in agreement with known experimental data regarding the behavior of peptides at the water–membrane interface. An analysis of the conformational dynamics of the signal sequence peptide at the interface was performed over the course of a few nanoseconds. ©1999 John Wiley & Sons, Inc. J Comput Chem 20: 971–982, 1999     

Water solvent and local anesthetics: A computational study

There are various experimental studies regarding the toxicity and the time of action of local anesthetics, which contain general insights about their pharmacological and physicochemical properties. Although a detailed microscopic analysis of the local anesthetics would contribute to understanding these properties, there are relatively few theoretical studies about these molecules. In this article, we present the results from calculations performed for three local anesthetics: tetracaine, procaine, and lidocaine, both in their charged and uncharged forms, in aqueous environment. We have used the density functional theory and molecular dynamics simulations to study the structural characteristics of these compounds. The radial distribution function g(r) was used to examine the structure of water molecules surrounding different regions of the local anesthetics. We demonstrated the nonhomogeneous character of the anesthetics with respect to their affinity to water solvent molecules as well as the modifications in their affinity to water caused by changes in their charge state. We also observed that the biological potency of the anesthetics is more related to the behavior of specific groups within the molecule, which are responsible for the interaction with the lipid phase of membranes, rather than the general properties of the molecule as a whole. © 2007 Wiley Periodicals, Inc. Int J Quantum Chem, 2007     

Molecular dynamics study of biomembrane/local anesthetics interactions

It is still controversial how local anesthetics (LAs) act upon the nervous system and how the membrane contributes to this process, since probably the most important active site of the LAs is located in the sodium channels, a trans-membrane protein. An important role of the bio-membrane would be the stabilization and orientation of local anesthetics molecules, reducing their translational and rotational degrees of freedom, which could reinforce the mechanisms which interrupt the nervous impulse. This study aims to perform a computational analysis of the LAs behaviour in the membrane, and the effect of the water/membrane interface on their stabilization and orientation. Analysis by molecular dynamics (MD) showed that the charged form of these drugs are oriented at the interface, while the neutral form can easily cross the interface, entering the membrane, in agreement with the most recent experimental results in the literature. In contrast, it is here suggested that benzocaine (BZC), which exists only in its uncharged form in physiological media, behaves like the charged anesthetics, remaining stabilized and oriented at the interface. This could explain the similar anesthetic effect of BZC and the charged forms of tetracaine (TTC) and lidocaine (LDC).     

Self-affine analysis of protein energy

We study the time series of the total energy of polypeptides and proteins. These time series were generated by molecular dynamics methods and analyzed by applying detrended fluctuation analysis to estimate the long-range power-law correlation, i.e. to measure scaling exponents α. Such exponents were calculated for all systems and their values follow environment conditions, i.e., they are temperature dependent and also, in a continuum medium approach, vary according to the dielectric constants (we simulated ?=2 and ?=80). The procedure was applied to investigate polyalanines, and other realistic models of proteins (Insect Defensin A and Hemoglobin). The present findings exhibit results that are consistent with previous ones obtained by other methodologies.     

In silico identification of noncompetitive inhibitors targeting an uncharacterized allosteric site of falcipain-2

Falcipain-2 (FP-2) is a Plasmodium falciparum hemoglobinase widely targeted in the search for antimalarials. FP-2 can be allosterically modulated by various noncompetitive inhibitors that have been serendipitously identified. Moreover, the crystal structures of two inhibitors bound to an allosteric site, termed site 6, of the homolog enzyme human cathepsin K (hCatK) suggest that the equivalent region in FP-2 might play a similar role. Here, we conduct the rational identification of FP-2 inhibitors through virtual screenings (VS) of compounds into several pocket-like conformations of site 6, sampled during molecular dynamics (MD) simulations of the free enzyme. Two noncompetitive inhibitors, ZINC03225317 and ZINC72290660, were confirmed using in vitro enzymatic assays and their poses into site 6 led to calculated binding free energies matching the experimental ones. Our results provide strong evidence about the allosteric inhibition of FP-2 through binding of small molecules to site 6, thus opening the way toward the discovery of new inhibitors against this enzyme.

     

Stochastic molecular optimization using generalized simulated annealing

We propose a stochastic optimization technique based on a generalized simulated annealing (GSA) method for mapping minima points of molecular conformational energy surfaces. The energy maps are obtained by coupling a classical molecular force field (THOR package) with a GSA procedure. Unlike the usual molecular dynamics (MD) method, the method proposed in this study is force independent; that is, we obtain the optimized conformation without calculating the force, and only potential energy is involved. Therefore, we do not need to know the conformational energy gradient to arrive at equilibrium conformations. Its utility in molecular mechanics is illustrated by applying it to examples of simple molecules (H₂O and H₂O₃) and to polypeptides. The results obtained for H₂O and H₂O₃ using Tsallis thermostatistics suggest that the GSA approach is faster than the other two conventional methods (Boltzmann and Cauchy machines). The results for polypeptides show that pentalanine does not form a stable α-helix structure, probably because the number of hydrogen bonds is insufficient to maintain the helical array. On the contrary, the icoalanine molecule forms an α-helix structure. We obtain this structure simulating all Φ, Ψ pairs using only a few steps, as compared with conventional methods. © 1998 John Wiley & Sons, Inc. J Comput Chem 19: 647–657, 1998