Urban Bren

Cytochrome P450 3A4 inhibition by ketoconazole: tackling the problem of ligand cooperativity using molecular dynamics simulations and free-energy calculations.

Cytochrome P450 3A4 (CYP3A4) metabolizes more than 50% of clinically used drugs and is often involved in adverse drug-drug interactions. It displays atypical binding and kinetic behavior toward a number of ligands characterized by a sigmoidal shape of the corresponding titration curves, which is indicative of a positive homotropic cooperativity. This requires a participation of at least two ligand molecules, whereby the binding of the first ligand molecule increases the affinity of CYP3A4 for the binding of the second ligand molecule. In the current study, a combination of molecular dynamics simulations and free-energy calculations was applied to elucidate the physicochemical origin of the observed positive homotropic cooperativity in ketoconazole binding to CYP3A4. The binding of the first ketoconazole molecule was established to increase the affinity for the binding of the second ketoconazole molecule by 5 kJ mol(-1), which explains and quantifies the experimentally observed cooperative behavior of CYP3A4. Shape complementarity through nonpolar van der Waals interactions was identified as the main driving force of this binding, which seems to be in line with the promiscuous nature of CYP3A4. Moreover, the calculated binding free energies were found to be in good agreement with the values predicted from a simple 2-ligand binding kinetic model as well as to successfully reproduce the experimental titration curve. This confirms the general applicability of rapid free-energy methods to study challenging biomolecular systems like cytochromes P450, which are characterized by a large flexibility and malleability of their active sites.

Development and Validation of Empirical Force Field Parameters for Netropsin

The netropsin molecule preferentially binds to the four consecutive A.T base pairs of the DNA minor groove and could therefore inhibit the expression of specific genes. The understanding of its binding on a molecular level is indispensable for computer-aided design of new antitumor agents. This knowledge could be obtained via molecular dynamics (MD) and docking simulations, but in this case appropriate force field parameters for the netropsin molecule should be explicitly defined. Our parametrization was based on the results of quantum chemical calculations. The resulting set of parameters was able to reproduce bond lengths, bond angles, torsional angles of the ab initio minimized geometry within 0.03 A, 3 deg and 5 deg, respectively, and its vibrational frequencies with a relative error of 4.3% for low and 2.8% for high energy modes. To show the accuracy of the developed parameters we calculated an IR spectrum of the netropsin molecule using MD simulation and found it to be in good agreement with the experimental one. Finally, we performed a 10 ns long MD simulation of the netropsin-DNA complex immersed in explicit water. The overall complex conformation remained stable at all times, and its secondary structure was well retained.

Cooperative Binding of Aflatoxin B1 by Cytochrome P450 3A4: A Computational Study

Aflatoxin B1 (AFB1)-the most potent natural carcinogen known to men-is metabolized by cytochrome P450 3A4 (CYP3A4), either to the genotoxic AFB1 exo-8,9-epoxide or to the detoxified 3α-hydroxy AFB1. The activation of the procarcinogen proceeds in a highly cooperative fashion, which differs from common allosteric regulation in the sense that it can be attributed to simultaneous occupancy of a single large and malleable active site by multiple ligand molecules. Unfortunately, unlike in the case of ketoconazole, there is currently no experimental structure available for the doubly ligated CYP3A4-AFB1 complex. Therefore, we employed a sequential molecular docking protocol to create various possible doubly ligated complexes and subsequently performed molecular dynamics simulations and free-energy calculations to check for their consistency with the available experimental data on regio- and stereoselectivity of both AFB1 oxidations as well as with available kinetic data. Only the system in which the first AFB1 molecule was bound in a face-on C8-C9 epoxidation mode and the second AFB1 molecule was bound in a side-on 3α-hydroxylation mode-a result of an unconstrained molecular docking protocol-has successfully fulfilled all the imposed criteria and is therefore proposed as the most likely structure of the doubly ligated complex of CYP3A4 with AFB1. The empirical Linear Interaction Energy method revealed that shape complementarity through nonpolar dispersion interactions between the two bound AFB1 molecules is the main source of the experimentally observed positive homotropic cooperativity. The reported study represents a nice example of how state-of-the-art molecular modeling techniques can be used to study complicated macromolecular complexes, whose structures have not yet been experimentally determined, and to validate these against the available experimental data. The proposed structure will facilitate future studies on the rational design of successful AFB1 modulators or on human subpopulations characterized by specific CYP3A4 polymorphisms that are especially sensitive to AFB1.

DNA polymerase β catalytic efficiency mirrors the Asn279–dCTP H-bonding strength

Ternary complexes of wild type or mutant form of human DNA polymerase β (pol β) bound to DNA and dCTP substrates were studied by molecular dynamics (MD) simulations. The occurrences of contact configurations (CC) of structurally important atom pairs were sampled along the MD trajectories, and converted into free‐energy differences, ΔG CC. ΔG CC values were correlated with the experimental binding and catalytic free energies for the wild type pol β and its Arg183Ala, Tyr271Ala, Asp276Val, Lys280Gly, Arg283Ala, and Glu295Ala mutants. The correlation coefficients show that the strength of the H‐bond between dCTP and Asn279 is a strong predictor of the mutation‐induced changes in the catalytic efficiency of pol β. This finding is consistent with the view that enzyme preorganization plays a major role in controlling DNA polymerase specific activity.

Carcinogenicity of Acrylamide: A Computational Study

This paper reports a series of ab initio, density functional theory (DFT), and semiempirical molecular orbital (MO) calculations concerning the reaction between the ultimate carcinogen of acrylamide and guanine. Acrylamide--a product of the Maillard reaction--is present in a variety of fried and oven-cooked food. After intake, it is epoxidized by cytochrome P450 2E1 to yield the ultimate carcinogen--glycidamide. Effects of solvation were considered using the Langevin dipoles (LD) model of Florian and Warshel and the solvent reaction field (SCRF) model of Tomasi and co-workers. In silico activation free energies are in very good agreement with the experimental value of 22.8 kcal/mol. This agreement presents strong evidence in favor of the validity of the proposed S N2 reaction mechanism and points to the applicability of quantum chemical methods to studies of reactions associated with carcinogenesis. In addition, insignificant stereoselectivity of the studied reaction was predicted. Finally, the competing reaction of glycidamide with adenine was simulated, and the experimentally observed regioselectivity was successfully reproduced.

Steric and Electrostatic Effects at the C2 Atom Substituent Influence Replication and Miscoding of the DNA Deamination Product Deoxyxanthosine and Analogs by DNA Polymerases

Deoxyinosine (dI) and deoxyxanthosine (dX) are both formed in DNA at appreciable levels in vivo by deamination of deoxyadenosine (dA) and deoxyguanosine (dG), respectively, and can miscode. Structure-activity relationships for dA pairing have been examined extensively using analogs but relatively few studies have probed the roles of the individual hydrogen-bonding atoms of dG in DNA replication. The replicative bacteriophage T7 DNA polymerase/exonuclease and the translesion DNA polymerase Sulfolobus solfataricus pol IV were used as models to discern the mechanisms of miscoding by DNA polymerases. Removal of the 2-amino group from the template dG (i.e., dI) had little impact on the catalytic efficiency of either polymerase, as judged by either steady-state or pre-steady-state kinetic analysis, although the misincorporation frequency was increased by an order of magnitude. dX was highly miscoding with both polymerases, and incorporation of several bases was observed. The addition of an electronegative fluorine atom at the 2-position of dI lowered the oligonucleotide T(m) and strongly inhibited incorporation of dCTP. The addition of bromine or oxygen (dX) at C2 lowered the T(m) further, strongly inhibited both polymerases, and increased the frequency of misincorporation. Linear activity models show the effects of oxygen (dX) and the halogens at C2 on both DNA polymerases as mainly due to a combination of both steric and electrostatic factors, producing a clash with the paired cytosine O2 atom, as opposed to either bulk or perturbation of purine ring electron density alone.

Molecular dynamics simulations give insight into d-glucose dioxidation at C2 and C3 by Agaricus meleagris pyranose dehydrogenase

The flavin-dependent sugar oxidoreductase pyranose dehydrogenase (PDH) from the plant litter-degrading fungus Agaricus meleagris oxidizes d-glucose (GLC) efficiently at positions C2 and C3. The closely related pyranose 2-oxidase (P2O) from Trametes multicolor oxidizes GLC only at position C2. Consequently, the electron output per molecule GLC is twofold for PDH compared to P2O making it a promising catalyst for bioelectrochemistry or for introducing novel carbonyl functionalities into sugars. The aim of this study was to rationalize the mechanism of GLC dioxidation employing molecular dynamics simulations of GLC–PDH interactions. Shape complementarity through nonpolar van der Waals interactions was identified as the main driving force for GLC binding. Together with a very diverse hydrogen-bonding pattern, this has the potential to explain the experimentally observed promiscuity of PDH towards different sugars. Based on geometrical analysis, we propose a similar reaction mechanism as in P2O involving a general base proton abstraction, stabilization of the transition state, an alkoxide intermediate, through interaction with a protonated catalytic histidine followed by a hydride transfer to the flavin N5 atom. Our data suggest that the presence of the two potential catalytic bases His-512 and His-556 increases the versatility of the enzyme, by employing the most suitably oriented base depending on the substrate and its orientation in the active site. Our findings corroborate and rationalize the experimentally observed dioxidation of GLC by PDH and its promiscuity towards different sugars.

Microwave catalysis revisited: an analytical solution.

In our previous work [Bren, U., et al. J. Phys. Chem. A 2008, 112, 166] we proposed a novel physical mechanism for microwave catalysis based on rotationally hot reactive species and verified its validity through a Monte Carlo simulation of a realistic chemical reaction: neutral ester hydrolysis. This article represents a continuation of our ongoing effort toward quantitative understanding of the microwave catalytic effect. It provides a derivation of an analytical solution for the microwave catalysis. The obtained expression is compared with the results of the Monte Carlo simulation and is applied to reproduce the microwave catalytic effect experimentally observed in the polyethylene terephthalate solvolysis. Implications for the interactions of microwaves with living organisms in the context of widespread mobile telephony are also discussed.

Uniform Free-Energy Profiles of the P–O Bond Formation and Cleavage Reactions Catalyzed by DNA Polymerases β and λ

Human X-family DNA polymerases β (Polβ) and λ (Polλ) catalyze the nucleotidyl-transfer reaction in the base excision repair pathway of the cellular DNA damage response. Using empirical valence bond and free-energy perturbation simulations, we explore the feasibility of various mechanisms for the deprotonation of the 3′-OH group of the primer DNA strand, and the subsequent formation and cleavage of P–O bonds in four Polβ, two truncated Polλ (tPolλ), and two tPolλ Loop1 mutant (tPolλΔL1) systems differing in the initial X-ray crystal structure and nascent base pair. The average calculated activation free energies of 14, 18, and 22 kcal mol–1 for Polβ, tPolλ, and tPolλΔL1, respectively, reproduce the trend in the observed catalytic rate constants. The most feasible reaction pathway consists of two successive steps: specific base (SB) proton transfer followed by rate-limiting concerted formation and cleavage of the P–O bonds. We identify linear free-energy relationships (LFERs) which show that the differences in the overall activation and reaction free energies among the eight studied systems are determined by the reaction free energy of the SB proton transfer. We discuss the implications of the LFERs and suggest pKa of the 3′-OH group as a predictor of the catalytic rate of X-family DNA polymerases.

Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates.

Monomeric Agaricus meleagris pyranose dehydrogenase (AmPDH) belongs to the glucose–methanol–choline family of oxidoreductases. An FAD cofactor is covalently tethered to His103 of the enzyme. AmPDH can double oxidize various mono‐ and oligosaccharides at different positions (C1 to C4). To study the structure/function relationship of selected active‐site residues of AmPDH pertaining to substrate (carbohydrate) turnover in more detail, several active‐site variants were generated, heterologously expressed in Pichia pastoris, and characterized by biochemical, biophysical and computational means. The crystal structure of AmPDH shows two active‐site histidines, both of which could take on the role as the catalytic base in the reductive half‐reaction. Steady‐state kinetics revealed that His512 is the only catalytic base because H512A showed a reduction in (kcat/KM)glucose by a factor of 105, whereas this catalytic efficiency was reduced by two or three orders of magnitude for His556 variants (H556A, H556N). This was further corroborated by transient‐state kinetics, where a comparable decrease in the reductive rate constant was observed for H556A, whereas the rate constant for the oxidative half‐reaction (using benzoquinone as substrate) was increased for H556A compared to recombinant wild‐type AmPDH. Steady‐state kinetics furthermore indicated that Gln392, Tyr510, Val511 and His556 are important for the catalytic efficiency of PDH. Molecular dynamics (MD) simulations and free energy calculations were used to predict d‐glucose oxidation sites, which were validated by GC‐MS measurements. These simulations also suggest that van der Waals interactions are the main driving force for substrate recognition and binding.