Unveiling Novel Urease Inhibitors for Helicobacter pylori: A Multi-Methodological Approach from Virtual Screening and ADME to Molecular Dynamics Simulations.

Helicobacter pylori (Hp) infections pose a global health challenge demanding innovative therapeutic strategies by which to eradicate them. Urease, a key Hp virulence factor hydrolyzes urea, facilitating bacterial survival in the acidic gastric environment. In this study, a multi-methodological approach combining pharmacophore- and structure-based virtual screening, molecular dynamics simulations, and MM-GBSA calculations was employed to identify novel inhibitors for Hp urease (HpU). A refined dataset of 8,271,505 small molecules from the ZINC15 database underwent pharmacokinetic and physicochemical filtering, resulting in 16% of compounds for pharmacophore-based virtual screening. Molecular docking simulations were performed in successive stages, utilizing HTVS, SP, and XP algorithms. Subsequent energetic re-scoring with MM-GBSA identified promising candidates interacting with distinct urease variants. Lys219, a residue critical for urea catalysis at the urease binding site, can manifest in two forms, neutral (LYN) or carbamylated (KCX). Notably, the evaluated molecules demonstrated different interaction and energetic patterns in both protein variants. Further evaluation through ADMET predictions highlighted compounds with favorable pharmacological profiles, leading to the identification of 15 candidates. Molecular dynamics simulations revealed comparable structural stability to the control DJM, with candidates 5, 8 and 12 (CA5, CA8, and CA12, respectively) exhibiting the lowest binding free energies. These inhibitors suggest a chelating capacity that is crucial for urease inhibition. The analysis underscores the potential of CA5, CA8, and CA12 as novel HpU inhibitors. Finally, we compare our candidates with the chemical space of urease inhibitors finding physicochemical similarities with potent agents such as thiourea.

Visualizing the protons in a metalloenzyme electron proton transfer pathway.

In redox metalloenzymes, the process of electron transfer often involves the concerted movement of a proton. These processes are referred to as proton-coupled electron transfer, and they underpin a wide variety of biological processes, including respiration, energy conversion, photosynthesis, and metalloenzyme catalysis. The mechanisms of proton delivery are incompletely understood, in part due to an absence of information on exact proton locations and hydrogen bonding structures in a bona fide metalloenzyme proton pathway. Here, we present a 2.1-Å neutron crystal structure of the complex formed between a redox metalloenzyme (ascorbate peroxidase) and its reducing substrate (ascorbate). In the neutron structure of the complex, the protonation states of the electron/proton donor (ascorbate) and all of the residues involved in the electron/proton transfer pathway are directly observed. This information sheds light on possible proton movements during heme-catalyzed oxygen activation, as well as on ascorbate oxidation.

Catalytic mechanism of the colistin resistance protein MCR-1.

The mcr-1 gene encodes a membrane-bound Zn2+-metalloenzyme, MCR-1, which catalyses phosphoethanolamine transfer onto bacterial lipid A, making bacteria resistant to colistin, a last-resort antibiotic. Mechanistic understanding of this process remains incomplete. Here, we investigate possible catalytic pathways using DFT and ab initio calculations on cluster models and identify a complete two-step reaction mechanism. The first step, formation of a covalent phosphointermediate via transfer of phosphoethanolamine from a membrane phospholipid donor to the acceptor Thr285, is rate-limiting and proceeds with a single Zn2+ ion. The second step, transfer of the phosphoethanolamine group to lipid A, requires an additional Zn2+. The calculations suggest the involvement of the Zn2+ orbitals directly in the reaction is limited, with the second Zn2+ acting to bind incoming lipid A and direct phosphoethanolamine addition. The new level of mechanistic detail obtained here, which distinguishes these enzymes from other phosphotransferases, will aid in the development of inhibitors specific to MCR-1 and related bacterial phosphoethanolamine transferases.

Multiscale Workflow for Modeling Ligand Complexes of Zinc Metalloproteins.

Zinc metalloproteins are ubiquitous, with protein zinc centers of structural and functional importance, involved in interactions with ligands and substrates and often of pharmacological interest. Biomolecular simulations are increasingly prominent in investigations of protein structure, dynamics, ligand interactions, and catalysis, but zinc poses a particular challenge, in part because of its versatile, flexible coordination. A computational workflow generating reliable models of ligand complexes of biological zinc centers would find broad application. Here, we evaluate the ability of alternative treatments, using (nonbonded) molecular mechanics (MM) and quantum mechanics/molecular mechanics (QM/MM) at semiempirical (DFTB3) and density functional theory (DFT) levels of theory, to describe the zinc centers of ligand complexes of six metalloenzyme systems differing in coordination geometries, zinc stoichiometries (mono- and dinuclear), and the nature of interacting groups (specifically the presence of zinc-sulfur interactions). MM molecular dynamics (MD) simulations can overfavor octahedral geometries, introducing additional water molecules to the zinc coordination shell, but this can be rectified by subsequent semiempirical (DFTB3) QM/MM MD simulations. B3LYP/MM geometry optimization further improved the accuracy of the description of coordination distances, with the overall effectiveness of the approach depending upon factors, including the presence of zinc-sulfur interactions that are less well described by semiempirical methods. We describe a workflow comprising QM/MM MD using DFTB3 followed by QM/MM geometry optimization using DFT (e.g., B3LYP) that well describes our set of zinc metalloenzyme complexes and is likely to be suitable for creating accurate models of zinc protein complexes when structural information is more limited.

Quantum Mechanics/Molecular Mechanics Simulations Show Saccharide Distortion is Required for Reaction in Hen Egg-White Lysozyme.

Hybrid quantum mechanics/molecular mechanics (QM/MM) calculations on lysozyme show significant distortion of the bound saccharide is required to facilitate the catalytic reaction.

Biocatalytic Routes to Lactone Monomers for Polymer Production.

Monoterpenoids offer potential as biocatalytically derived monomer feedstocks for high-performance renewable polymers. We describe a biocatalytic route to lactone monomers menthide and dihydrocarvide employing Baeyer-Villiger monooxygenases (BVMOs) from Pseudomonas sp. HI-70 (CPDMO) and Rhodococcus sp. Phi1 (CHMOPhi1) as an alternative to organic synthesis. The regioselectivity of dihydrocarvide isomer formation was controlled by site-directed mutagenesis of three key active site residues in CHMOPhi1. A combination of crystal structure determination, molecular dynamics simulations, and mechanistic modeling using density functional theory on a range of models provides insight into the origins of the discrimination of the wild type and a variant CHMOPhi1 for producing different regioisomers of the lactone product. Ring-opening polymerizations of the resultant lactones using mild metal-organic catalysts demonstrate their utility in polymer production. This semisynthetic approach utilizing a biocatalytic step, non-petroleum feedstocks, and mild polymerization catalysts allows access to known and also to previously unreported and potentially novel lactone monomers and polymers.

Structural Characterization of Arginine Fingers: Identification of an Arginine Finger for the Pyrophosphatase dUTPases.

Arginine finger is a highly conserved and essential residue in many GTPase and AAA+ ATPase enzymes that completes the active site from a distinct protomer, forming contacts with the γ-phosphate of the nucleotide. To date, no pyrophosphatase has been identified that employs an arginine finger fulfilling all of the above properties; all essential arginine fingers are used to catalyze the cleavage of the γ-phosphate. Here, we identify and unveil the role of a conserved arginine residue in trimeric dUTPases that meets all the criteria established for arginine fingers. We found that the conserved arginine adjacent to the P-loop-like motif enables structural organization of the active site for efficient catalysis via its nucleotide coordination, while its direct electrostatic role in transition state stabilization is secondary. An exhaustive structure-based comparison of analogous, conserved arginines from nucleotide hydrolases and transferases revealed a consensus amino acid location and orientation for contacting the γ-phosphate of the substrate nucleotide. Despite the structurally equivalent position, functional differences between arginine fingers of dUTPases and NTPases are explained on the basis of the unique chemistry performed by the pyrophosphatase dUTPases.

A Dynamic and Responsive Host in Action: Light-Controlled Molecular Encapsulation.

The rational design of a flexible molecular box, oAzoBox4+ , incoporating both photochromic and supramolecular recognition motifs is described. We exploit the E↔Z photoisomerization properties of azobenzenes to alter the shape of the cavity of the macrocycle upon absorption of light. Imidazolium motifs are used as hydrogen-bonding donor components, allowing for sequestration of small molecule guests in acetonitrile. Upon E→Z photoisomerization of oAzoBox4+ the guest is expelled from the macrocyclic cavity.

Introducing mutations to modify the C13/C9 ratio in linoleic acid oxygenations catalyzed by rabbit 15-lipoxygenase: a QM/MM and MD study.

Lipoxygenases (LOs) are a family of nonheme iron-containing enzymes that catalyze the hydroperoxidation of several polyunsaturated fatty acids with a huge regio- and stereospecificity. Mammalian 15-LO-1 yields almost exclusively oxygenation at the C13 position of the linoleic acid (LA), its preferred substrate. This is very important because metabolites derived from oxidation in distinct positions produce opposite physiological effects. We have combined here quantum mechanics/molecular mechanics calculations with molecular dynamics simulations to show how a suitable mutation of the rabbit 15-LO-1 enzyme can produce a significant amount of products derived from oxygenation at the C9 position of LA. In effect, the Leu597Val or Leu597Ala mutants are predicted to lead to a diminution of the oxygenation C13/C9 ratio in LA as huge as five orders of magnitude. This shows that the conserved residue Leu597 actually drives the regiospecific hydroperoxidation of LA catalyzed by 15-LO-1 enzyme.

Regio- and stereospecificity in the oxygenation of arachidonic acid catalyzed by Leu597 mutants of rabbit 15-lipoxygenase: a QM/MM study.

We combined quantum mechanics/molecular mechanics calculations with molecular dynamics simulations to study the addition of O2 to the pentadienyl radical of arachidonic acid (AA) catalyzed by the Leu597Val and Leu597Ala mutants of rabbit 15-lipoxygenase (15-rLO). In the Leu597Val mutant, the addition of O2 to C15 of AA is the predominant path, although it reduces the C15/C11 product ratio by almost ten times with respect to the wildtype enzyme. The S stereochemistry is kept. Mutation to Ala causes just the opposite effect: regiospecificity favoring addition to C15 is somewhat sharper than that in the wildtype, but the stereochemistry is R. This is because the extra space created by the mutation to Ala is big enough for AA to move so that it can adopt an alternative binding mode, and this opens new feasible paths for the attack of O2 . So, we showed that the Leu597Ala mutant of 15r-LO works as an aspirin-acetylated cyclooxygenase-2, which makes 15-(R)- hydroperoxyeicosatetraenoic acid.

On the regio- and stereospecificity of arachidonic acid peroxidation catalyzed by mammalian 15-lypoxygenases: a combined molecular dynamics and QM/MM study.

15-Lipoxygenases (15-LOs) catalyse the peroxidation reaction of arachidonic acid (AA) in mammals with remarkable regio- and stereospecificity. This positional-specific peroxidation is of paramount importance because it determines the nature and biological functions of the final metabolites generated by each LO as a result of the oxidative metabolism of AA. Although several hypotheses have been formulated concerning the regio- and stereospecificity of LOs, the molecular basis of such behaviour is still unclear. Herein, we combined quantum mechanics/molecular mechanics calculations with molecular dynamics simulations of the complete rabbit 15-LO/AA solvated model to examine the most accepted hypotheses for the regio- and stereospecificity of LOs. We have found that the clue to explain this specificity is the oxygen-targeting hypothesis through steric shielding of specific residues (mainly Leu597, Gln548 and Phe175, as well as the AA tail itself). Our deductions are based primarily on the analysis of the energy barrier heights from the oxygen addition reaction profiles.

A geometrical parametrization of C1'-C5' RNA ribose chemical shifts calculated by density functional theory.

It has been recently shown that NMR chemical shifts can be used to determine the structures of proteins. In order to begin to extend this type of approach to nucleic acids, we present an equation that relates the structural parameters and the (13)C chemical shifts of the ribose group. The parameters in the equation were determined by maximizing the agreement between the DFT-derived chemical shifts and those predicted through the equation for a database of ribose structures. Our results indicate that this type of approach represents a promising way of establishing quantitative and computationally efficient analytical relationships between chemical shifts and structural parameters in nucleic acids.

Calcium-Alginate-Chitosan Nanoparticle as a Potential Solution for Pesticide Removal, a Computational Approach.

Pesticides have a significant negative impact on the environment, non-target organisms, and human health. To address these issues, sustainable pest management practices and government regulations are necessary. However, biotechnology can provide additional solutions, such as the use of polyelectrolyte complexes to encapsulate and remove pesticides from water sources. We introduce a computational methodology to evaluate the capture capabilities of Calcium-Alginate-Chitosan (CAC) nanoparticles for a broad range of pesticides. By employing ensemble-docking and molecular dynamics simulations, we investigate the intermolecular interactions and absorption/adsorption characteristics between the CAC nanoparticles and selected pesticides. Our findings reveal that charged pesticide molecules exhibit more than double capture rates compared to neutral counterparts, owing to their stronger affinity for the CAC nanoparticles. Non-covalent interactions, such as van der Waals forces, π-π stacking, and hydrogen bonds, are identified as key factors which stabilized the capture and physisorption of pesticides. Density profile analysis confirms the localization of pesticides adsorbed onto the surface or absorbed into the polymer matrix, depending on their chemical nature. The mobility and diffusion behavior of captured compounds within the nanoparticle matrix is assessed using mean square displacement and diffusion coefficients. Compounds with high capture levels exhibit limited mobility, indicative of effective absorption and adsorption. Intermolecular interaction analysis highlights the significance of hydrogen bonds and electrostatic interactions in the pesticide-polymer association. Notably, two promising candidates, an antibiotic derived from tetracycline and a rodenticide, demonstrate a strong affinity for CAC nanoparticles. This computational methodology offers a reliable and efficient screening approach for identifying effective pesticide capture agents, contributing to the development of eco-friendly strategies for pesticide removal.

Combined nuclear magnetic resonance spectroscopy and molecular dynamics study of growth hormone releasing hexapeptide GHRP-6 and a cyclic analogue.

The Growth Hormone Releasing Hexapeptide, GHRP-6 was the first of a family of synthetic peptides that enhance the release of the Growth Hormone by the pituitary gland in a dose-dependent manner. Since its discovery, it has been used as a benchmark and starting point in numerous researches aiming to obtain new drugs. Complete resonance assignment of GHRP-6 NMR spectra in both open and cyclic forms are reported, showing some differences to random coil chemical shifts. Connectivities observed in the ROESY spectra indicate spatial proximity between the aromatic residues side-chains in both molecules, as well as between residues DPhe5 and Lys6 sidechains. An ensemble of 10 structures was generated for each one of the molecules, showing RMSD values indicative of nonrandom structures. Molecular Dynamics simulations, both with and without explicit solvent, were carried out for GHRP-6 and its cyclic analogue. Conformational analysis performed on the trajectories showed a nonrandom structure with a well preserved backbone. The presence of geometrical patterns resembling those typical of π-π interactions in both peptides, suggest that this kind of interactions may be relevant for the biological activity of GHRP-6. Same conclusion can be drawn from the spatial proximity of residues DPhe5 and Lys6 sidechains.

Steric and electronic situation in the 4-X-4'-[(4''-Y-phenyl)ethynyl]biphenyl homologous series: a joint theoretical and spectroscopic study.

In this work we have studied the rotational barriers, the polarization of the acetylenic triple bond, and the molecular dipole moments in the 4-X-4'-[(4''-Y-phenyl)ethynyl]biphenyl homologous series using the density functional theory (DFT) and 1D/2D NMR spectroscopy. This series of compounds constitutes an effective base for the acquisition of liquid crystals. The equilibrium angle (theta(eq)) and the torsional barriers DeltaE(0 degrees) and DeltaE(90 degrees) are not very sensitive to the substituent effects. We have found evidence for the similarity in the pi-conjugation of the Y-substituted and X,Y-disubstituted compounds, the latter with mesomorphic properties, by means of the graphic analysis of the relationship between the molecular dipolar moment mu(D) and the difference between the (13)C NMR chemical shifts of the acetylenic carbon atoms (Deltadelta(CI[triple bond]CII) [ppm]). The obtained results contribute to a better understanding of the structure-activity relationship for potential liquid crystalline systems.

Resistance to the "last resort" antibiotic colistin: a single-zinc mechanism for phosphointermediate formation in MCR enzymes.

MCR (mobile colistin resistance) enzymes catalyse phosphoethanolamine (PEA) addition to bacterial lipid A, threatening the "last-resort" antibiotic colistin. Molecular dynamics and density functional theory simulations indicate that monozinc MCR supports PEA transfer to the Thr285 acceptor, positioning MCR as a mono- rather than multinuclear member of the alkaline phosphatase superfamily.

Theoretical study of imidazole...NO complexes.

A set of weak complexes between imidazole (Imi) and nitric oxide (NO) were calculated with UMP2/6-31++G(d,p) and UMP2/6-311++G(2d,2p) levels of theory. Planar and nonplanar geometries were considered. Complexes of NO and protonated imidazole (ImiH(+)) were also studied due to the biological important effect of Imi protonation. It was found that ring protonation increases the stability of planar complexes and does not affect significantly nonplanar complexes. The Z-H...XY (Z = C, N and X, Y = N, O) interactions resulted as hydrogen bonds according to Koch and Popelier criteria based on AIM theory. Charge transfer was also found very important for complex stabilization within our theoretical framework. Planar NO...ImiH(+) complexes are more stable than those obtained with neutral Imi.

Molecular dynamics and NMR analysis of the configurational 13C assignment of epimeric 22,23-epoxides of stigmasterol.

The determination of the stereochemistry of brasinosteroid analogs with 22,23-epoxide groups can be easily achieved by means of (13)C NMR spectroscopy. Here, we provide a rationalization of the (13)C chemical shift pattern found in 22R,23R- and 22S,23S-epoxides of stigmasterol, based on the analysis of gamma effects. (22S,23S)- and (22R,23R)-3beta-acetoxystigmast-22,23-epoxy-5,6beta-diol were used in the study as model compounds. Our methodology starts with a conformational search by means of molecular dynamics and NMR (NOE contacts) spectroscopy, which is followed by the analysis of the different gamma interactions affecting the chemical shift of interest. We demonstrate that the differences between the (13)C chemical shift patterns of 22R,23R and 22S,23S isomers arise from gamma effects as the result of diverging local conformations around the C17-C20 and C20-C22 bonds.

On the unusual 2J(C2-H(f)) coupling dependence on syn/anti CHO conformation in 5-X-furan-2-carboxaldehydes.

A remarkable difference for (2)J(C(2)-H(f)) coupling constant in syn and anti conformers of 5-X-furan-2-carboxaldehydes (X = CH(3), Ph, NO(2), Br) and a rationalization of this difference are reported. On the basis of the current knowledge of the Fermi-contact term transmission, a rather unusual dual-coupling pathway in the syn conformer is presented. The additional coupling pathway resembles somewhat that of the J(H-H) in homoallylic couplings, which are transmitted by hyperconjugative interactions involving the pi(C=C) electronic system. The homoallylic coupling pathway can be labeled as sigma*(C-H) <-- pi(C=C) --> sigma*(C-H). In the present case, this additional coupling pathway, using an analogous notation, can be labeled as sigma*(C(2)-C(C)) <-- LP(1)(O(1))...LP(2)(O(C)) --> sigma*(C(C)-H(f)) (sigma*(C(2)-C(C))) where O(1) and O(C) stand for the ring and carbonyl O atoms, respectively. This additional coupling pathway is not activated in the anti conformers since both oxygen lone pairs do not overlap.