52 research outputs found
First-Principles Modeling of Biological Systems and Structure-Based Drug-Design
No full textMolecular modeling techniques play a relevant role in drug design providing detailed information at atomistic level on the structural, dynamical, mechanistic and electronic properties of biological systems involved in diseases’ onset, integrating and supporting commonly used experimental approaches. These information are often not accessible to the experimental techniques taken singularly, but are of crucial importance for drug design. Due to the enormous increase of the computer power in the last decades, quantum mechanical (QM) or first-principles-based methods have become often used to address biological issues of pharmaceutical relevance, providing relevant information for drug design. Due to their complexity and their size biological systems are often investigated by means of a mixed quantum-classical (QM/MM) approach, which treats at an accurate QM level a limited chemically relevant portion of the system and at the molecular mechanics (MM) level the remaining of the biomolecule and its environment. This method provides a good compromise between computational cost and accuracy, allowing to characterize the properties of the biological system and the (free) energy landscape of the process in study with the accuracy of a QM description. In this review, after a brief introduction of QM and QM/MM methods, we will discuss few representative examples, taken from our work, of the application of these methods in the study of metallo-enzymes of pharmaceutical interest, of metal-containing anticancer drugs targeting the DNA as well as of neurodegenerative diseases. The information obtained from these studies may provide the basis for a rationale structure-based drug design of new and more efficient inhibitors or drugs
The Structural Role of Mg2+ Ions in a Class I RNA Polymerase Ribozyme. A Molecular Simulation Study
No full textAccording to the RNA world hypothesis, self-replicating ribozymes, storing the genetic information and being able to perform catalysis, were the constituents of the first living organisms. In particular, RNA polymerase ribozymes, similar to current proteinaceous enzymatic polymerases, may have been able to promote the synthesis of RNA strands in a primitive world. Polymerase catalysis is usually assisted by Mg2+ ions, but it is not always trivial to find out experimentally the number of Mg2+ ions placed in the active site as well as the identity and the number of their coordination ligands. Here, we addressed this issue in an artificial class I ligase ribozyme. On the basis of a recently solved crystal structure, we constructed computational models of reactant and product states of this ribozyme, considering monometallic and bimetallic species. Our models were relaxed by force field based molecular dynamics (MD) simulations and mixed quantum-classical (QM/MM) MD. The structural and dynamical properties of these models were consistent with experimental data and were validated by a comparison with the catalytic sites of proteinaceous DNA and RNA polymerases. Consistently with enzymatic polymerases, our results suggest that class I RNA ligases most probably contain two magnesium ions in the active site and they may, therefore, catalyze the junction of two RNA strands via "a two Mg2+ ions" mechanism
Influence of the membrane lipophilic environment on the structure and on the substrate access/egress routes of the human aromatase enzyme. A computational study
No full textHuman aromatase (HA), an enzyme located on the membrane of the endoplasmatic reticulum, is of crucial biological importance in the biosynthesis of estrogens. High levels of estrogens are related with important pathologies, conferring to HA a key role as a pharmacological target. In this study we provide, for the first time, an atomistic model of HA embedded on a membrane model to understand the influence of the membrane lipophilic environment on the structural and dynamical properties of HA and on the access/egress pathways of the substrate (androstenedione, ASD) and of the oxygen molecule (involved in the enzymatic process) into/from the HA active site. To this end we used several computational techniques such as force field-based molecular dynamics (MD) simulations, Random Expulsion MD, Steered MD, and Implicit Ligand Sampling. Our results show that the membrane anchoring does not markedly affect the structural properties and the flexibility of the protein, but they clearly point out that the membrane has a marked effect on the access/egress routes of the reactants, stabilizing the formation of different channels for both ASD and O-2 with respect to those observed in pure water solution. Due to the importance of HA in medicine and since access/egress channels may influence its substrate selectivity, a detailed understanding of the role of the membrane in shaping these channels may be of valuable help in drug design
QM/MM MD simulations on the enzymatic pathway of the human flap endonuclease (hFEN1) elucidate common cleavage pathways to RNase H enzymes
No full textFlap endonucleases (FENs) are nucleic acid hydrolyzing enzymes in charge of excising S'-small DNA and RNA fragments (flaps) protruding from nucleic acid structures during the lagging strand DNA replication or the long-patch base excision repair (LP-BER) processes. In this work we report, for the first time, an atomistic and energetic rendering of the enzymatic catalysis promoted by the human FEN1. After reconstruction of a reactive hFEN/double strand (ds) DNA adduct we employed mixed quantum-classical (QM/MM) metadynamics and umbrella sampling free energy calculations, with the QM part treated with the AM1/d-PhoT Hamiltonian, to perform an extensive characterization of all possible reaction pathways underlying the enzymatic cycle. Our extensive investigation points to a most likely reaction pathway very similar to that recently proposed for ribonuclease H, in which the rate determining step is the nucleophilic attack of a water to the scissile phosphate, which occurs concomitantly with its activation by the pro-Rp oxygen of the nucleobase flanking the scissile phosphate. This step requires a free energy barrier in good agreement with experimental data (Delta G(exp)double dagger = 16.1 kcal/mol vs Delta F-calc double dagger = 16 +/- 2 kcal/mol). Due to the important role of FENs in maintaining nucleic acid fidelity and cell proliferation, a detailed understanding of its enzymatic mechanism has broad interest to elucidate a key enzymatic biological process for preserving genome integrity and has implications for medical and biotechnological applications
Theoretical Studies of Homogeneous Catalysts Mimicking Nitrogenase
No full textThe conversion of molecular nitrogen to ammonia is a key biological and chemical process and represents one of the most challenging topics in chemistry and biology. In Nature the Mo-containing nitrogenase enzymes perform nitrogen ‘fixation’ via an iron molybdenum cofactor (FeMo-co) under ambient conditions. In contrast, industrially, the Haber-Bosch process reduces molecular nitrogen and hydrogen to ammonia with a heterogeneous iron catalyst under drastic conditions of temperature and pressure. This process accounts for the production of millions of tons of nitrogen compounds used for agricultural and industrial purposes, but the high temperature and pressure required result in a large energy loss, leading to several economic and environmental issues. During the last 40 years many attempts have been made to synthesize simple homogeneous catalysts that can activate dinitrogen under the same mild conditions of the nitrogenase enzymes. Several compounds, almost all containing transition metals, have been shown to bind and activate N2 to various degrees. However, to date Mo(N2)(HIPTN)3N with (HIPTN)3N= hexaisopropyl-terphenyl-triamidoamine is the only compound performing this process catalytically. In this review we describe how Density Functional Theory calculations have been of help in elucidating the reaction mechanisms of the inorganic compounds that activate or fix N2. These studies provided important insights that rationalize and complement the experimental findings about the reaction mechanisms of known catalysts, predicting the reactivity of new potential catalysts and helping in tailoring new efficient catalytic compounds
Computational Approaches Elucidate the Allosteric Mechanism of Human Aromatase Inhibition: A Novel Possible Route to Small-Molecule Regulation of CYP450s Activities?
No full textHuman aromatase (HA) is a P450 cytochrome (CYP) with an essential role in estrogen biosynthesis. Since more than 70% of breast cancers are positive for estrogenic receptor (ER), the reduction of estrogen physiological concentrations through HA inhibition is one of most important therapeutic strategies against this cancer type. Recently, experimental evidence showed that selected taxmoxifen metabolites, which are typically used as estrogen receptor modulators (SERMs), inhibit HA through an allosteric mechanism. In this work, we present a computational protocol to (i) characterize the structural framework and (ii) define the atomistic details of the determinants for the noncompetitive inhibition mechanism. Our calculations identify two putative binding sites able to efficiently bind all tamoxifen metabolites. Analysis of long-scale molecular dynamics simulations reveal that endoxifen, the most effective noncompetitive inhibitor, induces significant enzyme rigidity by binding in one of the possible peripheral sites. The consequence of this binding event is the suppression of one of the functional enzymatic collective motions associated with breathing of the substrate access channel. Moreover, an internal dynamics-based alignment of HA with six other human cytochromes shows that this collective motion is common to other members of the CYP450 protein family. On this basis, our findings may thus be of help for the development of new (pan)inhibitors for the therapeutic treatment of cancer, targeting and modulating the activity of HA and of estrogen receptor, and may also stimulate the development of new drug design strategies for chemoprevention and chemoprotection via allosteric inhibition of CYP450 protein
Insight into the Mechanism of Hydrolysis of Meropenem by OXA-23 Serine-β-lactamase Gained by Quantum Mechanics/Molecular Mechanics Calculations
No full textThe fast and constant development of drug resistant bacteria represents a serious medical emergency. To overcome this problem, the development of drugs with new structures and modes of action is urgently needed. In this work, we investigated, at the atomistic level, the mechanisms of hydrolysis of Meropenem by OXA-23, a class D β-lactamase, combining unbiased classical molecular dynamics and umbrella sampling simulations with classical force field-based and quantum mechanics/molecular mechanics potentials. Our calculations provide a detailed structural and dynamic picture of the molecular steps leading to the formation of the Meropenem-OXA-23 covalent adduct, the subsequent hydrolysis, and the final release of the inactive antibiotic. In this mechanistic framework, the predicted activation energy is in good agreement with experimental kinetic measurements, validating the expected reaction path
Covalent docking of selected boron-based serine beta-lactamase inhibitors
No full textAmpC β-lactamase is a hydrolytic enzyme conferring resistance to β-lactam antibiotics in multiple Gram-negative bacteria. Therefore, identification of non-β-lactam compounds able to inhibit the enzyme is crucial for the development of novel antibacterial therapies. In general, AmpC inhibitors have to engage the highly solvent-exposed catalytic site of the enzyme. Therefore, understanding the implications of ligand–protein induced-fit and water-mediated interactions behind the inhibitor-enzyme recognition process is fundamental for undertaking structure-based drug design process. Here, we focus on boronic acids, a promising class of beta-lactamase covalent inhibitors. First, we optimized a docking protocol able to reproduce the experimentally determined binding mode of AmpC inhibitors bearing a boronic group. This goal was pursued (1) performing rigid and flexible docking calculations aiming to establish the role of the side chain conformations; and (2) investigating the role of specific water molecules in shaping the enzyme active site and mediating ligand protein interactions. Our calculations showed that some water molecules, conserved in the majority of the considered X-ray structures, are needed to correctly predict the binding pose of known covalent AmpC inhibitors. On this basis, we formalized our findings in a docking and scoring protocol that could be useful for the structure-based design of new boronic acid AmpC inhibitors
Role of Water in the Puzzling Mechanism of the Final Aromatization Step Promoted by the Human Aromatase Enzyme. Insights from QM/MM MD Simulations
No full textThe enzyme human aromatase (HA) catalyzes the conversion of androgens to estrogens via two hydroxylation reactions and a final unique aromatization step. Despite the great interest of HA as a drug target against breast cancer detailed structural and spectroscopic information on this enzyme became available only in the past few years. As such, the enigmatic mechanism of the final aromatization step is still a matter of debate. Here, we investigated the final step of the HA enzymatic cycle via hybrid quantum-classical (QM/MM) metadynamics and blue-moon ensemble simulations. Our results show that the rate-determining step of the aromatization process is the nucleophilic attack of the distal oxygen of a peroxo-ferric species on the formyl carbon of the enol-19-oxo-androstenedione, which occurs with a free energy barrier (ΔF(#)) of ∼ 16.7 ± 1.9 kcal/mol, in good agreement with experimental data. This reaction is followed by a water mediated 1β-hydrogen abstraction (ΔF(#) = 7.9 ± 0.8 kcal/mol) and by the formation of a hydroxo-ferric moiety. This latter may be finally protonated by a hydrogen delivery channel involving Asp309 and Thr310, both residues pointed out as crucial for HA activity. In the absence of the catalytic water in the active site the substrate does not assume a position suitable to undergo the nucleophilic attack. Our data not only reveal a novel possible mechanism for the aromatization process consistent with some of the spectroscopic and kinetic data available in the literature, complementing current knowledge on the mechanism of this enzyme, but also point out a remarkable influence of the level of theory used on the calculated free energy barriers. The structural information obtained in this study may be used for the rational structure-based drug design of HA inhibitors to be employed in breast cancer therapy
Structural and Dynamic Properties of Monoclonal Antibodies Immobilized on CNTs: A Computational Study
No full textDue to the widespread application of carbon nanotube (CNT)-based materials in nanomedicine, it is nowadays of paramount importance to unravel at the atomistic level of detail the structural properties of such bioconjugates in order to rationalize and predict the effect exerted by the graphitic framework on the bio-active counterpart. In this paper, we report for the first time all-atom explicit solvent molecular dynamics (MD) simulations investigating the structural and dynamic properties of a noncovalent bioconjugate in which the monoclonal Cetuximab antibody (Ctx) is adsorbed on a CNT surface. Upon selection of the three most representative adsorption modes as obtained by docking studies, force-field MD and DFT simulations unambiguously showed that hydrophobic interactions mainly govern the adsorption of the protein on the graphitic surface. Two main adsorption poses have been predicted: a pose-fab (p-fab) and pose-fc (p-fc) (fab = fragment antigen binding region; fc = fragment crystallizable region), the former being favored with small-diameter tubes (40 angstrom). In all the predicted poses, the secondary structure of Ctx is largely unaffected by the presence of the graphitic surface and, consistently with previous literature studies, our simulations reveal that positively charged amino acidic residues, such as Lys and Arg, predominantly contribute to the stabilization of the CNTCtx complex acting like surfactants. The predicted structural models are consistent with the experimental data, for which the immobilization of the antibody on CNTs does not disrupt the structural and recognition properties of the Ctx, consequently supporting the reliability of the used bioconjugation strategy for engineering stable and responsive hybrid nanomaterials for therapeutic applications. Moreover, a remarkable structural similarity of Ctx with antibodies of different isotypes suggests that in principle the CNT framework can interact in the same manner with all antibodies currently used in clinical applications
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