Biomedical subjects

Maria João Ramos

Publications and source records attributed to Maria João Ramos.

At least 19 recordsLinked to original sources

PubMed2007

Theoretical studies on farnesyltransferase: the distances paradox explained.

In spite of the enormous interest that has been devoted to its study, the mechanism of the enzyme farnesyltransferase (FTase) remains the subject of several crucial doubts. In this article, we shed a new light in one of the most fundamental dilemmas that characterize the mechanism of this puzzling enzyme commonly referred to as the "distances paradox", which arises from the existence of a large 8-A distance between the two reactive atoms in the reaction catalyzed by this enzyme: a Zn-bound cysteine sulphur atom from a peptidic substrate and the farnesyldiphosphate (FPP) carbon 1. This distance must be overcome for the reaction to occur. In this study, the two possible alternatives were evaluated by combining molecular mechanics (AMBER) and quantum chemical calculations (B3LYP). Basically, our results have shown that an activation of the Zn-bound cysteine thiolate with subsequent displacement from the zinc coordination sphere towards the FPP carbon 1 is not a realistic hypothesis of overcoming the large distance reported in the crystallographic structures of the ternary complexes between the two reactive atoms, but that a rotation involving the FPP molecule can bring the two atoms closer with moderate energetic cost, coherent with previous experimental data. This conclusion opens the door to an understanding of the chemical step in the farnesylation reaction.

Binding Sites↗

PubMed2006

Protein-ligand docking: current status and future challenges.

Understanding the ruling principles whereby protein receptors recognize, interact, and associate with molecular substrates and inhibitors is of paramount importance in drug discovery efforts. Protein-ligand docking aims to predict and rank the structure(s) arising from the association between a given ligand and a target protein of known 3D structure. Despite the breathtaking advances in the field over the last decades and the widespread application of docking methods, several downsides still exist. In particular, protein flexibility-a critical aspect for a thorough understanding of the principles that guide ligand binding in proteins-is a major hurdle in current protein-ligand docking efforts that needs to be more efficiently accounted for. In this review the key concepts of protein-ligand docking methods are outlined, with major emphasis being given to the general strengths and weaknesses that presently characterize this methodology. Despite the size of the field, the principal types of search algorithms and scoring functions are reviewed and the most popular docking tools are briefly depicted. Recent advances that aim to address some of the traditional limitations associated with molecular docking are also described. A selection of hand-picked examples is used to illustrate these features.

Algorithms↗

PubMed2006

Structural divergence and adaptive evolution in mammalian cytochromes P450 2C.

Cytochromes P450 (CYPs) comprise a superfamily of enzymes involved in various physiological functions, including the metabolism of drugs and carcinogenic compounds present in food, making them of great importance for human health. The possibility that CYPs could be broadening or changing substrate specificity in accordance to the high diversity of xenobiotics compounds environmentally available suggests that their metabolic function could be under adaptive evolution. We evaluated the existence of functional divergence and signatures of selection on mammalian genes from the drug-metabolizing CYP2 family. Thirteen of the sites found to be functionally divergent and the eight found to be under strong positive selection occurred in important functional domains, namely on the substrate entrance channel and within the active site. Our results provide insight into CYPs evolution and the role of molecular adaptation in enzyme substrate-specificity diversification.

Animals↗

PubMed2006

Molecular interactions between human cytochrome P450 1A2 and flavone derivatives.

Activation by human cytochrome P450 1A2 (hCYP1A2) of heterocyclic amines is assumed to trigger of a number of carcinogenic processes. In this work, a group of natural inhibitors of human cytochrome P450 1A2 reported in literature has been theoretically analysed. These consist of flavone hydroxylated derivatives, natural compounds that exist in plants and associated products. Different theoretical/computational tools were used to describe the specific molecular interactions between these compounds and hCYP1A2. Based on this analysis, a method is proposed for helping the selection of specific molecular features that enhance protein-inhibitor interaction.

Binding Sites↗

PubMed2005

Dehydration of ribonucleotides catalyzed by ribonucleotide reductase: the role of the enzyme.

This article focuses on the second step of the catalytic mechanism for the reduction of ribonucleotides catalyzed by the enzyme Ribonucleotide Reductase (RNR). This step corresponds to the protonation/elimination of the substrate's C-2' hydroxyl group. Protonation is accomplished by the neighbor Cys-225, leading to the formation of one water molecule. This is a very relevant step since most of the known inhibitors of this enzyme, which are already used in the fight against certain forms of cancer, are 2'-substituted substrate analogs. Even though some theoretical studies have been performed in the past, they have modeled the enzyme with minimal gas-phase models, basically represented by a part of the side chain of the relevant amino acids, disconnected from the protein backbone. This procedure resulted in a limited accuracy in the position and/or orientation of the participating residues, which can result in erroneous energetics and even mistakes in the choice of the correct mechanism for this step. To overcome these limitations we have used a very large model, including a whole R1 model with 733 residues plus the substrate and 10 A thick shell of water molecules, instead of the minimal gas-phase models used in previous works. The ONIOM method was employed to deal with such a large system. This model can efficiently account for the restrained mobility of the reactive residues, as well as the long-range enzyme-substrate interactions. The results gave additional information about this step, which previous small models could not provide, allowing a much clearer evaluation of the role of the enzyme. The interaction energy between the enzyme and the substrate along the reaction coordinate and the substrate steric strain energy have been obtained. The conclusion was that the barrier obtained with the present model was very similar to the one previously determined with minimal gas-phase models. Therefore, the role of the enzyme in this step was concluded to be mainly entropic, rather than energetic, by placing the substrate and the two reactive residues in a position that allows for the highly favorable concerted trimolecular reaction, and to protect the enzyme radical from the solvent.

Binding Sites↗

PubMed2005

Computational studies on class I ribonucleotide reductase: understanding the mechanisms of action and inhibition of a cornerstone enzyme for the treatment of cancer.

This review provides a synthesis of recent work, using computational methods, on the action and inhibition mechanisms of class I ribonucleotide reductase (RNR). This enzyme catalyzes the rate-limiting step of the pathway for the synthesis of DNA monomers and, therefore, has long been regarded as an important target for therapies aiming to control pathologies that depend strongly on DNA replication. In fact, over the last years, several molecules, which are able to impair RNR activity by different mechanisms, have been applied effectively in anti-cancer, anti-viral and anti-parasite therapies. A better understanding of the chemical mechanisms involved in normal catalysis and in inhibition of the enzyme is important for the rational design of more specific and effective inhibitor compounds. To achieve this goal, computational methods, particularly quantum chemical calculations, have been used more and more frequently. The ever-growing capabilities of these methods together with undeniable advantages make it a stimulating area for research purposes.

Animals↗

PubMed2005

Density-functional study of mechanisms for the cofactor-free decarboxylation performed by uroporphyrinogen III decarboxylase.

Uroporphyrinogen III decarboxylase catalyzes the fifth step in heme biosynthesis: the elimination of carboxyl groups from the four acetate side chains of uroporphyrinogen III to yield coproporphyrinogen III. The enzyme acts by successively protonating each of the four pyrrole rings present in the substrate, thereby allowing decarboxylation of their side chains, but the identity of the proton donors has not been established yet. Tyr164 has been suggested as a proton donor, and Asp86 has been proposed to act either as a proton donor or as an intermediate-stabilizing residue. We have performed density-functional calculations to study this reaction mechanism, and found that the rate-limiting step is substrate protonation, rather than decarboxylation. Surprisingly, whereas Tyr164 is unable to protonate the substrate, this protonation can be effected by a nearby arginine residue (Arg37), with a free energy barrier of 21.4 kcal.mol(-1), in remarkable agreement with the experimental value of 19.5 kcal.mol(-1). The central positioning of this residue in close proximity to all four pyrrole rings in the substrate may play a key role in the sequential activation of each of these moieties.

Coproporphyrinogens↗

PubMed2005

Discovery of a large number of previously unrecognized mitochondrial pseudogenes in fish genomes.

Nuclear inserted copies of mitochondrial origin (numts) vary widely among eukaryotes, with human and plant genomes harboring the largest repertoires. Numts were previously thought to be absent from fish species, but the recent release of three fish nuclear genome sequences provides the resource to obtain a more comprehensive insight into the extent of mtDNA transfer in fishes. From the sequence analyses of the genomes of Fugu rubripes, Tetraodon nigroviridis, and Danio rerio, we have identified 2, 5, and 10 recent numt integrations, respectively, which integrated into those genomes less than 0.6 million years (Myr) ago. Such results contradict the hypothesis of absence or rarity of numts in fishes, as (i) the ratio of numts to the total size of the nuclear genome in T. nigroviridis was superior to the ratio observed in several higher vertebrate species (e.g., chicken, mouse, and rat), and only surpassed by humans, and (ii) the mtDNA coverage transferred to the nuclear genome of D. rerio is exceeded only by human and mouse, within the whole range of eukaryotic genomes surveyed for numts. Additionally, 335, 336, and 471 old numts (>12.5 Myr) were detected in F. rubripes, T. nigroviridis, and D. rerio, respectively. Surprisingly, old numts are inserted preferentially into known or predicted genes, as inferred for recent numts in human. However, because in fish genomes such integrations are old, they are likely to represent evolutionary successes and they may be considered a potential important evolutionary mechanism for the enhancement of genomic coding regions.

Animals↗

PubMed2005

Theoretical study of the suicide inhibition mechanism of the enzyme pyruvate formate lyase by methacrylate.

The determination of pyruvate formate lyase crystallographic structure brought new insights to its mechanism of reaction and presented the possibility of a direct attack to the substrate from cysteine 418 as opposed to the previously expected cysteine 419. An inhibition study performed by Knappe and co-workers, using substrate-analogue methacrylate, confirms that cysteine 418 is most likely to add directly to pyruvate, since an inhibition product has been found as a substituent in this residue. The work presented here consists of a study of the inhibition mechanism of pyruvate formate lyase by methacrylate, using density functional theory with the hybrid B3LYP functional. We were able to determine all pertinent structures, confirm the proposed experimental mechanism, and add important detail to the energy profile associated with the mechanism of inhibition. Additionally, the obtained results provide the energy values for both the chemical reaction and the stereochemical reorganization necessary in order for the thiol-methacrylate adduct to come within reactional reach of Cys419.

Acetyltransferases↗

PubMed2005

Theoretical study on the inhibition of ribonucleotide reductase by 2'-mercapto-2'-deoxyribonucleoside-5'-diphosphates.

Ribonucleotide reductase (RNR) is responsible for the reduction of ribonucleotides into the correspondent 2'-deoxyribonucleotides in the only physiological process that yields the monomers of DNA. The enzyme has thus become an attractive target for chemotherapies that fight proliferation-based diseases, specifically cancer and infections by some viruses and parasites. 2'-Mercapto-2'-deoxyribonucleoside-5'-diphosphates (SHdNDP) are mechanism-based inhibitors of RNR and therefore potential chemotherapeutic agents for those indications. Previous experimental studies established the in vitro and in vivo activity of SHdNDP. In the in vitro studies, it was observed that the activity was dependent on the oxidative status of the medium, with the inactivation of RNR only occurring when molecular oxygen was available. To better understand the mechanism involved in RNR inactivation by SHdNDP, we performed theoretical calculations on the possible reactions between the inhibitors and the RNR active site. As a result, we propose the possible mechanistic pathways for the chemical events that occur in the absence and in the presence of O2. They correspond to a refinement and a complement of those proposed in the literature.

Deoxyribonucleosides↗

PubMed2005

Computational insight into anti-mutagenic properties of CYP1A flavonoid ligands.

Cytochrome P450 1A (CYP1A) is a subclass of enzymes involved in the biotransformation of heterocyclic amines present in cooked red meat to carcinogenic compounds. Anti-cancer properties have long been associated with flavonoids, and some compounds of this class have been shown to interact directly with CYP1A2. The understanding of this interaction is the purpose of this work. As the number of experimentally tested molecules is limited, two complementary methods in terms of information provided, are proposed for the study of protein-inhibitor interaction as alternatives to a QSAR analysis, using quantum mechanics as well as molecular mechanics.

Antimutagenic Agents↗

PubMed2004

Unraveling the mechanism of the farnesyltransferase enzyme.

Farnesyltransferase enzyme (FTase) is currently one of the most fascinating targets in cancer research. Studies in other areas, namely in the fight against parasites and viruses, have also led to very promising results. However, in spite of the thrilling achievements in the development of farnesyltransferase inhibitors (FTIs) over the past few years, the farnesylation mechanism remains, to some degree, a mystery. This review tries to shed some light on this puzzling enzyme by analyzing seven key mechanistic dilemmas, based on recent studies that have dramatically changed the way this enzyme is currently perceived.

Animals↗

PubMed2004

Evans Blue is an inhibitor of nuclear factor-kappa B (NF-kappaB)-DNA binding.

Nuclear factor-kappa B (NF-kappaB) is an important transcription factor, involved in many immune and inflammatory responses. It is critical in HIV gene expression as it has kappa B binding sites in the HIV-1 long-terminal repeat. Hence, targeting NF-kappaB to prevent its DNA binding holds a significant therapeutic potential. In this context, we report Evans Blue as a novel inhibitor of NF-kappaB-DNA binding. Evans Blue was found to be inhibiting DNA binding of NF-kappaB at a low concentration of 100 microM. Further, molecular modeling studies using docking and generation of electrostatic potential maps predicted a possible binding mode of EB to the DNA binding region of NF-kappaB, consistent with the experimental activity.

Cell Nucleus↗

PubMed2004

Farnesyltransferase--new insights into the zinc-coordination sphere paradigm: evidence for a carboxylate-shift mechanism.

Despite the enormous interest that has been devoted to the study of farnesyltransferase, many questions concerning its catalytic mechanism remain unanswered. In particular, several doubts exist on the structure of the active-site zinc coordination sphere, more precisely on the nature of the fourth ligand, which is displaced during the catalytic reaction by a peptide thiolate. From available crystallographic structures, and mainly from x-ray absorption fine structure data, two possible alternatives emerge: a tightly zinc-bound water molecule or an almost symmetrical bidentate aspartate residue (Asp-297beta). In this study, high-level theoretical calculations, with different-sized active site models, were used to elucidate this aspect. Our results demonstrate that both coordination alternatives lie in a notably close energetic proximity, even though the bidentate hypothesis has a somewhat lower energy. The Gibbs reaction and activation energies for the mono-bidentate conversion, as well as the structure for the corresponding transition state, were also determined. Globally, these results indicate that at room temperature the mono-bidentate conversion is reversible and very fast, and that probably both states exist in equilibrium, which suggests that a carboxylate-shift mechanism may have a key role in the farnesylation process by assisting the coordination/displacement of ligands to the zinc ion, thereby controlling the enzyme activity. Based on this equilibrium hypothesis, an explanation for the existing contradictions between the crystallographic and x-ray absorption fine structure results is proposed.

Alkyl and Aryl Transferases↗

PubMed2004

Mechanism for ribonucleotide reductase inactivation by the anticancer drug gemcitabine.

Gemcitabine (2',2'-difluoro-2'-deoxycytidine, dFdC) is a very promising anticancer drug, already approved for clinical use in three therapeutic indications. It is metabolized intracellularly to 5'-diphosphate (dFdCDP), which is known to be a potent inhibitor of ribonucleotide reductase (RNR). Although several nucleotide analogs show in vitro capacity of RNR inactivation, none has shown the in vivo efficacy of dFdCDP. Accordingly, the experimental data suggests that its mechanism of inhibition is different from the other known RNR suicide inhibitors. Enzyme inhibition in the absence of reductive species leads to complete loss of the essential radical in subunit R2, and formation of a new nucleotide-based radical. Interestingly, however, the presence of the reductants does not prevent inhibition--the radical is not lost but the targeted subunit of RNR becomes R1, which is inactivated possibly by alkylation. We have conducted a theoretical study, which led us to the first proposal of a possible mechanism for RNR inhibition by dFdCDP in the absence of reductants. This mechanism turned out to be very similar to the natural substrate reduction pathway and only deviates from the natural course after the formation of the well-known disulphide bridge. This deviation is caused precisely by the F atom in the beta-face, only present in this inhibitor. The essential radical in R2 is lost, and so is the enzyme catalytic activity. The nucleotide-based radical that constitutes the end product of our mechanism has been suggested in the literature as a possible candidate for the one detected experimentally. In fact, all experimental data available has been reproduced by the theoretical calculations performed here.

Antineoplastic Agents↗

PubMed2004

Definition of an electronic profile of compounds with inhibitory activity against hematin aggregation in malaria parasite.

Malaria is one of the most important parasitic diseases, affecting almost half of the world and posing a threat to the other half. Xanthone derivatives can behave as antimalarial drugs in the same mechanistic way as chloroquine and other related quinolines. This action is due to the inhibition of the detoxification pathway of the parasite, responsible for the production of hemozoin. We report a study of the electronic properties of the xanthonic and quinolinic compounds based on DFT calculations, in order to determine a pattern that could be applied to the development of new potentially active antimalarial molecules. As a result, a new interpretation of structure-activity relationship of the quinoline antimalarial drugs, and of the active hydroxylated xanthones is proposed here. We conclude that electronic features rather than steric factors control primarily the inhibitory activity of the studied compounds against hematin aggregation, concurring to a potential antimalarial activity.

Animals↗

PubMed2004

Theoretical study of ribonucleotide reductase mechanism-based inhibition by 2'-azido-2'-deoxyribonucleoside 5'-diphosphates.

2'-azido-2'-deoxyribonucleoside 5'-diphosphates are mechanism-based inhibitors of Ribonucleotide Reductase. Considerable effort has been made to elucidate their mechanism of inhibition, which is still controversial and not fully understood. Previous studies have detected the formation of a radical intermediate when the inhibitors interact with the enzyme, and several authors have proposed possible structures for this radical. We have conducted a theoretical study of the possible reactions involved, which allowed us to identify the structure of the new radical among the several proposals. A new reactional path is also proposed that is the most kinetically favored to yield this radical and ultimately inactivate the enzyme. The energetic involved in this mechanism, both for radical formation and radical decay, as well as the calculated Hyperfine Coupling Constants for the radical intermediate, are in agreement with the correspondent experimental values. This mechanistic alternative is fully coherent with remaining experimental data.

Deoxyribonucleosides↗

PubMed2004

Theoretical insights into the mechanism for thiol/disulfide exchange.

The mechanism for thiol/disulfide exchange has been studied with high-level theoretical calculations. Free energies, transition structures, charge densities, and solvent effects along the reaction pathway have been determined for the first time. Mechanistic results agree with experimental data, and support the idea that the thiolate is the reacting species and that the reaction indeed proceeds through an uncomplicated S(N)2 transition state. The transition structures have the charge density evenly concentrated in the attacking and leaving sulfur atoms. The charge densities allow us to rationalize the solvent effects. As transition structures have the charge density more widely distributed than reactants, hydrophobic environments catalyze the reaction. The effect can be so dramatic that disulfide exchange inside the active site of ribonucleotide reductase is estimated to be catalyzed 10(3) times faster than the reaction in water. It was also found that attack by thiol is much faster than previously assumed, if mediated through water chains. Although the present results, as well as experimental data, still suggest that thiolate is the main reaction species, water-mediated thiol attack is almost kinetically competitive, and can eventually become competitive under specific experimental conditions.

Journal Article↗