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Publications

Publications by Susana Rodrigues Pereira

2013

Computational study of the covalent bonding of microcystins to cysteine residues - a reaction involved in the inhibition of the PPP family of protein phosphatases

Authors
Pereira, SR; Vasconcelos, VM; Antunes, A;

Publication
FEBS JOURNAL

Abstract
Microcystins (MCs) are cyclic peptides, produced by cyanobacteria, that are hepatotoxic to mammals. The toxicity mechanism involves the potent inhibition of protein phosphatases, as the toxins bind the catalytic subunits of five enzymes of the phosphoprotein phosphatase (PPP) family of serine/threonine-specific phosphatases: Ppp1 (aka PP1), Ppp2 (aka PP2A), Ppp4, Ppp5 and Ppp6. The interaction with the proteins includes the formation of a covalent bond with a cysteine residue. Although this reaction seems to be accessory for the inhibition of PPP enzymes, it has been suggested to play an important part in the biological role of MCs and furthermore is involved in their nonenzymatic conjugation to glutathione. In this study, the molecular interaction of microcystins with their targeted PPP catalytic subunits is reviewed, including the relevance of the covalent bond for overall inhibition. The chemical reaction that leads to the formation of the covalent bond was evaluated in silico, both thermodynamically and kinetically, using quantum mechanical-based methods. As a result, it was confirmed to be a Michael-type addition, with simultaneous abstraction of the thiol hydrogen by a water molecule, transfer of hydrogen from the water to the alpha,beta-unsaturated carbonyl group of the microcystin and addition of the sulfur to the beta-carbon of the microcystin moiety. The calculated kinetics are in agreement with previous experimental results that had indicated the reaction to occur in a second step after a fast noncovalent interaction that inhibited the enzymes per se.

2011

The phosphoprotein phosphatase family of Ser/Thr phosphatases as principal targets of naturally occurring toxins

Authors
Pereira, SR; Vasconcelos, VM; Antunes, A;

Publication
CRITICAL REVIEWS IN TOXICOLOGY

Abstract
Phosphoprotein phosphatases (PPPs) constitute one of three otherwise unrelated families of enzymes that specialize in removing the phosphate group from phosphorylated serine and threonine residues. The involvement of PPP enzymes in the regulation of processes such as gene expression, DNA replication, morphogenesis, synaptic transmission, glycogen metabolism, and apoptosis has underscored their potential as targets for the treatment of a variety of conditions such as cancer, diabetes, or Alzheimer''s disease. Interestingly, PPP enzymes also constitute the physiological target of multiple naturally occurring toxins, including microcystins from cyanobacteria and cantharidin from beetles. This review is devoted to the PPP family of enzymes--with a focus on the human PPPs--and the naturally occurring toxins that are known to potently impair their activity. The interaction of the toxins with the enzymes is evaluated in atomic detail to obtain insight on two complementary aspects: (1) which specific structural differences within the similarly folded catalytic core of the PPP enzymes explain their diverse sensitivities to toxin inhibition and (2) which structural features presented by the various toxins account for the differential inhibitory potency towards each PPP. These analyses take advantage of numerous site-directed mutagenesis studies, structure-activity evaluations, and recent crystallographic structures of PPPs bound to different toxins.

2005

Overview of ribonucleotide reductase inhibitors: An appealing target in anti-tumour therapy

Authors
Cerqueira, NMFSA; Pereira, S; Fernandes, PA; Ramos, MJ;

Publication
CURRENT MEDICINAL CHEMISTRY

Abstract
This review provides up-to-date information on the inhibition of ribonucleotide reductase (RNR), the enzyme that catalyses the reduction of ribonucleotides into deoxyribonucleotides. Taking in account that DNA replication and repair are essential mechanisms for cell integrity and are dependent on the availability of deoxyribonucleotides, many researchers are giving special attention to this enzyme, since it is an attractive target to treat several diseases of our time specially cancer. This investment has already given some benefits since some of these inhibitors show potent chemotherapeutic efficacy against a wide range of tumours such as non-small cell lung cancer, adenocarcinoma of pancreas, bladder cancer, leukaemia and some solid tumours. In fact a few of them have already been approved for the clinical treatment of some kinds of cancer. All aspects of RNR inhibition and corresponding inhibitors are the subjects of this review. The inhibitors are divided in three main groups: translation inhibitors, which unable the formation of the enzyme; dimerization inhibitors that prevent the complexation of the two RNR subunits (R1 and R2); and catalytic inhibitors that inactivate subunit R1 and/or subunit R2, leading to RNR inactivity. In this last group special focus will be addressed to substrate analogues.

2005

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

Authors
Pereira, S; Fernandes, PA; Ramos, MJ;

Publication
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY

Abstract
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 O-2. They correspond to a refinement and a complement of those proposed in the literature.

2006

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

Authors
Pereira, S; Cerqueira, NMFSA; Fernandes, PA; Ramos, MJ;

Publication
EUROPEAN BIOPHYSICS JOURNAL WITH BIOPHYSICS LETTERS

Abstract
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.

2004

Mechanism for ribonucleotide reductase inactivation by the anticancer drug gemcitabine

Authors
Pereira, S; Fernandes, PA; Ramos, MJ;

Publication
JOURNAL OF COMPUTATIONAL CHEMISTRY

Abstract
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. (C) 2004 Wiley Periodicals, Inc.

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