Nuno M. F. S. A. Cerqueira

Overview of Ribonucleotide Reductase Inhibitors: An Appealing Target in Anti-Tumour Therapy

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.

The mechanism of formate oxidation by metal-dependent formate dehydrogenases.

Metal-dependent formate dehydrogenases (Fdh) from prokaryotic organisms are members of the dimethyl sulfoxide reductase family of mononuclear molybdenum-containing and tungsten-containing enzymes. Fdhs catalyze the oxidation of the formate anion to carbon dioxide in a redox reaction that involves the transfer of two electrons from the substrate to the active site. The active site in the oxidized state comprises a hexacoordinated molybdenum or tungsten ion in a distorted trigonal prismatic geometry. Using this structural model, we calculated the catalytic mechanism of Fdh through density functional theory tools. The simulated mechanism was correlated with the experimental kinetic properties of three different Fdhs isolated from three different Desulfovibrio species. Our studies indicate that the C–H bond break is an event involved in the rate-limiting step of the catalytic cycle. The role in catalysis of conserved amino acid residues involved in metal coordination and near the metal active site is discussed on the basis of experimental and theoretical results.

The effect of the sixth sulfur ligand in the catalytic mechanism of periplasmic nitrate reductase

The catalytic mechanism of nitrate reduction by periplasmic nitrate reductases has been investigated using theoretical and computational means. We have found that the nitrate molecule binds to the active site with the Mo ion in the +6 oxidation state. Electron transfer to the active site occurs only in the proton‐electron transfer stage, where the MoV species plays an important role in catalysis. The presence of the sulfur atom in the molybdenum coordination sphere creates a pseudo‐dithiolene ligand that protects it from any direct attack from the solvent. Upon the nitrate binding there is a conformational rearrangement of this ring that allows the direct contact of the nitrate with MoVI ion. This rearrangement is stabilized by the conserved methionines Met141 and Met308. The reduction of nitrate into nitrite occurs in the second step of the mechanism where the two dimethyl‐dithiolene ligands have a key role in spreading the excess of negative charge near the Mo atom to make it available for the chemical reaction. The reaction involves the oxidation of the sulfur atoms and not of the molybdenum as previously suggested. The mechanism involves a molybdenum and sulfur‐based redox chemistry instead of the currently accepted redox chemistry based only on the Mo ion. The second part of the mechanism involves two protonation steps that are promoted by the presence of MoV species. MoVI intermediates might also be present in this stage depending on the availability of protons and electrons. Once the water molecule is generated only the MoVI species allow water molecule dissociation, and, the concomitant enzymatic turnover.

Receptor-based virtual screening protocol for drug discovery

Computational aided drug design (CADD) is presently a key component in the process of drug discovery and development as it offers great promise to drastically reduce cost and time requirements. In the pharmaceutical arena, virtual screening is normally regarded as the top CADD tool to screen large libraries of chemical structures and reduce them to a key set of likely drug candidates regarding a specific protein target. This chapter provides a comprehensive overview of the receptor-based virtual screening process and of its importance in the present drug discovery and development paradigm. Following a focused contextualization on the subject, the main stages of a virtual screening campaign, including its strengths and limitations, are the subject of particular attention in this review. In all of these stages special consideration will be given to practical issues that are normally the Achilles heel of the virtual screening process.

Virtual screening in drug design and development.

Virtual screening (VS) is presently a key component in the process of drug design and development. VS is normally regarded as the selection of likely drug candidates from large libraries of chemical structures by using computational methodologies. However, the generic definition of VS is significantly wider and may encompass many different methods. This review tries to present a comprehensive overview of the virtual screening process and of its importance in the present drug discovery and development paradigm. Following a focused contextualization on the subject, an introduction to the general types of virtual screening methodologies is presented. The main stages of a virtual screening campaign, including its strengths and limitations, are the subject of particular attention in this review. This analysis is complemented with a careful selection of VS success stories. Finally, a reflection on the future challenges of this promising methodology is drawn.

Unraveling the enigmatic mechanism of L-asparaginase II with QM/QM calculations.

In this paper, we have studied the catalytic mechanism of L-asparaginase II computationally. The reaction mechanism was investigated using the ONIOM methodology. For the geometry optimization we used the B3LYP/6-31G(d):AM1 level of theory, and for the single points we used the M06-2X/6-311++G(2d,2p):M06-2X/6-31G(d) level of theory. It was demonstrated that the full mechanism involves three sequential steps and requires the nucleophilic attack of a water molecule on the substrate prior to the release of ammonia. There are three rate-limiting states, which are the reactants, the first transition state, and the last transition state. The energetic span is 20.2 kcal/mol, which is consistent with the experimental value of 16 kcal/mol. The full reaction is almost thermoneutral. The proposed catalytic mechanism involves two catalytic triads that play different roles in the reaction. The first triad, Thr12-Lys162-Asp90, acts by deprotonating a water molecule that subsequently binds to the substrate. The second triad, Thr12-Ty25-Glu283, acts by stabilizing the tetrahedral intermediate that is formed after the nucleophilic attack of the water molecule to the substrate. We have shown that a well-known Thr12-substrate covalent intermediate is not formed in the wild-type mechanism, even though our results suggest that its formation is expected in the Thr89Val mutant. These results have provided a new understanding of the catalytic mechanism of L-asparaginases that is in agreement with the available experimental data, even though it is different from all earlier proposals. This is of particular importance since this enzyme is currently used as a chemotherapeutic drug against several types of cancer and in the food industry to control the levels of acrylamide in food.

QSAR analysis of 2-benzoxazolyl hydrazone derivatives for anticancer activity and its possible target prediction

QSAR studies on a series of 2-benzoxazolyl hydrazone derivatives against various cancer cell lines were carried out to interpret the physicochemical properties responsible for the antitumor activity. The integy moments of the molecules (vsurf_ID8 and vsurf_IW6) reveals that the active site surface or the biological membrane where these compounds bind or penetrate must have a very specific and localized hydrophobic region. These integy moments reduce the interaction energy between the molecule and the water, which improve the antitumor activity. The potential energy descriptors indicate that the flexibility of the freely rotatable bonds is important for the interaction with the chemotherapeutic target and/or barriers to reach the target. Comparing the results obtained from this study and other QSAR studies addressed to similar compounds, we concluded that the benzoxazolyl derivatives may bind to the same target. The present analysis has shown that the antitumor activity can be improved with the presence of specific hydrophobic substituents and electro-donating groups nearby the hydrazone moiety. Moreover, the formation of an intramolecular hydrogen bond has a high impact on the pharmacological activity of these compounds. The information gathered from these studies provides useful information about the binding site of these compounds.

Molecular determinants of ligand specificity in family 11 carbohydrate binding modules – an NMR, X‐ray crystallography and computational chemistry approach

The direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi‐subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate‐binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X‐ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 Å in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies.

Ribonucleotide activation by enzyme ribonucleotide reductase: Understanding the role of the enzyme

This article focuses on the first step of the catalytic mechanism for the reduction of ribonucleotides catalyzed by the enzyme Ribonucleotide Reductase (RNR). This corresponds to the activation of the substrate. In this work a large model of the active site region involving 130 atoms was used instead of the minimal gas phase models used in previous works. The ONIOM method was employed to deal with such a large system. The results gave additional information, which previous small models could not provide, allowing a much clearer evaluation of the role of the enzyme in this step. Enzyme–substrate interaction energies, specific transition state stabilization, and substrate steric strain energies were obtained. It was concluded that the transition state is stabilized in 4.0 kcal/mol by specific enzyme–substrate interactions. However, this stabilization is cancelled by the cost in conformational energy for the enzyme to adopt the transition state geometry; the overall result is that the enzyme machinery does not lead to a rate enhancement in this step. It was also found that the substrate binds to the active site with almost no steric strain, emphasizing the complementarity and specificity of the RNR active site for nucleotide binding. The main role of the enzyme at the very beginning of the catalytic cycle was concluded to be to impose stereospecifity upon substrate activation and to protect the enzyme radical from the solvent, rather than to be an reaction rate enhancement.

Application of quantum mechanics/molecular mechanics methods in the study of enzymatic reaction mechanisms

Quantum mechanics/molecular mechanics (QM/MM) methods offer a very appealing option for the computational study of enzymatic reaction mechanisms, by separating the problem into two parts that can be treated with different computational methods. Hence, in a QM/MM formalism, the part of the system in which catalysis actually occurs and that involves the active site, substrates and directly participating amino acid residues is treated at an adequate quantum mechanical level to describe the chemistry taking place. For the remaining of the enzyme, which does not participate directly in the reaction, but that typically involves a much larger number of atoms, molecular mechanics is employed, traditionally through the application of a biomolecular force field. When applied with care, QM/MM methods can be used with great advantage in comparing, at a structural and energetic level, different mechanistic proposals, discarding mechanistic alternatives and proposing new mechanistic pathways that are consistent with the available experimental data. With time, diverse flavors within the QM/MM methods have emerged, differing in a variety of technical and conceptual aspects. Hence present alternatives differ between additive and subtractive QM/MM schemes, the type of boundary schemes, and the way in which the electrostatic interactions between the two regions are accounted for. Also, single‐conformation QM/MM, multi‐PES approaches, and QM/MM Molecular Dynamics coexist today, each type with its own advantages and limitations. This review focuses on the application of QM/MM methods in the study of enzymatic reaction mechanisms, briefly presenting also the most important technical aspects involved in these calculations. Particular attention is dedicated to the application of the single‐conformation QM/MM, multi‐PES QM/MM studies, and QM/MM‐FEP methods and to the advantages and disadvantages of the different types of QM/MM. Recent breakthroughs are also introduced. A selection of hand‐picked examples is used to illustrate such features. WIREs Comput Mol Sci 2017, 7:e1281. doi: 10.1002/wcms.1281