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Objectives: To evaluate the longevity of resin composite restorations placed in posterior teeth by dental students, using data from electronic records from 2008 to 2019., Materials and Methods: Demographic (gender and age) and clinical variables (dental group, position in dental arch, and the number of restored surfaces) were evaluated. The 5-year follow-up was assessed according to the day the restoration was placed. Kaplan-Meier curves were generated to calculate the annual failure rate. Data were analyzed by Chi-Square, Kruskal-Wallis, and Mann-Whitney tests (α = 0.05)., Results: In total, 3.883 records relative to return periodicity were analyzed. The final sample consisted of 900 restorations from 479 patients. The majority were females, aged between 31 and 60. In total, 256 failures were reported (success rate = 78%), showing an annual failure rate of 2.05%. The main reasons for failures were restoration replacement (55.5%), endodontics (21.9%), prosthetics (14.5%) and extraction (8.2%). There was a higher risk of failure in restorations involving three or more surfaces (p = 0.000) and in patients over 60 years (p < 0.001). In females (p = 0.030), molars (p = 0.044), and maxillary teeth (p = 0.038) failed in a shorter time., Conclusions: Resin composite restorations placed in permanent posterior teeth by dental students had high survival rates. The main reason for failure was the replacement of restorations. The age group and the number of restored surfaces significantly affected the success of the restorations., Clinical Relevance: The electronic health records over 12 years showed that 78% of the resin restorations in posterior teeth placed by dental students were successful for a minimum of five years., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Objectives: This study aimed to evaluate the effect of pomegranate solution ( Punica granatum ) on eroded dentin through antioxidant action, shear bond strength (SBS) and interface morphology., Materials and Methods: The 10% pomegranate peel extract was prepared by the lyophilization method. Punicalagin polyphenol was confirmed by high-performance liquid chromatography. Antioxidant activity was evaluated by capturing the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical. For the SBS, 48 dentin fragments were divided into sound or eroded, and subdivided according to the pretreatment ( n = 12): water or P. granatum . The surfaces were restored with self-etch adhesive and a bulk-fill resin (Ecosite; DMG). The SBS was done immediately (24 hours) and after thermal cycling + water storage (12 months). For scanning electron microscopy, 48 dentin fragments (24 sound and 24 eroded) received the same treatments as for SBS ( n = 6), and they were analyzed after 24 hours and 12 months., Results: The P. granatum had antioxidant action similar ( p = 0.246) to the phenolic standard antioxidants. After 24 hours, eroded dentin had lower SBS than sound dentin ( p < 0.001), regardless of the pretreatment. After 12 months, P. granatum maintained the SBS of sound dentin (13.46 ± 3.42 MPa) and eroded dentin (10.96 ± 1.90 MPa) statistically similar. The lowest values were found on eroded dentin treated with water (5.75 ± 1.65 MPa) ( p < 0.001). P. granatum on eroded dentin caused peritubular demineralization and hybrid layer with resin tags., Conclusions: The pomegranate extract had antioxidant action and preserved the adhesive interface of the eroded dentin., Competing Interests: Conflict of Interest: No potential conflict of interest relevant to this article was reported., (Copyright © 2024. The Korean Academy of Conservative Dentistry.)

Objectives: This study aimed to evaluate the level of knowledge, perception, and experience of teledentistry amongst Brazilian dentists with the ongoing COVID-19 pandemic., Methods: Between June and July 2020, an online questionnaire was used to collect information on the demographic and professional profiles of dentists and their levels of knowledge, perception, and attitudes towards teledentistry. Descriptive statistics were reported as percentages, and Chi-squared test was used to compare responses amongst general practitioners, specialists, and those with varying levels of professional experience., Results: This study included 575 dentists, the majority of whom were from the southeast region of Brazil (73.4%) and had a specialist degree (70.4%). Approximately 42.4% of the study sample had graduated more than 10 years ago, 39.3% between 2 and 10 years ago, and 18.3% up to 2 years ago. In general, the majority of dentists exhibited only a superficial awareness of the resolution that regulates teledentistry in Brazil. A greater number of specialists were aware of the resolution (P = .007) and felt confident performing teledentistry (P = .000) compared to general practitioners, whilst those with more experience (graduated more than 10 years ago) exhibited greater knowledge (P = .012) and preparation (P = .000). More than 60% of the dentists, regardless of dental specialty and clinical experience, had no knowledge of providing prescriptions via teledentistry. The majority of general practitioners (89.5%) and specialists (81.4%) had no previous experience using teledentistry., Conclusions: The findings of this study suggest that Brazilian dentists are inadequately prepared for the implementation of teledentistry., Competing Interests: Conflict of Interest None disclosed., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Objectives: This study aimed to evaluate the effects of different shaping protocols on the root canal geometry and remaining dentin thickness of mandibular premolars with radicular grooves (RG) and two canals by means of micro-CT., Materials and Methods: Mandibular premolars with RG and two canals were matched based on their similar morphological dimensions and assigned to 2 experimental groups according to the canal preparation (n=8): XP-endo Shaper (XPS) and ProTaper Next (PTN) systems. Before and after preparation, morphometric measurements of the volume, surface area, structure model index, area, perimeter, roundness, major and minor diameters, as well as untouched canal walls, canal transportation, and dentin wall thickness were evaluated. Data were statistically compared within and between groups using the independent sample t test (α=5%)., Results: XPS revealed less changes in volume, surface area, area, perimeter, and major and minor diameters (P<0.05). There was no significant difference in structure model index (SMI), roundness, and percentage of untouched canal walls between groups (P>0.05). XPS resulted in less apical canal transportation and a higher remaining dentin thickness toward the RG compared to PTN in lingual canals (P<0.05)., Conclusions: XPS revealed fewer changes in root canal geometry as well as lower transportation and a greater remaining dentin thickness in the lingual canals toward the RG compared to PTN., Clinical Relevance: Internal and external anatomical characteristics of mandibular premolars might have an influence on biomechanical preparation. Considering the risk of perforation, the canal preparation using XPS appears to be more conservative compared to PTN., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH, DE part of Springer Nature.)

Introduction: Amino acid depletion in the blood serum is currently being exploited and explored for therapies in tumors or viral infections that are auxotrophic for a certain amino acid or have a metabolic defect and cannot produce it. The success of these treatments is because normal cells remain unaltered since they are less demanding and/or can synthesize these compounds in sufficient amounts for their needs by other mechanisms. Areas covered: This review is focused on amino acid depriving enzymes and their formulations that have been successfully used in the treatment of several types of cancer and viral infections. Particular attention will be given to the enzymes L-asparaginase, L-arginase, L-arginine deiminase, and L-methionine-γ-lyase. Expert opinion: The immunogenicity and other toxic effects are perhaps the major limitations of these therapies, but they have been successfully decreased either through the expression of these enzymes from other organisms, recombination processes, pegylation of the selected enzymes or by specific mutations in the proteins. In 2006, FDA has already approved the use of L-asparaginase in the treatment of acute lymphoblastic leukemia. Other enzymes and in particular L-arginase, L-arginine deiminase, and L-methioninase have been showing promising results in vitro and in vivo studies.

A new d-erythrose 1,3-dioxane derivative was synthesized from d-glucose and found to be a highly stereoselective template as a dipolarophile. Different 1,3-dipoles of allenyl-type were employed, giving different regioselectivities, depending on its nature; the regioselectivity is complete with alkyl azides and phenyldiazomethane, but is inexistence with nitrile oxides. Computational studies were performed to understand the mechanisms of cycloadditions. All the studied cycloadditions were found to be concerted involving small free activation energies and are all exoenergonic. The stereoselectivity is due to a combined result of the steric effect H-8a and the hyperconjugative effect of the *C-O to the incoming 1,3-dipole. The regioselectivity observed in alkyl azides and phenyldiazomethane is mostly dependent on the distortion effect during the cycloaddition process. This distortion effect is however higher in the alkyl azide compounds than in phenyldiazomethane.

Cholesterol is an essential component of cell membranes and the precursor for the synthesis of steroid hormones and bile acids. The synthesis of this molecule occurs partially in a membranous world (especially the last steps), where the enzymes, substrates, and products involved tend to be extremely hydrophobic. The importance of cholesterol has increased in the past half-century because of its association with cardiovascular diseases, which are considered one of the leading causes of death worldwide. In light of the current need for new drugs capable of controlling the levels of cholesterol in the bloodstream, it is important to understand how cholesterol is synthesized in the organism and identify the main enzymes involved in this process. Taking this into account, this review presents a detailed description of several enzymes involved in the biosynthesis of cholesterol. In this regard, the structure and catalytic mechanism of the enzymes involved in cholesterol biosynthesis, from the initial two-carbon acetyl-CoA building block, will be reviewed and their current pharmacological importance discussed. We believe that this review may contribute to a deeper level of understanding of cholesterol metabolism and that it will serve as a useful resource for future studies of the cholesterol biosynthesis pathway.

In this paper, computational means were used to explain and predict the interaction of several odorant molecules, including three haloanisoles, 2,4,6-trichloroanisole (TCA), 2,4,6-tribromoanisole (TBA), and 2,4,6-trichlorophenol (TCP), with three olfactory receptors (ORs): OR1A1, OR1A2, and OR3A1. As the X-ray structure of these ORs is not known, the three-dimensional structure of each OR was modeled by homology modeling. The structures of these ORs were stabilized by molecular dynamic simulations and the complexes of the odorant molecules with each ORs were generated by molecular docking. The theoretical results have shown that each OR has distinct but well-defined binding regions for each type of odorant molecules (aldehydes and alcohols). In OR3A1, the aldehydes bind in the bottom region of the binding pocket nearby Ser257 and Thr249. In the paralogues OR1A1 and OR1A2, the aldehydes tend to interact in the top region of the binding pocket and close to a positively charged lysine. On the other hand, the alcohols interact in the bottom region of the active site and close to a negatively charged aspartate. These results indicate that when aldehydes and alcohols odorants compete in these two ORs, the aldehydes can block the access of the alcohols odorants to their specific binding site. This observation goes in line with the experimental data that reveals that when the odorant is an aldehyde, a lower quantity of ligand is needed to cause 50% of the maximum response (lower EC50), when compared with the alcohols. The theoretical results have also allowed to explain the differences in the activity of (S)-(-)-citronellol in the wild-type and mutated OR1A1. The theoretical results show that Asn109 has a preponderant role in this matter, since when it is mutated, it leads to a conformational rearrangement of the binding pocket that prevents the interaction of (S)-(-)-citronellol with Asp111 that was shown to be important for the OR activation. The good agreement between the theoretical and experimental results also lead us to study the potential interaction of the haloanisoles, TCA, TBA, and TCP with these ORs. The results have shown that these compounds can compete with other known agonists/antagonists for the access to the binding regions of ORs. These results may partially explain the capability of these compounds to give a musty odor to food and beverages at very low concentrations.

Although neglected by science for a long time, the olfactory sense is now the focus of a panoply of studies that bring new insights and raises interesting questions regarding its functioning. The importance in the clarification of this process is of interest for science, but also motivated by the food and perfume industries boosted by a consumer society with increasingly demands for higher quality standards. In this review, a general overview of the state of art of science regarding the olfactory sense is presented with the main focus on the peripheral olfactory system. Special emphasis will be given to the deorphanization of the olfactory receptors (ORs), a critical issue because the specificity and functional properties of about 90% of human ORs remain unknown mainly due to the difficulties associated with the functional expression of ORs in high yields., (© The Author 2015. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

It is remarkable how nature has been able to construct enzymes that, despite sharing many similarities, have simple but key differences that tune them for completely different functions in living cells. Periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) from the DMSOr family are representative examples of this. Both enzymes share almost identical three-dimensional protein foldings and active sites, in terms of coordination number, geometry and nature of the ligands. The substrates of both enzymes (nitrate and formate) are polyatomic anions that also share similar charge and stereochemistry. In terms of the catalytic mechanism, both enzymes have a common activation mechanism (the sulfur-shift mechanism) that ensures a constant coordination number around the metal ion during the catalytic cycle. In spite of these similarities, they catalyze very different reactions: Nap abstracts an oxygen atom from nitrate releasing nitrite, whereas FdH catalyzes a hydrogen atom transfer from formate and releases carbon dioxide. In this Account, a critical analysis of structure, function, and catalytic mechanism of the molybdenum enzymes periplasmic nitrate reductase (Nap) and formate dehydrogenase (Fdh) is presented. We conclude that the main structural driving force that dictates the type of reaction, catalyzed by each enzyme, is a key difference on one active site residue that is located in the top region of the active sites of both enzymes. In both enzymes, the active site is centered on the metal ion of the cofactor (Mo in Nap and Mo or W in Fdh) that is coordinated by four sulfur atoms from two pyranopterin guanosine dinucleotide (PGD) molecules and by a sulfido. However, while in Nap there is a Cys directly coordinated to the Mo ion, in FdH there is a SeCys instead. In Fdh there is also an important His that interacts very closely with the SeCys, whereas in Nap the same position is occupied by a Met. The role of Cys in Nap and SeCys in FdH is similar in both enzymes; however, Met and His have different roles. His participates directly on catalysis, and it is therefore detrimental for the catalytic cycle of FdH. Met only participates in substrate binding. We concluded that this small but key difference dictates the type of reaction that is catalyzed by each enzyme. In addition, it allows explaining why formate can bind in the Nap active site in the same way as the natural substrate (nitrate), but the reaction becomes stalled afterward.

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., (Copyright © 2015. Published by Elsevier Inc.)

In this work, a combination of homology modeling and molecular dynamics (MD) simulations was used to investigate the factors that modulate substrate specificity and activity of the mouse AOX isoforms: mAOX1, mAOX2 (previously mAOX3l1), mAOX3 and mAOX4. The results indicate that the AOX isoform structures are highly preserved and even more conserved than the corresponding amino acid sequences. The only differences are at the protein surface and substrate-binding site region. The substrate-binding site of all isoforms consists of two regions: the active site, which is highly conserved among all isoforms, and a isoform-specific region located above. We predict that mAOX1 accepts a broader range of substrates of different shape, size and nature relative to the other isoforms. In contrast, mAOX4 appears to accept a more restricted range of substrates. Its narrow and hydrophobic binding site indicates that it only accepts small hydrophobic substrates. Although mAOX2 and mAOX3 are very similar to each other, we propose the following pairs of overlapping substrate specificities: mAOX2/mAOX4 and mAOX3/mAXO1. Based on these considerations, we propose that the catalytic activity between all isoforms should be similar but the differences observed in the binding site might influence the substrate specificity of each enzyme. These results also suggest that the presence of several AOX isoforms in mouse allows them to oxidize more efficiently a wider range of substrates. This contrasts with the same or other organisms that only express one isoform and are less efficient or incapable of oxidizing the same type of substrates.

Modeling of molybdoenzymes began even before the knowledge of the three-dimensional structure of these enzymes. The theoretical and experimental knowledge on these enzymes is vast and newer investigation is regularly pursued to understand the electronic aspect of these proteins using computational means. The present review deals with some unique observation regarding the structure, function and reactivity of some models and native proteins in rationalizing the choice of diverse substrates in seemingly similar enzymes such as Nap (nitrate reductase) and Fdh (formate dehydrogenase) and the dual form of a specific substrate of an enzyme like trimethylamine N-oxide reductase (TAMOR) and providing the electronic reason for the inhibition in the oxypurinol-inhibited xanthine oxidase (XO).

New benzopsoralen analogues were synthesized and their inhibitory effect on the growth of tumourtumour cell lines (MDA MB231 and TCC-SUP) was evaluated. The in vitro antitumour activity of the new benzopsoralen analogues was discussed in terms of structure-activity relationship. Molecular docking studies with human-CYP2A6 enzymes were also carried out with the synthesized compounds to evaluate the potential of these molecules to interact with the haem group of the enzymes. The results demonstrated that the compounds that are able to interact with the iron ion of the haem cofactor and at the same time with active site Asn297 are those that have better anti-proliferative activity., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

The catalytic mechanism of carboxylesterases (CEs, EC 3.1.1.1) is explored by computational means. CEs hydrolyze ester, amide, and carbamate bonds found in xenobiotics and endobiotics. They can also perform transesterification, a reaction important, for instance, in cholesterol homeostasis. The catalytic mechanisms with three different substrates (ester, thioester, and amide) have been established at the M06-2X/6-311++G**//B3LYP/6-31G* level of theory. It was found that the reactions proceed through a mechanism involving four steps instead of two as is generally proposed: (i) nucleophilic attack of serine to the substrate, forming the first tetrahedral intermediate, (ii) formation of the acyl-enzyme complex concomitant with the release of the alcohol product, (iii) nucleophilic attack of a water or alcohol molecule forming the second tetrahedral intermediate, and (iv) the release of the second product of the reaction. The results agree very well with the available experimental data and show that the hydrolytic and the transesterification reactions are competitive processes when the substrate is an ester. In all the other studied substrates (thioester or amide), the hydrolytic and transesterification process are less favorable and some of them might not even take place under in vivo conditions.

Introduction: In the recent decades, the interest on glycosidases has dramatically increased, mainly because these enzymes play a key role in many biological processes. The importance of these enzymes is also reflected by a number of diseases, which result from the lack or dysfunction of a given glycosidase, as well as by the use of glycosidase inhibitors in the treatment of metabolic disorders or viral infections. Based on the biological potential associated to these enzymes, several glycosidase inhibitors have been developed. In this review, the most important inhibitors targeting these enzymes, including the disaccharides, iminosugars, carbasugars, thiosugars and other non-glycosidic compounds will be discussed and special attention will be given to the ones that are currently used clinically., Areas Covered: This review summarizes and characterizes the current knowledge regarding the classes of glycosidase inhibitors that have therapeutic potential in a wide range of diseases. It highlights the relevant research, patents and patent applications filed in the past years on the field., Expert Opinion: Since the glycosidase inhibitors are involved in several chronic diseases and possibly pandemic, the pharmaceutical research toward developing new generations of these molecules is very important to public health in the world. The unique combination of these compounds - for example, they share many properties with natural carbohydrates and also possess distinct specific characteristics - makes them precious for pharmaceutical companies in an attempt to search for potential new drugs. Currently, the most promising compounds are the iminosugars and thiosugars due to their better oral bioavailability.

In this article, we propose a method for locating functionally relevant chemical motifs in protein structures. The chemical motifs can be a small group of residues or structure protein fragments with highly conserved properties that have important biological functions. However, the detection of chemical motifs is rather difficult because they often consist of a set of amino acid residues separated by long, variable regions, and they only come together to form a functional group when the protein is folded into its three-dimensional structure. Furthermore, the assemblage of these residues is often dependent on non-covalent interactions among the constituent amino acids that are difficult to detect or visualize. To simplify the analysis of these chemical motifs and give access to a generalized use for all users, we developed chem-path-tracker. This software is a VMD plug-in that allows the user to highlight and reveal potential chemical motifs requiring only a few selections. The analysis is based on atoms/residues pair distances applying a modified version of Dijkstra's algorithm, and it makes possible to monitor the distances of a large pathway, even during a molecular dynamics simulation. This tool turned out to be very useful, fast, and user-friendly in the performed tests. The chem-path-tracker package is distributed as an independent platform and can be found at http://www.fc.up.pt/PortoBioComp/database/doku.php?id=chem-path-tracker., (© 2014 John Wiley & Sons A/S.)

The enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA-R) is the fundamental target for the treatment of hypercholesterolemia nowadays. The HMG-CoA-R clinical active site inhibitors (statins) are among the most widespread and profitable drugs ever sold but their side effects (myopathies, sometimes severe) still limit their use, which makes the finding of alternatives to statins a field of intense research. In this line, we address here a new strategy for inhibiting the homotetrameric HMG-CoA-R. The enzyme consists of a "dimer of dimers", each dimer having two active sites. We pursue here the inhibition of enzyme oligomerization, through drug binding to the dimer interface. We have computationally mutated 232 interfacial residues by alanine and calculated the loss in binding free energy among the monomers that build up each dimer of the homotetramer. This led to the identification of the (ten) key residues for the formation of the active dimer (Glu528, Ile531, Met534, Tyr644, Glu665, Asn686, Lys692, Lys735, Met742, and Val863). The results show that these residues are located in two specific spots of the protein with a cleft shape, whose shape and size is favorable for small drug binding. It is expectable that small molecules specifically bound to these druggable pockets will have a major effect on the oligomerization of the protein or/and in active site formation. This paves the way for the discovery of new families of inhibitors of HMG-CoA-R.

Numerous enzymes, such as the pyridoxal 5'-phosphate (PLP)-dependent enzymes, require cofactors for their activities. Using X-ray crystallography, structural snapshots of the L-serine dehydratase catalytic reaction of a bacterial PLP-dependent enzyme were determined. In the structures, the dihedral angle between the pyridine ring and the Schiff-base linkage of PLP varied from 18° to 52°. It is proposed that the organic cofactor PLP directly catalyzes reactions by active conformational changes, and the novel catalytic mechanism involving the PLP cofactor was confirmed by high-level quantum-mechanical calculations. The conformational change was essential for nucleophilic attack of the substrate on PLP, for concerted proton transfer from the substrate to the protein and for directing carbanion formation of the substrate. Over the whole catalytic cycle, the organic cofactor catalyzes a series of reactions, like the enzyme. The conformational change of the PLP cofactor in catalysis serves as a starting point for identifying the previously unknown catalytic roles of organic cofactors.

In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.

We have developed a computer program named 'VolArea' that allows for a rapid and fully automated analysis of molecular structures. The software calculates the surface area and the volume of molecular structures, as well as the volume of molecular cavities. The surface area facility can be used to calculate the solvent-exposed surface area of a molecule or the contact area between two molecules. The volume algorithm can be used to predict not only the space occupied by any molecular structure, but also the volume of cavities, such as tunnels or clefts. The software finds wide application in the characterization of systems, such as protein/ligand complexes, enzyme active sites, protein/protein interfaces, enzyme channels, membrane pores, solvent tunnels, among others. Some examples are given to illustrate its potential. VolArea is as a plug-in of the widely distributed software Visual Molecular Dynamics (VMD) and is freely available at http://www.fc.up.pt/PortoBioComp/Software/Volarea/Home.html., (© 2013 John Wiley & Sons A/S.)

Ribonucleotide reductase (RNR, E.C. 1.17.4.1), which is composed of two dissimilar proteins (subunits), often referred as R1 (containing polythiols) and R2 (containing non-heme iron and a free tyrosyl radical), which contribute to the role played by the enzyme. RNRs are one of the important targets in anticancer and antiviral drug development and many RNR inhibitors have been discovered at the end of the 20(th) century; many of them are already in clinical use. Triapine (3-AP) is one of the important RNR inhibitors belonging to the class of thiosemicarbazone derivatives, used in the treatment of various cancers. The structure activity relationship (SAR) studies on the investigated RNR inhibitors showed that the nitrogen atom in the pyridine (or other heterocycles) forms coordination complexes with the metal ions along with the imine, oxo and thio atoms of the thiosemicarbazone or semicarbazone pharmacophores. The computational analyses results in the adenine and purine derivatives suggest that the nitrogen atoms in the adenine rings make several hydrogen bonds with the water molecules present in the active site, as well as Gly249 and Glu288 residues. The OH group in third position of the sugar moiety interacts with the Ser217 (C=O) and the water molecules through hydrogen bonds. The aromatic rings in the molecules interact with the tyrosine residues. The thiosemicarbazone or semicarbazone derivatives explain that the flexibility and polar properties in the thiosemicarbazone or semicarbazone pharmacophoric regions allow the molecules to coordinate with the metal ion (especially iron) present in the RNR enzymes. This review concluded that RNR inhibitors composed of different fragments such as aryl, heteroaryl, sugar moiety, polar groups, flexible bonds, etc which are required for the binding of the molecules to the RNR enzymes. Further, the fragmental analysis of the RNR inhibitors on different toxicological and metabolic targets can provide significant novel molecules with acceptable pharmacokinetic properties.

A structural rearrangement known as sulfur shift occurs in some Mo-containing enzymes of the DMSO reductase family. This mechanism is characterized by the displacement of a coordinating cysteine thiol (or SeCys in Fdh) from the first to the second shell of the Mo-coordination sphere metal. The hexa-coordinated Mo ion found in the as-isolated state cannot bind directly any exogenous ligand (substrate or inhibitors), while the penta-coordinated ion, attained upon sulfur shift, has a free binding site for direct coordination of the substrate. This rearrangement provides an efficient mechanism to keep a constant coordination number throughout an entire catalytic pathway. This mechanism is very similar to the carboxylate shift observed in Zn-dependent enzymes, and it has been recently detected by experimental means. In the present paper, we calculated the geometries and energies involved in the sulfur-shift mechanism using QM-methods (M06/(6-311++G(3df,2pd),SDD)//B3LYP/(6-31G(d),SDD)). The results indicated that the sulfur-shift mechanism provides an efficient way to enable the metal ion for substrate coordination.

New tetracyclic benzofurocoumarin (benzopsoralen) analogues were synthesized and their inhibitory effect on the growth of tumor cell lines was evaluated. The human tumor cell lines used were MDA MB231 (breast adenocarcinoma), HeLa (cervix adenocarcinoma) and TCC-SUP (bladder transitional cell carcinoma). The in vitro antitumor activity of the new benzopsoralens was discussed in terms of structure-activity relationship. Molecular docking studies with human-CYP2A6 enzymes were also carried out with the synthesized compounds in order to evaluate the potential of these compounds to interact with the heme group of the enzymes. The results have demonstrated that the linear compounds have the most pronounced activity against tumor cell lines and this might be related to the better accessibility that these compounds have to the active site in relation to the angular ones that have shown in the majority of the cases multiple binding poses in the active site of CYP2A6., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

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.

In this review, we discuss the current patents concerning aryl/heteroaryl thiosemicarbazone derivatives as regards to their activities and properties, including coordination (chelation) properties. The mode of action of the aryl/heteroaryl thiosemicarbazone derivatives involves metal coordination with proteins or biological fluids that have metal ions in their structure. Additionally, these molecules can also form multiple hydrogen bonds through their (thio) amide and N3 nitrogen that ensure a strong interaction with the receptor. In some cases, strong π-π interactions can be observed too. Special attention is given to pyridyl, bis-pyridyl, benzoylpyridyl and isatin thiosemicarbazone derivatives that exhibit significant anticancer, antiviral and other activities in free and in metal complexed forms. This key biological role is often related with their capability to inhibit the enzyme ribonucleotide reductase, similar to what is observed with potent anticancer drugs such as Triapine and methisazone. Recent studies have revealed that thiosemicarbazone can also inhibit topoisomerase II α enzyme. Thiosemicarbazone derivatives form coordination complex with various metals such as Zn, Cu, Fe, Co, Ni, Pt, Pd, etc., and these complexes provide better activities than the free thiosemicarbazones. Recent patents show that the controlled or sustained release dosage form of the thiosemicarbazone derivatives along with ionizing radiations is used for the treatment of proliferative diseases (US20110152281, US20110245304, US20120172217).

Purpose: The aim of this study was to investigate the level of microstrain that is exerted during polymerization of acrylic resins used for splinting during implant impressions., Material and Methods: Two acrylic resins (GC Pattern Resin, Duralay II) and square transfer coping splinting methods were evaluated by means of strain gauge analysis. Two implants were embedded in a polyurethane block, and the abutments were positioned. Sixty specimens were prepared using two square transfer copings that were rigidly connected to each other using the acrylic resins. The specimens were randomly divided into three groups of 20 each for the splinting methods: Method 1 was a one-piece method; in method 2, the splint was separated and reconnected after 17 minutes; and in method 3, the splint was separated and reconnected after 24 hours. In each group, half the specimens were splinted with GC Pattern Resin and the other half were splinted with Duralay II. Three microstrain measurements were performed by four strain gauges placed on the upper surface of the polyurethane blocks at 5 hours after resin polymerization for all groups. The data were analyzed statistically., Results: Both resin type and splinting method significantly affected microstrain. Interaction terms were also significant. Method 1 in combination with Duralay II produced significantly higher microstrain (1,962.1 με) than the other methods with this material (method 2: 241.1 με; method 3: 181.5 με). No significant difference was found between splinting methods in combination with GC Pattern Resin (method 1: 173.8 με; method 2: 112.6 με; method 3: 105.4 με)., Conclusions: Because of the high microstrain generated, Duralay II should not be used for one-piece acrylic resin splinting, and separation and reconnection are suggested. For GC Pattern Resin, variations in splinting methods did not significantly affect the microstrain created.

The synthesis of five new tetracyclic benzofurocoumarin (benzopsoralen) analogues is described. Their inhibitory effects on the growth of three human tumor cell lines (MDA MB 231 (breast adenocarcinoma), HeLa (cervix adenocarcinoma) and TCC-SUP (bladder transitional cell carcinoma) were evaluated, and discussed in terms of structure-activity relationship., (Copyright © 2011 Elsevier Masson SAS. All rights reserved.)

Gemcitabine (dFdC, 2',2'-difluorodeoxycytidine) is a deoxycytidine nucleoside analogue of deoxycytidine in which two fluorine atoms have been inserted into the deoxyribose ring. Like other nucleoside analogues, gemcitabine is a prodrug. It is inactive in its original form, and depends on the intracellular machinery to gain pharmacological activity. What makes gemcitabine different from other nucleoside analogues is that it is actively transported across the cell membrane, it is phosphorylated more efficiently and it is eliminated at a slower rate. These differences, together with self-potentiation mechanisms, masked DNA chain termination and extensive inhibitory efficiency against several enzymes, are the source of gemcitabine's cytotoxic activity against a wide variety of tumors. This unique combination of metabolic properties and mechanistic characteristics is only found in very few other anticancer drugs, and both the FDA and the EMEA have already approved its use for clinical purposes, for the treatment of several types of tumors. In spite of the promising results associated with gemcitabine, the knowledge of its mode of action and of the enzymes it interacts with is still not fully documented. In this article we propose to review all these aspects and summarize the path of gemcitabine inside the cell.

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.

In this paper we studied the mechanism of formation of the internal aldimine, a common intermediate to most pyridoxal 5'-phosphate (PLP)-dependent enzymes. A large model based on the crystal structure from the human ornithine decarboxylase (ODC) enzyme was constructed and in total accounts for 504 atoms. The reaction mechanism was investigated using the ONIOM methodology (B3LYP/6-31G(d)//AM1), and the final energies were calculated with the M06/6-311++G(2d,2p)//B3LYP/6-31G(d) level of theory. It was demonstrated that the reaction is accomplished in three sequential steps: (i) the nucleophilic attack of Lysine69 to PLP, (ii) the carbinolamine formation, and (iii) a final dehydration step. For the carbinolamine formation, several mechanistic hypotheses were explored, and the preferred pathway assigns a key role for the conserved active site Cys360. The overall reaction is exergonic in -9.1 kcal/mol, and the rate-limiting step is the dehydration step (E(a) = 13.5 kcal/mol). For the first time, we provide an atomistic portrait of this mechanism in an enzymatic environment. Moreover, we were able to assign a novel role to Cys360 in the ODC reaction mechanism that was never proposed.

The pyridoxal-5'-phosphate-dependent enzymes (PLP enzymes) catalyze a myriad of biochemical reactions, being actively involved in the biosynthesis of amino acids and amino acid-derived metabolites as well as in the biosynthetic pathways of amino sugars and in the synthesis or catabolism of neurotransmitters. Although the scope of PLP-catalyzed reactions initially appears to be bewilderingly diverse, there is a simple unifying principle: In the resting state, the cofactor (PLP) is covalently bonded to the amino group of an active site lysine, forming an internal aldimine. Once the amino substrate interacts with the active site, a new Schiff base is generated, commonly referred to as the external aldimine. Only after this step, the mechanistic pathway for each PLP-catalyzed reaction diverges. In this paper, density functional methods have been applied to investigate this common step present in all PLP-dependent enzymes-the transimination reaction. The results indicate that the reaction involves three sequential steps: (i) formation of a tetrahedral intermediate with the active site lysine and the amino substrate bonded to the PLP cofactor; (ii) nondirect proton transfer between the amino substrate and the lysine residue; and (iii) formation of the external aldimine after the dissociation of the lysine residue. The overall reaction is exothermic (-12.0 kcal/mol), and the rate-limiting step is the second one with 12.6 kcal/mol for the activation energy.

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.

Ribonucleotide reductase (RNR) is the key enzyme in the biosynthesis of deoxyribonucleotides. Several different strategies for inactivation of RNRs have been reported, including the use of substrate analogues as mechanism-based inhibitors. This article undergoes a critical analysis on the current status of ribonucleotide reductase inhibitory mechanisms by substrate analogues highlighting experimental and theoretical/computational approaches. We have summarized a general portrait of the inhibitory mechanisms and classified the nucleoside analogue inhibitors in three main classes. The critical analysis undertaken will contribute in finding new and more effective ways of inhibiting RNR.

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 Mo(V) 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 Mo(VI) 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 Mo(V) species. Mo(VI) intermediates might also be present in this stage depending on the availability of protons and electrons. Once the water molecule is generated only the Mo(VI) species allow water molecule dissociation, and, the concomitant enzymatic turnover., (Copyright 2009 Wiley Periodicals, Inc.)

During the last decade, Virtual Screening (VS) has definitively established itself as an important part of the drug discovery and development process. VS involves the selection of likely drug candidates from large libraries of chemical structures by using computational methodologies, but the generic definition of VS encompasses many different methodologies. This chapter provides an introduction to the field by reviewing a variety of important aspects, including the different types of virtual screening methods, and the several steps required for a successful virtual screening campaign within a state-of-the-art approach, from target selection to postfilter application. This analysis is further complemented with a small collection important VS success stories.

Dealing with receptor flexibility in docking methodology is still a problem. The main reason behind this difficulty is the large number of degrees of freedom that have to be considered in this kind of calculations. In this paper, we present an automated procedure, called MADAMM, that allows flexibilization of both the receptor and the ligand during a multistaged docking with an automated molecular modeling protocol. We show that the orientation of particular residues at the interface between the protein and the ligand have a crucial influence on the way they interact during the docking process, and the standard docking methodologies failed to predict their correct mode of binding. We present some examples that demonstrate the capabilities of this approach when compared with traditional docking methodologies., ((c) 2008 Wiley-Liss, Inc.)

Nitrate reductase from Desulfovibrio desulfuricans ATCC 27774 (DdNapA) is a monomeric protein of 80 kDa harboring a bis(molybdopterin guanine dinucleotide) active site and a [4Fe-4S] cluster. Previous electron paramagnetic resonance (EPR) studies in both catalytic and inhibiting conditions showed that the molybdenum center has high coordination flexibility when reacted with reducing agents, substrates or inhibitors. As-prepared DdNapA samples, as well as those reacted with substrates and inhibitors, were crystallized and the corresponding structures were solved at resolutions ranging from 1.99 to 2.45 A. The good quality of the diffraction data allowed us to perform a detailed structural study of the active site and, on that basis, the sixth molybdenum ligand, originally proposed to be an OH/OH(2) ligand, was assigned as a sulfur atom after refinement and analysis of the B factors of all the structures. This unexpected result was confirmed by a single-wavelength anomalous diffraction experiment below the iron edge (lambda = 1.77 A) of the as-purified enzyme. Furthermore, for six of the seven datasets, the S-S distance between the sulfur ligand and the Sgamma atom of the molybdenum ligand Cys(A140) was substantially shorter than the van der Waals contact distance and varies between 2.2 and 2.85 A, indicating a partial disulfide bond. Preliminary EPR studies under catalytic conditions showed an EPR signal designated as a turnover signal (g values 1.999, 1.990, 1.982) showing hyperfine structure originating from a nucleus of unknown nature. Spectropotentiometric studies show that reduced methyl viologen, the electron donor used in the catalytic reaction, does not interact directly with the redox cofactors. The turnover signal can be obtained only in the presence of the reaction substrates. With use of the optimized conditions determined by spectropotentiometric titration, the turnover signal was developed with (15)N-labeled nitrate and in D(2)O-exchanged DdNapA samples. These studies indicate that this signal is not associated with a Mo(V)-nitrate adduct and that the hyperfine structure originates from two equivalent solvent-exchangeable protons. The new coordination sphere of molybdenum proposed on the basis of our studies led us to revise the currently accepted reaction mechanism for periplasmic nitrate reductases. Proposals for a new mechanism are discussed taking into account a molybdenum and ligand-based redox chemistry, rather than the currently accepted redox chemistry based solely on the molybdenum atom.

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 A 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.

Some pyrazolo[3,4-d]pyrimidines, structurally related with allopurinol, a well known xanthine oxidase inhibitor, clinically used in the therapy of gout, have also been reported as potent inhibitors of xanthine oxidase and the growth of several human tumour cell lines. Considering the potential interest of this family of compounds, the aim of the present study was to synthesise and provide a full chemical characterization of new N-aryl-5-amino-4-cyanopyrazole derivatives and their corresponding pyrazolo[3,4-d]pyrimidines. Their biological activity pertaining to the xanthine oxidase inhibition and effect on the growth of three tumour cell lines (MCF-7, NCI-H460, and SF-268) are also provided. With only one exception, the synthesised compounds showed no effect on the growth of the three tumour cell lines. However, a strong xanthine oxidase inhibitory activity was observed for almost all pyrazolo[3,4-d]pyrimidines tested, revealing some of them IC(50) values below 1 microM. The results of the molecular docking studies of these compounds, against xanthine oxidoreductase are also described, providing an atomistic explanation of the differences in the inhibitory efficiency. MEP calculations were used to explain different inhibitory efficiency of similar inhibitors.

Background and Objectives: Because brain death (BD) is a new concept and little divulged, it’s not well accepted in general population, including doctors and Medical students. This study aims to evaluate the knowledge of a sample of Medical students on the Brazilian BD diagnosis protocol., Methods: Descriptive cross-sectional survey that evaluated students from two medical schools in Salvador-BA. We used a questionnaire composed by questions about technical and ethical knowledge contained in the Federal Council of Medicine’s Resolution nº 1480/97 that establishes the criteria for BD diagnosis., Results: We evaluated 115 Medical students. In 14 questions about the knowledge of BD criteria, the mean of right answers were 6.7 ± 1.8, which were higher among the students that had attended some presentation on BD. Most of the students (87.4%) knew how to identify the candidates to the BD diagnosis protocol. However, only 5.2% and 16.1% of the students answered right, respectively, the clinical and complementary tests that should be accomplished during the diagnosis protocol. Facing a no-donor patient with confirmed diagnosis of BD, 66.4% referred that artificial life support should be suspended. Only 15% of the interviewed students had already evaluated a patient with BD, being this percentage higher among those who had already frequented ICU (38.2% versus 5.1%; p < 0.001)., Conclusions: The results of this study showed a limited knowledge of the evaluated students on BD diagnosis criteria, mainly in relation to the practical approach of this condition.

Ribonucleotide Reductase (RNR) plays a critical role in DNA synthesis, and is a well-recognized target for cancer chemotherapeutic and antiviral agents. RNR inhibition precludes DNA transcription and repair, from which results cell apoptosis. Many regulation checkpoints concerning RNR activity have been unravelled through the last two decades, with potential use to inhibit enzyme activity. This was accomplished by researchers from different but complementary areas, and from which several and different inhibitors have resulted. The volume of these studies has generated over 4000 articles since the discovery of RNR in 1960. This review summarises patents and papers during the period 1958 - 2005 dealing with the present understanding of ribonucleotide reductase biochemistry, mechanism of action and the most relevant data concerning RNR inhibition. Special attention is given to the inhibitors that have been patented and are currently in clinical use.

This paper focuses on the inhibition of ribonucleotide reductase (RNR) by gemcitabine, 2',2'-difluoro-2'-deoxycytidine (dFdC), a deoxycytidine analogue that is a very active drug against solid tumors and is currently commercialized as gemzar. RNR inactivation is reductant-dependent and occurs in a very different way from that of other known substrate analogues. In the presence of reductants monomer R1 of RNR is inhibited, whereas in the absence of reductants the radical is lost and monomer R2 is inhibited. As inside the cell reductants are available, it is likely that R1 inactivation is the most favorable mechanism responsible for drug cytotoxicity. This inhibition pathway has been unknown to date, but we have conducted a theoretical study that has led us to the first proposal of a mechanism for RNR inhibition by dFdC in the presence of reductants.

Several 2'-substituted-2'-deoxyribonucleotides are potent inactivators of the enzyme ribonucleotide reductase (RNR), by destroying the essential tyrosyl radical located in subunit R2 or/and covalently alkylating the subunit R1. In the absence of external reductants, the inactivation is achieved by alkylation of subunit R1 by a methylene-3(2H)-furanone. The furanone is generated in solution through degradation of a keto-deoxyribonucleotide intermediate, produced during the inhibitory mechanism of a wide group of 2'-substituted inhibitors, and is easily detected experimentally by UV spectroscopy. Interestingly, the same keto-deoxyribonucleotide is also a proposed intermediate of the normal substrate pathway, but by some unknown reason, it does not dissociate from the active site and does not inactivate the enzyme. Therefore, if the currently accepted mechanism for substrate reduction is correct, there must be some specific reason that makes such a reactive intermediate behave differently, not dissociating from the active site during substrate reduction. In this article, we propose to validate the current substrate mechanism by showing that the keto-deoxyribonucleotide dissociates from the active site only in the case of the inhibitors, and therefore, it corresponds to a viable intermediate in the substrate mechanism. Furthermore, we answer unexplained experimental observations that concern the predomination of the normal reduction mechanism over the abnormal ketone formation in the FdNDP and the release of F(-), either in the normal or in the abnormal turnover. For that purpose, we have investigated the interaction between the enzyme and this keto-deoxyribonucleotide generated from the normal substrate and from two widely studied representative inhibitors. A model containing 140 atoms was used to represent the desired structures. The results allowed us to conclude that the solvation free energy of the 2'-substituents, its influence inside the active site, and the charge transfer mechanism from a protein side chain to solution are the thermodynamic driving forces for the intermediate dissociation and subsequent RNR inhibition. Such charge transfer cannot be accomplished by the natural substrate, preventing its dissociation. These results elucidate a paradox which has been unexplained for more than 20 years and further validates both the proposed substrate and inhibition chemical mechanisms.

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.

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.

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.

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., ((c) 2004 Wiley Periodicals, Inc)