Montelukast and cefoperazone act as antiquorum sensing and antibiofilm agents against Pseudomonas aeruginosa.

AIMS: Drug repurposing is an attractive strategy to control biofilm-related infectious diseases. In this study, two drugs (montelukast and cefoperazone) with well-established therapeutic applications were tested on Pseudomonas aeruginosa quorum sensing (QS) inhibition and biofilm control. METHODS AND RESULTS: The activity of montelukast and cefoperazone was evaluated for Pqs signal inhibition, pyocyanin synthesis, and prevention and eradication of Ps. aeruginosa biofilms. Cefoperazone inhibited the Pqs system by hindering the production of the autoinducer molecules 2-heptyl-4-hydroxyquinoline (HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal or PQS), corroborating in silico results. Pseudomonas aeruginosa pyocyanin production was reduced by 50%. The combination of the antibiotics cefoperazone and ciprofloxacin was synergistic for Ps. aeruginosa biofilm control. On the other hand, montelukast had no relevant effects on the inhibition of the Pqs system and against Ps. aeruginosa biofilm. CONCLUSION: This study provides for the first time strong evidence that cefoperazone interacts with the Pqs system, hindering the formation of the autoinducer molecules HHQ and PQS, reducing Ps. aeruginosa pathogenicity and virulence. Cefoperazone demonstrated a potential to be used in combination with less effective antibiotics (e.g. ciprofloxacin) to potentiate the biofilm control action.

A Molecular Mechanics Energy Partitioning Software for Biomolecular Systems.

The partitioning of the molecular mechanics (MM) energy in calculations involving biomolecular systems is important to identify the source of major stabilizing interactions, e.g., in ligand-protein interactions, or to identify residues with considerable contributions in hybrid multiscale calculations, i.e., quantum mechanics/molecular mechanics (QM/MM). Here, we describe Energy Split, a software program to calculate MM energy partitioning considering the AMBER Hamiltonian and parameters. Energy Split includes a graphical interface plugin for VMD to facilitate the selection of atoms and molecules belonging to each part of the system. Energy Split is freely available at or can be easily installed through the VMD Store.

Inactivation Mechanism of the Fatty Acid Amide Hydrolase Inhibitor BIA 10-2474.

BIA 10-2474 is a time-dependent inhibitor of fatty acid amide hydrolase (FAAH) that was under clinical development for the treatment of neurological conditions when the program was terminated after one subject died and four were hospitalized with neurological symptoms during a first-in-human clinical study. The present work describes the mechanism of FAAH inhibition by BIA 10-2474 as a target-specific covalent inhibition, supported by quantum mechanics and molecular modelling studies. The inhibitor incorporates a weakly reactive electrophile which, upon specific binding to the enzyme's active site, is positioned to react readily with the catalytic residues. The reactivity is enhanced on-site by the increased molarity at the reaction site and by specific inductive interactions with FAAH. In the second stage, the inhibitor reacts with the enzyme's catalytic nucleophile to form a covalent enzyme-inhibitor adduct. The hydrolysis of this adduct is shown to be unlikely under physiological conditions, therefore leading to irreversible inactivation of FAAH. The results also reveal the important role played by FAAH Thr236 in the reaction with BIA 10-2474, which is specific to FAAH and is not present in other serine hydrolases. It forms a hydrogen bond with the imidazole nitrogen of the inhibitor and helps lowering the activation free energy of the first step of the reaction, by pre-orienting and stabilizing the inhibitor in a near-reactive configuration. In the second step, Thr236 can also serve as a mechanistic alternative to protonate the leaving group.

The Catalytic Mechanism of Pdx2 Glutaminase Driven by a Cys-His-Glu Triad: A Computational Study.

The catalytic mechanism of Pdx2 was studied with atomic detail employing the computational ONIOM hybrid QM/MM methodology. Pdx2 employs a Cys-His-Glu catalytic triad to deaminate glutamine to glutamate and ammonia - the source of the nitrogen of pyridoxal 5'-phosphate (PLP). This enzyme is, therefore, a rate-limiting step in the PLP biosynthetic pathway of Malaria and Tuberculosis pathogens that rely on this mechanism to obtain PLP. For this reason, Pdx2 is considered a novel and promising drug target to treat these diseases. The results obtained show that the catalytic mechanism of Pdx2 occurs in six steps that can be divided into four stages: (i) activation of Cys87 , (ii) deamination of glutamine with the formation of the glutamyl-thioester intermediate, (iii) hydrolysis of the formed intermediate, and (iv) enzymatic turnover. The kinetic data available in the literature (19.1-19.5 kcal mol-1 ) agree very well with the calculated free energy barrier of the hydrolytic step (18.2 kcal.mol-11 ), which is the rate-limiting step of the catalytic process when substrate is readily available in the active site. This catalytic mechanism differs from other known amidases in three main points: i) it requires the activation of the nucleophile Cys87 to a thiolate; ii) the hydrolysis occurs in a single step and therefore does not require the formation of a second tetrahedral reaction intermediate, as it is proposed, and iii) Glu198 does not have a direct role in the catalytic process. Together, these results can be used for the synthesis of new transition state analogue inhibitors capable of inhibiting Pdx2 and impair diseases like Malaria and Tuberculosis.

New insights into the catalytic mechanism of the SARS-CoV-2 main protease: an ONIOM QM/MM approach.

SARS-CoV-2 Mpro, also known as the main protease or 3C-like protease, is a key enzyme involved in the replication process of the virus that is causing the COVID-19 pandemic. It is also the most promising antiviral drug target targeting SARS-CoV-2 virus. In this work, the catalytic mechanism of Mpro was studied using the full model of the enzyme and a computational QM/MM methodology with a 69/72-atoms QM region treated at DLPNO-CCSD(T)/CBS//B3LYP/6-31G(d,p):AMBER level and including the catalytic important oxyanion-hole residues. The transition state of each step was fully characterized and described together with the related reactants and products. The rate-limiting step of the catalytic process is the hydrolysis of the thioester-enzyme adduct, and the calculated barrier closely agrees with the available kinetic data. The calculated Gibbs free energy profile, together with the full atomistic detail of the structures involved in catalysis, can now serve as valuable models for the rational drug design of transition state analogs as new inhibitors targeting the SARS-CoV-2 virus.

An Unsual Cys-Glu-Lys Catalytic Triad is Responsible for the Catalytic Mechanism of the Nitrilase Superfamily: A QM/MM Study on Nit2.

Nitrilase 2 (Nit2) is a representative member of the nitrilase superfamily that catalyzes the hydrolysis of α-ketosuccinamate into oxaloacetate. It has been associated with the metabolism of rapidly dividing cells like cancer cells. The catalytic mechanism of Nit2 employs a catalytic triad formed by Cys191, Glu81 and Lys150. The Cys191 and Glu81 play an active role during the catalytic process while the Lys150 is shown to play only a secondary role. The results demonstrate that the catalytic mechanism of Nit2 involves four steps. The nucleophilic attack of Cys191 to the α-ketosuccinamate, the formation of two tetrahedral enzyme adducts and the hydrolysis of a thioacyl-enzyme intermediate, from which results the formation of oxaloacetate and enzymatic turnover. The rate limiting step of the catalytic process is the formation of the first tetrahedral intermediate with a calculated activation free energy of 18.4 kcal/mol, which agrees very well with the experimental kcat (17.67 kcal/mol).

Multifunctional Enzymes as Targets for the Treatment of Tuberculosis: Paving the Way for New Anti-TB Drugs.

In spite of the medical and technological developments of the last centuries, Tuberculosis (TB) has remained a challenging disease, with a limited number of therapeutic options, particularly in light of the increase in drug-resistant cases. The search for new molecules continues, with several candidates currently in clinical testing and ongoing efforts to identify novel targets. This work summarizes recent developments on anti-TB therapy, starting by discussing the current epidemiologic status and presenting an overview of the history of anti-tuberculosis drug discovery. Special attention is dedicated to five multifunctional enzymes that are regarded as promising targets for new anti-TB drugs: 5-aminoimidazole-4-carboxamide ribonucleotide transformylase/IMP cyclohydrolase (ATIC); 3,4-dihydroxy-2-butanone 4-phosphate synthase (DHBPS)/GTP cyclohydrolase II (GCHII); glutamine dependent NAD+ Synthetase (NadE); chorismate synthase (CS); and Tryptophan synthase (TS). These enzymes are involved in metabolic pathways critical for the M. tuberculosis survival, growth or replication, but that are not expressed in humans or have significant differences in terms of functionality, which makes them appealing targets. Their function, structure, possible catalytic mechanisms, and current inhibition strategies and inhibitors are reviewed and discussed.

Alkaline phosphatase dual-binding sites for collagen dictate cell migration and microvessel assembly in vitro.

Interactions between cell types, growth factors, and extracellular matrix components involved in angiogenesis are crucial for new vessel formation leading to tissue regeneration. This study investigated whether cocultures of fibroblasts and endothelial cells (ECs; from macro- or microvasculature) play a role in the formation of microvessel-like structures by ECs, as well as modulate fibroblast differentiation and growth factors production (vascular endothelial cell growth factor, basic fibroblast growth factor, active transforming growth factor-ß1, and interleukin-8), which are important for vessel sprouting and maturation. Data obtained revealed that in vitro coculture systems of fibroblasts and human ECs stimulate collagen synthesis and growth factors production by fibroblasts that ultimately affect the formation and distribution of microvessel-like structures in cell cultures. In this study, areas with activated fibroblasts and high alkaline phosphatase (ALP) activity were also observed in cocultures. Molecular docking assays revealed that ALP has two binding positions for collagen, suggesting its impact in collagen proteins' aggregation, cell migration, and microvessel assembly. These findings indicate that bioinformatics and coculture systems are complementary tools for investigating the participation of proteins, like collagen and ALP in angiogenesis.

A Molecular Perspective on Sirtuin Activity.

The protein acetylation of either the α-amino groups of amino-terminal residues or of internal lysine or cysteine residues is one of the major posttranslational protein modifications that occur in the cell with repercussions at the protein as well as at the metabolome level. The lysine acetylation status is determined by the opposing activities of lysine acetyltransferases (KATs) and lysine deacetylases (KDACs), which add and remove acetyl groups from proteins, respectively. A special group of KDACs, named sirtuins, that require NAD+ as a substrate have received particular attention in recent years. They play critical roles in metabolism, and their abnormal activity has been implicated in several diseases. Conversely, the modulation of their activity has been associated with protection from age-related cardiovascular and metabolic diseases and with increased longevity. The benefits of either activating or inhibiting these enzymes have turned sirtuins into attractive therapeutic targets, and considerable effort has been directed toward developing specific sirtuin modulators. This review summarizes the protein acylation/deacylation processes with a special focus on the current developments in the sirtuin research field.

Benzo[a]phenoxazinium chlorides: Synthesis, antifungal activity, in silico studies and evaluation as fluorescent probes.

Four new benzo[a]phenoxazinium chlorides with combinations of chloride, ethyl ester and methyl as terminals of the amino substituents were synthesized. These compounds were characterized and their optical properties were studied in absolute dry ethanol and water. Their antiproliferative activity was tested against Saccharomyces cerevisiae in a broth microdilution assay, along with an array of 36 other benzo[a]phenoxazinium chlorides. Minimum Inhibitory Concentration (MIC) values between 1.56 and >200 µM were observed. Fluorescence microscopy studies, used to assess the intracellular distribution of the dyes, showed that these benzo[a]phenoxazinium chlorides function as efficient and site specific probes for the detection of the vacuole membrane. The added advantage of some of the compounds, that displayed the lower MIC values, was the simultaneous staining of both the vacuole membrane and the perinuclear membrane of endoplasmic reticulum (ER). Molecular docking studies were performed on the human membrane protein oxidosqualene cyclase (OSC), using the crystal structure available on PDB (code 1W6K). The results showed that these most active compounds accommodated better in the active sites of ER enzyme OSC suggesting this enzyme as a potential target. As a whole, the results demonstrate that the benzo[a]phenoxazinium chlorides are interesting alternatives to the available commercial dyes. Changes in the substituents of these compounds can tailor both their staining specificity and antimicrobial activity.

(3S,4R)-3,4-Dihydroxy-N-alkyl-l-homoprolines: synthesis and computational mechanistic studies.

This is the first synthetic report of (3S,4R)-dihydroxy-N-alkyl-l-homoprolines described so far. 2,4-O-Benzylidene-d-erythrose was obtained from d-glucose with an improved yield, and then transformed into the title (3S,4R)-dihydroxy-N-alkyl-l-homoprolines, in a two-step strategy, with excellent overall yields. Hydrogenolysis of the benzyl group led to the NH congener. The synthesis of final products from 1,4-lactone intermediates was studied by computational means either under acidic or basic conditions. The theoretical mechanism studies fully explain the experimental results: (a) an equilibrium between l-homoprolines and their bicyclic counterparts is established in acids; (b) the equilibrium suffers a complete displacement towards the l-homoproline side in a basic medium.

VMD Store-A VMD Plugin to Browse, Discover, and Install VMD Extensions.

Herein we present the VMD Store, an open-source VMD plugin that simplifies the way that users browse, discover, install, update, and uninstall extensions for the Visual Molecular Dynamics (VMD) software. The VMD Store obtains data about all the indexed VMD extensions hosted on GitHub and presents a one-click mechanism to install and configure VMD extensions. This plugin arises in an attempt to aggregate all VMD extensions into a single platform. The VMD Store is available, free of charge, for Windows, macOS, and Linux at https://biosim.pt/software/ and requires VMD 1.9.3 (or later).

Formation of Unstable and very Reactive Chemical Species Catalyzed by Metalloenzymes: A Mechanistic Overview.

Nature has tailored a wide range of metalloenzymes that play a vast array of functions in all living organisms and from which their survival and evolution depends on. These enzymes catalyze some of the most important biological processes in nature, such as photosynthesis, respiration, water oxidation, molecular oxygen reduction, and nitrogen fixation. They are also among the most proficient catalysts in terms of their activity, selectivity, and ability to operate at mild conditions of temperature, pH, and pressure. In the absence of these enzymes, these reactions would proceed very slowly, if at all, suggesting that these enzymes made the way for the emergence of life as we know today. In this review, the structure and catalytic mechanism of a selection of diverse metalloenzymes that are involved in the production of highly reactive and unstable species, such as hydroxide anions, hydrides, radical species, and superoxide molecules are analyzed. The formation of such reaction intermediates is very difficult to occur under biological conditions and only a rationalized selection of a particular metal ion, coordinated to a very specific group of ligands, and immersed in specific proteins allows these reactions to proceed. Interestingly, different metal coordination spheres can be used to produce the same reactive and unstable species, although through a different chemistry. A selection of hand-picked examples of different metalloenzymes illustrating this diversity is provided and the participation of different metal ions in similar reactions (but involving different mechanism) is discussed.

Small phospho-donors phosphorylate MorR without inducing protein conformational changes.

Phosphorylation is an essential mechanism of protein control and plays an important role in biology. The two-component system (TCS) is a bacterial regulation mechanism mediated by a response regulator (RR) protein and a kinase protein, which synchronize the regulatory circuit according to the environment. Phosphorylation is a key element in TCS function as it controls RR activity. In the present study, we characterize the behavior of MorR, an RR associated with Mo homeostasis, upon acetylphosphate and phosphoramidate treatment in vitro. Our results show that MorR was phosphorylated by both phospho-donors. Fluorescence experiments showed that MorR tryptophan emission is quenched by phosphoramidate. Furthermore, theoretical and computational results demonstrate that phosphorylation by phosphoramidate is more favorable than that by acetylphosphate. In conclusion, phosphorylated MorR is a monomeric protein and phosphorylation does not appear to induce observable conformational changes in the protein structure.

Total Stereoselective Michael Addition of N- and S- Nucleophiles to a d-Erythrosyl 1,5-Lactone Derivative. Experimental and Theoretical Studies Devoted to the Synthesis of 2,6-Dideoxy-4-functionalized-d- ribono-hexono-1,4-lactone.

The synthesis of a 1,5-lactone 2,4- O-alkylidene-d-erythrose derivative was found to be a highly stereoselective template in Michael addition trough the reaction of a d-erythrosyl 1,5-lactone derivative with nitrogen and sulfur nucleophiles. The sulfur adducts formed are 1 (d-erythrose derivative):1 (nucleophile), and the nitrogen adducts are 1:2. Both were then treated under HCl to give 2,6-dideoxy-4-functionalized-d- ribono-hexono-1,4-lactone by a reaction cascade in high overall yield. Reaction's scale up even improves the yield. The theoretical and computational results clearly explain the origin of the stereoselectivity, and the energetic course of reactions starting with nitrogen and sulfide nucleophiles. Considering that the 1,4-lactones obtained in this work offer a new molecular scaffold for organic synthesis, these new results provide a solid theoretical platform that can be used to speed up synthesis of other derivatives in a stereo- and regioselective way.

Parametrization of Molybdenum Cofactors for the AMBER Force Field.

Molybdenum-containing enzymes have been receiving significant attention from the scientific community since they participate in important biological processes and in the global biogeochemical cycles of carbon, nitrogen, and sulfur. Molecular modeling studies of these metalloproteins with classical force fields are, however, often hampered by the "missing parameter" problem. In this article, a set of parameters have been determined for the AMBER force field from nine different molybdenum cofactors. All of these cofactors were optimized and parametrized using a bonded model approach. All of the bonds and angles involving the Mo ion were parametrized. The Restrained Electrostatic Potential charges of the coordination sphere of each cofactor were also determined. Finally, we demonstrated the stability of our parameters in a showcase of nine enzymes. The parameters presented in this work provide a valuable resource for the molecular simulation community as they extend the range of metal ions that can be studied using classical approaches while also providing a starting point for subsequent parametrization of similar metal centers. All of the developed parameters can be found in the Supporting Information .

Visualizing the Microscopic World.

Visualization can be a motivating way of teaching students about the microscopic world. This can become even more exciting if the information is based on accurate computational results rather than on crude approximations that eventually might create unreal alternative perceptions. Here, we report on a VMD plug-in, named vmdMagazine, which can turn computational simulations into stunning high-impact video presentations, suitable for classes/lectures and even conferences. The software will help students/audience to understand atoms and molecules better and learn to like them. The present paper is meant to give a general idea of the software's potential, showing how it works and how it can be used for educational purposes. The software is freely available at: http://www.fc.up.pt/PortoBioComp/database/doku.php?id=vmdmagazine .

The Catalytic Mechanism of the Pyridoxal-5'-phosphate-Dependent Enzyme, Histidine Decarboxylase: A Computational Study.

The catalytic mechanism of histidine decarboxylase (HDC), a pyridoxal-5'-phosphate (PLP)-dependent enzyme, was studied by using a computational QM/MM approach following the scheme M06-2X/6-311++G(3df,2pd):Amber. The reaction involves two sequential steps: the decarboxylation of l-histidine and the protonation of the generated intermediate from which results histamine. The rate-limiting step is the first one (ΔG≠ =17.6 kcal mol-1 ; ΔGr =13.7 kcal mol-1 ) and agrees closely with the available experimental kcat (1.73 s-1 ), which corresponds to an activation barrier of 17.9 kcal mol-1 . In contrast, the second step is very fast (ΔG≠ =1.9 kcal mol-1 ) and exergonic (ΔGr =-33.2 kcal mol-1 ). Our results agree with the available experimental data and allow us to explain the role played by several active site residues that are considered relevant according to site-directed mutagenesis studies, namely Tyr334B, Asp273A, Lys305A, and Ser354B. These results can provide insights regarding the catalytic mechanism of other enzymes belonging to family II of PLP-dependent decarboxylases.

Total Facial Discrimination of 1,3-Dipolar Cycloadditions in a d-Erythrose 1,3-Dioxane Template: Computational Studies of a Concerted Mechanism.

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 Biosynthesis: A Mechanistic Overview.

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.