Exploring Rigid and Flexible Scaffolds to Develop Potent Glucuronic Acid Glycodendrimers for Dengue Virus Inhibition.

Multivalent glycodendrimers are valuable tools for studying carbohydrate-protein interactions, and their scaffolds represent important components to increase specificity and affinity. Previous work by our group described the preparation of a tetravalent glucuronic acid rigid dendron that binds with good affinity to the dengue virus envelope protein (KD = 22 µM). Herein, the chemical synthesis and binding analysis of three new sets of rigid, semirigid, and flexible glucuronic acid-based dendrimers bearing different levels of multivalency and their interactions with the dengue virus envelope protein are described. The different oligoalkynyl scaffolds were coupled to glucuronic acid azides by a copper-catalyzed azide-alkyne cycloaddition reaction through optimized synthetic strategies to afford the desired glycodendrimers with good yields. Surface plasmon resonance studies have demonstrated that glycodendrimers 12b and 12c, with flexible scaffolds, give the best binding interactions with the dengue virus envelope protein (12b: KD = 0.487 µM and 12c: KD = 0.624 µM). Their binding constant values were 45 and 35 times higher than the one obtained in previous studies with a rigid tetravalent glucuronic acid dendron (KD = 22 µM), respectively. Molecular modeling studies were carried out in order to understand the difference in behavior observed for 12b and 12c. This work reports an efficient glycodendrimer chemical synthesis process that provides an appropriate scaffold that offers an easy and versatile strategy to find new active compounds against the dengue virus.

Expanding the synthesis of a library of potent glucuronic acid glycodendrons for Dengue virus inhibition.

Multivalent glycodendrons are valuable tools to mimic many structural and functional features of cell-surface glycoconjugates and its focal position scaffolds represent important components to increase specificity and affinity. Previous work in our group described the preparation of a tetravalent glucuronic acid dendron that binds with good affinity to Dengue virus envelope protein (KD = 22 µM). Herein, the chemical synthesis and binding analysis of a new library of potent glucuronic acid dendrons bearing different functional group at the focal position and different level of multivalency are described. Their chemical synthesis was performed sequentially in three stages and with good yields. Namely a) the chemical synthesis of the oligo and polyalkynyl scaffolds, b) assembling with fully protected glucuronic acid-based azide units by using a microwave assisted copper-catalysed azide-alkyne cycloaddition reaction and c) sequential deprotection of hydroxyl and carboxylic acid groups. Surface Plasmon Resonance studies have demonstrated that the valency and the focal position functional group exert influence on the interaction with Dengue virus envelope protein. Molecular modelling studies were carried out in order to understand the binding observed. This work reports an efficient glycodendrons chemical synthesis that provides appropriate focal position functional group and multivalence, that offer an easy and versatile strategy to find new active compounds against Dengue virus.

Biocatalyzed Synthesis of Glycostructures with Anti-infective Activity.

Molecules containing carbohydrate moieties play essential roles in fighting a variety of bacterial and viral infections. Consequently, the design of new carbohydrate-containing drugs or vaccines has attracted great attention in recent years as means to target several infectious diseases.Conventional methods to produce these compounds face numerous challenges because their current production technology is based on chemical synthesis, which often requires several steps and uses environmentally unfriendly reactants, contaminant solvents, and inefficient protocols. The search for sustainable processes such as the use of biocatalysts and eco-friendly solvents is of vital importance. Therefore, their use in a variety of reactions leading to the production of pharmaceuticals has increased exponentially in the last years, fueled by recent advances in protein engineering, enzyme directed evolution, combinatorial biosynthesis, immobilization techniques, and flow biocatalysis. In glycochemistry and glycobiology, enzymes belonging to the families of glycosidases, glycosyltransferases (Gtfs), lipases, and, in the case of nucleoside and nucleotide analogues, also nucleoside phosphorylases (NPs) are the preferred choices as catalysts.In this Account, on the basis of our expertise, we will discuss the recent biocatalytic and sustainable approaches that have been employed to synthesize carbohydrate-based drugs, ranging from antiviral nucleosides and nucleotides to antibiotics with antibacterial activity and glycoconjugates such as neoglycoproteins (glycovaccines, GCVs) and glycodendrimers that are considered as very promising tools against viral and bacterial infections.In the first section, we will report the use of NPs and N-deoxyribosyltransferases for the development of transglycosylation processes aimed at the synthesis of nucleoside analogues with antiviral activity. The use of deoxyribonucleoside kinases and hydrolases for the modification of the sugar moiety of nucleosides has been widely investigated.Next, we will describe the results obtained using enzymes for the chemoenzymatic synthesis of glycoconjugates such as GCVs and glycodendrimers with antibacterial and antiviral activity. In this context, the search for efficient enzymatic syntheses represents an excellent strategy to produce structure-defined antigenic or immunogenic oligosaccharide analogues with high purity. Lipases, glycosidases, and Gtfs have been used for their preparation.Interestingly, many authors have proposed the use Gtfs originating from the biosynthesis of natural glycosylated antibiotics such as glycopeptides, macrolides, and aminoglycosides. These have been used in the chemoenzymatic semisynthesis of novel antibiotic derivatives by modification of the sugar moiety linked to their complex scaffold. These contributions will be described in the last section of this review because of their relevance in the fight against the spreading phenomenon of antibiotic resistance. In this context, the pioneering in vivo synthesis of novel derivatives obtained by genetic manipulation of producer strains (combinatorial biosynthesis) will be shortly described as well.All of these strategies provide a useful and environmentally friendly synthetic toolbox. Likewise, the field represents an illustrative example of how biocatalysis can contribute to the sustainable development of complex glycan-based therapies and how problems derived from the integration of natural tools in synthetic pathways can be efficiently tackled to afford high yields and selectivity. The use of enzymatic synthesis is becoming a reality in the pharmaceutical industry and in drug discovery to rapidly afford collections of new antibacterial or antiviral molecules with improved specificity and better metabolic stability.

Enzymatic Synthesis and Molecular Modelling Studies of Rhamnose Esters Using Lipase from Pseudomonas stutzeri.

Rhamnolipids are becoming an important class of glycolipid biosurfactants. Herein, we describe for the first time the enzymatic synthesis of rhamnose fatty acid esters by the transesterification of rhamnose with fatty acid vinyl esters, using lipase from Pseudomonas stutzeri as a biocatalyst. The use of this lipase allows excellent catalytic activity in the synthesis of 4-O-acylrhamnose (99% conversion and full regioselectivity) after 3 h of reaction using tetrahydrofuran (THF) as the reaction media and an excess of vinyl laurate as the acyl donor. The role of reaction conditions, such as temperature, the substrates molar ratio, organic reaction medium and acyl donor chain-length, was studied. Optimum conditions were found using 35 °C, a molar ratio of 1:3 (rhamnose:acyldonor), solvents with a low logP value, and fatty acids with chain lengths from C4 to C18 as acyl donors. In hydrophilic solvents such as THF and acetone, conversions of up to 99-92% were achieved after 3 h of reaction. In a more sustainable solvent such as 2-methyl-THF (2-MeTHF), high conversions were also obtained (86%). Short and medium chain acyl donors (C4-C10) allowed maximum conversions after 3 h, and long chain acyl donors (C12-C18) required longer reactions (5 h) to get 99% conversions. Furthermore, scaled up reactions are feasible without losing catalytic action and regioselectivity. In order to explain enzyme regioselectivity and its ability to accommodate ester chains of different lengths, homology modelling, docking studies and molecular dynamic simulations were performed to explain the behaviour observed.

Structural Determinants of the Neuronal Glycine Transporter 2 for the Selective Inhibitors ALX1393 and ORG25543.

The neuronal glycine transporter GlyT2 modulates inhibitory glycinergic neurotransmission by controlling the extracellular concentration of synaptic glycine and the supply of neurotransmitter to the presynaptic terminal. Spinal cord glycinergic neurons present in the dorsal horn diminish their activity in pathological pain conditions and behave as gate keepers of the touch-pain circuitry. The pharmacological blockade of GlyT2 reduces the progression of the painful signal to rostral areas of the central nervous system by increasing glycine extracellular levels, so it has analgesic action. O-[(2-benzyloxyphenyl-3-fluorophenyl)methyl]-l-serine (ALX1393) and N-[[1-(dimethylamino)cyclopentyl]methyl]-3,5-dimethoxy-4-(phenylmethoxy)benzamide (ORG25543) are two selective GlyT2 inhibitors with nanomolar affinity for the transporter and analgesic effects in pain animal models, although with deficiencies which preclude further clinical development. In this report, we performed a comparative ligand docking of ALX1393 and ORG25543 on a validated GlyT2 structural model including all ligand sites constructed by homology with the crystallized dopamine transporter from Drosophila melanogaster. Molecular dynamics simulations and energy analysis of the complex and functional analysis of a series of point mutants permitted to determine the structural determinants of ALX1393 and ORG25543 discrimination by GlyT2. The ligands establish simultaneous contacts with residues present in transmembrane domains 1, 3, 6, and 8 and block the transporter in outward-facing conformation and hence inhibit glycine transport. In addition, differential interactions of ALX1393 with the cation bound at Na1 site and ORG25543 with TM10 define the differential sites of the inhibitors and explain some of their individual features. Structural information about the interactions with GlyT2 may provide useful tools for new drug discovery.

Synthesis of Glycodendrimers with Antiviral and Antibacterial Activity.

Glycodendrimers are an important class of synthetic macromolecules that can be used to mimic many structural and functional features of cell-surface glycoconjugates. Their carbohydrate moieties perform key important functions in bacterial and viral infections, often regulated by carbohydrate-protein interactions. Several studies have shown that the molecular structure, valency and spatial organisation of carbohydrate epitopes in glycoconjugates are key factors in the specificity and avidity of carbohydrate-protein interactions. Choosing the right glycodendrimers almost always helps to interfere with such interactions and blocks bacterial or viral adhesion and entry into host cells as an effective strategy to inhibit bacterial or viral infections. Herein, the state of the art in the design and synthesis of glycodendrimers employed for the development of anti-adhesion therapy against bacterial and viral infections is described.

Distinct binding of cetirizine enantiomers to human serum albumin and the human histamine receptor H1.

Cetirizine, a major metabolite of hydroxyzine, became a marketed second-generation H1 antihistamine that is orally active and has a rapid onset of action, long duration of effects and a very good safety record at recommended doses. The approved drug is a racemic mixture of (S)-cetirizine and (R)-cetirizine, the latter being the levorotary enantiomer that also exists in the market as a third-generation, non-sedating and highly selective antihistamine. Both enantiomers bind tightly to the human histamine H1 receptor (hH1R) and behave as inverse agonists but the affinity and residence time of (R)-cetirizine are greater than those of (S)-cetirizine. In blood plasma, cetirizine exists in the zwitterionic form and more than 90% of the circulating drug is bound to human serum albumin (HSA), which acts as an inactive reservoir. Independent X-ray crystallographic work has solved the structure of the hH1R:doxepin complex and has identified two drug-binding sites for cetirizine on equine serum albumin (ESA). Given this background, we decided to model a membrane-embedded hH1R in complex with either (R)- or (S)-cetirizine and also the complexes of both ESA and HSA with these two enantiomeric drugs to analyze possible differences in binding modes between enantiomers and also among targets. The ensuing molecular dynamics simulations in explicit solvent and additional computational chemistry calculations provided structural and energetic information about all of these complexes that is normally beyond current experimental possibilities. Overall, we found very good agreement between our binding energy estimates and extant biochemical and pharmacological evidence. A much higher degree of solvent exposure in the cetirizine-binding site(s) of HSA and ESA relative to the more occluded orthosteric binding site in hH1R is translated into larger positional fluctuations and considerably lower affinities for these two nonspecific targets. Whereas it is demonstrated that the two known pockets in ESA provide enough stability for cetirizine binding, only one such site does so in HSA due to a number of amino acid replacements. At the histamine-binding site in hH1R, the distinct interactions established between the phenyl and chlorophenyl moieties of the two enantiomers with the amino acids lining up the pocket and between their free carboxylates and Lys179 in the second extracellular loop account for the improved pharmacological profile of (R)-cetirizine.

Efficient Synthesis of Muramic and Glucuronic Acid Glycodendrimers as Dengue Virus Antagonists.

Carbohydrates are involved in many important pathological processes, such as bacterial and viral infections, by means of carbohydrate-protein interactions. Glycoconjugates with multiple carbohydrates are involved in multivalent interactions, thus increasing their binding strengths to proteins. In this work, we report the efficient synthesis of novel muramic and glucuronic acid glycodendrimers as potential Dengue virus antagonists. Aromatic scaffolds functionalized with a terminal ethynyl groups were coupled to muramic and glucuronic acid azides by click chemistry through optimized synthetic strategies to afford the desired glycodendrimers with high yields. Surface Plasmon Resonance studies have demonstrated that the compounds reported bind efficiently to the Dengue virus envelope protein. Molecular modelling studies were carried out to simulate and explain the binding observed. These studies confirm that efficient chemical synthesis of glycodendrimers can be brought about easily offering a versatile strategy to find new active compounds against Dengue virus.

Reaction mechanism of nucleoside 2'-deoxyribosyltransferases: free-energy landscape supports an oxocarbenium ion as the reaction intermediate.

Insight into the catalytic mechanism of Lactobacillus leichmannii nucleoside 2'-deoxyribosyltransferase (LlNDT) has been gained by calculating a quantum mechanics-molecular mechanics (QM/MM) free-energy landscape of the reaction within the enzyme active site. Our results support an oxocarbenium species as the reaction intermediate and thus an SN1 reaction mechanism in this family of bacterial enzymes. Our mechanistic proposal is validated by comparing experimental kinetic data on the impact of the single amino acid replacements Tyr7, Glu98 and Met125 with Ala, Asp and Ala/norLeu, respectively, and accounts for the specificity shown by this enzyme on a non-natural substrate. This work broadens our understanding of enzymatic C-N bond cleavage and C-N bond formation.

Modification of a Putative Third Sodium Site in the Glycine Transporter GlyT2 Influences the Chloride Dependence of Substrate Transport.

Neurotransmitter removal from glycine-mediated synapses relies on two sodium-driven high-affinity plasma membrane GlyTs that control neurotransmitter availability. Mostly glial GlyT1 is the main regulator of glycine synaptic levels, whereas neuronal GlyT2 promotes the recycling of synaptic glycine and supplies neurotransmitter for presynaptic vesicle refilling. The GlyTs differ in sodium:glycine symport stoichiometry, showing GlyT1 a 2:1 and GlyT2 a 3:1 sodium:glycine coupling. Sodium binds to the GlyTs at two conserved Na+ sites: Na1 and Na2. The location of GlyT2 Na3 site remains unknown, although Glu650 has been involved in the coordination. Here, we have used comparative MD simulations of a GlyT2 model constructed by homology to the crystalized DAT from Drosophila melanogaster by placing the Na3 ion at two different locations. By combination of in silico and experimental data obtained by biochemical and electrophysiological analysis of GlyTs mutants, we provide evidences suggesting the GlyT2 third sodium ion is held by Glu-250 and Glu-650, within a region with robust allosteric properties involved in cation-specific sensitivity. Substitution of Glu650 in GlyT2 by the corresponding methionine in GlyT1 reduced the charge-to-flux ratio to the level of GlyT1 without producing transport uncoupling. Chloride dependence of glycine transport was almost abolished in this GlyT2 mutant but simultaneous substitution of Glu250 and Glu650 by neutral amino acids rescued chloride sensitivity, suggesting that protonation/deprotonation of Glu250 substitutes chloride function. The differential behavior of equivalent GlyT1 mutations sustains a GlyT2-specific allosteric coupling between the putative Na3 site and the chloride site.

In silico-designed mutations increase variable new-antigen receptor single-domain antibodies for VEGF165 neutralization.

The stability, binding, and tissue penetration of variable new-antigen receptor (VNAR) single-domain antibodies have been tested as part of an investigation into their ability to serve as novel therapeutics. V13 is a VNAR that recognizes vascular endothelial growth factor 165 (VEGF165). In the present study V13 was used as a parental molecule into which we introduced mutations designed in silico. Two of the designed VNAR mutants were expressed, and their ability to recognize VEGF165 was assessed in vitro and in vivo. One mutation (Pro98Tyr) was designed to increase VEGF165 recognition, while the other (Arg97Ala) was designed to inhibit VEGF165 binding. Compared to parental V13, the Pro98Tyr mutant showed enhanced VEGF165 recognition and neutralization, as indicated by inhibition of angiogenesis and tumor growth. This molecule thus appears to have therapeutic potential for neutralizing VEGF165 in cancer treatment.

Nonketotic hyperglycinemia: Functional assessment of missense variants in GLDC to understand phenotypes of the disease.

The rapid analysis of genomic data is providing effective mutational confirmation in patients with clinical and biochemical hallmarks of a specific disease. This is the case for nonketotic hyperglycinemia (NKH), a Mendelian disorder causing seizures in neonates and early-infants, primarily due to mutations in the GLDC gene. However, understanding the impact of missense variants identified in this gene is a major challenge for the application of genomics into clinical practice. Herein, a comprehensive functional and structural analysis of 19 GLDC missense variants identified in a cohort of 26 NKH patients was performed. Mutant cDNA constructs were expressed in COS7 cells followed by enzymatic assays and Western blot analysis of the GCS P-protein to assess the residual activity and mutant protein stability. Structural analysis, based on molecular modeling of the 3D structure of GCS P-protein, was also performed. We identify hypomorphic variants that produce attenuated phenotypes with improved prognosis of the disease. Structural analysis allows us to interpret the effects of mutations on protein stability and catalytic activity, providing molecular evidence for clinical outcome and disease severity. Moreover, we identify an important number of mutants whose loss-of-functionality is associated with instability and, thus, are potential targets for rescue using folding therapeutic approaches.

First-in-class inhibitor of the T cell receptor for the treatment of autoimmune diseases.

Modulating T cell activation is critical for treating autoimmune diseases but requires avoiding concomitant opportunistic infections. Antigen binding to the T cell receptor (TCR) triggers the recruitment of the cytosolic adaptor protein Nck to a proline-rich sequence in the cytoplasmic tail of the TCR's CD3ε subunit. Through virtual screening and using combinatorial chemistry, we have generated an orally available, low-molecular weight inhibitor of the TCR-Nck interaction that selectively inhibits TCR-triggered T cell activation with an IC50 (median inhibitory concentration) ~1 nM. By modulating TCR signaling, the inhibitor prevented the development of psoriasis and asthma and, furthermore, exerted a long-lasting therapeutic effect in a model of autoimmune encephalomyelitis. However, it did not prevent the generation of a protective memory response against a mouse pathogen, suggesting that the compound might not exert its effects through immunosuppression. These results suggest that inhibiting an immediate TCR signal has promise for treating a broad spectrum of human T cell-mediated autoimmune and inflammatory diseases.

Beyond a Fluorescent Probe: Inhibition of Cell Division Protein FtsZ by mant-GTP Elucidated by NMR and Biochemical Approaches.

FtsZ is the organizer of cell division in most bacteria and a target in the quest for new antibiotics. FtsZ is a tubulin-like GTPase, in which the active site is completed at the interface with the next subunit in an assembled FtsZ filament. Fluorescent mant-GTP has been extensively used for competitive binding studies of nucleotide analogs and synthetic GTP-replacing inhibitors possessing antibacterial activity. However, its mode of binding and whether the mant tag interferes with FtsZ assembly function were unknown. Mant-GTP exists in equilibrium as a mixture of C2'- and C3'-substituted isomers. We have unraveled the molecular recognition process of mant-GTP by FtsZ monomers. Both isomers bind in the anti glycosidic bond conformation: 2'-mant-GTP in two ribose puckering conformations and 3'-mant-GTP in the preferred C2' endo conformation. In each case, the mant tag strongly interacts with FtsZ at an extension of the GTP binding site, which is also supported by molecular dynamics simulations. Importantly, mant-GTP binding induces archaeal FtsZ polymerization into inactive curved filaments that cannot hydrolyze the nucleotide, rather than straight GTP-hydrolyzing assemblies, and also inhibits normal assembly of FtsZ from the Gram-negative bacterium Escherichia coli but is hydrolyzed by FtsZ from Gram-positive Bacillus subtilis. Thus, the specific interactions provided by the fluorescent mant tag indicate a new way to search for synthetic FtsZ inhibitors that selectively suppress the cell division of bacterial pathogens.

Interactions of bacterial cell division protein FtsZ with C8-substituted guanine nucleotide inhibitors. A combined NMR, biochemical and molecular modeling perspective.

FtsZ is the key protein of bacterial cell-division and target for new antibiotics. Selective inhibition of FtsZ polymerization without impairing the assembly of the eukaryotic homologue tubulin was demonstrated with C8-substituted guanine nucleotides. By combining NMR techniques with biochemical and molecular modeling procedures, we have investigated the molecular recognition of C8-substituted-nucleotides by FtsZ from Methanococcus jannaschii (Mj-FtsZ) and Bacillus subtilis (Bs-FtsZ). STD epitope mapping and trNOESY bioactive conformation analysis of each nucleotide were employed to deduce differences in their recognition mode by each FtsZ species. GMP binds in the same anti conformation as GTP, whereas 8-pyrrolidino-GMP binds in the syn conformation. However, the anti conformation of 8-morpholino-GMP is selected by Bs-FtsZ, while Mj-FtsZ binds both anti- and syn-geometries. The inhibitory potencies of the C8-modified-nucleotides on the assembly of Bs-FtsZ, but not of Mj-FtsZ, correlate with their binding affinities. Thus, MorphGTP behaves as a nonhydrolyzable analog whose binding induces formation of Mj-FtsZ curved filaments, resembling polymers formed by the inactive forms of this protein. NMR data, combined with molecular modeling protocols, permit explanation of the mechanism of FtsZ assembly impairment by C8-substituted GTP analogs. The presence of the C8-substituent induces electrostatic remodeling and small structural displacements at the association interface between FtsZ monomers to form filaments, leading to complete assembly inhibition or to formation of abnormal FtsZ polymers. The inhibition of bacterial Bs-FtsZ assembly may be simply explained by steric clashes of the C8-GTP-analogs with the incoming FtsZ monomer. This information may facilitate the design of antibacterial FtsZ inhibitors replacing GTP.

CRDOCK: an ultrafast multipurpose protein-ligand docking tool.

An ultrafast docking and virtual screening program, CRDOCK, is presented that contains (1) a search engine that can use a variety of sampling methods and an initial energy evaluation function, (2) several energy minimization algorithms for fine tuning the binding poses, and (3) different scoring functions. This modularity ensures the easy configuration of custom-made protocols that can be optimized depending on the problem in hand. CRDOCK employs a precomputed library of ligand conformations that are initially generated from one-dimensional SMILES strings. Testing CRDOCK on two widely used benchmarks, the ASTEX diverse set and the Directory of Useful Decoys, yielded a success rate of ~75% in pose prediction and an average AUC of 0.66. A typical ligand can be docked, on average, in just ~13 s. Extension to a representative group of pharmacologically relevant G protein-coupled receptors that have been recently cocrystallized with some selective ligands allowed us to demonstrate the utility of this tool and also highlight some current limitations. CRDOCK is now included within VSDMIP, our integrated platform for drug discovery.

MM-ISMSA: An Ultrafast and Accurate Scoring Function for Protein-Protein Docking.

An ultrafast and accurate scoring function for protein-protein docking is presented. It includes (1) a molecular mechanics (MM) part based on a 12-6 Lennard-Jones potential; (2) an electrostatic component based on an implicit solvent model (ISM) with individual desolvation penalties for each partner in the protein-protein complex plus a hydrogen bonding term; and (3) a surface area (SA) contribution to account for the loss of water contacts upon protein-protein complex formation. The accuracy and performance of the scoring function, termed MM-ISMSA, have been assessed by (1) comparing the total binding energies, the electrostatic term, and its components (charge-charge and individual desolvation energies), as well as the per residue contributions, to results obtained with well-established methods such as APBSA or MM-PB(GB)SA for a set of 1242 decoy protein-protein complexes and (2) testing its ability to recognize the docking solution closest to the experimental structure as that providing the most favorable total binding energy. For this purpose, a test set consisting of 15 protein-protein complexes with known 3D structure mixed with 10 decoys for each complex was used. The correlation between the values afforded by MM-ISMSA and those from the other methods is quite remarkable (r(2) ∼ 0.9), and only 0.2-5.0 s (depending on the number of residues) are spent on a single calculation including an all vs all pairwise energy decomposition. On the other hand, MM-ISMSA correctly identifies the best docking solution as that closest to the experimental structure in 80% of the cases. Finally, MM-ISMSA can process molecular dynamics trajectories and reports the results as averaged values with their standard deviations. MM-ISMSA has been implemented as a plugin to the widely used molecular graphics program PyMOL, although it can also be executed in command-line mode. MM-ISMSA is distributed free of charge to nonprofit organizations.

VSDMIP 1.5: an automated structure- and ligand-based virtual screening platform with a PyMOL graphical user interface.

A graphical user interface (GUI) for our previously published virtual screening (VS) and data management platform VSDMIP (Gil-Redondo et al. J Comput Aided Mol Design, 23:171-184, 2009) that has been developed as a plugin for the popular molecular visualization program PyMOL is presented. In addition, a ligand-based VS module (LBVS) has been implemented that complements the already existing structure-based VS (SBVS) module and can be used in those cases where the receptor's 3D structure is not known or for pre-filtering purposes. This updated version of VSDMIP is placed in the context of similar available software and its LBVS and SBVS capabilities are tested here on a reduced set of the Directory of Useful Decoys database. Comparison of results from both approaches confirms the trend found in previous studies that LBVS outperforms SBVS. We also show that by combining LBVS and SBVS, and using a cluster of ~100 modern processors, it is possible to perform complete VS studies of several million molecules in less than a month. As the main processes in VSDMIP are 100% scalable, more powerful processors and larger clusters would notably decrease this time span. The plugin is distributed under an academic license upon request from the authors.

Synthesis, biological assessment and molecular modeling of 14-aryl-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]quinolin-13-amines.

The synthesis and pharmacological evaluation of racemic 14-aryl-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]quinolin-13-amines (19-28), prepared by Friedländer reaction of 3-amino-1-aryl-1H-benzo[f]chromene-2-carbonitriles (10-18) with suitable cycloalkanones is described. These molecules are potent, in the nanomolar range [IC(50) (EeAChE)=7-101 nM], and selective inhibitors of acetylcholinesterase (AChE). The most potent inhibitor, 4-(13-amino-10,11,12,14-tetrahydro-9H-benzo[5,6]chromeno[2,3-b]quinolin-14-yl)phenol (20) [IC(50) (EeAChE)=7±2 nM] is four-fold more active than tacrine. Kinetic studies on compound 20 showed that this is a mixed-type inhibitor of EeAChE with a K(i) of 5.00 nM. However, racemic 20 was unable to displace propidium iodide, suggesting that the inhibitor does not strongly bind to the peripheral anionic site (PAS) of AChE. Docking, molecular dynamics stimulations, and MM-GBSA calculations agree well with this behavior.

Dopamine quinone chemistry: a study of the influence of amide, amidine and guanidine substituents [-NH-CX-Y] on the mode of reaction.

The influence of N-substituents on the mode of reaction of ortho-quinones generated by oxidation of N-substituted dopamine derivatives has been studied. Ortho-quinones with amide, urea or guanidine side chains are relatively stable, with evidence of rearrangement to para-quinomethanes. The N-methylthiourea derivative rapidly cyclises giving a bicyclic product . The trichloromethylamidine derivative also rapidly cyclises but in this case gives a spirocyclic derivative . In contrast to the transient formation of spirocyclic products by other ortho-quinones derived from dopamine derivatives, e.g., , the product is stable and has been isolated and fully characterised.