Molecular modelling studies of new potential human DNA polymerase α inhibitors.

The human polymerase α (pol α) is a promising target for the therapy of cancer e.g. of the skin. The authors recently built a homology model of the active site of human DNA pol α. This 3D model was now used for molecular modelling studies with eight novel analogues of 2-butylanilino-dATP, which is a highly selective nucleoside inhibitor of mammalian pol α. Our results suggest that a higher hydrophobicity of a carbohydrate side chain (pointing into a spacious hydrophobic cavity) may enhance the strength of the interaction with the target protein. Moreover, acyclic acyclovir-like derivatives outperformed those with a sugar-moiety, indicating that structural flexibility and higher conformational adaptability has a positive effect on the receptor affinity. Cytotoxicity tests confirmed our theoretical findings. Besides, one of our most promising compounds in the molecular modelling studies revealed high selectivity for the SCC-25 cell line derived from squamous cell carcinoma in man.

Dualsteric GPCR targeting: a novel route to binding and signaling pathway selectivity.

Selective modulation of cell function by G protein-coupled receptor (GPCR) activation is highly desirable for basic research and therapy but difficult to achieve. We present a novel strategy toward this goal using muscarinic acetylcholine receptors as a model. The five subtypes bind their physiological transmitter in the highly conserved orthosteric site within the transmembrane domains of the receptors. Orthosteric muscarinic activators have no binding selectivity and poor signaling specificity. There is a less well conserved allosteric site at the extracellular entrance of the binding pocket. To gain subtype-selective receptor activation, we synthesized two hybrids fusing a highly potent oxotremorine-like orthosteric activator with M(2)-selective bis(ammonio)alkane-type allosteric fragments. Radioligand binding in wild-type and mutant receptors supplemented by receptor docking simulations proved M(2) selective and true allosteric/orthosteric binding. G protein activation measurements using orthosteric and allosteric blockers identified the orthosteric part of the hybrid to engender receptor activation. Hybrid-induced dynamic mass redistribution in CHO-hM(2) cells disclosed pathway-specific signaling. Selective receptor activation (M(2)>M(1)>M(3)) was verified in living tissue preparations. As allosteric sites are increasingly recognized on GPCRs, the dualsteric concept of GPCR targeting represents a new avenue toward potent agonists for selective receptor and signaling pathway activation.

Human polymerase alpha inhibitors for skin tumors. Part 2. Modeling, synthesis and influence on normal and transformed keratinocytes of new thymidine and purine derivatives.

Recently, the three-dimensional structure of the active site of human DNA polymerase alpha (pol alpha) was proposed based on the application of molecular modeling methods and molecular dynamic simulations. The modeled structure of the enzyme was used for docking selective inhibitors (nucleotide analogs and the non-nucleoside inhibitor aphidicolin) in its active site in order to design new drugs for actinic keratosis and squamous cell carcinoma (SCC). The resulting complexes explained the geometrical and physicochemical interactions of the inhibitors with the amino acid residues involved in binding to the catalytic site, and offered insight into the experimentally derived binding data. The proposed structures were synthesized and tested in vitro for their influence on human keratinocytes and relevant tumor cell lines. Effects were compared to aphidicolin which inhibits pol alpha in a non-competitive manner, as well as to diclofenac and 5-fluorouracil, both approved for therapy of actinic keratosis. Here we describe three new nucleoside analogs inhibiting keratinocyte proliferation by inhibiting DNA synthesis and inducing apoptosis and necrosis. Thus, the combination of modeling studies and in vitro tests should allow the derivation of new drug candidates for the therapy of skin tumors, given that the agents are not relevant substrates of nucleotide transporters expressed by skin cancer cells. Kinases for nucleoside activation were detected, too, corresponding with the observed effects of nucleoside analogs.

Interactions of gallic acid, resveratrol, quercetin and aspirin at the platelet cyclooxygenase-1 level. Functional and modelling studies.

While resveratrol and quercetin possess antiplatelet activity, little is known on the effect of gallic acid on platelets. We studied the interactions of these three different polyphenols among themselves and with aspirin, at the level of platelet cyclooxygenase-1 (COX-1). Both functional (in vitro and in vivo) and molecular modelling approaches were used. All three polyphenols showed comparable antioxidant activity (arachidonic acid [AA]-induced intraplatelet ROS production); however, resveratrol and quercetin, but not gallic acid, inhibited AA-induced platelet aggregation. Gallic acid, similarly to salicylic acid, the major aspirin metabolite, prevented inhibition of AA-induced platelet function by aspirin but, at variance with salicylic acid, also prevented inhibition by the other two polyphenols. Molecular modelling studies, performed by in silico docking the polyphenols into the crystal structure of COX-1, suggested that all compounds form stable complexes into the COX-1 channel, with slightly different but functionally relevant interaction geometries. Experiments in mice showed that gallic acid administered before aspirin, resveratrol or quercetin fully prevented their inhibitory effect on serum TxB(2). Finally, a mixture of resveratrol, quercetin and gallic acid, at relative concentrations similar to those contained in most red wines, did not inhibit platelet aggregation, but potentiated sub-inhibitory concentrations of aspirin. Gallic acid interactions with other polyphenols or aspirin at the level of platelet COX-1 might partly explain the complex, and possibly contrasting, effects of wine and other components of the Mediterranean diet on platelets and on the pharmacologic effect of low-dose aspirin.

An indole-binding site is a major determinant of the ligand specificity of the GABA type A receptor-associated protein GABARAP.

The role of tryptophan as a key residue for ligand binding to the ubiquitin-like modifier GABA(A) receptor associated protein (GABARAP) was investigated. Two tryptophan-binding hydrophobic patches were identified on the conserved face of the GABARAP structure by NMR spectroscopy and molecular docking. GABARAP binding of indole and indole derivatives, including the free amino acid tryptophan was quantified. The two tryptophan binding sites can be clearly distinguished by mapping the NMR spectroscopy-derived residue-specific apparent dissociation constant, K(d), onto the three-dimensional structure of GABARAP. The biological relevance of tryptophan-binding pockets of GABARAP was supported by a highly conserved tryptophan residue in the GABARAP binding region of calreticulin, clathrin heavy chain, and the gamma2 subunit of the GABA(A) receptor. Replacement of tryptophan by alanine abolished ligand binding to GABARAP.

A three-dimensional model of CYP19 aromatase for structure-based drug design.

Aromatase (CYP450(arom), CYP19) is an enzyme responsible for converting the aliphatic androgens androstenedione and testosterone to the aromatic estrogens estrone and estradiol, respectively. These endogenous hormones are a key factor in cancer tumor formation and proliferation through a cascade starting from estrogen binding to estrogen receptor. To interfere with the overproduction of estrogens especially in tumor tissue, it is possible to inhibit aromatase activity. This can be achieved using aromatase inhibitors. In order to design novel aromatase inhibitors, it is necessary to have an understanding of the active site of aromatase. As no crystal structure of the enzyme has yet been published, we built a homology model of aromatase using the first crystallized mammalian cytochrome enzyme, rabbit 21-progesterone hydroxylase 2C5, as a template structure. The initial model was validated with exhaustive molecular dynamics simulation with and without the natural substrate androstenedione. The resulting enzyme-substrate complex shows very good stability and only two of the residues are in disallowed regions in a Ramachandran plot.

Metalloporphyrins inactivate caspase-3 and -8.

Activation of caspases represents one of the earliest biochemical indicators for apoptotic cell death. Therefore, measurement of caspase activity is a widely used and generally accepted method to determine apoptosis in a wide range of in vivo and in vitro settings. Numerous publications characterize the role of the heme-catabolizing enzyme heme oxygenase-1 (HO-1) in regulating apoptotic processes. Different metalloporphyrins representing inducers and inhibitors of this enzyme are often used, followed by assessment of apoptotic cell death. In the present work, we found that caspase-3-like activity, as well as activity of caspase-8 measured in either Fas (CD95) ligand-treated Jurkat T-lymphocytes or by the use of recombinant caspase-3 or -8, was inhibited by different metalloporphyrins (cobalt(III) protoporphyrin IX, tin and zinc(II) protoporphyrin-IX). Moreover, employing the mouse model of Fas-induced liver apoptosis these properties of porphyrins could also be demonstrated in vivo. The metalloporphyrins were shown to inhibit caspase-3-mediated PARP cleavage. Molecular modeling studies demonstrated that porphyrins can occupy the active site of caspase-3 in an energetically favorable manner and in a binding mode similar to that of known inhibitors. The data shown here introduce metalloporphyrins as direct inhibitors of caspase activity. This finding points to the need for careful employment of metalloporphyrins as modulators of HO-1.

Kappa-opioid receptor model in a phospholipid bilayer: molecular dynamics simulation.

A three-dimensional molecular model of the transmembrane domain of the kappa-opioid receptor in a phospholipid bilayer is presented. The endogenous ligand, dynorphin A (1), and synthetic ligands, benzomorphan-based compounds (2a, 2b) (Figure 1), are docked into the model. We report the results of a 500 ps molecular dynamics simulation of these protein-ligand complexes in a simplified bilayer of 97 molecules of the lipid dipalmitoylphosphatidylcholine and 26 water molecules per lipid. The simulations explore the stability and conformational dynamics of the model in a phospholipid bilayer; we also investigate the interactions of the protein with its ligands. Molecular simulation of the receptor-ligand complexes, endogenous and synthetic, has confirmed the existence of different binding domains for peptide and non-peptide ligands. Similarities are found in the dynamics and binding mode of all conformations of the synthetic ligands studied. The protonated hydrogen of the benzomorphan is always involved in an H-bond with Asp138, and other potentially stabilizing receptor-ligand interactions found involve the hydroxyl substituent on the benzomorphan, which may form an H-bond with Tyr139 or Gly190 according to the different molecules. The ester group of 2a may therefore form an H-bond with Ile316, while the carbonyl group of 2b forms an H-bond with Gln115 and Tyr312. The remaining part of the ligand is located in the extracellular portion of the pocket. It is surrounded by hydrophobic residues in the transmembrane region (TM), and it interacts with different sets of residues. The results obtained are in general agreement with site-directed mutagenesis data that have highlighted the importance of all TM regions for synthetic-ligand affinity with the kappa-opioid receptor.

A combined QM/MM approach to protein--ligand interactions: polarization effects of the HIV-1 protease on selected high affinity inhibitors.

HIV-1 protease inhibitors are one of the two widely used therapeutic agents for the treatment of HIV-infected patients. The investigation of HIV-1 protease-inhibitor interactions can provide further insight for developing new compounds that are still required due to the growing problem of drug resistance. To this end, a combined QM/MM approach was used to determine electrostatic and polarization interactions on three high affinity inhibitors, nelfinavir, mozenavir, and tipranavir. The present computational results show that explicit treatment of the polarization effect is particularly important since it can contribute as much as one-third of the total electrostatic interaction energy. Further, an amino acid decomposition analysis was applied to determine contributions of individual residues to the enzyme--inhibitor interactions. It was found that the 4-hydroxy-dihydropyrone substructure of tipranavir is especially suited for extended charge delocalization by interacting with the catalytic aspartates and isoleucines of the HIV-1 protease. The calculated electron density difference maps reaffirm and provide a means of visualizing these results.

N-(omega-(4-(2-methoxyphenyl)piperazin-1-yl)alkyl)carboxamides as dopamine D2 and D3 receptor ligands.

The dopamine D(3) receptor is recognized as a potential therapeutic target for the treatment of various neurological and psychiatric disorders. Targetting high affinity and D(3) versus D(2) receptor-preferring ligands, the partial agonist BP 897 was taken as a lead structure. Variations in the spacer and the aryl moiety led to N-alkylated 1-(2-methyoxyphenyl)piperazines with markedly improved affinity and selectivity. Molecular modeling studies supported the structural development. Pharmacophore models for dopamine D(2) and D(3) receptor ligands were developed from their potentially bioactive conformation and were compared in order to get insight into molecular properties of importance for D(2)/D(3) receptor selectivity. For the 72 compounds presented here, an extended and more linear conformation in the aliphatic or aryl spacers turned out to be crucial for dopamine D(3) receptor selectivity. Structural diversity in the aryl moiety (benzamides, heteroarylamides, arylimides) had a major influence on (sub)nanomolar D(3) receptor affinity, which was optimized with more rigid aryl acrylamide derivatives. Compound 38 (ST 280, (E)-4-iodo-N-(4-(4-(2-methoxyphenyl)piperazin-1-yl)butyl)cinnamoylamide) displayed a most promising pharmacological profile (K(i) (hD(3)) = 0.5 nM; K(i) (hD(2L)) = 76.4 nM; selectivity ratio of 153), and above that, compound 38 offered the prospect of a novel radioligand as a pharmacological tool for various D(3) receptor-related in vitro and in vivo investigation.

Design, synthesis, structure-activity relationships, and molecular modeling studies of 2,3-diaryl-1,3-thiazolidin-4-ones as potent anti-HIV agents.

Starting from 1H,3H-thiazolo[3,4-a]benzimidazoles (TBZs), we performed the design, synthesis, and the structure-activity relationship studies of a series of 2,3-diaryl-1,3-thiazolidin-4-ones. Some derivatives proved to be highly effective in inhibiting HIV-1 replication at nanomolar concentrations with minimal cytotoxicity, thereby acting as nonnucleoside HIV-1 RT inhibitors (NNRTIs). Computational studies were used to delineate the ligand-RT interactions and to probe the binding of the ligands to HIV-1 RT.

Molecular modeling and QSAR studies on K(ATP) channel openers of the benzopyran type.

The present paper describes our molecular modeling and quantitative structure-activity relationships (QSARs) studies on K(ATP) channel openers (KCOs) of the benzopyran type. In the first part we performed molecular modeling investigations with seven benzopyrans, varied at the C3- and C4-positions, in order to understand which molecular features at these positions are essentially effecting the biological activity. The impact of C6-substitution on biological activity was studied in the second part via HANSCH analysis. For this purpose physicochemical properties (charge distributions, lowest unoccupied molecular orbital (LUMO) energies, desolvation energies, volumes and dipole moments) were calculated for a set of 50 C6-varied benzopyrans. A QSAR equation was developed showing a relationship between the vasodilator activity and the direction of the dipole vector of the ligands. The conclusion can be drawn that a direct interaction between the C6-substituents and the receptor structure is not of primary importance. However, the substitutents influence the orientation of the whole ligand approaching the binding site. An unfavorably oriented ligand cannot bind to the binding site, thus exhibiting weak activity.

Glucocorticoid receptor interactions with glucocorticoids: evaluation by molecular modeling and functional analysis of glucocorticoid receptor mutants.

In the treatment of inflammatory skin diseases, there are some glucocorticoid (GC) double esters combining pronounced antiinflammatory activity and minor atrophogenic side effects. The reason, however, is only poorly understood. To investigate interactions of GCs with the ligand-binding domain of the glucocorticoid receptor (GR), we measured receptor-binding potency of a series of GC esters including their metabolites and performed a molecular modeling study using progesterone receptor crystal structure data. Ligand docking to the GR-binding pocket showed good fitting of GC 17-esters corresponding to their high receptor-binding affinity, and unfavorable sterical interactions for GC 21-esters with substituents larger than propionate. Molecular dynamics simulations served to visualize induced fit procedures. Ligand docked GC conformations after dynamics simulations were used for generation of a 3D quantitative structure-activity relationship model. Using a set of 11 steroids, this model showed a correlation coefficient (r(2)) of 0.98, a leave-one-out cross validation (q(2)) of 0.79 and was able to predict binding affinity of further six ligands with a standard error of prediction of 0.33. Moreover, interactions of Asn-564 and Met-639 with the steroids were investigated by studying GR mutants of these amino acids. Met-639 participates in hydrophobic interactions mainly with GC side chains, while Asn-564 forms a hydrogen bond to the C11-OH group of the steroid. Asn-564 is shown to be very important for ligand binding and even more for target gene activation and transcription factor repression.

Design, synthesis, inhibition studies, and molecular modeling of pepstatin analogues addressing different secreted aspartic proteinases of Candida albicans.

The family of secreted aspartic proteinases is known as an important virulence factor of yeast infections by Candida albicans in particular, which is the most common fungal pathogen for humans with respect to systemic disease. Due to the continuing increase of drug resistant strains, these proteinases are currently considered as promising drug target candidates. Based on the known Sap2-substrate specificity data and X-ray analyses of Sap/inhibitor complexes, three libraries of inhibitors were designed and synthesized by modifying the structure of pepstatin A, a common non-selective aspartic proteinase inhibitor, at the P3, P2, or P2' position. These novel inhibitors showed high inhibitory potencies for the isoenzymes Sap1, Sap3, Sap5 and Sap6. Then, the affinity and selectivity of the peptide ligands were investigated by molecular modeling, highlighting new key structural information for the design of potent and selective anti-virulence agents targeting Candida albicans.

Antitumor effects of guanosine-analog phosphonates identified by molecular modelling.

Aiming to address new drug targets, molecular modelling is gaining increasing importance although the prediction capability of the in silico method is still under debate. For an improved treatment of actinic keratosis and squamous cell carcinoma, inhibitors of human DNA polymerase alpha (pol alpha) are developed by docking nucleoside phosphonate diphosphates into the active site of pol alpha. The most promising prodrugs OxBu and OxHex were then prepared by total synthesis and tested in the squamous cancer cell line SCC25. OxBu and OxHex proved cytotoxic and antiproliferative in the nanomolar concentration range and thus exceeded activity of aphidicolin, the relevant model compound, and 5-fluorouracil, the current standard for the therapy of actinic keratosis. Interestingly, the cytotoxicity in normal human keratinocytes with OxHex was clearly less pronounced and even not detectable with OxBu. Moreover, cytotoxicity of OxBu in particular with the colorectal carcinoma cell line HT29 even surmounted cytotoxicity in SCC25, and other tumor cell lines were influenced, too, by both agents. Taken together, OxBu and OxHex may offer a new approach to cancer therapy, given the agents are sufficiently well tolerated in vivo which is to be suspected beside their chemical structure.

Key determinants of nucleotide-activated G protein-coupled P2Y(2) receptor function revealed by chemical and pharmacological experiments, mutagenesis and homology modeling.

The P2Y(2) receptor, which is activated by UTP, ATP, and dinucleotides, was studied as a prototypical nucleotide-activated GPCR. A combination of receptor mutagenesis, determination of its effects on potency and efficacy of agonists and antagonists, homology modeling, and chemical experiments was applied. R272 (extracellular loop EL3) was found to play a gatekeeper role, presumably responsible for recognition and orientation of the nucleotides. R272 is also directly involved in binding of dinucleotides, which behaved as partial agonists. Y118A (3.37) mutation led to dramatically reduced efficacy of agonists; it is part of the entry channel as well as the triphosphate binding site. While the Y114A (3.33) mutation did not have any effect on agonist activities, the antagonist Reactive Blue 2 (6) was completely inactive at that mutant. The disulfide bridge Cys25-Cys278 was found to be important for agonist potency but neither for agonist efficacy nor for antagonist potency.

Targeting human DNA polymerase alpha for the inhibition of keratinocyte proliferation. Part 1. Homology model, active site architecture and ligand binding.

In order to understand the binding modes of human DNA polymerase alpha (pol alpha) inhibitors on a molecular level, a 3D homology model of the active site of the enzyme was proposed based on the application of molecular modelling methods and molecular dynamic simulations using available crystal coordinates of pol alpha relatives. Docking results for a series of known nucleotide analogue inhibitors were consistent with reported experimental binding data and offered the possibility to elucidate structure-activity relationships via investigations of active site-inhibitor interactions. Furthermore, the study could explain, at least partially, the inhibitory effect of aphidicolin on pol alpha. In molecular dynamics simulations, aphidicolin occupied the catalytic centre, but acted in a not truly competitive manner with respect to nucleotides. It destabilized the replicating "closed" form of the pol alpha and transferred the enzyme into the inactive "open" conformation. This result is consistent with recent experiments on the binding mode of aphidicolin.

Alpha-lipoic acid as a directly binding activator of the insulin receptor: protection from hepatocyte apoptosis.

BACKGROUND AND AIM: Alpha-lipoic acid has cytoprotective potential which has previously been explained by its antioxidant properties. The aim of this study was to assess LA-induced-specific cytoprotective signalling pathways in hepatocytes. METHODS: Apoptosis of rat hepatocytes was induced by actinomycinD/TNF-alpha. Caspase-3-like activity was determined by a fluorometric; LDH by an enzymatic assay; and phosphorylation of the insulin receptor, Akt, and Bad by Western blot (after immunoprecipitation). Protein kinase and insulin receptor activities were measured by in vitro phosphorylation. Computer modeling studies were performed by using the program GRID. RESULTS: Alpha-lipoic acid decreased actinomycinD/TNF-alpha-induced apoptosis, as did the antioxidants Trolox and N-acetylcysteine. The activation of PI3-kinase/Akt involving phosphorlyation of Bad markedly contributed to the cytoprotective action of alpha-lipoic acid. Alpha-lipoic acid but not other antioxidants protected against actinomycinD/TNF-alpha-induced apoptosis via phosphorylation of the insulin receptor. Computer modeling studies revealed a direct binding site for alpha-lipoic acid at the tyrosine kinase domain of the insulin receptor, suggesting a stabilizing function in loop A that is involved in ATP binding. Treatment of immunoprecipitated insulin receptor with LA induced substrate phosphorylation. CONCLUSIONS: Alpha-lipoic acid mediates its antiapoptotic action via activation of the insulin receptor/PI3-kinase/Akt pathway. We show for the first time a direct binding site for alpha-lipoic acid at the insulin receptor tyrosine kinase domain, which might make alpha-lipoic acid a model substance for the development of insulin mimetics.

Generation of a homology model of the human histamine H(3) receptor for ligand docking and pharmacophore-based screening.

The human histamine H(3) receptor (hH(3)R) is a G-protein coupled receptor (GPCR), which modulates the release of various neurotransmitters in the central and peripheral nervous system and therefore is a potential target in the therapy of numerous diseases. Although ligands addressing this receptor are already known, the discovery of alternative lead structures represents an important goal in drug design. The goal of this work was to study the hH(3)R and its antagonists by means of molecular modelling tools. For this purpose, a strategy was pursued in which a homology model of the hH(3)R based on the crystal structure of bovine rhodopsin was generated and refined by molecular dynamics simulations in a dipalmitoylphosphatidylcholine (DPPC)/water membrane mimic before the resulting binding pocket was used for high-throughput docking using the program GOLD. Alternatively, a pharmacophore-based procedure was carried out where the alleged bioactive conformations of three different potent hH(3)R antagonists were used as templates for the generation of pharmacophore models. A pharmacophore-based screening was then carried out using the program Catalyst. Based upon a database of 418 validated hH(3)R antagonists both strategies could be validated in respect of their performance. Seven hits obtained during this screening procedure were commercially purchased, and experimentally tested in a [(3)H]N(alpha)-methylhistamine binding assay. The compounds tested showed affinities at hH(3)R with K ( i ) values ranging from 0.079 to 6.3 muM.

Allosteric small molecules unveil a role of an extracellular E2/transmembrane helix 7 junction for G protein-coupled receptor activation.

G protein-coupled receptors represent the largest superfamily of cell membrane-spanning receptors. We used allosteric small molecules as a novel approach to better understand conformational changes underlying the inactive-to-active switch in native receptors. Allosteric molecules bind outside the orthosteric area for the endogenous receptor activator. The human muscarinic M(2) acetylcholine receptor is prototypal for the study of allosteric interactions. We measured receptor-mediated G protein activation, applied a series of structurally diverse muscarinic allosteric agents, and analyzed their cooperative effects with orthosteric receptor agonists. A strong negative cooperativity of receptor binding was observed with acetylcholine and other full agonists, whereas a pronounced negative cooperativity of receptor activation was observed with the partial agonist pilocarpine. Applying a newly synthesized allosteric tool, point mutated receptors, radioligand binding, and a three-dimensional receptor model, we found that the deviating allosteric/orthosteric interactions are mediated through the core region of the allosteric site. A key epitope is M(2)Trp(422) in position 7.35 that is located at the extracellular top of transmembrane helix 7 and that contacts, in the inactive receptor, the extracellular loop E2. Trp 7.35 is critically involved in the divergent allosteric/orthosteric cooperativities with acetylcholine and pilocarpine, respectively. In the absence of allosteric agents, Trp 7.35 is essential for receptor binding of the full agonist and for receptor activation by the partial agonist. This study provides first evidence for a role of an allosteric E2/transmembrane helix 7 contact region for muscarinic receptor activation by orthosteric agonists.