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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Allosteric interactions with muscarinic acetylcholine receptors: complex role of the conserved tryptophan M2422Trp in a critical cluster of amino acids for baseline affinity, subtype selectivity, and cooperativity.

In general, the M2 subtype of muscarinic acetylcholine receptors has the highest sensitivity for allosteric modulators and the M5 subtype the lowest. The M2/M5 selectivity of some structurally diverse allosteric agents is known to be completely explained by M2 177Tyr and M2 423Thr in receptors whose orthosteric site is occupied by the conventional ligand N-methylscopolamine (NMS). This study explored the role of the conserved M2 422Trp and the adjacent M2 423Thr in the binding of alkane-bisammonio type modulators, gallamine, and diallylcaracurine V. Experiments were performed with human M2 or M5 receptors or mutants thereof. It was found that M2 422Trp and M2 423Thr independently influenced allosteric agent binding. The presence of M2 423Thr may enhance the affinity of binding, depending on the allosteric agent, either directly or indirectly (by avoiding sterical hindrance through its M5 counterpart 478His). Replacement of M2 422Trp and of the corresponding M5 477Trp by alanine revealed a pronounced contribution of these epitopes to subtype independent baseline affinity in NMS-bound and NMS-free receptors for all agents except diallylcaracurine V. In a few instances, this tryptophan also influenced cooperativity and subtype selectivity. Docking simulations using a three-dimensional M2 receptor model revealed that the aromatic rings of M2 177Tyr and M2 422Trp, in a concerted action, might fix one of the aromatic moieties of alkane-bisammonio compounds between them. Thus, M2 422Trp and the spatially adjacent M2 177Tyr, as well as M2 423Thr, form a cluster of amino acids within the allosteric binding cleft that is pivotal for both M2/M5 subtype selectivity and baseline affinity of allosteric agents.

Molecular mechanisms of antibiotic resistance: QM/MM modelling of deacylation in a class A beta-lactamase.

Modelling of the first step of the deacylation reaction of benzylpenicillin in the E. coli TEM1 beta-lactamase (with B3LYP/6-31G + (d)//AM1-CHARMM22 quantum mechanics/molecular mechanics methods) shows that a mechanism in which Glu166 acts as the base to deprotonate a conserved water molecule is both energetically and structurally consistent with experimental data; the results may assist the design of new antibiotics and beta-lactamase inhibitors.

Molecular dynamics simulations of bovine rhodopsin: influence of protonation states and different membrane-mimicking environments.

G-protein coupled receptors (GPCRs) are a protein family of outstanding pharmaceutical interest. GPCR homology models, based on the crystal structure of bovine rhodopsin, have been shown to be valuable tools in the drug-design process. The initial model is often refined by molecular dynamics (MD) simulations, a procedure that has been recently discussed controversially. We therefore analyzed MD simulations of bovine rhodopsin in order to identify contacts that could serve as constraints in the simulation of homology models. Additionally, the effect of an N-terminal truncation, the nature of the membrane mimic, the influence of varying protonation states of buried residues and the importance of internal water molecules was analyzed. All simulations were carried out using the program-package GROMACS. While N-terminal truncation negatively influenced the overall protein stability, a stable simulation was possible in both solvent environments. As regards the protonation state of titratable sites, the experimental data could be reproduced by the program UHBD (University of Houston Brownian Dynamics), suggesting its application for studying homology models of GPCRs. A high flexibility was observed for internal water molecules at some sites. Finally, interhelical hydrogen-bonding interactions could be derived, which can now serve as constraints in the simulations of GPCR homology models.

Atypical muscarinic allosteric modulation: cooperativity between modulators and their atypical binding topology in muscarinic M2 and M2/M5 chimeric receptors.

The binding and function of muscarinic acetylcholine receptors can be modulated allosterically. Some allosteric muscarinic ligands are "atypical", having steep concentration-effect curves and not interacting competitively with "typical" allosteric modulators. For atypical agents, a second allosteric site has been proposed. Different approaches have been used to gain further insight into the interaction with M2 receptors of two atypical agents, tacrine and the bispyridinium compound 4,4'-bis-[(2,6-dichloro-benzyloxy-imino)-methyl]-1,1'-propane-1,3-diyl-bispyridinium dibromide (Duo3). Interaction studies, using radioligand binding assays and the allosteric ligands obidoxime, Mg2+, and the new tool hexamethonium to antagonize the allosteric actions of the atypical ligands, showed different modes of interaction for tacrine and Duo3 at M2 receptors. A negatively cooperative interaction was observed between hexamethonium and tacrine (but not Duo3). A tacrine dimer that exhibited increased allosteric potency relative to tacrine but behaved like a typical allosteric modulator was competitively inhibited by hexamethonium. M2/M5-receptor mutants revealed a dependence of tacrine and Duo3 affinity on different receptor epitopes. This was confirmed by docking simulations using a three-dimensional model of the M2 receptor. These showed that the allosteric site could accommodate two molecules of tacrine simultaneously but only one molecule of Duo3, which binds in different mode from typical allosteric agents. Therefore, the atypical actions of tacrine and Duo3 involve different modes of receptor interaction, but their sites of attachment seem to be the "common" allosteric binding domain at the entrance to the orthosteric ligand binding pocket of the M2-receptor. Additional complex behavior may be rationalized by allosteric interactions transmitted within a receptor dimer.

Molecular design of two sterol 14alpha-demethylase homology models and their interactions with the azole antifungals ketoconazole and bifonazole.

Sterol 14alpha-demethylase (CYP51) is one of the known major targets for azole antifungals. Therapeutic side effects of these antifungals are based on interactions of the azoles with the human analogue enzyme. This study describes for the first time a comparison of a human CYP51 (HU-CYP51) homology model with a homology model of the fungal CYP51 of Candida albicans (CA-CYP51). Both models are constructed by using the crystal structure of Mycobacterium tuberculosis MT-CYP51 (PDB code: 1EA1). The binding mode of the azole ketoconazole is investigated in molecular dynamics simulations with the GROMACS force field. The usage of special parameters for the iron azole complex binding is necessary to obtain the correct complex geometry in the active site of the enzyme models. Based on the dynamics simulations it is possible to explain the enantioselectivity of the human enzyme and also to predict the binding mode of the isomers of ketoconazole in the active site of the fungal model.

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.

Different environments for a realistic simulation of GPCRs-application to the M2 muscarinic receptor.

A model of the human M(2) muscarinic receptor was taken as an example for a class A G-protein coupled receptor to explore the influence of different environments in a molecular dynamics simulation (MDS) on the protein structure. The most commonly used environment is the vacuum, although it is very unnatural for a transmembrane protein. As an alternative a membrane-like system, consisting of a lipophilic central layer and two aqueous flanking layers, was tested. The most realistic system that can be applied is a phospholipid bilayer with a surrounding physiological sodium chloride solution. From all systems good protein structures were received, nevertheless clear differences between the systems were detected in the structural comparison of the models. Subsequently it was analyzed whether the observed structural differences influence ligand binding. For this purpose the antagonist (S)-scopolamine was docked into the binding cavity, which is well known by many reported single and multiple point mutations. As expected from the observed structural variations triggered by the type of environment employed in MDS, also differences in the binding mode of (S)-scopolamine were detected, all contacts, however, which are known to be important were found.