Membrane protein native state discrimination by implicit membrane models.

Four implicit membrane models [IMM1, generalized Born (GB)-surface area-implicit membrane (GBSAIM), GB with a simple switching (GBSW), and heterogeneous dielectric GB (HDGB)] were tested for their ability to discriminate the native conformation of five membrane proteins from 450 decoys generated by the Rosetta-Membrane program. The energy ranking of the native state and Z-scores were used to assess the performance of the models. The effect of membrane thickness was examined and was found to be substantial. Quite satisfactory discrimination was achieved with the all-atom IMM1 and GBSW models at 25.4 Å thickness and with the HDGB model at 28.5 Å thickness. The energy components by themselves were not discriminative. Both van der Waals and electrostatic interactions contributed to native state discrimination, to a different extent in each model. Computational efficiency of the models decreased in the order: extended-atom IMM1 > all-atom IMM1 > GBSAIM > GBSW > HDGB. These results encourage the further development and use of implicit membrane models for membrane protein structure prediction.

Synthesis, biological activity and molecular modelling studies of tricyclic alkylimidazo-, pyrimido- and diazepinopurinediones.

Syntheses and biological activities of imidazo-, pyrimido- and diazepino[2,1-f]purinediones containing N-alkyl substituents (with straight, branched or unsaturated chains) are described. Tricyclic derivatives were synthesized by the cyclization of 8-bromo-substituted 7-(2-bromoethyl)-, 7-(3-chloropropyl)- or 7-(4-bromobutyl)-theophylline with primary amines under various conditions. Compound 22 with an ethenyl substituent was synthesized by dehydrohalogenation of 9-(2-bromoethyl)-1,3-dimethyltetrahydropyrimido[2,1-f]purinedione. The obtained derivatives (5-35) were initially evaluated for their affinity at rat A1 and A2A adenosine receptors (AR), showing moderate affinity for both adenosine receptor subtypes. The best ligands were diazepinopurinedione 28 (K i = 0.28 µM) with fivefold A2A selectivity and the non-selective A1/A2A AR ligand pyrimidopurinedione 35 (K i A1 = 0.28 µM and K i A2A = 0.30 µM). The compounds were also evaluated for their affinity at human A1, A2A, A2B and A3 ARs. All of the obtained compounds were docked to the A2A AR X-ray structure in complex with the xanthine-based, potent adenosine receptor antagonist-XAC. The likely interactions of imidazo-, pyrimido- and diazepino[2,1-f]purinediones with the residues forming the A2A binding pocket were discussed. Furthermore, the new compounds were tested in vivo as anticonvulsants in maximal electroshock, subcutaneous pentylenetetrazole (ScMet) and TOX tests in mice (i.p.). Pyrimidopurinediones showed anticonvulsant activity mainly in the ScMet test. The best derivative was compound 11, showing 100 % protection at a dose of 100 mg/kg without symptoms of neurotoxicity. Compounds 6, 7, 8 and 14 with short substituents showed neurotoxicity and caused death. In rat tests (p.o.), 9 was characterized by a high protection index (>13.3). AR affinity did not apparently correlate with the antiepileptic potency of the compounds.

Molecular modeling of A1 and A2A adenosine receptors: comparison of rhodopsin- and beta2-adrenergic-based homology models through the docking studies.

Adenosine receptors (ARs) are members of the superfamily of G protein-coupled receptors. The homology models of adenosine A1 and A2A receptors were constructed. The high-resolution X-ray structure of bovine rhodopsin and crystal structure of beta2-adrenergic receptor were used as templates. The binding sites of the A1 and A2A ARs were constructed by using data obtained from mutagenesis experiments as well as docking simulations of the respective AR antagonsists DPCPX and XAC. To compare rhodopsin- and beta2-adrenergic-based models, the binding mode of A1 (KW-3902, LUF-5437) and A2A (KW-6002, ZM-241385) ARs antagonists were also examined. The differences in the binding ability of both models were noted during the study. The beta2-adrenergic-based A2A AR model was much more capable to stabilize the ligand in the binding site cavity than the corresponding rhodopsin-based A2A AR model, however, such differences were not so clear in case of A1 AR models. It was suggested that for the A1 AR it is possible to use the crystal structure of rhodopsin as a template as well as beta2-adrenergic receptor, but for A2A AR, with the now available beta2-adrenergic receptor X-ray structure, docking studies should be avoided on the rhodopsin-based model. However, taking into account that the beta2AR shares about 31% of the residues with the AR in comparison to 21% in case of bRho, we suggest using beta2-adrenergic-based models for the A1 and A2A ARs for further in silico ligand screening also because of their generally better ability to stabilize ligands inside the binding pocket.

Interactions between ionizable amino acid side chains at a lipid bilayer-water interface.

Potentials of mean force (PMF) between ionizable amino acid side chains (Arg, Lys, His, Glu) in the headgroup area of a palmitoyl oleoyl phosphatidylcholine lipid bilayer were obtained from all-atom molecular dynamics simulations and the adaptive biasing force method. Simulations in bulk water were also performed for comparison. Side chains were constrained in collinear, stacking, and orthogonal (T-shaped) orientations. The most structured and attractive PMFs were observed for hydrogen-bonded side chains. Contact minima occurred at a distance of 2.6-3.1 Å between selected atoms or centers of mass with the most attractive interaction (-9.6 kcal/mol) observed between Arg(+) and Glu(-). Hydrogen bonds play a significant role in stabilizing these interactions. Interactions between like charged side chains can also be very attractive if the charges are screened by surrounding molecules or groups (e.g., the PMF value at the contact minimum for Arg(+)···Arg(+) is -7.6 kcal/mol). Like charged side chains can have contact minima as close as 3.6 Å. The PMFs depend strongly on the relative orientation of the side chains. In agreement with experimental studies and other simulations, we found the stacking arrangement of like charged side chains to be the most favorable orientation. Interaction energies and Lennard-Jones energies between side chains, headgroups, and water molecules were analyzed in order to rationalize the observed PMFs and their dependence on orientation. In general, the results cannot be explained by simple dielectric arguments.

Synthesis and biological activity of tricyclic cycloalkylimidazo-, pyrimido- and diazepinopurinediones.

Syntheses and physicochemical properties of N-cycloalkyl-substituted imidazo-, pyrimido- and 1,3-diazepino[2,1-f]purinediones are described. These derivatives were synthesized by cyclization of 7-halogenoalkyl-8-bromo-1,3-dimethylxanthine derivatives with aminocycloalkanes. The obtained compounds (1-33) were evaluated for their affinity to rat adenosine A(1) and A(2A) receptors. Selected compounds were additionally investigated for affinity to the human A(1), A(2A), A(2B) and A(3) receptor subtypes. The results of the radioligand binding assays at adenosine A(1) and A(2A) receptors showed that most of the compounds exhibited adenosine A(2A) receptor affinity at micromolar or submicromolar concentrations; an annelated pyrimidine ring was beneficial for A(2A) affinity. The most potent A(2A) ligands of the present series were compounds 6 (K(i) 0.33 µM rat A(2A), 0.31 µM human A(2A)), 8 (K(i) 0.98 µM rat A(2A), 0.42 µM human A(2A)) and 15 (K(i) 0.24 µM rat A(2A), 0.61 µM human A(2A)) with the latter one showing high A(2A) selectivity. In NaCl shift assay, 15 was shown to be an antagonist at A(2A) receptors. This result was confirmed for the best compounds 6, 8, 15 in cAMP accumulation studies. A 3D-QSAR equation with a good predicting power (q(2) = 0.88) for A(2A) AR affinity was obtained. The compounds were evaluated in vivo as anticonvulsants in MES and ScMet tests and examined for neurotoxicity in mice (i.p.). Most of them showed anticonvulsant activity in chemically induced seizures; among them the diazepinopurinediones were the best (e.g. 31) showing protection in both tests on short time symptoms, without signs of neurotoxicity. Five compounds, 8, 17, 20, 29, and 31, exhibited anticonvulsant activity after peroral application in rats. Structure-activity relationships are discussed including the analysis of lipophilic and spatial properties. The new compounds, which contain a basic nitrogen atom and can therefore be protonated, may be good starting points for obtaining A(2A) antagonists with good water-solubility.

N9-benzyl-substituted 1,3-dimethyl- and 1,3-dipropyl-pyrimido[2,1-f]purinediones: synthesis and structure-activity relationships at adenosine A1 and A2A receptors.

Synthesis and physicochemical properties of N-benzyl pyrimido[2,1-f]purinediones are described. These derivatives were synthesized by the cyclization of 7-chloropropylo-8-bromo-1,3-dimethyl- or 1,3-dipropyl xanthine derivatives with corresponding (un)substituted benzylamines. Dipropyl derivatives were obtained under microwave irradiation conditions either. The obtained compounds (1-20) were evaluated for their affinity to adenosine A1 and A2A receptors, selected compounds were additionally investigated for affinity to the A3 receptor subtype. The results of the radioligand binding assays to A1 and A2A adenosine receptors showed that most of the 1,3-dimethyl-9-benzylpyrimidopurinediones exhibited selective affinity to A2A receptors at micromolar or submicromolar concentrations (for example, derivative 9 with o-methoxy substituent displayed a Ki value of 0.699 microM at rat A2A receptor with more than 36-fold selectivity). Contrary to previously described arylpyrimido[2,1-f]purinediones dipropyl derivatives (compounds 15-20) showed affinity to both kinds of receptors increased, however A1 affinity increased to a larger extent, with the result that A2A selectivity was abolished. The best adenosine A1 receptor ligand was m-chlorobenzyl derivative 18 (Ki=0.089 microM and 5-fold A1 selectivity). Structure-activity relationships were discussed with the analysis of lipophilic and spatial properties of the investigated compounds. Pharmacophore model of adenosine A1 receptor antagonist was adopted for this purpose.

Phenylethyl-substituted pyrimido[2,1-f]purinediones and related compounds: structure-activity relationships as adenosine A(1) and A(2A) receptor ligands.

The synthesis of N-(un)substituted-phenylalkylpyrimido[2,1-f]purinediones was performed starting with 7-(3-chloropropyl)-8-bromotheophylline and 7-(3-chloropropyl)-8-bromo-1,3-dipropylxanthine. Compounds with unsubstituted or substituted ethylene spacer to an aromatic ring were synthesized. Additionally variations in the spacer-elongation of the linker containing more than two atoms, introduction of a double bond or heteroatoms were performed. Physicochemical properties of the synthesized compounds were described. The obtained compounds envisaged as sterically fixed and configurationally stable analogs of 8-styrylxanthines, were evaluated for their affinity to adenosine A(1) and A(2A) receptors, the receptor subtypes that are predominant in the brain. Selected compounds were also investigated for the affinity to the A(2B) and A(3) receptor subtypes. It was stated that phenylethyl pyrimido[2,1-f]purinediones and their analogs with variations of the ethylene spacer (substituted or extended) exhibit micromolar or submicromolar affinity for A(2A) ARs (adenosine receptors); for example compound 2Ac with p-hydroxy substituent displayed a K(i) value of 0.23 microM at the rat A(2A) receptor. In comparison to the previously obtained phenyl and benzyl pyrimido[2,1-f]purinediones compounds with a shorter spacer, phenethyl derivatives were optimal for A(2A) AR. The kind of substituent at the aromatic ring was important for the affinity. Oxygen and nitrogen atoms in the spacer resulted frequently in a slight decrease of the A(2A) AR affinity, introduction of more heteroatoms into the spacer-in carbamates-caused distinctly negative effect on the activity. In this series of compounds more frequently the adenosine A(1) activity was observed, also in submicromolar range as for dipropyl derivative 2Ba with K(i) value of 0.62 microM at the rat A(2A) AR. 3D-QSAR models were developed for the compounds presented in this paper as well as in the previous publications showing activity at adenosine A(1) and A(2A) ARs. It was concluded that for the activity at adenosine A(1) and A(2A) receptors lipophilicity, steric effects along with the molecule's electrostatic surface properties had greatest value. Chosen compounds were evaluated in vivo as anticonvulsants in MES, scMet tests and examined for neurotoxicity. Contrary to previously obtained phenyl and benzyl pyrimido[2,1-f]purinediones, all tested compounds were inactive as anticonvulsants.

Synthesis and biological activity of tricyclic aryloimidazo-, pyrimido-, and diazepinopurinediones.

Syntheses and physicochemical properties of N-aryl-substituted imidazo-, pyrimido-, and 1,3-diazepino[2,1-f]purinediones are described. These derivatives were synthesized by the cyclization of 7-haloalkyl-8-bromo-1,3-dimethyl- or 1,3-dipropyl-xanthine derivatives with corresponding arylamines. The obtained compounds (1-40), which can be envisaged as sterically fixed and configurationally stable analogs of 8-styrylxanthines, were evaluated for their affinity to adenosine A(1) and A(2A) receptors, the receptor subtypes that are predominant in the brain. Selected compounds were additionally investigated for affinity to the A(2B) and A(3) receptor subtypes. Many of the compounds showed adenosine A(2A) receptor affinity at micromolar or submicromolar concentrations and were A(2A)-selective, for example, compound 23 with p-fluoro substituent displayed K(i) value of 0.147 microM at the rat A(2A) receptor and more than 170-fold-A(2A) selectivity, compound 17 with naphthyl substituent had K(i) value of 0.219 microM and a more than 114-fold-A(2A) selectivity. The compounds were somewhat weaker and less selective at the human receptor subtypes. Elongation of the dimethyl substituent to dipropyl in xanthine moiety improved affinity but reduced selectivity. 1,3-Dimethylimidazo-, pyrimido-, and diazepinopurinediones were evaluated in vivo as anticonvulsants in MES, ScMet, TTE tests and examined for neurotoxicity in mice (ip). Substances with pyrimido ring displayed protective activity in ScMet or in MES and ScMet tests, showing also neurotoxicity. The pyrimidine annelated ring is beneficial for both receptor affinity and anticonvulsant activity.