Pharmacophore-directed Homology Modeling and Molecular Dynamics Simulation of G Protein-coupled Receptor： Study of Possible Binding Modes of 5-HT2c Receptor Agonists

A new pharmacophore-based modeling procedure, including homology modeling, pharmacophore study, flexible molecular docking, and long-time molecular dynamics (MD) simulations, was employed to construct the structure of the human 5-HT_(2C) receptor and determine the characteristics of binding modes of 5-HT_(2C) receptor agonists. An agonist-receptor complex has been constructed based on homology modeling and a pharmacophore hypothesis model based on some high active compounds. Then MD simulations of the ligand-receptor complex in an explicit membrane environment were carried out. The conformation of the 5- HT_(2C) receptor during MD simulation was explored, and the stable binding modes of the studied agonist were determined. Flexible molecular docking of several structurally diverse agonists of the human 5-HT_(2C) receptor was carried out, and the general binding modes of these agonists were investigated. According to the models presented in this work and the results of Flexi-Dock, the involvement of the amino acid residues Asp134, Ser138, Ash210, Asn331, Tyr358, Ile131, Ser132, Val135, Thr139, Ile189, Val202, Val208, Leu209, Phe214, Val215, Gly218, Ser219, Phe223, Trp324, Phe327, and Phe328 in agonist recognition was studied. The obtained binding modes of the human 5-HT_(2C) receptor agonists have good agreement with the site-directed mutagenesis data and other studies.     

Mutagenesis of the Human 5-HT1B Receptor: Differences from the Closely Related 5-HT1A Receptor and the Role of Residue F331 in Signal Transduction

Abstract

We have used a combination of sequence comparisons, computer-based modeling and site-directed mutagenesis to investigate the molecular interactions involved in ligand binding and signal transduction of the human 5-HT_1B receptor. Two amino acid residues, S212 in transmembrane region (TM) V and F331 in TM VI, were replaced by alanines. These amino acids are conserved in many G protein-coupled receptors and therefore likely to be important for receptor function. The mutant receptors were expressed in Chinese hamster ovary cells. The 5-HT-like agonist 5-carboxamido-tryptamine (5-CT) bound with 15-fold lower affinity to the S212A mutant as compared to wild-type receptor and the antagonist methiothepin bound with 17-fold lower affinity to the F331A mutant. No reduction in the affinity of 5-HT was seen for the S212A mutant, although an equivalent mutation in the 5-HT_1A receptor resulted in a 100-fold reduction of 5-HT binding. The inhibition of forskolin-stimulated cyclic AMP production by 5-HT was significantly reduced in cells expressing the F331A mutant, even though the endogenous ligand 5-HT bound with somewhat increased affinity. Methiothepin acted as an inverse agonist and increased the forskolin-stimulated cyclic AMP production at both the wild-type receptor and the mutants, and the effect was stronger on the F331A mutant. These results suggest that F331 is involved in the conformational changes necessary for signal transduction.     

Identification of the binding sites and selectivity of sarpogrelate,a novel 5-HT2 antagonist,to human 5-HT2A, 5-HT2B and 5-HT2C receptor subtypes by molecular modeling

The aim of the present study was to investigate the binding sites interactions and the selectivity of sarpogrelate to human 5-HT(2) receptor family (5-HT(2A), 5-HT(2B) and 5-HT(2C) receptor subtypes) using molecular modeling. Rhodopsin (RH) crystal structures were used as template to build structural models of the human serotonin-2A and -2C receptors (5-HT(2A)R, 5-HT(2C)R), whereas for 5-HT(2B)R, we used our previously published three-dimensional (3D) models based on bacteriorhodopsin (BR). Sarpogrelate, a novel 5-HT(2)R antagonist, was docked to the receptors. Molecular dynamics (MD) simulations produced the strongest interaction for 5-HT(2A)R/sarpogrelate complex. Upon binding, sarpogrelate constraints aromatic residues network (Trp(3.28), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2A)R; Phe(3.35), Phe(6.51), Trp(7.40) in 5-HT(2B)R; Trp(3.28), Phe(3.35), Phe(5.47), Trp(6.48), Phe(6.51), Phe(6.52) in 5-HT(2C)R) in a stacked configuration, preventing activation of the receptor. The models suggest that the structural origin of the selectivity of sarpogrelate to 5-HT(2A)R vs both 5-HT(2B)R and 5-HT(2C)R comes from the following results: (1) The tight interaction between the antagonist and the transmembrane domain (TMD) 3. Asp(3.32) neutralizes the cationic head and interacts simultaneously with carboxylic group hydrogen of the antagonist molecule. (2) Due to steric hindrance, Ser(5.46) (vs Ala(5.46) in 5HT(2B) and 5HT(2C)) prevents sarpogrelate to enter deeply inside the hydrophobic core of the helix bundle and to interact with Pro(5.50). (3) The side chain of Ile(4.56) (vs Ile(4.56) in 5HT(2B)R and Val(4.56) in 5HT(2C)R) constraints sarpogrelate to adjust its position by translating toward the strongly attractive Asp(3.32). These results are in good agreement with binding affinities (pKi) of sarpogrelate for 5-HT(2) receptor family expressed in transfected cell.     

Identification by comprehensive chimeric analysis of a key residue responsible for high affinity glucose transport by yeast HXT2

Hxt2 and Hxt1 are, respectively, high affinity and low affinity facilitative glucose transporter paralogs of Saccharomyces cerevisiae. We have previously investigated which amino acid residues of Hxt2 are important for high affinity transport activity. Studies with all the possible combinations of 12 transmembrane segments (TMs) of Hxt2 and Hxt1 revealed that TMs 1, 5, 7, and 8 of Hxt2 are necessary for high affinity transport. Systematic shuffling of the 20 amino acid residues that differ between Hxt2 and Hxt1 in these TMs subsequently identified 5 residues as important for such activity: Leu(59) and Leu(61) (TM1), Leu(201) (TM5), Asn(331) (TM7), and Phe(366) (TM8). We have now studied the relative importance of these 5 residues by individually replacing them with each of the other 19 residues. Replacement of Asn(331) yielded transporters with various affinities, with those of the Ile(331), Val(331), and Cys(331) mutants being higher than that of the wild type. Replacement of the Hxt2 residues at the other four sites yielded transporters with affinities similar to that of the wild type but with various capacities. A working homology model of the chimeric transporters containing Asn(331) or its 19 replacement residues indicated that those residues at this site that yield high affinity transporters (Ile(331), Val(331), Cys(331)) face the central cavity and are within van der Waals distances of Phe(208) (TM5), Leu(357) (TM8), and Tyr(427) (TM10). Interactions via these residues of the four TMs, which compose a half of the central pore, may thus play a pivotal role in formation of a core structure for high affinity transport.     

Binding interactions of antagonists with 5-hydroxytryptamine3A receptor models

Homology modeling was performed on the N-terminal extracellular regions of human, mouse, and guinea pig 5-hydroxytryptamine type 3A receptors (5-HT3R) based on the 24% sequence homology with and on the crystal structure of the snail acetylcholine binding protein (AChBP). Docking of 5-HT3 antagonists granisetron, tropisetron, ondansetron, dolasetron ('setrons), and (+)-tubocurarine suggests an aromatic binding cleft behind a hydrophilic vestibule. Several intra- and interface interactions, H-bonds, and salt bridges stabilize the pentameric structure and the binding cleft. The planar rings of antagonists are intercalated between aromatic side-chains (W183-Y234, Y143-Y153). S227 donates H-bonds to the carbonyl groups of 'setrons. The tertiary ammonium ions interact with E236, N128 or E129, and/or W90 (cation-pi interaction). This offers a molecular explanation of the pharmacophore models of 5-HT3R antagonists. Docking artifacts suggest some ambiguities in the binding loops A and C of the 5-HT3AR models. Lower potencies of (+)-tubocurarine for human, and those of tropisetron for guinea pig 5-HT3ARs can be attributed to steric differences of I/S230 in the binding cleft and to distinct binding interactions with E229 and S227, respectively. Ligand binding interferes with crucial intra- and interface interactions along the binding cleft.     

Virtual screening to identify <Emphasis Type="Italic">Leishmania braziliensis N</Emphasis>-myristoyltransferase inhibitors: pharmacophore models,docking, and molecular dynamics

Leishmaniasis is caused by several protozoa species belonging to genus Leishmania that are hosted by humans and other mammals. Millions of new cases are recorded every year and the drugs available on the market do not show satisfactory efficacy and safety. A hierarchical virtual screening approach based on the pharmacophore model, molecular docking, and molecular dynamics was conducted to identify possible Leishmania braziliensis N-misristoyltransferase (LbNMT) inhibitors. The adopted pharmacophore model had three main features: four hydrophobic centers, four hydrogen-bond acceptor atoms, and one positive nitrogen center. The molecules (n=15,000) were submitted to alignment with the pharmacophore model and only 27 molecules aligned to model. Six molecules were submitted to molecular docking, using receptor PDB ID 5A27. After docking, the ZINC35426134 was a top-ranked molecule (? 64.61 kcal/mol). The molecule ZINC35426134 shows hydrophobic interactions with Phe82, Tyr209, Val370, and Leu391 and hydrogen bonds with Asn159, Tyr318, and Val370. Molecular dynamics simulations were performed with the protein in its APO and HOLO forms for 37 ns in order to assess the stability of the protein–ligand complex. Results showed that the HOLO form was more stable than the APO one, and it suggests that the ZINC35426134 binding stabilizes the enzyme. Therefore, the selected molecule has the potential to meet the herein proposed target.     

Molecular modeling of the dopamine D2 and serotonin 5-HT1A receptor binding modes of the enantiomers of 5-OMe-BPAT.

Molecular modeling studies were undertaken in order to elucidate the possible dopamine D2 and serotonin 5-HT1A receptor binding modes of the enantiomers of 5-methoxy-2-[N-(2-benzamidoethyl)-N-n-propylamino]tetralin (5-OMe-BPAT, 1). For this purpose, a combination of indirect molecular modeling and direct construction of the seven transmembrane (7TM) domains of the receptors was employed in a stepwise, objective manner. Pharmacophore models and corresponding receptor maps were identified by superimposing selected sets of receptor agonists in their presumed pharmacologically active conformations, while taking the conformational freedom of the ligands into account. The 7TM models were then constructed around the agonist pharmacophore models, by adding the TM domains one-by-one. Initially, the relative positions of TM3, TM4, and TM5 were determined using the three-dimensional structure of bacteriorhodopsin, but subsequently the orientations of all TM domains were adjusted in order to mimic the topology of the TM domains of rhodopsin. The presumed dopamine D2 receptor binding conformations of (S)- and (R)-1 were determined by using the semirigid dopamine D2 receptor antagonist N-benzylpiquindone as a template for superposition. Similarly, the selective serotonin 5-HT1A receptor agonist flesinoxan was employed for identifying the serotonin 5-HT1A receptor binding conformations of the enantiomers of 1. After docking of the presumed pharmacologically active conformations in the 7TM models and subsequent optimization of the binding sites, specific interactions between the ligands and the surrounding amino acid residues, consistent with the structure-activity relationships, were observed. Thus, both enantiomers of 1 bound to the dopamine D2 receptor model in a similar fashion: a reinforced electrostatic interaction was present between the protonated nitrogen atoms and Asp114 in TM3; their carbonyl groups accepted a H-bond from Ser121 in TM3; their amide NH groups acted as H-bond donor to Tyr416 in TM7; and their benzamide phenyl rings were involved in a hydrophobic edge-to-face interaction with Trp386 in TM6. Differences were observed in the orientations of the 2-aminotetralin moieties, which occupied the agonist binding site. Whereas the (S)-enantiomer could form a H-bond between its 5-methoxy substituent and Ser193 in TM5, the (R)-enantiomer could not, which may account for the differences in their intrinsic efficacies at the dopamine D2 receptor. In the serotonin 5-HT1A receptor model, the benzamide phenyl rings of both enantiomers were involved in hydrophobic face-to-face interactions with Phe112 in TM3, while their protonated nitrogen atoms formed a reinforced electrostatic interaction with Asp116 in TM3. Consistent with the structure-affinity relationships of 1, the amide moieties were not involved in specific interactions. Both enantiomers of 1 could form a hydrogen bond between their 5-methoxy substituent and Thr200 in TM5, which may account for their full serotonin 5-HT1A receptor agonist properties.     

Amino acids important for ligand specificity of the human constitutive androstane receptor

The human constitutive androstane receptor (CAR, NR1I3) is an important ligand-activated regulator of oxidative and conjugative enzymes and transport proteins. Because of the lack of a crystal structure of the ligand-binding domain (LBD), wide species differences in ligand specificity and the scarcity of well characterized ligands, the factors that determine CAR ligand specificity are not clear. To address this issue, we developed highly defined homology models of human CAR LBD to identify residues lining the ligand-binding pocket and to perform molecular dynamics simulations with known human CAR modulators. The roles of 22 LBD residues for basal activity, ligand selectivity, and interactions with co-regulators were studied using site-directed mutagenesis, mammalian co-transfection, and yeast two-hybrid assays. These studies identified several amino acids within helices 3 (Asn(165)), 5 (Val(199)), 11 (Tyr(326), Ile(330), and Gln(331)), and 12 (Leu(343) and Ile(346)) that contribute to the high basal activity of human CAR. Unique residues within helices 3 (Ile(164) and Asn(165)), 5 (Cys(202) and His(203)), and 7 (Phe(234) and Phe(238)) were found control the selectivity for CAR activators and inhibitors. A single residue in helix 7 (Phe(243)) appears to explain the human/mouse species difference in response of CAR to 17alpha-ethynyl-3,17beta-estradiol.     

Diaryl substituted pyrrolidinones and pyrrolones as 5-HT2C inhibitors: synthesis and biological evaluation

Within the continuous quest for the discovery of novel compounds able to treat anxiety and depression, the generation of a pharmacophore model for 5-HT2C receptor antagonists and the discovery of a new class of potent and selective 5-HT2C molecules are reported.     

Mutations of Human NARS2, Encoding the Mitochondrial Asparaginyl-tRNA Synthetase,Cause Nonsyndromic Deafness and Leigh Syndrome

Here we demonstrate association of variants in the mitochondrial asparaginyl-tRNA synthetase NARS2 with human hearing loss and Leigh syndrome. A homozygous missense mutation ([c.637G>T; p.Val213Phe]) is the underlying cause of nonsyndromic hearing loss (DFNB94) and compound heterozygous mutations ([c.969T>A; p.Tyr323*] + [c.1142A>G; p.Asn381Ser]) result in mitochondrial respiratory chain deficiency and Leigh syndrome, which is a neurodegenerative disease characterized by symmetric, bilateral lesions in the basal ganglia, thalamus, and brain stem. The severity of the genetic lesions and their effects on NARS2 protein structure cosegregate with the phenotype. A hypothetical truncated NARS2 protein, secondary to the Leigh syndrome mutation p.Tyr323* is not detectable and p.Asn381Ser further decreases NARS2 protein levels in patient fibroblasts. p.Asn381Ser also disrupts dimerization of NARS2, while the hearing loss p.Val213Phe variant has no effect on NARS2 oligomerization. Additionally we demonstrate decreased steady-state levels of mt-tRNA^Asn in fibroblasts from the Leigh syndrome patients. In these cells we show that a decrease in oxygen consumption rates (OCR) and electron transport chain (ETC) activity can be rescued by overexpression of wild type NARS2. However, overexpression of the hearing loss associated p.Val213Phe mutant protein in these fibroblasts cannot complement the OCR and ETC defects. Our findings establish lesions in NARS2 as a new cause for nonsyndromic hearing loss and Leigh syndrome.     

The primary structure of the β-subunit of protocatechuate 3,4-dioxygenase from Pseudomonas aeruginosa

The complete amino acid sequence of the β-subunit of protocatechuate 3,4-dioxygenase was determined. The β-subunit contained four methionine residues. Thus, five peptides were obtained after cleavage of the carboxymethylated β-subunit with cyanogen bromide, and were isolated on Sephadex G-75 column chromatography. The amino acid sequences of the cyanogen bromide peptides were established by characterization of the peptides obtained after digestion with trypsin, chymotrypsin, thermolysin, or Staphylococcus aureus protease. The major sequencing techniques used were automated and manual Edman degradations. The five cyanogen bromide peptides were aligned by means of the amino acid sequences of the peptides containing methionine purified from the tryptic hydrolysate of the carboxymethylated β-subunit. The amino acid sequence of all the 238 residues was as follows: ProAlaGlnAspAsnSerArgPheValIleArgAsp ArgAsnTrpHis ProLysAlaLeuThrPro-Asp — TyrLysThrSerIleAlaArg SerProArgGlnAla LeuValSerIleProGlnSer — IleSerGluThrThrGly ProAsnPheSerHisLeu GlyPheGlyAlaHisAsp-His — AspLeuLeuLeuAsnPheAsn AsnGlyGlyLeu ProIleGlyGluArgIle-Ile — ValAlaGlyArgValValAsp GlnTyrGlyLysPro ValProAsnThrLeuValGluMet — TrpGlnAlaAsnAla GlyGlyArgTyrArg HisLysAsnAspArgTyrLeuAlaPro — LeuAspProAsn PheGlyGlyValGly ArgCysLeuThrAspSerAspGlyTyrTyr — SerPheArg ThrIleLysProGlyPro TyrProTrpArgAsnGlyProAsnAsp — TrpArgProAla HisIleHisPheGlyIle SerGlyProSerIleAlaThr-Lys — LeuIleThrGlnLeuTyr PheGluGlyAspPro LeuIleProMetCysProIleVal — LysSerIleAlaAsn ProGluAlaValGlnGln LeuIleAlaLysLeuAspMetAsnAsn — AlaAsnProMet AsnCysLeuAlaTyr ArgPheAspIleValLeuArgGlyGlnArgLysThrHis PheGluAsnCys. The sequence published earlier in summary form (Iwaki et al., 1979, J. Biochem.86, 1159–1162) contained a few errors which are pointed out in this paper.     

NMR-based substrate analog docking to Escherichia coli peptidyl-tRNA hydrolase

Escherichia coli peptidyl-tRNA hydrolase activity is inhibited by 3'-(L-[N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine, a stable mimic of the minimalist substrate 2'(3')-O-(L-[N,N-diacetyl-lysinyl)adenosine. The complex of this mimic with the enzyme has been analyzed by NMR spectroscopy, enabling experimental mapping of the catalytic center for the first time. Chemical shift variations point out the sensitivity of residues Asn10, Met67, Asn68, Gly111, Asn114, Leu116, Lys117, Gly147, Phe148, and Val149 to complex formation. Docking simulations based on ambiguous interaction restraints involving these residues show bondings of the peptide moiety of 3'-(l-[N,N-diacetyl-lysinyl)amino-3'-deoxyadenosine with Asn10, Asn68, and Asn114. A stacking interaction of Phe66 with the purine is also indicated. Drawn is a model of enzyme-bound peptidyl-tRNA substrate, in which: (i) the Asn114 δ(2) NH(2) group holds the water molecule that participates in the hydrolysis of the substrate, while Tyr15 binds the phosphate in the 5'-position of the 3'-terminal tRNA adenosine; (ii) the δ(2) NH(2) group of Asn68 holds the main-chain carbonyl of the C-terminal residue of the peptide esterified to tRNA; and (iii) the δ(2) NH(2) group of Asn10 holds the main-chain carbonyl of the penultimate C-residue. Functional value is given to this model by (i) showing that the enzyme becomes confusable with an aminoacyl-tRNA hydrolase upon mutagenesis of Asn10 and (ii) reinterpreting already obtained site-directed mutagenesis data.     

Elucidating the role of Trp105 in the KPC-2 ��-lactamase

The molecular basis of resistance to β‐lactams and β‐lactam‐β‐lactamase inhibitor combinations in the KPC family of class A enzymes is of extreme importance to the future design of effective β‐lactam therapy. Recent crystal structures of KPC‐2 and other class A β‐lactamases suggest that Ambler position Trp105 may be of importance in binding β‐lactam compounds. Based on this notion, we explored the role of residue Trp105 in KPC‐2 by conducting site‐saturation mutagenesis at this position. Escherichia coli DH10B cells expressing the Trp105Phe, ‐Tyr, ‐Asn, and ‐His KPC‐2 variants possessed minimal inhibitory concentrations (MICs) similar to E. coli cells expressing wild type (WT) KPC‐2. Interestingly, most of the variants showed increased MICs to ampicillin‐clavulanic acid but not to ampicillin‐sulbactam or piperacillin‐tazobactam. To explain the biochemical basis of this behavior, four variants (Trp105Phe, ‐Asn, ‐Leu, and ‐Val) were studied in detail. Consistent with the MIC data, the Trp105Phe β‐lactamase displayed improved catalytic efficiencies, k_cat/K_m, toward piperacillin, cephalothin, and nitrocefin, but slightly decreased k_cat/K_m toward cefotaxime and imipenem when compared to WT β‐lactamase. The Trp105Asn variant exhibited increased K_ms for all substrates. In contrast, the Trp105Leu and ‐Val substituted enzymes demonstrated notably decreased catalytic efficiencies (k_cat/K_m) for all substrates. With respect to clavulanic acid, the K_is and partition ratios were increased for the Trp105Phe, ‐Asn, and ‐Val variants. We conclude that interactions between Trp105 of KPC‐2 and the β‐lactam are essential for hydrolysis of substrates. Taken together, kinetic and molecular modeling studies define the role of Trp105 in β‐lactam and β‐lactamase inhibitor discrimination.     

Binding Interactions of Antagonists with 5‐Hydroxytryptamine3A Receptor Models

Abstract

Homology modeling was performed on the N‐terminal extracellular regions of human, mouse, and guinea pig 5‐hydroxytryptamine type 3A receptors (5‐HT₃R) based on the 24% sequence homology with and on the crystal structure of the snail acetylcholine binding protein (AChBP). Docking of 5‐HT₃ antagonists granisetron, tropisetron, ondansetron, dolasetron ('setrons), and (+)‐tubocurarine suggests an aromatic binding cleft behind a hydrophilic vestibule. Several intra‐ and interface interactions, H‐bonds, and salt bridges stabilize the pentameric structure and the binding cleft. The planar rings of antagonists are intercalated between aromatic side‐chains (W183‐Y234, Y143‐Y153). S227 donates H‐bonds to the carbonyl groups of 'setrons. The tertiary ammonium ions interact with E236, N128 or E129, and/or W90 (cation‐π interaction). This offers a molecular explanation of the pharmacophore models of 5‐HT₃R antagonists. Docking artifacts suggest some ambiguities in the binding loops A and C of the 5‐HT_3AR models. Lower potencies of (+)‐tubocurarine for human, and those of tropisetron for guinea pig 5‐HT_3ARs can be attributed to steric differences of I/S230 in the binding cleft and to distinct binding interactions with E229 and S227, respectively. Ligand binding interferes with crucial intra‐ and interface interactions along the binding cleft.     

Characterization of the functional insulin binding epitopes of the full-length insulin receptor

Mutational analyses of the secreted recombinant insulin receptor extracellular domain have identified a ligand binding site composed of residues located in the L1 domain (amino acids 1-470) and at the C terminus of the alpha subunit (amino acids 705-715). To evaluate the physiological significance of this ligand binding site, we have transiently expressed cDNAs encoding full-length receptors with alanine mutations of the residues forming the functional epitopes of this binding site and determined their insulin binding properties. Insulin bound to wild-type receptors with complex kinetics, which were fitted to a two-component sequential model; the Kd of the high affinity component was 0.03 nM and that of the low affinity component was 0.4 nM. Mutations of Arg14, Phe64, Phe705, Glu706, Tyr708, Asn711, and Val715 inactivated the receptor. Alanine mutation of Asn15 resulted in a 20-fold decrease in affinity, whereas mutations of Asp12, Gln34, Leu36, Leu37, Leu87, Phe89, Tyr91, Lys121, Leu709, and Phe714 all resulted in 4-10-fold decreases. When the effects of the mutations were compared with those of the same mutations of the secreted recombinant receptor, significant differences were observed for Asn15, Leu37, Asp707, Leu709, Tyr708, Asn711, Phe714, and Val715, suggesting that the molecular basis for the interaction of each form of the receptor with insulin differs. We also examined the effects of alanine mutations of Asn15, Gln34, and Phe89 on insulin-induced receptor autophosphorylation. They had no effect on the maximal response to insulin but produced an increase in the EC50 commensurate with their effect on the affinity of the receptor for insulin.     

Synthesis and structure–affinity relationships of novel small molecule natural product derivatives capable of discriminating between serotonin 5-HT1A, 5-HT2A, 5-HT2C receptor subtypes

Efforts to develop ligands that distinguish between clinically relevant 5-HT2A and 5-HT2C serotonin receptor subtypes have been challenging, because their sequences have high homology. Previous studies reported that a novel aplysinopsin belonging to a chemical class of natural products isolated from a marine sponge was selective for the 5-HT2C over the 5-HT2A receptor subtype. Our goal was to explore the 5-HT2A/2C receptor structure–affinity relationships of derivatives based on the aplysinopsin natural product pharmacophore. Twenty aplysinopsin derivatives were synthesized, purified and tested for their affinities for cloned human serotonin 5-HT1A, 5-HT2A, and 5-HT2C receptor subtypes. Four compounds in this series had >30-fold selectivity for 5-HT2A or 5-HT2C receptors. The compound (E)-5-((5,6-dichloro-1H-indol-3-yl)methylene)-2-imino-1,3-dimethylimidazolidin-4-one (UNT-TWU-22, 16) had approximately 2100-fold selectivity for the serotonin 5-HT2C receptor subtype: an affinity for 5-HT2C equal to 46 nM and no detectable affinity for the 5-HT1A or 5-HT2A receptor subtypes. The two most important factors controlling 5-HT2A or 5-HT2C receptor subtype selectivity were the combined R1,R3-alkylation of the imidazolidinone ring and the type and number of halogens on the indole ring of the aplysinopsin pharmacophore.     

The serotonin binding site of human and murine 5-HT2B receptors: molecular modeling and site-directed mutagenesis

Bacteriorhodopsin and rhodopsin crystal structures were used as templates to build structural models of the mouse and human serotonin (5-HT)-2B receptors (5-HT(2B)Rs). Serotonin was docked to the receptors, and the amino acids predicted to participate to its binding were subjected to mutagenesis. 5-HT binding affinity and 5-HT-induced inositol triphosphate production were measured in LMTK(-) cells transfected with either wild-type or mutated receptor genes. According to these measurements, the bacteriorhodopsin-based models of the 5-HT(2B)Rs appear more confident than the rhodopsin-based ones. Residues belonging to the transmembrane domains 3 and 6, i.e. Asp(3.32), Ser(3.36), Phe(6.52), and Asn(6.55), make direct contacts with 5-HT. In addition, Trp(3.28), Phe(3.35), Phe(6.52), and Phe(7.38) form an aromatic box surrounding 5-HT. The specificity of human and mouse 5-HT(2B)Rs may be reflected by different rearrangements of the aromatic network upon 5-HT binding. Two amino acids close to Pro(5.50) in the human transmembrane domain 5 sequence were permuted to introduce a "mouse-like" sequence. This change was enough to confer the human 5-HT(2B)R properties similar to those of the mouse. Taken together, the computed models and the site-directed mutagenesis experiments give a structural explanation to (i) the different 5-HT pK(D) values measured with the human and mouse 5-HT(2B)Rs (7.9 and 5.8, respectively) and (ii) the specificity of 5-HT binding to 5-HT(2B)Rs as compared with other serotonergic G-protein coupled receptors.     

In silico characterization of binding mode of CCR8 inhibitor: homology modeling,docking and membrane based MD simulation study

Human CC-chemokine receptor 8 (CCR8) is a crucial drug target in asthma that belongs to G-protein-coupled receptor superfamily, which is characterized by seven transmembrane helices. To date, there is no X-ray crystal structure available for CCR8; this hampers active research on the target. Molecular basis of interaction mechanism of antagonist with CCR8 remains unclear. In order to provide binding site information and stable binding mode, we performed modeling, docking and molecular dynamics (MD) simulation of CCR8. Docking study of biaryl-ether-piperidine derivative (13C) was performed inside predefined CCR8 binding site to get the representative conformation of 13C. Further, MD simulations of receptor and complex (13C-CCR8) inside dipalmitoylphosphatidylcholine lipid bilayers were performed to explore the effect of lipids. Results analyses showed that the Gln91, Tyr94, Cys106, Val109, Tyr113, Cys183, Tyr184, Ser185, Lys195, Thr198, Asn199, Met202, Phe254, and Glu286 were conserved in both docking and MD simulations. This indicated possible role of these residues in CCR8 antagonism. However, experimental mutational studies on these identified residues could be effective to confirm their importance in CCR8 antagonism. Furthermore, calculated Coulombic interactions represented the crucial roles of Glu286, Lys195, and Tyr113 in CCR8 antagonism. Important residues identified in this study overlap with the previous non-peptide agonist (LMD-009) binding site. Though, the non-peptide agonist and currently studied inhibitor (13C) share common substructure, but they differ in their effects on CCR8. So, to get more insight into their agonist and antagonist effects, further side-by-side experimental studies on both agonist (LMD-009) and antagonist (13C) are suggested.     

Amino acid sequence of a protease inhibitor isolated from Sarcophaga bullata determined by mass spectrometry.

The amino acid sequence of a protease inhibitor isolated from the hemolymph of Sarcophaga bullata larvae was determined by tandem mass spectrometry. Homology considerations with respect to other protease inhibitors with known primary structures assisted in the choice of the procedure followed in the sequence determination and in the alignment of the various peptides obtained from specific chemical cleavage at cysteines and enzyme digests of the S. bullata protease inhibitor. The resulting sequence of 57 residues is as follows: Val Asp Lys Ser Ala Cys Leu Gln Pro Lys Glu Val Gly Pro Cys Arg Lys Ser Asp Phe Val Phe Phe Tyr Asn Ala Asp Thr Lys Ala Cys Glu Glu Phe Leu Tyr Gly Gly Cys Arg Gly Asn Asp Asn Arg Phe Asn Thr Lys Glu Glu Cys Glu Lys Leu Cys Leu.     

The signaling pathway of dopamine D2 receptor (D2R) activation using normal mode analysis (NMA) and the construction of pharmacophore models for D2R ligands

G-protein-coupled receptors (GPCRs) are targets of more than 30% of marketed drugs. Investigation on the GPCRs may shed light on upcoming drug design studies. In the present study, we performed a combination of receptor- and ligand-based analysis targeting the dopamine D2 receptor (D2R). The signaling pathway of D2R activation and the construction of universal pharmacophore models for D2R ligands were also studied. The key amino acids, which contributed to the regular activation of the D2R, were in detail investigated by means of normal mode analysis (NMA). A derived cross-correlation matrix provided us an understanding of the degree of pair residue correlations. Although negative correlations were not observed in the case of the inactive D2R state, a high degree of correlation appeared between the residues in the active state. NMA results showed that the cytoplasmic side of the TM5 plays a significant role in promoting of residue–residue correlations in the active state of D2R. Tracing motions of the amino acids Arg219, Arg220, Val223, Asn224, Lys226, and Ser228 in the position of the TM5 are found to be critical in signal transduction. Complementing the receptor-based modeling, ligand-based modeling was also performed using known D2R ligands. The top-scored pharmacophore models were found as 5-sited (AADPR.671, AADRR.1398, AAPRR.3900, and ADHRR.2864) hypotheses from PHASE modeling from a pool consisting of more than 100 initial candidates. The constructed models using 38 D2R ligands (in the training set) were validated with 15 additional test set compounds. The resulting model correctly predicted the pIC₅₀ values of an additional test set compounds as true unknowns.