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Abby L Parrill

Publications and source records attributed to Abby L Parrill.

14 recordsLinked to original sources
PubMed2006

Identification of the hydrophobic ligand binding pocket of the S1P1 receptor.

Sphingosine 1-phosphate (S1P), a naturally occurring sphingolipid mediator and also a second messenger with growth factor-like actions in almost every cell type, is an endogenous ligand of five G protein-coupled receptors (GPCRs) in the endothelial differentiation gene family. The lack of GPCR crystal structures sets serious limitations to rational drug design and in silico searches for subtype-selective ligands. Here we report on the experimental validation of a computational model of the ligand binding pocket of the S1P1 GPCR surrounding the aliphatic portion of S1P. The extensive mutagenesis-based validation confirmed 18 residues lining the hydrophobic ligand binding pocket, which, combined with the previously validated three head group-interacting residues, now complete the mapping of the S1P ligand recognition site. We identified six mutants (L3.43G/L3.44G, L3.43E/L3.44E, L5.52A, F5.48G, V6.40L, and F6.44G) that maintained wild type [32P]S1P binding with abolished ligand-dependent activation by S1P. These data suggest a role for these amino acids in the conformational transition of S1P1 to its activated state. Three aromatic mutations (F5.48Y, F6.44G, and W6.48A) result in differential activation, by S1P or SEW2871, indicating that structural differences between the two agonists can partially compensate for differences in the amino acid side chain. The now validated ligand binding pocket provided us with a pharmacophore model, which was used for in silico screening of the NCI, National Institutes of Health, Developmental Therapeutics chemical library, leading to the identification of two novel nonlipid agonists of S1P1.

Binding Sites↗
PubMed2005

Synthesis and pharmacological evaluation of second-generation phosphatidic acid derivatives as lysophosphatidic acid receptor ligands.

Short-chain phosphatidic acid derivatives, dioctanoyl glycerol pyrophosphate (DGPP 8:0, 1) and phosphatidic acid 8:0 (PA 8:0, 2), were previously identified as subtype-selective LPA(1) and LPA(3) receptor antagonists. Recently, we reported that the replacement of the phosphate headgroup by thiophosphate in a series of fatty alcohol phosphates (FAP) improves agonist as well as antagonist activities at LPA GPCR. Here, we report the synthesis of stereoisomers of PA 8:0 analogs and their biological evaluation at LPA GPCR, PPARgamma, and ATX. The results indicate that LPA receptors stereoselectively interact with glycerol backbone modified ligands. We observed entirely stereospecific responses by dioctyl PA 8:0 compounds, in which (R)-isomers were found to be agonists and (S)-isomers were antagonists of LPA GPCR. From this series, we identified compound 13b as the most potent LPA(3) receptor subtype-selective agonist (EC(50)=3 nM), and 8b as a potent and selective LPA(3) receptor antagonist (K(i)=5 nM) and inhibitor of ATX (IC(50)=600 nM). Serinediamide phosphate 19b was identified as an LPA(3) receptor specific antagonist with no effect on LPA(1), LPA(2), and PPARgamma.

Animals↗
PubMed2005

Sphingosine 1-phosphate analogue recognition and selectivity at S1P4 within the endothelial differentiation gene family of receptors.

Synergistic computational and experimental studies provided previously unforeseen details concerning the structural basis of S1P (sphingosine 1-phosphate) recognition by the S1P4 G-protein-coupled receptor. Similarly to reports on the S1P1 receptor, cationic and anionic residues in the third transmembrane domain (R3.28 and E3.29 at positions 124 and 125) form ion pairs with the phosphate and ammonium of S1P, and alanine mutations at these positions abolished specific S1P binding, S1P-induced receptor activation and cell migration. Unlike findings on the S1P1 receptor, no cationic residue in the seventh transmembrane domain interacts with the phosphate. Additionally, two previously undiscovered interactions with the S1P polar headgroup have been identified. Trp186 at position 4.64 in the fourth transmembrane domain interacts by a cation-pi interaction with the ammonium group of S1P. Lys204 at position 5.38 forms an ion pair with the S1P. The S1P4 and S1P1 receptors show differences in binding-pocket shape and electrostatic distributions that correlate with the published structure-activity relationships. In particular, the binding pocket of mS1P4 (mouse S1P4) has recognition sites for the anionic phosphate and cationic ammonium groups that are equidistant from the end of the non-polar tail. In contrast, the binding pocket of hS1P1 (human S1P4) places the ammonium recognition site 2 A (1 A=0.1 nm) closer to the end of the non-polar tail than the phosphate recognition site.

Amino Acid Sequence↗
PubMed2005

S1P1-selective in vivo-active agonists from high-throughput screening: off-the-shelf chemical probes of receptor interactions, signaling, and fate.

The essential role of the sphingosine 1-phosphate (S1P) receptor S1P(1) in regulating lymphocyte trafficking was demonstrated with the S1P(1)-selective nanomolar agonist, SEW2871. Despite its lack of charged headgroup, the tetraaromatic compound SEW2871 binds and activates S1P(1) through a combination of hydrophobic and ion-dipole interactions. Both S1P and SEW2871 activated ERK, Akt, and Rac signaling pathways and induced S1P(1) internalization and recycling, unlike FTY720-phosphate, which induces receptor degradation. Agonism with receptor recycling is sufficient for alteration of lymphocyte trafficking by S1P and SEW2871. S1P(1) modeling and mutagenesis studies revealed that residues binding the S1P headgroup are required for kinase activation by both S1P and SEW2871. Therefore, SEW2871 recapitulates the action of S1P in all the signaling pathways examined and overlaps in interactions with key headgroup binding receptor residues, presumably replacing salt-bridge interactions with ion-dipole interactions.

Animals↗
PubMed2004

A single amino acid determines preference between phospholipids and reveals length restriction for activation of the S1P4 receptor.

BACKGROUND: Sphingosine-1-phosphate and lysophosphatidic acid (LPA) are ligands for two related families of G protein-coupled receptors, the S1P and LPA receptors, respectively. The lysophospholipid ligands of these receptors are structurally similar, however recognition of these lipids by these receptors is highly selective. A single residue present within the third transmembrane domain (TM) of S1P receptors is thought to determine ligand selectivity; replacement of the naturally occurring glutamic acid with glutamine (present at this position in the LPA receptors) has previously been shown to be sufficient to change the specificity of S1P1 from S1P to 18:1 LPA. RESULTS: We tested whether mutation of this "ligand selectivity" residue to glutamine could confer LPA-responsiveness to the related S1P receptor, S1P4. This mutation severely affected the response of S1P4 to S1P in a [35S]GTP gamma S binding assay, and imparted sensitivity to LPA species in the order 14:0 LPA > 16:0 LPA > 18:1 LPA. These results indicate a length restriction for activation of this receptor and demonstrate the utility of using LPA-responsive S1P receptor mutants to probe binding pocket length using readily available LPA species. Computational modelling of the interactions between these ligands and both wild type and mutant S1P4 receptors showed excellent agreement with experimental data, therefore confirming the fundamental role of this residue in ligand recognition by S1P receptors. CONCLUSIONS: Glutamic acid in the third transmembrane domain of the S1P receptors is a general selectivity switch regulating response to S1P over the closely related phospholipids, LPA. Mutation of this residue to glutamine confers LPA responsiveness with preference for short-chain species. The preference for short-chain LPA species indicates a length restriction different from the closely related S1P1 receptor.

Amino Acid Substitution↗
PubMed2004

Functional dissection and molecular characterization of calcium-sensitive actin-capping and actin-depolymerizing sites in villin.

All proteins of the villin superfamily, which includes the actin-capping and -severing proteins such as gelsolin, scinderin, and severin, are calcium-regulated actin-modifying proteins. Like some of these proteins, villin has morphologically distinct effects on actin assembly depending on the free calcium concentrations. At physiological calcium (Ca2+) villin nucleates and bundles actin, whereas at higher concentrations it caps (>50 microm) and severs (>200 microM) actin filaments. Although Ca(2+)-binding sites have been described in villin, the functional characterization of these sites has not been done previously. In the present study we functionally dissect the calcium-dependent actin-capping and -depolymerizing sites in villin. Our analysis reveals that villin binds Ca2+ with a Kd of 80.5 microM, a stoichiometry of 5.97, and a Hill's coefficient of 1.2. Using the NMR structure of villin 14T and the gelsolin-actin/Ca2+ crystal structure, six putative sites that result in Ca(2+)-induced conformational changes were identified in human villin and confirmed by mutational analysis. Molecular dynamics studies support the mutational analysis and provide a model for structural difference in the A93G mutant that prevents the calcium-induced conformational changes in the S1 domain of villin. Furthermore, we determined that villin expresses at least two types of Ca(2+)-sensitive sites that determine separate functional properties; site 1 (Glu-25, Asp-44, and Glu-74) regulates actin-capping, whereas sites 1 and 2 (Asp-86, Ala-93, and Asp-61), together with the intra-domain calcium-sensitive sites in villin, regulate actin depolymerization by villin. This is the first study that employs sequential mutagenesis to biochemically and functionally characterize the calcium-sensitive sites in villin. Such mutational analysis and functional characterization of the actin-capping and -depolymerizing sites are unknown for other proteins of the villin family.

Actins↗
PubMed2004

Sphingosine 1-phosphate and lysophosphatidic acid receptors: agonist and antagonist binding and progress toward development of receptor-specific ligands.

Sphingosine 1-phosphate and lysophosphatidic acid are two phospholipid growth factors whose importance in physiology and pathophysiology is becoming more and more apparent. Structure-activity relationships for agonism and antagonism at the thirteen known cell-surface and one intracellular receptor are described. Particular emphasis is placed on ligands having different selectivity than the parent molecules. Structural insights regarding agonist and antagonist recognition by the receptors from both computational modeling studies and crystallography are also discussed.

Animals↗
PubMed2003

Fatty alcohol phosphates are subtype-selective agonists and antagonists of lysophosphatidic acid receptors.

A more complete understanding of the physiological and pathological role of lysophosphatidic acid (LPA) requires receptor subtype-specific agonists and antagonists. Here, we report the synthesis and pharmacological characterization of fatty alcohol phosphates (FAP) containing saturated hydrocarbon chains from 4 to 22 carbons in length. Selection of FAP as the lead structure was based on computational modeling as a minimal structure that satisfies the two-point pharmacophore developed earlier for the interaction of LPA with its receptors. Decyl and dodecyl FAPs (FAP-10 and FAP-12) were specific agonists of LPA(2) (EC(50) = 3.7 +/- 0.2 microM and 700 +/- 22 nM, respectively), yet selective antagonists of LPA(3) (K(i) = 90 nM for FAP-12) and FAP-12 was a weak antagonist of LPA(1). Neither LPA(1) nor LPA(3) receptors were activated by FAPs; in contrast, LPA(2) was activated by FAPs with carbon chains between 10 and 14. Computational modeling was used to evaluate the interaction between individual FAPs (8 to 18) with LPA(2) by docking each compound in the LPA binding site. FAP-12 displayed the lowest docked energy, consistent with its lower observed EC(50). The inhibitory effect of FAP showed a strong hydrocarbon chain length dependence with C12 being optimum in the Xenopus laevis oocytes and in LPA(3)-expressing RH7777 cells. FAP-12 did not activate or interfere with several other G-protein-coupled receptors, including S1P-induced responses through S1P(1,2,3,5) receptors. These data suggest that FAPs are ligands of LPA receptors and that FAP-10 and FAP-12 are the first receptor subtype-specific agonists for LPA(2).

Animals↗
PubMed2002

Regulation of actin dynamics by tyrosine phosphorylation: identification of tyrosine phosphorylation sites within the actin-severing domain of villin.

We have previously shown that villin, an epithelial cell actin-binding protein, is tyrosine phosphorylated both in vitro and in vivo and that villin's actin-modifying functions are regulated by phosphorylation. Here as a first step toward understanding the role of villin tyrosine phosphorylation, we sought to identify the major phosphorylation site(s) in human villin and study its role in actin filament assembly. We generated a series of carboxyl-terminal truncation mutants of villin and cloned them in the prokaryotic expression vector pGEX-2T. Full-length villin and the truncation mutants were expressed in TKX1 cells, which carry an inducible tyrosine kinase gene. Using this approach, we identified a region in the amino-terminal actin-severing domain of villin as the site of phosphorylation (amino acids 1-261). Five phosphorylation sites were identified by direct mutation of candidate tyrosines (Y) to phenylalanine (F), namely, Y46, -60, -64, -81, and -256. Changing all of these sites to phenylalanine resulted in a villin mutant that neither was phosphorylated in TKX1 cells nor was a substrate for c-src kinase in an in vitro kinase assay. Using a pyrene actin-based fluorescence assay, we mapped the various phosphorylated tyrosine residues with the actin-nucleating and -depolymerizing functions of villin. Phosphorylation of any one of the identified sites inhibited the actin-nucleating function of villin, whereas phosphorylation at Y46 and/or Y60 increased the actin-severing activity of villin. Since there is significant homology between the amino-terminal end of villin and other actin-severing proteins, the results provide a structural basis for the actin-severing mechanism and help understand the relationship of phosphorylation with this function.

Actins↗
PubMed2002

Molecular basis for lysophosphatidic acid receptor antagonist selectivity.

Recent characterization of lysophosphatidic acid (LPA) receptors has made possible studies elucidating the structure-activity relationships (SAR) for agonist activity at individual receptors. Additionally, the availability of these receptors has allowed the identification of antagonists of LPA-induced effects. Two receptor-subtype selective LPA receptor antagonists, one selective for the LPA1/EDG2 receptor (a benzyl-4-oxybenzyl N-acyl ethanolamide phosphate, NAEPA, derivative) and the other selective for the LPA3/EDG7 receptor (diacylglycerol pyrophosphate, DGPP, 8:0), have recently been reported. The receptor SAR for both agonists and antagonists are reviewed, and the molecular basis for the difference between agonism and antagonism as well as for receptor-subtype antagonist selectivity identified by molecular modeling is described. The implications of the newly available receptor-subtype selective antagonists are also discussed.

Amino Acid Sequence↗
PubMed2002

QSAR studies of HIV-1 integrase inhibition.

Compounds from a wide variety of structural classes inhibit HIV-1 integrase. However, a single unified understanding of the relationship between the structures and activities of these compounds still eludes researchers. We report herein the development of QSAR models for integrase inhibition. The genetic function approximation (GFA) was utilized to select descriptors for the development of the QSAR models. The best QSAR model derived for the complete set of 11 structural classes had a correlation coefficient (r(2)) of only 0.54 and a cross-validated correlation coefficient (q(2)) of only 0.42. This indicated that the compounds studied may differ in the exact relationship between structure and inhibition, perhaps through interactions with different subsets of amino acids in the binding pocket, or through the presence of non-overlapping binding pockets. Descriptor-based cluster analysis indicated that the 11 structural classes of integrase inhibitors studied belonged to two clusters, one consisting of five structural classes, and the other six. QSAR models for these two clusters had r(2) values of 0.79 and 0.82 and q(2) values of 0.71 and 0.74, a significant improvement over models obtained for the complete set of compounds. The two models were applied to predict the activities of compounds from the same structural classes as those used to build the models, giving r(2) values of 0.65 and 0.78. The models were also used to predict the activities of compounds shown in crystallographic or docking studies to interact near the active site metal ion. The model describing the larger cluster of structural classes was better able to reproduce the biological activities of these five structures with an average percent residual error of 7.9 compared with the 19.3% residual error for predictions from the other model. This indicated that the six structural classes comprising the larger cluster may bind near the metal ion in a fashion similar to that observed in one publicly available co-crystal structure of an inhibitor bound to HIV-1 integrase. Flexible alignment of inhibitors in the two clusters found different pharmacophores that are consistent with previously published pharmacophores developed on the basis of individual structural classes that have produced novel inhibitory compounds. Thus we expect that these two QSAR models can be used in the search for novel HIV-1 integrase inhibitors as well as to provide insight into the binding modes of such diverse chemical compounds.

Anti-HIV Agents↗
PubMed2002

Three-dimensional quantitative structure-activity relationship and comparative molecular field analysis of dipeptide hydroxamic acid Helicobacter pylori urease inhibitors.

A homology model of Helicobacter pylori urease was developed by using the crystal structure of urease from Klebsiella aerogenes (EC 3.5.1.5) as a template. The acetohydroxamic acid moiety was docked into the active pocket of the enzyme model, followed by relaxation of the complex by use of molecular dynamics. The resulting conformation was used as a template to construct 24 potential dipeptide hydroxamic acid inhibitors with which comparative molecular field analysis (CoMFA) was performed. The resulting model provided a cross-validation correlation coefficient (q(2)(L00)) of 0.610, a conventional r(2) value of 0.988, and an F (Fisher indication of statistical significance) value of 294.88. We were able to validate the CoMFA model by using the 50% inhibitory concentrations of six compounds that were not included in the construction of the model. A very good structural correlation was observed between the amino acids in the model urease's active pocket and the contour maps derived from the CoMFA model. This correlation, accompanied by the validation supplied by use of the CoMFA data, illustrates that the model can aid in the prediction and design of novel H. pylori urease inhibitors.

Computational Biology↗
PubMed2002

Interaction of the novel anthracycline antitumor agent N-benzyladriamycin-14-valerate with the C1-regulatory domain of protein kinase C: structural requirements, isoform specificity, and correlation with drug cytotoxicity.

Anthracycline antibiotics like doxorubicin (DOX) are known to exert their antitumor effects primarily via DNA intercalation and topoisomerase II inhibition. By contrast, the noncross-resistant cytoplasmically localizing DOX analogue, N-benzyladriamycin-14-valerate (AD 198), only weakly binds DNA and does not inhibit topoisomerase II, yet it displays superior antitumor activity, strongly suggesting a distinct cytotoxic mechanism. In recent modeling studies, we reported a structural similarity between AD 198 and commonly accepted ligands for the C1-domain of protein kinase C (PKC), and we hypothesized that the unique biological activity of AD 198 may derive, in part, through this kinase. Consistent with this hypothesis, the present biochemical studies demonstrate that AD 198 competes with [3H]phorbol-12,13-dibutyrate ([3H]PDBu) for binding to phorbol-responsive PKC isoforms, the isolated C1b domain of PKC-delta (delta C1b), and the nonkinase phorbol ester receptor, beta2-chimaerin. In NIH/3T3 cells, AD 198 competitively blocks PKC activation by C1-ligands. Importantly, neither DOX nor N-benzyladriamycin, the principal AD 198 metabolite, inhibits basal or phorbol-stimulated PKC activity or appreciably competes for [3H]PDBu binding. In CEM cells, structure activity studies with 14-acyl congeners indicate that the rapid induction of apoptosis correlates with competition for [3H]PDBu binding, strongly implicating phorbol-binding proteins in drug activity. Collectively, these studies support the conclusion that AD 198 is a C1-ligand and that C1-ligand receptors are selective drug targets. These studies provide the impetus for continuing efforts to understand the molecular basis for the unique biological activity of AD 198 and provide for the design of analogues with improved affinity for C1-domains and potentially greater antitumor activity.

3T3 Cells↗