Computational identification and experimental characterization of substrate binding determinants of nucleotide pyrophosphatase/phosphodiesterase 7

<p>Abstract</p> <p>Background</p> <p>Nucleotide pyrophosphatase/phosphodiesterase 7 (NPP7) is the only member of the mammalian NPP enzyme family that has been confirmed to act as a sphingomyelinase, hydrolyzing sphingomyelin (SM) to form phosphocholine and ceramide. NPP7 additionally hydrolyzes lysophosphatidylcholine (LPC), a substrate preference shared with the NPP2/autotaxin(ATX) and NPP6 mammalian family members. This study utilizes a synergistic combination of molecular modeling validated by experimental site-directed mutagenesis to explore the molecular basis for the unique ability of NPP7 to hydrolyze SM.</p> <p>Results</p> <p>The catalytic function of NPP7 against SM, LPC, platelet activating factor (PAF) and para-nitrophenylphosphorylcholine (pNPPC) is impaired in the F275A mutant relative to wild type NPP7, but different impacts are noted for mutations at other sites. These results are consistent with a previously described role of F275 to interact with the choline headgroup, where all substrates share a common functionality. The L107F mutation showed enhanced hydrolysis of LPC, PAF and pNPPC but reduced hydrolysis of SM. Modeling suggests this difference can be explained by the gain of cation-pi interactions with the choline headgroups of all four substrates, opposed by increased steric crowding against the sphingoid tail of SM. Modeling also revealed that the long and flexible hydrophobic tails of substrates exhibit considerable dynamic flexibility in the binding pocket, reducing the entropic penalty that might otherwise be incurred upon substrate binding.</p> <p>Conclusions</p> <p>Substrate recognition by NPP7 includes several important contributions, ranging from cation-pi interactions between F275 and the choline headgroup of all substrates, to tail-group binding pockets that accommodate the inherent flexibility of the lipid hydrophobic tails. Two contributions to the unique ability of NPP7 to hydrolyze SM were identified. First, the second hydrophobic tail of SM occupies a second hydrophobic binding pocket. Second, the leucine residue present at position 107 contrasts with a conserved phenylalanine in NPP enzymes that do not utilize SM as a substrate, consistent with the observed reduction in SM hydrolysis by the NPP7-L107F mutant.</p

Lysophosphatidic acid receptor agonists and antagonists (WO2010051053)

Lysophosphatidic acid (LPA) is a bioactive lipid involved in signaling pathways that result in cell survival, proliferation, migration and invasion. These cellular responses are a critical element of both normal development as well as pathophysiology. In particular, dysregulated LPA production and function have been linked with cancer and cardiovascular disease. RxBio, Inc. has generated several series of LPA analogs with varied agonist/antagonist function at the LPA1-3 GPCR targets of LPA signaling. These analogs are simplified relative to LPA through deletion of the glycerol moiety linking the LPA phosphate and fatty acid groups. One of the example compounds was shown to protect intestinal crypt cells from radiation-induced apoptosis in mice when whole body irradiation occurred 2 h after oral dosing. © 2011 Informa UK, Ltd

Synthetic lipids and their role in defining macromolecular assemblies

Lipids have a variety of physiological roles, ranging from structural and biophysical contributions to membrane functions to signaling contributions in normal and abnormal physiology. This review highlights some of the contributions made by Robert Bittman to our understanding of lipid assemblies through the production of synthetic lipid analogs in the sterol, sphingolipid, and glycolipid classes. His contributions have included the development of a fluorescent cholesterol analog that shows strong functional analogies to cholesterol that has allowed live imaging of cholesterol distribution in living systems, to stereospecific synthetic approaches to both sphingolipid and glycolipid analogs crucial in defining the structure-activity relationships of lipid biological targets

Comparative modeling of lipid receptors

Comparative modeling is a powerful technique to generate models of proteins from families already represented by members with experimentally characterized three-dimensional structures. The method is particularly important for modeling membrane-bound receptors in the G Protein-Coupled Receptor (GPCR) family, such as many of the lipid receptors (such as the cannabinoid, prostanoid, lysophosphatidic acid, sphingosine 1-phosphate, and eicosanoid receptor family members), as these represent particularly challenging targets for experimental structural characterization methods. Although challenging modeling targets, these receptors have been linked to therapeutic indications that vary from nociception to cancer, and thus are of interest as therapeutic targets. Accurate models of lipid receptors are therefore valuable tools in the drug discovery and optimization phases of therapeutic development. This chapter describes the construction and evaluation of comparative structural models of lipid receptors beginning with the selection of template structures. © 2012 Springer Science+Business Media, LLC

Crystal structures of a second G protein-coupled receptor: Triumphs and implications

Long sought: The recently reported crystallographic structures of the β2-adrenergic receptor demonstrate that GPCR exhibit the strong structural conservation expected, in addition to some surprising structural differences. These structures were obtained through a number of methodological advances that are broadly applicable to studies of other GPCR. (Figure Presented). © 2008 Wiley-VCH Verlag GmbH & Co. KGaA

Lysophospholipid interactions with protein targets

Bioactive lysophospholipids include lysophosphatidic acid (LPA), sphingosine 1-phosphate (S1P), cyclic-phosphatidic acid (CPA) and alkyl glycerolphosphate (AGP). These lipid mediators stimulate a variety of responses that include cell survival, proliferation, migration, invasion, wound healing, and angiogenesis. Responses to lysophospholipids depend upon interactions with biomolecular targets in the G protein-coupled receptor (GPCR) and nuclear receptor families, as well as enzymes. Our current understanding of lysophospholipid interactions with these targets is based on a combination of lysophospholipid analog structure activity relationship studies as well as more direct structural characterization techniques such as X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, and experimentally-validated molecular modeling. The direct structural characterization studies are the focus of this review, and provide the insight necessary to stimulate structure-based therapeutic lead discovery efforts in the future. © 2008 Elsevier B.V. All rights reserved

Evolutionary and genetic methods in drug design

Many phases of rational drug design involve finding solutions to large combinatorial problems for which an exhaustive search is intractable. A simulation of the evolutionary pressure of natural selection can be incorporated into artificial intelligence algorithms to rapidly find good, if not optimal, solutions to such problems. This review describes implementations and select applications of genetic algorithms and evolutionary programming in various aspects of rational drug design. Evolutionary methods have been developed in the areas of pharmacophore elucidation, lead discovery and lead optimization, as well as in many areas of peripheral importance to rational drug design