The autotaxin (ATX) enzyme exhibits lysophospholipase D activity responsible for the conversion of lysophosphatidyl choline to lysophosphatidic acid (LPA). ATX and LPA have been linked to the initiation of atherosclerosis, cancer invasiveness, and neuropathic pain. ATX inhibition therefore offers currently unexploited therapeutic potential, and substantial interest in the development of ATX inhibitors is evident in the recent literature. Here we report the performance-based comparison of ligand-based pharmacophores developed on the basis of different combinations of ATX inhibitors in the training sets against an extensive database of compounds tested for ATX inhibitory activity, as well as with docking results of the actives against a recently reported ATX crystal structure. In general, pharmacophore models show better ability to select active ATX inhibitors binding in a common location when the ligand-based superposition shows a good match to the superposition of actives based on docking results. Two pharmacophore models developed on the basis of competitive inhibitors in combination with the single inhibitor crystallized to date in the active site of ATX were able to identify actives at rates over 40%, a substantial improvement over the <10% representation of active site-directed actives in the test set database.
Autotaxin; pharmacophore; docking
A structurally diverse dataset of 119 compounds was used to develop and validate a 2D binary QSAR model for the LPA3 receptor. The binary QSAR model was generated using an activity threshold of greater than 15% inhibition at 10µM. The overall accuracy of the model on the training set was 82%. It had accuracies of 55% for active and 91% for inactive compounds, respectively. The model was validated using an external test set of 10 compounds. The accuracy on the external test set was 60% overall, identifying three out of seven actives and all three inactive compounds. This model was combined with similarity searching to rapidly screen libraries and select 14 candidate LPA3 antagonists. Experimental assays confirmed 13 of these (93%) met the 15% inhibition threshold defining actives. The successful application of the model to select candidates for screening demonstrates the power of this binary QSAR model to prioritize compound selection for experimental consideration.
binary QSAR; lysophosphatidic acid receptor; similarity searching
Compound 5 ([5-(3-nitrophenoxy)-1,3-dioxo-1,3-dihydro-2-isoindol-2-yl]acetic acid) was identified as a weak selective LPA3 antagonist (IC50=4504 nM) in a virtual screening effort to optimize a dual LPA2&3 antagonist. Structure-based drug design techniques were used to prioritize similarity search matches of compound 5. This strategy rapidly identified 10 novel antagonists. The two most efficacious compounds identified inhibit activation of the LPA3 receptor by 200 nM LPA with IC50 values of 752 nM and 2992 nM. These compounds additionally define changes to our previously reported pharmacophore that will improve its ability to identify more potent and selective LPA3 receptor antagonists. The results of the combined computational and experimental screening are reported.
Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to form the bioactive lipid lysophosphatidic acid (LPA). LPA stimulates cell proliferation, cell survival, and cell migration and is involved in obesity, rheumatoid arthritis, neuropathic pain, atherosclerosis and various cancers, suggesting that ATX inhibitors have broad therapeutic potential. Product feedback inhibition of ATX by LPA has stimulated structure activity studies focused on LPA analogs. However, LPA displays mixed mode inhibition, indicating it can bind to both the enzyme and the enzyme-substrate complex. This suggests that LPA may not interact solely with the catalytic site. In this report we have prepared LPC analogs to help map out substrate structure activity relationships. The structural variances include length and unsaturation of the fatty tail, choline and polar linker presence, acyl versus ether linkage of the hydrocarbon chain, and methylene and nitrogen replacement of the choline oxygen. All LPC analogs were assayed in competition with the synthetic substrate, FS-3, to show the preference ATX has for each alteration. Choline presence and methylene replacement of the choline oxygen were detrimental to ATX recognition. These findings provide insights into the structure of the enzyme in the vicinity of the catalytic site as well as suggesting that ATX produces rate enhancement, at least in part, by substrate destabilization.
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.
Phospholipid; G protein-coupled receptor; lysophosphatidic acid; sphingosine 1-phosphate; autotaxin; PPAR
In the present study, we utilized virtual screening to identify LPA3 antagonists. We have developed a three point structure-based pharmacophore model based on known LPA3 antagonists. This model was used to mine the NCI database. Docking, pharmacophore development, and database mining produced new, non-lipid leads. Experimental testing of seven computationally-selected pharmacophore hits produced one potentiator and three antagonists, one of which displays both LPA3 selectivity and nanomolar potency. Similarity searching in the ChemBridge database using the most promising lead as the search target produced five additional LPA3 antagonists and a potent dual LPA1&2 antagonist.
lysophosphatidic acid; GPCR; database mining; pharmacophore
Computational modeling and its application in ligand screening and ligand receptor interaction studies play important roles in structure-based drug design. A series of sphingosine 1-phosphate (S1P) receptor ligands with varying potencies and receptor selectivities were docked into homology models of the S1P1-5 receptors. These studies provided molecular insights into pharmacological trends both across the receptor family as well as at single receptors. This study identifies ligand recognition features that generalize across the S1P receptor family, features unique to the S1P4 and S1P5 receptors, and suggests significant structural differences of the S1P2 receptor. Docking results reveal a previously unknown sulfur-aromatic interaction between the S1P4 C5.44 sulfur atom and the phenyl ring of benzimidazole as well as π-π interaction between F3.33 of S1P1,4,5 and aromatic ligands. The findings not only confirm the importance of a cation-π interaction between W4.64 and the ammonium of S1P at S1P4 but also predict the same interaction at S1P5. S1P receptor models are validated for pharmacophore development including database mining and new ligand discovery and serve as tools for ligand optimization to improve potency and selectivity.
Sphingosine 1-phosphate (S1P); G protein-coupled receptor (GPCR); Endothelial differentiation gene (EDG); Computational Model; Ligand Recognition
A homology model of Helicobacter pylori urease was developed by using the crystal structure of urease from Klebsiella aerogenes (EC 126.96.36.199) 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 (q2L00) of 0.610, a conventional r2 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.
A compelling question of how phospholipids interact with their target receptors has been of interest since the first receptor-mediated effects were reported. The recent report of a crystal structure for the S1P1 receptor in complex with an antagonist phospholipid provides interesting perspective on the insights that had previously been gained through structure–activity studies of the phospholipids, as well as modeling and mutagenesis studies of the receptors. This review integrates these varied lines of investigation in the context of their various contributions to our current understanding of phospholipid–receptor interactions. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
S1P; LPA; GPCR structure; Structure–activity relationships
The sphingosine 1-phosphate receptor 1 (S1P1) and its ligand, sphingosine 1-phosphate (S1P), have now emerged as critical regulators of lymphocyte trafficking, vascular development and integrity, and immunity. S1P1 is targeted by the phosphorylation product of fingolimod, which has been approved for the treatment of multiple sclerosis. The recent progress in the structural biology of heterotrimeric guanine nucleotide–binding protein (G protein)–coupled receptors has now enabled the elucidation of the structure of S1P1. Analysis of the structure, along with structure activity and mutagenesis analysis, highlighted key interactions associated with the binding of S1P and agonists and suggested that the ligand may gain access to the binding pocket by lateral diffusion within the plasma membrane. The S1P1 crystal structure will be helpful for designing ligands that specifically target S1P1.
G protein-coupled receptor (GPCR) structures are of interest as a means to understand biological signal transduction and as tools for therapeutic discovery. The growing number of GPCR crystal structures demonstrates that the extracellular loops (EL) connecting the membrane-spanning helices show tremendous structural variability relative to the more structurally-conserved seven transmembrane α-helical domains. The EL of the LPA1 receptor have not yet been conclusively resolved, and bear limited sequence identity to known structures. This study involved development of a peptide to characterize the intrinsic structure of the LPA1 GPCR second EL. The loop was embedded between two helices that assemble into a coiled-coil, which served as a receptor-mimetic folding constraint (LPA1-CC-EL2 peptide). The ensemble of structures from multi-dimensional NMR experiments demonstrated that a robust coiled-coil formed without noticeable deformation due to the EL2 sequence. In contrast, the EL2 sequence showed well-defined structure only near its C-terminal residues. The NMR ensemble was combined with a computational model of the LPA1 receptor that had previously been validated. The resulting hybrid models were evaluated using docking. Nine different hybrid models interacted with LPA 18:1 as expected, based on prior mutagenesis studies, and one was additionally consistent with antagonist affinity trends.
G protein-coupled receptor; GPCR; lysophosphatidic acid; LPA; NMR; GPCR segment model
Autotaxin (ATX, NPP2) is a member of the nucleotide pyrophosphate phosphodiesterase enzyme family. ATX catalyzes the hydrolytic cleavage of lysophosphatidylcholine (LPC) via a lysophospholipase D activity that leads to the generation of the growth factor-like lipid mediator lysophosphatidic acid (LPA). ATX is highly upregulated in metastatic and chemotherapy-resistant carcinomas and represents a potential target to mediate cancer invasion and metastasis. Here we report the synthesis and pharmacological characterization of inhibitors of ATX based on the 4-tetradecanoylaminobenzyl phosphonic acid scaffold that was previously found to lack sufficient stability in cellular systems. The new 4-substituted benzyl phosphonic acid and 6-substituted naphthalen-2-yl-methyl phosphonic acid analogs blocked ATX with Ki values in the low-micromolar-nanomolar range against FS-3, LPC, and nucleotide substrates through a mixed-mode mechanism of inhibition. None of the compounds tested inhibited the activity of related enzymes (NPP6 and NPP7). In addition, the compounds were evaluated as agonists or antagonists of seven LPA receptor subtypes. Analogs 22 and 30b, the two most potent ATX inhibitors, dose-dependently inhibited the invasion of MM1 hepatoma cells across murine mesothelial and human vascular endothelial monolayers in vitro. The average terminal half-life for compound 22 was 10h ± 5.4h and it caused a long-lasting reduction plasma LPA levels. Compounds 22 and 30b significantly reduced lung metastasis of B16-F10 syngeneic mouse melanoma in a post-inoculation treatment paradigm. The described 4-substituted benzyl phosphonic acids and 6-substituted naphthalen-2-yl-methyl phosphonic acids represent new lead compounds that effectively inhibit the ATX-LPA-LPA receptor axis both in vitro and in vivo.
ATX inhibitors; LPA receptors; 4-substituted benzyl phosphonic acids; 6-substituted naphthalen-2-yl-methyl phosphonic acids; structure-activity relationships
Signal transduction modifiers that modulate the lysophosphatidic acid (LPA) pathway have potential as anticancer agents. Herein, we describe metabolically stabilized LPA analogues that reduce cell migration and invasion and cause regression of orthotopic breast tumors in vivo. Two diastereoisomeric α-bromophosphonates (BrP-LPA) were synthesized, and the pharmacology was determined for five LPA G protein–coupled receptors (GPCRs). The syn and anti diastereomers of BrP-LPA are pan-LPA GPCR antagonists and are also nanomolar inhibitors of the lysophospholipase D activity of autotaxin, the dominant biosynthetic source of LPA. Computational models correctly predicted the diastereoselectivity of antagonism for three GPCR isoforms. The anti isomer of BrP-LPA was more effective than syn isomer in reducing migration of MDA-MB-231 cells, and the anti isomer was superior in reducing invasion of these cells. Finally, orthotopic breast cancer xenografts were established in nude mice by injection of MB-231 cells in an in situ cross-linkable extracellular matrix. After 2 weeks, mice were treated with the BrP-LPA alone (10 mg/kg), Taxol alone (10 mg/kg), or Taxol followed by BrP-LPA. All treatments significantly reduced tumor burden, and BrP-LPA was superior to Taxol in reducing blood vessel density in tumors. Moreover, both the anti- and syn-BrP-LPA significantly reduced tumors at 3 mg/kg.
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.
Cyclic phosphatidic acid (1-acyl-2,3-cyclic-glycerophosphate, CPA), one of nature’s simplest phospholipids, is found in cells from slime mold to humans and has largely unknown function. We find here that CPA is generated in mammalian cells in a stimulus coupled-manner by Phospholipase D2 (PLD2), and binds to and inhibits the nuclear hormone receptor PPARγ with nanomolar affinity and high specificity through stabilizing its interaction with the corepressor SMRT. CPA production inhibits the PPARγ target-gene transcription that normally drives adipocytic differentiation of 3T3-L1 cells, lipid accumulation in RAW264.7 cells and primary mouse macrophages, and arterial wall remodeling in a rat model in vivo. Inhibition of PLD2 by shRNA, a dominant negative mutant, or a small molecule inhibitor blocks CPA production and relieves PPARγ inhibition. We conclude that CPA is a second messenger and a physiological inhibitor of PPARγ, revealing that PPARγ is regulated by endogenous agonists as well as by antagonists.
FTY720 (Fingolimod™), a synthetic analogue of sphingosine 1-phosphate (S1P), activates four of the five EDG-family S1P receptors and is in a phase-III clinical study for the treatment of multiple sclerosis. (S)-FTY720-phosphate (FTY720-P) causes S1P1 receptor internalization and targeting to the proteasomal degradative pathway, and thus acts as a functional antagonist of S1P1 by depleting the functional S1P1 receptor from the plasma membrane. Here we describe the pharmacological characterization of two unsaturated phosphonate enantiomers of FTY720, (R)- and (S)-FTY720-vinylphosphonate. (R)-FTY720-vinylphosphonate was a full agonist of S1P1 (EC50 20 ± 3 nM). In contrast, the (S) enantiomer failed to activate any of the five S1P GPCRs and was a full antagonist of S1P1,3,4 (Ki 384 nM, 39 nM, and 1190 nM, respectively) and a partial antagonist of S1P2, and S1P5. Both enantiomers dose-dependently inhibited lysophospholipase D (recombinant autotaxin) with Ki values in the low micromolar range, although with different enzyme kinetic mechanisms. When injected into mice, both enantiomers caused transient peripheral lymphopenia. (R)- and (S)-FTY720-vinylphosphonates activated ERK1/2, AKT, and exerted an antiapoptotic effect in camptothecin-treated IEC-6 intestinal epithelial cells, which primarily express S1P2 transcripts and traces of S1P5. (S)-FTY720-vinylphosphonate is the first pan-antagonist of S1P receptors and offers utility in probing S1P responses in vitro and in vivo. The biological effects of the (R)- and (S)-FTY720-vinylphosphonate analogues underscore the complexity of FTY720 cellular targets.
FTY720; sphingosine 1-phosphate; lysophosphatidic acid; autotaxin; lysophospholipase D; lymphocyte egress; EDG receptor; inhibitor
Background & Aims
We recently identified lysophosphatidic acid (LPA) as a potent antiapoptotic agent for the intestinal epithelium. The objective of the present study was to evaluate the effect of octadecenyl thiophosphate (OTP), a novel rationally designed, metabolically stabilized LPA mimic, on radiation-induced apoptosis of intestinal epithelial cells in vitro and in vivo
The receptors and signaling pathways activated by OTP were examined in IEC-6 and RH7777 cell lines and wild-type and LPA1 and LPA2 knockout mice exposed to different apoptotic stimuli
OTP was more efficacious than LPA in reducing gamma irradiation–, camptothecin-, or tumor necrosis factor α/cycloheximide–induced apoptosis and caspase-3-8, and caspase-9 activity in the IEC-6 cell line. In RH7777 cells lacking LPA receptors, OTP selectively protected LPA2 but not LPA1 and LPA3 transfectants. In C57BL/6 and LPA1 knockout mice exposed to 15 Gy gamma irradiation, orally applied OTP reduced the number of apoptotic bodies and activated caspase-3–positive cells but was ineffective in LPA2 knockout mice. OTP, with higher efficacy than LPA, enhanced intestinal crypt survival in C57BL/6 mice but was without any effect in LPA2 knockout mice. Intraperitoneally administered OTP reduced death caused by lethal dose (LD)100/30 radiation by 50%.
Our data indicate that OTP is a highly effective antiapoptotic agent that engages similar prosurvival pathways to LPA through the LPA2 receptor subtype.
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, disregulated LPA production and function have been linked with cancer and cardiovascular disease. RxBio, Inc., have generated several series of LPA analogs with varied agonist/antagonist function at the LPA1–3 G protein-coupled receptor 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 two hours after oral dosing.
lysophosphatidic acid; radioprotection; chemoprotection; cancer; LPA2 receptor
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.
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.
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.
Cyclic phosphatidic acid (CPA) is a naturally occurring analog of lysophosphatidic acid (LPA) in which the sn-2 hydroxy group forms a 5-membered ring with the sn-3 phosphate. Here we describe the synthesis of R-3-CCPA and S-3-CCPA along with their pharmacological properties as inhibitors of lysophospholipase D/autotaxin, agonists of the LPA5 GPCR, and blockers of lung metastasis of B16-F10 melanoma cells in a C57BL/6 mouse model. S-3CCPA was significantly more efficacious in the activation of LPA5 compared to the R stereoisomer. In contrast, no stereoselective differences were found between the two isomers toward the inhibition of autotaxin or lung metastasis of B16-F10 melanoma cells in vivo. These results extend the potential utility of these compounds as potential lead compounds warranting evaluation as cancer therapeutics.
lysophosphatidic acid; NPP2; autotaxin; GPR92; lysophospholipase D
The lysophospholipase D enzyme, autotaxin (ATX), has been linked to numerous human diseases including cancer, neurophatic pain, obesity, and Alzheimer’s disease. Although the ATX protein was initially purified and characterized in 1992, a link to bioactive lipid metabolism was not made until 2002. In the past decade, metal chelators, lysophospholipid product analogs, and more recently small non-lipid inhibitors of the enzyme were successfully identified. The majority of these inhibitors have been characterized using recombinant purified ATX in vitro, with very few examples studied in more complex systems. Translation of ATX inhibitors from the hands of medicinal chemists to clinical use will require substantially expanded characterization of ATX inhibitors in vivo.
Autotaxin; lysophosphatidic acid; lysophospholipase D; NPP2; cancer
G protein-coupled receptors (GPCRs) comprise a large class of transmembrane proteins that play critical roles in both normal physiology and pathophysiology. These critical roles offer targets for therapeutic intervention, as exemplified by the substantial fraction of current pharmaceutical agents that target members of this family. Tremendous contributions to our understanding of GPCR structure and dynamics have come from both indirect and direct structural characterization techniques. Key features of GPCR conformations derived from both types of characterization techniques are reviewed.
G protein-coupled receptor; GPCR; structure; crystallography; NMR; modeling
GPCR; crystallography; diffusible ligand; drug design
The dissociation constant for an ionizable ligand binding to a receptor is dependent on its charge and therefore on its environmentally-influenced pKa value. The pKa values of sphingosine 1-phosphate (S1P) were studied computationally in the context of the wild type S1P1 receptor and the following mutants: E3.29Q, E3.29A, and K5.38A. Calculated pKa values indicate that S1P binds to S1P1 and its site mutants with a total charge of −1, including a +1 charge on the ammonium group and a −2 charge on the phosphate group. The dissociation constant of S1P binding to these receptors was studied as well. The models of wild type and mutant proteins originated from an active receptor model that was developed previously. We used ab initio RHF/6–31+G(d) to optimize our models in aqueous solution, where the solvation energy derivatives are represented by conductor-like polarizable continuum model (C-PCM) and integral equation formalism polarizable continuum model (IEF-PCM). Calculation of the dissociation constant for each mutant was determined by reference to the experimental dissociation constant of the wild type receptor. The computed dissociation constants of the E3.29Q and E3.29A mutants are 3–5 orders of magnitude higher than those for the wild type receptor and K5.38A mutant, indicating vital contacts between the S1P phosphate group and the carboxylate group of E3.29. Computational dissociation constants for K5.38A, E3.29A and E3.29Q mutants were compared with experimentally determined binding and activation data. No measurable binding of S1P to the E3.29A and E3.29Q mutants was observed, supporting the critical contacts observed computationally. These results validate the quantitative accuracy of the model.
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.
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]GTPγS 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.
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.