A single amino acid determines lysophospholipid specificity of the S1P1 (EDG1) and LPA1 (EDG2) phospholipid growth factor receptors.

The phospholipid growth factors sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA) are ligands for the related G protein-coupled receptors S1P(1)/EDG1 and LPA(1)/EDG2, respectively. We have developed a model of LPA(1) that predicts interactions between three polar residues and LPA. One of these, glutamine 125, which is conserved in the LPA receptor subfamily (LPA(1)/EDG2, LPA(2)/EDG4, and LPA(3)/EDG7), hydrogen bonds with the LPA hydroxyl group. Our previous S1P(1) study identified that the corresponding glutamate residue, conserved in all S1P receptors, ion pairs with the S1P ammonium. These two results predict that this residue might influence ligand recognition and specificity. Characterization of glutamate/glutamine interchange point mutants of S1P(1) and LPA(1) validated this prediction as the presence of glutamate was required for S1P recognition, whereas LPA recognition was possible with either glutamine or glutamate. The most likely explanation for this dual specificity behavior is a shift in the equilibrium between the acid and conjugate base forms of glutamic acid due to other amino acids surrounding that position in LPA(1), producing a mixture of receptors including those having an anionic glutamate that recognize S1P and others with a neutral glutamic acid that recognize LPA. Thus, computational modeling of these receptors provided valid information necessary for understanding the molecular pharmacology of these receptors.

Sphingosylphosphocholine is a naturally occurring lipid mediator in blood plasma: a possible role in regulating cardiac function via sphingolipid receptors.

Blood plasma and serum contain factors that activate inwardly rectifying GIRK1/GIRK4 K+ channels in atrial myocytes via one or more non-atropine-sensitive receptors coupled to pertussis-toxin-sensitive G-proteins. This channel is also the target of muscarinic M(2) receptors activated by the physiological release of acetylcholine from parasympathetic nerve endings. By using a combination of HPLC and TLC techniques with matrix-assisted laser desorption ionization-time-of-flight MS, we purified and identified sphingosine 1-phosphate (SPP) and sphingosylphosphocholine (SPC) as the plasma and serum factors responsible for activating the inwardly rectifying K+ channel (I(K)). With the use of MS the concentration of SPC was estimated at 50 nM in plasma and 130 nM in serum; those concentrations exceeded the 1.5 nM EC(50) measured in guinea-pig atrial myocytes. With the use of reverse-transcriptase-mediated PCR and/or Western blot analysis, we detected Edg1, Edg3, Edg5 and Edg8 as well as OGR1 sphingolipid receptor transcripts and/or proteins. In perfused guinea-pig hearts, SPC exerted a negative chronotropic effect with a threshold concentration of 1 microM. SPC was completely removed after perfusion through the coronary circulation at a concentration of 10 microM. On the basis of their constitutive presence in plasma, the expression of specific receptors, and a mechanism of ligand inactivation, we propose that SPP and SPC might have a physiologically relevant role in the regulation of the heart.

Identification of Edg1 receptor residues that recognize sphingosine 1-phosphate.

Originating from its DNA sequence, a computational model of the Edg1 receptor has been developed that predicts critical interactions with its ligand, sphingosine 1-phosphate. The basic amino acids Arg(120) and Arg(292) ion pair with the phosphate, whereas the acidic Glu(121) residue ion pairs with the ammonium moiety of sphingosine 1-phosphate. The requirement of these interactions for specific ligand recognition has been confirmed through examination of site-directed mutants by radioligand binding, ligand-induced [(35)S]GTPgammaS binding, and receptor internalization assays. These ion-pairing interactions explain the ligand specificity of the Edg1 receptor and provide insight into ligand specificity differences within the Edg receptor family. This computational map of the ligand binding pocket provides information necessary for understanding the molecular pharmacology of this receptor, thus underlining the potential of the computational method in predicting ligand-receptor interactions.

Organization-dependent effects of toxic bivalent ions microtubule assembly and glycolysis.

The effects of bivalent ions on tubulin dynamics and the upper phase of glycolysis were investigated at different organization levels in vitro. Cu2+, Cd2+, Hg2+ and CrO4(2-) inhibit the tubulin polymerization at an IC50 of 14-24 microM with high cooperativity and also induce microtubule disassembly. The apparent binding constants of the ions to tubulin, estimated by fluorescence quenching, vary between 6 and 28 microM. BIAcore measurements for tubulin-tubulin interaction suggest that the presence of Cu2+ affects neither koff nor kon, but the amount of the bound tubulin. While the inhibitory effect of Cu2+ on tubulin polymerization is partially abolished by cross-linking of microtubules with substoichiometric amounts of phosphofructokinase or decoration of tubules with cytosolic proteins, in the presence of kinase but not with cytosolic proteins the tubules are resistant to CrO4(2-). No inhibitory effect of Cu2+ or CrO4(2-) on microtubule assembly was detected in the MAP-containing cytosolic fraction. Electron microscopy revealed that tubules assembled in the presence of Cu2+ or CrO4(2-) ions contain aggregates of thread-like oligomers that are less conspicuous in the presence of cytosolic proteins. Cu2+, Cd2+, and Hg2+ inhibit the glycolytic flux in the cytosolic fraction characterized at equilibrium by an IC50 of 10-14 microM with high cooperativity. Tubulin diminishes the inhibitory effect of the cations. These data indicate that the responses elicited by the bivalent ions are highly dependent on the supramolecular organization of the systems.

Pharmacological characterization of phospholipid growth-factor receptors.

The phospholipid growth-factor (PLGE) terminology is proposed to describe a group of endogenous glycerol- and sphingolipid mediators that regulate cell proliferation through plasma membrane receptors. In addition to LPA and SPP, multiple PLGFs are present in blood plasma and serum. PLGF activity is regulated by its stimulus-coupled production and by endogenous inhibitors. In addition to LPA and SPP, alkenyl-glycerophosphate, cyclic-phosphatidic acid, and sphingosylphosphorylcholine were detected in biological fluids using mass spectrometry. Heterologous desensitization studies indicate the expression of multiple LPA-activated receptors in a variety of cell types, which are differentially activated by the different PLGFs. Northern blot and RT-PCR results reinforce the coexpression of PSP24 alpha and different members of the EDG1-7 receptors in the same cell. Stable heterologous expression of the PSP24 alpha, EDG2, and EDG4 receptors in HEK293 cells show distinct PLGF specificities and dose-response properties for each receptor subtype. Thus, both the controlled availability of the different agonists/inhibitors and the regulated expression of their receptors regulate the biological effects of PLGFs.

A new potent calmodulin antagonist with arylalkylamine structure: crystallographic, spectroscopic and functional studies.

An arylalkylamine-type calmodulin antagonist, N-(3, 3-diphenylpropyl)-N'-[1-R-(3, 4-bis-butoxyphenyl)ethyl]-propylene-diamine (AAA) is presented and its complexes with calmodulin are characterized in solution and in the crystal. Near-UV circular dichroism spectra show that AAA binds to calmodulin with 2:1 stoichiometry in a Ca(2+)-dependent manner. The crystal structure with 2:1 stoichiometry is determined to 2.64 A resolution. The binding of AAA causes domain closure of calmodulin similar to that obtained with trifluoperazine. Solution and crystal data indicate that each of the two AAA molecules anchors in the hydrophobic pockets of calmodulin, overlapping with two trifluoperazine sites, i.e. at a hydrophobic pocket and an interdomain site. The two AAA molecules also interact with each other by hydrophobic forces. A competition enzymatic assay has revealed that AAA inhibits calmodulin-activated phosphodiesterase activity at two orders of magnitude lower concentration than trifluoperazine. The apparent dissociation constant of AAA to calmodulin is 18 nM, which is commensurable with that of target peptides. On the basis of the crystal structure, we propose that the high-affinity binding is mainly due to a favorable entropy term, as the AAA molecule makes multiple contacts in its complex with calmodulin.

Enhanced association of mutant triosephosphate isomerase to red cell membranes and to brain microtubules.

In a Hungarian family with triosephosphate isomerase (TPI; D-glyceraldehyde-3-phosphate keto-isomerase, EC 5.3.1.1) deficiency, two germ-line identical, but phenotypically differing compound heterozygote brothers (one of them with neurological disorder) have been identified with the same very low (<5%) TPI activity and 20- or 40-fold higher erythrocyte dihydroxyacetone phosphate levels as compared with normal controls. Our present studies with purified TPI and hemolysates revealed the binding of TPI, and the binding of human wild-type and mutant TPIs in hemolysate, to the red cell membrane, and the interference of binding with other hemolysate proteins. The binding of the mutant TPI is enhanced as compared with the wild-type enzyme. The increased binding is influenced by both the altered structure of the mutant and the changes in the red cell membrane. Compared with binding of glyceraldehyde-3-phosphate dehydrogenase, the isomerase binding is much less sensitive to ionic strength or blocking of the N-terminal tail of the band-3 transmembrane protein. The binding of TPIs to the membrane decreases the isomerase activity, resulting in extremely high dihydroxyacetone phosphate levels in deficient cells. In cell-free brain extract, tubulin copolymerizes with TPI and with other cytosolic proteins forming highly decorated microtubules as shown by immunoblot analysis with anti-TPI antibody and by electron microscopic images. The efficacy order of TPI binding to microtubules is propositus > brother without neurological disorder > normal control. This distinct microcompartmentation of mutant proteins may be relevant in the development of the neurodegenerative process in TPI deficiency and in other, more common neurological diseases.

Combined enhancement of microtubule assembly and glucose metabolism in neuronal systems in vitro: decreased sensitivity to copper toxicity.

Brain cell-free extract greatly stimulates the polymerization rate of purified tubulin with a reduction of the nucleation period and without a significant alteration of the final assembly state. This effect is mimicked by neuroblastoma extract at 10-fold lower extract concentration, but not by excess muscle extract. Copper inhibits microtubule assembly in vitro but in the presence of brain extract the copper effect is suspended. Electron microscopic images showed that intact microtubules are formed and decorated by cytosolic proteins in the absence and presence of copper, while the copper alone induces the formation of S-shaped sheets and oligomeric threads. The flux of triosephosphate formation from glucose is enhanced by microtubules in brain extract, but not in muscle extract. Copper inhibits the glycolytic flux; however, the presence of microtubules not only suspends the inhibition by copper but the activation of glycolysis by microtubules is also preserved. We conclude that the organization of neuronal proteins modifies both the rates of microtubule assembly and glycolysis, and reduces their sensitivities against the inhibition caused by copper.

Naturally occurring analogs of lysophosphatidic acid elicit different cellular responses through selective activation of multiple receptor subtypes.

Lysophosphatidic acid (LPA), plasmalogen-glycerophosphate (alkenyl-GP) and, cyclic-phosphatidic acid (cyclic-PA) are naturally occurring phospholipid growth factors (PLGFs). PLGFs elicit diverse biological effects via the activation of G protein-coupled receptors in a variety of cell types. In NIH3T3 fibroblasts, LPA and alkenyl-GP both induced proliferation, whereas cyclic-PA was antiproliferative. LPA and alkenyl-GP decreased cAMP in a pertussis toxin-sensitive manner, whereas cyclic-PA caused cAMP to increase. LPA and alkenyl-GP both stimulated the activity of the mitogen-actived protein kinases extracellular signal regulated kinases 1 and 2 and c-Jun NH2-terminal kinase, whereas cyclic-PA did not. All three PLGFs induced the formation of stress fibers in NIH3T3 fibroblasts. To determine whether these lipids activated the same or different receptors, heterologous desensitization patterns were established among the three PLGFs by monitoring changes in intracellular Ca2+ in NIH3T3 fibroblasts. LPA cross-desensitized both the alkenyl-GP and cyclic-PA responses. Alkenyl-GP cross-desensitized the cyclic-PA response, but only partially desensitized the LPA response. Cyclic-PA only partially desensitized both the alkenyl-GP and LPA responses. We propose that pharmacologically distinct subsets of PLGF receptors exist that distinguish between cyclic-PA and alkenyl-GP, but are all activated by LPA. We provide evidence that the PSP24 receptor is selective for LPA and not activated by the other two PLGFs. RT-PCR and Northern blot analysis indicate the co-expression of mRNAs encoding the EDG-2, EDG-4, and PSP24 receptors in a variety of cell lines and tissues. However, the lack of mRNA expression for these three receptors in the LPA-responsive Rat-1 and Sp2-O-Ag14 cells suggests that a number of PLGF receptor subtypes remain unidentified.

Identification of a novel growth factor-like lipid, 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) that is present in commercial sphingolipid preparations.

Lysophosphatidic acid, a member of the acidic phospholipid autacoid (APA) family of lipid mediators, elicits diverse cellular effects that range from mitogenesis to the prevention of programmed cell death. Sphingosine 1-phosphate and sphingosylphosphorylcholine have also been proposed to be ligands of the APA receptors. However, key observations that provide the foundation of this hypothesis have not been universally reproducible, leading to a controversy in the field. We provide evidence that 1-O-cis-alk-1'-enyl-2-lyso-sn-glycero-3-phosphate (alkenyl-GP) is present in some commercial sphingolipid preparations and is responsible for many of their APA-like effects, which were previously attributed to sphingosylphosphorylcholine. Alkenyl-GP was generated by acidic and basic methanolysis from ethanolamine lysoplasmalogen, which was present in the sphingomyelin fraction that is used to manufacture sphingosylphosphorylcholine. We present the structural identification of alkenyl-GP, using 1H and 13C NMR, Fourier transform infrared spectrometry, and mass spectrometry. Alkenyl-GP was a potent activator of the mitogen-activated protein kinases ERK1/2 and elicited a mitogenic response in Swiss 3T3 fibroblasts. In contrast, sphingosylphosphorylcholine at a concentration of 10 microM was only a weak mitogen and only weakly activated the extracellular signal-regulated protein kinases. Alkenyl-GP has recently been detected as an injury-induced component in the anterior chamber of the eye (Liliom, K., Guan, Z., Tseng, H., Desiderio, D. M., Tigyi, G., and Watsky, M. (1998) Am. J. Physiol. 274, C1065-C1074), indicating that this lipid is a naturally occurring member of the APA mediator family.

Growth factor-like phospholipids generated after corneal injury.

The present study provides evidence that growth factor-like glycerophosphate mediators of the lysophosphatidic acid (LPA) family are present in the aqueous humor and the lacrimal gland fluid of the rabbit eye. By use of a combination of HPLC, two-dimensional TLC, mass spectrometry, and the Xenopus oocyte bioassay, the LPA-like phospholipids LPA, cyclic PA, alkenyl-glycerophosphate (GP), lysophosphatidylserine, and phosphatidic acid were detected as physiological constituents of the fluids bathing the cornea. Corneal injury resulted in an increased production of some of these mediators. Alkenyl-GP, a novel member of the LPA family, has been identified in postinjury aqueous humor, establishing that it is generated endogenously. LPA and its homologues were found to be mitogenic in freshly dissociated keratocytes from uninjured corneas. There appears to be a link between the occurrence of LPA responsiveness in keratocytes activated by injury and the increase in LPA-like activity in aqueous humor. These data suggest that LPA and its homologues are involved in maintaining the integrity of the normal cornea and in promoting cellular regeneration of the injured cornea.

Role of lysophosphatidic acid in endothelin-1- and hematoma-induced alteration of cerebral microcirculation.

Cerebral hematoma increases cerebrospinal fluid (CSF) endothelin-1 (ET-1). Inhibitors of ET-1 synthesis prevent this increment and hematoma-induced modification of cerebral arteriolar reactivity. We hypothesized that intrathecal ET-1 injection could 1) modify pial arteriolar reactivity similarly to hematoma; 2) increase CSF lysophosphatidic acid (LPA), a potential contributor to altered cerebrovascular reactivity; and 3) reduce the level of adenosine 3',5'-cyclic monophosphate (cAMP) in the CSF. Either ET-1 (10(-7) M) or artificial CSF was injected over the left parietal cortex of newborn pigs. Four days later, cranial windows were implanted. CSF ET was increased from a basal level of 11 fmol/ml to 18 fmol/ml 4 days after ET-1 injection, whereas CSF cAMP was reduced from 2,700 to 950 fmol/ml. The mean diameter of pial arterioles was reduced 31%. In control animals, 10(-12) M ET caused dilation, and higher concentrations induced vasoconstriction. Four days after ET-1 injection topical ET-1 caused constriction instead of dilation at 10(-12) M, and constrictions at higher doses were enhanced. Norepinephrine-induced constrictions were potentiated in the ET-1-injected group. Dilations to cAMP-dependent (but not independent) vasodilators were attenuated after ET-1. The concentration of the vasoconstrictor lipid mediator LPA increased approximately fourfold. Thus intrathecal injection of ET-1 mimics hematoma-induced modification of cerebral vascular reactivity and increase in LPA production. The mechanism(s) of ET-1- and hematoma-induced modifications may involve LPA, which is known to contribute to the loss of dilator responses by inhibition of cAMP product on. The present study further suggests that ET-1 together with LPA could be causing changes in cerebrovascular reactivity following cerebral hemorrhage. ET-1 stimulates the release of LPA from brain parenchyma independent of serum so that LPA could serve as a secondary mediator.

Local anesthetics inhibit receptors coupled to phosphoinositide signaling in Xenopus oocytes.

Effects and the mechanism of action of quaternary amine local anesthetics on ligand- and voltage-activated ion currents were studied using voltage-clamped ovarian follicles and oocytes from Xenopus laevis. The fast inward and slow outward currents in response to acetylcholine were unaltered by procaine, whereas the oscillatory and smooth inward chloride currents (ICl) were abolished. Potassium currents (IK) elicited by norepinephrine and oscillatory ICl elicited by lysophosphatidic acid were blocked. Procaine caused a noncompetitive inhibition of oscillatory ICl mediated by heterologously expressed neurotransmitter receptors from the rat brain. Threefold differences were found in the procaine sensitivity of the 5-HT2a and 5-HT2c receptors. The rank order of intrinsic inhibitory activity of local anesthetics was: procaine > lidocaine > dibucaine > tetracaine. Extra- or intracellular application of procaine did not alter the Ca2+-activated Cl- current, indicating that neither the endogenous voltage-gated Ca2+ nor the Ca2+-activated Cl- channels account for the inhibition. Procaine caused only a slight reduction in ICl elicited by photolysis of caged inositol 1,4,5-trisphosphate (InsP3) and did not abolish ICl triggered by GTP[gamma-S]-induced direct activation of G proteins. For receptors coupling to the phosphoinositide/Ca2+ signal transduction pathway, the primary and physiologically relevant site of procaine action appears to be on the extracellular surface, upstream from the G protein, presumably on the receptor.

Molecular cloning of a high-affinity receptor for the growth factor-like lipid mediator lysophosphatidic acid from Xenopus oocytes.

Lysophosphatidic acid (1-acyl-2-lyso-snglycero-3-phosphate, LPA) is a multifunctional lipid mediator found in a variety of organisms that span the phylogenetic tree from humans to plants. Although its physiological function is not clearly understood, LPA is a potent regulator of mammalian cell proliferation; it is one of the major mitogens found in blood serum. In Xenopus laevis oocytes, LPA elicits oscillatory Cl- currents. This current, like other effects of LPA, is consistent with a plasma membrane receptor-mediated activation of G protein-linked signal transduction pathways. Herein we report the identification of a complementary DNA from Xenopus that encodes a functional high-affinity LPA receptor. The predicted structure of this protein of 372 amino acids contains features common to members of the seven transmembrane receptor superfamily with a predicted extracellular amino and intracellular carboxyl terminus. An antisense oligonucleotide derived from the first 5-11 predicted amino acids, selectively inhibited the expression of the endogenous high-affinity LPA receptors in Xenopus oocytes, whereas the same oligonucleotide did not affect the low-affinity LPA receptor. Expression of the full-length cRNA in oocytes led to an increase in maximal Cl- current due to increased expression of the high-affinity LPA receptor, but activation of the low-affinity receptor was, again, unaffected. Oocytes expressing cRNA prepared from this clone showed no response to other lipid mediators including prostaglandins, leukotrienes, sphingosine 1-phosphate, sphingosylphosphorylcholine, and platelet-activating factor, suggesting that the receptor is highly selective for LPA.

A novel membrane receptor with high affinity for lysosphingomyelin and sphingosine 1-phosphate in atrial myocytes.

Activation of IK(ACh) is the major effect of the vagal neutrotransmitter acetylcholine in the heart. We report that both lysosphingomyelin (D-erythro-sphingosyl-phosphorylcholine; SPC) and sphingosine 1-phosphate (SPP) activate IK(ACh) in guinea pig atrial myocytes through the same receptor with an EC50 of 1.5 and 1.2 nM, respectively. Pertussis toxin abolished the activation of IK(ACh) by either lipid. The putative receptor showed an exquisite stereoselectivity for the naturally occurring D-erythro-(2S,3R)-SPC stereoisomer, the structure of which was confirmed by mass spectroscopy and NMR. These lipids caused complete homologous and heterologous desensitization with each other but not with ACh, indicating that both act on the same receptor. This receptor displays a distinct structure-activity relationship: it requires an unsubstituted amino group because N-acetyl-SPC, lysophosphatidic acid and lysophosphatidylcholine were inactive. Because SPP and SPC are naturally occurring products of membrane lipid metabolism, it appears that these compounds might be important extracellular mediators acting on a family of bona fide G protein-coupled receptors. Expression of these receptors in the heart raises the possibility that sphingolipids may be a part of the physiological and/or pathophysiological regulation of the heart. Based on their ligand selectivity we propose a classification of the sphingolipid receptors.

N-palmitoyl-serine and N-palmitoyl-tyrosine phosphoric acids are selective competitive antagonists of the lysophosphatidic acid receptors.

Lysophosphatidic acid is the best characterized member of a lipid mediator family with growth factor-like activities that act through a class of G protein-coupled plasma membrane receptors. In Xenopus laevis oocytes, lysophosphatidate activates at least two pharmacologically distinct receptor subtypes distinguished by 1-acyl-sn-glycero-2,3-cyclic phosphate. Both of these naturally occurring ligands elicit oscillatory Cl- currents in the oocyte through G protein-coupled activation of the phosphoinositide/Ca2+ second messenger system, which in turn leads to the opening of Ca(2+)-activated Cl- channels. We developed an improved chemical synthesis and purification procedure for two N-acylated amino acid phosphates. N-Palmitoyl-serine and N-palmitoyl-tyrosine phosphoric acids inhibited the lysophosphatidate-activated Cl- currents with IC50 values of 5.4 +/- 0.7 and 6.5 +/- 1.5 nM at the high affinity site and 805 +/- 97 and 172 +/- 36 nM at the low affinity receptor site, respectively. In selective activation of the cyclic lysophosphatidate receptor, IC50 values of 330 +/- 30 and 490 +/- 40 nM were obtained, respectively. The D- and L-stereoisomers were equally effective when applied extracellularly. In contrast, they were ineffective when microinjected into the oocyte, indicating an extracellular site of inhibition. The inhibitors did not alter currents elicited by the different acetylcholine, serotonin, and glutamate receptors expressed heterologously in the oocyte. Pharmacological analysis of the results indicates that N-palmitoyl-serine and N-palmitoyl-tyrosine phosphoric acids are potent and specific competitive inhibitors of the lysophosphatidate receptors in the X. laevis oocyte.

Xenopus oocytes express multiple receptors for LPA-like lipid mediators.

In Xenopus laevis oocytes, both lysophosphatidic acid (LPA) and a cyclic phosphate-containing analogue 1-acyl-sn-glycero-2,3-cyclic phosphate (cLPA) isolated from Physarum polycephalum activated oscillatory Cl- currents. cLPA elicited oscillatory currents only when applied extracellularly and, similarly to LPA, evoked homologous desensitization. cLPA applied to oocytes previously desensitized b y LPA failed to elicit a current, indicating that LPA completely desensitized the cLPA receptors. In contrast, when oocytes were desensitized by cLPA, LPA still evoked large currents. The lack of heterologous desensitization between cLPA and LPA indicates that the former acts on a distinct receptor subpopulation(s), which is also activated by LPA. The alkyl-ether analogue 1-hexadecyl-2-lyso-sn-glycero-3-phosphate (16:0-GP) and dioleoyl-phosphatidic acid (18:1-PA) showed heterologous desensitization patterns similar to that of LPA with regard to cLPA. Complete heterologous desensitization was obtained between LPA and 16:0-GP or 18:1-PA. These observations demonstrate the simultaneous expression of at least two different types of receptors for LPA-like lipid mediators on Xenopus oocytes and that these receptors show different pharmacological properties in their selectivity to cLPA.

Inhibitors of lipid phosphatidate receptors: N-palmitoyl-serine and N-palmitoyl-tyrosine phosphoric acids.

An improved synthesis of two lipid phosphoric acids, N-palmitoyl-L-serine phosphoric acid (NP-Ser-PA) and N-palmitoyl-L-tyrosine phosphoric acid (NP-Tyr-PA), from the benzyl esters of L-serine and L-tyrosine is described. The sequence of N-acylation, followed by phosphitylation with N, N-diisopropyl dibenzyl phosphoramidite, oxidation to the corresponding phosphate triesters, and simultaneous debenzylation of the dibenzyl phosphate and benzyl carboxylic esters gave NP-Ser-PA and NP-Tyr-PA in high overall yields. NP-Ser-PA and NP-Tyr-PA and their D stereoisomers were potent reversible inhibitors of the lysophosphatidic acid receptors expressed in Xenopus oocytes, thus providing prototypic structures for the development of inhibitors of the lysophosphatidate family of phospholipid growth factors.

Anti-calmodulin potency of indol alkaloids in in vitro systems.

We have demonstrated that bis-indol Vinca alkaloids of anti-mitotic activities (vinblastine, vincristine, and navelbine) bind to calmodulin in a Ca(2+)-dependent manner. We designed direct binding tests (fluorescence energy transfer and circular dichroism measurements) to quantify the interactions of bis-indol derivatives with calmodulin. The dissociation constants of calmodulin-navelbine and calmodulin-vinblastine complexes with 1:1 stoichiometry are 0.5 microM and 3 microM, respectively. These values indicate that the binding affinities of these Vinca alkaloids to calmodulin and tubulin are comparable. Immunological, enzyme kinetic and fluorescence anisotropy measurements showed that bis-indol alkaloids inhibit the interactions of calmodulin with target proteins. The results of indirect enzyme-linked immunosorbent assay showed that bis-indol alkaloids effectively antagonize with anti-calmodulin antibody for calmodulin binding (IC50 = 90 microM, 400 microM, and 430 microM for navelbine, vincristine and vinblastine, respectively). According to the fluorescence anisotropy and enzyme kinetic measurements, vinblastine, vincristine and vinblastine, similarly to trifluoperazine, the classic calmodulin antagonist, compete with target enzyme [phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11)] for an inhibitory effect either on immunocomplex formation or on calmodulin-enzyme interaction. Navelbine appeared in our tests as the most potent drug in inhibiting the association of calmodulin to target proteins in comparison to other bis-indol derivatives. Since navelbine and vinblastine possess identical vindoline moiety, although they differ in the catharantine part, the difference in anti-calmodulin potencies is suggested to reside predominantly on this portion of the molecules. These findings might establish the pharmacological importance of these activities in the specificity and toxicity of the drugs.