Small peptides as potent mimetics of the protein hormone erythropoietin.

Random phage display peptide libraries and affinity selective methods were used to isolate small peptides that bind to and activate the receptor for the cytokine erythropoietin (EPO). In a panel of in vitro biological assays, the peptides act as full agonists and they can also stimulate erythropoiesis in mice. These agonists are represented by a 14- amino acid disulfide-bonded, cyclic peptide with the minimum consensus sequence YXCXXGPXTWXCXP, where X represents positions allowing occupation by several amino acids. The amino acid sequences of these peptides are not found in the primary sequence of EPO. The signaling pathways activated by these peptides appear to be identical to those induced by the natural ligand. This discovery may form the basis for the design of small molecule mimetics of EPO.

Peptide agonist of the thrombopoietin receptor as potent as the natural cytokine.

Two families of small peptides that bind to the human thrombopoietin receptor and compete with the binding of the natural ligand thrombopoietin (TPO) were identified from recombinant peptide libraries. The sequences of these peptides were not found in the primary sequence of TPO. Screening libraries of variants of one of these families under affinity-selective conditions yielded a 14-amino acid peptide (Ile-Glu-Gly-Pro-Thr-Leu-Arg-Gln-Trp-Leu-Ala-Ala-Arg-Ala) with high affinity (dissociation constant approximately 2 nanomolar) that stimulates the proliferation of a TPO-responsive Ba/F3 cell line with a median effective concentration (EC50) of 400 nanomolar. Dimerization of this peptide by a carboxyl-terminal linkage to a lysine branch produced a compound with an EC50 of 100 picomolar, which was equipotent to the 332-amino acid natural cytokine in cell-based assays. The peptide dimer also stimulated the in vitro proliferation and maturation of megakaryocytes from human bone marrow cells and promoted an increase in platelet count when administered to normal mice.

Increased potency of an erythropoietin peptide mimetic through covalent dimerization.

We have synthesized a chemically defined, dimeric form of an erythropoietin mimetic peptide (EMP) that displays 100-fold increased affinity for the erythropoietin receptor (EPOR) and correspondingly elevated potency in cell-based assays and in mice. The dimeric EMP1 was synthesized using a C-terminal lysine residue as a branch point. A beta-alanine residue was coupled to the main-chain (alpha) amino group of the lysine residue in order to provide a pseudosymmetrical scaffold where both the side-chain and main-chain were of approximately equal length. Using an orthogonal protection system, independently disulphide-cylized EMP1 moieties were synthesized upon this scaffold. The proposed mechanism of increased potency of the dimer over the parental compound EMP1 is consistent with the structure of a cocrystal of EMP1 and the extracellular domain of the EPOR in which a noncovalent peptide dimer is seen spanning the cleft between two molecules of the EPOR extracellular domain.

A chimeric D2 dopamine/m1 muscarinic receptor with D2 binding specificity mobilizes intracellular calcium in response to dopamine.

Using PCR methodology, a chimeric receptor cDNA was constructed in which the entire third cytoplasmic loop of the human D2 dopamine receptor was replaced by the analogous portion of the human m1 muscarinic receptor. When expressed in CHO cells, the chimeric D2/m1 receptor bound dopaminergic ligands with affinities similar to the native D2(414) receptor. Intracellular calcium levels (measured with Fura-2) were not altered when CHO cells expressing the D2(414) receptor were exposed to dopamine. In contrast, dopamine elevated intracellular calcium levels in cells expressing the D2/m1 chimeric receptor in a dose-dependent manner which was blocked by the D2 antagonist, fluphenazine. The ability to construct G-protein-linked receptor chimeras which mobilize calcium with nearly unaltered pharmacologic specificity raises the possibility of a generic strategy for creating non-radioisotopic reporter systems for use in drug discovery.

Crystals of soluble interleukin-1 receptor complexed with its natural antagonist reveal a 1:1 receptor-ligand complex.

Interleukin-1 is a cytokine involved in the acute phase response against infection and injury. We obtained crystals of a complex of soluble, recombinant human interleukin-1 receptor and recombinant human interleukin-1 receptor antagonist, a naturally occurring antagonist. The crystals are suitable for X-ray analysis and diffract to 2.7 A resolution. Solvent content calculations indicate that the crystals contain one receptor and one antagonist molecule per asymmetric unit. Other receptor to antagonist ratios are highly unlikely. These results suggest that the interleukin-1 antagonist binds a single receptor molecule and does not cause receptor aggregation.

A monoclonal antibody specific for a dynorphin precursor.

Murine monoclonal antibodies were produced against dynorphin-32 (D32), a naturally occurring opioid peptide containing the sequence of dynorphin A (Dyn A) at its amino terminus, followed by Lys-Arg, then by the sequence of dynorphin B (Dyn B). Initial characterization using an enzyme-linked immunosorbent assay revealed that one clone (17.M) recognized D32 but not Dyn A or Dyn B. A solid-phase radioimmunoassay using 125I-labeled antibody 17.M was developed and showed a maximum sensitivity of 45 fmol for synthetic D32. Dyn B crossreacted only 0.05%; Dyn A and other related opioid peptides crossreacted less than 0.001%. Immunoreactive D32 was detected in extracts of rat brain and anterior pituitary but not neurointermediate lobe of pituitary. The unique specificity of monoclonal antibody 17.M should make it useful in investigations of the differential localization and release of Dyn A and Dyn B and the precursor D32.

Multidimensional analysis of ligand binding data: application to opioid receptors.

The existence of distinct mu and delta opioid receptors is now well accepted. Most investigators favor the hypothesis that these receptors are physically distinct and that the enkephalins are only 2-10 fold selective for the delta receptor. Rothman and Westfall (Mol. Pharmacol. 21:548-557) recently challenged this hypothesis, proposing that at least some population of mu and delta receptors coexist in an opioid receptor complex and that the enkephalins are at least 100 fold selective for the delta receptor. In this paper we describe a generally applicable method we have used to design and analyze ligand binding experiments which distinguish between the two different models.

Biochemical evidence for somatostatin receptors in murine neuroblastoma clone N1E-115.

Radioligand binding and functional assays were employed to demonstrate the existence of somatostatin receptors in the murine neuroblastoma clone N1E-115. Saturation experiments with [125I][Tyr11]somatostatin-14 indicated the presence of a single class of binding sites in membranes prepared from N1E-115 cells (Kd = 83 pM; Bmax = 21,000 receptors/cell). Somatostatin-14, somatostatin-28 and L363586 (cyclo(N-Me-ALA-TYR-D-TRP-LYS-VAL-PHE] all displaced the 125I-ligand monophasically in N1E-115 cells (Ki values were 28, 82 and 34 pM, respectively), which contrasted with the binding heterogeneity apparent with L363586 in rat brain membranes. The binding of [125I][Tyr11]somatostatin-14 was reduced by GppNHp, indicating that N1E-115 somatostatin receptors interacted with guanine nucleotide binding protein(s). Somatostatin agonists decreased by 30-50% the levels of [3H]cyclic AMP induced in intact cells by forskolin, prostaglandin E1, or vasoactive intestinal polypeptide. The EC50 values for inhibition of the [3H]cyclic AMP response to PGE1 by L363586, somatostatin-14, and somatostatin-28 were 0.24, 0.63 and 1.0 nM, respectively. Pertussis toxin treatment of N1E-115 cells reduced both binding to the receptor and the functional response to somatostatin-14. These data suggest that a single class of somatostatin receptors in N1E-115 cells are linked to the inhibition of adenylate cyclase through a Gi protein.

The effects of receptor selective opioid peptides on morphine-induced analgesia.

Utilizing the mouse tail-flick assay, four opioid peptides, which have been reported to be selective for either mu- or delta-opioid receptors, were examined for their analgesic potency and for their ability to modify morphine-induced analgesia. [D-Ala2,D-Leu5]enkephalin and [D-Ser2,Thr6]leucine-enkephalin, putative delta-receptor selective peptides, produced a potent analgesic response and at subanalgesic doses potentiated morphine-induced analgesia. Morphiceptin and [D-Ala2,Pro5]enkephalinamide, putative mu-receptor selective peptides, were similarly found to produce analgesia. However, in contrast to the delta-receptor selective peptides, three mu-receptor selective peptides were unable to alter the potency of morphine. Thus, it would appear that the potentiation of morphine analgesia is a unique property of delta-receptor selective peptides.

Dopamine D1 receptor stimulation of cyclic AMP accumulation in COS-1 cells.

Dopamine is shown to stimulate cAMP accumulation in COS-1 cells via endogenously expressed dopamine D1 receptors. A dissociation of dopamine and beta-adrenoceptor responses is demonstrated by the use of selective antagonists and different desensitization patterns following exposure of the cells to dopamine or the beta-adrenoceptor agonist, isoproterenol. The dopamine response in COS-1 cells exhibits a pharmacological profile similar to that found in dopamine D1 tissues such as rat striatum and fish retina. The presence of DARPP-32 (dopamine- and cAMP-regulated phosphoprotein, Mr 32,000) immunoreactivity in COS-1 cells is shown by Western blotting and is consistent with the endogenous expression of a dopamine D1 receptor in these cells. It is concluded that a dopamine D1 receptor is expressed in COS-1 cells and the implications of this are discussed.

Evaluation of the interactions of mu and delta selective ligands with [3H]D-Ala2-D-Leu5-enkephalin binding to mouse brain membranes.

The interactions of putative mu and delta selective ligands with [3H]D-ala2-D-leu5 enkephalin (DADLE) binding to mouse brain membranes were investigated. Computerized curve fitting of displacement curves performed at three different concentrations of 3H-DADLE indicated that a one site competitive model was sufficient to explain the interactions of leu-enkephalin (LE) and D-ser2-thr6-leucine enkephalin with 3H-DADLE binding. Similar experiments with morphine and morphiceptin were unique in that the multiple displacement curves crossed over one another. A two-site competitive model was required to adequately describe the interactions of these mu selective ligands with 3H-DADLE. This two-site model was one in which the inhibitor had higher affinity for the site labeled with lower affinity by 3H-DADLE. However, this two site model did not correctly predict the interaction of LE with 3H-DADLE in the presence of morphiceptin. These data indicate that: 1) putative mu and delta selective ligands do not bind to a common high affinity site; 2) mu selective ligands are not simple mixed inhibitors of a single site labeled by 3H-DADLE; and 3) competitive binding models may not explain the interaction of mu ligands with 3H-DADLE binding.

A generic method for expression and use of "tagged" soluble versions of cell surface receptors.

A general method for expression, purification, immobilization, detection and radiolabeling of extracellular domains (ECD) of type I membrane proteins. The type I interleukin-1 receptor (IL-1RtI), the alpha-subunit of interleukin-2 receptor (IL-2R alpha) and E-selectin are used as illustrative examples of cell surface receptors. DNA encoding the ECD of the proteins are fused at their 3' end to a chimeric DNA which serves to generically "tag" the recombinant ECD. The resulting fusion protein contains a substrate sequence for protein kinase-A (PKA) adjacent to the signal sequence from human placental alkaline phosphatase (HPAP), The HPAP signal sequence directs the formation of the phosphatidylinositol-glycan (PI-G) anchorage of the protein at the cell surface. When these chimeric genes are expressed in CHO cells, the ECDs are detected on the cell surface and can be released by treatment with phosphatidylinositol-specific phospholipase-C (PI-PLC). Based on protein processing known to occur for native HPAP, twenty amino acids from the HPAP signal sequence remain at the C-terminus of the ECD. A high affinity monoclonal antibody was generated against this common epitope. This antibody can be used to detect, purify and immobilize the ECDs. In addition, the ECDs can be radiolabeled with 32P by treatment with PKA and maintain the ability to bind their natural ligands. This "tagging" method has been successfully applied to many other type I proteins which serve as cell surface receptors.

Randomized, double-blind, placebo-controlled study of XP13512/GSK1838262 in patients with RLS.

OBJECTIVE: To assess the efficacy and tolerability of the nondopaminergic agent XP13512/GSK1838262 in adults with moderate to severe primary restless legs syndrome (RLS). METHODS: Patient Improvements in Vital Outcomes following Treatment in Restless Legs Syndrome I was a 12-week, multicenter, randomized, double-blind, placebo-controlled trial of XP13512 1,200 mg or placebo taken once daily at 5:00 pm with food. Coprimary endpoints were mean change from baseline International Restless Legs Scale (IRLS) total score and proportion of investigator-rated responders (very much improved or much improved on the Clinical Global Impression-Improvement scale) at week 12 (last observation carried forward). Tolerability was assessed using adverse events, vital signs, and clinical laboratory parameters. RESULTS: A total of 222 patients were randomized (XP13512 = 114, placebo = 108) and 192 patients (XP13512 = 100, placebo = 92) completed the study. At week 12, the mean change from baseline IRLS total score was greater with XP13512 (-13.2) compared with placebo (-8.8). Analysis of covariance, adjusted for baseline score and pooled site, demonstrated a mean treatment difference of -4.0 (95% confidence interval [CI], -6.2 to -1.9; p = 0.0003). More patients treated with XP13512 (76.1%) were responders compared with placebo (38.9%; adjusted OR 5.1; 95% CI, 2.8 to 9.2; p < 0.0001). Significant treatment effects for both coprimary measures were identified at week 1, the earliest time point measured. The most commonly reported adverse events were somnolence (XP13512 27%, placebo 7%) and dizziness (XP13512 20%, placebo 5%), which were mild to moderate in intensity and generally remitted. CONCLUSIONS: XP13512 1,200 mg, taken once daily, significantly improved restless legs syndrome (RLS) symptoms compared with placebo and was generally well tolerated in adults with moderate to severe primary RLS.

Comparison of the filtration and centrifugation methods for assaying [3H](D-ala2, D-leu5)enkephalin binding to mouse brain membranes.

The binding of [3H](D-ala2, D-leu5)enkephalin (DADLE) to mouse brain membranes was studied via rapid filtration with GF/C filters and via the centrifugation method. The amount of specific binding determined by filtration was found to be dependent on the length of time between the initiation of vacuum and the actual time of filtration. Vacuum strength alone also significantly affected the amount of specifically bound [3H]DADLE measured by filtration. The centrifugation method increased the number of experimentally determined binding sites when compared to the filtration method. The difference in the number of binding sites was only partially attributable to passage of binding material through the GF/C filter. The ability of morphine to displace [3H]DADLE was also found to differ slightly between the two methods. These data indicate that centrifugation is a better method of determining the binding parameters of [3H]DADLE in mouse brain membrane preparations.

Ligand dissociation constants from competition binding assays: errors associated with ligand depletion.

The dissociation constant of a ligand at a binding site (e.g., receptor, antibody) is often determined indirectly by competitive displacement of a radioligand. It is well known that such a determination may be seriously in error unless the free concentrations of both the radioligand and the unlabeled ligand can be measured. By means of computer simulations we have explored the conditions under which this error may occur and its magnitude. We offer guidelines for recognizing a probably inaccurate dissociation constant, and we show how, in some cases, a correction can be made. The problem addressed here is not only a theoretical one; it can arise in the ordinary performance of competition binding assays.

Comparative immuno-removal: a novel method for estimating the concentration of an unknown immunoreactive compound.

We describe a method for estimating the molar concentration of an unknown immunoreactive compound. The amount of antibody required to bind half of a standard is compared to the amount required to bind half of an equal number of immunoreactive equivalents of the unknown. We demonstrate the utility of the method using a morphine antibody affinity resin and compounds structurally related to morphine.

Type-A cholecystokinin binding sites in cow brain: characterization using (-)-[3H]L364718 membrane binding assays.

(-)-[3H]L364718 membrane binding assays were employed to localize and characterize cholecystokinin (CCK)-A binding sites in rat and cow brain. Specific binding was detected in all brain areas tested, but in all areas of rat brain and most areas of cow brain the level was too low to allow characterization of the ligand binding specificity of these sites. Membranes prepared from cow nucleus accumbens and striatum contained higher levels of (-)-[3H]L364718 specific binding which represented 55-70% of total binding. Characterization of the ligand binding properties of (-)-[3H]L364718 binding sites in cow nucleus accumbens revealed that these sites are similar to CCK-A sites found in pancreatic membranes. Binding of (-)-[3H]L364718 was saturable and had high affinity (Kd = 45 pm). Sites labeled by (-)-[3H]L364718 displayed stereospecificity for the stereoisomers of CR1409. The competition curve for CCK8 was shallow and was steepened and shifted to the right by the presence of the stable GTP analog guanosine 5'-(beta,delta-imido)triphosphate. The potency of CCK8, but not (-)-L36478, was also affected by the buffer in which the assay was conducted. Future use of (-)-[3H]L364718 membrane binding assays using cow nucleus accumbens and/or striatum will help explore the possibility of differences in ligand recognition among CCK-A sites found in brain and peripheral tissues.

Type-A cholecystokinin receptors in CHP212 neuroblastoma cells: evidence for association with G protein and activation of phosphoinositide hydrolysis.

125I-Bolton Hunter-cholecystokinin octapeptide (BH-CCK8) and (-)-[3H]L-364718 membrane binding assays were used to identify and characterize cholecystokinin (CCK) receptors in CHP212 human neuroblastoma cells. The ligand binding properties of CCK receptors in these cells are similar to those found in pancreas (CCK-A sites) and differ from the predominant type of CCK binding site found in brain (CCK-B sites). The specific binding of 125I-BH-CCK8 but not (-)-[3H]L-364718 was reduced by the metabolically stable GTP analog guanosine 5'-(beta-delta-imido)trisphosphate. A substantial difference in the Bmax for the radiolabeled agonist (125I-BH-CCK8) and antagonist [(-)-[3H]L-364718] was noted. These observations are consistent with CCK receptors existing in guanine nucleotide-binding protein-coupled and -uncoupled states. Similar to its action in pancreatic acinar cells, CCK8(S) stimulated the accumulation of [3H]inositol phosphates in cells prelabeled with [3H]myo-inositol (EC50 = 3.2 +/- 0.4 nM; maximum response = 4.5 +/- 0.4 x basal). The intrinsic activity of CCK analogues in stimulating phosphoinositide hydrolysis was substantially less than their reported intrinsic activity in stimulating phosphoinositide hydrolysis in pancreatic acinar cells. The CHP212 neuroblastoma cell may serve as a useful model for the recently reported CCK-A binding site found in the central nervous system.