Mimicry of erythropoietin by a nonpeptide molecule.

Erythropoietin (EPO) controls the proliferation and differentiation of erythroid progenitor cells into red blood cells. EPO induces these effects by dimerization of the EPO receptors (EPOR) present on these cells. To discover nonpeptide molecules capable of mimicking the effects of EPO, we identified a small molecule capable of binding to one chain of EPOR and used it to synthesize molecules capable of inducing dimerization of the EPOR. We first identified compound 1 (N-3-[2-(4-biphenyl)-6-chloro-5-methyl]indolyl-acetyl-L-lysine methyl ester) by screening the in-house chemical collection for inhibitors of EPO binding to human EPOR and then prepared compound 5, which contains eight copies of compound 1 held together by a central core. Although both compounds inhibited EPO binding of EPOR, only compound 5 induced dimerization of soluble EPOR. Binding of EPO to its receptor in cells results in activation of many intracellular signaling molecules, including transcription factors like signal transducer and activator of transcription (STAT) proteins, leading to growth and differentiation of these cells. Consistent with its ability to induce dimerization of EPOR in solution, compound 5 exhibited much of the same biological activities as EPO, such as (i) the activation of a STAT-dependent luciferase reporter gene in BAF3 cells expressing human EPOR, (ii) supporting the proliferation of several tumor cell lines expressing the human or mouse EPOR, and (iii) the in vitro differentiation of human progenitor cells into colonies of erythrocytic lineage. These data demonstrate that a nonpeptide molecule is capable of inducing EPOR dimerization and mimicking the biological activities of EPO.

Arginine 206 of the C5a receptor is critical for ligand recognition and receptor activation by C-terminal hexapeptide analogs.

C5a is a 74-amino-acid glycoprotein whose receptor is a member of the rhodopsin superfamily. While antagonists have been generated to many of these receptors, similar efforts directed at family members whose natural ligands are proteins have met with little success. The recent development of hexapeptide analogs of C5a has allowed us to begin elucidation of the molecular events that lead to activation by combining a structure/activity study of the ligand with receptor mutagenesis. Removal of the hexapeptide's C-terminal arginine reduces affinity by 100-fold and eliminates the ability of the ligand to activate the receptor. Both the guanidino side chain and the free carboxyl of the arginine participate in the interaction. The guanidino group makes the energy-yielding contact with the receptor, while the free carboxylate negates "electrostatic" interference with Arg-206 of the receptor. It is the apparent movement Arg-206 induced by this set of interactions that is responsible for activation, since conversion of Arg-206 to alanine eliminates the agonist activity of the hexapeptides. Surprisingly, activation is a nearly energy-neutral event and may reflect the binding process rather than the final resting site of the ligand.

Quantification of a matrix metalloproteinase-generated aggrecan G1 fragment using monospecific anti-peptide serum.

Several members of the matrix metalloproteinase family have been reported to cleave aggrecan in the interglobular domain between Asn-341 and Phe-342. An antiserum was prepared against a peptide conjugate corresponding to the C-terminal sequence of the matrix metalloproteinase-generated aggrecan G1 fragment (Phe335-Val-Asp-Ile-Pro-Glu-Asn341). A quantitative radioimmunoassay, with a limit of detection of about 80 pM, was developed using this antiserum. This antiserum requires the free carboxyl group of the C-terminal asparagine for optimal recognition. If the C-terminal asparagine is excised from the sequence, replaced with closely related amino acids, or extended across the matrix metalloproteinase cleavage site, there is a 40-10,000-fold loss in detection. Using peptides cleaved from the N-terminus, it was determined that the antiserum requires the entire Phe-Val-Asp-Ile-Pro-Glu-Asn sequence for optimal recognition. The radioimmunoassay detects matrix metalloproteinase-generated G1 fragments with similar sensitivity to the Phe-Val-Asp-Ile-Pro-Glu-Asn peptide, but it does not recognize intact aggrecan. Immunoreactive aggrecan G1 fragments of molecular mass 50 kDa are generated by the matrix metalloproteinases stromelysin and gelatinase A. In contrast, under identical conditions, the closely related metalloproteinases, gelatinase B and collagenase, as well as cathepsin G, cathepsin B and human leucocyte elastase, did not generate a G1 fragment recognized by the antiserum. The anti-Phe-Val-Asp-Ile-Pro-Glu-Asn serum detects stromelysin-generated aggrecan G1 fragments from mouse, guinea pig, rabbit and human, indicating that the detection is not species-specific. This antiserum and radio-immunoassay should be useful for quantifying and characterizing matrix metalloproteinase-generated aggrecan G1 fragments in articular cartilage and synovial fluids from humans and various animal models of articular-cartilage destruction.

Development of C5a receptor antagonists. Differential loss of functional responses.

C5a is a 74-amino acid glycoprotein generated on activation of the C system. The responses evoked by C5a, both in vitro and in vivo, and its association with inflammatory diseases, suggest that a receptor antagonist would be of considerable therapeutic importance. However, efforts at generating antagonists have so far been unsuccessful. Structure/activity studies of the C terminus of C5a have generated peptide analogues with nanomolar affinities, but all of these retain strong agonist properties. We now report hexapeptides of the form NMePhe-Lys-Pro-dCha-X-dArg in which increasing aromaticity at position 5 leads to a progressive loss of agonism with little change in binding affinity. The different responses induced by C5a are lost in the order: degranulation before Ca(2+)-flux before chemotaxis. We also describe the first full antagonist of C5a, because the peptide in which x = Trp is not only devoid of all agonist properties, but it inhibits C5a induced degranulation and C5a stimulated G protein activation.

The amino terminus of the human C5a receptor is required for high affinity C5a binding and for receptor activation by C5a but not C5a analogs.

The binding domain of the human C5a receptor consists of two distinct and physically separable subsites. One of these sites binds the C-terminal 8 amino acids of C5a and is as yet undefined, while the second site lies in the N terminus of the receptor and interacts with the core of C5a. Two deletion mutants were prepared to probe the importance of this second site. Removal of residues 2-22 decreased the binding affinity for C5a by 600-fold, while extending the deletion through residue 30 caused a further 75-fold decrease. Thus, the N terminus is responsible for at least 45% of the total energy for the binding of C5a. The five aspartic acids present in the deleted segments appear to be critical residues, as their conversion to alanines accounts for most of the affinity lost in the two truncations. Despite its importance for binding, the N terminus is not necessary for signal transduction, as a C-terminal peptide analog of C5a was able to stimulate G protein activation and to generate a Ca2+ flux through a receptor lacking residues 2-22. However, intact C5a was a very poor activator of this truncated receptor. These results imply that interaction between the N terminus of the receptor and C5a produces a conformational change in C5a that allows it's C terminus to properly interact with and activate the receptor.

Two-site binding of C5a by its receptor: an alternative binding paradigm for G protein-coupled receptors.

The guanine nucleotide-binding protein-coupled receptor superfamily binds a vast array of biological messengers including lipids, odorants, catecholamines, peptides, and proteins. While some small molecules bind to these receptors at a single interhelical site, we find that the binding domain on the receptor for the inflammatory protein C5a is more complex and consists of two distinct subsites. This more elaborate motif appears to be an evolutionary adaptation of the simpler paradigm to which a second interaction site has been added in the receptor N terminus. Surprisingly, occupation of only one of the subsites is required for receptor activation. The two-site motif is not unique to the C5a receptor but appears to be widely used by the superfamily to accommodate macromolecular ligands.

D-amino acid and alanine scans of the bioactive portion of porcine motilin.

A recent systematic study of porcine motilin fragments has clearly shown that biological activity resides in the amino-terminal end. The amino-terminal tetradecapeptide retains more than 90% of the potency of the full molecule. We now examined the effect of replacement of residues 1 through 11 by either their D-isomer or by alanine in [Leu13]pMOT(1-14). Peptides were synthesized using Fmoc solid phase methodology, purified by HPLC, and assayed for their ability to displace bound motilin (rabbit antral smooth muscle homogenate) and to induce contractions (isolated rabbit duodenal segments). The negative logarithm of the concentration displacing 50% of the tracer (pIC50), or producing 50% of the maximal contractile response (pEC50), was determined. All compounds were still full agonists. A reduction in potency of more than two log units was seen for the compounds in which residues 1 (Phe), 4 (Ile), and 7 (Tyr) were replaced by Ala and residues 3 (Pro), 4 (Ile), and 6 (Thr) by their D-isomer. The largest drop was noted for the analogs substituted at position 4. For all compounds there was an almost perfect correlation between the pIC50 and the pEC50 values (r = 0.96), although the pEC50 was consistently smaller. These results show that the biological activity of motilin is mainly determined by the first seven residues. The pharmacophore consists of the aromatic rings from Phe1 and Tyr7 and the aliphatic side chains from Val2 and Ile4. Pro3, Phe5, and Thr6 may stabilize the bioactive conformation.

Synthesis and in vitro evaluation of [Leu13]porcine motilin fragments.

Several peptide fragments representing N-terminal, C-terminal, and internal sequences of [Leu13]porcine motilin ([Leu13]pMOT) were synthesized using Fmoc solid phase methodology. Peptides were assayed for motilin receptor binding activity in a rabbit antrum smooth muscle preparation and for stimulation of contractile activity in segments of rabbit duodenum. In vitro activity was directly correlated with motilin receptor binding affinity for all [Leu13]pMOT fragments examined. N-Terminal fragments of just over half the length of the native peptide are nearly equipotent as full-length motilin. These results suggest that the N-terminal segment, together with residues from the mid-portion of the molecule, constitutes the bioactive portion of pMOT. The C-terminal segment, in contrast, contributes little to receptor binding affinity or in vitro activity.

Capillary zone electrophoresis studies of motilin peptides. Effects of charge, hydrophobicity, secondary structure and length.

Motilin is a gut hormone, which is involved in gastrointestinal motility. Capillary electrophoresis studies were made on 24 peptides that are N-terminal, C-terminal or internal fragments of motilin. The isoelectric point, total charge and hydrophobicity were calculated for all of the peptides. The effects of buffers and pH on migration time and resolution were studied. These included citrate buffer, pH 2.5; phosphate buffer, pH 7.0 and borate buffer, pH 10.0. A capillary zone electrophoresis method was developed to resolve 14 of the motilin peptides. Secondary structure predictions were made using the Chou-Fasman method. Circular dichroism spectra were collected to confirm presence of alpha-helix in several fragments. Effects of charge, hydrophobicity, secondary structure and length of the motilin fragments on migration time were studied.

Chiral high-performance liquid chromatography of aromatic cyclic dipeptides using cyclodextrin stationary phases.

A series of enantiomers of cyclic and linear dipeptides containing aromatic amino acids was prepared and chromatographed on beta- and gamma-cyclodextrin (CD) columns. The retention times, separation factor alpha and resolution values were calculated. The relevance of the distance of the chiral center from the phenyl ring for chiral resolution was studied. A model was developed using X-ray crystallographic data for an inclusion complex of beta-CD and the enantiomers of cyclic (Phe-Gly).

High-performance liquid chromatographic separation of peptide and amino acid stereoisomers.

Several high-performance liquid chromatographic (HPLC) methods are described for separation of peptide stereoisomers which are not well resolved by traditional reversed-phase chromatography. These chiral HPLC methods include investigations with a beta-cyclodextrin column, a Pirkle D-Phenyl Glycine column and a Chiral-Pak WH column. A method based on derivatization of dipeptides with a chiral reagent, N-acetyl-L-cysteine and o-phthalaldehyde, is also discussed. A series of linear and cyclic dipeptides and modified amino acids were chromatographed on the four systems. Resolution varied for the four different systems depending on the types of compounds that were chromatographed.