Synthetic glucagon antagonists and partial agonists.

This paper reports the synthesis and the biological activities of six new glucagon analogues. In these compounds N-terminal modifications of the glucagon sequence were made, in most cases combined with changes in the C-terminal region which had been shown previously to enhance receptor affinity. The design of these analogues was based on [Lys17,18,Glu21]glucagon,1 a superagonist, which binds five times better than glucagon to the glucagon receptor, and on the potent glucagon antagonist [D-Phe4,Tyr5,Arg12]glucagon, which does not stimulate adenylate cyclase system even at very high concentrations. The N-terminal modifications involved substitution of His1 by the unnatural conformationally constrained residue, 4,5,6,7-tetrahydro-1H-imidazo[c]pyridine-6-carboxylic acid (Tip) and by desaminohistidine (dHis). In addition we prepared two analogues (6 and 7), in which we deleted the Phe6 residue, which was suggested to be part of a hydrophobic patch and involved in receptor binding. The following compounds were synthesized: [Tip1, Lys17,18,Glu21]glucagon (2); [Tip1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (3); [dHis1,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21]glucagon (4); [dHis1,Asp3,D-Phe4,Tyr5,Arg12,Lys17,18,Glu21+ ++]glucagon (5); des-Phe6-[Tip1,D-Phe4,Tyr5,Arg12,Glu21]glucagon (6); des-Phe6-[Asp3,D-Phe4,Tyr5,Arg12,Glu21]glucagon (7). The binding potencies of these new analogues relative to glucagon (= 100) are 3.2 (2), 2.9 (3), 10.0 (4), 1.0 (5), 8.5 (6), and 1.7 (7). Analogue 2 is a partial agonist (maximum stimulation of adenylate cyclase (AC) approximately 15% and a potency 8.9% that of glucagon, while the remaining compounds 3-7 are antagonists unable to activate the AC system even at concentrations as high as 10(-5) M. In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve.(ABSTRACT TRUNCATED AT 250 WORDS)     

Design and synthesis of glucagon partial agonists and antagonists

The hyperglycemia and ketosis of diabetes mellitus are generally associated with elevated levels of glucagon in the blood. This suggests that glucagon is a contributing factor in the metabolic abnormalities of diabetes mellitus. A glucagon-receptor antagonist might provide important evidence for glucagons's role in this disease. In this work we describe how we combined structural modifications that led to glucagon analogues with partial agonist activity to give glucagon analogues that can act as competitive antagonists of glucagon-stimulated adenylate cyclase activity. Using solid-phase synthesis methodology and preparative reverse-phase high-performance liquid chromatography, we synthesized the following seven glucagon analogues and obtained them in high purity: [D-Phe4,Tyr5,Arg12]glucagon (2); [D-Phe4,Tyr5,Lys17,18]glucagon (3); [Phe1,Glu3,Lys17,18]glucagon (4); [Glu3,Val5,Lys17,18]glucagon (5); [Asp3,D-Phe4,Ser5,Lys17,18]glucagon (6); I4-[Asp3,D-Phe4,Ser5,Lys17,18]glucagon (7); [Pro3]glucagon (8). Purity was assessed by enzymatic total hydrolysis, by chymotryptic peptide mapping, and by reverse-phase high-performance liquid chromatography. The new analogues were tested for specific binding, for their effect on the adenylate cyclase activity in rat liver membranes, and for their effect on the blood glucose levels in normal rats relative to glucagon. Analogues showing no adenylate cyclase activity were examined for their ability to act as antagonists by displacing glucagon-stimulated adenylate cyclase dose-response curves to the right (higher concentrations). The binding potencies of the new analogues relative to glucagon (= 100) were respectively 1.0 (2), 1.3 (3), 3.8 (4), 0.4 (5), 1.3 (6), 5.3 (7), and 3 (8). Glucagon analogues 3-5 and 8 were all weak partial agonists with EC50 values of 500 (3), 250 (4), 1600 (5), and 395 nM (8), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Receptor binding and adenylate cyclase activities of glucagon analogues modified in the N-terminal region

In this study, we determined the ability of four N-terminally modified derivatives of glucagon, [3-Me-His1,Arg12]-, [Phe1,Arg12]-, [D-Ala4,Arg12]-, and [D-Phe4]glucagon, to compete with 125I-glucagon for binding sites specific for glucagon in hepatic plasma membranes and to activate the hepatic adenylate cyclase system, the second step involved in producing many of the physiological effects of glucagon. Relative to the native hormone, [3-Me-His1,Arg12]glucagon binds approximately twofold greater to hepatic plasma membranes but is fivefold less potent in the adenylate cyclase assay. [Phe1,Arg12]glucagon binds threefold weaker and is also approximately fivefold less potent in adenylate cyclase activity. In addition, both analogues are partial agonists with respect to adenylate cyclase. These results support the critical role of the N-terminal histidine residue in eliciting maximal transduction of the hormonal message. [D-Ala4,Arg12]glucagon and [D-Phe4]glucagon, analogues designed to examine the possible importance of a beta-bend conformation in the N-terminal region of glucagon for binding and biological activities, have binding potencies relative to glucagon of 31% and 69%, respectively. [D-Ala4,Arg12]glucagon is a partial agonist in the adenylate cyclase assay system having a fourfold reduction in potency, while the [D-Phe4] derivative is a full agonist essentially equipotent with the native hormone. These results do not necessarily support the role of an N-terminal beta-bend in glucagon receptor recognition. With respect to in vivo glycogenolysis activities, all of the analogues have previously been reported to be full agonists.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of the 10-13 region of glucagon for its receptor interactions and activation of adenylate cyclase

The role of the Tyr10-Ser11-Lys12-Tyr13 region of glucagon in the binding interaction and activation of the glucagon receptor was investigated by means of the synthetic glucagon analogues [Phe13]glucagonamide, [Phe10]glucagonamide, [Phe10]glucagon, [Phe10,13]glucagon, [Pro11]glucagon, [Pro11,Gly12]glucagonamide, [Ala11]glucagon, and [Oac11-13]glucagonamide. These analogues were synthesized by solid-phase peptide synthesis on p-methylbenzhydrylamine or Merrifield resins with protected N alpha-tert-butyloxycarbonyl amino acids. Purification by dialysis, cation-exchange chromatography, gel filtration, and preparative reverse-phase high-performance liquid chromatography (HPLC) gave products that proved homogeneous by thin-layer chromatography and HPLC and on analysis by amino acid analysis, by sequencing, and by alpha-chymotryptic peptide mapping with HPLC. Biological activities were examined by measurement of the stimulation of liver plasma membrane adenylate cyclase and by specific displacement of [125I]glucagon from glucagon receptors. The results of these studies indicate that while the biological "message" region of glucagon is located elsewhere, the 10-13 region has multiple roles in the glucagon-glucagon receptor interaction: this region provides functional groups for direct binding interaction with the receptor, and this region interacts with the receptor in such a way as to allow the "transduction message" portion of glucagon to interact and activate the receptor.     

Glucagon antagonists. Synthesis and inhibitory properties of Asp3-containing glucagon analogs

In an effort to find analogs of glucagon that would bind to the glucagon receptor of the rat liver membrane but would not activate membrane-bound adenyl cyclase, several hybrid molecules were synthesized which contained sequences from both glucagon and secretin. [Asp3, Glu9]Glucagon and [Asp3, Glu9, Arg12]glucagon were inactive in the adenyl cyclase assay even at high concentrations but retained some binding affinity for the receptor. They were able to displace 125I-glucagon completely from its receptor and could completely inhibit the activation of adenyl cyclase by natural or synthetic glucagon. The inhibition index [I/A]50 was approximately 110 for both analogs. [Asp3]Glucagon, [Glu3]glucagon and [Asp3, Lys17, 18, Glu21]glucagon were weak partial agonists, while [Asp3, Glu21]glucagon was inactive and a poor inhibitor. The peptides were synthesized by solid-phase methods and purified to homogeneity by reverse-phase high-performance liquid chromatography on C18 silica columns. These are the first fully synthetic competitive glucagon antagonists to be reported.     

Glucagon: structure-function relationships investigated by sequence deletions

A series of glucagon analogues, des-(1-4)-glucagon, des-(5-9)-glucagon, des-(10-15)-glucagon, des-(16-21)-glucagon, des-(22-26)-glucagon and des-(27-29)-glucagon, were prepared by condensation of synthetic fragments and characterized biologically and immunologically. Fully synthetic glucagon was also characterized. The potencies with regard to glucagon receptor binding in purified rat liver plasma membranes were, in decreasing order: synthetic glucagon 108%, des-(1-4)-glucagon 5.7%, des-(27-29)-glucagon 0.92%, des-(5-9)-glucagon 0.47%, des-(10-15)-glucagon 0.0028%, des-(16-21)-glucagon 0.0017% and des-(22-26)-glucagon 0.00060% relative to that of natural porcine glucagon. Des-(27-29)-glucagon was the only analogue that activated the adenylate cyclase in rat liver plasma membranes or stimulated the lipolysis in isolated free fat cells from rat epididymal fat pad. The potencies were 0.16% and 0.20% of that of glucagon, respectively. Des-(1-4)-glucagon was a glucagon antagonist in the adenylate cyclase assay. The immunoreactivities of the glucagon analogues were determined with two commonly used anti-glucagon sera, K 5563 and K 4023, directed towards the C-terminus and some segment in the sequence 2-23, respectively. In the K 5563 assay, des-(27-29)-glucagon and des-(22-26)-glucagon had potencies of 0.0009% and less than 0.09% of that of glucagon, respectively. The remaining analogues had potencies varying from 45% to 141% of that of glucagon. In the K 4023 assay, the analogues showed a non-linear dilution effect. The combined results indicate a partition within the glucagon molecule with regard to receptor binding and adenylate cyclase activation. The region 10-26 appears to be the most important for receptor binding, whereas 1-4 is essential for adenylate cyclase activation. The C-terminal segment 27-29 is important for the maintenance of full receptor binding but non-essential for adenylate cyclase activation.     

Glucagon antagonists: contribution to binding and activity of the amino-terminal sequence 1-5, position 12, and the putative alpha-helical segment 19-27

Glucagon binding to and recognition by its cell surface receptor is the necessary first step in the cascade of events leading to the activation of adenylate cyclase by the hormone. It has long been presumed that glucagon adopts an ordered conformation upon binding to its membrane-bound receptor. A recent model of this three-dimensional structure based on biophysical data, predicts beta-turns at positions 2-5, 10-13, and 15-18, and an alpha-helical region between residues 19-27. Our approach in the design of antagonists of glucagon was to elucidate the steric and electronic features that stabilize these secondary structures to obtain analogs that bind with high affinity to the receptor but do not activate adenylate cyclase. Nineteen glucagon analogs incorporating structural changes at the amino-terminal sequence 1-5, at positions 9 and 12, and at the carboxyl-terminal helical region were synthesized. Des-His1-[Glu9]glucagon amide was recently shown to be a competitive inhibitor. Our synthetic studies in combination with this modification have resulted in seven new glucagon antagonists. The implications for the structural and conformational properties required for binding and activity of glucagon and the glucagon peptide family are discussed.     

Structure-activity studies at the rat tachykinin NK2 receptor: effect of substitution at position 5 of neurokinin A.

A series of analogues of neurokinin A(4-10) was synthesized using solid phase techniques with Chiron pins, and purified by HPLC. The potencies of 10 peptides with substitution at Ser5 were assessed at rat fundus NK2 receptors. In membrane binding studies with [125I]-[Lys5,Tyr(I2)7,MeLeu9,Nle10]-NKA(4-10), all compounds except [Asp5]NKA(4-10) showed reasonable affinity, and analogues with Lys and Arg substitutions were five-fold more potent than NKA(4-10). In functional studies, all peptides were able to contract the rat isolated fundus strips. Analogues with Phe, His and Asn substitutions were substantially weaker in functional than in binding studies, whereas there was an excellent correlation (r = 0.95) between binding and functional potency for the remaining seven peptides. [Phe5]NKA(4-10) is in fact neurokinin B(4-10) and this residue may be critical in determining selectivity between NK2 and NK3 receptors. Analogues with a basic residue (Lys, Arg) at position 5 showed both increased affinity and functional potency, whereas the neutral [Asn5]NKA(4-10) was equally as weak in contractile studies as the acidic [Asp5]NKA(4-10). However, [Glu5]NKA(4-10) and [Gln5]NKA(4-10) were no different from NKA(4-10). Our results could indicate the presence of a negative charge on the NK2 receptor, close to position 5 of NKA. This would facilitate interaction with positively charged side chains and impede interaction with negatively charged side chains, particularly the inflexible side chain of aspartic acid. Thus, not only the charge, but also the length of the side chain of the residue at position 5, seems to be important for interaction with the rat NK2 receptor.     

Position 9 replacement analogs of glucagon uncouple biological activity and receptor binding.

Recent studies on the glucagon antagonist des-His1-[Glu9]glucagon amide have resulted in pure inhibitors of the hormone, suggesting that the inhibitory properties may be centered around position 9. The present study was designed to investigate the chemical characteristics of substitutions in position 9 of glucagon that determine binding affinity and biological activity. Twenty replacement analogs of position 9 of glucagon were synthesized and assessed for their ability to bind to the glucagon receptor in rat hepatocyte membranes and to activate adenylate cyclase. Any substitution of aspartic acid 9 was accompanied by a severely diminished capacity to transmit the biological signal, while retaining receptor binding affinity. These results are an indication of an uncoupling of receptor binding and biological activity at this locus and define a central role of aspartic acid 9 in glucagon activity. Single replacement or deletion of either His1 or Asp9 in glucagon caused a 20- to 50-fold decrease in cyclase activity, whereas these same changes made in tandem caused virtually complete loss of activity, with decreases of 10(4)-to 10(6)-fold. These observations have led us to speculate that, at the molecular level, the region of glucagon required for transduction of the biological response may be distinct from the binding region and is mediated by a coupled interaction between His1 and Asp9 of the hormone and a complementary functional site of the glucagon receptor.     

Insulin-like growth factor I and its binding proteins: a study of the binding interface using B-domain analogues

The biological activity of the insulin-like growth factors (IGF-I and IGF-II) is regulated by six IGF binding proteins (IGFBPs 1-6). To examine the surface of IGF-I that associates with the IGFBPs, we created a series of six IGF-I analogues, [His(4)]-, [Gln(9)]-, [Lys(9)]-, [Ser(16)]-, [Gln(9),Ser(16)]-, and [Lys(9),Ser(16)]IGF-I, that contained substitutions for residues Thr(4), Glu(9), or Phe(16). Substitution of Ser for Phe(16) did not affect secondary structure but significantly decreased the affinity for all IGFBPs by between 14-fold and >330-fold, indicating that Phe(16) is functionally important for IGFBP association. While His(4) or Gln(9) substitutions had little effect on IGFBP affinity, changing the negative charge of Glu(9) to a positive Lys(9) selectively decreased the affinities of IGFBP-2 and -6 by 140- and 30-fold, respectively. Furthermore, the effects of mutations to both residues 9 and 16 appear to be additive. The analogues are biologically active in rat L6 myoblasts and they retain native structure as assessed by their far-UV circular dichroism (CD) profiles. We propose that Phe(16) and adjacent hydrophobic residues (Leu(5) and Leu(54)) form a functional binding pocket for IGFBP association.     

Streptokinase binds preferentially to the extended conformation of plasminogen through lysine binding site and catalytic domain interactions

Binding of streptokinase (SK) to plasminogen (Pg) activates the zymogen conformationally and initiates its conversion into the fibrinolytic proteinase, plasmin (Pm). Equilibrium binding studies of SK interactions with a homologous series of catalytic site-labeled fluorescent Pg and Pm analogues were performed to resolve the contributions of lysine binding site interactions, associated changes between extended and compact conformations of Pg, and activation of the proteinase domain to the affinity for SK. SK bound to fluorescein-labeled [Glu]Pg(1) and [Lys]Pg(1) with dissociation constants of 624 +/- 112 and 38 +/- 5 nM, respectively, whereas labeled [Lys]Pm(1) bound with a 57000-fold tighter dissociation constant of 11 +/- 2 pM. Saturation of lysine binding sites with 6-aminohexanoic acid had no effect on SK binding to labeled [Glu]Pg(1), but weakened binding to labeled [Lys]Pg(1) and [Lys]Pm(1) 31- and 20-fold, respectively. At low Cl(-) concentrations, where [Glu]Pg assumes the extended conformation without occupation of lysine binding sites, a 23-fold increase in the affinity of SK for labeled [Glu]Pg(1) was observed, which was quantitatively accounted for by expression of new lysine binding site interactions. The results support the conclusion that the SK affinity for the fluorescent Pg and Pm analogues is enhanced 13-16-fold by conversion of labeled [Glu]Pg to the extended conformation of the [Lys]Pg derivative as a result of lysine binding site interactions, and is enhanced 3100-3500-fold further by the increased affinity of SK for the activated proteinase domain. The results imply that binding of SK to [Glu]Pg results in transition of [Glu]Pg to an extended conformation in an early event in the SK activation mechanism.     

Analogues of parathyroid hormone modified at positions 3 and 6. Effects on receptor binding and activation of adenylyl cyclase in kidney and bone.

Predictive and spectroscopic methods were used to develop a model of the structures of the 1-34 peptides of parathyroid hormone (PTH) and the PTH-related protein (PTHrP). Circular dichroism (CD) studies of bovine PTH-(1-34) and human PTHrP-(1-34)amide in the presence of trifluoroethanol suggest the presence of 24-26 alpha-helical residues. For both peptides, interactions between amino- and carboxyl-region alpha-helices are predicted to result in a hydrophobic core with externally facing hydrophilic residues that include probable determinants of receptor binding and activation. Two such residues, Ser3 and Gln6, are conserved in all known members of the PTH/PTHrP family. We have synthesized 13 novel analogues of bovine PTH-(1-34) monosubstituted at positions 3 and 6 and have determined their biological activities in renal and bone cell radioreceptor and adenylyl cyclase assays. Position 3 analogues displayed biological activity that was reduced in direct proportion to the volume of the substituent side-chain. Position 6 analogues also displayed reduced biological activity, but no simple correlation with side-chain volume or hydrophobicity was evident. The analogues fully displaced labeled PTH from binding sites in renal membranes and bone cells, but [Phe3]bPTH-(1-34), [Tyr3]bPTH-(1-34), [Phe6] bPTH-(1-34), and [Ser6]bPTH-(1-34) were only partial agonists in one or both adenylyl cyclase assays. Of these, [Phe3]bPTH-(1-34) and [Phe6]bPTH-(1-34) were tested for antagonist activity and were found to inhibit the activation of adenylyl cyclase in response to bPTH-(1-34) or hPTHrP-(1-34)amide. These results indicate that positions 3 and 6 contribute important determinants of PTH receptor binding and activation. Modification at these positions represents a novel approach to the development of antagonists of PTH action.     

Receptor occupancy and adenylate cyclase activation in rat liver and heart membranes by 10 glucagon analogs modified in position 2,3, 4, 25, 27 and/or 29

Rat liver and heart membranes were tested for adenylate cyclase activation by glucagon and 10 glucagon analogs mono- or polysubstituted in positions 2-4, 25, 27 and/or 29. The first membranes were, in addition, examined for the capacity of glucagon analogs to inhibit the binding of [125I]iodoglucagon. The monophasic slope of dose-effect curves suggested interaction with one class of glucagon receptors in both tissues, receptors in liver being more sensitive to the ligands and more efficiently coupled to adenylate cyclase than heart receptors. Structure-activity studies on liver membranes revealed that modifications of the beta-turn potential in the 2-4 region by single residue substitutions could lead to partial agonists (with D-Gln3 or Phe4) or to a superagonist (with D-Phe4). The importance of a proper alpha-helix conformation in the C-terminal part of glucagon for binding affinity was also obvious: replacing Trp25, Met27 and Thr29 in combination by Phe25, Leu27 and Thr29-NH2 increased the affinity while single or combined substitutions with Gly25 and/or Nle27 sharply decreased the affinity. Similar trends were less evident but still obvious on heart membranes.     

The relation of predicted structure to observed conformation and activity of glucagon analogs containing replacements at positions 19, 22, and 23

Six new analogs of glucagon have been synthesized containing replacements at positions 19, 22, and 23. They were designed to study the correlation between predicted conformation in the 19-27 segment of the hormone and the conformation calculated from circular dichroism measurements and the observed activation of adenylate cyclase in the liver membrane. The analogs were [Val19]glucagon, [Val22]glucagon, [Glu23]glucagon, [Val19,Glu23]glucagon, [Glu22,Glu23]glucagon, and [Ala22,Ala23]glucagon. The structures predicted for the 19-27 segment ranged from strongly alpha helical to weakly beta sheet. The observed conformations varied as functions of amino acid composition, solvent, concentration, pH, and temperature but did not correlate well with prediction. There was, however, a correlation between predicted structure and activation of adenylate cyclase in rat liver membranes.     

Position 3 analogues of a crustacean pigment-dispersing hormone: synthesis and biological activity

In an effort to explain the difference in potencies between the two characterized crustacean pigment-dispersing hormones (alpha-PDH; beta-PDH) and to define a role for residue 3 in these octadecapeptide hormones, we have synthesized and purified seven position 3 alpha-PDH analogues ([Ala3], [Ile3], [Asn3], [Gln3], [Asp3], [Glu3], and [Lys3]alpha-PDH). When tested for melanophore pigment-dispersing activity in destalked Uca, [Glu3]alpha-PDH was found to be 325% more potent than alpha-PDH. Reduced potencies were observed for the [Asp3] (58%), [Asn3] (26%), [Gln3] (11%), and [Ala3] (8%) derivatives. Much lower potencies were displayed by the [Lys3] and [Ile3] analogues (0.73% and 0.66%, respectively). These results suggest that the position 3 side chain carboxylate anion of [Glu3]alpha-PDH stabilizes the active receptor-bound conformer through a charge-charge interaction.     

Interaction of a cationic polymer with negatively charged proteoliposomes

In the present study, we have analyzed a previously identified constitutively active pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptor with a deletion of the single amino acid residue Glu(261) (Y.-J. Cao, G. Gimpl, F. Fahrenholz, A mutation of second intracellular loop of pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation, FEBS Lett. 469 (2000)). This glutamic acid residue is highly conserved within the second intracellular loop of class II G protein-coupled receptors and may thus be of importance for many members of this receptor class. To explore the molecular characteristics of this mutant receptor, we performed photoaffinity labeling using previously defined photoreactive PACAP analogues. In COS cells, the PAC1 receptor was expressed in two differently glycosylated forms: a M(r) 75,000 and a M(r) 55,000 form. According to partial deglycosylation, at least three carbohydrate chains may exist in the rat PAC1 receptor expressed in COS cells. The constitutively active PAC1 receptor was expressed at the surface of COS-7 cells at the same density as the wild-type receptor. With respect to the different photoreactive PACAP analogues, the labeling specificity was the same for the wild-type versus mutant receptor: (125)I-[Lys(15)(pBz(2))]-PACAP-27 and (125)I-[Bpa(22)]-PACAP-27 were efficiently incorporated into each of the receptors, whereas (125)I-[Bpa(6)]-PACAP-27 labeled each of the receptors only to a negligible extent. This suggests that both receptors have the same or at least a very similar hormone binding site which is in close contact to Tyr(22) and Lys(15) located in the carboxy-terminal alpha-helical region of the PACAP-27 molecule. However, in comparison with the wild-type PAC1 receptor, the constitutively active receptor showed a markedly (approx. 6--8-fold) enhanced photoaffinity labeling efficiency in particular of the high glycosylated form. The enzymatically deglycosylated rat PAC1 receptor was efficiently labeled by photoreactive PACAP analogues. In contrast, nonglycosylated PAC1 receptors produced by tunicamycin treatment of the transfected COS-7 cells showed a 30-fold lower affinity for PACAP-27 and were capable of signal transduction with 30--50-fold lower potency as compared with the glycosylated PAC1 receptors.     

Differential response to adrenocorticotropic hormone analogs of bovine adrenal plasma membranes and cells.

The ability of three analogs of ACTH1-24 ([Gln5, Phe9] ACTH1-24, [Gln5, Ala9[Acth1-24, and [Gln5, Lys8, Phe9[ ACTH1-24) embodying tryptophan substitutions to activate the adenylate cyclase system of a bovine adrenal plasma membrane preparation was compared to the effect of the analogs on adenosine 3':5'-monophosphate (cyclic AMP) accumulation and steroidogenesis in viable bovine adrenocortical cells. The results were not comparable. Whereas the analogs antagonized the ACTH1-24-activated membrane cyclase they stimulated cyclic AMP accumulation as well as steroid production of the cells. None of the analogs inhibited steroidogenesis of ACTH1-24-stimulated cells, but two of them, at very high dose levels, inhibited cyclic AMP production. The ability of the analogs to stimulate steroidogenesis of the adrenal cells half-maximally decreased in the order tryptophan greater than phenylalanine greater than alanine, indicating that the aromaticity of the indole ring of tryptophan is necessary for maximal interaction between hormone and receptor. Both the absolute and relative steroidogenic potencies were the same for several analogs when assayed with rat adrenal cells. Although only a small fraction of the cell's potential to produce cyclic AMP was necessary to induce maximum steroid production, the relative activities of a series of analogs were the same for steroidogenesis as for cyclic AMP accumulation. Furthermore, the concentration of cyclic AMP necessary for full steroidogenesis was practically identical for a series of peptides that differed widely in potency. These findings support the postulate that cyclic AMP accumulation and steroidogenesis in adrenocortical cells are coupled processes. The differential behavior of bovine adrenal plasma membranes and bovine adrenocortical cells toward ACTH analogs indicates that structure-function studies using cyclase assays may not reflect events that take place in the intact adrenal or in cell preparations derived therefrom.     

Functional evidence for an intramolecular side chain interaction between residues 6 and 10 of receptor-bound parathyroid hormone analogues

The N-terminal domain of PTH(1-34) is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated the possibility that the side chains of residues 6 (Gln) and 10 (Gln or Asn) of PTH analogues, which would align on the same face of the predicted alpha-helix, could interact and thereby contribute to the PTH/P1R interaction process. We utilized PTH(1-11), PTH(1-14), and PTH(1-34) analogues substituted with alanine at one or both of these positions and functionally evaluated the peptides in cell lines (HKRK-B7 and HKRK-B28) stably expressing the P1R, as well as in COS-7 cells transiently expressing either the P1R or a P1R construct that lacks the amino-terminal extracellular domain (P1R-DelNt). In HKRK-B7 cells, the single substitutions of Gln(6) --> Ala and Gln(10) --> Ala reduced the cAMP-stimulating potency of [Ala(3),Gln(10),Arg(11)]rPTH(1-11)NH(2) approximately 60- and approximately 2-fold, respectively, whereas the combined Ala(6,10) substitution resulted in a approximately 2-fold gain in potency, relative to the single Ala(6) substitution. Similar effects on P1R-mediated cAMP-signaling potency and P1R-binding affinity were observed for these substitutions in [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]rPTH(1-14)NH(2). Installation of a lactam bridge between the Lys(6) and the Glu(10) side chains of [Ala(3,12),Lys(6),Glu(10),Har(11),Trp(14)]rPTH(1-14)NH(2) increased signaling potency 6-fold, relative to the nonbridged linear analogue. Alanine substitutions at positions 6 and/or 10 of [Tyr(34)]hPTH(1-34)NH(2) did not affect signaling potency nor binding affinity on the intact P1R; however, Ala(6) abolished PTH(1-34) signaling on P1R-DelNt, and this effect was reversed by Ala(10). The overall data support the hypothesis that the N-terminal portion of PTH is alpha-helical when bound to the activation domain of the PTH-1 receptor and they further suggest that intrahelical side chain interactions between residues 6 and 10 of the ligand can contribute to the receptor interaction process.     

Dermorphin-based potential affinity labels for mu-opioid receptors.

Dermorphin and [Lys7]dermorphin, selective micro -opioid receptor ligands originating from amphibian skin, have been modified with various electrophiles in either the 'message' or 'address' sequences as potential peptide-based affinity labels for micro -receptors. Introduction of the electrophilic isothiocyanate and bromoacetamide groups on the para position of Phe3 and Phe5 was accomplished by incorporating Fmoc-Phe(p-NHAlloc) into the peptide followed by selective deprotection and modification. The corresponding amine-containing peptides were also prepared. The pure peptides were evaluated in radioligand binding experiments using Chinese hamster ovary (CHO) cells expressing micro - and delta-opioid receptors. In dermorphin, introduction of the electrophilic groups in the 'message' domain lowered the binding affinity by > 1000-fold; only [Phe(p-NH2)3]dermorphin retained nanomolar affinity for micro -receptors. Modifications in the 'address' region of both dermorphin and [Lys7]dermorphin were relatively well tolerated. In particular, [Phe(p-NH2)5,Lys7]dermorphin showed similar affinity to dermorphin, with almost 2-fold higher selectivity for micro -receptors. [Phe(p-NHCOCH2Br)5]- and [Phe(p-NHCOCH2Br)5,Lys7]dermorphin exhibited relatively high affinity (IC50 = 27.7 and 15.1 nm, respectively) for micro -receptors. However, neither of these peptides inhibited [3H]DAMGO binding in a wash-resistant manner.     

Cyclic parathyroid hormone related protein antagonists: lysine 13 to aspartic acid 17 [i to (i + 4)] side chain to side chain lactamization.

Cyclization of parathyroid hormone related protein (7-34)amide [PTHrP(7-34)NH2] via covalent bond formation between the epsilon-amino of Lys13 and the beta-carboxyl of Asp17 yielded a 20-membered ring lactam. This analogue, [Lys13,Asp17]PTHrP(7-34)NH2, was 5-10-fold more potent than the linear parent peptide (Kb = 15 and 18 nM in PTH receptor binding assays, and Ki = 130 and 17 nM in PTH-stimulated adenylate cyclase assays in bovine renal cortical membrane and in human bone derived B10 cells, respectively). In contrast, a linear analogue in which charges in positions 13 and 17 were eliminated and other stereoisomers of the above-mentioned lactam in which either Lys13 and/or Asp17 were replaced by the corresponding D-amino acids were much less potent with regard to antagonist bioactivity than the parent peptide. The rationale for the design of the lactam as well as the conformational implications for the PTHrP sequence in light of reported models suggested for the 1-34 peptide are described. The potential use of conformationally constrained analogues for elucidating the "bioactive conformation" of antagonists and for the design of substantially simplified molecular structures for antagonists is discussed.