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 [Lys^17.18,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, Lys^17.18,Glu²¹]glucagon (2); [Tip¹,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17.18,Glu²¹ glucagon (3); [dHis¹,d -Phe⁴,Tyr⁵,Arg¹², Lys^17.18,Glu²¹ glucagon (4); [dHis¹,Asp³,d -Phe⁴,Tyr⁵,Arg¹²,Lys^17.18,Glu²¹]glucagon (5); des-Phe⁶-[Tip¹,D-Phe⁴,Tyr⁵Arg¹²,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^?5m . In addition, in competition experiments, analogues 3-7 caused a right-shift of the glucagon stimulated adenylate cyclase dose-response curve. Hence these compounds are glucagon receptor antagonists with respect to the glucagon receptor coupled to the adenylate cyclase system.     

Structure-activity studies of hydrophobic amino acid replacements at positions 9, 11 and 16 of glucagon

We have designed and synthesized eight compounds 2-9 which incorporate neutral, hydrophobic amino acid residues in positions 9, 11 and 16 of the glucagon molecule: (2) [desHis¹,Va1⁹,11e^11,16] glucagon amide, (3) [desHis¹,Val^9,11,16]glucagon amide, (4) [desHis¹,Va1⁹,Leu^11,16]glucagon amide, (5) [desHis¹,Nle⁹,11e^11,16]glucagon amide, (6) [desHis¹,Nle⁹,Val^11,16]glucagon amide, (7) [desHis¹,Nle⁹,Leu^11,16]glucagon amide, (8) [desHis¹,Val⁹,Leu^11,16,Lys^17,18,Glu²¹]glucagon amide and (9) [desHis¹,Nle⁹,Leu^11,16,Lys^17,18,Glu²¹]glucagon amide. The effect of neutral, hydrophobic residues at positions 9, 11 and 16 led to good binding to the glucagon receptor. Compared to glucagon (IC₅₀= 1.5 nM), analogues 2-9 were found to have IC₅₀ values of 6.0, 6.0, 11.0, 9.0, 2.5, 2.8, 6.5 and 7.0 nM, respectively. When these compounds were tested for their ability to block adenylate cyclase (AC) activity, they were found to be antagonists having no stimulation of adenyl cyclase, with PA₂, values of 6.15, 6.20, 6.30, 7.25, 6.10, 7.30, 6.25 and 7.25, respectively. © Munksgaard 1997.     

Importance of the C-terminal α-helical structure for glucagon's biological activity

The synthetic glucagon analogues [Glu²¹]glucagon, 2 , and [Lys^17.18.Glu²¹]glucagon, 3 , were designed using Chou-Fasman calculations for the purpose of enhancing the probability for the formation of a C-terminal amphipathic α-helical conformation. Circular dichroism indicates increased α-helical content for these analogues in solution relative to glucagon. Analogues 2 and 3 also exhibit a 3-fold and 5-fold increase in receptor binding potency, respectively. The adenylate cyclase stimulating potencies of 2 and 3 relative to glucagon are 2.1 and 7 times greater, respectively. Attempts were made at further α-helical enhancement by further substitutions in the 10–13 region of glucagon. as represented by the glucagon analogues [Phe¹³,Lys^17.18 Glu²¹]glucagon, 4 , and [Phe^10.13, Lys^17.18,Glu²¹]glucagon. 5 . These latter substitutions resulted in lowered receptor binding and adenylate cyclase potencies for 4 and 5 relative to 3 despite increased α-helical content in solution as observed by circular dichroism spectroscopy.     

Synthesis and hormonal activity of [Tyr 22] glucagon and [desHis 1, Tyr 22] glucagon

[Tyr²²] glucagon and [desHis¹, Tyr²²] glucagon were synthesized by an improved solid phase procedure on a Pam-resin. The course of the synthesis was monitored by quantitative ninhydrin analysis and preview sequencing. Following cleavage by the low/high HF method the peptides were purified by ion exchange chromatography and reverse phase HPLC. The overall yield of homogeneous isolated peptide from the first amino acid was 41%. Circular dichroism measurements on dilute solutions in mixed aqueous organic solvents at pH 2, 6.9 and 9.2 showed increased β-sheet structure relative to glucagon. [Tyr²²]glucagon was a full agonist with 20–30% activity in the rabbit blood glucose assay and 10% activity in the rat liver membrane adenyl cyclase assay. [desHis¹, Tyr²²] glucagon had only a trace of activity in the adenyl cyclase assay (>0.002%) but bound to membranes in a competitive [¹²⁵I] glucagon assay 1.0% as well as glucagon. The analog completely inhibited formation of cAMP by natural glucagon, with 50% inhibition at a ratio of 83:1 and pA₂ = 6.7. The data are discussed in terms of models of glucagon structure in dilute solution.     

Syntheses,opioid binding affinities,and potencies of dynorphin A analogues substituted in positions 1, 6, 7, 8 and 10

Structural, stereochemical, stereoelectronic and conformational requirements for biological activity of dynorphin A_1–11-NH₂ analogues at opioid receptors were explored by substitution of Tyr¹, Arg⁶, Arg⁷, Ile⁸ and Pro¹⁰ with other amino acid residues. Interestingly, substitution of Tyr¹ with N^α-Ac-Tyr^l, D-Tyr¹, Phe¹ or p-BrPhe¹ led to analogues that were quite potent at κ opioid receptors, and additional substitution of Ile⁸with D-Ala⁸ and/or Pro¹⁰ with D-Pro¹⁰ retained high potency in brain binding assay: [N^α-Ac-Tyr¹]- (1), [D-Tyr¹]- (2) [Phe¹]- (3), [Phe¹. D-Ala⁸]- (5), [p-BrPhe¹, D-Ala^s]- (6), [Phe¹, D-Pro¹⁰]- (7) and [Phe¹, D-Ala⁸, D-Pro¹⁰]-Dyn A_1–11-NH₂ (8) had IC₅₀(nM) binding affinities of 13.2, 18.6, 1.64, 1.26, 1.84, 2.44 and 1.62 nM, respectively. The D-Phe¹ analogue 4, however, was only weakly active (610 nM). All of the analogues except 4 were modestly selective for κ vs. μ guinea pig brain opioid receptor (11- to 88–fold) and quite selective for κ vs. δ receptors (65–576). However, all of the analogues appeared to have very low or essentially no activity in the guinea pig ileum and mouse vas deference functional bioassays, and one analogue, 5, appeared to have weak antagonist activities. On the other hand, if constrained amino acids such as β-methylphenylalanine or 1,2,3,4-tetrahydroisoquinoline carboxylic acid, and hydroxyproline were placed in the 1 position, inactive analogues or analogues with greatly reduced potency and biological activity were obtained (compounds 12–14). It had previously been suggested that the Arg⁶ and Arg⁷ residues were critical for biological activity. However, when we replace either one of these residues, [Nle⁶]Dyn A_1–11 (9) and [Nle⁷]Dyn A_1–11-NH₂ (10) were both highly potent binders in κ receptor binding studies (IC₅₀= 0.95 and 0.43 nM, respectively), and interestingly also were potent in μ and δ binding studies. Furthermore, both of the analogues were modestly potent in the GPI and MVD assays (94, 65 nM; 31, 81 nM, respectively). These results demonstrate that basic residues at positions 6 and 7 in dynorphin are not very important for binding to κ opioid receptors. Finally, many of the compounds reported here showed high selectivity for central vs. peripheral κ opioid receptors, with compound 4 being the most selective (63 000-fold).     

Synthesis and receptor binding analysis of dermorphin hepta-, hexa- and pentapeptide analogues

Sixteen dermorphin analogues were synthesized and characterized for μ- and δ-opioid receptor binding properties using [³H]DAGO and [³H]DPDPE, respectively. The analogues included the following: substitutions at position 4 and/or the C-terminal residue; deletions of Gly⁴ or Pro⁶-Ser⁷; inclusion of Z or an acetyl group on the β-amino group of Lys⁷; and the presence of either a C-terminal amide or free acid group. Two peptides, [Lys7-OH]- and [Lys⁷-NH₂]dermorphin, had μ-affinities (K_iμ= 0.15–0.13 nm ) and μ-selectivities (K_iδ/K_iμ= 1158–1482) higher than dermorphin (K_iμ= 0.28 nm ; K_iδ/K_iμ= 295) and best fitted a one-site binding model similar to dermorphin. Significantly better (P <0.0001) fits to a two-site binding model vs. a one-site model were observed with four dermorphin analogues: [Lys(Z)⁷-OH]heptapeptide, [des-Gly⁴(Tyr⁴,Pro⁵,Asn⁶-OH)]hexapeptide and two pentapeptides, [Tyr⁵-NH₂] and [Trp⁴,Asn⁵-OH]. Our data revealed a complex binding pattern for dermorphin analogues to brain μ-receptors in which Hill coefficients less than 0.85 generally suggest heterogeneity of μ-receptors; however, only detailed analyses of the data derived from the non-linear regression fits for one- or two-components gave evidence for the possible existence of two separate [³H]DAGO binding sites. Eight of our dermorphin analogues had significantly better fits for a two-site model (P <0.05), but only four seemed to have two distinct Kⁱ, values (P <0.0001).     

Structure and protein kinase C stimulating activities of lactam analogues of human parathyroid hormone fragment

Five analogues of human parathyroid hormone (hPTH-(20-34)-NH₂, I; cyclo[Lys²⁶-Asp³⁰]-hPTH-(20-34)-NH₂, II; cyclo[Glu²²-Lys²⁶]-hPTH-(20-34)-NH₂, III; cyclo[Lys²⁷-Asp³⁰]-hPTH-(20-34)-NH₂, IV; and [Leu²⁷]-hPTH-(20-34)-NH₂, V) were tested for their ability to promote membrane-bound protein kinase C (PKC) activity in a rat osteosarcoma cell line (ROS 17/2). Analogues I, II and V stimulated PKC activity in the picomolar range, whereas analogues III and IV did not stimulate this activity at any concentration tested. The circular dichroism spectra in neutral, aqueous buffer showed an increase in α-helix in analogues II, III and V as compared to I; this increase appeared to be in the region of the cyclic lactam structure. Analogue IV did not adopt a helical structure, even in the presence of 40% trifluoroethanol, a helix-promoting solvent. The remaining analogues showed a three- to four-fold enhancement of α-helix in this solvent. Analogues II and III had increased retention times in reversed-phase chromatography, as compared to I and IV, This is consistent with a stabilization of amphiphilic helix in analogues II and III compared with I and IV, The data suggest that in the region bounded approximately by residues 24–32, an amphiphilic α-helix is important for correct functional binding to the PTH receptor.     

促性腺激素释放多肽(GRP)对体外培养小鼠垂体分泌LH的影响

合成的促性腺激素释放多肽(GRP)及其类似物GRp～NH₂,[Glu^7.9.14Lys^6.10]GRP(6～14),[phe¹⁴]GRP(5～14)和[phe¹⁴]GRP浓度在0.05mmol·L^-1时,具有刺激体外培养的小鼠垂体分泌LH的作用。其活性依此相当对照垂体的115.4,114.2,140,160和179%。小鼠于妊振第7～9天或第1～5天,每只sc[phe¹⁴]GRP1mg·d^-1,或于妊娠第2～4天每只sc[phe¹⁴]GRP(5～14)1mg·d^-1,有40～60%的妊娠动物出现死胎。     

The 10th and 11th residues of short length N‐terminal PTH(1–12) analogue are important for its optimum potency

Abstract: The 10th and 11th residues of parathyroid hormone PTH(1–12) analogues were substituted to study the structure and function of PTH analogues. The substitution of Ala¹⁰ of [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1–12)NH₂ with Glu¹⁰ and/or the Arg¹¹ with Ile¹¹ markedly decreased cAMP generating activity. Data from circular dichroism (CD) and the nuclear magnetic resonance (NMR) structural analysis of [Ala^3,10,12(Leu⁷/Phe⁷)Arg¹¹]rPTH(1–12)NH₂ revealed tight α‐helical structures, while the Glu¹⁰ and/or Ile¹¹ substituted analogues showed unstable α‐helical structures. We conclude that 10th and 11th residues are important for stabilizing its helical conformation and that destabilization of the α‐helical structure, induced by substituting the above residues, remarkably affect its biological potency.     

Comparative biological activities of potent active-site analogues of α-melanotropin. Effect of tyrosine substitution at position-4

We have prepared several α-melanotropin (α-MSH) analogues with tyrosine substituted for methionine at the 4-position and determined their melanotropic activities on the frog (Rana pipiens), lizard (Anolis carolinensis) and S-91 (Cloudman) mouse melanoma adenylate cyclase bioassays. The potencies of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ and Ac-[Tyr⁴]-α-MSH_4–11-NH₂ were compared with α-MSH and with their corresponding methionine and norleucine substituted analogues. The Tyr-4 analogues were found to be less active than the Nle-4 analogues on both the frog and lizard assays. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ was found to be less active than Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on the lizard bioassay, but more active than the longer fragment on the frog skin assay. Ac-[Tyr⁴]-α-MSH_4–10-NH₂ exhibited extremely prolonged biological activity on frog skin, but not on lizard skin, while the melanotropic activity of Ac-[Tyr⁴]-α-MSH_4–11-NH₂ was rapidly reversed on both assay systems. The increased potency of Ac-[Tyr⁴]-α-MSH_4–10-NH₂ over Ac-[Tyr⁴]-α-MSH_4–11-NH₂ on frog melanocytes may be related to the fact that the shorter 4–10 analogue exhibits prolonged biological activity. Interestingly, it was found that both Tyr-4 analogues were partial agonists on the mouse melanoma adenylate cyclase bioassay, and stimulated the enzyme to only about 50% of the maximal activity of α-MSH. We reported previously that replacement of L-Phe-7 by its D-enantiomer in [Nle⁴]-α-MSH and its Nle-4 containing analogues resulted in peptides with increased potency and in some instances prolonged activity. Similarly, incorporation of D-Phe-7 into Tyr-4 containing melanotropin fragments produced analogues Ac-[Tyr⁴, D-Phe⁷]-αMSH_4–10-NH₂ and Ac-[Tyr⁴, D-Phe⁷]-α-MSH_4–11-NH₂, which also exhibited greatly increased biological activity in all three assay systems. Both of these analogues were also found to have prolonged activity in the frog skin bioassay but little or no prolonged activity in the lizard skin bioassay. These two analogues turned out to be full agonists in the mouse melanoma adenylate cyclase bioassay and were equipotent to α-MSH. These results demonstrate that substitution of tyrosine for methionine at position-4 dramatically affects the potency and prolonged activity of these melanotropin analogues and the melanotropic activities observed as a result of such substitutions are themselves affected by concomitant substitutions at the 7(Phe) and 11 (Lys) positions of the analogues.     

Some pharmacological properties of cyclic and linear analogs obtained by substituting each residue of an oxytocin antagonist with d-tryptophan

We synthesized 10 analogs (1–10) derived from the sequence of [Pmp¹, D-Trp², Arg⁸]oxytocin, (parent antagonist or PA), (Pmp =β,β-pentamethylene-β-mercaptopropionic acid) which is a potent antagonist (pA₂= 7.77) of the uterotonic effect of oxytocin (OT) in rats, as determined in our uterotonic assay. Eight of the following analogs were designed by replacement of each residue in the PA sequence, other than the residue at position 2, with d -tryptophan: Ac-d -Trp-d -Trp-Ile-Gln-Asn-Val-Pro-Arg-Gly-NH₂, (1); [Pmp¹, d -Trp(For)², Arg⁸] OT, (2); [Pmp¹, d -Trp², d -Trp³, Arg⁸] OT, (3); [Pmp¹, D-Trp², d -Trp⁴, Arg⁸] OT, (4); [Pmp¹, d -Trp², d -Trp⁵, Arg⁸] OT, (5); Aaa-d -Trp-Ile-Gln-Asn-d -Trp-Pro-Arg-Gly-NH₂, (6); [Pmp¹, d -Trp², d -Trp⁷, Arg⁸] OT, (7); [Pmp¹, d -Trp², d -Trp⁸] OT, (8); [Pmp¹, d -Trp², Arg⁸, d -Trp⁹] OT, (9); [Pmp¹d -Trp², Arg⁸, d -Trp(For)⁹] OT, (10). To avoid free mercaptan groups, Val⁶ was chosen in analog 1 instead of Cys and Aaa¹ (Aaa = 1-adamantaneacetic acid) in analog 6 instead of Pmp¹. Of the linear analogs, 1 was inactive as an OT antagonist and 6 was a very poor antagonist, with a pA₂= 5.66, but it was more potent than Aaa-d -Trp-Ile-Gln-Asn-Val-Pro-Arg-Gly-NH₂, which has a pA₂= 5.33, as we had previously reported. Analog 2, featuring d -Trp(For)², pA₂= 7.37, was weaker than PA, indicating that the formyl group lowers potency. Analogs 3 and 4 were much weaker than PA, and analog 5 was inactive. Hence, other than at position 2, d -Trp is undesirable in the ring sequence of PA. However, analogs featuring d -substituents in the tail portion of PA were good antagonists. Replacement with d -Trp⁷ gave antagonist 7, pA₂= 7.92, which is somewhat more potent than PA. Replacement with d -Trp⁹ gave antagonist 9, pA₂= 8.18, which is about 2.5 times more potent than PA, although d -Trp(For)⁹, introduced in analog 10, pA₂= 8.10, was not as potent. Replacement with d -Trp⁸ results in analog 8, pA₂= 7.83, equipotent with PA. In the antidiuretic assay analogs 7, 8, and 9, each with a pA₂ equal to or less than < 5.6, were very weak antagonists of the antidiuretic hormone, arginine vasopressin (AVP). Hence, the potent analogs 7, 9, and especially 8 which, unlike AVP or the usual OT antagonists, lacks a basic amino acid, are important leads for future design of highly potent and probably, more selective OT antagonists.     

NMR and quenched molecular dynamics studies of superpotent linear and cyclic α-melanotropins

Conformational searching, computer simulations, synthesis and NMR are used on a variety of α melanocyte-stimulating hormone (α-MSH) analogues to understand the physical characteristics required for biological potency. Peptides I (AC-[Nle⁴,Asp⁵,d -Phe⁷,Lys¹⁰]α-MSH(4-10)-NH₂), II (Ac-c[Nle⁴,Asp⁵,d -Phe⁷,Lys¹⁰]α-MSH(4-10)-NH₂) and III (Ac-[Nle⁴,Asp⁵,d -Phe⁷,Dap¹⁰]α-MSH(4-10)-NH₂ all show very similar conformational properties (backbone and side-chain torsional angles), and all display high biological potencies. The modeling results for these compounds are supported by the NMR data. Peptide IV (Ac-c[Nle⁴,Asp⁵,d -Phe⁷,Dap¹⁰]α-MSH(4-10)-NH₂) appears to have a markedly different conformation and has decreased biological potency.     

Demonstration of a neurokinin A receptor subtype in transfected fibroblasts

In competitive radioligand binding assays, the NK₂ receptor antagonists [Tyr⁵,D-Trp^6,8,9,Arg¹⁰]NKA(4–10) (MEN 10207) and [Tyr⁵,D-Trp^6,8,9,Arg¹⁰]NKA(3–10) (MEN 10208) had high and low affinity, respectively, in bovine stomach membranes and SKLKB82#3 cells, a murine fibroblast cell line transfected with a cDNA encoding for the bovine NK₂ receptor. These antagonists also had different affinities when inhibiting neurokinin A-induced polyphosphoinositide hydrolysis in SKLKB82#3 murine fibroblasts. Thus, the de novo protein expressed by the SKLKB82#3 murine fibroblasts may represent a distinct NK₂ receptor subtype.     

Potentiation of glucose-induced insulin release in islets by desHis1[Glu9]glucagon amide

Glucagon and secretin and some of their hybrid analogs potentiate glucose-induced release of insulin from isolated mouse pancreatic islets. It was recently shown that the synthetic glucagon analog, desHis¹[Glu⁹]glucagon amide, does not stimulate the formation of cyclic adenosine monophosphate in the rat hepatocyte membrane, but binds well to the glucagon receptor and is a good competitive antagonist of glucagon. In the present study the effect of this analog on isolated islets was examined. desHis¹-[Glu⁹]glucagon amide at 3 x 10^?7m , in the presence of 0.01 m d -glucose, increased the release of insulin by 30% and maintained that level for the full 30-min test period. The rate of insulin release returned to the glucose-induced base line after removal of the peptide. The same insulin level was produced by 3 x 10^?9m glucagon, and at 3 x 10^?7m glucagon insulin release was enhanced 290% above the glucose base line.     

Roles of Basic Amino Acid Residues in the Activity of μ-Conotoxin GIIIA and GIIIB,Peptide Blockers of Muscle Sodium Channels

To study in detail the roles of basic amino acid residues in the activity of μ-conotoxin GIIIA (μ-GIIIA) and GIIIB (μ-GIIIB), specific blockers of muscle sodium channels, seven analogs of μ-GIIIA, and two analogs of μ-GIIIB were synthesized. μ-GIIIA analogs were synthesized by replacing systematically the three Arg residues (Arg¹, Arg¹³, and Arg¹⁹) with one, two, and three Lys residues. μ-GIIIB analogs were synthesized by replacing simultaneously all four Lys residues (Lys⁹, Lys¹¹, Lys¹⁶, and Lys¹⁹) with Arg residues and further replacement of acidic Asp residues with neutral Ala residues. Circular dichroism spectra of the synthesized analogs suggested that the replacement did not affect the three dimensional structure. The inhibitory effects on the twitch contractions of the rat diaphragm showed that the side chain guanidino group of Arg¹³ of μ-GIIIA was important for the activity, whereas that of Arg¹⁹ had little role for biological activity. Although [Arg^9,11,16,19]μ-GIIIB showed higher activity than native μ-GIIIB, highly basic [Ala^2,12, Arg^9,11,16,19]μ-GIIIB showed lower activity, suggesting that there was an appropriate molecular basicity for the maximum activity.     

Specificity of antisera obtained to Substance P and its C-terminal hexapeptide

Synthetic fragments and analogs were used to characterize specificity of antisera toSP and SP_6–11. [Tyr⁸]SP and [Lys⁶]SP_6–11 were both used as radioiodinated ligands. The latter was conjugated with Bolton-Hunter reagents before labelling. In both systems, the C-terminal pentapeptide SP_7–11 was the shortest fragment showing antigenic identity with Substance P molecule. Substitution of different amino acid residues in SP_6–11 by His or Gly showed that all but Glu⁶ take part in the structure of the antigenic determinant.     

Structure–activity relationships of arodyn,a novel acetylated kappa opioid receptor antagonist*,†

Abstract: We previously reported that the novel dynorphin A (Dyn A, Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐Arg‐Ile‐Arg‐Pro‐Lys‐Leu‐Lys‐Trp‐Asp‐Asn‐Gln) analog arodyn (Ac[Phe^1,2,3,Arg⁴,d ‐Ala⁸]Dyn A‐(1–11)NH₂, Bennett, M.A., Murray, T.F. & Aldrich, J.V. (2002) J. Med. Chem. vol. 45, pp. 5617–5619) is a κ opioid receptor‐selective peptide [K_i(κ) = 10 nm , K_i ratio (κ/μ/δ) = 1/174/583] which exhibits antagonist activity at κ opioid receptors. In this study, a series of arodyn analogs was prepared and evaluated to explore the structure–activity relationships (SAR) of this peptide; this included an alanine scan of the entire arodyn sequence, sequential isomeric d ‐amino acid substitution in the N‐terminal ‘message’ sequence, NMePhe substitution individually in positions 1–3, and modifications in position 1. The results for the Ala‐substituted derivatives indicated that Arg⁶ and Arg⁷ are the most important residues for arodyn's nanomolar binding affinity for κ opioid receptors. Ala substitution of the other basic residues (Arg⁴, Arg⁹ and Lys¹¹) resulted in lower decreases in affinity for κ opioid receptors (three‐ to fivefold compared with arodyn). Of particular interest, while [Ala¹⁰]arodyn exhibits similar κ opioid receptor binding as arodyn, it displays higher κ vs. μ opioid receptor selectivity [K_i ratio (κ/μ) = 1/350] than arodyn because of a twofold loss in affinity at μ opioid receptors. Surprisingly, the Tyr¹ analog exhibits a sevenfold decrease in κ opioid receptor affinity, indicating that arodyn displays significantly different SAR than Dyn A; [Tyr¹]arodyn also unexpectedly exhibits inverse agonist activity in the adenylyl cyclase assay using Chinese hamster ovary cells stably expressing κ opioid receptors. Substitution of NMePhe in position 1 gave [NMePhe¹]arodyn which exhibits high affinity [K_i(κ) = 4.56 nm ] and exceptional selectivity for κ opioid receptors [K_i ratio (κ/μ/δ) = 1/1100/>2170]. This peptide exhibits antagonistic activity in the adenylyl cyclase assay, reversing the agonism of 10 nm Dyn A‐(1–13)NH₂. Thus [NMePhe¹]arodyn is a highly κ opioid receptor‐selective antagonist that could be a useful pharmacological tool to study κ opioid receptor‐mediated activities.     

New analogs of human growth hormone-releasing hormone (1-29) with high and prolonged antagonistic activity

Based on our previous results, in conjunction with various structural considerations, 19 new analogs of the GHRH antagonist [PhAc-Tyr¹,D-Arg²,Phe(pCl)⁶,Abu¹⁵,Nle²⁷,Agm²⁹]hGHRH(1-29) (MZ-5-156) were synthesized by the solid-phase method. These compounds were designed to develop further analogs of this class with increased receptor-binding affinity. All analogs had Abu¹⁵ and Nle²⁷ modifications and were acylated with phenylacetic acid at the N-terminus. Most of the analogs had d -Arg² and Phe(pCl)⁶ substituents and Agm²⁹ or Arg²⁹-NH₂ at the C-terminus. Additional single substitutions consisted of the incorporation of d - or l -Tic¹, d -Tic², Tic⁶ or Phe(pNO₂)⁶ and Arg²⁹-NH₂. The Arg²⁹-NH₂ analog of MZ-5-156 (KT-48) was further modified by single substitutions using Pal¹; d -Tpi²; d - or l -Phe⁴; Phe(pX)⁶×= F, Cl, I; Tyr⁷; Aib⁸; Tyr(Me)¹⁰ or Phe(pCl)¹⁰. Four peptides had multiple substitutions. All the analogs were evaluated for their ability to inhibit GH release induced by hGHRH(1-29)NH₂in vitro and some were also tested in vivo. Peptides [PhAc-Tyr¹,D-Arg²,Phe(pI)⁶,Abu¹⁵,Nle²⁷]hGHRH(1-29)NH₂ (KT-30), [PhAc-Tyr¹,D-Arg²,Phe(pCl)⁶,Aib⁸,Abu¹⁵,Nle²⁷]hGHRH(1-29)NH₂ (KT-50) and [PhAc-Tyr¹,D-Arg²,Phe(pCl)⁶,Tyr(Me)¹⁰,Abu¹⁵,Nle²⁷]hGHRH(1-29)NH₂ (KT-40) with Phe(pI)⁶, Aib⁸ or Tyr(Me)¹⁰modifications, respectively, showed high and prolonged inhibitory effect in superfused rat pituitary system. Analog KT-50 also exhibited a strong and long-term inhibitory activity in vivo in rats. Most of the new analogs showed high binding affinities to rat pituitary GHRH receptors.     

Angiotensin II analogues with N‐terminal lactam bridge cyclization: an overview on AT1 receptor activation and tachyphylaxis

Angiotensin II (AngII) is the final active product of the renin enzymatic cascade, which is responsible for sustaining blood pressure. To investigate the effect of N‐terminal cyclization on AT₁ activation and tachyphylaxis, we designed conformationally constrained analogues with an i‐(i + 1) lactam bridge. All analogues presented the same binding coefficient and tachyphylactic index, but some of them such as Cyclo (0‐1a) [Glu⁰, endo‐(Lys^1a)]‐AngII and Cyclo (0‐1a) [Asp⁰, endo‐(Orn^1a)]‐AngII showed higher potency. The same tachyphylactic index presented by AngII and cyclic analogues was surprising. We expected a variation after the modification of AngII N‐terminal region.     

CARBON-13-N.M.R.-SPECTRA OF ANTAMANIDE,SEVERAL ANALOGUES AND THEIR ALKALI ION COMPLEXES

Natural abundance carbon-13 Fourier transform n.m.r.-spectra were obtained of the cyclic decapeptides [Phe⁴ Val⁶] antamanide (J); antamanide (II); [Tyr⁵] antamanide (III); [Ala¹] antamanide (IV) and their ion complexes (I)-Na⁺, (II)-Na⁺, (II)-Li⁺, (III)-Na⁺ and (IV)-Na⁺, Based upon literature data, systematic comparisons and model compounds, a line assignment approach was performed for the majority of the aliphatic carbons. The spectra of the [Ala Val⁶] analogues (V) (Bystrov et al., 1972) and II (measured in CD₃CN Patel, 1973a, b) and their complexes were also assigned. Characteristic chemical shift variations observed upon complex formation were calculated (free peptide/Me⁺ complex) for a number of corresponding carbons, revealing shift changes up to 2.4 p.p.m. Preliminary calculations of the angle at selected prolines for some ion complexes are included.