Comparison of insect kinin analogs with cis-peptide bond motif 4-aminopyroglutamate identifies optimal stereochemistry for diuretic activity

The insect kinins are present in a wide variety of insects and function as potent diuretic peptides, though they are subject to rapid degradation by internal peptidases. Insect kinin analogs incorporating stereochemical variants of (2S,4S)-4-aminopyroglutamate (APy), a cis-peptide bond motif, demonstrate significant activity in a cricket diuretic assay. Insect kinin analogs containing (2R,4R)-APy, (2S,4R)-APy and (2S,4S)-APy are essentially equipotent on an insect diuretic assay, with EC(50) values of about 10(-7)M, whereas the (2R,4S)-APy analog is at least 10-fold more potent (EC(50) = 7 x 10(-9)M). Conformational studies in aqueous solution indicate that the (2R,4S)-APy analog is considerably more flexible than the other three variants, which may explain its greater potency. The work identifies the optimal stereochemistry for the APy scaffold with which to design biostable, peptidomimetic analogs with the potential to disrupt critical insect kinin-regulated processes in insects.     

Bioactivity of growth hormone releasing hormone (1-29) analogues after SC injection in man

The bioactivity of growth hormone releasing hormone 1-29 [GHRH(1-29)NH2] has been compared with that of an agonist analogue [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2, in normal male volunteers. Using a submaximal dose of 3 micrograms/kg administered subcutaneously, peak growth hormone (GH) response and area under the GH curve were similar for the native and agonist analogue. In addition, no significant differences were found in peak GHRH(1-29) immunoreactivity, area under the GHRH(1-29) curves or plasma disappearance rates of the two peptides. The results suggest that, in keeping with the relative activities of other "superactive" analogues tested so far, the greatly enhanced activity of [Ac-D-Tyr1,D-Ala2]-GHRH(1-29)NH2 observed in the rat is not found in humans. It is possible that this species difference is due to differences in the interaction of GHRH peptides with the rat and the human somatotroph GHRH receptor.     

Phosphonamidates as transition-state analogue inhibitors of thermolysin

Six phosphorus-containing peptide analogues of the form Cbz-NHCH2PO2--L-Leu-Y (Y = D-Ala, NH2, Gly, L-Phe, L-Ala, L-Leu) have been prepared and evaluated as inhibitors of thermolysin. The Ki values for these compounds range from 1.7 microM to 9.1 nM and correlate well with the Km/kcat values for the corresponding peptide substrates [Morihara, K., & Tsuzuki, H. (1970) Eur. J. Biochem. 15, 374-380] but not with the Km values alone. The correlation noted between inhibitor Ki and substrate Km/kcat is the most extensive one of this type, providing strong evidence that the phosphonamidates are transition-state analogues and not simply multisubstrate ground-state analogues. Cbz-NH2CH2PO2--L-Leu-L-Leu (Ki = 9.1 nM) is the most potent inhibitor yet reported for thermolysin.     

Structure-activity analysis of truncated orexin-A analogues at the orexin-1 receptor

Truncated peptide analogues of orexin-A were prepared and their biological activity assesed at the orexin-1 receptor. Progressive N-terminal deletions identified the minimum C-terminal sequence required for maintaining a significant agonist effect, whilst an alanine scan and other pertinent substitutions identified key side-chain and stereochemical requirements for receptor activation.     

Covalently cyclized agonist and antagonist analogues of bombesin and related peptides.

During a search for possible cyclization points in shortened, potent bombesin agonists and antagonists, it was found that the joining of amino acid residues in positions 6 and 14 by various means resulted in retention of significant binding affinity for rat pancreatic acini and murine Swiss 3T3 cells. In one series of analogues, Cys residues in these positions were used for bridging via a disulfide bond. (D)-C-Q-W-A-V-G-H-L-C-NH2 retained significant binding affinity for rat pancreatic acini cells and was a full amylase releasing agonist (EC50 187 nM). Potency was markedly increased by substituting D-Ala for Gly (EC50 67 nM compared to 10 nM for its linear counterpart) and was decreased by substituting L-Cys for D-Cys in this analogue (EC50 214 nM), thus strongly suggesting stabilization of peptide folding by the D residues. Elimination of the COOH-terminal amino acid produces competitive antagonists in the linear analogues; however, (D)-C-Q-W-A-V-G-H-C-NH2 was devoid of activity. Likewise, cyclization to position 13 with the 14 amino acids intact to give (D)-C-Q-W-A-V-G-H-C-L-NH2 resulted in an almost inactive peptide. On the other hand, as in the linear series, the reduced peptide bond analogue, (D)-C-Q-W-A-V-(D)-A-H-L-psi (CH2NH)-C-NH2, was a receptor antagonist (IC50 5.7 mM), albeit much weaker than the corresponding linear analogues, but with no residual agonist activity. Direct head-to-tail cyclization was also tried. Both cyclo[(D)-F-Q-W-A-V-G-H-L-L] (EC50 346 nM) and the shorter cyclo [Q-W-A-V-G-H-L-L] (EC50 1236 nM) were full agonists. Elimination of the COOH-terminal residue in cyclo[(D)-p-Cl-F-Q-W-A-V-(D)-A-H-L] produced an agonist (EC50 716 nM) rather than an antagonist. These results provide support for the proposal that both bombesin agonists and antagonists adopt a folded conformation at their receptor(s). Furthermore, the retention of appreciable potencies using several cyclization strategies and chain lengths suggests that further optimization of these structures both in terms of potency and ring size is possible. Since these peptides have increased conformational restriction, they should begin to serve as useful substrates for NMR and molecular modeling studies aimed at comparing the obviously subtle differences between agonist and antagonist structures.     

Novel mu-selective Met-enkephalinamide analogs with antagonist activity. Synthesis, receptor binding, analgesic properties and conformational studies

Four novel mu-selective peptide antagonists have been synthesized and examined for receptor binding, analgesic agonist and antagonist activity and energy conformational properties. These peptides were designed by analogy to results of molecular modeling of 3-phenyl piperidines which led to incorporating four modified tyrosine residues, m-Tyr, beta-methyl-m-Tyr, N-phenethyl-m-Tyr and alpha, beta-dimethyl-m-Tyr into D-Ala2-Met5-enkephalinamide. Peptides were synthesized by stepwise solution synthesis using an active ester coupling procedure. Receptor binding assays were performed on rat brain homogenates and data were analyzed by a modified version of the program LIGAND. Analgesic agonist and antagonist activity was evaluated by the mouse tail-flick test. Energy-optimized conformations were obtained using a program called Molecule-AIMS. The results demonstrate that relative ratios of in vivo agonist and antagonist potencies in D-Ala2-Met5-enkephalinamides can be modulated by chemical modification of the tyrosine residue. A shift in the phenolic-OH position from para to meta significantly enhances relative antagonist versus agonist activity; addition of a beta-CH3 group to the m-Tyr enhances mu-selectivity and leads to nearly equal agonist/antagonist activity. Energy conformational studies indicate that all analogs with high mu-receptor affinity examined have a common energy accessible B'II 2-3 turn conformation similar to that previously identified for high mu-affinity binding in peptides, lending further support to this candidate conformer. This conformer also has tyrosine side-chain angles which allowed total overlap with the amine and phenolic groups of a known structure of 3-(m-OH phenyl)-piperidine. This structural similarity together with the observation of mixed agonist antagonist activity in both types of opioids confirms the rationale upon which design of these peptides was based.     

Stereochemical requirements for receptor recognition of the mu-opioid peptide endomorphin-1

A series of diastereoisomers of endomorphin-1 (EM1, Tyr(1)-Pro(2)-Trp(3)-Phe(4)-NH(2)) have been synthesized and their potency measured using the guinea pig ileum assay. [D-Phe(4)]EM1 possessed 1/10 the potency of EM1, while potencies of [D-Tyr(1)]EM1 and [D-Trp(3)]EM1 were 50- and 100-fold lower, respectively. Drastic loss of activity occurred in the [D-Pro(2)]EM1 peptide. The structural determinants for the inactivity and reduced potency of the diastereoisomers were investigated using NMR spectroscopy and conformational analysis. Simulations of trans-[D-Pro(2)]EM1 using NOE-derived distance constraints afforded well-defined structures in which Tyr and Trp side chains stack against the proline ring. The inactivity of [D-Pro(2)]EM1 was explained by structural comparison with EM1 (, FEBS Lett. 439:13-20). The two peptides showed an opposite orientation of the Trp(3) residue with respect to Tyr(1), thus suggesting a role of Pro(2) as a stereochemical spacer in orienting Trp(3) and Phe(4) toward regions suitable for mu-receptor interaction. The agonist activity of [D-Tyr(1)]EM1 and [D-Trp(3)]EM1 was attributed to their ability to adopt low-energy conformations that mimic those of EM1. The requirements for mu-receptor activation were examined further by comparing EM1 with the mu-peptide [D-Ala(2), MePhe(4), Gly-ol]-enkephalin (DAMGO). Conformations of DAMGO with a Tyr(1)-MePhe(4) phenyl ring separation of approximately 12 A were found to mimic Tyr(1)-Phe(4) of EM1, thus suggesting overlapping binding modes between these two peptides.     

Structure-activity relationships for in vitro diuretic activity of CAP2b in the housefly

A series of truncated and Ala-replacement analogs of the peptide Manse-CAP2b (pELYAFPRV-NH(2)) were assayed for diuretic activity on Malpighian tubules of the housefly Musca domestica (M. domestica). The C-terminal hexapeptide proved to be the active core, the minimum sequence required to retain significant diuretic activity. However, full activity required the C-terminal heptapeptide, which was equipotent with the most active of the native housefly CAP2b peptides. Replacement of Arg(7) and Val(8) with Ala led to inactivity and a large 70-fold drop in potency, respectively, indicating that these were critical residues. The Leu(2) was semicritical, where a six-fold loss in potency was observed. Conversely, the replacement of all other residues with Ala led to much smaller effects on potency and these positions were considered to be noncritical. This structure-activity relationship data can aid in the design of mimetic agonist/antagonist analogs of this diuretic peptide family with enhanced biostability and bioavailability, as tools for arthropod endocrinologists and as potential pest management agents capable of disrupting the water balance in pest flies.     

Antinociceptive effects of [D-Ala2]deltorphin II, a highly selective delta agonist in vivo

The present study has characterized the antinociceptive actions of [D-Ala2]deltorphin II following intracerebroventricular (i.c.v.) administration in the mouse tail-flick test. [D-Ala2]deltorphin II produced dose- and time-related antinociception, with maximal effects at +10 min and significant antinociception which lasted for 40-60 min. [D-Ala2]deltorphin II was 13-fold more potent than i.c.v. [D-Pen2, D-Pen5]enkephalin (DPDPE), a second highly selective delta agonist, and approximately equipotent with i.c.v. morphine in producing antinociception. The antinociceptive effects of i.c.v. [D-Ala2]deltorphin II and DPDPE, but not those of morphine, were antagonized by the selective delta antagonist, ICI 174,864. In contrast, pretreatment with the non-equilibrium mu antagonist, beta-funaltrexamine blocked morphine antinociception, but failed to antagonize [D-Ala2]deltorphin II and DPDPE antinociception. These data indicate that [D-Ala2]deltorphin II produced its antinociceptive effects at a supraspinal delta receptor. [D-Ala2]deltorphin II appears to be the most appropriate delta opioid agonist currently available for studies in vivo and support the involvement of delta receptors in supraspinal antinociception.     

Comparative biological activities of potent analogues of alpha-melanotropin. Effect of nonaromatic and para substituted aromatic amino acids at position 7

Several alpha-melanotropin (alpha-MSH) analogues with para substituted aromatic and nonaromatic amino acids in the 7-position of the hormone were prepared and their melanotropic activities determined in the frog (Rana pipiens) and lizard (Anolis carolinensis) skin bioassays. D and L-Phe(p-NO2), D- and L-Tyr, D- and L-Ala, and Gly were substituted in the 7-position. The use of substituted D or L-aromatic amino acids in the 7th position of the central Ac-[Nle4]-alpha-MSH4-11-NH2 fragment resulted in a loss in potency relative to the corresponding phenylalanine-containing analogue. The loss in potency cannot be due entirely to steric hindrance at the melanophore receptor, since nonaromatic amino acids substituted in the 7th position of this octapeptide fragment also generally led to a loss in biological activity. We reported previously that replacement of phenylalanine-7 by its D enantiomer led to a marked increase in potency in each fragment analogue tested. Analogues containing other D amino acids in the 7th position also were more potent than their L amino acid-containing analogues with one exception: Ac-[Nle4, Ala7]-alpha-MSH4-11-NH2 was more potent than Ac-[Nle4, D-Ala7]-alpha-MSH4-11-NH2 in the frog skin bioassay. Replacement of phenylalanine-7 by glycine resulted in a large decrease in potency in both bioassays, illustrating the importance of the side chain group, in this position of alpha-MSH, to biological potency of the hormone.     

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)     

Regulation of amino acid-sensitive TOR signaling by leucine analogues in adipocytes

In adipocytes, amino acids stimulate the target of rapamycin (TOR) signaling pathway leading to phosphorylation of the translational repressor, eIF-4E binding protein-I (4E-BP1), and ribosomal protein S6. L-leucine is the primary mediator of these effects. The structure-activity relationships of a putative L-leucine recognition site in adipocytes (LeuR(A)) that regulates TOR activity were analyzed by examining the effects of leucine analogues on the rapamycin-sensitive phosphorylation of the translational repressor, eIF-4E binding protein-I (4E-BP1), an index of TOR activity. Several amino acids that are structurally related to leucine strongly stimulated 4E-BP1 phosphorylation at concentrations greater than the EC(50) value for leucine. The order of potency was leucine > norleucine > threo-L-beta-hydroxyleucine approximately Ile > Met approximately Val. Other structural analogues of leucine, such as H-alpha-methyl-D/L-leucine, S-(-)-2-amino-4-pentenoic acid, and 3-amino-4-methylpentanoic acid, possessed only weak agonist activity. However, other leucine-related compounds that are known agonists, antagonists, or ligands of other leucine binding/recognition sites did not affect 4E-BP1 phosphorylation. We conclude from the data that small lipophilic modifications of the leucine R group and alpha-hydrogen may be tolerated for agonist activity; however, leucine analogues with a modified amino group, a modified carboxylic group, charged R groups, or bulkier aliphatic R groups do not seem to possess significant agonist activity. Furthermore, the leucine recognition site that regulates TOR signaling in adipocytes appears to be different from the following: (1) a leucine receptor that regulates macroautophagy in liver, (2) a leucine recognition site that regulates TOR signaling in H4IIE hepatocytes, (3) leucyl tRNA or leucyl tRNA synthetase, (4) the gabapentin-sensitive leucine transaminase, or (5) the system L-amino acid transporter.     

Structural requirement for the agonist activity of the TLR2 ligand Pam2Cys

Synthetic lipopeptides have demonstrated great potential as a vaccine strategy for eliciting cellular and humoral immunity. One of the most potent lipid moieties used is S-[2,3-bis(palmitoyloxy)propyl]cysteine (Pam2Cys). Pam2Cys binds to and activates dendritic cells by engagement of Toll like receptor 2 (TLR 2). In this study, we have investigated the structural requirement of the agonist activity of Pam2Cys by varying the three structural elements of the core structure S-(2,3-dihydroxypropyl)-cysteine namely (1) the α-amino group of the cysteine residue (2) the sulphur atom of the cysteine residue and (3) the 2,3-dihydroxypropyl moiety. Four novel analogues of Pam2Cys were made and each of these analogues were incorporated into vaccine constructs and examined for immunogenicity. Our results demonstrate that (1) the potency of the peptide vaccine is least affected by removal of the amino group (2) substitution of the sulphur atom with an amide bond leads to significant reduction of biological activity (3) removal of the amino group and at the same time substitution of the sulphur with an amide bond significantly decreases the biological activity (4) in the two analogues in which the sulphur atom is replaced with an amide bond the analogue containing the 1,3-dihydroxypropyl moiety demonstrates higher activity than the one which contains 2,3-dihydroxypropyl. In conclusion, the results demonstrate strict structural requirements for agonist activity of the TLR2 ligand Pam2Cys.     

Diuretic and myotropic activities of N-terminal truncated analogs of Musca domestica kinin neuropeptide

Musca kinin (Musdo-K; NTVVLGKKQRFHSWG-NH(2)) and N-terminal truncated analogs of 4-14 residues in length were assayed for diuretic and myotropic activity on housefly Malpighian tubules and hindgut, respectively. The pentapeptide was the minimum sequence required for biological activity, but it was > 5 orders of magnitude less potent than the intact peptide. The pharmacological profiles of the different analogs in the two assays were very similar, suggesting the same receptor is present on both tissues. Potency was little affected by the deletion of Asn(1), but was reduced > 10-fold after the removal of Thr(2). Deletion of the next 5 residues had relatively little effect, but after the second lysyl residue (Lys(8)) was removed potency fell by one to two orders of magnitude. There was a similar drop in potency after the removal of Arg(10), and at 100 microM the pentapeptide had only 20% of the diuretic activity of the intact peptide. The importance of Arg(10) was confirmed by comparing dose-response curves for Musdo-K [6-15] and Acheta kinin-V (AFSHWG-NH(2)) in the diuretic assay; the substitution of arginine by alanine produced a significant reduction in potency and some loss of activity.     

Discovery of potent and selective peptide agonists at the GRP-preferring bombesin receptor (BB2).

Analogues of the nonselective bombesin receptor synthetic agonist H-D-Phe-Gln-Trp-Ala-Val-betaAla-His-Phe-Nle-NH2 were prepared and their biological activity assessed at the NMB-preferring/bombesin receptor (NMB-R: BB1), the GRP-preferring/bombesin receptor (GRP-R: BB2) and the orphan receptor bombesin receptor subtype-3 (BRS-3; BB3). Progressive N-terminal deletions identified the minimum C-terminal sequences required for maintaining a significant agonist effect, whilst an alanine scan, targeted changes in stereochemistry and other pertinent substitutions identified key side-chain and stereochemical requirements for activation. Key structural elements required for functional potency at BB1 BB2 and BB3, and for selectivity between these receptor subtypes were established. Synthetic peptides were discovered. which were highly potent agonists at BB2 and extremely selective over both BB1 and BB3.     

Development of a potent bombesin receptor antagonist with prolonged in vivo inhibitory activity on bombesin-stimulated amylase and protein release in the rat.

Of the various types of potent bombesin(Bn)/gastrin releasing peptide receptor antagonists that have been discovered, the desMet14-methyl ester peptides are devoid of residual agonist activity and are among the most potent in terms of in vitro receptor blockade and also in terms of their prolonged inhibition of bombesin-stimulated amylase and protein release in the rat. We have now examined the in vitro and in vivo properties of a new series of methyl ester analogues, [D-Phe6]Bn(6-13)OMe, [D-Phe6,D-Ala11]Bn(6-13)OMe, N alpha-propionyl-[D-Ala24]GRP(20-26)OMe, and [D-pentafluoro-Phe6,D-Ala11]Bn(6-13)OMe, which have an additional D-amino acid substituent and some highly lipophilic moieties at the N-terminus. All analogues were able to potently antagonize the ability of Bn to stimulate amylase release from rat acinar cells, with IC50 values of 2.4, 2.5, 0.6, and 1.3 nM, respectively. The four peptides were found to have binding affinities for these cells comparable to Bn itself, with K(i)s of 10.3, 2.8, 5.5, and 3.6 nM, respectively, but all had little or no affinity for neuromedin B receptors on murine C6 cells. Single bolus IV injections of these peptides were found to potently inhibit amylase and protein release caused by IV infusion of bombesin into the rat. Generally the peptides containing the D-Ala substituent were longer acting than [D-Phe6]Bn(6-13)OMe, so that [D-Phe6,D-Ala11]Bn(6-13)OMe and N alpha-propionyl-[D-Ala24]GRP(20-26)OMe displayed significant inhibitory effects for up to 1.5 h after administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Biological activities of glucagon-like peptide-1 analogues in vitro and in vivo

Studies support a role for glucagon-like peptide 1 (GLP-1) as a potential treatment for diabetes. However, since GLP-1 is rapidly degraded in the circulation by cleavage at Ala(2), its clinical application is limited. Hence, understanding the structure-activity of GLP-1 may lead to the development of more stable and potent analogues. In this study, we investigated GLP-1 analogues including those with N-, C-, and midchain modifications and a series of secretin-class chimeric peptides. Peptides were analyzed in CHO cells expressing the hGLP-1 receptor (R7 cells), and in vivo oral glucose tolerance tests (OGTTs) were performed after injection of the peptides in normal and diabetic (db/db) mice. [D-Ala(2)]GLP-1 and [Gly(2)]GLP-1 showed normal or relatively lower receptor binding and cAMP activation but exerted markedly enhanced abilities to reduce the glycemic response to an OGTT in vivo. Improved biological effectiveness of [D-Ala(2)]GLP-1 was also observed in diabetic db/db mice. Similarly, improved biological activity of acetyl- and hexenoic-His(1)-GLP-1, glucagon((1-5)-, glucagon((1-10))-, PACAP(1-5)-, VIP(1-5)-, and secretin((1-10))-GLP-1 was observed, despite normal or lower receptor binding and activation in vitro. [Ala(8/11/12/16)] substitutions also increased biological activity in vivo over wtGLP-1, while C-terminal truncation of 4-12 amino acids abolished receptor binding and biological activity. All other modified peptides examined showed normal or decreased activity in vitro and in vivo. These results indicate that specific N- and midchain modifications to GLP-1 can increase its potency in vivo. Specifically, linkage of acyl-chains to the alpha-amino group of His(1) and replacement of Ala(2) result in significantly increased biological effects of GLP-1 in vivo, likely due to decreased degradation rather than enhanced receptor interactions. Replacement of certain residues in the midchain of GLP-1 also augment biological activity.     

Beta-amino acid analogs of an insect neuropeptide feature potent bioactivity and resistance to peptidase hydrolysis

Insect neuropeptides of the insect kinin class share a common C-terminal pentapeptide sequence F(1)X(1)(2)X(2)(3)W(4)G(5)-NH(2) (X(2)(3) = P, S, A) and regulate such critical physiological processes as water balance and digestive enzyme release. Analogs of the insect kinin class, in which the critical residues of F(1), P(3), and W(4) were replaced with beta(3)-amino acid or their beta(2)-homo-amino acid variants, have been synthesized by the solid phase peptide strategy. The resulting single- and double-replacement analogs were evaluated in an insect diuretic assay and enzyme digestion trials. Analogs modified in the core P(3) position produce a potent and efficacious diuretic response that is not significantly different from that obtained with the endogenous achetakinin peptides. The analogs also demonstrate enhanced resistance to hydrolysis by ACE and NEP, endopeptidases that inactivate the natural insect neuropeptides. This paper describes the first instance of beta-amino acids analogs of an arthropod peptide that demonstrate significant bioactivity and resistance to peptidase degradation.     

New antagonists of LHRH. II. Inhibition and potentiation of LHRH by closely related analogues

Modifications of the previously described LHRH antagonists, [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and the corresponding D-Hci6 analogue, have been made to alter the hydrophobicity of the N-terminal acetyl-tripeptide portion. Substitution of D-Trp3 with the less hydrophobic D-Pal(3) had only marginal effects on the antagonistic activities and receptor binding potencies of the D-Cit/D-Hci6 analogues, but it appeared to further improve the toxicity lowering effect of D-Cit/D-Hci6 substitution. Antagonists containing D-Pal(3)3 and D-Cit/D-Hci6 residues, i.e. [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH (SB-75) and [Ac-D-Nal(2)1, D-Phe(4Cl)2, D-Pal(3)3, D-Hci6, D-Ala10]LHRH (SB-88), were completely free of the toxic effects, such as cyanosis and respiratory depression leading to death, which have been observed in rats with the D-Trp3, D-Arg6 antagonist and related antagonists. Replacement of the N-acetyl group with the hydrophilic carbamoyl group caused a slight decrease in antagonistic activities, particularly in vitro. Introduction of urethane type acyl group such as methoxycarbonyl (Moc) or t-butoxycarbonyl (Boc) led to analogues that showed LHRH-potentiating effect. The increase in potency induced by these analogues, e.g. [Moc-D-Nal(2)1, D-Phe(4Cl)2, D-Trp3, D-Cit6, D-Ala10]LHRH and [Boc-D-Phe1, D-Phe(4Cl)2, D-Pal(3)3, D-Cit6, D-Ala10]LHRH, was 170-260% and persisted for more than 2 h when studied in a superfused rat pituitary system.     

Structure-activity relationships of benzylidene anabaseines in nicotinic acetylcholine receptors of cockroach nerve cords

Ten analogues of 6'-chloro-3-benzylideneanabaseine (CBA) bearing substituents at the ortho- and the para-positions of the phenyl group were synthesized, together with two related compounds. The affinity of the synthesized compounds for nicotinic acetylcholine receptors (nAChRs) in the nerve cord of the American cockroach (Periplaneta americana L.) was examined by the radioligand binding assay using [(3)H]epibatidine (EPI), a nAChR agonist. All 12 tested compounds inhibited [(3)H]EPI binding, showing K(i) values ranging from 14.6 to 6830nM. The potency variation of para-substituted CBA analogues was explained by the steric (Delta B(1)) and electronic (sigma(p)) parameters of the para-substituents, or by the steric parameter and the charge of the N1 nitrogen atom (qN(1)). Among the CBA analogues, only two compounds containing a dimethylamino group and a methoxy group at the para-position showed high insecticidal activity against the German cockroach (Blattella germanica) when injected after pretreatment with metabolic inhibitors. High-affinity analogues of CBA might be suitable probes for use in classifying and characterizing insect nAChR subtypes.