PBAN selective antagonists: inhibition of PBAN induced cuticular melanization and sex pheromone biosynthesis in moths

A D-Phe scan (sequential D-Phe replacement) library of linear peptides, synthesized on the basis of a slightly modified active sequence of PBAN (YFSPRL-amide) was employed to detect potential inhibitors of cuticular melanization in Spodoptera littoralis larvae and to compare their stimulatory and inhibitory melanization activity with their pheromonotropic agonistic and antagonistic activities. A quantitative melanotropic assay was used to monitor the extent of cuticular melanization elicited by Hez-PBAN1-33NH2 in S. littoralis larvae in the presence and absence of the D-Phe peptides. The data revealed the presence of two partial melanotropic antagonists, and disclosed the presence of selective pure melanotropic agonists and pure pheromonotropic antagonists indicating differences in the inhibitory and stimulatory patterns of the library with respect to both activities. The differences between the pheromonotropic and melanotropic inhibitory patterns of the peptides hints at the possibility that sex pheromone biosynthesis in the pheromone gland of Heliothis peltigera females and induction of cuticular melanization in S. littoralis may be mediated by different receptors (that may result either from presence of different receptor sub-types or may reflect species differences in receptor structure and/or properties) despite the fact that they are induced by the same peptide (PBAN1-33NH2).     

Inhibition of PK/PBAN-mediated functions in insects: discovery of selective and non-selective inhibitors

The antagonistic properties of a few linear and backbone cyclic (BBC) conformationally constraint peptide libraries and their analogs, were tested for the ability to inhibit pyrokinin/pheromone biosynthesis activating neuropeptide (PK/PBAN) mediated functions: sex pheromone biosynthesis in Heliothis peltigera female moths, cuticular melanization in Spodoptera littoralis larvae, pupariation in the fleshfly Neobellieria bullata and hindgut contraction in Leucophaea maderae, elicited by exogenously injected PBAN, pheromonotropin (PT), leucopyrokinin (LPK), myotropin (MT) or by the endogenous peptides. The data revealed differential inhibitory patterns within the same assay with different elicitors (in both the pheromonotropic and melanotropic assays) and among the different functions and disclosed selective antagonists, hinting at the possibility that the receptors that mediate those functions may differ from one another structurally.     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists.

A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C-terminal hexapeptide sequence Tyr-Phe-Ser-Pro-Arg-Leu-NH2 of PBAN1-33NH2; whereas library II (D-Phe library) was based on the sequence of the PBAN lead linear antagonist Arg-Tyr-Phe-d-Phe-Pro-Arg-Leu-NH2. In both libraries the Pro residue was replaced by the BBC building unit Nalpha-(omega-aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N-terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl3I for selective deprotection of the Boc group from the building unit prior to on-resin amino-end to backbone-nitrogen (AE-BN) cyclization, during solid-phase synthesis with Fmoc chemistry.     

Backbone cyclic peptide antagonists, derived from the insect pheromone biosynthesis activating neuropeptide, inhibit sex pheromone biosynthesis in moths.

We describe an application of the backbone cyclization and cycloscan concept for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) antagonists capable of inhibiting sex pheromone biosynthesis in Heliothis peltigera female moths. Two backbone cyclic (BBC) sub-libraries were designed and synthesized. The structure of the first sub-library ([Arg27]PBAN27-33NH2, termed the Ser sub-library) was based on the active C-terminal hexapeptide sequence (Tyr-Phe-Ser-Pro-Arg-Leu-NH2) of PBAN1-33NH2, which was found to comprise its active core. The second sub-library ([Arg27, D-Phe30]PBAN27-33NH2, termed the D-Phe sub-library) was based on the sequence of the lead antagonist Arg-Tyr-Phe-(D)Phe-Pro-Arg-Leu-NH2. In both sub-libraries the Pro residue was replaced by an Nalpha(omega-amino-alkyl)Gly building unit having various lengths of the alkyl chain. All the cyclic peptides in each sub-library had the same primary sequence and the same location of the ring. The members of each library differed from each other by the bridge size and bridge chemistry. Screening of the two libraries for pheromonotropic antagonists resulted in the disclosure of four compounds that fully inhibited sex pheromone biosynthesis at 1 nmol and were devoid of agonistic activity. All antagonistic peptides originated from the D-Phe sub-library. Substitution of the D-Phe30 amino acid with a Ser resulted in a loss of antagonistic activity. Agonistic activities were exhibited by peptides from both sub-libraries.     

Discovery of 7alpha-substituted dihydrotestosterones as androgen receptor pure antagonists and their structure-activity relationships

A series of 7alpha-substituted dihydrotestosterone derivatives were synthesized and evaluated for androgen receptor (AR) pure antagonistic activity. From reporter gene assay (RGA), the compound with a side chain containing N-n-butyl-N-methyl amide (19a) showed pure antagonistic activity (IC(50)=340nM, FI(5)>10,000nM), whereas known AR antagonists showed partial agonistic activities. The optimization of 19a led to compound 23 (CH4892280), which showed more potent pure antagonistic activity (IC(50)=190nM, FI(5)>10,000nM). The SARs of tested compounds suggested that the length of the side chain and the substituents on the amide nitrogen are important for pure antagonistic activities.     

Insect neuropeptide antagonists

The development of a new integrated approach to the generation of a novel type of insect neuropeptide (Np) antagonists and putative insect control agents based on backbone cyclic compounds is described. The approach, termed the backbone cyclic neuropeptide-based antagonist (BBC-NBA), was applied to the insect pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) family as a model, and led to the discovery of a potent linear lead antagonist and several highly potent, metabolically stable BBC antagonists, devoid of agonistic activity, which inhibited PBAN-mediated activities in moths in vivo. This review briefly summarizes our knowledge of insect Nps, describes the PK/PBAN Np family, presents the basic concepts behind the BBC-NBA approach, and introduces the advantages of this method for generation of Np agonists, antagonists and insecticide prototype molecules.     

Discovery of a linear lead antagonist to the insect pheromone biosynthesis activating neuropeptide (PBAN)

We report the discovery of a linear lead antagonist for the insect pheromone biosynthesis activating neuropeptide (PBAN) which inhibits sex pheromone biosynthesis in the female moth Heliothis peltigera. Two approaches have been used in attempting to convert PBAN agonists into antagonists. The first involved omission of the C-terminal amide and reduction of the sequence from the N-terminus in a linear library based on PBAN 1-33NH(2.) The second involved replacement of L amino-acids by the D hydrophobic amino acid D-Phe in a linear library based on PBAN28-33NH(2.) Screening of the two libraries for pheromonotropic antagonists resulted in the disclosure of one compound out of the D-Phe library (Arg-Tyr-Phe-D-Phe-Pro-Arg-Leu-NH(2)) which inhibited sex pheromone production by 79 and 64% at 100 pmol in two moth colonies and exhibited low agonistic activity. Omission of the C-terminal amide in PBAN 1-33NH(2) and its shorter analogs did not lead to the discovery of an antagonistic compound.     

Identification of a series of tetrahydroisoquinoline derivatives as potential therapeutic agents for breast cancer

A series of tetrahydroisoquinoline-N-phenylamide derivatives were designed, synthesized, and tested for their relative binding affinities, and antagonistic activities against estrogen receptor (ER). Compound 1f (relative binding affinity, RBA=5) showed higher binding affinity than tamoxifen (RBA=1), a potent ER antagonist and currently being used for breast cancer therapy. Compound 1f also exerted optimal antagonistic activity against ER in reporter and cell proliferation assays. Interestingly, compound 1j, which only has a minor agonistic effect against ER, acted as a progesterone receptor (PR) antagonist and exerted agonistic activity against AP-1 through ER pathway. Our results show that these new compounds can be employed as leading pharmacophore for further development of potent selective ER and/or PR modulators or antagonists.     

Structure-activity relationship of [Nphe1]-NC-(1-13)-NH2, a pure and selective nociceptin/orphanin FQ receptor antagonist.

A series of analogs of the ORL1 receptor antagonist [Nphe1]-NC(1-13)-NH2 was prepared and tested for agonistic and antagonistic activities in the mouse vas deferens, a preparation that shows high sensitivity to nociceptin and related peptides. The purpose of this study was to determine the role of the aromatic residue at the N-terminal for antagonism and eventually identify compounds with improved potency. Results indicated that all 23 compounds are inactive as agonists, and the antagonistic potency of the initial template [Nphe1]-NC(1-13)-NH2 is high (pKB 6.43) compared with those of all other compounds except [(S)(betaMe)Nphe1]NC(1-13)-NH2 (pK(B) 6.48). The other 22 compounds can be divided into two groups: 10 show antagonistic potencies (pK(B)) ranging from 5.30 to 5.86, whereas the other 12 compounds are inactive. This study clearly shows that the aromatic ring of Nphe is very critical for the interaction with the ORL1 receptor and can not be enlarged or sterically modified without significant loss of antagonistic potency.     

Effects of structural modifications of N-CPM-normorphine derivatives on agonist and antagonist activities in isolated organs

The agonistic and antagonistic properties of N-cyclopropylmethyl (N-CPM) morphine derivatives were observed in mouse vas deferens (MVD), longitudinal muscle of guinea pig ileum (GPI) and rabbit vas deferens (LVD). In MVD the K(e) values of the titled compounds (N-CPM-morphine, N-CPM-isomorphine, N-CPM-dihydromorphine, N-CPM-dihydroisomorpPhine, N-CPM-dihydromorphone and naltrexone) were measured for mu-, kappa- and delta-receptors using normorphine, ethylketocyclazocine (EKC) and D-Pen2-D-Pen5-enkephaline (DPDPE) as selective agonists on the receptors, respectively. For mu-receptors of MVD the tested compounds showed similar affinity. For kappa-receptors the non-iso-6-OH derivatives possessed much less affinity than the iso-derivatives. Similar difference could be observed for delta-receptors. The agonistic activities of these compounds in MVD were observed to be between 0-20% of the inhibition of muscle contractions. In GPI the compounds except naltrexone possessed strong agonistic activities effectively antagonized by nor-binaltorphimine (nor-BNI) (K(e) of nor-BNI was 0.23 nM) suggesting that they were strong kappa-receptor agonists. We investigated these agents in LVD too, which contains kappa-receptors, but they did not produce any agonist potencies. It raises the possibility that the kappa-receptor subtypes of LVD and MVD are different from the kappa-receptor subtype of GPI or the vasa deferentia contain much fewer kappa-receptors than GPI and the intrinsic activities of these compounds are too small to reach the 50% inhibition of the contractions.     

Structure-activity relationship of cyclic peptide penta-c[Asp-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys]-NH(2) at the human melanocortin-1 and -4 receptors: His(6) substitution

A series of MT-II related cyclic peptides, based on potent but non-selective hMC4R agonist (Penta-c[Asp-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys]-NH(2)) was prepared in which His(6) residue was systematically substituted. Two of the most interesting peptides identified in this study are Penta-c[Asp-5-ClAtc-DPhe-Arg-Trp-Lys]-NH(2) and Penta-c[Asp-5-ClAtc-DPhe-Cit-Trp-Lys]-NH(2) which are potent hMC4R agonists and are either inactive or weak partial agonists (not tested for their antagonist activities) in hMC1R, hMC3R and hMC5R agonist assays.     

An amphiphilic, PK/PBAN analog is a selective pheromonotropic antagonist that penetrates the cuticle of a heliothine insect

A linear pyrokinin (PK)/pheromone biosynthesis activating neuropeptide (PBAN) antagonist lead (RYF[dF]PRLa) was structurally modified to impart amphiphilic properties to enhance its ability to transmigrate the hydrophobic cuticle of noctuid moth species and yet retain aqueous solubility in the hemolymph to reach target PK/PBAN receptors within the internal insect environment. The resulting novel PK/PBAN analog, Hex-Suc-A[dF]PRLa (PPK-AA), was synthesized and evaluated as an antagonist in a pheromonotropic assay in Heliothis peltigera against 4 natural PK/PBAN peptide elicitors (PBAN; pheromonotropin, PT; myotropin, MT; leucopyrokinin, LPK) and in a melanotropic assay in Spodoptera littoralis against 3 natural PK/PBAN peptide elicitors (PBAN, PT, LPK). The analog proved to be a potent and efficacious inhibitor of sex pheromone biosynthesis elicited by PBAN (84% at 100 pmol) and PT (54% at 100 pmol), but not by MT and LPK. PPK-AA is a selective pure antagonist (i.e., does not exhibit any agonistic activity) as it failed to inhibit melanization elicited by any of the natural PK/PBAN peptides. The analog was shown to transmigrate isolated cuticle dissected from adult female Heliothis virescens moths to a high extent of 25-30% (130-150 pmol), representing physiologically significant quantities. PPK-AA represents a significant addition to the arsenal of tools available to arthropod endocrinologists studying the endogenous mechanisms of PK/PBAN regulated processes, and a prototype for the development of environmentally friendly pest management agents capable of disrupting the critical process of reproduction.     

Benzothiazole-based N-(phenylsulfonyl)amides as a novel family of PPARα antagonists

The discovery of PPAR antagonists is emerging as an useful tool for elucidating the biological role of the receptor. Here we report the identification of N-(phenylsulfonyl)amides containing the benzothiazole scaffold, a novel class of potent PPARα antagonists obtained from chemical modification of carboxylic acid agonists. In this work, a group of phenylsulfonamides were synthesized and in vitro evaluated against the agonistic effect of GW7647; they showed an inhibitory effect on PPARα activation, with best compounds revealing a dose-dependent antagonistic profile. Some of these antagonists showed also an inhibitory effect on CPT1A pattern expression.     

Evaluation of a focused library of N-aryl L-homoserine lactones reveals a new set of potent quorum sensing modulators

A focused library of N-aryl l-homoserine lactones was designed around known lactone leads and evaluated for antagonistic and agonistic activity against quorum-sensing receptors in Agrobacterium tumefaciens, Pseudomonas aeruginosa, and Vibrio fischeri. Several compounds were identified with significantly heightened activities relative to the lead compounds, and new structure-activity relationships (SARs) were delineated. Notably, 4-substituted N-phenoxyacetyl and 3-substituted N-phenylpropionyl l-homoserine lactones were identified as potent antagonists of TraR and LuxR, respectively.     

Discovery and design of novel vasopressin hypotensive peptide agonists

This presentation will trace the serendipitous discovery of novel vasopressin (VP) hypotensive agonists d(CH2)5[D-Tyr(Et)2,X3]VAVP (where X = Arg, Lys). These peptides were uncovered as part of an ongoing program aimed at the design of potent and selective VP antidiuretic (V2 receptor) antagonists. We will also present highlights of our subsequent preliminary studies seeking (i) to design high affinity radioiodinatable ligands for the localization and characterization of the putative VP vasodilatory (V1c?) receptor; (ii) to identify the structural features of selective and non-selective cyclic and linear VP and oxytocin (OT) antagonists of the V2 receptor, the vascular (V1a) receptor and of the uterine (OT) receptor required for hypotensive agonism and; (iii) to enhance hypotensive potency. These novel VP hypotensive agonists could serve as valuable research tools in studies on the roles of VP in blood pressure regulation and may also lead to the development of a new class of therapeutically useful antihypertensives.     

Novel linear antagonists of the antidiuretic (V2) and vasopressor (V1) responses to vasopressin

We report the solid phase synthesis of a series of 16 linear analogues of the cyclic antagonist of the antidiuretic (V2) and the vasopressor (V1) responses to arginine vasopressin (AVP), d(CH2)5[D-Tyr(Et)2, Val4]AVP(A). Peptide 1, the linear precursor of (A), (CH2)5(SH)-CH2-CO-D-Tyr(Et)-Phe-Val-Asn-Cys-Pro-Arg-Gly-NH2 was modified at position six with alpha-L-aminobutyric acid (Abu) to give peptide 2. Further modifications of the Abu6 analogue (No. 2) at position one by substituting cyclohexylacetic acid (Caa), cyclohexylpropionic acid (Cpa), 1-adamantaneacetic acid (Aaa), phenylacetic acid (Phaa), tert.-butylacetic acid (t-Baa), isovaleric acid (Iva), propionic acid (Pa), L-penicillamine (P), tert.-butoxycarbonyl (Boc) or omitting any substituent at this position, and/or in combination with Arg-NH2(9), Ala-NH2(9), D-Arg8-Arg-NH2(9), and desGly9 modifications yielded the remaining 14 peptides. All 16 peptides were examined for agonistic and antagonistic potencies in AVP V2 and V1 assays in rats. Apart from the Cpa analogue and the analogue lacking any substituent in the 1-position, all exhibit substantial V2 and V1 antagonism. A number are as potent as (A) as V2 antagonists. With an anti-V2 pA2 = 8.11 +/- 0.07, Aaa-D-Tyr(Et)-Phe-Val-Asn-Abu-Pro-Arg-Arg-NH2 (No. 6) is as potent as any cyclic AVP V2 antagonist reported to date. The PaI analogue of No. 6 exhibits promising anti-V2/anti-V1 selectivity. These findings prove conclusively that a ring structure is not a requirement for recognition of or for binding to AVP V2 or V1 receptors. This discovery thus offers a promising new approach to the design of peptide and non-peptide antagonists of AVP and perhaps also to other cyclic peptides such as somatostatin, atrial-natriuretic factor, insulin, and the recently discovered endothelin. Some of these linear antagonists may be of value as pharmacological tools and as therapeutic agents.     

Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues.

A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C- and N-terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N-carboxy alkylated and N-amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc-glycine to the N-alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre-built in solution. For backbone cyclization at the C-terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea-pig ileum (GPI) assays. Additionally, the potentiation of the BK-induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [D-Phe7]-BK. Side chain as well as backbone cyclization in the N-terminus of [D-Phe7]-BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin-induced contraction of guinea-pig ileum.