Substance P analogues function as bombesin receptor antagonists and inhibit small cell lung cancer clonal growth

Human small cell lung cancer (SCLC) produces and secretes BN/GRP (bombesin/gastrin releasing peptide). Because BN stimulates the growth of SCLC cells and these cells have receptors for BN-like peptides, it is important to define agents which disrupt this self-promoting autocrine growth cycle. Here, substance P analogues were evaluated as BN receptor antagonists using SCLC cell lines. (D-Arg¹, D-Pro², D-Trp^7,9, Leu¹¹) substance P [(APTTL)SP] was one of the more potent analogues tested in inhibiting BN-like peptide receptor binding with an IC₅₀ value of 1 μM. Micromolar concentrations of (APTTL)SP antagonized BN receptor mediated elevation of cytosolic Ca²⁺ levels and decreased the colony formation in soft agarose. These data suggest that SP analogues function as SCLC BN receptor antagonists and may be useful in disrupting the autocrine growth function of BN-like peptides.     

High affinity receptors for bombesin/GRP-like peptides on human small cell lung cancer

The binding of a radiolabeled bombesin analogue to human small cell lung cancer (SCLC) cell lines was investigated. (125I-Tyr4)bombesin bound with high affinity (Kd = 0.5 nM) to a single class of sites (2,000/cell) using SCLC line NCI-H446. Binding was reversible, saturable and specific. The pharmacology of binding was investigated using NCI-H466 and SCLC line NCI-H345. Bombesin and structurally related peptides, such as gastrin releasing peptide (GRP), but not other peptides, such as substance P or vasopressin, inhibited high affinity (125I-Tyr4)BN binding activity. Finally, the putative receptor, a 78,000 dalton polypeptide, was identified by purifying radiolabeled cell lysates on bombesin or GRP affinity resins and then displaying the bound polypeptides on sodium dodecylsulfate polyacrylamide gels. Because SCLC both produces bombesin/GRP-like peptides and contains high affinity receptors for these peptides, they may function as important autocrine regulatory factors for human SCLC.     

Influence of degradation on binding properties and biological activity of endomorphin 1

The recently-isolated endogenous peptide endomorphin 1 has high affinity for the mu opioid receptor and plays an important role in analgesia. Several of its degradation products have been isolated from the central nervous system. Degradation products present structural similarities and may influence the receptor binding properties and biological activity of the parent compound. Therefore, we investigated how degradation of endomorphin 1 might influence ligand binding to the mu opioid receptor, the consequent activation of G proteins and its antinociceptive effect. Both N- and C-terminal truncation of endomorphin 1 resulted in peptides presenting considerably lower opioid receptor binding potency. None of these peptides had an effect on GTP binding, nor was able to produce analgesia, suggesting that degradation destroys the biological activity of endomorphin 1.     

Peptide E and other proenkephalin-derived peptides are potent kappa opiate receptor agonists

Various proenkephalin-derived peptides such as peptide E and the bovine adrenal medulla peptides BAM-12P and BAM-22P are potent competitors on mu and kappa binding sites in guinea pig brain sections. Moreover, they are all potent agonists in the rabbit vas deferens, a specific kappa opiate receptor bioassay. As described before, dynorphin and some of its fragments are also potent kappa agonists. Our results suggest that not only prodynorphin-derived peptides could act as endogenous kappa ligands but also some proenkephalin-derived peptides such as peptide E.     

Pharmacological and immunohistochemical characterization of the APJ receptor and its endogenous ligand apelin

Apelin peptides have recently been identified to be the endogenous ligands for the G protein-coupled receptor APJ. However, little is known about the physiological roles of this ligand-receptor pairing. In the present study we investigated the pharmacology of several apelin analogues at the human recombinant APJ receptor using radioligand binding and functional assays. This has led to the identification of key residues in the apelin peptide required for functional potency and binding affinity through structure-activity studies. In particular, we have identified that replacement of leucine in position 5, or arginine in position 2 and 4 of the C-terminal apelin peptide, apelin-13, resulted in significant changes in pharmacology. We also investigated the detailed localization of pre-proapelin and APJ receptor mRNA in a wide range of human, rat and mouse tissues using quantitative RT-PCR, and carried out a detailed immunohistochemical study of the distribution of the APJ receptor in rat brain and spinal cord. Interestingly, the APJ receptor was not only co-localized in white matter with GFAP in the spinal cord, but was also clearly localized on neurones in the brain, suggesting that this receptor and its peptide may be involved in a wide range of biological process yet to be determined.     

Thymosin alpha 1-like peptides: localization and biochemical characterization in the rat brain and pituitary gland

Using a radioimmunoassay for thymosin alpha 1, endogenous thymosin-like peptides were characterized in the rat brain and pituitary gland. Thymosin alpha 1-like peptides were present in high concentrations in hypothalamus and pituitary extracts. These peptides were characterized using gel filtration techniques and the main peak of immunoreactive thymosin had a molecular weight similar to that of thymosin alpha 1 (3108 daltons). Using HPLC techniques, one main peak of immunoreactivity was present in brain extracts, whereas two peaks were present in pituitary extracts, one of which coeluted with thymosin alpha 1. The discrete regional distribution of thymosin alpha 1-like peptides was investigated and the highest densities of immunoreactive thymosin were present in the median eminence and arcuate nucleus of the hypothalamus, as well as the neurointermediate lobe of the pituitary. Due to the anatomical proximity of immunoreactive thymosin to loci containing known releasing factors and hormones, thymosin alpha 1-like peptides may function as neuroendocrine regulatory agents.     

Distribution of bombesin binding sites in the rat gastrointestinal tract

In an attempt to identify potential target sites for the satiety action of bombesin (BN), the distribution and pharmacological specificity of bombesin binding sites were examined in the rat gastrointestinal tract by in vitro autoradiography utilizing (125I-Tyr4) bombesin. Specific BN binding was localized to the circular muscle level of the gastric fundus and antrum, submucosal layer of the small intestine and longitudinal and circular muscle and submucosal layers of the colon. Pharmacological studies indicated that gastrin releasing peptide (GRP), Ac-GRP20-27 and BN-like compounds, litorin and ranatensin, inhibited the binding of (125I-Tyr4)BN with high affinity while compounds which lacked COOH-terminal homology with BN demonstrated a low affinity for BN binding sites. The wide distribution of BN binding sites in the gastrointestinal tract provides a number of potential sites for the mediation of the satiety action of BN.     

Binding specificity of the mouse cerebral cortex receptor for small cholecystokinin peptides

Prior studies have shown that the cerebral cortex cholecystokinin (CCK) receptor can bind CCK and gastrin analogs with high affinity. In the present work the brain CCK receptor had approximately a three times greater affinity for CCK₈ than its C-terminal tetrapeptide (CCK₄) while the C-terminal tripeptide (CCK₃) was 1000-fold less potent than CCK₄. Thus the C-terminal tetrapeptide appears to be the minimal C-terminal CCK sequence required for high affinity binding. Since brain membranes degrade various peptides including CCK, we also evaluated the stability of CCK analogs under the conditions used to measure receptor binding by the following three methods: (1) Studies of degradation-resistant analogs in binding assays; (2) analysis of analog degradation by high performance liquid chromatography (HPLC); and (3) determination of the change in potency of CCK analogs in competitive binding studies subsequent to preincubation with brain membranes. These studies indicated that degradation of analogs by the brain membranes although significant did not account for the differences in potency of analogs in competitive binding studies. Therefore, the observed differences in potencies of the analogs tested are due to the receptor affinity and not sensitivity of the analog to degradation.     

Side chain modifications change the binding and agonist properties of endomorphin 2.

Side chain modifications were introduced to endomorphin 2 (E2) to improve its binding properties and biological activity. A number of C-terminal modifications decreased the binding affinity to the mu-opioid receptor and the intrinsic activity in rat brain membranes. The exception was E2-ol, which showed increased binding affinity to MOR and higher potency in stimulating [(35)S]GTPgammaS binding. N-methylation of Phe(3) (MePhe(3)) attenuated the binding affinity and produced a rightward shift of [(35)S]GTPgammaS binding curves. All derivatives had lower intrinsic activity than E2. Some of the modified peptides partially inhibited, while YPF-benzyl-allyl-amide fully inhibited, the E2 or [d-Ala(2),MePhe(4),Gly(5)ol]enkephalin stimulated [(35)S]GTPgammaS binding. Marked differences were found between the results obtained using tritiated E2, tritiated naloxone, and [(35)S]GTPgammaS binding, indicating the possible involvement of multiple binding sites. The data presented demonstrate that the C-terminal amide group has an essential role in the regulation of the binding and the agonist/antagonist properties of E2.     

Inhibition of serotonin release by bombesin-like peptides in rat hypothalamus in vitro

We investigated the activity of bombesin (BN), neuromedin-C (NM-C) and neuromedin-B (NM-B) on serotonin (5-HT) release and reuptake in rat hypothalamus (HYP) in vitro. BN and NM-C but not NM-B (all 1 microM) decreased K+ evoked 3H-5-HT release from superfused HYP slices by 25%. Bacitracin (BCN, 2 micrograms/ml), a nonspecific peptidase inhibitor, reversed the inhibitory effect of BN on K+ evoked 3H-5-HT release. Phosphoramidon (PAN, 10 microM) an endopeptidase 24.11 inhibitor, abolished the inhibitory effect of BN, but not NM-C, on K+ evoked 3H-5-HT release. The peptidyl dipeptidase A inhibitor enalaprilat (ENP, 10 microM), enhanced both BN and NM-C inhibition of 3H-5-HT release. Bestatin (BST, 10 microM) had no effect on BN or NM-C inhibitory activity on 3H-5-HT release. Neither BN, NM-C nor NM-B affected reuptake of 3H-5-HT into HYP synaptosomes alone or in combination with any of the peptidase inhibitors, nor did these peptides alter the ability of fluoxetine to inhibit 3H-5-HT uptake. These data suggest: a) that BN-like peptides may alter neurotransmission in the HYP by acting presynaptically on the 5-HT release mechanism; b) a similarity in the structural requirements for the BN induced inhibition of 5-HT release and BN evoked thermoregulatory disturbances; and c) that peptidases may selectively augment or reduce pharmacologic activity of BN-like peptides upon CNS administration.     

Spermine modulation of the glutamate(NMDA) receptor is differentially responsive to conantokins in normal and Alzheimer's disease human cerebral cortex

The pharmacology of the N -methyl-d-aspartate (NMDA) receptor site was examined in pathologically affected and relatively spared regions of cerebral cortex tissue obtained at autopsy from Alzheimer's disease cases and matched controls. The affinity and density of the [(3)H]MK-801 binding site were delineated along with the enhancement of [(3)H]MK-801 binding by glutamate and spermine. Maximal enhancement induced by either ligand was regionally variable; glutamate-mediated maximal enhancement was higher in controls than in Alzheimer's cases in pathologically spared regions, whereas spermine-mediated maximal enhancement was higher in controls in areas susceptible to pathological damage. These and other data suggest that the subunit composition of NMDA receptors may be locally variable. Studies with modified conantokin-G (con-G) peptides showed that Ala(7)-con-G had higher affinity than Lys(7)-con-G, and also defined two distinct binding sites in controls. Nevertheless, the affinity for Lys(7)-con-G was higher overall in Alzheimer's brain than in control brain, whereas the reverse was true for Ala(7)-con-G. Over-excitation mediated by specific NMDA receptors might contribute to localized brain damage in Alzheimer's disease. Modified conantokins are useful for identifying the NMDA receptors involved, and may have potential as protective agents.     

A structure function study of C-terminal extensions of bombesin.

Synthetic C-terminal extensions of BN were synthesized and the biological potency evaluated using Swiss 3T3 and small cell lung cancer cells. BN, which has an amidated C-terminal, inhibited specific [125I-Tyr4]BN binding activity to Swiss 3T3 cells with an IC50 value of 1 nM, whereas the IC50 of BN-OH, which has a free C-terminal, was 1800 nM. The IC50 values of BNG, BNGK and BNGKK were 1400, 4700 and 500 nM, respectively. Similar binding data were obtained using SCLC cell line NCI-H345 and the bombesin analogues functioned as agonists based on the ability to elevate cytosolic Ca2+ in Fura-2 AM loaded SCLC cells. Also, the bombesin analogues stimulated 3H-thymidine uptake in Swiss 3T3 cells and the ED50 values for BN, BNG, BNGK and BNGKK were 1, 1300, 3900 and 400 nM. These data suggest that an amidated C-terminal is essential for high affinity binding and potency of BN.     

Inhibition of gastric emptying by bombesin-like peptides is dependent upon cholecystokinin-A receptor activation.

The amphibian peptide bombesin (BN) and the related mammalian peptides gastrin-releasing peptide (GRP) and neuromedin B (NMB) inhibit gastric emptying in rats. Exogenous administration of BN stimulates the release of cholecystokinin (CCK), a gastrointestinal peptide that also potently inhibits gastric emptying. To determine whether the inhibition of gastric emptying by BN-like peptides is mediated by a CCK-dependent mechanism, we examined the ability of the CCK-A receptor antagonist, devazepide, to block the inhibition of saline gastric emptying produced by BN, GRP18-27 and NMB. Using the same dosages as in the gastric emptying experiment, we also evaluated the effect of devazepide on feeding suppression produced by systemically administered BN. Our results showed that devazepide completely blocked the suppression of gastric emptying produced by BN, GRP18-27 and NMB but had no effect on BN-induced suppression of food intake. These results suggest that BN-like peptides inhibit gastric emptying through an indirect mechanism that is dependent upon CCK-A receptor activation. In contrast, the suppression of food intake by BN, in this experimental paradigm, is independent of CCK-A receptors.     

In vitro binding and functional studies of Ac-RYYRIK-ol and its derivatives, novel partial agonists of the nociceptin/orphanin F/Q receptor

Following the discovery of nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) and its endogenous ligand, an extensive search has started to find selective agonists and antagonists targeting this novel receptor-ligand system due to their therapeutic potentials. By the help of the combinatorial chemistry a series of hexapeptides with a general formula of Ac-RYY-R/K-W/I-R/K-NH(2) having high NOP receptor affinity and selectivity were identified. On the basis of this information we developed a number of novel compounds. The detailed structure-activity studies on the partial agonist Ac-RYYRIK-NH(2) are reported in this communication. Besides the modifications on N- and C-terminal, Arg-Cit exchange was performed on the template structure. The novel hexapeptides were analyzed in radioligand binding, functional biochemical [(35)S]GTPgammaS binding assays by using membranes from rat brains and Chinese hamster ovary cells expressing human NOP receptor. The agonist/antagonist properties were also tested on in the mouse vas deferens bioassay. C-terminal modification yielded a high affinity, selective and potent NOP ligand (Ac-RYYRIK-ol) with a partial agonist property. Several analogs of this compound were synthesized. The presence of the positively charged arginine residue at the first position turned out to be crucial for the biological activity of the hexapeptide. The N-terminal modifications with various acyl groups (ClAc, pivaloyl, formyl, benzoyl, mesyl) decreased the affinity of the ligand towards the receptor and the intrinsic activity for stimulating the G-protein activation was also decreased. The structure-activity studies on the hexapeptide derivatives provided some basic information on the structural requirements for receptor binding and activation.     

Synthesis and evaluation of conformationally constrained peptide analogues as the Src SH3 domain binding ligands

Src kinase activity is regulated by the interaction of SH3 domain with protein sequences that are rich in proline residues. Identification of more potent SH3 domain binding ligands that can regulate Src kinase activity is a subject of major interest. Conformationally constrained peptides have been previously used for improving the binding potency of the Src SH2 domain binding peptide ligands and peptide substrates of the substrate-binding site of Src. A series of peptide analogues of Ac-VSLARRPLPPLP (1, Ac-VSL12, K_d = 0.34 μM) were synthesized by introducing conformational constraints to improve the binding affinity towards the Src SH3 domain. Peptides synthesized through cyclization between N-terminal to C-terminal [VSLARRPLPPLP] or N-terminal to side chain flanking residues (i.e., [^βAVS]LARRPLPPLP and [VSLE]RRPLPPLP) exhibited at least 6.4-fold less binding affinity (K_d = 2.19–4.85 μM) when compared to 1. The data suggest upon N-terminal cyclization with C-terminal or flanking residues, the interactions of the amino acids in the core RPLPPLP reduce significantly with the residues within the Src SH3 domain. Conformationally constrained peptide V[SLARRPLPPLP] (5) was synthesized through cyclization of C-terminal to the serine side chain and displayed a comparable binding affinity (K_d = 0.35 μM) towards the Src SH3 domain versus that of 1. Thus, this template may be used to optimize and generate more potent analogues with higher stability.     

Molecular determinants of receptor affinity and selectivity of the natural delta-opioid agonist, dermenkephalin

Processing of the polyprotein precursor pro-dermorphin generates two distantly related D-amino acid-containing peptides, dermorphin and dermenkephalin, which are among the most selective high affinity agonists described, respectively, for the mu- and delta-opioid receptors. Dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2, is a linear, potentially flexible peptide devoid of structural homology with either enkephalins, endorphins, or dynorphins and, as such, represents a useful tool for identifying determinants of high affinity and selective binding of opioids to the delta-receptor. A series of selected dermenkephalin analogs and homologs was investigated for affinity at the mu- and delta-sites in the brain. Whereas dermenkephalin has high affinity and specificity for the delta-opioid receptors, its tetrapeptide amino end, dermenkephalin-[1-4]-NH2 binds almost exclusively at the mu-receptors. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2, is only marginally more selective for the u-sites than is dermenkephalin-[1-4]-NH2. Using dermorphin-dermenkephalin peptide hybrids and C-terminal deletion analogs of dermenkephalin, we showed the critical role that the C-terminal residues Met6 and Asp7 play in specifying correct addressing of dermenkephalin toward delta-receptors. The potent mu-deteminant located within the amino end of dermenkephalin is over-whelmed by the powerful delta-directing ability of the carboxy end. The negatively charged side chain of Asp7 makes a significant contribution to the delta-addressing ability of the C-terminal region, a finding consistent with Schwyzer's membrane selection model (Schwyzer, R. (1986) Biochemistry 25, 6335-6342). The Leu residue in position 5 and D-configuration about the alpha-carbon of Met2 were found to be of crucial importance for high affinity binding to delta-receptors. Whereas the Met residue in position 6 in dermenkephalin could safely be oxidized or replaced with D-Met, oxidation of Met2 led to deleterious effects, this analog being 1/100 as potent as dermenkephalin at delta-sites. Overall, the data collected demonstrate that highest levels of selectivity and affinity for the delta-opioid receptors can be achieved with small-sized, potentially flexible, linear peptides and further support the model according to which, in addition to optimum accommodation at the receptor, selection for delta-receptors is reduced by the effective positive charge of the molecule. Dermenkephalin may provide a starting point for the design of agonists and antagonists with nearly total specificity for the delta-sites. Such pharmacological agents could be used to explore the ill-defined physiological role and behavioral actions conveyed by delta-opioid receptors.     

Differential contribution of C-terminal regions of dermorphin and dermenkephalin to opioid-sites selection and binding potency

Dermorphin and dermenkephalin are D-aminoacid containing peptides generated from processing of the plurifonctional biosynthetic precursor pro-dermorphin. Dermorphin, Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH2 (DRM) and dermenkephalin, Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (DREK), are among the most selective and potent agonists described respectively for the mu- and delta-opioid receptors. In order to identify determinants of selectivity and high-affinity receptor binding of dermorphin and dermenkephalin, a series of analogs was investigated for their affinity at the mu- and delta-receptors in the brain. The tetrapeptide amino end of both DRM and DREK were found to display high affinity and selectivity towards mu-receptors. Substitution of the C-terminal tripeptide of DREK with that of DRM reversed the receptor selectivity of DREK from delta to mu. Replacement of the C-terminal tripeptide of DRM with the C-terminal counterpart of DREK shifted the selectivity of DRM from mu to delta. These data emphasize the critical contribution of the carboxy end of DREK to delta-selectivity. They further suggest that the potent mu-address lying in the N terminus of DREK is overwhelmed by the powerful delta-directing ability of the carboxy end. Unlike DREK, the C-terminus of DRM is not involved in opioid receptor sites selection but is important insofar as it serves to stabilize interactions of DRM with the mu-receptor binding site.     

Physiological roles of urocortins, human homologues of fish urotensin I, and their receptors

Urocortin 1, a human homologue of fish urotensin I, together with its related-compounds (urocortins 2 and 3), comprises a distinct family of stress peptides. Urocortin 1 has a high affinity for both corticotropin-releasing factor (CRF) type 1 receptor (CRF1) and CRF type 2 receptor (CRF2), and urocortins 2 and 3 have a high affinity for CRF2, while CRF has a low affinity for CRF2 and a high affinity for CRF1. These differences of the binding affinity with receptors make the biological actions of these peptides. Besides the binding affinity with receptors, the limited overlap of the distribution of CRF and urocortins may also contribute to the differences of physiological roles of each peptide. Urocortins show 'stress-coping' responses such as anxiolysis and dearousal in the brain. In the periphery, recent studies show the potent effects of urocortins on the cardiovascular and immune systems. In this review article, we take a look over the series of peptides included in this family, especially in terms of the versatility of biological actions, along with the various characters of the receptors.     

From nature to creation: Going around in circles,the art of peptide cyclization

Cyclic peptides and cyclotides are becoming common identities within the present efforts seen in peptide engineering – as we seek approaches to achieve potent biological activity, pharmacological selectivity, structurally stability and oral bioavailability. Yet this unique family of peptides has faced uncommon hurdles in their discovery, synthesis and bioengineering, retaining to characteristics that truly deviate these from their linear counterparts. In this mini-review we take a board spectrum approach to introduce this novel family of biomolecules and the troubles that they face in their sequence and disulfide connectivity assignment, together highlighting the present combined strategies involved in cyclic peptide/cyclotide synthesis and modification. These efforts have circumvented otherwise impossible hurdles in their manipulation and production that are only now advancing cyclic peptides/cyclotides as research probes and future pharmaceutical templates.     

Structural properties and peptide ligand binding of the capsid homology domains of human Arc

The activity-regulated cytoskeleton-associated protein (Arc) is important for synaptic plasticity and the normal function of the brain. Arc interacts with neuronal postsynaptic proteins, but the mechanistic details of its function have not been fully established. The C-terminal domain of Arc consists of tandem domains, termed the N- and C-lobe. The N-lobe harbours a peptide binding site, able to bind multiple targets. By measuring the affinity of human Arc towards various peptides from stargazin and guanylate kinase-associated protein (GKAP), we have refined its specificity determinants. We found two sites in the GKAP repeat region that bind to Arc and confirmed these interactions by X-ray crystallography. Phosphorylation of the stargazin peptide did not affect binding affinity but caused changes in thermodynamic parameters. Comparison of the crystal structures of three high-resolution human Arc-peptide complexes identifies three conserved C–H…π interactions at the binding cavity, explaining the sequence specificity of short linear motif binding by Arc. We further characterise central residues of the Arc lobe fold, show the effects of peptide binding on protein dynamics, and identify acyl carrier proteins as structures similar to the Arc lobes. We hypothesise that Arc may affect protein-protein interactions and phase separation at the postsynaptic density, affecting protein turnover and re-modelling of the synapse. The present data on Arc structure and ligand binding will help in further deciphering these processes.