David H. Coy

Cdna Cloning, Characterization, and Brain Region Specific Expression of a Neuromedin-b Preferring Bombesin Receptor

Recent binding studies in the central nervous system and other tissues provide evidence that the mammalian bombesin-like peptides, gastrin-releasing peptide (GRP) and neuromedin-B (NMB), exert their numerous physiological effects through at least two different receptors. We describe the structure and expression of a cloned NMB-preferring bombesin receptor (NMB-R) with properties distinct from a GRP-preferring bombesin receptor (GRP-R) reported previously. In particular, the NMB-R shows higher affinity binding to NMB than to GRP in BALB 3T3 fibroblasts expressing the cloned NMB-R. The distinct regional distribution of NMB-R and GRP-R mRNA in the brain suggests that both bombesin receptor subtypes play independent roles in mediating many of the dramatic effects of bombesin-like peptides in the central nervous system.

Progress in the development of potent bombesin receptor antagonists

Bombesin and the mammalian-related peptides gastrin-releasing peptide (GRP), GRP and neuromedin B have been shown to have numerous actions in the CNS, gastrointestinal tract and on growth. However, the role of the peptides in various physiological processes has remained unclear because of the lack of potent antagonists. Recent in vitro studies have described four different classes of bombesin receptor antagonist, some of which are active in the nanomolar range and in vivo. Robert Jensen and David Coy describe recent insights into peptide structural determinants of biological activity. Evidence from structure-function studies have resulted in identification of some analogues that function as potent antagonists in all systems examined. Furthermore, various subtypes of bombesin receptors can now be differentiated by these various classes of antagonist.

Synthesis and opioid activities of stereoisomers and other D-amino acid analogs of methionine-enkephalin

Abstract A series of D-amino acid-substituted analogs of the opiate peptide, methionine5-enkephalin, were synthesized by solid-phase methods and tested for their abilities to inhibit electrically-evoked contractions of mouse vasa deferentia and to compete with tritiated enkephalin for opiate receptors on particulate fractions isolated from homogenates of rat brain. [D-Ala2]-enkephalin and [D-Ala2]-enkephalin amide were found to be the most potent peptides in both assay systems, being about 1000% active in the vas deferens bioassay and 120% and 150% active, respectively, in the stereospecific binding test relative to methionine5-enkephalin itself. In comparison, [D-Met5]-, [D-Tyr1]-, [D-Leu2]-, [D-Phe2]-, [D-Ala3]-, and [D-Phe4]-enkephalin had not more than 10% activity. The stabilization of the β-bend conformation of methionine5-enkephalin by the substitution of D-alanine in position 2 of the peptide chain may contribute to the high activities of the [D-Ala2]-analogs.

Adrenomedullin is a potent stimulator of osteoblastic activity in vitro and in vivo

Adrenomedullin is a 52-amino acid vasodilator peptide produced in many tissues, including bone. It has 20% sequence identity with amylin, a regulator of osteoblast growth, and circulates in picomolar concentrations. The present study assesses whether adrenomedullin also acts on osteoblasts. At concentrations of 10-12 M and greater, adrenomedullin produced a dose-dependent increase in cell number and [3H]thymidine incorporation in cultures of fetal rat osteoblasts. This effect was also seen with adrenomedullin-(15-52), -(22-52), and -(27-52), but adrenomedullin-(40-52) was inactive. These effects were lost in the presence of amylin blockers, suggesting they were mediated by the amylin receptor. Adrenomedullin also increased [3H]thymidine incorporation into cultured neonatal mouse calvaria but, unlike amylin, did not reduce bone resorption in this model. Adrenomedullin stimulated phenylalanine incorporation into both isolated osteoblasts and calvaria. When injected daily for 5 days over the calvariae of adult mice, it increased indexes of bone formation two- to threefold ( P < 0.0001) and increased mineralized bone area by 14% ( P = 0.004). It is concluded that adrenomedullin regulates osteoblast function and that it increases bone mass in vivo. The potential of this family of peptides in the therapy of osteoporosis should be further evaluated.

A novel bombesin receptor antagonist inhibits autocrine signals in a small cell lung carcinoma cell line

The human small cell lung carcinoma (SCLC) cell line NCI-H345 constitutively produces gastrin-releasing peptide (GRP), a peptide homologous to the mitogen bombesin. In addition, NCI-H345 cells express bombesin receptors and respond to bombesin with rapid activation of phospholipase C and mobilization of intracellular Ca2+. Treatment of NCI-H345 cells with a novel potent bombesin receptor antagonist [Leu13-psi-CH2NH-Leu14]bombesin blocked the increase in phosphatidylinositol turnover and cytoplasmic free Ca2+ ([Ca2+]i) stimulated by bombesin. Furthermore [Leu13-psi-CH2NH-Leu14]bombesin inhibited NCI-H345 colony formation in defined semisolid medium in the absence of exogenous GRP. The rapid, hormone-induced accumulation of inositol(1,4,5)trisphosphate was markedly more sensitive to antagonist inhibition than the hormone-induced Ca2+ transient, the sustained accumulation of inositol monophosphates, or colony formation in soft agarose. These data demonstrated inhibition of transmembrane signals associated with autocrine growth control in SCLC by a novel peptide receptor antagonist.

Ability of various bombesin receptor agonists and antagonists to alter intracellular signaling of the human orphan receptor BRS-3.

Bombesin (Bn) receptor subtype 3 (BRS-3) is an orphan receptor that is a predicted member of the heptahelical G-protein receptor family and so named because it shares a 50% amino acid homology with receptors for the mammalian bombesin-like peptides neuromedin B (NMB) and gastrin-releasing peptide. In a recent targeted disruption study, in which BRS-3-deficient mice were generated, the mice developed obesity, diabetes, and hypertension. To date, BRS-3’s natural ligand remains unknown, its pharmacology unclear, and cellular basis of action undetermined. Furthermore, there are few tissues or cell lines found that express sufficient levels of BRS-3 protein for study. To define the intracellular signaling properties of BRS-3, we examined the ability of [d-Phe6,β-Ala11,Phe13,Nle14]Bn-(6–14), a newly discovered peptide with high affinity for BRS-3, and various Bn receptor agonists and antagonists to alter cellular function in hBRS-3-transfected BALB 3T3 cells and hBRS-3-transfected NCI-H1299 non-small cell lung cancer cells, which natively express very low levels of hBRS-3. This ligand stimulated a 4–9-fold increase in [3H]inositol phosphate formation in both cell lines under conditions where it caused no stimulation in untransfected cells and also stimulated an increase in [3H]IP1, [3H]IP2, and 3H]IP3. The elevation of [3H]IP was concentration-dependent, with an EC50 of 20–35 nm in both cell lines. [d-Phe6,β-Ala11,Phe13,Nle14]Bn-(6–14) stimulated a 2–3-fold increase in [Ca2+] i , a 3-fold increase in tyrosine phosphorylation of p125FAK with an EC50 of 0.2–0.7 nm, but failed to either stimulate increases in cyclic AMP or inhibit forskolin-stimulated increases. None of nine naturally occurring Bn peptides or three synthetic Bn analogues reported to activate hBRS-3 did so with high affinity. No high affinity Bn receptor antagonists had high affinity for the hBRS-3 receptor, although two low affinity antagonists for gastrin-releasing peptide and NMB receptors, [d-Arg1,d-Trp7,9,Leu11]substance P and [d-Pro4,d-Trp7,9,10]substance P-(4–11), inhibited hBRS-3 receptor activation. The NMB receptor-specific antagonistd-Nal,Cys,Tyr,d-Trp,Lys,Val, Cys,Nal-NH2 inhibited hBRS-3 receptor activation in a competitive fashion (K i = 0.5 μm). Stimulation of p125FAK tyrosine phosphorylation by hBRS-3 activation was not inhibited by the protein kinase C inhibitor, GF109203X, or thapsigargin, alone or in combination. These results show that hBRS-3 receptor activation increases phospholipase C activity, which causes generation of inositol phosphates and changes in [Ca2+] i and is also coupled to tyrosine kinase activation, but is not coupled to adenylate cyclase activation or inhibition. hBRS-3 receptor activation results in tyrosine phosphorylation of p125FAK, and it is not dependent on activation of either limb of the phospholipase C cascade. Although the natural ligand is not a known bombesin-related peptide, the availability of [d-Phe6,β-Ala11,Phe13,Nle14]Bn-(6–14), which functions as a high affinity agonist in conjunction with hBRS-3-transfected cell lines and the recognition of three classes of receptor antagonists including one with affinity of 0.5 μm, should provide important tools to assist in the identification of its natural ligand, the development of more potent selective receptor antagonists and agonists, and further exploration of the signaling properties of the hBRS-3 receptor.

Increased analgesic activities of a fluorinated and a dimeric analogue of [D-Ala-2]-methionine enkephalinamide

Abstract [D-Ala-2, pentafluorophenylalanine-4]-Met-enkephalinamide and N α , N e -bis (D-Ala-2-Met-enkephalin)-Lys amide were synthesized by solid-phase techniques. They were found to be at least 10 and 4 times more potent respectively, and longer lasting, than the highly active parent peptide, [D-Ala-2]-Met-enkephalinamide, in producing analgesia in the rat after central injection. In the mouse vas deferens in vitro assay, the results from which frequently do not correlate well with in vivo assessment of the opiate activity of peptides, both analogues were slightly less active than [D-Ala-2]-enkephalinamide. Thus, a fluorinated and a dimeric analogue of enkephalin were devised and found to have markedly increased analgesic activity.

Receptor-specific somatostatin analogs: Correlations with biological activity

A number of cyclic and linear somatostatin (SRIF) analogs have now been found to have promising levels of selectivity for rodent somatostatin receptors (rsst2,3,5), but not sst1 and sst4. Comparisons between binding affinities for these and transfected human receptors are just beginning to emerge and we present results from a comparison of affinities of several key families of peptides for sst2 present on rat AR42J cells and on cells transfected with human (h)sst2. The typical cyclic octapeptide analogs, octreotide, lanreotide, and RC-160, exhibited similar affinities to SRIF for rsst2, but somewhat lower affinities for the human receptor. Affinities of several analogs for transfected hsst5 were also measured. As with the rat receptor, octreotide-related analogs had low affinity for hsst5. The highly specific rsst5 analog, DC-23-99, was less so for the human receptor; however, a D-Tyr1 version of DC-23-99 had subnanomolar affinity (Ki, 0.68 nmol/L) and high selectivity. A new extended-ring analog, BIM-23268D, showed superior affinity to DC-23-99 and even to SRIF and SRIF-28 for hsst5 (K(i), 0.38 nmol/L), and had the highest sst5/sst2 selectivity ratio of any analog that we have tested thus far.

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)

Dissociation of the effects of amylin on osteoblast proliferation and bone resorption

This study assesses the structure-activity relationships of the actions of amylin on bone. In fetal rat osteoblasts, only intact amylin and amylin-(1-8) stimulated cell proliferation (half-maximal concentrations 2.0 x 10(-11) and 2.4 x 10(-10) M, respectively). Amylin-(8-37), COOH terminally deamidated amylin, reduced amylin, and reduced amylin-(1-8) (reduction results in cleavage of the disulfide bond) were without agonist effect but acted as antagonists to the effects of both amylin and amylin-(1-8). Calcitonin gene-related peptide-(8-37) also antagonized the effects of amylin and amylin-(1-8) on osteoblasts but was substantially less potent in this regard than amylin-(8-37). In contrast, inhibition of bone resorption in neonatal mouse calvariae only occurred with the intact amylin molecule and was not antagonized by any of these peptides. The rate of catabolism of the peptides in calvarial cultures was not accelerated in comparison with that of intact amylin. This dissociation of the actions of amylin suggests that it acts through two separate receptors, one on the osteoclast (possibly the calcitonin receptor) and a second on the osteoblast.This study assesses the structure-activity relationships of the actions of amylin on bone. In fetal rat osteoblasts, only intact amylin and amylin-(1-8) stimulated cell proliferation (half-maximal concentrations 2.0 × 10-11 and 2.4 × 10-10 M, respectively). Amylin-(8-37), COOH terminally deamidated amylin, reduced amylin, and reduced amylin-(1-8) (reduction results in cleavage of the disulfide bond) were without agonist effect but acted as antagonists to the effects of both amylin and amylin-(1-8). Calcitonin gene-related peptide-(8-37) also antagonized the effects of amylin and amylin-(1-8) on osteoblasts but was substantially less potent in this regard than amylin-(8-37). In contrast, inhibition of bone resorption in neonatal mouse calvariae only occurred with the intact amylin molecule and was not antagonized by any of these peptides. The rate of catabolism of the peptides in calvarial cultures was not accelerated in comparison with that of intact amylin. This dissociation of the actions of amylin suggests that it acts through two separate receptors, one on the osteoclast (possibly the calcitonin receptor) and a second on the osteoblast.