Pegylated peptides. IV. Enhanced biological activity of site-directed pegylated GRF analogs

Conditions have been developed for the site-specific pegylation (NH2-terminus, side-chain and carboxy-terminus) of a potent analog of growth hormone-releasing factor, [Ala15]-hGRF(1-29)-NH2. These pegylated peptides were prepared by solid-phase peptide synthesis using the Fmoc/tBu strategy, and were fully characterized by analytical HPLC, amino-acid analysis, 1H-NMR spectroscopy and laser desorption mass spectrometry. Biological activities of hGRF analogs were determined in vitro utilizing stimulation of growth hormone release by cultured rat pituitary cells as an index. GH-releasing potencies of the pegylated hGRF analogs were compared to a series of model analogs of [Ala15]-hGRF(1-29)-NH2 that were acetylated or protected as the ethylamides at the pegylation sites. It was found that acetylation at the NH2-terminus resulted in reduced potency, which was not further affected when the NH2-terminus was pegylated, regardless of the size of poly(ethyleneglycol) (PEG) employed (e.g. PEG2000 or PEG5000). Pegylation at Asp8 or Lys12 decreased biological potency, a situation which was exacerbated by increasing the molecular weight of PEG. Pegylation at Lys21 or Asp25 did not significantly affect biological activity. The C-terminal model peptide, [Ala15,Orn(Ac)30]-hGRF(1-29)-NH2, was the most potent analog identified in this series (ca. 4-5-fold that of hGRF(1-44)-NH2. The COOH-terminal pegylated analogs retained this increased level of biological activity independent of PEG molecular weight. These studies demonstrate that a biologically active peptide can be pegylated and retain the full in vitro potency of the peptide. However, the biological activity is highly dependent on the site of pegylation and, in some cases, the molecular weight of PEG (degree of pegylation) moiety used.     

Human growth hormone-releasing hormone hGHRH(1-29)-NH2: systematic structure-activity relationship studies

Two complete and two partial structure-activity relationship scans of the active fragment of human growth hormone-releasing hormone, [Nle27]-hGHRH(1-29)-NH2, have identified potent agonists in vitro. Single-point replacement of each amino acid by alanine led to the identification of [Ala8]-, [Ala9]-, [Ala15]- (Felix et al. Peptides 1986 1986, 481), [Ala22]-, and [Ala28, Nle27]-hGHRH(1-29)-NH2 as being 2-6 times more potent than hGHRH(1-40)-OH (standard) in vitro. Nearly complete loss of potency was seen for [Ala1], [Ala3], [Ala5], [Ala6], [Ala10], [Ala11], [Ala13], [Ala14], and [Ala23], whereas [Ala16], [Ala18], [Ala24], [Ala25], [Ala26], and [Ala29] yielded equipotent analogues and [Ala7], [Ala12], [Ala17], [Ala20], [Ala21], and [Ala27] gave weak agonists with potencies 15-40% that of the standard. The multiple-alanine-substituted peptides [MeTyr1,Ala15,22,Nle27]-hGHRH(1-29)-NH2 (29) and [MeTyr1,Ala8,9,15,22,28,Nle 27]-hGHRH(1-29)-NH2 (30) released growth hormone 26 and 11 times, respectively, more effectively than the standard in vitro. Individual substitution of the nine most potent peptides identified from the Ala series with the helix promoter alpha-aminoisobutyric acid (Aib) produced similar results, except for [Aib8] (doubling vs [Ala8]), [Aib9] (having vs [Ala9]), and [Aib15] (10-fold decrease vs [Ala15]). A series of cyclic analogues was synthesized having the general formula cyclo(25-29)[MeTyr1,-Ala15,Xaa25,Nle27,Yaa29+ ++]-GHRH(1-29)-NH2, where Xaa and Yaa represent the bridgehead residues of a side-chain cystine or [i-(i + 4)] lactam ring. The ring size, bridgehead amino acid chirality, and side-chain amide bond location were varied in this partial series in an attempt to maximize potency. Application of lactam constraints in the C-terminus of GHRH(1-29)-NH2 identified cyclo(25-29)[MeTyr1,Ala15,DAsp25,Nle27,Orn29+ ++]-hGHRH(1-29)-NH2 (46) as containing the optimum bridging element (19-membered ring) in this region of the molecule. This analogue (46) was 17 times more potent than the standard. Equally effective was an [i-(i + 3)] constraint yielding the 18-membered ring cyclo(25-28)[MeTyr1,Ala15,Glu25,Nle,27Lys28]- hGHRH-(1-29)-NH2 (51) which was 14 times more potent than the standard. A complete [i-(i + 3)] scan of cyclo(i,i + 3)[MeTyr1,Ala15,Glui,Lys(i + 3),Nle27]-hGHRH(1-29)-NH2 was then produced in order to test the effects of a Glu-to-Lys lactam bridge at all points in the peptide. Of the 26 analogues in the series, 11 had diminished potencies of less than 10% that of the agonist standard, 4 were weak agonists (15-40% relative potency), and 4 analogues were equipotent to the standard. The 7 most potent analogues ranged in potency from 3 to 14 times greater than that of the standard and contained the [i-(i + 3)] cycles between residues 4-7, 5-8, 9-12, 16-19, 21-24, 22-25, and 25-28. The combined results from these systematic studies allowed for an analysis of structural features in the native peptide that are important for receptor activation. Reinforcement of the characteristics of amphiphilicity, helicity, and peptide dipolar effects, using recognized medicinal chemistry approaches including introduction of conformational constraints, has resulted in several potent GHRH analogues.     

Structure-activity relationships of a series of analogues of the octadecaneuropeptide ODN on calcium mobilization in rat astrocytes

The octadecaneuropeptide ODN (QATVGDVNTDRPGLLDLK), originally characterized as an endogenous ligand for central-type benzodiazepine receptors, increases intracellular calcium concentration ([Ca2+]i) in rat astroglial cells. A series of ODN analogues was synthesized, and each compound was studied for its ability to induce Ca2+ mobilization in cultured rat astrocytes. Replacement of each amino acid by an L-alanine residue (AlaScan) showed that the N-terminal region of the molecule was relatively tolerant to alanine substitution (2-8, 10), except for the Ala9-substituted analogue (9) which was totally devoid of activity. Pyroglutamization (21) and acetylation (22) of the Gln1 residue reduced the Ca2+ response suggesting that a free N-terminal amine function is required for full activity of ODN. Alanine substitution of the residues in the C-terminal region of the molecule (11-14, 16-18) significantly reduced the biological activity of ODN. In particular, modifications of the Leu15 residue (15, 20) abolished the Ca2+-mobilizing activity. The analogues [Ala9]ODN (9), [Ala15]ODN (15), [D-Thr9]ODN (19), and [D-Leu15]ODN (20) partially antagonized the Ca2+ response evoked by ODN. Most importantly, the octapeptide ODN11-18 (OP, 24) produced a dose-response curve that was superimposable to that obtained with ODN, indicating that the C-terminal region of the molecule possesses full biological activity. Finally, the AlaScan of OP revealed that replacement of the Leu5 residue by Ala (29) or D-Leu (33) totally suppressed the calcium response, confirming the crucial contribution of the Leu15 residue of ODN to the biological activity of the neuropeptide.     

Design and characterisation of long-R3-insulin-like growth factor-I muteins which show resistance to pepsin digestion

Site-directed mutagenesis was used to construct pepsin-resistant, single-point mutations of the N-terminal extended IGF-I analogue, long-R3-IGF-I. In order to identify the most susceptible sites, the kinetics of long-R3-IGF-I digestion by purified porcine pepsin were determined. Pepsin initially cleaved the Leu10-Phe11 bond in the N-terminal extension peptide to generate FVN-R3-IGF-I, followed in rapid succession by cleavage at Gln15-Phe16, Tyr24-Phe25, Leu10-Val11 and Met59-Tyr60 in the IGF-I moiety. Single-point mutations at these sites were designed on the basis of the preferred cleavage bonds for pepsin, as well as amino acid substitutions less likely to disturb protein structure. These included Leu10Val, Phe16Ala, Phe25Leu, Asp53Glu and Met59Gln. All five muteins retained growth-promoting activity equivalent to or higher than that of IGF-I. In terms of pepsin susceptibility, Leu10Val and Asp53Glu were degraded as rapidly as the parent long-R3-IGF-I, Met59Gln and Phe25Leu were partially stabilised, and Phe16Ala showed a marked improvement in stability over a wide range of pepsin:substrate ratios. Accordingly, the Phe16Ala mutein, long-R3A16-IGF-I, has potential for oral applications to enhance gastric growth and repair.     

Characterization of VIP receptor-effector system antagonists in rat and mouse peritoneal macrophages

In the present study we show that the synthetic peptides [4-Cl-D-Phe6,Leu17]VIP and the growth hormone releasing factor (GRF) analog [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 inhibit in a competitive manner the specific [125I]VIP binding to both rat and mouse peritoneal macrophages. In rat peritoneal macrophages, the order of potency of the different peptides, as expressed by the IC50 values was: VIP (IC50 = 1.90 +/- 0.16 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 125.8 +/- 13.2 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 354.8 +/- 21.2 nM). In mouse peritoneal macrophages a similar pattern of potency was observed: VIP (IC50 = 1.58 +/- 0.12 nM) > [4-Cl-D-Phe6,Leu17]VIP (IC50 = 110.8 +/- 10.7 nM) > [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 (IC50 = 251 +/- 19.2 nM). The behavior as VIP receptor antagonists of both [4-Cl-D-Phe6,Leu17]VIP and [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2 in rat and mouse peritoneal macrophages was confirmed by: (a) the shift to the right of VIP dose-stimulated cyclic AMP production curves in the presence of the two antagonists; (b) the agreement between the order of efficacy of the two peptides in competition experiments with the corresponding inhibition of cyclic AMP production; (c) the inefficiency of the two antagonists on the stimulation of cyclic AMP production by the beta-adrenoceptor agonist isoproterenol, which indicates the specificity of the interaction; (d) the synergic effect of VIP on isoproterenol-stimulated cyclic AMP production was completely abolished by [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2, suggesting that both antagonists acted via specific VIP receptors. Moreover, propranolol, a beta-adrenoceptor antagonist, did not affect the VIP-stimulated cyclic AMP production and the antagonist role of [4-Cl-D-Phe6,Leu17]VIP or [Ac-Tyr1,D-Phe2]GRF-(1-29)-NH2; (e) in cross-linking experiments, the intensity of the labeling of the [125I]VIP/receptor complexes was significantly lower with the antagonists than in the control experimental situation in both mouse and rat peritoneal macrophage membranes.     

Modulation of gramicidin channel structure and function by the aliphatic "spacer" residues 10, 12, and 14 between the tryptophans

In the linear gramicidins, the four aromatic residues at positions 9, 11, 13, and 15 are well-known to be important for the structure and function of membrane-spanning gramicidin channels. To investigate whether the "spacer" residues between the tryptophans in gramicidin A (gA) are important for channel structure and function, D-Leu-10, -12. and -14 of gA were replaced by Ala, Val, or Ile. (For practical reasons, the Ile substitutions were introduced into the enantiomeric gramicidin A-, gA-.) Circular dichroism spectra of [D-Ala10,12,14]gA, [D-Val10,12,14]gA, or [Ile10,12,14]gA- incorporated into sodium dodecyl sulfate micelles or 1, 2-dimyristoyl-sn-glycero-3-phosphocholine vesicles differ from the spectrum of the native [D-Leu10,12,14]gA. All the analogue spectra display reduced ellipticity at both 218 and 235 nm, indicating the presence of double-stranded conformers with the Ala analogue spectra showing the largest departure from the native gA spectra. Size-exclusion chromatograms of the Val and Ile analogues show both monomer and dimer peaks, accompanied by peak broadening; the chromatograms for the Ala analogue show broad, overlapping peaks and suggest the presence of higher oligomers and/or (rapidly) interconverting conformations. All three analogues form membrane-spanning channels, with the channel-forming potency of the Ala analogue being much less than that of gA or the other analogues. In 1.0 M CsCl, the conductance of each analogue channel is approximately 25% less than that of [D-Leu10,12,14]gA channels. The lifetimes of the analogue channels also are less than of [D-Leu10,12, 14]gA channels, with the largest (8-fold) reduction being for [D-Ala10,12,14]gA channels. Hybrid channel experiments show that the beta6.3-helical backbone folding pattern is retained in the channel-forming subunits and that the substitutions primarily influence ion entry. Both the bulk and the stereochemistry of the aliphatic residues between the tryptophans of gA are important for channel structure and function.     

Nociceptin receptor binding in mouse forebrain membranes: thermodynamic characteristics and structure activity relationships

The present study describes the labelling of the nociceptin (NC) receptor, ORL1, in mouse forebrain membranes with a new ligand partially protected from metabolic degradation at the C-terminal; the ligand, [3H]-NC-NH2, has a specific activity of 24.5 Ci mmol(-1). Saturation experiments revealed a single class of binding sites with a KD value of 0.55 nM and Bmax of 94 fmol mg(-1) of protein. Non specific binding was 30% of total binding. Kinetic binding studies yielded the following rate constants: Kobs = 0.104 min(-1); K1 =0.034 min(-1): T1/2=20 min; K(+1)=0.07 min nM(-1). Thermodynamic analyses indicated that [3H]-NC-NH2 binding to the mouse ORL1 is totally entropy driven, similar to what has been observed for the labelled agonists to the opioid receptors OP1(delta), OP2(kappa) and OP3(mu). Receptor affinities of several NC fragments and analogues, including the newly discovered ORL-1 receptor antagonist [Phe1psi(CH2-NH)Gly2]NC(1-13)-NH2([F/G]NC(1-13)-NH2), were also evaluated in displacement experiments. The competition curves for these compounds were found to be parallel to that of NC and the following order of potency was determined for NC fragments: NC-OH = NC-NH2-NC(1-13)-NH2 > > NC(1-12)-NH2 > NC(1-13)-OH > > NC(1-11)-NH2, and for NC and NC(1-13)-NH2 analogues: [Tyr1]NC-NH2 > or = [Leu1]NC(1-13)-NH2 > or = [Tyr1]NC(1-13)-NH2 > or = [F/G]NC(1-13)-NH2 > > [Phe3]NC(1-13)-NH2 > [DF/G]NC(1-13)-NH2. Standard opioid receptor ligands (either agonists or antagonists) were unable to displace [3H]-NC-NH2 binding when applied at concentrations up to 10 microM indicating that this new radioligand interacts with a non opioid site, probably the ORL1 receptor.     

The mechanism of autooxidation of myoglobin

Time courses for the autooxidation of native and mutant sperm whale and pig myoglobins were measured at 37 degrees C in the presence of catalase and superoxide dismutase. In sperm whale myoglobin, His64(E7) was replaced with Gln, Gly, Ala, Val, Thr, Leu, and Phe; Val68(E11) was replaced with Ala, Ile, Leu, and Phe; Leu29(B10) was replaced with Ala, Val, and Phe. In pig myoglobin, His64(E7) was replaced with Val; Val68(E11) was replaced with Thr and Ser; Thr67(E10) was replaced with Ala, Val, Glu, and Arg; Lys45(CD3) was replaced with Ser, Glu, His, and Arg. The observed pseudo-first order rate constants varied over 4 orders of magnitude, from 58 h-1 (H64A) to 0.055 h-1 (native) to 0.005 h-1 (L29F) at 37 degrees C, pH 7, in air. The dependences of the observed autooxidation rate constant on oxygen concentration and pH were measured for native and selected mutant myoglobins. In the native proteins and in most mutants still possessing the distal histidine, autooxidation occurs through a combination of two mechanisms. At high [O2], direct dissociation of the neutral superoxide radical (HO2) from oxymyoglobin dominates, and this process is accelerated by decreasing pH. At low [O2], autooxidation occurs by a bimolecular reaction between molecular oxygen and deoxymyoglobin containing a weakly coordinated water molecule. The neutral side chain of the distal histidine (His64) inhibits autooxidation by hydrogen bonding to bound oxygen, preventing both HO2 dissociation and the oxidative bimolecular reaction with deoxymyoglobin. Replacement of His64 by amino acids incapable of hydrogen bonding to the bound ligand markedly increases the rate of autooxidation and causes the superoxide mechanism to predominate. Increasing the polarity of the distal pocket by substitution of Val68 with Ser and Thr accelerates autooxidation, presumably by facilitating protonation of the Fe(II).O2 complex. Increasing the net anionic charge at the protein surface in the vicinity of the heme group also enhances the rate of autooxidation. Decreasing the volume of the distal pocket by replacing small amino acids with larger aliphatic or aromatic residues at positions 68 (E11) and 29 (B10) inhibits autooxidation markedly by decreasing the accessibility of the iron atom to solvent water molecules.     

Dynorphin A(6-12) analogs suppress thermal edema

Dynorphin A (Dyn A) is a 17-residue opioid peptide derived from prodynorphin precursors found in mammalian tissues. Removal of Tyr1 from Dyn A produces a peptide that is more potent than Dyn A in attenuating the acute phase of the inflammatory response, as measured by inhibition of heat-induced edema in the anesthetized rat's paw (exposure to 58 degrees C water for 1 min). Dyn A(2-17), however, no longer interacts with opioid receptors. It was postulated that the non-opioid anti-inflammatory actions of Dyn A(2-17) may reside in Dyn A(6-12); that is, Arg-Arg-Ile-Arg-Pro-Lys-Leu. here we report on the activities of Dyn A(6-12) analogs modified by substitutions on the N terminus, by single N-methyl substitution and by single replacement of residues by alanine. The results indicated that the minimal sequence required for an anti-edema ED50 of <1.0 micromol/kg i.v. was anisoyl-Arg6-Arg7-Xaa8-Arg9-Pro10)-Xaa11-+ ++Xaa12-NH2. A prototype, p-anisoyl-[D-Leu12] Dyn A(6-12)-NH2, with an ED50 of 0.20 micromol/kg i.v. compared to an ED50 of 0.08 micromol/kg i.v. for Dyn A(2-17), was selected for further tests of biological activity. This analog, like Dyn A(2-17), lowered blood pressure in anesthetized rats. In a model of neurogenic inflammation, produced by antidromic stimulation of the vagus in the anesthetized rat, p-anisoyl-[D-Leu12] Dyn A(6-12)-NH2, 0.23 micromol/kg i.v., attenuated the negativity of tracheal tissue interstitial pressure (Pif), which normally develops after nerve stimulation. Modulation of interstitial pressure may be the mechanistic basis for the anti-edema properties of these Dyn A(6-12) analogs.     

Structural characteristics for biological activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli: X-ray crystallography of weakly toxic and nontoxic analogs

Heat-stable enterotoxin (ST) produced by a pathogenic strain of Escherichia coli exerts its function by binding to a membrane-bound guanylyl cyclase on intestinal epithelial cell membranes, which in turn catalyzes the production of cyclic GMP as a second messenger in the cells. To elucidate the structural requirements for the biological activities of ST, we synthesized [Mpr5,Gly13]STp(5-17) and [Mpr5,Leu13]STp(5-17), which are weakly toxic and nontoxic analogs of STp, in which the toxic domain consists of the sequence from Cys at position 5 to Cys at position 17. In these analogs, Cys at position 5 is replaced by Mpr (beta-mercaptopropionic acid) and Ala at position 13 by Gly and Leu, respectively. We examined these analogs by X-ray diffraction analysis using direct methods and refined the structures to crystallographic R factors of 7.3% and 6.6% using 5492 and 5122 data, respectively, observed > 3 sigma (Fo) with a resolution of 0.89 A. These peptides have a right-handed spiral structure consisting of three structural segments: an N-terminal 3(10) helix, a central type I beta-turn, and a C-terminal type II beta-turn. These structures show minor differences from that of [Mpr5]STp(5-17), the fully toxic analog of heat-stable enterotoxin [Ozaki et al. (1991) J. Biol. Chem. 266, 5934-5941], suggesting that the decrease and loss of the biological activities of [Mpr5,Gly13]STp(5-17) and [Mpr5,Leu13]STp(5-17), respectively, are not caused by structural changes but are associated with the direct interaction of Ala13 with the receptor protein. Careful comparison of these structures in crystalline states revealed that ST has the following structural characteristics: (i) inherent flexibility at the junctions of the three segments and in the central segment, which includes the putative receptor-binding residues, Ala13, (ii) a specific hydrophobic character around the central segment, and (iii) an unexpected C-terminal folding similar to those of functionally unrelated peptides that are known to be ionophores.     

Angiotensin II modulates ANP-R2/ANP-C receptor-mediated inhibition of adenylyl cyclase in vascular smooth muscle cells: role of protein kinase C

In the present studies, we have investigated the modulation of atrial natriuretic peptide (ANP) receptor of R2 subtype (ANP-R2/ANP-C) coupled to adenylyl cyclase/cAMP signal transduction system by angiotensin II (angII). C-ANF4-23 [des(Gln18, Ser19, Gln20, Leu21, Gly22)ANF4-23-NH2] and AngII inhibited adenylyl cyclase activity in a concentration-dependent manner in vascular smooth muscle cells (VSmc A-10). The maximal inhibitions observed were about 40 and 30%, respectively, with an apparent Ki of about 1 and 10 nm. Pretreatment of the cells with AngII resulted in the attenuation of both C-ANF4-23 and AngII-mediated inhibitions of adenylyl cyclase, without altering [125I]-ANF binding. The levels of Gialpha-2 and Gialpha-3 proteins as determined by immunoblotting were also augmented by AngII treatment. In addition, AngII treatment stimulated the phosphorylation of Gialpha2 but not of Gialpha3 or ANP-C receptor, as revealed by immunoprecipitation of the proteins using specific antibodies after prelabelling the cells with [32P]orthophosphate. Staurosporine and chelerythrine, protein kinase C (PKC) inhibitors at 1 and 100 nm, respectively, prevented the AngII-mediated desensitization of C-ANF 4-23-sensitive adenylyl cyclase. In addition, the AngII-mediated phosphorylation of Gialpha2 protein was also inhibited partially by about 35% by staurosporine treatment. These results suggest that the attenuation of C-ANF4-23-mediated inhibition of adenylyl cyclase activity by AngII may not be attributed to the downregulation of receptors or to the decreased levels of G-proteins, and may involve PKC-dependent mechanisms.     

Purification and characterization of animal porphobilinogen synthases. I. Bovine liver porphobilinogen synthase

Porphobilinogen synthase was purified from ox liver by ammonium sulfate fractionation, heat denaturation and column chromatography (purification: 400-fold; specific activity 4.72 nkat). The molecular weight of the native enzyme obtained by thin-layer gel filtration is about 280 000. Using 8M urea in the presence of dithiothreitol as reducing agent, the molecule breaks down into 8 subunits of molecular weight 36 000 (dodecylsulfate gel electrophoresis); the preparation of aminoethylated subunit is described. According to the above-mentioned molecular weight and to the above-mentioned molecular weight and to the quantitative amino acid analysis after total hydrolysis, the following compositon of the enzymes subunit was calculated ASX23-25 Thr7 Ser23-24 Glx29-31 Pro22-23 Gly22-24 Ala36-37 Val23-26 Met7 Ile9 Leu34-35 Tyr10 Phe11-12 Lys11-12 Cys6-7 His6-8 Arg22 Trp1-2. The subunits, having two free sulfhydryl groups, therefore consists of a chain of about 306 amino acids. The Dansyl-Edman procedure did not enable identification of any free N-terminal amino acid. The acyl group blocking the N-terminus is an acetyl group. It was identified, after hydrazinolysis of the enzyme, by means of chromatographic comparison with 1-formyl-2-dansyl-hydrazine and 1-acetyl-2-dansylhydrazine, whose syntheses and UV spectra are described.     

Classification of amino acid spin systems using PFG HCC(CO)NH-TOCSY with constant-time aliphatic 13C frequency labeling

We have developed a useful strategy for identifying amino acid spin systems and side-chain carbon resonance assignments in small 15N-, 13C-enriched proteins. Multidimensional constant-time pulsed field gradient (PFG) HCC(CO)NH-TOCSY experiments provide side-chain resonance frequency information and establish connectivities between sequential amino acid spin systems. In PFG HCC(CO)NH-TOCSY experiments recorded with a properly tuned constant-time period for frequency labeling of aliphatic 13C resonances, phases of cross peaks provide information that is useful for identifying spin system types. When combined with 13C chemical shift information, these patterns allow identification of the following spin system types: Gly, Ala, Thr, Val, Leu, Ile, Lys, Arg, Pro, long-type (i.e., Gln, Glu and Met), Ser, and AMX-type (i.e., Asp, Asn, Cys, His, Phe, Trp and Tyr).     

Antinociceptive analogs of orphanin FQ/nociceptin(1-11)

The presence of pairs of basic amino acids within the sequence of orphanin FQ/nociceptin (OFQ/N) peptide, the endogenous ligand for the ORL1/KOR-3 receptor, has raised the possibility that processing might generate pharmacologically important truncated peptides, including OFQ/N(1-11). OFQ/N(1-11) is pharmacologically active in vivo with a potency comparable to OFQ/N. Several tyrosine-containing analogs of OFQ/N(1-11) have been synthesized and examined for antinociceptive activity. Like OFQ/N(1-11), [Tyr1]OFQ/N(1-11), [Tyr10]OFQ/N(1-11) and [IodoTyr10]OFQ/N(1-11) given supraspinally in mice were antinociceptive in the tailflick assay in mice. The tyrosine analogs showed similar potencies as OFQ/N(1-11) but longer durations of action. This response was readily reversed by the opioid antagonist naloxone despite poor affinities for these analogs at opioid receptors. Another compound, [Tyr11]OFQ/N(1-11) was highly epileptogenic, inducing naloxone-sensitive seizures in greater than 50% of the mice tested at doses comparable to those examined with the other analogs. These results indicate that it is possible to make analgesic OFQ/N(1-11) analogs. The activity of [IodoTyr10]OFQ/N(1-11) suggests that it may prove useful as a radioligand in exploring potential OFQ/N(1-11) binding sites.     

Residues Val254, His256, and Phe259 of the angiotensin II AT1 receptor are not involved in ligand binding but participate in signal transduction

The role of the external third of helix VI of the angiotensin II (AII) AT1 receptor for the interaction with its ligand and for the subsequent signal transduction was investigated by individually replacing residues 252-256 by Ala, and residues 259 or 261 by Tyr, and permanently transfecting the resulting mutants to Chinese hamster ovary (CHO) cells. Binding experiments showed no great changes in affinity of any of the mutants for AII, [Sar1]-AII, or [Sar1, Leu8]-AII, but the affinity for the nonpeptide antagonist DuP753 was significantly decreased. The inositol phosphate response to AII was remarkably decreased in mutants V254A, H256A, and F259Y. These results indicate that AT1 residues Val254, His256, and Phe259 are not involved in ligand binding but participate in signal transduction. Based in these results and in others from the literature, it is suggested that, in addition to the His256 imidazole ring, the Phe259 aromatic ring interacts with the AII's Phe8, thus contributing to the signal-triggering mechanism.     

Structural modifications of the N-terminal tetrapeptide segment of [D-Ala2]deltorphin I: effects on opioid receptor affinities and activities in vitro and on antinociceptive potency

A series of deltorphin I analogs containing D- or L-N-methylalanine (MeAla), D- or L-proline (Pro), alpha-aminoisobutyric acid (Aib), sarcosine (Sar) or D-tert-leucine (Tle) in place of D-Ala2, or phenylalanine in place of Tyr1, was synthesized. The opioid activity profiles of these peptides were determined in mu and delta opioid receptor-representative binding assays and bioassays in vitro as well as in the rat tail flick test in vivo. In comparison with the deltorphin I parent, both the L- and the D-MeAla2-analog were slightly more potent delta agonists in the mouse vas deferens (MDV) assay, and the D-MeAla2-analog showed two-fold higher antinociceptive potency in the analgesic test. In view of the fact that deltorphin analogs with an unsubstituted L-amino acid residue in the 2-position generally lack opioid activity, the observed high delta opioid potency of [L-MeAla2]deltorphin I is postulated to be due to the demonstrated presence of a conformer with a cis Tyr1-MeAla2 peptide bond, since the cis conformer allows for a spatial arrangement of the pharmacophoric moieties in the N-terminal tripeptide segment similar to that in active deltorphin analogs containing a D-amino acid residue in the 2-position. Substitution of Aib in the 2-position led to a compound, H-Tyr-Aib-Phe-Asp-Val-Val-Gly-NH2, which displayed lower delta receptor affinity than the parent peptide but higher delta selectivity and, surprisingly, three times higher antinociceptive potency. The D- and L-Pro2-, Sar2- and D-Tle2-analogs showed much reduced delta receptor affinities and were inactive in the tail flick test. Replacement of Tyr1 in deltorphin I with Phe produced a 32-fold decrease in delta receptor affinity but only a 7-fold drop in antinociceptive potency.     

NMDA-receptor antagonist requirements in conantokin-G

A series of variants of the neuroactive 17-residue gamma-carboxyglutamate-(Gla)-containing polypeptide, conantokin-G (con-G), were synthesized with the intention of determining those features that were important for its N-methyl-D-aspartate (NMDA) receptor-targeted antagonist activity and for adoption of its divalent cation-dependent alpha-helical conformation. Employing the binding of [3H]dizolcipine (MK-801) as an assay for open receptor ion channels in rat brain membranes, which displays inhibition by con-G (IC50 = 0.48 microM), it was found that replacement by an Ala residue of Gla4 led to complete inactivation of the peptide, whereas a similar replacement of Gla3 resulted in a 20-fold decreased potency. Ala substitutions for Gla10 and Gla14 did not substantially affect [3H]MK-801 binding. This same substitution at Gla7 appeared to slightly enhance binding. Ala replacements of non-Gla residues demonstrated that four of them, viz. Glu2, Leu5, Gln9, and Ile12, possessed at least 200-fold decreases in inhibitory potency, whereas similar replacements at Gly1, Leu11, and Arg13 resulted in peptides with 8- to 12-fold increases in the IC50 values. The remaining amino acid residues tested in the single Ala replacement series showed no significant changes in the inhibitory characteristics of wild-type con-G. Additional studies with carboxyl-terminal truncated peptides revealed that the carboxyl-terminal 4 amino acids were unimportant for this activity. There was no strict correlation of inhibition of [3H]MK-801 binding with the ability of these peptides to form cation-dependent alpha-helices. Peptides with notably low alpha-helical content in the presence of these cations were lacking at least one, or both, of Gla10 and Gla14. Con-G[Gla3,4,7,10,14E] and con-G[Gla7,10,14E] were the only peptides that remained in a completely random conformation upon metal ion addition.     

Nonpeptide peptidomimetic antagonists of the neuropeptide Y receptor: benextramine analogs with selectivity for the peripheral Y2 receptor

We synthesized a new series of benextramine analogs as neuropeptide Y (NPY) functional group mimetics and tested them for N-[propionyl-3H]NPY ([3]NPY) displacement activity in rat brain membrane homogenates and for NPY receptor antagonist activity in the rat femoral artery. The tetraamine, carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-1,6-hexanediamine (15) was equipotent with benextramine (based on comparison of the relevant IC50's) in a rat brain [3H]NPY displacement assay, suggesting that the disulfide is not a necessary feature of benextramine's [3H]NPY displacement activity, although this analog maintained selectivity for the benextramine-sensitive binding site population. The bis(N,N-dialkylguanyl) disulfide and carbon analogs 14a-c were 3-4 times more potent than their respective controls in displacing [3H]NPY from rat brain membrane homogenates with IC50's ranging from 15 to 18 microM and maintained selectivity for the benextramine-sensitive, Y1 binding site population. However, the activity of the carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-N,N'-(1,6- hexanediyl)diguanidine tetrahydrochloride (14b) showed a different profile in a femoral artery vasoconstriction assay; at 1.0 nM, this analog shifted the concentration-effect curve of the Y2-selective agonist NPY13-36 to the right (pA2 = 9.2; Kd = 0.63 nM) without a significant change in the maximum effect, while even at 1.0 mM it had no effect on the vasoconstrictive activity of the Y1-selective agonist [Leu31,Pro34]NPY. Thus, the bis(N,N-dialkylguanidine) analogs of benextramine are selective, competitive antagonists of the postsynaptic NPY receptor in the femoral artery.     

1H NMR investigation of distal mutant deoxy myoglobins. Interpretation of proximal His contact shifts in terms of a localized distal water molecule

1H NMR spectra of a series of distal point mutants of human and sperm whale deoxy myoglobin have been recorded and their spectral parameters compared with those of wild type. The substitutions investigated include His64(E7)-->Gly, Ala, Val, Leu, Ile, and Gln and Val68(E11)-->Ala, Ile. The three resonances from the proximal His F8 imidazole ring, as well as two heme methyl signals, are identified in each of the proteins. Significant perturbations of the NMR spectra of mutant deoxy myoglobins (Mbs) occurred only upon substitution of His64(E7) by any non-polar residue, with only minor variation in parameters throughout the range of side chains. These spectral changes are attributed to the elimination of a non-coordinated ordered water molecule in the distal pocket found hydrogen bonded to His64(E7) in crystals of wild-type deoxy Mb, but abolished in the His64(E7)-->Leu mutant deoxy Mb crystal (Quillin, M. L., Arduini, R. M., Olson, J. S., and Philips, G. N., Jr. (1993) J. Mol. Biol. 234, 140-155). The observed spectral changes, increased His F8 ring spin delocalization, and decreased heme in-plane asymmetry, can be directly attributed to the weakening of the effective axial field and a decrease in the asymmetry in the rhombic ligand field resulting from removal of the water molecule. The hyperfine shift patterns for the mutants His64(E7)-->Gln and Val68(E11)-->Ile deoxy Mbs are minimally perturbed from that of wild type and are interpreted to reflect a conserved distal water-binding site. In the point mutant Val68(E11)-->Ala, the decreased covalency to the axial His F8 is interpreted as reflecting a conserved distal water molecule that can interact more strongly with the iron due to the reduced steric bulk of the E11 side chain. The differential 1H NMR spectral parameters for the His F8 resonances in the two subunits of T state deoxy Hb A are shown to be similarly consistent with the known occupation of the distal water binding site in the alpha-, but not beta-subunit.     

Glycodermorphins: opioid peptides with potent and prolonged analgesic activity and enhanced blood-brain barrier penetration

1. In order to improve the in vivo stability of the opioid peptide dermorphin we synthesized O-betaglucosylated analogs ([Ser7-O-betaGlc]dermorphin and [Ser7-O-betaGlc(Ac)4]-dermorphin) and C-alphagalactosylated analogs ([Ala7-C-alphaGal]dermorphin and [Ala7-C-alphaGal(Ac)4]-dermorphin). 2. O- and C-glycosylation of dermorphin halved the peptide affinity for brain mu-opioid receptors and the biological potency in guinea-pig ileum assay (GPI). Despite their lower opioid receptor affinity, when administered intracerebroventricularly (i.c.v., 8-40 pmol) and subcutaneously (s.c., 0.5-3 micromol kg(-1)) in rats, glycosylated analogs were two times more potent than dermorphin in reducing the nociceptive response to radiant heat. Acetylation of sugar hydroxyl groups reduces 5-10 times both biological activity on GPI and mu-receptor affinity, whereas the antinociceptive potency was equal to (i.c.v.) or only two-three times lower (s.c.) than dermorphin potency. 3. Blood-Brain Barrier Permeability Index (BBB-PI) of the glycodermorphins was significantly higher than that of dermorphin, indicating a facilitated entry into the brain: O-beta-linked glucoconiugates are expected to enter CNS by the glucose transporter GLUT-1 of the endothelial barrier. However the calculated BBB-PI for the C-alphagalactoside was about two times higher than that of the O-betaglucoside, excluding the implication of GLUT-1 that is known to be selective for O-beta-links and preferring for the exose glucose. 4. The enhanced brain permeability with the subsequent decrease in peripheral dosage of these opioid peptides did not result in lowering constipation.