Replacement of Tyr-SO3H by a p-carboxymethyl-phenylalanine in a CCK8-derivative preserves its high affinity for CCK-B receptor.

The sulfated tyrosine present in the sequence of CCK8 Asp26-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-PheNH2, seems to play a critical role in the recognition of CCK-A binding sites. In this work, we have investigated whether the presence of an anionic charge on the tyrosine moiety is strictly necessary and whether the sulfate moiety interacts with a divalent cation in the receptor subsite. For this purpose, the novel amino acids (L,D)Phe(p-CH2CO2H) and (L,D) Phe(p-CH2CONHOH), as well as their L-resolved forms were introduced into the sequence of Ac[X27, Nle28, Nle31]-CCK27-33 by solid phase method. The biological activities of these new derivatives were compared to two almost equiactive analogues of CCK8, Ac[Phe(p-CH2SO3H)27, Nle28, Nle31]-CCK27-33 and Boc[Nle28, Nle31]-CCK27-33 (BDNL) and to the nonsulfated analogue of the latter peptide (BDNL NS). All these new CCK-related analogues behave as agonists in stimulating pancreatic amylase release and display high affinity for brain binding sites (KI approximately 3-11 nM) but the only peptides which retain affinity for CCK-A receptors (KI approximately 20 nM) are those containing a p-carboxymethyl phenylalanine. Thus, introduction of this amino acid under an esterified form on the side chain, into specific and potent CCK-B agonists could allow compounds endowed with good bioavailabilities to be obtained.     

Stabilization of a tyrosine O-sulfate residue by a cationic functional group: formation of a conjugate acid-base pair.

Sulfated tyrosine [Tyr(SO3H)]-containing peptides showed characteristic peak patterns in their liquid secondary-ion mass spectrometry (LSIMS) spectra. Protonated molecules were desulfated more easily than their deprotonated counterparts. Therefore, the stabilities of the Tyr(SO3H) residues were well-reflected by peak patterns in their positive-ion spectra. These intrinsic peak patterns were investigated by comparing the behavior of each Tyr(SO3H) residue in acidic solution. As the peptide chain was lengthened and the number of cationic functional groups increased, the peak representing the [MH]+ of a Tyr(SO3H)-containing peptide became more prominent than that representing the desulfated [MH-SO3]+. These alterations in peptide structure also increased the stability of the Tyr(SO3H) residue in acidic solution. Based on the desulfation mechanism of an aryl monosulfate, we predicted that intramolecular cationic functional groups would stabilize Tyr(SO3H) residues by forming conjugate acid-base pairs (or salt bridges) both in the gaseous phase and in acidic solution. In accordance with this theory, Arg residues would take primary responsibility for this self-stabilization within Tyr(SO3H)-containing peptides. Moreover, a long peptide backbone was expected to have a weak protective effect against desulfation of the [MH]+ in the gaseous phase. Tyr(SO3H) residues were also stabilized by adding an external basic peptide containing multiple Arg residues. Formation of such intermolecular acid-base pairs was demonstrated by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) which detected conjugated peptide ions. The energetically favorable formation of conjugate acid-base pairs prompted by Tyr(SO3H) residues might be a driving force for protein folding and protein-protein interaction.     

Novel glycosylated [Lys(7)]-dermorphin analogues: synthesis, biological activity and conformational investigations.

Syntheses of the [Lys(7)]- and [Hyp(6),Lys(7)]-dermorphin analogues in which either Tyr(5) or Hyp(6) are O-glucosylated are described. For comparison, the carbohydrate-free peptides have also been prepared. Structural investigations by FT-IR and CD measurements were carried out on the synthetic analogues and some preliminary pharmacological experiments were also performed.The biological potency of the glucosylated analogues was compared with that of the micro-opioid receptor agonist dermorphin in GPI preparations. Glucosylation of either Tyr(5) or Hyp(6) reduces the potency of both [Lys(7)]-dermorphin and [Hyp(6),Lys(7)]-dermorphin. The effect induced by the Tyr(5) glucosylation is quite strong and the potency of both peptides is reduced by about 150 times. A similar but less dramatic effect is induced by the glucosylation of the Hyp(6) residue, and the potency of the parent peptide is reduced by about 15 times. The presence of acetyl groups on the sugar hydroxyl functions further reduces the agonistic potency of the glucosylated analogues. The analgesic potency of [Hyp(6),Lys(7)]-, [Hyp(betaGlc)(6),Lys(7)]- and [Tyr(betaGlc)(5),Lys(7)]-dermorphin were also tested in vivo by the tail-flick test. The glucosylated hydroxyproline-containing analogue is 8-10 times less active than the parent peptide, but its analgesic effect lasts significantly longer.     

Surface-enhanced Raman difference between bombesin and its modified analogues on the colloidal and electrochemically roughen silver surfaces

In this article, surface-enhanced Raman scattering (SERS) spectra of bombesin (BN) and its six modified analogues ([D-Phe(12)]BN, [Tyr(4)]BN, [Tyr(4),D-Phe(12)]BN, [D-Phe(12),Leu(14)]BN, [Leu(13)-(R)-Leu(14)]BN, and [Lys(3)]BN) on a colloidal silver surface are reported and compared with SERS spectra of these species immobilized onto an ellectrochemically roughen silver electrode. Changes in enhancement and wavenumber of proper bands upon adsorption on different silver surfaces are consistent with BN and its analogues adsorption primarily through Trp(8). Slightly different adsorption states of these molecules are observed depending upon natural amino acids substitution. For example, the indole ring in all the peptides interacts with silver nanoparticles in a edge-on orientation. It is additionally coordinated to the silver through the N(1)--H bond for all the peptides, except [Phe(12)]BN. This is in contrary to the results obtained for the silver roughen electrode that show direct but not strong N(1)--H/Ag interaction for all peptides except [D-Phe(12),Leu(14)]BN and [Leu(13)-(R)-Leu(14)]BN. For BN only C==O is not involved in the chemical coordination with the colloidal surface. [Lys(3)]BN and BN also adsorb with the C--N bond of NH(2) group normal and horizontal, respectively, to the colloidal surface, whereas C--NH(2) in other peptides is tilted to this surface. Also, the Trp(8) --CH(2)-- moiety of only [Tyr(4)]BN, [Lys(3)]BN, and [Tyr(4),D-Phe(12)]BN coordinates to Ag, whereas the Phe(12) ring of [Phe(12)]BN, [Tyr(4),D-Phe(12)]BN, and [D-Phe(12),Leu(14)]BN assists in the peptides binding only on the colloidal silver.     

Circular dichroic properties of the tyrosine residues in tetrazole analogues of opioid peptides.

CD studies on tetrazole analogues of opioid peptides show that peptides sharing the same N-terminal sequence, H-TyrPsi[CN(4)]Gly-, give very large Cotton effects of the Tyr side chain in the near-UV region. CD spectra of five such peptides: H-TyrPsi[CN(4)]Gly-Gly-Phe-Leu-OH (I), H-TyrPsi[CN(4)]Gly-Phe-Pro-Gly-Pro-Ile-NH(2) (II), H-TyrPsi[CN(4)]Gly-Phe-Pro-NH(2) (III), H-TyrPsi[CN(4)]Gly-Phe-Gly-Tyr-Pro-Ser-NH(2) (IV), and H-TyrPsi[CN(4)]Gly-Phe-Asp-Val-Val-Gly-NH(2) (V), and two others for comparison: H-Tyr-GlyPsi[CN(4)]Gly-Phe-Leu-OH (VI) and H-TyrPsi[CN(4)]Ala-Phe-Gly-Tyr-Pro-Ser-NH(2) (VII), were measured in methanol, 2,2,2-trifluoroethanol, and water at different pH values. The spectra show that the conformations of the Tyr(1) residue in peptides I-V are very similar in all solvents used but differ distinctly from those observed for VI and VII. Strong Tyr bands in the aromatic region result probably from the rigid structure of the common N-terminal part of peptides I-V. These bands are weaker for IV, which maybe due to the presence of a second Tyr residue in that peptide, giving an opposite contribution to the CD spectrum as that arising from Tyr1. It seems that the rigid structure of the N-terminal part of I-V results from the interaction of the Tyr(1) side chain and the tetrazole ring. The CD bands of the Tyr residues of VI and VII are much smaller than those of I-V in all solvents, except VII in trifluoroethanol (TFE) where Tyr bands comparable in intensity to those of I-V are observed. This spectral property may derive from the same sign contribution of both Tyr residues of VII to the CD spectrum.     

Analogues of human parathyroid hormone (1-31)NH(2): further evaluation of the effect of conformational constraint on biological activity

A series of conformationally-restricted analogues of hPTH was prepared, based on the parent peptide agonist, cyclo(Lys(18)-Asp(22))[Ala(1),Nle(8),Lys(18),Asp(22),Leu(27)]hPTH(1-31)NH(2) (2, EC(50)=0.29nM). Truncation of 2 at either the N- or C-termini resulted in peptides with reduced agonist activity as measured by stimulation of adenylate cyclase activity in the rat osteosarcoma cell line (ROS 17/2.8). Alanine- and glycine-scanning at the N-terminus of 2 was consistent with data previously obtained on linear hPTH(1-34). Other locations within the primary sequence of hPTH(1-31)NH(2) were evaluated by the placement of the [i, i+4] lactam constraining element. Ring size and lactam orientations at the 18-22 positions were also examined.     

Phase transfer catalysis in solid phase peptide synthesis. Preparation of cyclo[Xxx-Pro-Gly-Yyy-Pro-Gly] model peptides and their conformational analysis.

Relatively small cyclic peptides that contain functionalized side chains provide interesting model compounds for studying side chain-side chain interactions, peptide backbone flexibility (especially if X-Pro bonds are included), and as potential enzyme mimetics. In order to develop more efficient synthetic routes to compounds such as cyclo(Xxx-Pro-Gly-Yyy-Pro-Gly), using the Merrifield method, we have investigated several orthogonal solid phase synthesis strategies and contrasted the use of two solid phase peptide-resin cleavage techniques for preparing partially protected linear sequences. Phase transfer catalysis using tetrabutyl ammonium hydrogen sulfate in THF with saturated aqueous K2CO3 provides peptide acid salts in which most of the common protecting groups (Arg(NO2), Tyr(Bzl), Z-Lys, Lys(Boc), and Glu(tBu)) are not affected. Using 500 MHz proton NMR, peptides having a cyclo (L-L-Gly-L-L-Gly) sequence generally display two conformers in DMSO-d6 with the major isomer being the bis-cis conformer, while the minor form contains two beta turns. For peptides with a cyclo(D-L-Gly-L-L-Gly) sequence, the major conformer contains one cis and one trans X-Pro bond and one Type II beta turn, as previously predicted for related structure by Kopple and others.     

In vitro and in vivo characterization of three 68Ga- and 111 In-labeled peptides for cholecystokinin receptor imaging

Cholecystokinin (CCK) receptors are overexpressed in several human tumor types, such as medullary thyroid carcinomas and small cell lung cancers. Several ligands for the CCK2 receptor (CCK2R) have been developed for radionuclide targeting of these tumors. In this study, we evaluated whether radiolabeled DOTA-sCCK8 and its stabilized derivative, DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)], are suitable for imaging of CCK2R-positive tumors, using DOTA-MG0 as a reference. In vivo targeting of CCK2R-positive tumors with DOTA-sCCK8, DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)], and DOTA-MG0, labeled with (111)In or (68)Ga, was evaluated in BALB/c nude mice with a subcutaneous A431-CCK2R tumor. Biodistribution studies and single-photon emission computed tomography (SPECT) and positron emission tomography (PET) were performed at 1 hour postinjection. All peptides specifically accreted in the CCK2R-expressing tumors. Both (111)In-DOTA-sCCK8 and (111)In-DOTA-sCCK8[Phe(2)(p-CH2SO3H), Nle(3,6)] showed good tumor retention (4.65% ID/g and 5.44% ID/g, respectively, at 4 hours postinjection). On PET/computed tomographic (CT) and SPECT/CT scans, subcutaneous A431-CCK2R tumors were clearly visualized with low uptake of sCCK8 peptides in the intestines. Whereas radiolabeled DOTA-MG0 showed high kidney uptake (70% ID/g), the sCCK8 peptides showed low uptake in the kidneys. Sulfated CCK8 analogues combined high tumor uptake with low retention in the kidney and are therefore promising tracers for imaging of CCK2R-positive tumors.     

Conformational analysis of a potent SSTR3-selective somatostatin analogue by NMR in water solution.

The three-dimensional structure of a potent SSTR3-selective analogue of somatostatin, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-Agl(8)(N(beta) Me, 2-naphthoyl)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-Agl(8)(N(beta) Me, 2-naphthoyl)]-SRIF) (peptide 1) has been determined by (1)H NMR in water and molecular dynamics (MD) simulations. The peptide exists in two conformational isomers differing mainly by the cis/trans isomerization of the side chain in residue 8. The structure of 1 is compared with the consensus structural motifs of other somatostatin analogues that bind predominantly to SSTR1, SSTR2/SSTR5 and SSTR4 receptors, and to the 3D structure of a non-selective SRIF analogue, cyclo(3-14)H-Cys(3)-Phe(6)-Tyr(7)-D-2Nal(8)-Lys(9)-Thr(10)-Phe(11)-Cys(14)-OH (des-AA(1, 2, 4, 5, 12, 13)[Tyr(7), D-2Nal(8)]-SRIF) (peptide 2). The structural determinant factors that could explain selectivity of peptide 1 for SSTR3 receptors are discussed.     

NMR conformational analyses on (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23]: the importance of alpha-helices, a cation-pi interaction and a beta-turn

The preferred conformations of the orphan G-protein coupled receptor agonists (des-bromo) neuropeptide B [1-23] and neuropeptide W [1-23], referred to as NPB and NPW, have been determined by (1)H NMR, CD, and molecular modeling. The sequences of NPB and NPW are WYKPAAGHSSYSVGRAAGLLSGL and WYKHVASPRYHTVGRAAGLLMGL, respectively. These are hypothalamic peptides that exert their biological actions on GPR7 and GPR8 receptors. Micellar solutions using the membrane mimetic, sodium dodecylsulphate-d(25) (SDS), were used to mimic a physiological environment for the peptides. The secondary structure of NPB consists of a type II beta-turn involving residues Lys(3) to Ala(6). The C-terminal region of NPB exists in a conformational equilibrium between different secondary structures, including an alpha-helix from residues Arg(15) to Ser(21), and a 3(10)-helix from residues Ser(12) to Ser(21). The N-terminus of NPW exhibits a cation-pi interaction between the Lys(3) side chain and the quadrupole moment of the Trp(1) indole group. At the C-terminus of NPW, a well-defined alpha-helical conformation exists from Arg(15) to Met(21). As NPB and NPW have 91% sequence homology from residues Val(13) to Leu(23), with only residue 21 differing between the two peptides, the similar C-terminal secondary structures of these two peptides are consistent with the sequences. This is supported by the similar CD spectra. The different secondary structures at the N-termini for NPB and NPW point to the importance of the N-terminus in receptor binding. This is consistent with the work of Fujii et al. [J. Biol. Chem. 277, 34010-34016 (2002)] who observed that iodination of the NPB Tyr(2) resulted in decreased agonistic activity at GPR7. In addition, Tanaka et al. [Proc. Natl. Acad. Sci. USA 100, 6251-6256 (2003)] showed that deletion of Trp(1) from NPB or NPW drastically decreased activity at GPR7 for NPB and GPR7 and GPR8 for NPW. Therefore, we postulate that the N-terminus is involved in membrane recognition and receptor binding.     

Treatment of experimental allergic encephalomyelitis (EAE) induced by guinea pig myelin basic protein epitope 72-85 with a human MBP(87-99) analogue and effects of cyclic peptides

Experimental autoimmune encephalomyelitis (EAE) is an inflammatory and demyelinating disease of the central nervous system and is an animal model of multiple sclerosis (MS). In the present report, a linear analogue and a series of cyclic semi-mimetic peptides were designed and synthesized based on the human myelin basic protein (MBP(87-99)) epitope (Val87-His-Phe-Phe-Lys-Asn-Ile-Val-Thr-Pro-Arg-Thr-Pro90) and on Copolymer I (a mixture of random polymers of Ala, Gln, Lys and Tyr used to treat MS). These analogues were designed looking for suppressors of EAE induced by guinea pig MBP(72-85) epitope (Gln-Lys-Ser-Gln-Arg-Ser-Gln-Asp-Glu-Asn-Pro-Val) in Lewis rats. The linear analogue [Arg91,Ala96]MBP(87-99), in which Arg substitutes Lys91 and Ala substitutes Pro96, was found to be a strong inhibitor which when administered to Lewis rats together with the encephalitogenic agonist MBP(72-85) completely prevented the induction of EAE. In contrast, three N- and C-termini amide-linked cyclic semi-mimetic peptides, [cyclo-Phe-Arg-Asn-Ile-Val-Thr-Ala-Acp (1), cyclo-Phe-Ala-Arg-Gln-Acp (2), cyclo-Tyr-Ala-Lys-Gln-Acp (3)] as well as a Lys side chain and C-terminous cyclic semi mimetic peptide cyclo(Lys, Acp)-Phe-Lys-Asn-Ile-Val-Thr-Ala-Acp (4) which contain segments of MBP(87-99) or are constituted from immunophoric residues of copolymer 1, were ineffective in inducing or inhibiting EAE in Lewis rats. However co-injection of cyclic analogues with MBP(72-85) delayed the onset of EAE indicating a modulatory effect on the EAE activity of MBP(72-85). These findings suggest that molecule length, size of cyclic moiety and backbone conformation are important elements for immunogenic activity. Moreover blockade of MBP(72-85) induced EAE by the unrelated peptide [Arg91,Ala56]MBP(87-99) could indicate that the mechanism of inhibition is not due to binding competition but rather due to the delivery of a negative signal by the antagonist which overcomes the agonist response possibly through the activation of antigen specific regulatory T cells.     

Functional evidence for an intramolecular side chain interaction between residues 6 and 10 of receptor-bound parathyroid hormone analogues

The N-terminal domain of PTH(1-34) is critical for PTH-1 receptor (P1R) activation and has been postulated to be alpha-helical when bound to the receptor. We investigated the possibility that the side chains of residues 6 (Gln) and 10 (Gln or Asn) of PTH analogues, which would align on the same face of the predicted alpha-helix, could interact and thereby contribute to the PTH/P1R interaction process. We utilized PTH(1-11), PTH(1-14), and PTH(1-34) analogues substituted with alanine at one or both of these positions and functionally evaluated the peptides in cell lines (HKRK-B7 and HKRK-B28) stably expressing the P1R, as well as in COS-7 cells transiently expressing either the P1R or a P1R construct that lacks the amino-terminal extracellular domain (P1R-DelNt). In HKRK-B7 cells, the single substitutions of Gln(6) --> Ala and Gln(10) --> Ala reduced the cAMP-stimulating potency of [Ala(3),Gln(10),Arg(11)]rPTH(1-11)NH(2) approximately 60- and approximately 2-fold, respectively, whereas the combined Ala(6,10) substitution resulted in a approximately 2-fold gain in potency, relative to the single Ala(6) substitution. Similar effects on P1R-mediated cAMP-signaling potency and P1R-binding affinity were observed for these substitutions in [Aib(1,3),Gln(10),Har(11),Ala(12),Trp(14)]rPTH(1-14)NH(2). Installation of a lactam bridge between the Lys(6) and the Glu(10) side chains of [Ala(3,12),Lys(6),Glu(10),Har(11),Trp(14)]rPTH(1-14)NH(2) increased signaling potency 6-fold, relative to the nonbridged linear analogue. Alanine substitutions at positions 6 and/or 10 of [Tyr(34)]hPTH(1-34)NH(2) did not affect signaling potency nor binding affinity on the intact P1R; however, Ala(6) abolished PTH(1-34) signaling on P1R-DelNt, and this effect was reversed by Ala(10). The overall data support the hypothesis that the N-terminal portion of PTH is alpha-helical when bound to the activation domain of the PTH-1 receptor and they further suggest that intrahelical side chain interactions between residues 6 and 10 of the ligand can contribute to the receptor interaction process.     

Interaction of linear and cyclic peptide antagonists at the human B(2) kinin receptor

The ligand receptor interactions involving the C-terminal moiety of kinin B(2) receptor antagonists Icatibant (H-DArg-Arg-Pro-Hyp-Gly-Thi-Ser-Dtic-Oic-Arg-OH), MEN 11270 (H-DArg-Arg-Pro-Hyp-Gly-Thi-c(Dab-Dtic-Oic-Arg)c(7gamma-10alpha)) and a series of analogs modified in position 10 were investigated by radioligand-binding experiments at the wild type (WT) and at the Ser(111)Ala and Ser(111)Lys mutant human kinin B(2) receptors. Icatibant and [Lys(10)]-Icatibant maintained the same high affinity towards the three receptors. For Icatibant-NH(2), [Ala(10)]-Icatibant, MEN 11270 and [Glu(10)]-MEN 11270, the changes in affinity at the WT and Ser(111)Lys receptors indicated that the presence of a net positive or negative charge at the C-terminal moiety of these peptides caused a decrease in affinity to the WT receptor and that Ser(111) residue is in proximity of the side chain of residue 10. The changes in affinity measured with [desArg(10)]-Icatibant and [desArg(10)]-Icatibant-NH(2), moreover, confirmed that a C-terminal charge compensation between the positive charge of Arg(10) side chain and the C-terminal free carboxylic function favours a high affinity interaction.     

Zebrafish express the common parathyroid hormone/parathyroid hormone-related peptide receptor (PTH1R) and a novel receptor (PTH3R) that is preferentially activated by mammalian and fugufish parathyroid hormone-related peptide.

To further explore the evolution of receptors for parathyroid hormone (PTH) and PTH-related peptide (PTHrP), we searched for zebrafish (z) homologs of the PTH/PTHrP receptor (PTH1R). In mammalian genes encoding this receptor, exons M6/7 and M7 are highly conserved and separated by 81-84 intronic nucleotides. Genomic polymerase chain reaction using degenerate primers based on these exons led to two distinct DNA fragments comprising portions of genes encoding the zPTH1R and the novel zPTH3R. Sequence comparison of both full-length teleost receptors revealed 69% similarity (61% identity), but less homology with zPTH2R. When compared with hPTH1R, zPTH1R showed 76% and zPTH3R 67% amino acid sequence similarity; similarity with hPTH2R was only 59% for both teleost receptors. When expressed in COS-7 cells, zPTH1R bound [Tyr(34)]hPTH-(1-34)-amide (hPTH), [Tyr(36)]hPTHrP-(1-36)-amide (hPTHrP), and [Ala(29),Glu(30), Ala(34),Glu(35), Tyr(36)]fugufish PTHrP-(1-36)-amide (fuguPTHrP) with a high apparent affinity (IC(50): 1.2-3.5 nM), and was efficiently activated by all three peptides (EC(50): 1.1-1.7 nM). In contrast, zPTH3R showed higher affinity for fuguPTHrP and hPTHrP (IC(50): 2.1-11.1 nM) than for hPTH (IC(50): 118.2-127.0 nM); cAMP accumulation was more efficiently stimulated by fugufish and human PTHrP (EC(50): 0.47 +/- 0.27 and 0.45 +/- 0.16, respectively) than by hPTH (EC(50): 9.95 +/- 1.5 nM). Agonist-stimulated total inositol phosphate accumulation was observed with zPTH1R, but not zPTH3R.     

Preparation of a photoreactive analog of parathyroid hormone [Nle8, Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]bovine parathyroid hormone-(1-34)NH2, a selective, high-affinity ligand for characterization of parathyroid hormone receptors

Photoaffinity radiolabeling techniques have been widely used to characterize the properties of peptide hormone receptors. However, the identity of authentic receptors is often uncertain because many macromolecules are labeled. These ambiguities are due, in part, to the use of a heterogeneous mixture of photoreactive photoligands, many of which have no or low affinity for the relevant hormone receptor. In this report, we describe the synthesis, purification, and structural analysis of the photoreactive parathyroid hormone analog, [Nle8,Lys(N-epsilon-4-azido-2-nitrophenyl)13,Nle18,Tyr34]-bovine parathyroid hormone-(1-34)NH2. The sulfur-free, oxidation-resistant, synthetic analog of bovine parathyroid hormone (PTH), [Nle8,Nle18,Tyr34]bovine PTH-(1-34)NH2 (NlePTH), was reacted with 4-fluoro-3-nitrophenylazide under nonaqueous conditions to yield several derivatives which were separated by reverse-phase high-performance liquid chromatography and analyzed by amino acid compositional analysis, thin-layer chromatography, and ultraviolet and visible absorption spectroscopy. Among the NlePTH derivatives generated, one of the least hydrophobic was shown to retain the highest potency as assessed in the canine renal cortical membrane radioreceptor assay. Sequence analysis of this peptide, after it had been derivatized with 4-fluoro-3-nitro-[2,6-3H]phenylazide and purified to homogeneity, permitted us to determine that the structure of this analog is [Nle8,Lys(N-epsilon-4-azide-2-nitrophenyl)13,Nle18,Tyr34]bovine PTH-(1-34)NH2. We emphasize the importance of using photoreactive ligands which are purified and subjected to detailed chemical and biological analyses for characterizing the properties of parathyroid hormone receptors and receptors for other peptide hormones.     

Long-range effects in protein--ligand interactions mediate peptide specificity in the human major histocompatibilty antigen HLA-B27 (B*2701).

B*2701 differs from all other HLA-B27 subtypes of known peptide specificity in that, among its natural peptide ligands, arginine is not the only allowed residue at peptide position 2. Indeed, B*2701 is unique in binding many peptides with Gln2 in vivo. However, the mutation (Asp74Tyr) responsible for altered selectivity is far away from the B pocket of the peptide binding site to which Gln/Arg2 binds. Here, we present a model that explains this effect. It is proposed that a new rotameric state of the conserved Lys70 is responsible for the unique B*2701 binding motif. This side chain should be either kept away from pocket B through its interaction with Asp74 in most HLA-B27 subtypes, or switched to this pocket if residue 74 is Tyr as in B*2701. Involvement of Lys70 in pocket B would thus allow binding of peptides with Gln2. Binding of Arg2-containing peptides to B*2701 is also possible because Lys70 could adopt another conformation, H-bonded to Asn97, which preserves the same binding mode of Arg2 as in B*2705. This model was experimentally validated by mutating Lys70 into Ala in B*2701. Edman sequencing of the B*2701(K70A) peptide pool showed only Arg2, characteristic of HLA-B27-bound peptides, and no evidence for Gln2. This supports the computational model and demonstrates that allowance of B*2701 for peptides with Gln2 is due to the long-range effect of the polymorphic residue 74 of HLA-B27, by inducing a conformational switch of the conserved Lys70.     

Analgesic dipeptide derivatives. II. Synthetic dipeptides containing a C-terminal 2-(2-nitrophenylsulfenyl) tryptophan residue

The syntheses of dipeptide derivatives of the general formula X-Trp(Nps)-OR (R = H or Me; X = Lys, Gly, Ala, Leu, Ser, Glu, His, Arg, Orn, Dpr, Gpr and Har) are described. The analgesic activities of Gpr- and Har-containing dipeptides have been evaluated and, in the light of these results, the influence of the side chain length of the basic amino acid on the analgesic effect is studied.     

Electrophysiological studies with new CCK analogs: correlation with binding affinity on B-type receptors

The electrophysiological effects of Boc-D-Asp-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-Phe-NH2 (compound I) and Boc-gamma-D-Glu-Tyr(SO3H)-Nle-D-Lys-Trp-Nle-Asp-Phe-NH2 (compound II), two cyclic cholecystokinin analogs with high selectivity for CCK-B receptors, as well as the effects of the linear enzyme-resistant analog Boc-[Nle28,Nle31]-CCK7 (BDNL), were compared with those of CCK8 using extracellular recordings in rat hippocampal slices in vitro. Bath applications of the three synthetic compounds resulted in concentration-dependent and reversible increases in single-unit activity. Comparison of equieffective concentrations yielded the following potency rank order: BDNL greater than CCK8 greater than compound II greater than compound I. There was a close correlation (r = .96, slope = 0.98) between the excitatory activities of the analogs and their potencies in displacing radiolabelled CCK8 from CCK-B receptors on rat brain membranes.     

Synthesis, biological activity and conformational analysis of cyclic GRF analogs

A novel cyclic GRF analog, cyclo(Asp8-Lys12)-[Asp8,Ala15]-GRF(1-29)-NH2, i.e. cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2, was synthesized by the solid phase procedure and found to retain significant biological activity. Solid phase cyclization of Asp8 to Lys12 proceeded rapidly (approximately 2 h) using the BOP reagent. Substitution of Ala2 with D-Ala2 and/or NH2-terminal replacement (desNH2-Tyr1 or N-MeTyr1) in the cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 system resulted in highly potent analogs that were also active in vivo. Conformational analysis (circular dichroism and molecular dynamics calculations based on NOE-derived distance constraints) demonstrated that cyclo8,12[Asp8,Ala15]-GRF(1-29)-NH2 contains a long alpha-helical segment even in aqueous solution. A series of cyclo8,12 stereoisomers containing D-Asp8 and/or D-Lys12 were prepared and also found to be highly potent and to retain significant alpha-helical conformation. The high biological activity of cyclo8,12[N-MeTyr1,D-Ala2,Asp8,Ala15]-GRF(1-29)- NH2 may be explained on the basis of retention of a preferred bioactive conformation.     

Preparation and characterization of [N alpha-(4-azido-2-nitrophenyl)Ala1,Tyr36]-parathyroid hormone related peptide (1-36)amide: a high-affinity, partial agonist having high cross-linking efficiency with its receptor on ROS 17/2.8 cells

The synthesis, purification, and structural analysis of the major compounds resulting from photoderivatization of [Tyr36]-parathyroid hormone related peptide (1-36)amide [[Tyr36]PTHrP(1-36)amide] are described. The reaction of the synthetic peptide with 4-fluoro-3-nitrophenyl azide under nonaqueous conditions yields three major products (peaks D-1, D-2, and G), which were purified to homogeneity by reverse-phase high-performance liquid chromatography. Subsequent amino acid analysis showed that the peptides of peaks D-1 and G each lack one lysine residue, while the peptide in peak D-2 lacks one alanine residue, suggesting that these residues are chemically modified by photoderivatization. Sequence analysis of the photoderivatized peptides revealed that compounds D-1 and G were derivatized on Lys13 and Lys11, respectively. Compound D-2 was N-blocked, indicating that this compound is derivatized on the alpha-amino function of Ala1. Both Lys residues of D-2 were quantitatively recovered upon sequencing after digestion with endoproteinase Glu-C. Compounds D-2 and G had apparent KdS of 1 X 10(-9) M and 0.6 X 10(-9) M, respectively, for their receptors on ROS 17/2.8 cells, which are identical with or similar to that of the underivatized [Tyr36]PTHrP(1-36)amide. Compound G had the same adenylate cyclase stimulating potency as the underivatized, synthetic [Tyr36]PTHrP(1-36)amide, whereas compound D-2 was only a partial agonist, having about 25% of the maximal cAMP production. Compound D-1, which is modified on Lys13, retained only 2-4% of its receptor binding affinity and biological activity relative to that of its parent compound.(ABSTRACT TRUNCATED AT 250 WORDS)