Analysis of the adrenal angiotensin II receptor with the photoaffinity labeling method

The angiotensin II (Asp-Arg-Val-Tyr-Ile-His-Pro-Phe, AT) receptor of bovine adrenocortex has been investigated with photosensitive analogues of AT. In a first series of experiments, we have shown that isolated cortical cells secrete aldosterone in a permanent and specific manner if they have been photolyzed in the presence of the photolabel [Sar1,(4'-N3)Phe8]AT. This permanent stimulation is in contrast to the smooth muscle assays where under similar conditions a permanent and specific block was always observed. It is assumed that the irreversible occupation of the AT receptor produces this effect. In a second type of experiment, we have shown that the AT binding site on adrenocortical membranes can be specifically and irreversibly occupied under similar conditions and that this occupation can be prevented in a competitive manner by the presence of nonphotosensitive hormone. Using a radioactive label, [Sar1,(3'-125I)Tyr4,(4'-N3)Phe8]AT, we have identified the AT receptor as a 300-kDa protein by means of gel filtration under nonreducing and nondenaturating conditions. Under reducing and denaturing conditions, a subunit of 60 kDs was found by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. The AT receptor is proposed to be a 300-kDa protein with one binding subunit of 60 kDa.     

In vitro and in vivo characterization of opioid activities of endomorphins analogs with novel constrained C-terminus: evidence for the important role of proper spatial disposition of the third aromatic ring

In the present study, the C-terminus of endomorphin (Tyr(1)-Pro(2)-Trp/Phe(3)-Phe(4)-NH(2), EMs) analogs [Xaa(4)-R]EMs, modified by substitution of a non-aromatic residue for Phe(4) and ending up with -NH-benzyl, were designed to generate an atypical conformationally constrained peptide set. We investigated the effects of these analogs on the opioid receptors affinity, guinea pig ileum (GPI) and mouse vas deferens (MVD) activity, system arterial pressure (SAP), heart rate (HR), antinociception and colonic motility. Analogs 5 ([D-V(4)-Bzl]EM1) and 10 ([D-V(4)-Bzl]EM2), which exhibit appropriate spatial orientations of the third aromatic ring, were about 3-4 times more potent than their parents both in vivo and in vitro. However, a drastic loss of activity was found in analogs 2 ([A(4)-Bzl]EM1) and 7 ([A(4)-Bzl]EM2), which possess improper spatial orientations of the third aromatic ring. Interestingly, analog 7 or 3 ([G(4)-Bzl]EM1), when injected intravenously (i.v.), produced significantly different changes in SAP from their parents. Surprisingly, analog 4 displayed relatively higher vasodepressor activity but significantly less potent colonic contractile activity than analog 5. This may be elicited by the differences in the spatial disposition of the third aromatic ring, which were verified by molecular modeling. Our results indicate that the proper spatial disposition of the third aromatic ring plays an important role in the regulation of pharmacological activities of EMs.     

Synthesis and biological activities of angiotensin II and Sarmesin analogues containing cyclohexylalanine

Analogues of angiotensin II with cyclohexylalanine (Cha) at position 4 or 8, and analogues of the competitive (type II) angiotensin antagonist [Sar1,Tyr(Me)4]ANG II (Sarmesin) with Cha at position 8, have been prepared by the solid phase method and purified by reversed-phase HPLC. Analogues of ANG II with Cha at position 8 in which the position 1 residue was substituted with sarcosine (Sar) or amino-isobutyric acid (Aib) or was deleted (Des), were slowly reversing (Type I) antagonists with "pA2" values in the rat isolated uterus assay of approximately 8.5. The additional substitution of Tyr(Me) for Tyr at position 4 of these peptides gave reversible competitive (Type I/II) antagonists with pA2 values of 6.7, 5.8, and less than 5, while substitution of Phe for Tyr gave pA2 values of 7.4, 6.7, and less than 5, respectively. All 19 peptides synthesized in this study had low intrinsic agonist activity in the rat isolated uterus assay except for the type I antagonists [Sar1, Cha8]ANG II (7%), [Aib1, Cha8]ANG II (12%) and [Des1, Cha8]ANG II (20%). These data illustrate that the substitution of Cha at position 8 of ANG II analogues produces potent antagonists; however, Type I antagonists retain significant agonist activity whereas Type I/II antagonists do not. In contrast, substitution of Cha at position 4 in a variety of ANG II analogues resulted in severely diminished biological activity, illustrating that the presence of an aromatic ring quadrupole at position 4 is obligatory for receptor binding and activity.     

Photoaffinity labeling of a viral induced protein from tobacco. Characterization of nucleotide-binding properties

We have used the photoaffinity analogs 8-azidoadenosine 5'-triphosphate (8-N3ATP) and 8-azidoguanosine 5'-triphosphate (8-N3GTP) to investigate the relationship between a viral induced protein (Mr = 120,000) in tobacco mosaic virus (TMV)-infected tobacco and the TMV-induced RNA-dependent RNA polymerase activity. When the radioactive analogs [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP were incubated with the tobacco tissue homogenate from TMV-infected plants, incorporation of label occurred into the viral induced protein in the presence of UV light. The incorporation was found to be totally dependent on UV-illumination and greatly enhanced by Mg2+. Saturation of photoincorporated label indicates an apparent Kd of 16 microM (+/- 3 microM) and 12 microM (+/- 3 microM) for 8-N3ATP and 8-N3GTP, respectively. Protection against photolabeling by [gamma-32P]8-N3ATP and [gamma-32P]8-N3GTP with various nonradioactive nucleotides and nucleosides suggests that the photolabeled site is protected best by nucleoside triphosphates. At 200 microM both deoxyribonucleoside triphosphates and ribonucleoside triphosphates were very effective at protecting the site from photolabeling. These data suggest that the photolabeled protein may be part of an RNA-dependent RNA polymerase. The utility of nucleotide photoaffinity analogs as a method to study viral induced nucleotide-binding proteins is discussed.     

Study on the influence of tyrosine deprotonation on the myotropic action of angiotensin II.

The influence of phenolic tyrosine ionization in angiotensin II on the myotropic action of this peptide has been investigated in vitro on rabbit aortic strips. [Sar1, Tyr4]angiotensin II and [Sar1, (4'-amino) Phe4]angiotensin II (as a reference which cannot undergo the same ionization) were tested over a pH range from 6.8 to 9.0 and their activities compared. The results clearly indicate that angiotensin II with a deprotonated phenolic hydroxyl group on Tyr in position 4 is not the most active or only active form of angiotensin II.     

Long-acting oxytocin antagonists: effects of 2-D-stereoisomer substitution on antagonistic potency and duration of action

Recently we reported the discovery of a series of 2-O-alkyltyrosine- (or 2-p-alkylphenylalanine), 4-threonine-, and 8-ornithine-substituted analogs of [1-penicillamine]oxytocin [( Pen1]OT) which possess prolonged anti-OT activity. In this study, we attempt to improve the potency and the duration of action of this series of OT antagonists by exploring the effects of D-stereoisomer substitution in the 2 position. We compare the in vitro anti-OT potency, expressed in pA2 values, and the duration of in vivo inhibitory action, expressed in recovery t1/2, of [Pen1]OT, [Pen1,Orn8]OT, [Pen1,Thr4]OT, [Pen1,Tyr(OMe)2,Thr4, Orn8]OT, [Pen1, Tyr(OEt)2,Thr4,Orn8]OT, [Pen1,D-Tyr(OEt)2,Thr4,Orn8]OT, [Pen1,Phe2,Thr4]OT, [Pen1,Phe(Me)2,Thr4,Orn8]OT, [Pen1,D-Phe(Me)2,Thr4,Orn8]OT, [Pen1,Phe(Et)2,Thr4,Orn8]OT, and [Pen1,D-Phe(Et)2,Thr4,Orn8]OT. The results show that modifications of the amino acid in position 2 by alkylation of the aromatic ring and use of D-stereoisomerism produce nonparallel effects on the in vitro potency and duration of action of OT antagonists. Time-action curve determinations show that long-acting OT antagonists exhibit delayed peak inhibitory action. Long action is not coupled with high potency in all cases. This dissociation between potency and duration of action gives support to our hypothesis that the potency and duration of action of these peptides may each have different conformational structure requirements.     

Identification of selective and non-selective, biostable beta-amino acid agonists of recombinant insect kinin receptors from the southern cattle tick Boophilus microplus and mosquito Aedes aegypti

The multifunctional arthropod 'insect kinins' share the evolutionarily conserved C-terminal pentapeptide motif Phe-X1-X2-Trp-Gly-NH2, where X1=His, Asn, Ser, or Tyr and X2=Ser, Pro, or Ala. Eight different analogs of the insect kinin C-terminal pentapeptide active core in which the critical residues Phe 1, Pro3 and Trp 4 are replaced with beta 3-amino acid and/or their beta2-amino acid counterparts were evaluated on recombinant insect kinin receptors from the southern cattle tick, Boophilus microplus (Canestrini) and the dengue vector, the mosquito Aedes aegypti (L.). A number of these analogs previously demonstrated enhanced resistance to degradation by peptidases. Single-replacement analog beta 2 Trp 4 and double-replacement analog [beta 3 Phe 2, beta 3 Pro 3] of the insect kinins proved to be selective agonists for the tick receptor, whereas single-replacement analog beta 3 Pro 3 and double-replacement analog [beta 3 Phe, beta 3 Pro 3] were strong agonists on both mosquito and tick receptors. These biostable analogs represent new tools for arthropod endocrinologists and potential leads in the development of selective, environmentally friendly arthropod pest control agents capable of disrupting insect kinin-regulated processes.     

Structure-desensitization relationships of angiotensin analogues in the rat isolated uterus

The desensitizing potencies of angiotensin II (ANG II) analogues modified at positions 1, 2, 4, 7, and 8 have been examined in the rat isolated uterus assay by determining the time of recovery of the half-maximal concentration (EC50) response to angiotensin II after treatment of the tissues with a high dose (10(-5) M) of each analogue for 2 min. The magnitude of the desensitization effect was substituent dependent in the following manner: position 1, sarcosine (Sar) greater than Asp greater than des-Asp; position 2, Arg greater than Sar; position 4, Tyr greater than Tyr(Me) approximately Phe; position 7, 3,4-dehydroproline (Dpr) greater than Pro greater than thioproline (Tpr) greater than Sar; position 8, Ile greater than D-Trp greater than Ala greater than Phe. The "additivity" rule applied to these structure-desensitization relationships and the most potent desensitizer, requiring 3 h for reestablishment of the EC50 response, was [Sar1, Dpr7, Ile8]-ANG II. The desensitizing potencies of these analogues did not correlate with agonist or antagonist activities and demonstrated that the angiotensin-mediated tissue desensitization process has unique structural determinants. Methylation or elimination of the tyrosine hydroxyl group of strong desensitizers virtually eliminated the desensitization effect, implicating the phenoxyl moiety in the mechanism of desensitization. The initial phase of recovery of angiotensin responsiveness after desensitization by several analogues appeared to obey first-order kinetics. The results are discussed in the contexts of both one- and two-site receptor models.     

Proton magnetic resonance studies of angiotensin II conformation: cis-trans isomerism in sarcosine7-containing analogs

1H-NMR spectra for the angiotensin agonist sarcosine-(Sar)Arg-Val-Tyr-Ile-His-Sar-Phe [( Sar1,Sar7]Ang II) and the antagonist Sar-Arg-Val-Tyr-Ile-His-Sar-Ile in dimethylsulfoxide-d6 were examined at 400 MHz. Splitting of the resonances for Tyr, His, and Sar protons revealed that the His6-Sar7 peptide bond existed in both cis and trans forms, with one isomer predominating in the ratio 5:1 in both peptides. Comparison of the chemical shifts for the His6 and Phe8 ring protons in these peptides suggested a His/Phe stacking interaction in [Sar1,Sar7]Ang II which is important for agonist activity.     

1H-NMR studies of [Sar1]angiotensin II conformation by nuclear Overhauser effect spectroscopy in the rotating frame (ROESY): clustering of the aromatic rings in dimethylsulfoxide

The conformational properties of the octapeptide [Sar1]ANG II in dimethylsulfoxide-d6 were investigated by rotating frame nuclear Overhauser effect spectroscopy (ROESY). Interresidue ROESY interactions were observed between Tyr ortho and Phe ring protons, between Phe ring and Pro C gamma protons, and also between His C alpha and Pro C delta protons. A weak connectivity was also observed between the Sar N-CH3 protons and a Tyr ortho proton. Intraresidue interactions between alpha and beta protons in Tyr, His and Phe indicated restricted rotation for the side-chains of the three aromatic residues. These findings suggest that [Sar1]ANG II takes up a folded conformation in DMSO in which the three aromatic rings form a cluster. Connectivities between the His C alpha proton and the two Pro C delta protons illustrated a preferred conformation for angiotensin II in DMSO in which the His-Pro bond exists as the trans isomer. The NMR spectroscopic evidence is consistent with the presence of a Tyr charge relay system in the biologically active conformation of angiotensin II and with the postulated role of the Tyr hydroxyl group in angiotensin II for receptor activation.     

Design,synthesis and biological evaluation of cyclic angiotensin II analogues with 3,5 side-chain bridges. Role of C-terminal aromatic residue and ring cluster for activity and implications in the drug design of AT1 non-peptide antagonists

The novel amide linked angiotensin II (ANG II) cyclic analogues: gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Ile(8)] ANG II (I) and gamma, epsilon -cyclo(3, 5)-[Sar(1)-Glu(3)-Lys(5)-Phe(8)] ANG II (II) have been designed, synthesized and bioassayed in anesthetized rabbits in order to unravel structural ring cluster characteristics important for receptor activation. Analogue I with Ile at position 8 was an inhibitor of Angiotensin II while analogue II with Phe at position 8 was found to be an agonist. Similar results were reported for cyclic compounds that have reversed the linking between positions 3 and 5. The overall results show that positions 3 and 5 do not govern the biological activity of the synthetic analogues. It also appears that the aromatic ring cluster (Tyr-His-Phe) in agonist peptides is an essential stereo-electronic feature for Angiotensin II to exert its biological activity. A non-peptide mimetic of ANG II, 1-[2'-[(N-benzyl)tetrazol-5-yl]biphenyl-4-yl]methyl]-2-hydroxymethylbenzimidazole (BZI8) has been designed and synthesized. This molecule is more rigid and much less active than AT(1) non-peptide mimetic losartan probably because it lacks to mimic the orientation of tetrazole and the pharmacophore segments of butyl chain and imidazole ring.     

Theoretical conformational analysis of six arginine vasopressin analogs with the L-naphthylalanine in position 3.

Introduction of the naphthylalanine residue into either position 3 of arginine vasopressin (AVP), or its analogs results in peptides with interesting pharmacological properties. The single substituted analog of AVP with L-2-Nal in position 3 causes moderate antiduretic activity, whereas [Mpa1, (L-1-Nal)3, (D-Arg)8] VP and [Mpa1, (L-2-Nal)3, (D-Arg)8] VP are potent and selective V2 agonists. Moreover [(L-2-Nal)3, (D-Arg)8] VP is among the most potent and selective antagonists of V1a receptors. In this study we carried out conformational calculations on [(L-1-Nal)3] AVP, [(L-2-Nal)3] AVP, [(L-1-Nal)3, (D-Arg)8] VP, [(L-2-Nal)3, (D-Arg)8] VP, [Mpa1, (L-1-Nal)3, (D-Arg)8] VP, [Mpa1, (L-2-Nal)3, (D-Arg)8] VP, using the ECEPP/3 force field with and without including hydration to simulate aqueous and nonpolar environments. It was found that in all six compound studied, the low-energy conformations have common geometry and relative energies. Therefore, the modifications of the Phe in position 3 influence the binding to the receptor by changing the size of the third residue, rather than by changing the conformational space. The lowest-energy conformations in the presence and absence of water had beta-turns at residues Phe3-Gln4 and Gln4-Asn5 and Gln4-Asn5, respectively. The conformation at the Gln4-Asn5 turn was most similar to the crystal structure of the pressinoic acid (the cyclic moiety of vasopressin).     

The design and synthesis of a potent Angiotensin II cyclic analogue confirms the ring cluster receptor conformation of the hormone Angiotensin II

The novel amide linked Angiotensin II potent cyclic analogue, c-[Sar1,Lys3,Glu5] ANG II 19 has been designed and synthesized in an attempt to test the aromatic ring clustering and the charge relay bioactive conformation we have recently suggested for ANG II. This constrained cyclic analogue was synthesized by connecting the Lys3 amino and Glu5 carboxyl side chain groups, and it was found to be potent in the rat uterus assay and in anesthetized rabbits. The central part of the molecule is fixed covalently in the conformation predicted according to the backbone bend conformational model proposed for Angiotensin II. The obtained results using a combination of 2D NMR, 1D NOE spectroscopy and molecular modeling revealed a similar Tyr4-Ile5-His6 bend, a His6-Pro7 trans configuration and a side chain aromatic ring cluster of the key aminoacids Tyr4, His6, Phe8 for c-[Sar1,Lys3,Glu5] ANG II as it has been found for ANG II (Matsoukas, J. H.; Hondrelis, J.; Keramida, M.; Mavromoustakos, T.; Markriyannis, A.; Yamdagni, R.; Wu, Q.; Moore, G. J. J. Biol. Chem. 1994, 269, 5303). Previous study of the conformational properties of the Angiotensin II type I antagonist [Hser(gamma-OMe)8] ANG II (Matsoukas, J. M.; Agelis, G.; Wahhab, A.; Hondrelis, J.; Panagiotopoulos. D.; Yamdagni, R.; Wu, Q.; Mavromoustakos, T.; Maia, H.; Ganter, R.; Moore, G. J. J. Med. Chem. 1995, 38, 4660) using 1-D NOE spectroscopy coupled with the present study of the same type of lead antagonist Sarilesin revealed that the Tyr4-Ile5-His6 bend, a conformational property found in Angiotensin II is not present in type I antagonists. The obtained results provide an important conformational difference between Angiotensin II agonists and type I antagonists. It appears that our synthetic attempt to further support our proposed model was successful and points out that the charge relay system and aromatic ring cluster are essential stereoelectronic features for Angiotensin II to exert its biological activity.     

Photoaffinity labeling of the rabbit reticulocyte guanine nucleotide exchange factor and eukaryotic initiation factor 2 with 8-azidopurine nucleotides. Identification of GTP- and ATP-binding domains

We have covalently modified rabbit reticulocyte polypeptide chain initiation factor 2 (eIF-2) and the guanine nucleotide exchange factor (GEF) with the 8-azido analogs of GTP (8-N3GTP) and ATP (8-N3ATP). Of the five subunits of GEF, the Mr 40,000 polypeptide binds 8-[gamma-32P]N3GTP, and the Mr 55,000 and 65,000 polypeptides bind 8-[gamma-32P]N3ATP. Both 8-N3GTP and 8-N3ATP specifically label the beta-subunit of eIF-2. Covalent binding of 8-azidopurine analogs to the eukaryotic initiation factors is dependent on UV irradiation. Binding of 8-N3GTP and 8-N3ATP is specific for the guanine- and adenine-binding sites on the protein, respectively. GDP and GTP, but not ATP, inhibit the photoinsertion of 8-N3GTP to the protein. Similarly, ATP, but not GTP, inhibits the photoinsertion of 8-N3ATP. The inclusion of NADP+ in the reaction mixtures also interferes with the binding of 8-N3ATP to GEF. Mg2+ inhibits the binding of the 8-azido analogs of GTP and ATP to both eIF-2 and GEF, whereas EDTA stimulates the photoinsertion of these nucleotides. Identical results are obtained when the binding of GTP and ATP to these proteins, in the presence of Mg2+ or EDTA, is estimated by nitrocellulose membranes. In enzymatic assays, 8-N3GTP supports the activity of eIF-2 and GEF, indicating that the interaction of 8-N3GTP is catalytically relevant.     

Further studies on proctolin analogues modified in position 2 of the peptide chain and their influence on heart-beat frequency of insects

Seven proctolin analogues (I-VII) modified in position 2 of the peptide chain by Phe (p-guanidino) (I), Phe (p-OEt) (II), Tyr (3'-NH2) (III), Tyr (3'-NO2) (IV), Afb (p-OH) (V) (Afb = 3-amino-4-phenyl-L-butyric acid), Afb (p-NH2) (VI), Afb (p-NO2) (VII), and the tetrapeptide Tyr (3'-NH2)-Leu-Pro-Thr (VIII) were synthesized by the classic liquid-phase method. The biological effects of the peptides were investigated in cardioexcitatory tests on two insect species, the cockroach Periplaneta americana L., and the yellow mealworm, Tenebrio molitor L. Within physiological concentrations (10(-9)-10(-7) M) peptides II, III, and IV stimulated the heart action of P. americana like proctolin itself. Under identical conditions, in the case of T. molitor, only peptide III showed cardiostimulatory properties, whereas other compounds (including II and IV) were inactive at concentrations up to 10(-7) M. Results reported here reflect, with reference to the analogues I-VII, selective recognition of receptors on myocardium of both insect species. The tetrapeptide VIII revealed a weak deacceleratory effect on P. americana and T. molitor heart action.     

Importance of the N-terminal domain of the type I angiotension II antagonist [Sar1,Ile8]ANG II for receptor blockade

Analogues of the Type I angiotensin (ANG) antagonist, [Sar1,Ile8]ANG II, in which the N-terminal dipeptide was modified were synthesized by the solid phase method and purified by reversed-phase HPLC. Antagonist potencies (pA2) of the peptides were determined on the rat isolated uterus using ANG II as the agonist. Substitution of the Arg residue occupying position 2 of [Sar1,Ile8]ANG II (pA2 8.1) by Gly, Ala, Nle, Phe, Pro or Sar reduced the antagonist potency to pA2 = 7.0, 6.8, 6.7, 6.8, 5.8 and 5.3, respectively. Deletion of the N-terminal Sar residue in these same peptides gave pA2 = 6.8, 5.7, 5.5, 5.9, 6.1 and 7.5, respectively. The characteristically long duration of action of [Sar1,Ile8] was absent for all of these analogues including (des1, Sar2, Ile8]ANG II. These findings demonstrate that the antagonist potencies of Type I angiotensin antagonists for smooth muscle receptors, and also the long duration of action, are dependent on the location of positive charges within the peptide and on the conformation of the molecule in determining favorable electrostatic interactions with the receptor. A model is proposed in which the two positively charged loci on the angiotensin molecule (N-terminus and Arg) interact with two corresponding anionic binding sites on the smooth muscle receptor. The possibility that the prolonged duration of action of [Sar1, Ile8]ANG II results from binding to a different site on the angiotensin receptor from that occupied by ANG II is discussed in relation to the present findings.     

Synthesis of new head-activator analogues and their application for improved radioimmunoassays

New analogues of head activator were produced for receptor and radioimmunoassay studies. The precursor molecules [(4'-I)Phe11] head activator and [Tyr11] head activator were synthesised for catalytic tritiation and iodination, respectively. With the tracer [(3,5-125 I2)Tyr11] head activator the sensitivity range of the radioimmunoassay was 5-100 fmol.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs.

Ascidiacyclamide (ASC), cyclo(-Ile1-Oxz2-d-Val3-Thz4-)2 (Oxz=oxazoline and Thz=thiazole) has a C2-symmetric sequence, and the relationships between its conformation and symmetry have been studied. In a previous study, we performed asymmetric modifications in which an Ile residue was replaced by Gly, Leu or Phe to disturb the symmetry [Doi et al. (1999) Biopolymers49, 459-469]. In this study, the modifications were extended. The Ile1 residue was replaced by Gly, Ala, aminoisobutyric acid (Aib), Val, Leu, Phe or d-Ile, and the d-Val3 residue was replaced by Val. The structures of these analogs were analyzed by X-ray diffraction, 1H NMR and CD techniques. X-Ray diffraction analyses revealed that the [Ala1], [Aib1] and [Phe1]ASC analogs are folded, whereas [Val1]ASC has a square form. These structures are the first examples of folded structures for ASC analogs in the crystal state and are similar to the previously reported structures of [Gly1] and [Phe1]ASC in solution. The resonances of amide NH and Thz CH protons linearly shift with temperature changes; in particular, those of [Aib1], [d-Ile1] and [Val3]ASCs exhibited a large temperature dependence. DMSO titration caused nonlinear shifts of proton resonances for all analogs and largely affected [d-Ile1] and [Val3]ASCs. A similar tendency was observed upon the addition of acetone to peptide solutions. Regarding peptide concentration changes, amide NH and Thz CH protons of [Gly1]ASC showed a relatively large dependence. CD spectra of these analogs indicated approximately two patterns in MeCN solution, which were related to the crystal structures. However, all spectra showed a similar positive Cotton effect in TFE solution, except that of [Val3]ASC. In the cytotoxicity test using P388 cells, [Val1]ASC exhibited the strongest activity, whereas the epimers of ASC ([d-Ile1] and [Val3]ASCs), showed fairly moderate activities.     

Position 4 substituted somatostatin analogs: increased binding to somatostatin receptors in pituitary and brain

Somatostatin (S-14) analogs with Phe4 substitutions bound to pituitary and cerebrocortical S-14 receptors more avidly than did S-14. The 2-4 fold greater affinities of the Phe4 S-14 as well as analogs with structural modification of the Phe4 residue for binding to pituitary S-14 receptors showed good correlation with their reported potencies for in vivo Gh inhibition. In the cerebral cortex, [Phe4] S-14, [Phe4, D-Trp8] S-14 and [F5-Phe4] S14 were 2-3 times more potent while [p-NH2-Phe4] S-14 was 6 times more potent compared to S-14 in binding to S-14 receptors. The increased binding affinities of the Phe4 analogs in these two tissues does not appear to be due to differential stability of the analogs compared to S-14 under the experimental conditions used. [Thr4] S-14 exhibited very low binding in both these tissues. Thus structural modification of the position 4 moiety of the S-14 molecule does not result in dissociated affinities for binding to S-14 receptors in the brain and the pituitary. The increased receptor binding affinities of the Phe4 analogs in the cerebral cortex suggest that they may be more potent than S-14 in the CNS.