Clinical outcome of patients with various advanced cancer types vaccinated with an optimized cryptic human telomerase reverse transcriptase (TERT) peptide: results of an expanded phase II study.

BACKGROUND: TERT (telomerase reverse transcriptase) plays a critical role in tumor cell growth and survival. In an expanded phase II study, we evaluated the immunological and clinical responses to the TERT-targeting Vx-001 vaccine in patients with advanced solid tumors. METHODS: HLA-A*0201-positive patients received two subcutaneous injections of the optimized TERT(572Y) peptide followed by four injections of the native TERT(572) peptide, every 3 weeks. Peptide-specific immune responses were evaluated by enzyme-linked immunosorbent spot at baseline, and after the second and the sixth vaccinations. RESULTS: Fifty-five patients were enrolled and 34 (62%) completed the six vaccinations. A TERT-specific T-cell immune response was observed in 55% and 70% of patients after the second and the sixth vaccinations, respectively. The disease control rate (DCR) was 36% [95% confidence interval (CI) 24% to 49%], including one complete and one partial response. Immunologically responding patients had a better clinical outcome than nonresponders [DCR: 44% versus 14% (P = 0.047); progression-free survival (PFS): 5.2 versus 2.2 months (P = 0.0001) and overall survival: 20 versus 10 months (P = 0.041)]. Multivariate analysis revealed that the immunological response was an independent variable associated with increased PFS (hazard ratio = 3.35; 95% CI 1.7-6.7). CONCLUSION: Vx-001 vaccine was well tolerated and induced a TERT-specific immunological response, which was significantly correlated with improved clinical outcome.

Sulphonamide-based bombesin prodrug analogues for glutathione transferase, useful in targeted cancer chemotherapy.

Glutathione transferases (GSTs) are enzymes involved in cellular detoxification by catalysing the nucleophilic attack of glutathione (GSH) on the electrophilic centre of a number of toxic compounds and xenobiotics, including certain chemotherapeutic drugs. The encountered chemotherapeutic resistant of tumour cells, thus, has been associated with the increase of total GST expression. GSTs, in addition to GSH-conjugating activity, exhibit sulphonamidase activity, catalyzing the GSH-mediated hydrolysis of sulphonamide bonds. Such reactions are of interest as potential tumour-directed prodrug activation strategies. In the present work we report the design and synthesis of novel chimaeric sulphonamide derivatives of bombesin, able to be activated by the model human isoenzyme GSTA1-1 (hGSTA1-1). These derivatives bear a peptidyl-moiety (analogues of bombesin peptide: R-[Lue(13)]-bombesin, R-[Phe(13)]-bombesin and R-[Ser(3),Arg(10),Phe(13)]-bombesin, where R=C(6)H(5)SO(2)NH-) as molecular recognition element for targeting the drug selectively to tumour cells. The released S-alkyl-glutathione, after hGSTA1-1-mediated cleavage of the sulphonamide bond, provides an inhibitor of varied strength against GSTs from different sources. These prodrugs are envisaged as a plausible means to sensitize drug-resistant tumours that overexpress GSTs.

A phase I study of the optimized cryptic peptide TERT(572y) in patients with advanced malignancies.

OBJECTIVE: It was the aim of this study to evaluate the safety of the optimized cryptic peptide TERT(572Y) in pretreated patients with advanced cancer. METHODS: Nineteen patients with progressive and chemotherapy-refractory tumors received escalated doses (2-6 mg) of 2 subcutaneous injections of the optimized TERT(572Y) peptide followed by 4 subcutaneous injections of the native TERT(572) peptide every 3 weeks. Both TERT peptides were coinjected with adjuvant Montanide ISA51. Toxicity was evaluated every 3 weeks and peptide-specific CD8+ cells were detected by flow cytometry using TERT(572Y) tetramers. RESULTS: Fourteen out of 19 patients completed the vaccination program. No grade III/IV toxicity was observed. Grade I anemia was observed in 4 patients and local skin reaction at the injection site in 11 patients. Other nonhematologic toxicities were mild, and no late toxicity was observed after a median postvaccination follow-up period of 10.7 months. There was no dose-limiting toxicity. Peripheral blood TERT(572Y)-specific CD8+ lymphocytes were detected in 13 out of 14 evaluable patients after 2 injections with the optimized TERT(572Y) peptide. There was no complete or partial response, but 4 patients (21%) with persistent TERT(572Y)-specific CD8+ experienced stable disease for a median of 10.5 months. CONCLUSION: TERT(572Y) peptide vaccine is well tolerated and effective in eliciting specific TERT(572Y) CD8+ lymphocytes in pretreated cancer patients, demonstrating that cryptic peptides could be used in cancer immunotherapy.

Synthesis, liposomal formulation and thermal effects on phospholipid bilayers of leuprolide.

A novel liposomal formulation was developed for the encapsulation of the oligopeptide leuprolide (GlpHisTrpSerTyr-D-LeuLeuArgProNHEt), a potent analogue of gonadotropin releasing hormone used in the treatment of advanced prostate cancer, endometriosis and precocious puberty. Leuprolide was synthesized using solid phase methodology on a {3-[(ethyl-Fmoc-amino)-methyl]-1-indol-1-yl}-acetyl AM resin and Fmoc/tBu chemistry. The new liposomal formulation, called 'liposomes in liposomes' is composed of egg phosphatidylcholine:dipalmitoylphosphatidylglycerol in a molar ratio of 98.91:1.09 (internal liposomes) and egg phosphatidylcholine:dipalmitoylphosphatidylglycerol:cholesterol in a molar ratio of 68.71:0.76:30.53 (external liposomes). It offers high encapsulation efficiency (73.8% for leuprolide); it can provide new delivery characteristics and it may have possible advantages in future applications regarding the encapsulation and delivery of bioactive peptides to target tissues. Furthermore, the physicochemical characteristics (size distribution and zeta-potential) of the liposomal formulations and the thermal effects on leuprolide in model lipidic bilayers composed of dipalmitoylphosphatidylcholine were studied using differential scanning calorimetry. Finally, the dynamic effects of leuprolide in an egg phosphatidylcholine/cholesterol system were examined using solid state 13C MAS NMR spectroscopy.

3D solution structure of [Tyr3]octreotate derivatives in DMSO: structure differentiation of peptide core due to chelate group attachment and biologically active conformation.

The solution models of [Tyr3]octreotate (DPhe1-Cys2-Tyr3-DTrp4-Lys5-Thr6-Cys7-Thr8-COOH, disulfide bridged) (I), its analogs functionalized with an open chain tetraamine chelator, N4-[Tyr3]octreotate (II), and the N4-(Asp)2-[Tyr3]octreotate (III) peptide have been determined through 2D 1H NMR spectroscopy in DMSO. Chemical shift analysis has been performed in an attempt to elucidate structural changes occurring during attachment of the tetraamine to the peptide backbone. NMR-derived geometrical constraints have been used in order to calculate high resolution conformers of the above peptides. Conformational analysis of the three synthetic analogues, have shown that these somatostatin analoges adopt a predominant antiparallel beta-sheet conformation characterized by a beta-like turn spanning residues DTrp4 and Lys5 which is supported in the case of N4-(Asp)2-[Tyr3]octreotate and N4-[Tyr3]octreotate by medium range NOEs. These data indicate that the above-mentioned molecules adopt a rather constrained structure in the 4-residue loop Tyr3-Thr6. Additionally, the C-terminal of [Tyr3]octreotate, comprising Cys7 and Thr8, appears to form a turn-like structure manifested by characteristic side-chain NOEs between Lys5 and Thr8, which have not been detected for the other two compounds. These data are discussed in the light of previous structural data of Sandostatin (octreotide) and suggest that attachment of the N4-chelator and two Asp residues at the N-end of [Tyr3]octreotate impose considerable structural changes and affect the binding properties of these peptides. Indeed, the IC50 values determined during competition binding assays against the sst2 (somatostatin subtype 2 receptor) suggest that the presence of the N4 group enhances receptor affinity, while extension of peptide chain by two negatively-charged Asp residues impairs receptor affinity at approximately one order of magnitude.

Carotid atherosclerosis in type 2 diabetes mellitus: potential role of endothelin-1, lipoperoxides, and prostacyclin.

Factors were studied that may initiate macroangiopathy or enhance or aggravate its pathogenesis in patients with type 2 diabetes mellitus. A total of 151 diabetics were compared with healthy controls (n=50); all patients and subjects were normotensive and without renal failure. Plasma endothelin-1 and free radical levels were measured. In addition, plasma prostacyclin levels were assessed by assaying its stable, spontaneous, breakdown product 6-keto-prostaglandin-F1a. Diabetics were divided into three groups: those with clinically evident macroangiopathy and those with early or without atherosclerosis (as determined by the carotid intima-media thickness. Plasma endothelin-1 levels were increased in all diabetics with atherosclerosis. Plasma free radical levels were increased in diabetics with macroangiopathy when compared with control subjects. The plasma levels of 6-keto-prostaglandin-F1a were slightly, but significantly, decreased in the diabetics with macroangiopathy when compared with control subjects. The carotid intima-media thickness was significantly greater in diabetics without macroangiopathy when compared with the controls. Furthermore, the intima-media thickness increased significantly in this group of diabetics but not in the controls over a 30-month follow-up period. Several factors may contribute to atherogenesis in diabetics. These include increased plasma endothelin-1 and free radical levels as well as a deficiency of prostacyclin. These factors may become targets for intervention as well as markers of disease progression.

A general strategy to enhance immunogenicity of low-affinity HLA-A2. 1-associated peptides: implication in the identification of cryptic tumor epitopes.

Low-affinity MHC class I-associated cryptic epitopes derived from self proteins overexpressed in a wide variety of human tumors or derived from antigens of viruses exhibiting a high mutation rate, could be interesting candidates for tumor and virus immunotherapy, respectively. However, identification of low-affinity MHC-associated epitopes comes up against their poor immunogenicity. Here we describe an approach that enhances immunogenicity of nonimmunogenic low-affinity HLA-A2.1-binding peptides. It consists of modifying their sequence by introducing a tyrosine in the first position (P1Y). P1Y substitution enhances affinity of HLA-A2.1-associated peptides without altering their antigenic specificity. In fact, P1Y variants of ten nonimmunogenic low-affinity peptides exhibited a 2.3- to 55-fold higher binding affinity and/or stabilized the HLA-A2.1 for at least 2 h more than the corresponding native peptides. More importantly, P1Y variants efficiently triggered in vivo native peptide-specific CTL which also recognized the corresponding naturally processed epitope. The possibility for generating CTL against any low-affinity HLA-A2.1-associated peptide provides us with the necessary tool for the identification of cryptic tumor and virus epitopes which could be used for peptide-based immunotherapy.

Angiotensin II analogues with sulphur-containing side-chains in position 5. A structure-activity relationship study.

Four sets of angiotensin II (AngII) analogues with position 5 modifications, two agonist series with either Asp or Sar in position 1 and L-Phe in position 8, and two antagonist series with again Asp or Sar in position 1 and Leu in position 8 were synthesized. Modifications in positions 5 were introduced successively: Ile, Nle, Met, S-ethyl Cys, S-n-propyl-Cys, S-n-butyl Cys, S-t-butyl Cys and S-benzyl Cys in all four series. The study was undertaken in order to investigate the 5-position residue of AngII by replacing the hydrophobic side-chain by another containing an electrophilic moiety. The analogues were synthesised by solid phase synthesis using the Boc/Bzl or Fmoc/But strategy. All analogues were evaluated by their binding properties to the AT1 receptor on bovine adrenocortical membranes (bAT1). The results indicate that AngII analogues bind, irrespective of their agonistic or antagonistic nature or of their position 1 modification, in a similar manner and that position 5 modifications without beta-branching behave in an additive manner towards their affinity.

Synthetic peptides as putative therapeutic agents in transplantation medicine: application of PEPSCAN to the identification of functional sequences in the extracellular domain of the interleukin-2 receptor beta chain (IL-2Rbeta).

A desired treatment strategy in transplantation medicine is the selective targeting of alloreactive T cells without impairing antileukemic and antiviral activities. One approach is the synthesis of peptides that interfere with the binding of interleukin-2 (IL-2) to its high affinity receptor (IL-2R). This blocks the activation and proliferation of the antigen-activated T cells and the secretion of IL-2. The latter binds to its receptor, via the extracellular domain of the IL-2Rbeta chain, while its cytoplasmic domain is required for intracellular signal transduction. In this study, the PEPSCAN method was applied in order to identify antigenic sequences (epitopes) in the extracellular domain of the IL-2Rbeta. Based on the primary amino acid (aa) sequence of the IL-2Rbeta, a total of 239 overlapping dodecapeptides, spanning the entire sequence of IL-2Rbeta, were synthesized by PEPSCAN and their immunoreactivity was tested by ELISA using monoclonal antibodies (mAbs) specific for IL-2Rbeta such as TU11, Mikbeta1, HuMikbeta1 and TU27. TU11 recognized a linear epitope located in the region 85R-Q(96). None of the 239 synthetic peptides was recognized by TU27. Mikbeta1 (and HuMikbeta1) recognized a discontinuous epitope formed by aa located in the IL-2Rbeta domains L(106) to P(148) and E(170) to A(202). Subsequently, synthetic peptides corresponding to the identified putative epitopic sequences were prepared by solid phase synthesis and their immunogenicity in vivo was assessed by raising polyclonal antibodies. Given that Mikbeta1 and HuMikbeta1 inhibit binding of IL-2 on the IL-2Rbeta, we addressed the question of whether the identified antigenic sequences serve as putative IL-2 binding domains. Synthetic peptides corresponding to these sequences were tested for their ability to compete with IL-2 for binding and, thereby, inhibit IL-2-induced proliferation of mitogen-stimulated human peripheral blood T cells. Sequences 107M-E(118) and 178Y-Q(199) probably represent functional IL-2 binding domains on IL-2Rbeta, since these synthetic peptides significantly inhibited the proliferation of activated T cells and secretion of IL-2.

Progression of carotid atherosclerosis and the role of endothelin in diabetic patients.

An increased thickness of the carotid artery wall is thought to be a sign of early atherosclerosis. Since vascular endothelium is the site of formation of several substances, we have investigated the rate of progression of carotid atherosclerosis and the contribution of endothelin (ET-1), lipid peroxides [measured as thiobarbituric acid reacting species (TBARS)] and 6-keto-Prostaglandin-F1A (6-keto-PG-F1A) at baseline and after 30-months. Fifty patients with Type 2 diabetes without evidence of macroangiopathy, hypertension, proteinuria or proliferative retinopathy, and 27 healthy, non-diabetic persons were studied. Arterial wall thickness was measured as the mean of the maximum intimal-medial thickness (IMT) in 16 carotid segments by b-mode ultrasound. The IMT values was significantly increased in diabetic subjects (at baseline: 1110 +/- 310 microm, after 30 months: 1260 +/- 280 microm, p < 0.01), but not in control subjects (1100 +/- 280 microm, 1200 +/- 290 microm, respectively). At baseline time both groups had similar levels of ET-1, TBARS and 6-keto-PG-F1A. In 30-months follow-up, the ET-1 level 8.0 pmol/l (5.8-10.7) was significantly elevated in diabetic subjects, compared with the level at baseline time 7.43 pmol/l (4.8-11.1) p < 0.01. No significant differences were found in the other examined parameters in the studied groups. Although insulin levels remained unchanged in the two studied groups, in 30 months follow-up, the insulin level in the diabetic subjects, 92.4 +/- 35.1 pmol/l was significantly elevated compared with those of control subjects 76.0 +/- 31.0 pmol/l, p < 0.05. In conclusion, endothelis is the main associate of the change of IMT value over 30 months in diabetic patients, in whom the extent of atherosclerosis was significantly greater than in control subjects.

Plasma levels of endothelin and early carotid atherosclerosis in diabetic patients.

An increased thickness of the carotid artery wall is thought to be a sign of early atherosclerosis. Since plasma endothelin concentrations were released from vascular endothelial cells, we have investigated the possible relationship between endothelin 1 (ET-1) and arterial wall thickness. Ninety-eight patients with Type 2 diabetes without evidence of macroangiopathy, hypertension, proteinuria or proliferative retinopathy, and 50 non-diabetic subjects were studied. After an overnight fast, blood was taken for ET-1, glucose, HbA1c, lipids, insulin and C-peptide. Arterial wall thickness was measured as the mean of the maximum intimal-medial thickness (IMT) in 16 carotid segments by B-mode ultrasound. ET-1 levels were significantly elevated in diabetic patients with IMT>1100 microm, 8.3 pmol/l (5.2-12.9) compared with control subjects, 7.6 pmol/l (5.0-11.0), p<0.01 and with diabetic subjects with IMT<500 microm, 7.43 pmol/l (4.8-11.1), p<0.01. The diabetic (IMT>1100 microm) study group had also significantly higher levels of insulin, 102.8 +/- 46.4 pmol/l vs control subjects, 77.5 +/- 32.4 pmol/l, p<0.01. In diabetic subjects, no correlation was found between ET-1 and IMT with glucose, HbA1c, lipids, age or duration of diabetes, respectively. We conclude that ET-1 levels are elevated in Type 2 diabetic patients with increased IMT. Thus providing further support for the role of endothelin in atherosclerosis.

Insertion of an electronegative sulfur atom in the side chain of position 5 of angiotensin II: changes in the tachyphylactic properties of the peptide.

Angiotensin II (AII) analogs bearing n-Leu, Met or S-substituted groups for cysteine at position 5 were studied regarding their agonistic and tachyphylactic properties. It was shown that these analogs lowered the relative affinity towards the AT1 receptor as determined by contractile responses, which could be due to the removal of the beta-branching residue at position 5. Insertion of a sulfur atom in a different position away from the attached backbone carbon atom presented no significant difference in EC50 values for these analogs. Interestingly, the S-bearing analogs at position 5 were full agonists but the tachyphylactic property was lost, in contrast to [n-Leu5]AII, which still induced reduction of the contractile responses. Nevertheless after replacing the Asp with Sar in position 1 (Sar1) tachyphylaxis was again established. It is concluded that the insertion of Met or an S-substituted cysteine into the side chain at position 5 of AII may promote interactions with its receptor due to the slight electronegative character of the sulfur atom and changes in the restricted conformational freedom of the Ile5 residue in the AII molecule. This was overcome by Sar1, probably through interactions due to its fully protonated N-terminal amino group and favoring the conformation responsible for the tachyphylaxis phenomenon.

Chimeric peptides: a new approach to enhancing the immunogenicity of peptides with low MHC class I affinity: application in antiviral vaccination.

Recruitment of the CTL repertoire specific for subdominant epitopes that have a low MHC class I-binding affinity could be the way to achieve an efficient protective immunity against spontaneous tumors and viruses with high mutation rate. However, we have reported recently that subdominant peptides of influenza A Puerto Rico/8/34 (flu PR8) nucleoprotein (NP) with low Db affinity are only partially able to protect mice against lethal influenza infection. This seems to be due to their inability to recruit the specific CTL repertoire, and suggests that subdominant peptides could be used for vaccination only if they become highly immunogenic. In this work, we describe an approach that allows enhancement of the immunogenicity of every low affinity peptide presented by the Db molecule. It consists in producing chimeric peptides composed by amino acids from a high Db affinity peptide (NP366) in positions that interact with the MHC, and amino acids from low Db affinity nonimmunogenic influenza NP-derived peptides (NP17, NP97, NP330, and NP469) in positions that are exposed to the TCR. All chimeric peptides tested exhibited a high Db affinity and efficiently recruited the CTL repertoire specific for the corresponding low Db affinity peptide. Furthermore, vaccination with chimeric peptides that corresponded to subdominant NP17 and NP97 peptides induced a very potent anti-flu PR8 protective immunity.

Protection against lethal viral infection by vaccination with nonimmunodominant peptides.

CTL response of H-2b mice to influenza PR8 virus is directed against the nucleoprotein (NP)-derived immunodominant 366-374 (NP366PR8) peptide presented by the Db molecule. However, NP has three nonimmunodominant peptides corresponding to the 17-25 (NP17), 55-63 (NP55), and 97-105 (NP97) sequences that have the Db consensus motifs and bind to the Db molecule with an intermediate (NP55) or low (NP17 and NP97) affinity. In a previous report, we have shown that NP55 peptide is naturally processed by infected cells. In the present work, we studied whether nonimmunodominant peptides can protect mice against viral infection. Antiviral protection was evaluated by measuring three parameters: survival after inoculation of a lethal dose of mouse-adapted PR8 virus, percentage of pulmonary lesions in surviving mice, and virus clearance from lungs of infected mice. Our results showed that immunization of B6 mice with nonimmunodominant peptides protected from PR8 virus infection, although less efficiently than immunization with the immunodominant NP366PR8 peptide. Protection was mediated by CD8 T cells. The efficacy of nonimmunodominant peptides correlated with their Db binding affinity; the low affinity binders NP17 and NP97 induced a weaker protection than the intermediate affinity binder NP55. A mixture of NP366PR8 and nonimmunodominant peptides gave a higher protection than NP366PR8 peptide alone. In conclusion, nonimmunodominant peptides protect against a viral infection with an efficacy that is proportional to their affinity for the restricting class I molecule.

Methylation in positions 1 and 7 of angiotensin II. A structure-activity relationship study.

Six analogues of angiotensin II (Ang) were synthesized with modifications in positions 1 and 7. The study was undertaken in order to learn more about the influence of alkylations in positions 1 and 7 and their interdependence. Previous studies have shown that alpha, alpha-dimethylation of Gly (aminoisobutyric acid, Aib) in position 1 produces quite potent analogues, as does N-methylation of Gly (sarcosine). Combination of both C alpha- and N alpha-methylations to N-Me-Aib1, however, did not produce an affinity increase. Decyclisation of the Pro7-residue produced moderately active analogues with position 7 N-methylation and inactive analogues if the N-alkylation was suppressed. In order to investigate a possible stereochemical interdependence of positions 1 and 7, a group of peptides with combinations of position 1 and 7 alkylations were investigated. The following analogues were prepared: [Sar1,Aib7]Ang, [Sar1,Aib,Leu8]Ang, [Aib1,7,Leu8]Ang, [Aib1,7,Leu8]Ang, [N-Me-Aib1,Aib7]Ang, [N-Me-Aib1,Aib7,Leu8]Ang. They were synthesized by classical solid phase synthesis using the BOC-TFA-HF scheme. The biological properties of these peptides were assessed on the rabbit aorta preparation and their binding potencies were measured on bovine adrenal membranes. Both on agonistic and antagonistic [Leu8]Ang analogues single Aib substitutions in position 1 or 7 induced affinity reduction in both bioassays. Simultaneous Aib modifications in positions 1 and 7 induced more important affinity loss in a synergic manner in both bioassays and as well for agonists and antagonists. The N-Me-Aib1 modifications induce similar affinity loss with or without concomitant Aib7 modification.(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis and biological activity of substance P C-terminal hexapeptide and heptapeptide analogues.

The analogues [Glu(OBzl)11]SP6-11 and [Glu(OBzl)11]SP5-11 of the C-terminal hexapeptide and heptapeptide of Substance P have been synthesized by conventional solution methods. In each analogue the SCH3 group of Met11 is replaced by the COOCH2C6H5 group. The in vitro activity of both analogues has been determined on three biological preparations: guinea pig ileum (GPI), rat vas deferens (RVD), and rat portal vein (RPV). The selectivity for the different receptors has been studied by utilizing atropine-treated guinea pig ileum (GPI + At). The results showed that both analogues are mainly active on GPI through the NK-1 receptor and that both analogues are equipotent to Substance P.

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.

Changes in residues 1 and 4 of angiotensin II affect differently its agonist and tachyphylactic properties.

Two series of angiotensin II analogues with modifications at positions 1 or 4 of the peptide chain were studied with respect to their tachyphylactic properties and to the kinetics of relaxation of the guinea-pig ileum after a contractile response to maximally effective concentrations. Tachyphylaxis was measured by the decrease in response amplitude after three successive treatments ("tachyphylactic index") and the relaxation rate was evaluated by the time taken for the tonus to reach half of its value at the moment of agonist washout ("half relaxation time"). A correlation between tachyphylactic index and half relaxation time was found for the series of position 1 analogues, but not for the position 4 analogues. For the two series, the half relaxation times of the tachyphylactic analogues decreased from the first to the third of a series of successive treatments. Bulky substituents at position 1, which did not greatly affect the agonist activity, suppressed the tachyphylactic property. The results provide evidence that the agonist and tachyphylactic properties of angiotensin II are due to its interaction, respectively, with an agonist site and a "tachyphylaxis" site on the receptor and that the structural requirements for binding to the two sites are different.

Importance of the N-terminal domain of the type II angiotensin antagonist sarmesin for receptor blockade.

Analogues of the competitive angiotensin antagonist [Sar1,Tyr(ME)4]angiotensin II (sarmesin) with modifications at the N-terminus have been prepared by the solid-phase method and purified by reversed-phase HPLC. Substitution of the Sar1 residue of sarmesin with N,N-dimethyl-Gly, N-ethyl-Gly, aminoisobutyric, (methylamino)isobutyric, aminocaproic, and oxamic acids gave analogues that had the following respective antagonist activities (pA2) in the rat isolated uterus assay: less than 6, 6.9, 5.5, 6.0, less than 6, and 5.3. The additional substitution of Ile for Phe at the C-terminus of the latter four peptides gave pA2 values of 7.1, 5.1, less than 5, and 5. Substitution of the Arg2 residue of sarmesin with Nle or Sar abolished antagonist activity. These data emphasize the stringent and discriminating structural requirements in the N-terminal domain of sarmesin that endow this analogue with its antagonist properties and suggest the presence of defined steric constraints in this region of the molecule during receptor blockade.