B2-kinin receptor like binding in rat glomerular membranes

Incubation of a radiolabeled bradykinin analog, [125I]-Tyr8-BK with a crude membrane preparation obtained from isolated rat glomeruli revealed a time dependent binding. The binding was saturable, reversible and was a linear function of protein membrane concentration. The radiolabeled Tyr8-BK bound to a single class of binding sites with an equilibrium dissociation constant (KD) of 3.9 +/_ 0.7 nM and a density (Bmax) of 31 +/- 5 fmol/mg protein. The BK-receptor complex was not affected by angiotensin II or by arginine vasopressin and atrial natriuretic factor. BK binding was reversed by bradykinin (Ki = 0.3 10(-9) M), and by other kinin analogs in the following order of potency: Lys-BK, Met-Lys-BK, Thi5,8-D Phe7-BK. However, Des-Arg9-BK had no effect on binding of the radiolabelled BK. These results are consistent with the presence of a B2-kinin like receptor in rat glomeruli.     

On the role of basic residues of head-to-tail cyclic bradykinin analogues on rat duodenum relaxation

Summary Three linear bradykinin (BK) analogues, Lys-Lys-BK, Nle-Lys-BK and Lys-Nle-BK and their head-to-tail cyclic analogues, along with cyclo-Nle-Nle-BK and cyclo-Lys-Lys-[Trp⁵]BK, were synthesized and tested on an isolated rat duodenum preparation. All kinins, except the [Trp⁵]-analogue, cause relaxation with EC₅₀ values in the picomolar range. The most potent linear analogue (Lys-Nle-BK) is about 40 times more active than BK and the most potent cyclic kinin (cyclo-Nle-Lys-BK) is about 6 times more active. Present results suggest that the significant potency of cyclo-Lys-Lys-BK, the earlier most potent cyclic kinin which is only a little less potent than linear BK, depends on the ring size rather than on the presence of the extra basic residues.     

On the role of basic residues of head-to-tail cyclic bradykinin analogues on rat duodenum relaxation

Three linear bradykinin (BK) analogues, Lys-Lys-BK, Nle-Lys-BK and Lys-Nle-BK and their head-to-tail cyclic analogues,along with cyclo-Nle-Nle-BK and cyclo-Lys-Lys-[Trp⁵]BK, weresynthesized and tested on an isolated rat duodenum preparation.All kinins, except the [Trp⁵]-analogue, cause relaxation withEC₅₀ values in the picomolar range. The most potent linearanalogue (Lys-Nle-BK) is about 40 times more active than BK andthe most potent cyclic kinin (cyclo-Nle-Lys-BK) is about 6 timesmore active. Present results suggest that the significant potencyof cyclo-Lys-Lys-BK, the earlier most potent cyclic kinin which isonly a little less potent than linear BK, depends on the ringsize rather than on the presence of the extra basic residues.     

Peptomeric analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds

A series of linear and monocyclic analogues of trypsin inhibitor SFTI-1 isolated from sunflower seeds, modified by N-(4-aminobutyl)glycine (Nlys) and N-benzylglycine (Nphe), were obtained by the solid-phase method. Some of these peptomers displayed trypsin or chymotrypsin inhibitory activity. In contradiction to the literature data, in most analogues peptide bonds formed by these peptoid monomers were at least partially hydrolyzed by the experimental enzymes at two different pH (3.5 and 8.3). Nevertheless, the replacement of Phe present in the P(1) substrate specificity of linear inactive SFTI-1 analogue with Nphe, yielded a potent chymotrypsin inhibitor. The introduction of one cyclic element (a disulfide bridge or head-to-tail cyclization) to the analogues synthesized significantly increased their proteinase resistance.     

Sulfamide antifolates inhibiting thymidylate synthase: synthesis,enzyme inhibition and cytotoxicity

Synthesis and biological evaluation are described of seven new analogues (3-9) of two potent thymidylate synthase inhibitors, 10-propargyl-5,8-dideazafolate (1) and its 2-methyl-2-deamino congener ICI 198583 (2). While the new compunds 3 and 4 were analogues of 1 and 2, respectively, containing a p-aminobenzenesulfonyl residue in place of the p-aminobenzoic acid residue, the remaining 5 new compounds were analogues of 4 with the L-glutamic acid residue replaced by glycine (5), L-valine (6), L-alanine (7), L-phenylglycine (8) or L-norvaline (9). The new analogues were tested as inhibitors of thymidylate synthases isolated from tumour (Ehrlich carcinoma), parasite (Hymenolepis diminuta) and normal tissue (regenerating rat liver) and found to be weaker inhibitors than the parent 10-propargyl-5,8-dideazafolic acid. Selected new analogues, tested as inhibitors of growth of mouse leukemia L 5178Y cells, were less potent than the parent 10-propargyl-5,8-dideazafolic acid. Substitution of the glutamyl residue in compound 4 with L-norvaline (9) resulted in only a 5-fold stronger thymidylate synthase inhibitor, but a 40-fold weaker cell growth inhibitor.     

Synthesis and study of cyclic and linear analogs of neurotensin

New cyclic analogues of neurotensin (NT): [cyclo (13----8), Gly8]NT-(8-13), [cyclo (13----7), Gly7]NT-(7-13), [cyclo (13----5 epsilon), Lys5]NT-(5-13), [cyclo (13----4 epsilon), Lys4]NT-(4-13), and their linear precursors have been synthesized. The latter (protected linear compounds) were prepared by solid-phase peptide synthesis, and cyclization was attained by using diphenylphosphoryl azide. Cyclization of C-terminal hexa- and octapeptide fragments of NT was found to lead to cycloanalogues possessing high depressor activity. As judged by CD spectral data in aqueous solution, the cyclohexapeptide analogue has a relatively rigid conformation different from its linear counter-part and the NT-(9-13) fragment, whereas NT, its cyclohepta- and cyclononapeptides have random structure.     

Potent bradykinin antagonists containing N-benzylglycine or N-benzyl-l-alanine in position 8.

Two new analogues of a previously designed bradykinin (BK) antagonist, d-Arg-Arg-Pro-Hyp-Gly-Thi-Ser-d-Phe-Thi-Arg, substituted in position 8 by N-benzylglycine and N-benzyl-l-alanine were designed, synthesized and bioassayed. The results show an impressive enhancement of B2 antagonistic potencies of both peptides in comparison with the model. In two further analogues these modifications were combined with acylation of the N-terminus with 1-adamantanacarboxylic acid. Acylated analogues exhibited higher antagonistic potency in comparison with the parent compounds, however, the range of effect was not as high as in previously described cases. The activity of analogues was assessed by their ability to inhibit vasodepressor response to exogenous BK (rat blood pressure test). Our results may be of value in the design of more potent BK antagonists.     

Biologically active loop-shaped analogs of bradykinin and polisteskinin

The classical methods of peptide chemistry have been employed to synthesize loop-shaped derivatives of bradykinin and polisteskinin, Lys-Lys-Lys-[cyclo (9----1 epsilon), Lys1, Gly6]bradykinin and Lys-Lys-Lys-Leu-Arg-Gly[cyclo (9----1 epsilon)Lys1, Gly6] bradykinin. In the course of synthesis, the linear "tail" fragments were attached to partially deblocked cyclopeptide. Protective groups were removed by treating with hydrogen fluoride, the end products were purified using reversed-phase and ion exchange chromatography. Biological experiments in vivo have revealed that the two compounds elicit a prolonged hypotensive effect in rats which is characteristic of cyclic bradykinin analogues. With the latter compound, a decrease in arterial pressure is preceded by a brief hypertensive action. The loop-shaped analogues are slightly myotropic when applied to rat uterus preparations in vitro.     

Synthesis and biological evaluation of analogues of the potent ADAM8 inhibitor cyclo(RLsKDK) for the treatment of inflammatory diseases and cancer metastasis

The metalloproteinase ADAM8 serves as a pivotal catalyst in the development of inflammatory diseases and cancer metastasis. The cyclic peptide cyclo(RLsKDK) has been shown to inhibit the enzymatic activity of ADAM8 with high specificity and potency. Herein we report a structure–activity relationship (SAR) study of cyclo(RLsKDK) that involves the synthesis and biological evaluation of the lead compound and structural analogues thereof. This study provides insight into the ligand–receptor interactions that govern the binding of cyclo(RLsKDK) to the ADAM8 disintegrin domain and represents a stepping stone for the development of new treatments for inflammatory diseases and cancer metastasis.     

Bradykinin-induced biphasic response in the rat isolated stomach fundus: Functional evidence for a novel bradykinin receptor

The responses of the rat isolated stomach fundus to bradykinin (BK) and des-Arg⁹-BK (DA-BK) have been examined. In rat isolated stomach fundus pre-contracted with BaCl₂ (0.5-1 mM), BK caused concentration-dependent biphasic responses characterized by relaxation followed by contraction. DA-BK also caused marked relaxations, but, unlike BK, induced only small contractions. Removal of the mucosal layer initially abolished the relaxant responses to BK and both responses to DA-BK without affecting BK-induced contractions, but repeated challenges with BK or DA-BK revealed a time-depeendent reappearance of the relaxant responses, suggesting “de novo” synthesis of BK receptors. Pretreatment of rat stomach fundus with tetrodotoxin (1 μM), atropine (1 μM), captopril (3 μM), prazosin (1 μM) or glibenclamide (1 μM) did not significantly modify the biphasic responses to BK (300 nM). The biphasic responses to DA-BK were antagonized selectivley by the B₁ receptor antagonist des-Arg⁹-[Leu ⁸]-BK (DAL- BK) (1 μM). In contrast, the biphasic responses to BK were unaffected by DAL-BK or by several selective peptide antagonists of B₂ receptors including NPC 431 (Thi^5,8, D-Phe⁷)-BK, NPC 349 (D-Arg Hyp³, Thi^5,8, D-Phe⁷)-BK, NPC 567 (D-Arg -Hyp³, D-Phe⁷)-BK and NPC 361 (D-Phe⁷)-BK (3 to 10 μM). These results are consistent with the view that the biphasic responses of the rat isolated stomach fundus to BK appear to be mediated by a novel BK receptor which is insensitive to blockade by B₁ and B₂ selective BK receptor antagonists.     

Characterization of bradykinin receptors in canine cultured corneal epithelial cells: Pharmacological and functional studies

The pharmacological properties of bradykinin (BK) receptors were characterized in canine cultured corneal epithelial cells (CECs) using [(3)H]-BK as a radioligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant of 0.34 +/- 0.07 nM and a maximum receptor density of 179 +/- 23 fmol/mg protein. Neither a B(1) receptor-selective agonist (des-Arg(9)-BK) nor antagonist ([Leu(8), des-Arg(9)]-BK) significantly inhibited [(3)H]-BK binding to CECs, thus excluding the presence of B(1) receptors in canine CECs. The specific binding of [(3)H]-BK to CECs was inhibited by B(2) receptor-selective agonists (BK and kallidin) and antagonists (Hoe 140 and [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK), with a best fit using a one-binding-site model. The order of potency for the inhibition of [(3)H]-BK binding was BK = Hoe 140 > kallidin > [D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK. Stimulation of CECs by BK produced a concentration-dependent accumulation of inositol phosphates (IP) and an initial transient peak of intracellular Ca(2+). B(2) receptor-selective antagonist ([D-Arg(0), Hyp(3), Thi(5,8), D-Phe(7)]-BK) significantly antagonized the BK-induced responses with dissociation constants of 6.0-6.1. Pretreatment of CECs with pertussis toxin (PTX) or cholera toxin did not alter the BK-induced IP accumulation. Incubation of CECs in the absence of external Ca(2+) led to a significant attenuation of the IP accumulation induced by BK. These results demonstrate that BK directly stimulates phospholipase C-mediated signal transduction through BK B(2) receptors via a PTX-insensitive G protein in canine CECs. This effect may function as the transducing mechanism for BK-mediated cellular responses.     

Bradykinin and its receptors in non-mammalian vertebrates

The generation of bradykinin (BK) in blood by the action of the kallikrein-kinin system has been studied intensively in mammals but the system has received relatively little attention in non-mammalian vertebrates. The plasma of crocodilians and Testudines (turtles and tortoises) contains all the components of the kallikrein-kinin system found in mammals (prekallikrein activator, prekallikrein, kininogen, and kininases) and activation results in generation of [Thr6]-BK. Plasma of birds and snakes probably lacks a prekallikrein activator analogous to mammalian Factor XII but treatment with exogenous proteases (pig pancreatic kallikrein and/or trypsin) generates [Thr6, Leu8]-BK (chicken), [Ala1, Thr6]-BK (python) and [Val1, Thr6]-BK (colubrid snakes). The skins of certain frogs, particularly of the genus Rana, contain very high concentrations of BK-related peptides but their pathway of biosynthesis involves the action of cellular endoproteinase(s) cleaving at the site of single arginyl residues rather than by the action of the kallikrein-kinin system. Evidence for a prekallikrein activator in fish plasma is lacking but treatment with exogenous proteases generates [Arg0, Trp5, Leu8]-BK (trout and cod), [Trp5]-BK (bowfin and gar), [Met1, Met5]-BK (sturgeon). The cardiovascular actions and effects upon gastrointestinal smooth muscle of these peptides in their species of origin differ markedly. For example, intra-arterial injections of the native BK peptides into unanesthetized fish produce transient hypertension in the cod, complex depressor and pressor responses in the trout and bowfin and hypotension in the sturgeon. Pharmacological studies in snakes and fish and with the recombinantally expressed chicken BK receptor have demonstrated that the BK receptors in the tissues of non-mammalian vertebrates have appreciably different ligand binding properties from the well-characterized mammalian B1 and B2 receptors.     

Synthesis and biological activity of seventeen analogues of human insulin

We synthesized seventeen analogues of human insulin, applying the principle of stepwise, selective formation of the disulphide bonds. Most of these analogues only differ from human insulin in the replacement of a single amino acid in positions 2, 5, 6, 7, 8 and 11 of the A chain and 5, 7, 13 and 16 of the B-chain. The influence of these modifications on the physicochemical properties of the analogues is discussed. Eight analogues could be crystallized. All the analogues produce the same biological effects as insulin, but differ markedly in their potency. In isolated fat cells in vitro, [HisA8]insulin showed a relative potency of 2.46 in stimulating glucose oxidation (human insulin = 1), whereas [D-CysA6,A11]insulin had a potency of only 0.00027. Very low potency was observed when IleA2 or the half-cystines A6, A7, A11 or B7 were modified. Replacement of the invariant GlnA5 by alanine only reduced potency slightly. All the analogues are full agonists. The effects of the analogues on glucose oxidation and lipolysis are correlated, supporting the view that they are mediated by a common receptor on the fat-cell membrane. Hypoglycaemic potencies in the rat were similar to potencies in vitro. As expected, no correlation was demonstrable between antiserum binding--measured in the radioimmunoassay--and biological activity. Several results of this investigation are difficult to reconcile with the current view regarding the structure-activity relationship of insulin which appears to require further refinement.     

Antagonists of oxytocin featuring replacement with modified beta-mercaptopropionic acids at position 1.

Twenty analogues were synthesized of [Pmp1, D-Trp2, Arg8]oxytocin, PA, (Pmp = beta,beta-pentamethylene-beta-mercaptopropionic acid), a potent antagonist of the uterotonic effect of oxytocin in the rat (uterotonic test in vitro, pA2 = 7.77) and in the baboon. Systematic substitution of Pmp1 was made with beta-mercaptopropionic acids featuring replacement of the 4-methylene group of the cyclohexyl ring of Pmp with isosteric O, S, NH or with C=O. Since the more hydrophilic NH and C=O substitutions showed a sharply decreased antagonistic potency (rat uterotonic test in vitro), additional modifications were made to reduce their hydrophilicity. Acylation of the NH group with various acyl groups, and ketalization or thioketalization of C=O with more or less bulky substituents led to a partial restoration of potency, the N-carbamyl- and the 2-mercapto-2-adamantaneacetyl analogues being equipotent with PA. Internal cyclization by amidation of the NH-group with Gly-9, resulted in a bicyclic analogue, (cyclo 1-9)[(HN)Pmp1, Gly9]PA which was equipotent with PA. When Pen-6 was introduced into the bicyclic derivative instead of Cys-6, to reduce the flexibility of the rings, the resulting (cyclo 1-9)[(HN)Pmp1, Pen6, Gly9]PA had somewhat better potency (pA2 = 8.17) in the uterotonic test and no detectable activity in the antidiuretic assay. In the case of substitution of PA with beta,beta-(3-thiapentamethylene)-beta-mercaptopropionic acid, (S)Pmp, there was also an increase in inhibitory potency in the uterotonic test (pA2 = 8.08): the analogue had extremely weak antidiuretic activity. To establish the importance of the steric effects of the Pen-6 substitution, analogues [Pen6]PA and [(S)Pmp1, Pen6]PA were made and found to be very potent, with a pA2 of 8.72 and 8.86, respectively. The high potency of the latter analogue and its extremely weak action in the diuretic assay makes it an attractive candidate for studies on the inhibition of the biological effects of oxytocin and for the prevention of preterm labour.     

Synthesis and biological activity of adipokinetic hormone analogues modified at the C-terminus

A series of Locusta adipokinetic hormone I (AKH-I), C-terminal threonine residue using a combination of solid- and liquid-phase methodology and evaluated in Locusta migratoria, in a lipid mobilization assay in vivo and an acetate uptake assay in vitro. Modifications at Thr¹⁰ of AKH-I involved replacement of its C-terminal amide by the groups -OH, -OCH₃, -NHCH₃, -N(CH₃)₂, and -NHC₆H₅; the last three groups were also applied to the amide of AKH-I-[Thr(Brl)¹⁰]. The methyl ester, monomethyl, and dimethyl analogues were all of lower activity than the parent in the lipid mobilization assay, but lost less than two orders of potency. In the acetate uptake assay, again the methyl ester analogue showed the greatest retention of biological activity of all modified peptides. A cyclic analogue, cyclo (PLNFTPNWGT), was active in both assay, but only at very high concentrations. Almost all analogues were more active in the acetate uptake assay than in the lipid assay, but unusually, AKH-I-NHCH₃ and AKH-I-N(CH₃)₂, together with cyclo (PLNFTPNWGT), were more active in the lipid mobilization assay. In addition, the acid AKH-I analogue did not suffer as large a loss in potency in the lipid mobilization assay as in the acetate uptake assay, although it was less potent in the former. The relative potencies of these two methyl analogues contrast with those for AKH AKH-I-[Thr(Bzl)¹⁰]-NHCH₃ and AKH-I-[Thr(Bzl)¹⁰]-N(CH₃)₂, which, together with both phenyl analogues, were significantly more active in the acetate uptake assay. We conclude that the acetate uptake assay has a greater preference for a hydrophobic C-terminus, compared with the lipid mobilization assay.     

Structure-activity studies with somatostatin: role of lysine in positions 4 and 9 for gastric activity

The gastric inhibitory activity of somatostatin analogues modified in position 4 or 9, was investigated in conscious cats prepared with gastric fistulae. Gastric secretion was stimulated with pentagastrin. Deletion of Lys4, or substitution with an alcoholic (Thr) or acidic (Glu) residue yielded analogues with reduced (10% or less) potency compared with somatostatin. In comparison, [Phe4]somatostatin and modified Phe4 analogues [( p-NH2-Phe4]-, [F5Phe4]- and [Phe4,D-Trp8]somatostatin) were approximately equipotent with somatostatin. The high potency of the Phe4 analogues illustrates that a basic side-chain in position 4 is not essential for gastric activity. In contrast, [Thr9]-, [Glu9]-, [Phe9]- and [p-NH2-Phe9]somatostatin were all inactive (less than 5% potency of somatostatin) in the stomach. Thus the lysyl residue in position 9 is more critical than that in position 4 for somatostatin's gastric activity.     

Synthesis of [cyclo(Glu gamma-----epsilon Lys(Gly)]ACTH-(5-14) undecapeptide. Biological and physico-chemical properties of analogs of ACTH-(5-10)- and ACTH-(5-14)-peptides

Modified corticotropin fragment - [Lys11 (Gly)]ACTH-(5-14)- and its cyclic analogue - [cyclo (Glu gamma----epsilon Lys (Gly)] ACTH-(5-14)-undecapeptides have been synthesized by classical approach. The cyclic structure has been fixed by amide bond between gamma-COOH group of glutamic acid and alpha-NH2 group of glycine coupled to the epsilon-NH2 group of lysine. Fragment condensation has been achieved by azide or dicyclohexylcarbodiimide methods. Cyclization has been performed using diphenylphosphorylazide. The melanotropic activity of the cyclicanalogue on isolated frog skin exceeds by two orders of magnitude that of the linear undecapeptide, however the steroidogenic activity in isolated cells of rat adrenal cortex is diminished by an order of magnitude as compared with that of the linear precursor. A similarity of the CD spectra for the cyclic ACTH peptides and their linear counterparts in water and trifluoroethanol points to the similarity and relative rigidity of their structures.     

Structural characterization of cyclic kallidin analogues in DMSO by nuclear magnetic resonance and molecular dynamics.

The conformational properties in DMSO of two head-to-tail cyclic analogues of kallidin ([Lys(0)]-bradykinin, KL) as well as those of the corresponding linear peptides were studied by NMR and molecular dynamics (MD) simulations. The modifications in the sequence were introduced at position 6, resulting in the four peptides, [Tyr(6)]-KL (YKL), [Trp(6)]-KL (WKL), cyclo-([Tyr(6)]-KL) (YCKL) and cyclo-([Trp(6)]-KL) (WCKL).The linear WKL analogue was significantly more potent than kallidin on rat duodenum preparations, whereas YKL was significantly less potent. Both cyclic peptides, YCKL and WCKL displayed similar activity, lower than that of the linear analogues and also of cyclo-KL.The two linear analogues display high conformational flexibility in DMSO. In the predominant conformer, for both peptides, all three X-Pro bonds adopt a trans configuration. Three out of four conformers present in YCKL and WCKL were completely assigned. The configurations at the X-Pro bonds are the same for the two analogues. All cyclic conformers show a cis configuration in at least one X-Pro bond and always opposite configuration for the two consecutive X-Pro bonds.The NOE-restrained MD calculations resulted in the detection of several elements of secondary structure in each of the conformers. Such elements are described and their possible relevance to biological activity is discussed.     

Effects of acyclic analogs of atrial natriuretic factor on proliferative processes of the epithelial tissue in white rats]

The effect of atrial natriuretic factor (ANF) synthetic linear truncated analogues AP-H-6-OH and AP-FOR-6-OH on corneal, skin, duodenum and colon epithelium proliferation has been studied on male rats. The epithelium mitotic activity and DNA synthesis were evaluated 4 and 24 h after intraperitoneal injection of 10 or 100 micrograms/kg peptides. In a dose of 10 micrograms/kg both ANF synthetic analogues inhibited proliferation processes in corneal epithelium, but activated the DNA synthesis in duodenum and colon epithelium. AP-FOR-6-OH (10 micrograms/kg) decreased the mitotic activity of skin epithelium and increased the silver grain density over the cell nuclei at the same time. 100 micrograms/kg ANF analogues stimulated cell mitogenesis in all organs studied. According to the data obtained ANF linear truncated analogues influence on epithelium proliferation is similar to effector of previously studied cyclic atriopeptin AP II.     

Solution structures of cyclic and dicyclic analogues of growth hormone releasing factor as determined by two-dimensional NMR and CD spectroscopies and constrained molecular dynamics.

Solution structures were determined for a linear analogue of growth hormone releasing factor (GRF), and cyclic and dicyclic analogues in which the side chains of aspartyl and lysyl residues spaced at positions i-(i + 4) were joined to form a lactam. The four analogues were [Ala15]-GRF-(1-29)-NH2 and its cyclo8-12, cyclo21-25, and dicyclo8-12;21-25 derivatives. The peptides were studied in two solvent systems: 75% methanol/25% water at pH 6.0; and 100% water at pH 3.0. CD spectroscopy was used to assess the overall alpha-helical content. Nuclear magnetic resonance spectroscopy was used to determine the structures in more detail. Nearly complete proton resonance assignments were made for each of the peptides, in both solvents. Nuclear Overhauser effects were converted into distance constraints and applied in the molecular dynamics program CHARMM to evaluate the range of low-energy structures that satisfied the nmr data. In 75% methanol, all of the peptides are comprised of a single alpha-helical segment with fraying of one to three residues at each end. The linear analogue has a tendency to kink. In water, the analogues have two helical segments with flexible regions between them and at the termini of the peptides. The linear analogue is helical at residues 7-14 and 21-28. In the cyclo8-12 analogue, the N-terminal helical region extends to include residues 7-19, while the other helical region is slightly shortened. In the cyclo21-25 analogue, the C-terminal helical region is extended to include residues 19-28, while the N-terminal helical region is destabilized. The dicyclic analogue has the largest N-terminal helix, spanning residues 7-20, but its helical segment at residues 21-28 is not well ordered. All of the analogues exhibit substantial biological activity. The cyclic and dicyclic analogues show dramatically increased resistance to degradation during incubation with human plasma. The i-(i + 4) lactam, therefore, appears to be a synthetic means of stabilizing a local alpha-helical conformation, which may be of general use in the design of active, stable peptides.