Furanoid sugar amino acids as dipeptide mimics in design of analogs of vasoactive intestinal peptide receptor binding inhibitor

Abstract: In this study we describe the development of peptidomimetic analogs of the potent vasoactive intestinal peptide receptor binding inhibitor, Leu¹ ‐Met² ‐Tyr³ ‐Pro⁴ ‐Thr⁵ ‐Tyr⁶ ‐Leu⁷ ‐Lys⁸ ‐OH 1, by incorporating furanoid sugar amino acids (SAAs) 2‐4 into the molecule. The furanoid SAAs 2‐4 were used as dipeptide isosteres to replace Tyr³ ‐Pro⁴ or Pro⁴ ‐Thr⁵ in sequence 1 . The resulting analogs 5 ‐ 9 were tested for their anti‐cancer activities in vitro, following the standard MTT assay on a panel of human cancer cell lines. One of the potent analogs, 6a was tested in vivo for tumor regression on primary colon tumor xenografted nude mice. Our experimental results suggest that many of these analogs show either retention or enhancement of biological activity.     

HUMAN SOMATOTROPIN

Three analogs of the carboxyl terminal plasmin fragment of human somatotropin, [Nle¹⁷⁰, Ala^165,182,189]-human somatotropin-(145–191), [Nle¹⁷⁰, Ala^165,182,189]-human somatotropin-(140–191), and [Lys^135,136,138, Glu^137,139, Nle¹⁷⁰, Ala^165,182,189]-human somatotropin-(135–191) have been synthesized by the solid-phase method. The synthetic peptides were assayed for growth-promoting activity and their potency was found to be comparable to that of [S-carbamideomethylcysteine^165,182,189]-human somatotropin-(141–191) which was derived from the native hormone.     

Design,Synthesis, and Biological Evaluation of Novel Peptide Gly3‐MC62 Analogues as Potential Antidiabetic Agents

Two series of conformationally constrained analogues from Gly ³ ‐ MC 62 were designed by scanning the residues Lys¹, Thr², Met⁴, Lys⁵, Met^7, and Ala⁸ with an i‐(i + 2) lactam bridge consisting of a Glutamic acid–xaa–lysine (Glu–Xaa–Lys) scaffold and a diproline fragment. They were synthesized and evaluated for their antihyperglycemic effects. Through screening in normal and mice with diabetes mellitus, peptides II ‐5 , III ‐3 , III ‐4 , and III ‐5 showed significant improvement in antihyperglycemic and antioxidative activities compared with Gly ³ ‐ MC 62 , especially the compound III ‐4 . The primary mechanism of the compounds ( II ‐5 , III ‐3 , III ‐4 , and III ‐5 ) underlying this effect is the islet β‐cells against oxidative damage induced by STZ, and III ‐4 ‐treated mice showed considerable improvement in the preservation of beta cells in the pancreatic islets of DM mice. These data suggested that III ‐4 could be candidate for the future treatment of diabetes mellitus.     

Biological and conformational study of β‐substituted prolines in MT‐II template: steric effects leading to human MC5 receptor selectivity*

Abstract: To investigate the molecular basis for the interaction of the χ‐constrained conformation of melanotropin peptide with the human melanocortin receptors, a series of β‐substituted proline analogs were synthesized and incorporated into the Ac‐Nle‐c[Asp‐His‐d ‐Phe‐Arg‐Trp‐Lys]‐NH₂ (MT‐II) template at the His⁶ and d ‐Phe⁷ positions. It was found that the binding affinities generally diminished as the steric bulk of the p‐substituents of the 3‐phenylproline residues increased. From (2S, 3R)‐3‐phenyl‐Pro⁶ to (2S, 3R)‐3‐(p‐methoxyphenyl)‐Pro⁶ analogs the binding affinity decreased 23‐fold at the human melanocortin‐3 receptor (hMC3R), 17‐fold at the hMC4R, and eight‐fold at the hMC5R, but selectivity for the hMC5R increased. In addition, the substitution of the d ‐Phe⁷ residue with a (2R, 3S)‐3‐phenyl‐Pro resulted in greatly reduced binding affinity (10³–10⁵) at these melanocortin receptors. Macromodel's Large Scale Low Mode (LLMOD) with OPLS‐AA force field simulations revealed that both MT‐II and SHU‐9119 share a similar backbone conformation and topography with the exception of the orientation of the side chains of d ‐Phe⁷/d ‐Nal (2′)⁷ in χ space. Introduction of the dihedrally constrained phenylproline analogs into the His⁶ position (analogs 2 – 6 ) caused topographical changes that might be responsible for the lower binding affinities. Our findings indicate that hMC3 and hMC4 receptors are more sensitive to steric effects and conformational constraints than the hMC5 receptor. This is the first example for melanocortin receptor selectivity where the propensity of steric interactions in χ space of β‐modified Pro⁶ analogs of MT‐II has been shown to play a critical role for binding as well as bioefficacy of melanotropins at hMC3 and hMC4 receptors, but not at the hMC5 receptor.     

Probing the functional conformation of the tridecapeptide mating pheromone of Saccharomyces cerevisiae through study of disulfide-constrained analogs

Analogs of the Saccharomyces cerevisiaeα-mating factor, Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr, where Lys⁷ and Gln¹⁰ were replaced with Cys, Cys(CH₃), or Ser, were synthesized using solid-phase procedures on a phenylacetamidomethyl resin. Cyclo^7,10[Cys⁷,X⁹,Cys¹⁰,Nle¹²]α-factor, where X = D-Val, D-Ala, l -Ala and Gly, were prepared by on-resin cyclization using thallic trifluoroacetate in yields of 20–30%. Linear sulfhydryl-containing peptides were generated from their corresponding cyclic peptide by treatment with dithioerythritol in basic solution. In the linear analogs, replacement of both Lys⁷ and Gln¹⁰ with a cysteine residue resulted in an over 100-fold loss of the biological activity when compared with the native pheromone. The corresponding cyclic disulfides were 5–10-fold more active than their sulfhydryl-contaihing homologs, and cyclo^7,10[Cys⁷,L-Ala⁹,Cys¹⁰,Nle¹²] α-factor was 50-fold more potent than linear analogs containing Ser or Cys(CH₃) in positions 7 and 10. Binding competition studies indicated that all analogs had low affinity for the α-factor receptor and there was a poor correlation between binding and activity in a growth arrest assay. A cyclic analog in which residues 8 and 9 were replaced by 5-aminopentanoic acid was not biologically active. Based on NMR studies, all cyclic peptides have a higher tendency to form β-turns spanning residues 7–10 than their less active linear counterparts. The results provide strong evidence that this β-turn is important for optimal signal transduction by α-factor.     

Synthesis and biologic activity of conformationally constrained analogs of L‐363,301

Abstract: We report the synthesis, biological activity and conformational analysis of analogs of the cyclic hexapeptide L‐363,301, c[Pro⁶‐Phe⁷‐d ‐Trp⁸‐Lys⁹‐Thr¹⁰‐Phe¹¹] (numbering as in the native hormone somatostatin‐14). The d ‐Trp in position 8 was replaced with (2R,3S)‐ and (2R,3R)‐β‐MeTrp respectively, with an added methyl group in the beta position of Trp. The objective of our study was to determine the potency and selectivity generated by the added constraint in the beta position of the d ‐Trp upon binding to human somatostatin receptors hsst1‐5. We synthesized the building blocks enantioselectively and incorporated them into the peptides by SPPS. Competition binding assays revealed that both compounds 2 and 3 were selective for hsst2 over hsst5. The (2R,3S) analog 2 was approximately 30 times more potent at hsst2 than the (2R,3R) analog 3 . Interestingly, the (2R,3R) compound showed no binding affinity at hsst5.     

Synthesis and in vitro and in vivo activity of analogs of growth hormone-releasing hormone (GH-RH) with C-terminal agmatine

In the search for more active analogs of human growth hormone-releasing hormone (GH-RH), 37 new compounds were synthesized by solid phase methodology, purified, and tested biologically. Most of the analogs contained a sequence of 27 amino acids and N-terminal desaminotyrosine (Dat) and C-terminal agmatine (Agm), which are not amino acids. In addition to Dat in position 1 and Agm in position 29, the majority of the analogs had Ala¹⁵ and Nle²⁷ substitutions and one or more additional L- or D-amino acid modifications. [Dat¹, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-51) was the most active analog. Its in vitro GH-releasing potency was 10.5 times higher than that of GH-RH(1-29)NH₂ and in the i.v. in vivo assay, MZ-2-51 was 4-5 times more active than the standard. After s.c. administration to rats, MZ-2-51 showed an activity 34 times higher at 15min and 179 times greater at 30min than GH-RH(1-29)NH₂ and also displayed a prolonged activity. D-Tyr¹⁰, D-Lys¹², and D-Lys¹² homologs of MZ-2-51 also showed enhanced activities. Thus, [Dat¹, D-Tyr¹⁰, Ala¹⁵, Nle²⁷]GH-RH(1-28)Agm (MZ-2-159), [Dat¹, D-Lys¹², Ala¹⁵, Nle²⁷]GH-RH(1-28)AGM (MZ-2-57), and [Dat¹, Ala¹⁵, D-Lys²¹, Nle²⁷]GH-RH(1-28)Agm (MZ-2-75) were 4-6 times more active in vitro than GH-RH(1-29)NH₂. In vivo, after i.v. administration, analog MZ-2-75 was equipotent and analogs MZ-2-159 and MZ-2-57 about twice as potent as the standard. After S.C. administration, the potencies of MZ-2-57 and MZ-2-75 were 10-14 times higher than the standard at 15 min and 45-89 times greater when determined at 30 min. Most of the analogs containing two or more D-amino acid substitutions were less active than GH-RH(1-29)NH₂ or inactive. Our studies indicate that very potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.     

Potent agonists of growth hormone-releasing hormone

Analogs of the 29 amino acid sequence of growth hormone-releasing hormone (GH-RH) with agmatine (Agm) in position 29 have been synthesized by the solid phase method, purified, and tested in vitro and in vivo. The majority of the analogs contained desaminotyrosine (Dat) in position 1, but a few of them had Tyr¹, or N-MeTyr¹. Some peptides contained one or more additional l - or d -amino acid substitutions in positions 2, 12, 15, 21, 27, and/or 28. Compared to the natural sequence of GH-RH(1-29)NH₂, [Dat′,Ala¹⁵]GH-RH(1-28)Agm (MZ-3-191) and [d -Ala²,Ala¹⁵]GH-RH(l-28)Agm (MZ-3-201) were 8.2 and 7.1 times more potent in vitro, respectively. These two peptides contained Met²⁷. Their Nle²⁷ analogs, [Dat^I,Ala¹⁵,Nle²⁷]GH-RH(1-28)Agm(MZ-2-51), prepared previously (9), and [^D-Ala²,Ala¹⁵,Nle²⁸]GH-RH(l-28)Agm(MZ-3-195) showed relative in vitro potencies of 10.5 and 2.4, respectively. These data indicate that replacement of Met²⁷ by Nle²⁷ enhanced the GH-releasing activity of the analog when the molecule contained Dat¹-Ala² residues at the N-terminus, but peptides containing Tyr¹-D-Ala² in addition to Nle²⁷ showed decreased potencies. Replacement of Ser²⁸ with Asp in multi-substituted analogs of GH-RH(l-28)Agm resulted in a decrease in in vitro potencies compared to the parent compound. Thus, the Ser²⁸-containing MZ-2-51, and [Dat¹,Ala¹⁵,d -Lys²¹,Nle²⁷]GH-RH(l-28)Agm, its Asp²⁸ homolog (MZ-3-149), possessed relative activities of 10.5 and 5.6, respectively. In vivo after the iv injection, the analogs [Dat¹,Ala¹⁵,Nle²⁷,Asp²⁸]GH-RH(l-28)Agm (MZ-3-149), [Dat¹, Ala¹⁵]GH-RH(l-28)Agm, (MZ-3-191) and [d -Ala²,Ala^,5]GH-RH(l-28)Agm (MZ-3-201) showed a potency equivalent to 7.6, 4.9 and 3.3 times that of GH-RH(1-29)NH₂, respectively, at 5 min and 20.3, 4.3 and 1.7 times higher, respectively, at 15 min. After sc administration, analogs MZ-3-149, MZ-3-191, and MZ-3-201 were shown to be 63.7, 55.2 and 56.8 times more potent than the parent hormone at 15 min and 57.6, 60.6, and 42.6 times more active, respectively, at 30 min. In addition, MZ-3-149 had prolonged GH-releasing activity as compared to the standard, and proved to be more potent than MZ-2-51, the most active member of our previous series (8, 9). Our studies indicate that very potent GH-RH analogs can result from the combination of agmatine in position 29 with other substitutions.     

Glycation Methods for Bombesin Analogs Containing the (NαHis)Ac chelator for 99mTc(CO)3 Radiolabeling

The overexpression of peptide receptors in a variety of human carcinomas has generated considerable interest in peptide‐based radiopharmaceuticals for peptide receptor imaging and peptide receptor radiotherapy. The gastrin‐releasing peptide receptor is overexpressed in human prostate‐, breast‐, colon‐ and small cell lung carcinoma cells. We have developed metabolically stable ^99mTc‐radiolabeled bombesin ([Cha¹³, Nle¹⁴]BBS(7–14)) analogs, which bind with high affinity to the gastrin‐releasing peptide receptors. However, because of their lipophilicity, they showed unfavorable biodistribution with high hepatic accumulation and hepatobiliary excretion. We now report a study of different glycation methods for [Cha¹³, Nle¹⁴]BBS(7–14) analogs to improve their biodistribution profile. Whereas the glycation using the Maillard reaction was problematic, resulting in low yields, selective introduction of the glycomimetic shikimic acid to the side chain of a Lys residue was possible. A chemoselective ligation of α‐d ‐glucose to an amino‐oxyacetylated [Cha¹³, Nle¹⁴]BBS(7–14) analog could be achieved, but was complicated by the co‐elution of starting peptide and glycopeptide. The best procedure consisted of the [1,3]‐cycloaddition of N₃‐β‐d ‐glucose to a propargylglycine‐containing [Cha¹³, Nle¹⁴]BBS(7–14) analog, using a catalytic amount of Cu(I)I. All glycated [Cha¹³, Nle¹⁴]BBS(7–14) analogs showed high affinity for the gastrin‐releasing peptide receptor and rapid accumulation into PC‐3 tumor cells.     

NMR and quenched molecular dynamics studies of superpotent linear and cyclic α-melanotropins

Conformational searching, computer simulations, synthesis and NMR are used on a variety of α melanocyte-stimulating hormone (α-MSH) analogues to understand the physical characteristics required for biological potency. Peptides I (AC-[Nle⁴,Asp⁵,d -Phe⁷,Lys¹⁰]α-MSH(4-10)-NH₂), II (Ac-c[Nle⁴,Asp⁵,d -Phe⁷,Lys¹⁰]α-MSH(4-10)-NH₂) and III (Ac-[Nle⁴,Asp⁵,d -Phe⁷,Dap¹⁰]α-MSH(4-10)-NH₂ all show very similar conformational properties (backbone and side-chain torsional angles), and all display high biological potencies. The modeling results for these compounds are supported by the NMR data. Peptide IV (Ac-c[Nle⁴,Asp⁵,d -Phe⁷,Dap¹⁰]α-MSH(4-10)-NH₂) appears to have a markedly different conformation and has decreased biological potency.     

β-ENDORPHIN: SYNTHESIS AND ANALGESIC ACTIVITY OF SEVERAL ANALOGS MODIFIED IN POSITIONS 2 AND 5

The solid-phase syntheses of [Sar²]-, [Ala²]-, [D-Leu²]-, [D-Lys²]-β- endorphins and [Pro⁵]-, [Leu⁵]-, [D-Leu⁵]-, [D-Ala², D-Leu⁵]-β-endorphins are described. The synthetic peptides were purified by chromatography on carboxymethylcellulose and partition chromatography on Sephadex G-50. They were characterized by partition chromatography on agarose, thin-layer chromatography, paper electrophoresis, and amino acid analyses of acid and enzymic hydrolysates. Bioassay of the synthetic analogs for analgesic activity by the tail-flick method showed the D-Leu² analog to be 48% as potent as β_h-endorphin while the Ala², D-Lys², Leu⁵, and [D-Ala², D-Leu⁵] analogs were 8 to 17% as active. The Sar², D-Leu⁵, and Pro⁵ analogs were less than 1% as potent.     

Exploring relationships between mimic configuration,peptide conformation and biological activity in indolizidin‐2‐one amino acid analogs of gramicidin S*

Abstract: Indolizidin‐2‐one amino acids (I²aas, 6S‐ and 6R‐ 1 ) possessing 6S‐ and 6R‐ring‐fusion stereochemistry were introduced into the antimicrobial peptide gramicidin S (GS) to explore the relationships between configuration, peptide conformation and biological activity. Solution‐phase and solid‐phase techniques were used to synthesize three analogs with I²aa residues in place of the d ‐Phe‐Pro residues at the turn regions of GS: [(6S)‐I²aa^{4?5,4′?5′}]GS ( 2 ), [Lys^2,2′,(6S)‐I²aa^{4?5,4′?5′}]GS ( 3 ) and [(6R)‐I²aa^{4?5,4′?5′}]GS ( 4 ). Although conformational analysis of [I²aa^{4?5,4′?5′}]GS analogs 2?4 indicated that both ring‐fusion stereoisomers of I²aa gave peptides with CD and NMR spectral data characteristic of GS, the (6S)‐I²aa analogs 2 and 3 exhibited more intense CD curve shapes, as well as greater numbers of nonsequential NOE between opposing Val and Leu residues, relative to the (6R)‐I²aa analog 4 , suggesting a greater propensity for the (6S)‐diastereomer to adopt the β‐turn/antiparallel β‐pleated sheet conformation. In measurements of antibacterial and antifungal activity, the (6S)‐I²aa analog 2 exhibited significantly better potency than the (6R)‐I²aa diastereomer 4 . Relative to GS, [(6S)‐I²aa^{4?5,4′?5′}]GS ( 2 ) exhibited usually 1/2 to 1/4 antimicrobial activity as well as 1/4 hemolytic activity. In certain cases, antimicrobial and hemolytic activities of GS were shown to be dissociated through modification at the peptide turn regions with the (6S)‐I²aa diastereomer. The synthesis and evaluation of GS analogs 2?4 has furnished new insight into the importance of ring‐fusion stereochemistry for turn mimicry by indolizidin‐2‐one amino acids as well as novel antimicrobial peptides.     

An affinity label for δ‐opioid receptors derived from [d‐Ala2]deltorphin I*

Abstract: A series of potential affinity label derivatives of the amphibian opioid peptide [d ‐Ala²]deltorphin I were prepared by incorporation at the para position of Phe³ (in the ‘message’ sequence) or Phe⁵ (in the ‘address’ sequence) of an electrophilic group (i.e. isothiocyanate or bromoacetamide). The introduction of the electrophile was accomplished by incorporating Fmoc‐Phe(p‐NHAlloc) into the peptide, followed later in the synthesis by selective deprotection of the Alloc group and modification of the resulting amine. While para substitution decreased the δ‐opioid receptor affinity, selected analogs retained nanomolar affinity for δ receptors. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited moderate affinity (IC₅₀ = 83 nm ) and high selectivity for δ receptors, while the corresponding amine and bromoacetamide derivatives showed pronounced decreases in δ‐receptor affinity (80‐ and >1200‐fold, respectively, compared with [d ‐Ala²]deltorphin I). In the ‘address’ sequence, the Phe(p‐NH₂)⁵ derivative showed the highest δ‐receptor affinity (IC₅₀ = 32 nm ), while the Phe(p‐NHCOCH₂Br)⁵ and Phe(p‐NCS)⁵ peptides displayed four‐ and tenfold lower δ‐receptor affinities, respectively. [d ‐Ala²,Phe(p‐NCS)³]deltorphin I exhibited wash‐resistant inhibition of [³H][d ‐Pen²,D‐Pen⁵]enkephalin (DPDPE) binding to δ receptors at a concentration of 80 nm . [d ‐Ala², Phe(p‐NCS)³]deltorphin I represents the first affinity label derivative of one of the potent and selective amphibian opioid peptides, and the first electrophilic affinity label derivative of an agonist containing the reactive functionality in the ‘message’ sequence of the peptide.     

Assignments of the 270 MHz PMR spectrum of somatostatin using pH titration,synthetic analogs and double resonance difference spectroscopy

We report the assignment procedure for the 270 MHz PMR spectrum in D²O of the 14 amino acid peptide hormone somatostatin, Ala¹ Gly² Cys³ Lys⁴ Asn⁵ Phe⁶ Phe⁷ Trp⁸ Lys⁹ Thr¹⁰ Phe¹¹ Thr¹² Ser¹³ Cys¹⁴ using a series of synthetic analogs in which a single amino acid residue was replaced by an Ala residue. The principal methods used were pH titration and extensive double resonance experiments (difference scalar decouplings and nuclear Overhauser effect measurements).     

Antimicrobial Properties of Brevinin‐2‐Related Peptide and its Analogs: Efficacy Against Multidrug‐Resistant Acinetobacter baumannii

Brevinin‐2 related peptide (B2RP; GIWDTIKSMG¹⁰KVFAGKILQN²⁰L.NH₂), first isolated from skin secretions of the mink frog Lithobates septentrionalis, shows broad‐spectrum antimicrobial activity but its therapeutic potential is limited by moderate hemolytic activity. The peptide adopts an α‐helical conformation in a membrane‐mimetic solvent but amphipathicity is low. Increasing amphipathicity together with hydrophobicity by the substitutions Lys¹⁶→Leu and Lys¹⁶→Ala increased hemolytic activity approximately fivefold without increasing antimicrobial potency. The substitution Leu¹⁸→Lys increased both cationicity and amphipathicity but produced decreases in both antimicrobial potency and hemolytic activity. In contrast, increasing cationicity of B2RP without changing amphipathicity by the substitution Asp⁴→Lys resulted in a fourfold increase in potency against Escherichia coli [minimal inhibitory concentration (MIC) = 6 μm ) and twofold increases in potency against Staphylococcus aureus (MIC = 12.5 μm ) and Candida albicans (MIC = 6 μm ) without changing significantly hemolytic activity against human erythrocytes (LC₅₀ = 95 μm ). The emergence of antibiotic‐resistant strains of the Gram‐negative bacterium Acinetobacter baumannii constitutes a serious risk to public health. B2RP (MIC = 3–6 μm ) and [Lys⁴]B2RP (MIC = 1.5–3 μm ) potently inhibited the growth of nosocomial isolates of multidrug‐resistant Acinetobacter baumannii. Although the analogs [Lys⁴, Lys¹⁸]B2RP and [Lys⁴, Ala¹⁶, Lys¹⁸]B2RP showed reduced potency against Staphylococcus aureus, they retained activity against Acinetobacter baumannii (MIC = 3–6 μm ) and had very low hemolytic activity (LC₅₀ > 200 μm ).     

Interaction of gonadotropin-releasing hormone and its agonist analogs with Ca2+ in a nonpolar milieu. Correlation with biopotencies

Extracellular Ca²⁺ is necessary for the action of gonadotropin-releasing hormone (GnRH). Assuming that this partly because of the interaction of the hormone with the relatively abundant extracellular Ca²⁺ in the low dielectric milieu of the bilayer plasma membrane, we studied the interaction of GnRH and five of its agonist analogs with Ca²⁺ under membrane-mimetic conditions. The peptides used, in increasing order of their reported gonadotropin-releasing activities, were: des-amide GnRH (or GnRH-OH); [Ala⁶]GnRH; [d -Ala⁶]GnRH; des-Gly¹⁰[d -Ala⁶,Pro⁹-NHEt]GnRH and, des-Gly¹⁰[d -Trp⁶,Pro⁹-NHEt]GnRH. Changes in the far-UV CD and fluorescence spectra of these peptides in trifluoroethanol were used to monitor conformational changes and obtain the Ca²⁺-binding isotherms. The data show that GnRH and its active analogs contain two Ca²⁺ binding sites, whereas the inactive analogs have only one. The extent of Ca²⁺ binding by the agonist peptides paralleled their biological potency ranking. The superactive analog des-Gly¹⁰[d -Trp⁶,Pro⁹-NHEt]GnRH exhibited the ability to transport Ca²⁺ ions across large unilamellar vesicles of dimyristoylphosphatidylcholine. Our study shows that significant differences among the GnRH and its analog peptides, suggestive of differences in their conformations, are manifested only in the presence of Ca²⁺. This observation would provide a basis for understanding GnRH action in terms of the hormone's interaction with Ca²⁺ in the lipid milieu.     

Structure-function analysis of the Saccharomyces cerevisiae tridecapeptide pheromone using alanine-scanned analogs

Twenty-six peptide analogs of the Saccharomyces cerevisiaeα-factor, a tridecapeptide mating pheromone (W¹H²W³L⁴Q⁵L⁶K⁷P⁸G⁹Q¹⁰p¹¹M¹²Y¹³) with either l - or D-alanine replacement of each amino acid residue (Ala-scanned) and with the isosteric replacement of methionine at position 12 by norleucine, were synthesized, purified to homogeneity and assayed for biological activity and receptor binding. Two new and effective antagonists. [D-Ala³,Nle¹²]α-factor and [D-Ala⁴,Nle¹²]α-factor, were found among the series, and the [D-Ala¹⁰,Nle¹²]α-factor demonstrated a marked ability to increase the biological activity of [Nle¹²]α-factor without having any effect by itself. One analog, the [L-Ala¹α-factor, showed a 3-fold increase in bioactivity over the [Nle¹²]α-factor, although its binding to the α-factor receptor was about 70-fold less than [Nle¹²]α-factor. Residues near the carboxyl terminus contributed more strongly to receptor binding than other residues, whereas those near the amine terminus of the α-factor played an important role in signal transduction. The effect of insertion of D-Ala residues at positions 7, 8, 9 and 10 on bioactivity and receptor binding of the peptide suggested a specific positioning role of the central loop in establishing optimal contacts between the receptor and the ends of the pheromone. We conclude that the α-factor may be divided into segments with dominant roles in forming the biologically active pheromone conformation, in receptor binding and in initiating signal transduction. The discovery of such relationships was made possible by the systematic variation of each residue in the peptide and by the testing of each analog in highly defined biological and binding assays.     

Effect of asymmetric modification on the conformation of ascidiacyclamide analogs

Abstract: 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) Biopolymers 49 , 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.     

Incorporation of vinylogous scaffolds in the C‐terminal tripeptide of substance P

Abstract: Glycine‐9 and leucine‐10 of substance P (SP) are critical for (NK)‐1 receptor recognition and agonist activity. Proψ(Z)‐CH=CH(CH₃)‐CONH)Leu (or Met) and Proψ((E)‐CH=CH(CH₃)‐CONH)Leu (or Met) have been introduced in the sequence of SP, in order to restrict the conformational flexibility of the C‐terminal tripeptide, Gly‐Leu‐Met‐NH₂, of SP. Proψ((Z)‐CH=C(CH₂CH(CH₃)₂)‐CONH)Met‐NH₂, with an isobutyl substituent to mimic the Leu side‐chain, was also incorporated in place of the C‐terminal tripeptide. The substituted‐SP analogs were tested for their affinity to human NK‐1 receptor specific binding sites (NK‐1M and NK‐1m) and their potency to stimulate adenylate cyclase and phospholipase C in Chinese Hamster ovary (CHO) cells transfected with the human NK‐1 receptor. The most potent SP analogs [Pro⁹ψ((Z)CH=C(CH₃)CONH)Leu¹⁰]SP and [Pro⁹ψ ((E)CH=C(CH₃)CONH)Leu¹⁰]SP, are about 100‐fold less potent than SP on both binding sites and second messenger pathways. These vinylogous (Z)‐ or (E)‐CH=C(CH₃)‐ or (Z)‐CH=C(CH₂CH(CH₃)₂) moieties hamper the correct positioning of the C‐terminal tripeptide of SP within both the NK‐1M‐ and NK‐1m‐specific binding sites. The origin of these lower potencies is related either to an incorrect peptidic backbone conformation and/or an unfavorable receptor interaction of the methyl or isobutyl group.     

D ˙ Ala3 analog of elastin polypentapeptide.

The polypentapeptide, H(L˙Val₁-L˙Pro₂-D·Ala₃-L˙Val₄-Gly₅)_n Val-OMe which is the D·Ala₃ analog of the elastomeric polypentapeptide (PPP) of elastin, (L˙Val₁-L˙Pro₂-Gly₃-L˙Val₄-Gly₅)_n, has been synthesized. Its conformation is compared to that of the PPP and found to be similar with a somewhat stabilized β-turn. The D·Ala₃ analog coacervates to form a more cohesive viscoelastic material and the coacervate when cross-linked by γ-irradiation exhibits an approximate doubling of the Young's modulus of elasticity. These results are discussed in connection with other related analogs of the polypentapeptide of elastin, which are non-elastomeric, and found to be consistent with a proposed conformationally based librational entropy mechanism of elasticity.