Modifications of the cyclic mu receptor selective tetrapeptide Tyr‐c[d‐Cys‐Phe‐d‐Pen]NH2 (Et): effects on opioid receptor binding and activation

Abstract: The previously described cyclic mu opioid receptor‐selective tetrapeptide Tyr‐c[d ‐Cys‐Phe‐d ‐Pen]NH₂ (Et) (JOM‐6) was modified at residues 1 and 3 by substitution with various natural and synthetic amino acids, and/or by alteration of the cyclic system. Effects on mu and delta opioid receptor binding affinities, and on potencies and efficacies as measured by the [³⁵S]‐GTPγS assay, were evaluated. Affinities at mu and delta receptors were not influenced dramatically by substitution of Tyr¹ with conformationally restricted phenolic amino acids. In the [³⁵S]‐GTPγS assay, all of the peptides tested exhibited a maximal response comparable with that of fentanyl at the mu opioid receptor, and all showed high potency, in the range0.4–9 nm . However, potency changes did not always correlate with affinity, suggesting that the conformation required for binding and the conformation required for activation of the opioid receptors are different. At the delta opioid receptor, none of the peptides were able to produce a response equivalent to that of the full delta agonist BW 373,U86 and only one had an EC₅₀ value of less than 100 nm . Lastly, we have identified a peptide, d ‐Hat‐c[d ‐Cys‐Phe‐d ‐Pen]NH₂ (Et), with high potency and > 1000‐fold functional selectivity for the mu over delta opioid receptor as measured by the [³⁵S]‐GTPγS assay.     

Topography of the neurotensin (NT)(8−9) binding site of human NT receptor‐1 probed with NT(8–13) analogs

Abstract: A series of neurotensin (NT)(8–13) analogs featuring substitution of the Arg⁸ and/or Arg⁹ residues with non‐natural cationic amino acids was synthesized and evaluated for binding to the human NT receptor‐1 (hNTR‐1). The modifications were designed to probe specific steric and electrostatic requirements in the N‐terminal cationic region of NT(8–13) for receptor binding as a general evaluation of the feasibility of incorporating minor structural changes into a peptide at a crucial polar receptor binding site. Many of the non‐natural amino acids are more or less isosteric to Arg but more lipophilic as a result of addition of alkyl groups or through removal or replacement of NH character with methylene or methyl substituents, whereas others vary the distance between the cation and the α–amino acid carbon. Substitution of Arg⁸ with N^G‐alkylated Arg derivatives or homolysine (Hlys) maintained the subnanomolar affinity of NT(8–13) to the hNTR‐1. Position 8 incorporation of Hlys produced the most favorable primary amine side‐chain substitution to date. Moderate losses in affinity observed with position 9 substitutions were attributed to adverse steric effects. Doubly substituted [Hlys⁸, DAB⁹]NT(8–13), in which DAB is 2,4‐diaminobutyric acid, was also prepared and tested as the shorter side‐chain of DAB is known to be favored in position 9 of NT(8–13). This analog maintained 60% of NT(8–13) binding affinity making it the most favored des‐guanidinium‐containing analog known. These results demonstrate that adequate receptor binding affinity can be maintained over a structural range of Arg analogs, thus providing a range of peptides expected to exhibit altered pharmacokinetic properties. From the standpoint of the hNTR‐1 cationic binding sites, these results help to map out the structural stringency inherent in the formation of a tight binding complex with NT(8–13) and related analogs.     

13C-n.m.r. characterization of formaldehyde bonds in model mixtures and proteins containing lysine

Reactions involving formaldehyde, lysine and another amino acid (tyrosine, arginine, asparagine, glutamine or cysteine) were studied by ¹³C-n.m.r. analysis. The products of the crosslinking between lysine and tyrosine were found to be acid-resistant and were isolated using ion exchange chromatography. With the other selected amino acids, the crosslinked products with lysine are acid-labile and could not be isolated; however, n.m.r. allows the determination of their structure. In addition, the formation of methyllysine and of formyllsine have been observed. The ¹³C-n.m.r. chemical shifts of the formaldehyde derived hydroxymethyl, methylene, methyl and formyl carbons with the amino acids side chains help in the interpretation of ¹³C-enriched formaldehyde treated protein ¹³C-n.m.r. spectra. With BSA, we have observed the formation of hydroxymethyllysine, hydroxymethylhistidine, hydroxymethyl-asparagine or -glutamine, methyllysine, and a crosslink between lysine and arginine. These results show the great importance of lysine in the treatment of protein with formaldehyde. Use of ¹³C-n.m.r. is considered highly suitable for obtaining more knowledge about formaldehyde binding to peptides and proteins.     

The Biological Consequences of Replacing d‐Ala in Biphalin with Amphiphilic α‐Alkylserines

Biphalin, a synthetic opioid peptide with a broad affinity for all opioid receptors (δ, μ, and κ) and high antinociceptive activity, has been under extensive study as a potential analgesic drug. This study presents the synthesis and biological properties of four new analogues of biphalin containing amphiphilic α‐alkylserines in position 2 and 2′. The incorporation of bulky α,α‐disubstituted amino acids in the peptide chain using standard peptide chemistry is often unsuccessful. We synthesized depsipeptides, and then, the desired peptides were obtained by internal O,N‐migration of the acyl residue from the hydroxyl to the amino group under mild basic conditions. The potency and selectivity of the new analogues were evaluated by a competitive receptor‐binding assay in the rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). Their binding affinity is strongly dependent on the chirality of α‐alkylserine, as analogues containing (R)‐α‐alkylserines displayed higher μ receptor affinity and selectivity than those incorporating the (S)‐isomers.     

Development of highly potent and ective dynorphin A analogues as new medicines

Abstract: Dynorphin A (Dyn A), a 17 amino acid peptide H‐Tyr‐Gly‐Gly‐Phe‐Leu‐Arg‐Arg‐Ile‐Arg‐Pro‐Lys‐Leu‐Lys‐Trp‐Asp‐Asn‐Gln‐OH, is a potent opioid peptide which interacts preferentially with κ‐opioid receptors. Research in the development of selective and potent opioid peptide ligands for the κ‐receptor is important in mediating analgesia. Several cyclic disulphide bridge‐containing peptide analogues of Dyn A, which were conformationally constrained in the putative message or address segment of the opioid ligand, were designed, synthesized and assayed. To further investigate the conformational and topographical requirements for the residues in positions 5 and 11 of these analogues, a systematic series of Dyn A_1?11‐NH₂ cyclic analogues incorporating the sulphydryl‐containing amino acids l ‐ and d ‐Cys and l ‐ and d ‐Pen in positions 5 and 11 were synthesized and assayed. Cyclic lactam peptide analogues were also synthesized and assayed. Several of these cyclic analogues, retained the same affinity and selectivity (vs. the μ‐ and δ‐receptors) as the parent Dyn A_1?11‐NH₂ peptide in the guinea‐pig brain (GPB), but exhibited a much lower activity in the guinea‐pig ileum (GPI), thus leading to centrally vs. peripherally selective peptides. Studies of the structure–activity relationship of Dyn A peptide provide new insights into the importance of each amino acid residue (and their configurations) in Dyn A analogues for high potency and good selectivity at κ‐opioid receptors. We report herein the progress towards the development of Dyn A peptide ligands, which can act as agonists or antagonists at cell surface receptors that modulate cell function and animal behaviour using various approaches to rational peptide ligand‐based drug design.     

Alanine Scan of an Immunosuppressive Peptide (CP): Analysis of Structure–Function Relationships

Core peptide is a hydrophobic peptide, the sequence of which is derived from the T‐cell antigen receptor alpha‐chain transmembrane region. Previous studies have shown that core peptide can inhibit T‐cell‐mediated immune responses both in vitro and in vivo. Here, we report the role each constituent amino acid plays within core peptide using an alanine scan and the amino acid effect on function using a biological antigen presentation assay. The biophysical behaviour of these analogues in model membranes was analysed using surface plasmon resonance studies and then binding correlated with T‐cell function. Removal of any single hydrophobic amino acid between the two charged amino acids in core peptide (R, K) resulted in lower binding. Changing the overall net charge of core peptide, by removing either of the positively charged residues (R or K), had varying effects on peptide binding and IL‐2 production. There was a direct correlation (ρ = 0.718) between peptide binding to model membranes and peptide ability to inhibit IL‐2. Except for IL‐2 inhibition, production of other T‐cell cytokines such as GM‐CSF, IFN‐γ, IL‐1α, IL‐4, IL‐5, IL‐6, IL‐10, IL‐17 and T‐cell antigen receptor alpha‐chain was not detected using a fluorescent bead immunoassay. This study provides important structure–function relationships essential for further drug design.     

Peptide analogs from E‐cadherin with different calcium‐binding affinities

Abstract: Cadherins are a family of calcium‐dependent cell‐surface proteins that are fundamental in controlling the development and maintenance of tissues. Motif B of E‐cadherin seems to be a crucial calcium‐binding site as single point mutations (D134A and D134K) completely inactivate its adhesion activity. We analyzed peptide models corresponding to motif B (amino acids 128–144) as well as selected mutations of this motif. Our NMR studies showed that this motif B sequence is actually an active calcium‐binding region, even in the absence of the rest of the cadherin molecule. We found that the binding affinity of this motif is very sensitive to mutations. For example, our peptide P128‐144 with the native calcium‐binding sequence has an affinity of K_d 0.4 mm , whereas the mutants P128‐144/D134A and P128‐144/D134K containing the replacement of Asp¹³⁴ by Ala and Lys, have K_d values of only 1.5 and 11 mm , respectively. Removing Asp at position 134, which correlates with the loss of adhesion activity, decreases calcium‐binding affinity 20‐fold. Ala¹³², along with residues Asp¹³⁴, Asp¹³⁶ and Asn¹⁴³, is involved in calcium binding in solution. We also demonstrated that the calcium‐binding affinity can be increased ≈ 3‐fold when an additional Asp is introduced at position 132. In 50% organic solvent, this binding affinity of peptide P128‐144/A132D (17‐mer) from E‐cadherin is similar to that of peptide P72–100/C73–77–91A (29‐mer) from α‐lactalbumin.     

An internal histidine residue from the bacterial surface protein,PAM, mediates its binding to the kringle‐2 domain of human plasminogen

Abstract: The determinants of binding of a peptide lacking C‐termini‐exposed lysine residues to a kringle domain were investigated using an up‐regulated lysine binding kringle (K2_Pg[C⁴G/E⁵⁶D/K⁷²Y]) of plasminogen and a peptide (a1‐PAM) with a sequence derived from a surface‐exposed M‐like streptococcal protein. Significant kringle‐induced chemical shifts in a His side‐chain of a1‐PAM were revealed by two‐dimensional NMR. Further studies using isothermal titration calorimetry (ITC) provided support for the involvement of His¹² in the peptide/protein complex. In an effort to screen a1‐PAM‐derived truncation peptides, a combinatorial mixture, a1Δa2‐PAM[H¹²X] (where X = Pro, Arg, His, Trp, Lys, Ala, Phe, Asp and Gly), was analyzed using the surface‐enhanced laser desorption ionization time‐of‐flight mass spectrometry (SELDI) platform. The major peptide that remained bound to the surface of the K2_Pg[C⁴G/E⁵⁶D/K⁷²Y]‐containing chip was that containing His¹², corresponding to the wild‐type sequence. Minor peaks, representing binding, were obtained for Lys¹²‐, Arg¹²‐ and Trp¹²‐containing peptides. Individual peptides containing these amino acids were then examined using ITC and the binding constants obtained correlated with the relative strengths of binding estimated from the SELDI‐based screen.     

Structure–activity studies on high affinity NOP‐active hexapeptides

Abstract: Nociceptin/orphanin FQ (N/OFQ) is a 17 amino acid peptide that is the endogenous ligand for the G‐protein coupled receptor ORL1 (NOP), a member of the opioid receptor family. Although it is clear that this receptor system is involved in a variety of physiologic functions, including analgesia, the precise actions of N/OFQ remain largely uncharacterized. One reason for this has been limited number of high‐affinity ligands to NOP, and particularly the lack of availability of useful specific antagonists. Herein, we describe the pharmacologic activity of a series of modified amino acid containing modifications of the hexapeptide Ac‐RYYRWR‐NH₂, with high affinity for NOP. These compounds were tested for binding affinity using [³H]N/OFQ binding to human NOP in CHO cells, and functional activity by measuring stimulation of [³⁵S]GTPγS‐binding in CHO cell membranes. These studies suggest that each Arg of the hexapeptide is required to maintain high‐binding affinity. The peptide maintains high affinity if the Tyr² or Tyr³ are modified, but at least one of these residues must maintain its hydroxyl group or there is a large decrease in intrinsic activity of the peptide.     

Synthesis and AT2 receptor‐binding properties of angiotensin II analogues

Abstract: The present study investigates the importance of the amino acid side chains in the octapeptide angiotensin II (Ang II) for binding to the AT₂ receptor. A Gly scan was performed where each amino acid in Ang II was substituted one‐by‐one with glycine. The resulting set of peptides was tested for affinity to the AT₂ receptor (porcine myometrial membranes). For a comparison, the peptides were also tested for affinity to the AT₁ receptor (rat liver membranes). Only the substitution of Arg² reduced affinity to the AT₂ receptor considerably (92‐fold when compared with Ang II). For the other Gly‐substituted analogues the affinity to the AT₂ receptor was only moderately affected. To further investigate the role of the Arg² side chain for receptor binding, we synthesized some N‐terminally modified Ang II analogues. According to these studies a positive charge in the N‐terminal end of angiotensin III [Ang II (2–8)] is not required for high AT₂ receptor affinity but seems to be more important in Ang II. With respect to the AT₁ receptor, [Gly²]Ang II and [Gly⁸]Ang II lacked binding affinity (K_i > 10 μm ). Replacement of the Val³ or Ile⁵ residues with Gly produced only a slight decrease in affinity. Interestingly, substitution of Tyr⁴ or His⁶, which are known to be very important for AT₁ receptor binding, resulted in only 48 and 14 times reduction in affinity, respectively.     

Preclinical evaluation of new GnRH‐I receptor radionuclide therapy with 177Lu‐peptide tracer

The purpose of this study was to develop preclinical evaluation of a novel radiolabeled gonadotropin‐releasing hormone (GnRH) receptor targeting peptide for prostate cancer therapy. The new antiproliferative agent of GnRH‐I analogue was developed on the basis of the D‐Trp⁶‐GnRH‐I scaffold, and in vivo pharmacokinetics and receptor binding affinity were enhanced by the substitution of Gly‐NHNH₂ for Gly‐NH₂ at position 10 in D‐Trp⁶‐GnRH‐I. To evaluate ¹⁷⁷Lu‐DOTA‐triptorelin‐hydrazide as radionuclide therapy of tumor, the quality control tests and preclinical stage assessment were carried out. Solid‐phase method was used to synthesize new peptide. Characterization and purity of peptide were done by mass spectroscopy and high‐performance liquid chromatography (HPLC). In order to be utilized in targeted therapy, the new GnRH‐I agonist was coupled with pSCN‐Bn‐DOTA. The precipitate crude of DOTA‐triptorelin‐hydrazide was then purified via preparative HPLC. At optimal conditions of time, temperature, ligand amount, and lutetium content, DOTA‐triptorelin‐hydrazide was labeled with ¹⁷⁷Lu (specific activity not less than 925 GBq/mg). Investigation of the in vivo biodistribution and in vitro studies for ¹⁷⁷Lu‐DOTA‐TRPHYD was performed in three different ways, and the binding of radiopeptide to GnRH receptors was expressed on the human cell lines using ¹²⁵I‐labeled D‐TRP⁶GnRH‐I as a tracer, respectively. Synthesized novel GnRH‐I was obtained with purity greater than 98%. Paper chromatography was found to be the most suitable with R_f of the complex and observed radiochemical purity of RTLC and HPLC greater than 97%. For in vivo studies, ¹⁷⁷Lu‐DOTA‐triptorelin‐hydrazide showed promising results with fast clearance from the blood and resulted in good T/NT ratios at 1, 4, and 24 hours postinjection and satisfactory biodistribution with no significant activity seen in normal tissue. The values of internalization efficiency and receptor affinity of new radiopeptide binding were IC₅₀ = 0.47 ± 0.06 vs 0.13 ± 0.01 nM for triptorelin and cellular uptake: 3.4 ± 0.7% at 1 hour and 6.8 ± 1.17% at 4 hours of the internal reference. The results showed a good stability and radiochemical purity of the obtained radioconjugate. For in vivo and in vitro studies, new radiopeptide showed a high uptake of ¹⁷⁷Lu conjugate in tumor and rapid clearance from the blood stream almost entirely via the renal/urinary pathway and binding to the GnRH receptors with high specificity and affinity, respectively.     

Synthesis and evaluation of potential affinity labels derived from endomorphin‐2

Abstract: In an attempt to identify potential peptide‐based affinity labels for opioid receptors, endomorphin‐2 (Tyr‐Pro‐Phe‐PheNH₂), a potent and selective endogenous ligand for µ‐opioid receptors, was chosen as the parent peptide for modification. The tetrapeptide analogs were prepared using standard Fmoc‐solid phase peptide synthesis in conjunction with incorporation of Fmoc‐Phe(p‐NHAlloc) and modification of the p‐amino group. The electrophilic groups isothiocyanate and bromoacetamide were introduced into the para position on either Phe³ or Phe⁴; the corresponding free amine‐containing peptides were also prepared for comparison. The peptides bearing an affinity label group and their free amine analogs were evaluated in a radioligand‐binding assay using Chinese hamster ovary (CHO) cells expressing µ‐ and δ‐opioid receptors. Modification on Phe⁴ was better tolerated than on Phe³ for µ‐receptor binding. Among the analogs tested, [Phe(p‐NH₂)⁴]endomorphin‐2 showed the highest affinity (IC₅₀ = 37 nm ) for µ‐receptors. The Phe(p‐NHCOCH₂Br)⁴ analog displayed the highest µ‐receptor affinity (IC₅₀ = 158 nm ) among the peptides containing an affinity label group. Most of the compounds exhibited negligible binding affinity for δ‐receptors, similar to the parent peptide.     

Synthesis and biological activities of new side chain and backbone cyclic bradykinin analogues

Abstract: A series of conformationally constrained cyclic analogues of the peptide hormone bradykinin (BK, Arg‐Pro‐Pro‐Gly‐Phe‐Ser‐Pro‐Phe‐Arg) was synthesized to check different turned structures proposed for the bioactive conformation of BK agonists and antagonists. Cycles differing in the size and direction of the lactam bridge were performed at the C‐ and N‐terminal sequences of the molecule. Glutamic acid and lysine were introduced into the native BK sequence at different positions for cyclization through their side chains. Backbone cyclic analogues were synthesized by incorporation of N‐carboxy alkylated and N‐amino alkylated amino acids into the peptide chain. Although the coupling of Fmoc‐glycine to the N‐alkylated phenylalanine derivatives was effected with DIC/HOAt in SPPS, the dipeptide building units with more bulky amino acids were pre‐built in solution. For backbone cyclization at the C‐terminus an alternative building unit with an acylated reduced peptide bond was preformed in solution. Both types of building units were handled in the SPPS in the same manner as amino acids. The agonistic and antagonistic activities of the cyclic BK analogues were determined in rat uterus (RUT) and guinea‐pig ileum (GPI) assays. Additionally, the potentiation of the BK‐induced effects was examined. Among the series of cyclic BK agonists only compound 3 with backbone cyclization between positions 2 and 5 shows a significant agonistic activity on RUT. To study the influence of intramolecular ring closure we used an antagonistic analogue with weak activity, [d ‐Phe⁷]‐BK. Side chain as well as backbone cyclization in the N‐terminus of [d ‐Phe⁷]‐BK resulted in analogues with moderate antagonistic activity on RUT. Also, compound 18 in which a lactam bridge between positions 6 and 9 was achieved via an acylated reduced peptide bond has moderate antagonistic activity on RUT. These results support the hypothesis of turn structures in both parts of the molecule as a requirement for BK antagonism. Certain active and inactive agonists and antagonists are able to potentiate the bradykinin‐induced contraction of guinea‐pig ileum.     

Functional preference of the constituent amino acid residues in a phage‐library‐based nonphosphorylated inhibitor of the Grb2‐SH2 domain

Abstract: A nonphosphorylated disulfide‐bridged peptide, cyclo(Cys‐Glu¹‐Leu‐Tyr‐Glu‐Asn‐Val‐Gly‐Met‐Tyr⁹‐Cys)‐amide (termed G1) has been identified, by phage library, that binds to the Grb2‐SH2 domain but not the src SH2 domain. Synthetic G1 blocks the Grb2‐SH2 domain association (IC₅₀ of 15.5 µm ) with natural phosphopeptide ligands. As a new structural motif that binds to the Grb2‐SH2 domain in a pTyr‐independent manner, the binding affinity of G1 is contributed by the highly favored interactions of its structural elements interacting with the binding pocket of the protein. These interactions involve side‐chains of amino acids Glu¹, Tyr³, Glu⁴, Asn⁵, and Met⁸. Also a specific conformation is required for the cyclic peptide when bound to the protein. Ala scanning within G1 and molecular modeling analysis suggest a promising model in which G1 peptide binds in the phosphotyrosine binding site of the Grb2‐SH2 domain in a β‐turn‐like conformation. Replacement of Tyr³ or Asn⁵ with Ala abrogates the inhibitory activity of the peptide, indicating that G1 requires a Y‐X‐N consensus sequence similar to that found in natural pTyr‐containing ligands, but without Tyr phosphorylation. Significantly, the Ala mutant of Glu¹, i.e. the amino acid N‐terminal to Y³, remarkably reduces the binding affinity. The position of the Glu¹ side‐chain is confirmed to provide a complementary role for pTyr³, as demonstrated by the low micromolar inhibitory activity (IC₅₀ = 1.02 µm ) of the nonphosphorylated peptide 11 , G1(Gla¹), in which Glu¹ was replaced by γ‐carboxy‐glutamic acid (Gla).     

Hydrogen fluoride catalyzed migration of side chain protecting groups onto Fmoc during solid phase peptide synthesis

The solid-phase synthesis of the N^α-Fmoc analog of protein kinase C substrate (PKCS, Lys-Arg-Ala-Lys-Ala-Lys-Thr-Thr-Lys-Lys-Arg) was characterized by low recovery from the resin and the concomitant appearance of four impurities. FAB-MS revealed molecular weights for two of these impurities that corresponded to the desired peptide plus Tos or Bzl. The other two were justified by invoking a CO₂ elimination of the Clz protecting group to yield: 1) peptide plus 2-chlorobenzyl (CIBzl) and 2) peptide plus ClBzl and Tos. A CF-FAB analysis of carboxypeptidase digestions allowed observation of peptide cleavage down to an ion corresponding to lysine, Fmoc, and the corresponding protecting group(s). These data revealed that the impurities were not the result of incomplete deprotection but the result of migration of the protecting groups to the N-terminal end of the peptide. NMR experiments were subsequently performed and revealed the exact site of substitution: the meta positions of the N-terminal Fmoc. These impurities are presumed to arise by electrophilic aromatic substitution of the fluorene group during HF treatment. The desired Fmoc analog served as a convenient, albeit low-yielding, intermediate for purification of the highly charged PKCS by preparative self-displacement HPLC.     

Synthetic peptide derived from α‐fetoprotein inhibits growth of human breast cancer: investigation of the pharmacophore and synthesis optimization

Abstract: A synthetic peptide that inhibits the growth of estrogen receptor positive (ER+) human breast cancers, growing as xenografts in mice, has been reported. The cyclic 9‐mer peptide, cyclo[EMTOVNOGQ], is derived from α‐fetoprotein (AFP), a safe, naturally occurring human protein produced during pregnancy, which itself has anti‐estrogenic and anti‐breast cancer activity. To determine the pharmacophore of the peptide, a series of analogs was prepared using solid‐phase peptide synthesis. Analogs were screened in a 1‐day bioassay, which assessed their ability to inhibit the estrogen‐stimulated growth of uterus in immature mice. Deletion of glutamic acid, Glu1, abolished activity of the peptide, but glutamine (Gln) or asparagine (Asn) could be substituted for Glu1 without loss of activity. Methionine (Met2) was replaced with lysine (Lys) or tyrosine (Tyr) with retention of activity. Substitution of Lys for Met2 in the cyclic molecule resulted in a compound with activity comparable with the Met2‐containing cyclic molecule, but with a greater than twofold increase in purity and corresponding increase in yield. This Lys analog demonstrated anti‐breast cancer activity equivalent to that of the original Met‐containing peptide. Therefore, Met2 is not essential for biologic activity and substitution of Lys is synthetically advantageous. Threonine (Thr3) is a nonessential site, and can be substituted with serine (Ser), valine (Val), or alanine (Ala) without significant loss of activity. Hydroxyproline (Hyp), substituted in place of the naturally occurring prolines (Pro4, Pro7), allowed retention of activity and increased stability of the peptide during storage. Replacement of the first Pro (Pro4) with Ser maintains the activity of the peptide, but substitution of Ser for the second Pro (Pro7) abolishes the activity of the peptide. This suggests that the imino acid at residue 7 is important for conformation of the peptide, and the backbone atoms are part of the pharmacophore, but Pro4 is not essential. Valine (Val5) can be substituted only with branched‐chain amino acids (isoleucine, leucine or Thr); replacement by d ‐valine or Ala resulted in loss of biologic activity. Thus, for this site, the bulky branched side chain is essential. Asparagine (Asn6) is essential for activity. Substitution with Gln or aspartic acid (Asp), resulted in reduction of biologic activity. Removal of glycine (Gly8) resulted in a loss of activity but nonconservative substitutions can be made at this site without a loss of activity indicating that it is not part of the pharmacophore. Cyclization of the peptide is facilitated by addition of Gln9, but this residue does not occur in AFP nor is it necessary for activity. Gln9 can be replaced with Asn, resulting in a molecule with similar activity. These data indicate that the pharmacophore of the peptide includes side chains of Val5 and Asn6 and backbone atoms contributed by Thr3, Val5, Asn6, Hyp7 and Gly8. Met2 and Gln9 can be modified or replaced. Glu1 can be replaced with charged amino acids, and is not likely to be part of the binding site of the peptide. The results of this study provide information that will be helpful in the rational modification of cyclo[EMTOVNOGQ] to yield peptide analogs and peptidomimetics with advantages in synthesis, pharmacologic properties, and biologic activity.     

DiSSiMiL: Diverse Small Size Mini‐Libraries applied to simple and rapid epitope mapping of a monoclonal antibody

Abstract: Methods for screening protein–protein interactions are useful in protein science and for the generation of drug leads. We set out to develop a simplified assay to rapidly test protein–protein interactions, with a library of 400 pentapeptides comprising the 20 natural amino acids at two variable positions followed by three glycines (NH₂‐X₁X₂GGG). The library was used to identify the epitope of monoclonal antibody (mAb) 10D11 directed against the HOXD4 protein. Three pentapeptide ‘hits’ were selected (VYGGG, PWGGG and WKGGG) from direct binding assays screening for pentapeptide?mAb interactions; and from assays using pentapeptides in solution to competitively block HOXD4?mAb interactions. Alignment of the three ‘hit’ pentapeptides to the HOXD4 sequence predicts the mAb 10D11 epitope as NH₂‐VYPWMK. Synthesis of NH₂‐VYPWMK hexapeptide confirmed this prediction; and an alanine scan of HOXD4 ablated binding by mAb 10D11 when amino acids in the putative epitope were mutated. We propose that these simplified but diverse libraries can be used for rapid epitope mapping of some mAbs, and for generating lead small peptide analogs that interfere with receptor–ligand or other protein–protein interactions, or with enzymatic activity.     

Integrin α5β1: a new purification strategy based on immobilized peptides

Abstract: Novel efficient and robust affinity chromatography material: There are several strategies known for the purification of integrins by affinity chromatography, but the disadvantages of common strategies like insufficient selectivity or compelling conditions for the elution still require alternatives. A new strategy, based on the immobilized C‐terminally modified peptide Ac‐Gly‐Ala‐c‐(Cys^SS‐Arg‐Arg‐Glu‐Thr‐Ala‐Trp‐Ala‐Cys^SS)‐Gly‐Ala‐O(CH₂CH₂O)₂CH₂CH₂‐NH₂ allows for the affinity purification of the integrin α₅β₁. While RGD peptides have been proven in the past to be inappropriate for selective purification of integrins by affinity chromatography, the new peptide can be efficiently used for selective enrichment of the integrin α₅β₁. It is a specific ligand of the target protein, but does not contain an RGD sequence. The application of well‐characterized affinity chromatography material with a site‐specifically immobilized peptide allows to obtain integrin α₅β₁ in a single chromatography step without contamination by other integrins. This process combines the advantages of a selective and monospecific protein‐ligand recognition with mild elution conditions and a low sensitivity of the immobilized ligand with respect to column regeneration.     

Chaperone‐like activity of a synthetic peptide toward oxidized γ‐crystallin

αA‐Crystallin can function like a molecular chaperone. We recently reported that the αA‐crystallin sequence, KFVIFLDVKHFSPEDLTVK (peptide‐1, residues 70–88) by itself possesses chaperone‐like (anti‐aggregating) activity during a thermal denaturation assay. Based on the above data we proposed that the peptide‐1 sequence was the functional site in αA‐crystallin. In this study we investigated the specificity of peptide‐1 against γ‐crystallin aggregation in the presence of H₂O₂ and CuSO_4. Peptide‐1 was able to completely protect against the oxidation‐induced aggregation of γ‐crystallin. Removal of N‐terminal Lys or the replacement of Lys with Asp ( D FVIFLDVKHFSPEDLTVK, peptide‐2) did not alter the anti‐aggregation property of peptide‐1. However, deletion of KF residues from the N‐terminus of peptide‐1 resulted in a significant loss of its anti‐aggregation property. Bio‐gel P‐30 size‐exclusion chromatography of γ‐crystallin incubated with peptide‐2 under oxidative conditions revealed that a major portion of the peptide elutes in the void volume region along with γ‐crystallin, suggesting the binding of the peptide to the protein. Peptide‐1 and ‐2 were also able to prevent the UV‐induced aggregation of γ‐crystallin. These data indicate that the same amino acid sequence in αA‐crystallin is likely to be responsible for suppressing the heat‐denatured, oxidatively modified and UV‐induced aggregation of proteins.     

Design of chimeric peptide ligands to galanin receptors and substance P receptors

Several chimeric peptides were synthesized and found to be high-affinity ligands for both galanin and substance P receptors in membranes from the rat hypothalamus. The peptide galantide, composed of the N-terminal part of galanin and C-terminal part of substance P (SP), galanin-(1-12)-Pro-SP-(5-11) amide, which is the first galanin antagonist to be reported, recognizes two classes of galanin binding sites (K_D(1)<0.1 nM and K_D(2)∼ 6 nM) in the rat hypothalamus, while it appears to bind to a single population of SP receptors (K_D∼ 40 nM). The chimeric peptide has higher affinity towards galanin receptors than the endogenous peptide galanin-(1-29) (KD ∼ 1 nm ) or its N-terminal fragment galanin-(1-13) (K_D∼ 1,nm ), which constitutes the A′-terminus of the chimeric peptide. Galantide has also higher affinity for the SP receptors than the C-terminal SP fragment-(4-11) amide (K_D= 0.4μm ), which constitutes its C-terminal portion. Substitution of amino acid residues, which is of importance for recognition of galanin by galanin receptors, such as [Trp²], in the galanin portion of the chimeric peptide or substitution of ([Phe⁷] or [Met¹¹]-amide) in the SP portion of chimeric peptide both cause significant loss in affinity of the analogs of galantide for both the galanin- and the SP-receptors. These results suggest that the high affinity of the chimeric peptide, galantide, may in part be accounted for by simultaneous recognition/binding to both receptors. In line with this suggestion is the finding that the binding of the chimeric ligands to the galanin receptor is strongly influenced by the presence of SP (1 μm ) or spantide (1 μm ). We have performed the synthesis and binding studies with 11 chimeric peptides, all composed of the N-terminal galanin-(1-13) fragment or of its analogs, linked to the C-terminal portion of SP or its peptide antagonist, spantide. Our results, similar to earlier reports on chimeric peptides, suggest that high-affinity ligands to peptide receptors can be produced by linking biologically active N-terminal and C-terminal portions of peptides via linkers, enabling a) independent recognition of the chimeric peptide by the relevant receptors and b) intramolecular interactions between the joined N- and C-terminal peptide fragments. These two phenomena may also explain why some of the chimeric peptides have higher affinity than the endogenous peptide(s) and why galantide, and some of its analogs presented here, behave(s) as a galanin receptor antagonist(s).