Isolation and characterization of insulin from the Brockmann body of Dissostichus mawsoni,an Antarctic teleost fish

Abstract: The Brockmann body of fish synthesizes and secretes insulin. The Brockmann body of Antarctic fish has been described anatomically and shown to contain insulin immunoreactive sites, however, the primary structure of an Antarctic fish insulin has yet to be reported. Insulin was isolated from the Brockmann bodies of the Antarctic perciform teleost, Dissostichus mawsoni. The peptide was purified to homogeneity by gel filtration and reversed‐phase HPLC. Insulin‐containing fractions were identified by radioimmunoassay using antisera raised against porcine insulin. Electrospray ionization‐mass spectrometry determined the mass of the isolated product to be 5725.27 a.m.u. The amino acid composition and primary structure were determined for the pyridylethylated A‐ and B‐ chains. The amino acid sequences of the A chain and B chain were H‐Gly‐Ile‐Val‐Glu‐Gln‐Cys‐Cys‐His‐Gln‐Pro¹⁰‐Cys‐Asn‐Ile‐Phe‐Asp‐Leu‐Gln‐Asn‐Tyr‐Cys²⁰‐Asn‐OH and H‐Ala‐Pro‐Gly‐Pro‐Gln‐His‐Leu‐Cys‐Gly‐Ser¹⁰‐His‐Leu‐Val‐Asp‐Ala‐Leu‐Tyr‐Leu‐Val‐Cys²⁰‐Gly‐Glu‐Arg‐Gly‐Phe‐Phe‐Tyr‐Asn‐Pro‐Lys³⁰‐OH, respectively. The primary structure of insulin from Antarctic fish is compared with known structures of insulin from other vertebrates.     

Molecular Cloning of a Novel Tryptophyllin Peptide from the Skin of the Orange‐Legged Monkey Frog,Phyllomedusa hypochondrialis

Tryptophyllins are a group of small (4–14 amino acids), heterogenous peptides, mostly from the skins of hylid frogs from the genera, Phyllomedusa and Litoria. To date, more than forty TPHs have been discovered in species from these two genera. Here, we describe the identification of a novel tryptophyllin type 3 peptide, PhT‐3, from the extracts of skin of the orange‐legged monkey frog, Phyllomedusa hypochondrialis, and molecular cloning of its precursor‐encoding cDNA from a cDNA library constructed from the same skin sample. Full primary structural characterization was achieved using a combination of direct Edman degradation, mass spectrometry and deduction from cloned skin‐derived cDNA. The open‐reading frame of the precursor cDNA was found to consist of 63 amino acid residues. The mature peptide arising from this precursor contains a post‐translationally modified N‐terminal pyroglutamate (pGlu) residue, formed from acid‐mediated cyclization of an N‐terminal Gln (Q) residue, and with the structure: pGlu‐Asp‐Lys‐Pro‐Phe‐Trp‐Pro‐Pro‐Pro‐Ile‐Tyr‐Pro‐Met. Pharmacological assessment of a synthetic replicate of this peptide on phenylephrine preconstricted rat tail artery segments, revealed a reduction in relaxation induced by bradykinin. PhT‐3 was also found to mediate antiproliferative effects on human prostate cancer cell lines.     

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.     

Characterization of the solution conformations of leuprolide acetate

Abstract: Leuprolide acetate (pGlu‐His‐Trp‐Ser‐Tyr‐d ‐Leu‐Leu‐Arg‐Pro‐NHEt), a potent LHRH agonist in wide clinical use, was characterized conformationally by NMR and circular dichroism. It displayed quite different preferred conformations under different solution conditions: two low population β‐turns in water, a nascent helix in TFE/water at low pH, and a high population β‐turn in TFE/water at slightly acidic pH. The pH‐related conformational change in TFE/water is attributed to the pK_a of the acetate counterion, not to ionizable groups on the peptide. None of these conformations are in exact agreement with previous computational predictions.     

Cyclization of several linear penta‐ and heptapeptides with different metal ions studied by CD spectroscopy*

Abstract: A cyclic pentapeptide c(Tyr‐Leu‐Ala‐Gly‐Pro) ( I ), which was isolated and identified from Pseudostellaria heterophylla medicinal herbs, and two cyclic heptapeptides, c(Gly‐Tyr‐Gly‐Gly‐Pro‐Phe‐Pro) ( II ) and c(Gly‐Ile‐Pro‐Tyr‐Ile‐Ala‐Ala) ( III ), which were isolated and identified from Stellaria yunnanensis Franch (M), were synthesized by using 3‐(diethoxyphosphoryloxy)‐1,2,3‐benzotriazin‐4(3 H)‐one (DEPBT) as a coupling reagent in solution, and mediated by different metal ions, from their linear peptide precursors H‐Tyr‐Leu‐Ala‐Gly‐Pro‐OH ( I‐1 ) and H‐Ala‐Gly‐Pro‐Tyr‐Leu‐OH ( I‐2 ), H‐Gly‐Tyr‐Gly‐Gly‐Pro‐Phe‐Pro‐OH ( II‐1 ) and H‐Gly‐Ile‐Pro‐Tyr‐Ile‐Ala‐Ala‐OH ( III‐1 ), respectively. The results show that alkali metal ions can improve the cyclization yields and/or the cyclization rates of linear peptide precursors, such as Na⁺ ion is favorable for the cyclization of linear pentapeptides and Cs⁺ ion is favorable for the cyclization of linear heptapeptides, while some bivalent and trivalent metal ions, such as Mg²⁺, Ca²⁺, Zn²⁺, Fe²⁺, Ni²⁺ and Cr³⁺ reduced/inhibited both the cyclization yields and the cyclization rates of the linear peptide precursors. The circular dichroism spectra of I‐1 , II‐1 and III‐1 with different metal ions were studied to elucidate the changes in their secondary structures. It is shown that Cs⁺ can induce and stabilize the type I β‐turn conformation in the linear heptapeptide II‐1 and the type II β‐turn conformation in the linear heptapeptide III‐1 .     

Synthesis and evaluation of a new series of peptide‐based endothelin receptor antagonists

Abstract: Novel peptide‐based endothelin (ET) receptor antagonists were designed and synthesized in our laboratory. BQ‐485, HIM‐CO‐Leu‐d ‐Trp‐d ‐Trp‐OH, was selected as the leading compound. The primary structures of these new tripeptides were ABO‐CO‐Leu‐d ‐Trp‐d ‐AA(X)‐OH. The introduction of unnatural aromatic amino acids into these tripeptides was useful in the structure–activity relationship studies. Among the 20 tripeptides, 16 of them showed high activities against the contraction of rat aortic smooth muscles induced by ET‐1.     

Ammonia cleaves polypeptides at asparagine proline bonds

Abstract: Polypeptides that contain the sequence Asn‐Pro undergo complete cleavage at this amide bond with ammonia. One cleavage product possesses Pro as the new amino terminus and the other Asn or isoAsn as the new C‐terminus, the formation of the latter probably arising by way of a cyclic succinimide intermediate. Other Asn‐X bonds where X = Tyr, Gln, Ile, Glu, Ala, Gly, Asn or Phe did not exhibit any peptide bond cleavage, whereas when X = Leu, Thr and Ser partial cleavage was observed. Asn residues not involved in chain‐cleavage underwent deamidation to Asp as shown by MALDI‐ToF mass spectrometry (MS) analysis. The partial conversion of in‐chain Asp residues to isoAsp under the reaction conditions was inferred from RP‐HPLC and MS analysis of reaction mixtures.     

Tryptophan‐containing peptide helices: interactions involving the indole side chain*

Abstract: Two designed peptide sequences containing Trp residues at positions i and i + 5 (Boc‐Leu‐Trp‐Val‐Ala‐Aib‐Leu‐Trp‐Val‐OMe, 1 ) as well as i and i + 6 (Boc‐Leu‐Trp‐Val‐Aib‐Ala‐Aib‐Leu‐Trp‐Val‐OMe, 2 ) containing one and two centrally positioned Aib residues, respectively, for helix nucleation, have been shown to form stable helices in chloroform solutions. Structures derived from nuclear magnetic resonance (NMR) data reveal six and seven intramolecularly hydrogen‐bonded NH groups in peptides 1 and 2 , respectively. The helical conformation of octapeptide 1 has also been established in the solid state by X‐ray diffraction. The crystal structure reveals an interesting packing motif in which helical columns are stabilized by side chain–backbone hydrogen bonding involving the indole N?1H of Trp(2) as donor, and an acceptor C=O group from Leu(6) of a neighboring molecule. Helical columns also associate laterally, and strong interactions are observed between the Trp(2) and Trp(7) residues on neighboring molecules. The edge‐to‐face aromatic interactions between the indoles suggest a potential C‐H…π interaction involving the Cζ3H of Trp(2). Concentration dependence of NMR chemical shifts provides evidence for peptide association in solution involving the Trp(2) N?1H protons, presumably in a manner similar to that observed in the crystal.     

Insect neuropeptide antagonist. Part II. Synthesis and biological activity of backbone cyclic and precyclic PBAN antagonists

Abstract: A new approach for the design and synthesis of pheromone biosynthesis activating neuropeptide (PBAN) agonists and antagonists using the backbone cyclization and cycloscan concepts is described. Two backbone cyclic (BBC) libraries were synthesized: library I (Ser library) was based on the active C‐terminal hexapeptide sequence Tyr‐Phe‐Ser‐Pro‐Arg‐Leu‐NH₂ of PBAN1‐33NH₂; whereas library II (d ‐Phe library) was based on the sequence of the PBAN lead linear antagonist Arg‐Tyr‐Phe‐d ‐Phe‐Pro‐Arg‐Leu‐NH₂. In both libraries the Pro residue was replaced by the BBC building unit N^α‐(ω‐aminoalkyl) Gly having various lengths of alkyl chain. The peptides of the two libraries were tested for agonistic and antagonistic activity. Four precyclic peptides based on two of the BBC antagonists were also synthesized; their activity revealed that a negative charge at the N‐terminus of the peptide abolished antagonistic activity. We also describe the use of the reagent SiCl₃I for selective deprotection of the Boc group from the building unit prior to on‐resin amino‐end to backbone‐nitrogen (AE‐BN) cyclization, during solid‐phase synthesis with Fmoc chemistry.     

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.     

Conformational comparison of µ‐selective endomorphin‐2 with its C‐terminal free acid in DMSO solution,by 1H NMR spectroscopy and molecular modeling calculation

Abstract: In order to make clear the structural role of the C‐terminal amide group of endomorphin‐2 (EM2, H‐Tyr‐Pro‐Phe‐Phe‐NH₂), an endogenous µ‐receptor ligand, in the biological function, the solution conformations of endomorphin‐2 and its C‐terminal free acid (EM2OH, H‐Tyr‐Pro‐Phe‐Phe‐OH), studied using two‐dimensional ¹H NMR measurements and molecular modeling calculations, were compared. Both peptides were in equilibrium between the cis and trans isomers around the Tyr‐Pro ω bond in a population ratio of ≈ 1 : 2. The lack of significant temperature and concentration dependence of NH protons suggested that the NMR spectra reflected the conformational features of the respective molecules themselves. Fifty possible 3D structures for the each isomer were generated by the dynamical simulated annealing method under the proton?proton distance constraints derived from the ROE cross‐peaks. These energy‐minimized conformers, which were all in the φ torsion angles estimated from J_NHCαH coupling constants within ± 30°, were then classified in groups one or two according to the folding backbone structures. All trans and cis EM2 conformers adopt an open conformation in which their extended backbone structures are twisted at the Pro²–Phe³ moiety. In contrast, the trans and cis conformers of EM2OH show conformational variation between the ‘bow’‐shaped extended and folded backbone structures, although the cis conformers of its zwitterionic form are refined into the folded structure of the close disposition of C‐ and N‐terminal groups. These results indicate clearly that the substitution of carboxyl group for C‐terminal amide group makes the peptide flexible. The conformational requirement for µ‐receptor activation has been discussed based on the active form proposed for endomorphin‐1 and by comparing conformational features of EM2 and EM2OH.     

Detection of bioactive peptides including gonadotrophin‐releasing factors (GnRHs) in horse urine using ultra‐high performance liquid chromatography–high resolution mass spectrometry (UHPLC/HRMS)

The use of bioactive peptides as a doping agent in both human and animal sports has become increasingly popular in recent years. As such, methods to control the misuse of bioactive peptides in equine sports have received attention. This paper describes a sensitive accurate mass method for the detection of 40 bioactive peptides and two non‐peptide growth hormone secretagogues (< 2 kDa) at low pg/mL levels in horse urine using ultra‐high performance liquid chromatography‐high resolution mass spectrometry (UHPLC/HRMS). A simple mixed‐mode cation exchange solid‐phase extraction (SPE) cartridge was employed for the extraction of 42 targets and/or their in vitro metabolites from horse urine. The final extract was analyzed using UHPLC/HRMS in positive electrospray ionization (ESI) mode under both full scan and data independent acquisition (DIA, for MS²). The estimated limits of detection (LoD) for most of the targets could reach down to 10 pg/mL in horse urine. This method was validated for qualitative detection purposes. The validation data, including method specificity, method sensitivity, extraction recovery, method precision, and matrix effect were reported. A thorough in vitro study was also performed on four gonadotrophin‐releasing factors (GnRHs), namely leuprorelin, buserelin, goserelin, and nafarelin, using the S9 fraction isolated from horse liver. The identified in vitro metabolites have been incorporated into the method for controlling the misuse of GnRHs. The applicability of this method was demonstrated by the identification of leuprorelin and one of its metabolites, Leu M4, in urine obtained after intramuscular administration of leuprorelin to a thoroughbred gelding (castrated horse).     

Peptide‐doxorubicin conjugates specifically degraded by matrix metalloproteinases expressed from tumor

Specific peptide‐doxorubicin conjugates were developed for targeting matrix metalloproteinases (MMPs) expressed from tumors. The peptide‐doxorubicin conjugates were designed to be cleaved by MMP‐2 and MMP‐9 in order that doxorubicin or the active form that acts as an anticancer agent was released free from the peptide fragment at the tumor site. Three types of peptide‐doxorubicin conjugates were synthesized using the peptides: GPLG (Gly‐Pro‐Leu‐Gly), GPLGV (Gly‐Pro‐Leu‐Gly‐Val), and GPLGPAG (Gly‐Pro‐Leu‐Gly‐Pro‐Ala‐Gly). The synthesized peptide‐doxorubicin conjugates were characterized for their degradation behavior and bioactivity in vitro, and their antitumoral activity was assessed using the Lewis lung carcinoma (LLC) model, which expresses MMP‐2 and MMP‐9. After incubation with active MMP‐2 for 24 h, GPLG‐doxorubicin was barely degraded, whereas GPLGV‐doxorubicin and GPLGPAG‐doxorubicin were considerably degraded by active MMP‐2. Consequently, all peptide‐doxorubicin conjugates had significantly low cytotoxicity compared to doxorubicin, but tumor growth suppression was exhibited only by GPLGV‐doxorubicin and GPLGPAG‐doxorubicin. The tumor growth suppression by the two conjugates was higher compared to control, although it did not exceed the suppression level shown by doxorubicin. The low toxicity exhibited by peptide‐doxorubicin conjugates resulted in only slight body weight loss in mice, whereas doxorubicin greatly reduced body weight and induced severe side effects. Therefore, we propose MMPs‐specific peptide‐doxorubicin conjugates in targeting anti‐cancer drug delivery that could reduce systemic toxicities. Drug Dev. Res. 67:438–447, 2006. © 2006 Wiley‐Liss, Inc.     

Stabilization of a tyrosine O‐sulfate residue by a cationic functional group: formation of a conjugate acid–base pair

Abstract: Sulfated tyrosine [Tyr(SO₃H)]‐containing peptides showed characteristic peak patterns in their liquid secondary‐ion mass spectrometry (LSIMS) spectra. Protonated molecules were desulfated more easily than their deprotonated counterparts. Therefore, the stabilities of the Tyr(SO₃H) residues were well‐reflected by peak patterns in their positive‐ion spectra. These intrinsic peak patterns were investigated by comparing the behavior of each Tyr(SO₃H) residue in acidic solution. As the peptide chain was lengthened and the number of cationic functional groups increased, the peak representing the [MH]⁺ of a Tyr(SO₃H)‐containing peptide became more prominent than that representing the desulfated [MH–SO₃]⁺. These alterations in peptide structure also increased the stability of the Tyr(SO₃H) residue in acidic solution. Based on the desulfation mechanism of an aryl monosulfate, we predicted that intramolecular cationic functional groups would stabilize Tyr(SO₃H) residues by forming conjugate acid–base pairs (or salt bridges) both in the gaseous phase and in acidic solution. In accordance with this theory, Arg residues would take primary responsibility for this self‐stabilization within Tyr(SO₃H)‐containing peptides. Moreover, a long peptide backbone was expected to have a weak protective effect against desulfation of the [MH]⁺ in the gaseous phase. Tyr(SO₃H) residues were also stabilized by adding an external basic peptide containing multiple Arg residues. Formation of such intermolecular acid–base pairs was demonstrated by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) which detected conjugated peptide ions. The energetically favorable formation of conjugate acid–base pairs prompted by Tyr(SO₃H) residues might be a driving force for protein folding and protein–protein interaction.     

Synthesis and radiobiological evaluation of a new 99mTc‐labeled small peptide: 99mTc‐YGGSLAK as imaging agent

Peptides are known as receptor‐specific molecules that play an important role not only in diagnosis and therapy of neoplastic diseases, but also in the pathogenesis of other diseases. In an effort to develop a peptide‐based radiopharmaceutical for scintigraphic studies, a small peptide Tyr‐Gly‐Gly‐Ser‐Leu‐Ala‐Lys (YGGSLAK) was synthesized by Fmoc solid‐phase peptide synthesis using an automated synthesizer. This peptide was analyzed by TLC, HPLC and mass spectroscopy. Then, we investigated its analytical and pharmacokinetic study after radiolabeling with technetium‐99 m (^99mTc) using SnCl₂. The radiochemical purity of the complex was over 95% and log p value was 1.46. In vivo biodistribution studies in rat showed that most activity of this injected radiolabeled peptide (^99mTc‐YGGSLAK) was accumulated in the liver and followed by gallbladder, intestines and kidney. Scintigraphy studies on rabbits also showed very high uptake and retention in the liver and gallbladder, and after 1 h slowly excreted through the hepatobiliary system and a little amount was also excreted through the renal system. The radiochemical and in vitro and in vivo characterization indicates that ^99mTc‐YGGSLAK has certain favorable properties and it might be used as a new radiopharmaceutical for hepatobiliary scintigraphy. Copyright © 2011 John Wiley & Sons, Ltd.     

In vitro evaluation of the effects of anti‐fungals,benzodiazepines and non‐steroidal anti‐inflammatory drugs on the glucuronidation of 19‐norandrosterone: implications on doping control analysis

We have studied whether the phase II metabolism of 19‐norandrosterone, the most representative metabolite of 19‐nortestosterone (nandrolone), can be altered in the presence of other drugs that are not presently included on the Prohibited List of the World Anti‐Doping Agency. In detail, we have evaluated the effect of non‐prohibited drugs belonging to the classes of anti‐fungals, benzodiazepines, and non‐steroidal anti‐inflammatory drugs on the glucuronidation of 19‐norandrosterone. In vitro assays based on the use of either pooled human liver microsomes or specific recombinant isoforms of uridine diphosphoglucuronosyl‐transferase were designed and performed to monitor the formation of 19‐norandrosterone glucuronide from 19‐norandrosterone. Determination of 19‐norandrosterone (free and conjugated fraction) was performed by gas chromatography – mass spectrometry after sample pretreatment consisting of an enzymatic hydrolysis (performed only for the conjugated fraction), liquid/liquid extraction with tert‐butylmethyl ether, and derivatization to form the trimethylsilyl derivative. In parallel, a method based on reversed‐phase liquid chromatography coupled to tandem mass spectrometry in positive electrospray ionization with acquisition in selected reaction monitoring mode was also developed to identify the non‐prohibited drugs considered in this study. Incubation experiments have preliminarily shown that the glucuronidation of 19‐norandrosterone is principally carried out by UGT2B7 (39%) and UGT2B17 (31%). Inhibition studies have shown that the yield of the glucuronidation reaction is reduced in the presence of the anti‐fungals itraconazole, ketoconazole, and miconazole, of the benzodiazepine triazolam and of the non‐steroidal anti‐inflammatory drugs diclofenac and ibuprofen, while no alteration was recorded in the presence of all other compounds considered in this study. Copyright © 2015 John Wiley & Sons, Ltd.     

Aromatic interactions in tryptophan‐containing peptides: crystal structures of model tryptophan peptides and phenylalanine analogs*

Abstract: The crystal structures of the peptides, Boc‐Leu‐Trp‐Val‐OMe ( 1) , Ac‐Leu‐Trp‐Val‐OMe ( 2a and 2b), Boc‐Leu‐Phe‐Val‐OMe ( 3 ), Ac‐Leu‐Phe‐Val‐OMe ( 4 ), and Boc‐Ala‐Aib‐Leu‐Trp‐Val‐OMe ( 5 ) have been determined by X‐ray diffraction in order to explore the nature of interactions between aromatic rings, specifically the indole side chain of Trp residues. Peptide 1 adopts a type I β‐turn conformation stabilized by an intramolecular 4→1 hydrogen bond. Molecules of 1 pack into helical columns stabilized by two intermolecular hydrogen bonds, Leu(1)NH…O(2)Trp(2) and IndoleNH…O(1)Leu(1). The superhelical columns further pack into the tetragonal space group P4₃ by means of a continuous network of indole–indole interactions. Peptide 2 crystallizes in two polymorphic forms, P2₁ ( 2a ) and P2₁2₁2₁ ( 2b ). In both forms, the peptide backbone is extended, with antiparallel β‐sheet association being observed in crystals. Extended strand conformations and antiparallel β‐sheet formation are also observed in the Phe‐containing analogs, Boc‐Leu‐Phe‐Val‐OMe ( 3 ) and Ac‐Leu‐Phe‐Val‐OMe ( 4 ). Peptide 5 forms a short stretch of 3₁₀‐helix. Analysis of aromatic–aromatic and aromatic–amide interactions in the structures of peptides, 1 , 2a , 2b are reported along with the examples of 14 Trp‐containing peptides from the Cambridge Crystallographic Database. The results suggest that there is no dramatic preference for a preferred orientation of two proximal indole rings. In Trp‐containing peptides specific orientations of the indole ring, with respect to the preceding and succeeding peptide units, appear to be preferred in β‐turns and extended structures.     

Advantages of a Synthetic Peptide Immunogen Over a Protein Immunogen in the Development of an Anti‐Pilus Vaccine for Pseudomonas aeruginosa

The type IV pilus is an important adhesin in the establishment of infection by Pseudomonas aeruginosa. We have previously reported on a synthetic peptide vaccine targeting the receptor‐binding domain of the main structural subunit of the pilus, PilA. The receptor‐binding domain is a 14‐residue disulfide loop at the C‐terminal end of the pilin protein. The objective of this study was to compare the immunogenicity of a peptide‐conjugate to a protein subunit immunogen to determine which was superior for use in an anti‐pilus vaccine. BALB/c mice were immunized with the native PAK strain pilin protein and a synthetic peptide of the receptor‐binding domain conjugated to keyhole limpet haemocyanin. A novel pilin protein with a scrambled receptor‐binding domain was used to characterize receptor‐binding domain‐specific antibodies. The titres against the native pilin of the animals immunized with the synthetic peptide‐conjugate were higher than the titres of animals immunized with the pilin protein. In addition, the affinities of anti‐peptide sera for the intact pilin receptor‐binding domain were significantly higher than affinities of anti‐pilin protein sera. These results have significant implications for vaccine design and show that there are significant advantages in using a synthetic peptide‐conjugate over a subunit pilin protein for an anti‐pilus vaccine.     

Synthesis of Novel Oxazolo[4,5‐b]pyridine‐2‐one based 1,2,3‐triazoles as Glycogen Synthase Kinase‐3β Inhibitors with Anti‐inflammatory Potential

A novel series of oxazolo[4,5‐b]pyridine‐2‐one based 1,2,3‐triazoles has been synthesized by click chemistry approach and evaluated for in vitro GSK‐3β inhibitory activity. Compound 4g showed maximum inhibition with IC₅₀ value of 0.19 μ m . Keeping in view the effect of GSK‐3β inhibition on inflammation, compounds 4g , 4d , 4f , 4i , 4n and 4q exhibiting significant GSK‐3β inhibition were examined for in vivo anti‐inflammatory activity in rat paw edema model. The compounds 4g , 4d , 4f and 4i showed pronounced in vivo anti‐inflammatory activity (76.36, 74.54, 72.72 and 70.90%, respectively, after 5h post‐carrageenan administration) and were further found to inhibit the pro‐inflammatory mediators, viz. NO, TNF‐ α , IL‐1 β , and IL‐6 substantially in comparison with indomethacin, an anti‐inflammatory drug as well as SB216763, a GSK‐3 β inhibitor, reported to exert a similar effect. Histopathology studies confirmed the tolerance of gastric mucosa to these compounds.