Chemical synthesis and receptor binding of catfish somatostatin: a disulfide‐bridged β‐d‐Galp‐(1→3)‐α‐d‐GalpNAc O‐glycopeptide*

Abstract: The glycopeptide hormone catfish somatostatin (somatostatin‐22) has the amino acid sequence H‐Asp‐Asn‐Thr‐Val‐Thr‐Ser‐Lys‐Pro‐Leu‐Asn‐Cys‐Met‐Asn‐Tyr‐Phe‐Trp‐Lys‐Ser‐Arg‐Thr‐Ala‐Cys‐OH; it includes a cyclic disulfide connecting the two Cys residues, and the major naturally occurring glycoform contains d ‐GalNAc and d ‐Gal O‐glycosidically linked to Thr⁵. The linear sequence was assembled smoothly starting with an Fmoc‐Cys(Trt)‐PAC‐PEG‐PS support, using stepwise Fmoc solid‐phase chemistry. In addition to the nonglycosylated peptide, two glycosylated forms of somatostatin‐22 were accessed by incorporating as building blocks, respectively, N^α‐Fmoc‐Thr(Ac₃‐α‐D‐GalNAc)‐OH and N^α‐Fmoc‐Thr(Ac₄‐β‐D‐Gal‐(1→3)‐Ac₂‐α‐D‐GalNAc)‐OH. Acidolytic deprotection/cleavage of these peptidyl‐resins with trifluoroacetic acid/scavenger cocktails gave the corresponding acetyl‐protected glycopeptides with free sulfhydryl functions. Deacetylation, by methanolysis in the presence of catalytic sodium methoxide, was followed by mild oxidation at pH 7, mediated by N^α‐dithiasuccinoyl (Dts)‐glycine, to provide the desired monomeric cyclic disulfides. The purified peptides were tested for binding affinities to a panel of cloned human somatostatin receptor subtypes; in several cases, presence of the disaccharide moiety resulted in 2‐fold tighter binding.     

Secondary structure of a dynamic type I’β‐hairpin peptide

Abstract: A spontaneously folding β‐hairpin peptide (Lys‐Lys‐Tyr‐Thr‐Val‐Ser‐Ile‐Asn‐Gly‐Lys‐Lys‐Ile‐Thr‐Val‐Ser‐Ile) and related cyclic (cyclo‐Gly‐Lys‐Tyr‐Ile‐Asn‐Gly‐Lys‐Ile‐Ile‐Asn) and linear (Ser‐Ile‐Asn‐Gly‐Lys) controls were studied to determine the effects of various factors on secondary structure. Secondary structure was evaluated using circular dichroism (CD) and 1D and 2D ¹H nuclear magnetic resonance (NMR). The effects of chemical modifications in the peptide and various solution conditions were investigated to determine their impact on peptide structure. The β‐hairpin peptide displayed a CD minimum at 216 nm and a TOCSY i + 1 ? i + 2 and i + 2 ?i + 3 interaction, confirming the expected structure. Using NMR α‐proton (H) chemical shifts, the extents of folding of the β‐hairpin and linear control were estimated to be 51 and 25% of the cyclic control (pH 4, 37 °C), which was taken to be maximally folded. Substitution of iso‐aspartic acid for Asn reduced the secondary structure dramatically; substitution of aspartic acid for Asn also disrupted the structure. This result suggests that deamidation in unconstrained β‐turns may have adverse effects on secondary structure. N‐terminal acetylation and extreme pH conditions also reduced structure, while the addition of methanol increased structure.     

A series of simple detection systems for genetic variants of flavin-containing monooxygenase 3 (FMO3) with impaired function in Japanese subjects

Increasing numbers of single-nucleotide substitutions of the human flavin-containing monooxygenase 3 (FMO3) gene are being recorded in mega-databases. Phenotype–gene analyses revealed impaired FMO3 variants associated with the metabolic disorder trimethylaminuria. Here, a series of reliable FMO3 genotyping confirmation methods was assembled and developed for 45 impaired FMO3 variants, mainly found in Japanese populations, using singleplex or duplex polymerase chain reaction (PCR)–restriction fragment length polymorphism (RFLP) methods and singleplex, duplex, or tetraplex allele-specific PCR methods. Nine PCR-RFLP procedures with single restriction enzymes and fourteen duplex PCR-RFLP procedures (for p.Trp41Ter and p.Thr329Ala, p.Met66Val and p.Leu163Pro, p.Pro70Leu and p.Glu308Gly, p.Asn114Ser and p.Ser195Leu, p.Glu158Lys and p.Ile441Thr, p.Cys197Ter and p.Trp388Ter, p.Arg205Cys and p.Val257Met, p.Arg205His and p.Cys397Ser, p.Met211ArgfsTer10 and p.Arg492Trp, p.Arg223Gln and p.Leu473Pro, p.Met260Val and p.Thr488Ala, p.Tyr269His and p.Ala311Pro, p.Ser310Leu and p.Gly376Glu, and p.Gln470Ter and p.Arg500Ter) were newly established along with eight singleplex (for p.Pro153GlnfsTer14, p.Gly191Cys, p.Pro248Thr, p.Ile486Met, and p.Pro496Ser, among others), one duplex (p.Ile199Ser and p.Asp286Tyr), and one tetraplex (p.Ile7Thr, p.Val58Ile, p.Thr201Lys, and p.Gly421Val) allele-specific PCR systems. This series of systems should facilitate the easy detection in a clinical setting of FMO3 variants in Japanese subjects susceptible to low drug clearances or drug reactions possibly caused by impaired FMO3 function.     

Effects of single d‐amino acid substitutions on disruption of β‐sheet structure and hydrophobicity in cyclic 14‐residue antimicrobial peptide analogs related to gramicidin S

Abstract: Gramicidin S (GS) is a 10‐residue cyclic β‐sheet peptide with lytic activity against the membranes of both microbial and human cells, i.e. it possesses little to no biologic specificity for either cell type. Structure–activity studies of de novo‐designed 14‐residue cyclic peptides based on GS have previously shown that higher specificity against microbial membranes, i.e. a high therapeutic index (TI), can be achieved by the replacement of a single l ‐amino acid with its corresponding d ‐enantiomer [Kondejewski, L.H. et al. (1999) J. Biol. Chem. 274 , 13181]. The diastereomer with a d ‐Lys substituted at position 4 caused the greatest improvement in specificity vs. other l to d substitutions within the cyclic 14‐residue peptide GS14, through a combination of decreased peptide amphipathicity and disrupted β‐sheet structure in aqueous conditions [McInnes, C. et al. (2000) J. Biol. Chem. 275 , 14287]. Based on this information, we have created a series of peptide diastereomers substituted only at position 4 by a d ‐ or l ‐amino acid (Leu, Phe, Tyr, Asn, Lys, and achiral Gly). The amino acids chosen in this study represent a range of hydrophobicities/hydrophilicities as a subset of the 20 naturally occurring amino acids. While the d ‐ and l ‐substitutions of Leu, Phe, and Tyr all resulted in strong hemolytic activity, the substitutions of hydrophilic d ‐amino acids d ‐Lys and d ‐Asn in GS14 at position 4 resulted in weaker hemolytic activity than in the l ‐diastereomers, which demonstrated strong hemolysis. All of the l ‐substitutions also resulted in poor antimicrobial activity and an extremely low TI, while the antimicrobial activity of the d ‐substituted peptides tended to improve based on the hydrophilicity of the residue. d ‐Lys was the most polar and most efficacious substitution, resulting in the highest TI. Interestingly, the hydrophobic d ‐amino acid substitutions had superior antimicrobial activity vs. the l ‐enantiomers although substitution of a hydrophobic d ‐amino acid increases the nonpolar face hydrophobicity. These results further support the role of hydrophobicity of the nonpolar face as a major influence on microbial specificity, but also highlights the importance of a disrupted β‐sheet structure on antimicrobial activity.     

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.     

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.     

Functional characterization of human xanthine oxidase allelic variants

OBJECTIVE: Xanthine oxidase (XO) catalyzes the oxidation of endogenous and exogenous purines and pyrimidines. In this study, we speculated that individual variations in XO activity are caused by genetic variations in the XO gene. METHODS: To investigate the genetic variations in XO in 96 Japanese participants, denaturing high-performance liquid chromatography was used. To assess the effects of these variations on enzymatic activity, wild-type XO and 21 types of variant XO--including those in the database and those just discovered--were transiently expressed in COS-7 cells. RESULTS: Three nonsynonymous single nucleotide polymorphisms, including 514G>A (Gly172Arg), 3326A>C (Asp1109Thr), and 3662A>G (His1221Arg) were identified in Japanese participants. Functional characterization of 21 XO variants showed a deficiency in enzyme activity in two variants (Arg149Cys and Thr910Lys); low activity (intrinsic clearance, CLint: 22-69% compared with the wild-type) in six variants (Pro555Ser, Arg607Gln, Thr623Ile, Asn909Lys, Pro1150Arg, and Cys1318Tyr); and high activity (CLint: approximately two-fold higher than that in the wild-type) in two variants (Ile703Val and His1221Arg). CONCLUSION: These results suggest that several single nucleotide polymorphisms in the XO gene are involved in individual variations in XO activity. In addition, such findings will be useful to identify xanthinuria patients.     

Helix‐stabilizing effects of the pentapeptide KIFMK and its related peptides on the sodium channel inactivation gate peptides

Abstract: We have previously found by NMR and CD spectroscopic studies that the helical content of the sodium channel inactivation gate‐related peptide (Ac‐GGQDIFMTEEQK‐NH₂; MP‐1A) in 80% trifluoroethanol solutions was increased by adding a pentapeptide, KIFMK. In order to study in further detail whether the presence of the IFM motif and the two lysine residues is a prerequisite for stabilizing the helical conformation, we examined interactions between various oligopeptides (RIFMR, KIFMTK, KIQMK, KAFAK, KIIIK) and MP‐1A and its related peptides; that is, MP‐2A in which Phe was replaced by Gln, MP‐1MMA in which Thr was replaced by Met, MP‐1TA in which Thr was removed from MP‐1A, and MP‐1A′ in which l ‐Phe was replaced by d ‐Phe. It was found that the IFM motif was absolutely necessary in both the oligopeptide and the inactivation gate peptide. This finding means that hydrophobic interactions are operative between KIFMK and MP‐1A. In contrast, KIFMK destabilized the helical structure of MP‐1MMA, MP‐1TA, and MP‐1A′, showing that the conformation around the IFM motif in the inactivation gate peptides is an important factor. It was concluded that the IFM motif and the two Lys residues are a prerequisite for effectively stabilizing the α‐helix of MP‐1A.     

Development of a synthetic cyclized peptide derived from α‐fetoprotein that prevents the growth of human breast cancer

Abstract: The peptide, EMTPVNPG, derived from alpha‐fetoprotein, inhibits estrogen‐stimulated growth of immature mouse uterus and estrogen‐dependent proliferation of human breast cancer cells. However, the biological activities of the peptide diminish over time in storage, even when in the lyophilized state, probably because of peptide aggregation through hydrophobic interaction among monomers. Two analogs of EMTPVNPG were designed with the intent of minimizing aggregation and retaining biological activity during prolonged storage. EMTOVNOG, where O is 4‐hydroxyproline, is a linear peptide generated by substituting 4‐hydroxyproline for the two prolines, thereby increasing peptide hydrophilicity. This analog exhibited a dose‐dependent inhibition of estrogen‐stimulated growth of immature mouse uterus similar to that of EMTPVNPG (maximal activity at 1 µg/mouse). A second analog, cyclo‐(EMTOVNOGQ), a hydrophilic, cyclic analog with increased conformational constraint, was as potent as the other peptides in its inhibition of estrogen‐dependent growth of immature mouse uterus, and had an expanded effective dose range. Both linear and cyclized hydroxyproline‐substituted analogs exhibited indefinite shelf‐life. Furthermore, both analogs inhibited the estrogen‐dependent growth of MCF‐7 human breast cancer growing as a xenograft in SCID mice. These analogs may become significant, novel agents for the treatment of breast cancer.     

The detection of a synthetic Interleukin‐1 receptor antagonist peptide in a seized product from a racing stable

A synthetic Interleukin‐1 receptor antagonist peptide with the sequence Acetyl‐Phe‐Glu‐Trp‐Thr‐Pro‐Gly‐Tyr‐Trp‐Gln‐Pro‐Tyr‐Ala‐Leu‐Pro‐Leu‐OH has been identified in a vial seized during a stable inspection. The use of peptide‐based Interleukin‐1 receptor antagonists as anti‐inflammatory agents has not been previously reported, making this peptide the first in a new class of sports doping peptides. The peptide has been characterized by high‐resolution mass spectrometry and a detection method developed based on solid‐phase extraction and liquid chromatography ‐ triple quadrupole mass spectrometry. Using in vitro and in vivo models to study the properties of the peptide after administration, the peptide was shown to be highly unstable in plasma and was not detected in urine after administration in a rat. The poor stability of the peptide makes detection challenging but also suggests that it has limited effectiveness as an anti‐inflammatory drug. Copyright © 2015 John Wiley & Sons, Ltd.     

Synthetic protein design: construction of a four‐stranded β‐sheet structure and evaluation of its integrity in methanol–water systems

Abstract: The characterization of a four‐stranded β‐sheet structure in a designed 26‐residue peptide Beta‐4 is described. The sequence of Beta‐4 (Arg‐Gly‐Thr‐Ile‐Lys‐^Dpro‐Gly‐Ile‐Thr‐Phe‐Ala‐^DPro‐Ala‐Thr‐Val‐Leu‐Phe‐Ala‐Val‐^DPro‐Gly‐Lys‐Thr‐Leu‐Tyr‐Arg) was chosen such that three strategically positioned ^DPro‐Xxx segments nucleate type II′β‐turns, which facilitate hairpin extension. A four‐stranded β‐sheet structure is determined in methanol from 500 MHz ¹H NMR data using a total of 100 observed NOEs, 11 dihedral restraints obtained from vicinal J_CαH‐NH values and 10 hydrogen bonding constraints obtained from H/D exchange data. The observed NOEs provide strong evidence for a stable four‐stranded sheet and a nonpolar cluster involving Ile⁸, Phe¹⁰, Val¹⁵ and Phe¹⁷. Circular dichroism studies in water–methanol mixtures provide evidence for melting of the β‐sheet structure at high water concentrations. NMR analysis establishes that the four‐stranded sheet in Beta‐4 is appreciably populated in 50% (v/v) aqueous methanol. In water, the peptide structure is disorganized, although the three β‐turn nuclei appear to be maintained.     

Three‐dimensional structure of the α‐MSH‐derived candidacidal peptide [Ac‐CKPV]2

Abstract: Previous research has shown that the immunomodulatory peptide α‐melanocyte‐stimulating hormone (α‐MSH) and its carboxy‐terminal tripeptide KPV (Lys‐Pro‐Val α‐MSH_11?13) have antimicrobial influences. By inserting a Cys‐Cys linker between two units of KPV, we designed the dimer [Ac‐CKPV]₂ that showed excellent candidacidal effects in pilot tests and was the subject of further investigations. [Ac‐CKPV]₂ was active against azole‐resistant Candida spp. Therefore, the molecule appeared a promising candidate for therapy of fungal infections and was the subject of a structural study. ¹H‐NMR and restrained mechanic and dynamic calculations suggest that the peptide adopts an extended backbone structure with a β‐turn‐like structure. These results open a pathway to development of additional novel compounds that have candidacidal effects potentially useful against clinical infections.     

Genetic variants of flavin-containing monooxygenase 3 (FMO3) derived from Japanese subjects with the trimethylaminuria phenotype and whole-genome sequence data from a large Japanese database

Flavin-containing monooxygenase 3 (FMO3) is a polymorphic xenobiotic- and dietary compound-metabolizing enzyme associated with the genetic disorder trimethylaminuria. We phenotyped 428 Japanese subjects using traditional urinary phenotyping assays and identified two subjects with <20% FMO3 metabolic capacity. Both subjects had novel frameshift mutations. Proband 1 harbored a novel CC deletion resulting in p.[(Pro153Gln fs; Phe166Ter)] FMO3, which was in trans configuration with p.(Cys197Ter). Proband 2 harbored a novel T deletion resulting in p.[(Met211Arg fs; Val220Ter)] FMO3, which was in trans configuration with p.[(Val257Met; Met260Val)]. We also analyzed a new large Japanese database for novel single nucleotide substitutions of FMO3 and identified the following variants with very low frequencies (<∼0.1%): p.(Lys56Glu), p.(Ser112Asn), p.(Asn164Lys), p.(Gly191Cys), p.(Ile199Ser), p.(Pro248Thr), p.(Pro248Leu), p.(Asp286Tyr), and p.(Ala311Pro). Recombinant FMO3 proteins of the above and unanalyzed variants underwent kinetic analysis of their trimethylamine/benzydamine N-oxygenation activities. Gly191Cys, Ile199Ser, Asp286Tyr, and Ala311Pro variant FMO3 proteins exhibited severely decreased activities (V_max/K_m <5% of wild-type). Although these new variants were rare alleles in Japanese self-reported trimethylaminuria sufferers and in the large genomic database, we found that most Japanese individuals compound heterozygous or homozygous for any of these missense FMO3 variants or known severe mutations [e.g., p.(Cys197Ter)] had impaired FMO3-dependent N-oxygenation of malodorous trimethylamine.     

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.     

Solution structures of the inactivation gate particle peptides of rat brain type‐IIA and human heart sodium channels in SDS micelles

Abstract: The Ile‐Phe‐Met (IFM) motif located in the III–IV linker of voltage‐gated sodium channels has been identified as a major component of the fast inactivation gate. If Gln was substituted for Phe, the role in the gate was disrupted completely. If Ile was replaced by Gln inactivation became slightly incomplete and if the Thr, which is adjacent to the IFM motif (‐IFMT‐), was replaced by Met, inactivation became much more incomplete than in the I/Q mutation, but not as vigorous as in the F/Q mutation. Previously, we studied the structures of the inactivation gate‐related peptide (K1480–K1496 in rat brain type‐IIA, MP‐3A) and its F1489/Q substituted one (MP‐4A) in SDS micelles and found that the conformational change of the IFM hydrophobic cluster due to the F/Q substitution may be a reason for disrupting the gate. In this study, in order to obtain supporting evidence for this view and also to further knowledge of the effect of I/Q and T/M mutations on the structure of the IFM cluster, we studied the structures of I1488Q [MP(rb)‐3QFMT] and T1491M [MP(rb)‐3IFMM] substituted peptides. The fragment peptide K1477–K1493 [MP(hh)‐3A] and its T1488M substituted peptide [MP(hh)‐3IFMM] in the human heart sodium channel were also studied. It was found that the backbone structures around the IMF motif of MP‐3A, MP(hh)‐3A and MP(rb)‐3QFMT resemble one another in such a manner that the residues Ile(Gln) and Thr are brought so close together that they form a unique type of lid to occlude the pore. In contrast, the residues between Ile and M1491 of MP(rb)‐3IFMM or M1488 of MP(hh)‐3IFMM were fairly far apart from each other. We conclude that Thr plays an important role in forming a structure of the IFM hydrophobic cluster for inactivation.     

Multiple anchoring of myelopeptides on sequential oligopeptide carriers (SOCn): synthesis,conformation and studies in human leukemia cells

Abstract: Myelopeptides, MP‐6 (Val‐Asp‐Pro‐Pro) and MP‐4 (Phe‐Arg‐Pro‐Arg‐Ile‐Met‐Thr‐Pro), induce metabolic changes in human leukemia cells, HL‐60, characteristic of the differentiation process, which should be regarded as a promising therapeutic approach in cancer and related diseases. With the aim to optimize the differentiation effect of MPs, they were coupled to the Lys‐N^εH₂ groups of a sequential oligopeptide carrier Ac‐(Lys‐Aib‐Gly)₄, SOC₄, and the constructs obtained were studied. The rigid 3₁₀ secondary structure of the carrier is preserved even after linkage of the MPs, which also maintain their initial conformations without interacting either with each other or with the carrier, as demonstrated by ¹H nuclear magnetic resonance (NMR) spectroscopy. It is concluded that the carrier accommodates the presentation of MPs, thus improving their differentiation effect on human leukemia cells.     

Anticancer activity of VmCT1 analogs against MCF‐7 cells

Antimicrobial peptides are considered promising drug candidates due to their broad range of activity. VmCT1 (Phe–Leu–Gly–Ala–Leu–Trp–Asn–Val–Ala–Lys–Ser–Val–Phe–NH₂) is an α‐helical antimicrobial peptide that was obtained from the Vaejovis mexicanus smithi scorpion venom. Some of its analogs showed to be as antimicrobial as the wild type, and they were designed for understanding the influence of physiochemical parameters on antimicrobial and hemolytic activity. Some cationic antimicrobial peptides exhibit anticancer activity so VmCT1 analogs were tested to verify the anticancer activity of this family of peptides. The analogs were synthesized, purified, characterized, and the conformational studies were performed. The anticancer activity was assessed against MCF‐7 mammary cancer cells. The results indicated that [Glu]⁷‐VmCT1‐NH₂, [Lys]³‐VmCT1‐NH₂, and [Lys]⁷‐VmCT1‐NH₂ analogs presented moderated helical tendency (0.23–0.61) and tendency of anticancer activity at 25 μmol/L in 24 hr of experiment; and [Trp]⁹‐VmCT1‐NH₂ analog that presented low helical tendency and moderated anticancer activity at 50 μmol/L. These results demonstrated that single substitutions on VmCT1 led to different physicochemical features and could assist on the understanding of anticancer activity of this peptide family.     

STUDIES ON PEPTIDES LXXXI. Application of a New Arginine Derivative,NG-Mesitylene-2-Sulfonylarginine,to the Synthesis of Substance P and Neurotensin

A new devised arginine derivative, N^G-mesitylene-2-sulfonylarginine, Arg(Mts), was employed for the synthesis of hypothalamic substance P and neurotensin. The former was obtained in 74% yield by treatment of the protected undecapeptide amide, Z - Arg(Mts) - Pro - Lys(Z) - Pro - Gln - Gln - Phe - Phe - Gly - Leu - Met(O)-NH₂, with methanesulfonic acid in the presence of anisole followed by reduction of the sulfoxide with 2-mercaptoethanol. The latter was obtained in 54% yield by the similar treatment of the protected tridecapeptide ester, Z-Pyr-Leu - Tyr - Glu(OBzl) - Asn - Lys(Z)-Pro-Arg(Mts) - Arg(Mts) - Pro - Tyr-Ile-Leu-OBzl, with methanesulfonic acid. As scavenger, a mixture of anisole-thioanisole-o-cresol (1:1:1, by vol.) was employed to suppress the side reaction, O-mesitylene-2-sulfonation of the Tyr residue.     

Amino acid sequence of the tryptic SH-peptide of thermitase,a thermostable serine proteinase from Thermoactinomyces vulgaris. Relation to the subtilisins

The following amino acid sequence of the tryptic SH-peptide of thermitase, a thermostable serine proteinase from Thermoactinomyces vulgaris, was determined: Val - Val - Gly - Gly - Trp - Asp - Phe - Val-Asp-Asn-Asp-Ser-Thr-Pro-Gln-Asn - Gly - Asn - Gly -His64- Gly - Thr - His -Cys68 - Ala-Gly-Ile-Ala-Ala-Ala-Val-Thr-Asn - Asn - Ser - Thr - Gly - Ile - Ala - Gly - Thr - Ala - Pro - Lys. This sequence shows homology with the highly conservative part of the subtilisin sequences around the active site His-64. The single cysteine residue of thermitase is localized near this histidine residue thus replacing valine in position 68 (according to the numbering of the subtilisins). This becomes evident also from the specific labeling of the active site histidine with a radioactive inhibitor (Z-Ala-Ala-Phe-¹⁴CH₂-Cl). The tryptic SH-peptide isolated from the modified enzyme contains all the radioactivity and has the same end group and amino acid composition as the tryptic peptide isolated from the tryptic digest of the unlabeled enzyme and subjected to sequential analysis. From sequence homology as well as from secondary structure predictions it may be concluded that the geometry of the active site of thermitase is very similar to that of the subtilisins with the cysteine residue nearby. The inactivation of thermitase by labeling of the SH-group with mercury compounds may then be due to a sterical hindrance or to a more direct interaction of the mercury atom with the charge relay system of the enzyme.