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Chemical synthesis of a polypeptide backbone derived from the primary sequence of the cancer protein NY-ESO-1 enabled by kinetically controlled ligation and pseudoprolines.
Harris PW, Brimble MA., Biopolymers. 2015 Feb 5. doi: 10.1002/bip.22621. [Epub ahead of print]

The cancer protein NY-ESO-1 has been shown to be one of the most promising vaccine candidates although little is known about its cellular function. Using a chemical protein strategy, the 180 amino acid polypeptide, tagged with an arginine solubilising tail, was assembled in a convergent manner from four unprotected peptide α- thioester peptide building blocks and one cysteinyl polypeptide, which were in turn prepared by Boc and Fmoc SPPS respectively. To facilitate the assembly by ligation chemistries, non-native cysteines were introduced as chemical handles into the polypeptide fragments; pseudoproline dipeptides and microwave assisted Fmoc SPPS were crucial techniques to prepare the challenging hydrophobic Cterminal fragment. Three sequential kinetically controlled ligations, which exploited the reactivity between peptide arylthioesters and peptide alkylthioesters, were then used in order to assemble the more tractable N-terminal region of NY-ESO-1. The ensuing 147 residue polypeptide thioester then underwent successful final native chemical ligation with the very hydrophobic C-terminal polypeptide bearing an N-terminal cysteine affording the 186 residue polypeptide as an advanced intermediate en route to the native NY-ESO-1 protein. This article is protected by copyright. All rights reserved. © 2015 Wiley Periodicals, Inc.

Oxytocin prevents ethanol actions at {delta} subunit-containing GABAA receptors and attenuates ethanol-induced motor impairment in rats.Bowen MT, Peters ST, Absalom N, Chebib M, Neumann ID, McGregor IS., PNAS, 2015 Feb 23. doi:10.1073/pnas.1416900112 [Epub ahead of print]

Even moderate doses of alcohol cause considerable impairment of motor coordination, an effect that substantially involves potentiation of GABAergic activity at δ subunit-containing GABAA receptors (δ-GABAARs). Here, we demonstrate that oxytocin selectively attenuates ethanol-induced motor impairment and ethanol-induced increases in GABAergic activity at δ-GABAARs and that this effect does not involve the oxytocin receptor. Specifically, oxytocin (1 μg i.c.v.) given before ethanol (1.5 g/kg i.p.) attenuated the sedation and ataxia induced by ethanol in the open-field locomotor test, wire-hanging test, and righting-reflex test in male rats. Using two-electrode voltage-clamp electrophysiology in Xenopus oocytes, oxytocin was found to completely block ethanol-enhanced activity at α4β1d and α4β3d recombinant GABAARs. Conversely, ethanol had no effect when applied to α4β1 or α4β3 cells, demonstrating the critical presence of the d subunit in this effect. Oxytocin had no effect on the motor impairment or in vitro effects induced by the δ-selective GABAAR agonist 4,5,6,7-tetrahydroisoxazolo(5,4- c)pyridin-3-ol, which binds at a different site on δ-GABAARs than ethanol. Vasopressin, which is a nonapeptide with substantial structural similarity to oxytocin, did not alter ethanol effects at δ-GABAARs. This pattern of results confirms the specificity of the interaction between oxytocin and ethanol at δ- GABAARs. Finally, our in vitro constructs did not express any oxytocin receptors, meaning that the observed interactions occur directly at δ-GABAARs. The profound and direct interaction observed between oxytocin and ethanol at the behavioral and cellular level may have relevance for the development of novel therapeutics for alcohol intoxication and dependence.

Engineering Potent and Selective Analogs of GpTx-1, a Tarantula Venom Peptide Antagonist of the NaV1.7 Sodium Channel Murray JK, Ligutti J, Liu D, Zou A, Poppe L, Li H, Andrews KL, Moyer BD, McDonough SI, Favreau P, Stöcklin R, Miranda LP., J. Med. Chem., Just Accepted Manuscript DOI: 10.1021/jm501765v Publication Date (Web): February 6, 2015 Copyright © 2015 American Chemical Society

NaV1.7 is a voltage-gated sodium ion channel implicated by human genetic evidence as a therapeutic target for the treatment of pain. Screening fractionated venom from the tarantula Grammostola porteri led to the identification of a 34- residue peptide, termed GpTx-1, with potent activity on NaV1.7 (IC50 = 10 nM) and promising selectivity against key NaV subtypes (20x and 1000x over NaV1.4 and NaV1.5, respectively). NMR structural analysis of the chemically synthesized three disulfide peptide was consistent with an inhibitory cystine knot motif. Alanine scanning of GpTx-1 revealed that residues Trp29, Lys31, and Phe34 near the C-terminus are critical for potent NaV1.7 antagonist activity. Substitution of Ala for Phe at position 5 conferred three hundred-fold selectivity against NaV1.4. A structure-guided campaign afforded additive improvements in potency and NaV subtype selectivity, culminating in the design of [Ala5,Phe6,Leu26,Arg28]GpTx-1 with a NaV1.7 IC50 value of 1.6 nM and >1000x selectivity against NaV1.4 and NaV1.5.

Glucagon-like peptide-1 protects the murine hippocampus against stressors via Akt and ERK1/2 signaling. Yoshino Y, Ishisaka M, Tsujii S, Shimazawa M, Hara H., Biochem Biophys Res. Commun. ,2015 Feb 7. doi: 10.1016/j.bbrc.2015.01.098. [Epub ahead of print]

Alzheimer's disease (AD) is a common neurodegenerative disease characterized by cognitive dysfunction and neuronal cell death in the hippocampus and cerebral cortex. Glucagonlike peptide-1 (GLP-1) is an insulinotropic peptides. GLP-1- associated medicines are widely used as treatments for type 2 diabetes. In addition, they have been shown to ameliorate pathology in AD mouse models. Here, we investigated the effects of GLP-1 on different stressors in murine hippocampal HT22 cells. GLP-1 (7-36)prevented H2O2-, l-glutamate-, tunicamycin-, thapsigargin-, and amyloid β1-42-induced neuronal cell death in a concentration-dependent manner. GLP-1 (7-36) treatment for 1 h significantly increased phosphorylated Akt and extracellular signal-regulated kinase 1 and 2 (ERK1/2) when compared with vehicle-treatment. These results suggest that GLP-1 (7-36) is protective against these stressors via activation of survival signaling molecules, such as Akt and ERK1/2 in HT22 cells. In conclusion, GLP-1 and activators of the GLP-1 receptor might be useful targets for the treatment of AD. Copyright © 2015 Elsevier Inc. All rights reserved.

Glucagon-Like Peptide-1 (7-36) [GLP-1 (7-36)] has shown potential in the treatment of type II diabetes. For example, it restores myocardial insulin sensitivity and prevents progressive heart failure.

(Chen M, et al. Cardiovasc. Diabetol., 2014, 13, 115)

GLP-(7-36) also has protective effects in the brain. It has protective effects on brain ischemia/reperfusion damage in diabetic rats.

(Zhao L, et al., Brain Res., 2015 Jan. 16. doi: 10.1016/j.brainres.2015.01.014 [Epub. ahead of print]) and protects synaptic and learning functions from neuroinflammation in rodents (Iwai T, et al., J. Neurosci. Res., 2014, 92, 946-54).

Administered intrathecally, GLP-1(7-36) suppresses pain hypersensitivity.

(Gong N, et al, J. Neurosci, 2014, 34, 5322-34)

AAPPTec offers glucagon-like peptide-1 (7-36) for research applications.

4-Cyanophenylalanine residues may be utilized as non-invasive fluorescent and IR probes. 4-Cyanophenylalanine residues have been used as probes in 2D IR spectroscopy (Urbanek DC, et al., J. Phys. Chem. Lett., 2010, 1, 3311-5; Chung JK, et al., J. Am. Chem. Soc. , 2012, 134, 12118-24). The 4-cyanophenylalanine is easily substituted for a phenylalanine residue in synthetic peptides. Methods of genetically encoding 4-cyanophenylalanine have also been developed ( Schultz KC, et al., J. Am. Chem. Soc., 2006, 128, 13984-5). EJ Petersson and coworkers have utilized 4- cyanophenylalanine with backbone thioamides as FRET pairs (Goldberg JM, et al., J. Am. Chem. Soc., 2010, 132, 14718-20; Wissner, EF, et al., J. Am. Chem. Soc., 2013, 135, 6529-40).

AAPPTec offers Boc-protected, Fmoc-protected, or unprotected L-4-cyanophenylalanine for use in structural studies. These products are available in convenient catalog quantities or you may request a bulk quotation by email to sales@aapptec.com.

Disomers are also available from AAPPTec.

AAPPTec provides high quality custom peptides quickly at very competitive prices. Our peptide chemists have prepared peptides with up to 80 to 100 residues in high purity. They have decades of experience preparing and purifying all types of peptides, including hydrophobic peptides, cyclized peptides and peptides conjugated to biotin, fatty acids or dyes. Most modifications, such as phosphorylation, unusual amino acids, side chain labels and fluorescent tags, can be incorporated.

To request a quotation for custom peptides, email your sequences, the quantity of each peptide and the purity you require to info@aapptec.com or use our convenient online quotation request form at www.aapptec.com.

Pseudoproline dipeptides have many uses in peptide synthesis. They are often utilized in the synthesis of difficult, hydrophobic sequences. The pseudoproline structure imposes a bend in the peptide backbone that disrupts hydrogen bonding patterns of the peptide and reduces the tendency of the peptide chain to aggregate.

The ability of the pseudoproline to promote a bend in the peptide structure facilitates ring formation in peptides. The pseudoproline can be incorporated as a temporary turn-inducing element to promote the cyclization of a peptide. Final deprotection of the peptide converts the pseudoproline back to a native serine or threonine residue.

Since pseudoprolines can be coupled with little to no racemization, pseudoprolines are very useful as the Cterminal residue in peptide fragments that will be coupled in solution phase reactions.

AAPPTec has the largest selection of pseudoproline dipeptides at very competitive prices. To view AAPPTec’s prices and complete list of pseudoproline dipeptides,
please go to our web site at http://www.aapptec.com/-c-
57.html.

Oxytocin acts both as a hormone and a neurotransmitter in the brain. The primary effects of oxytocin are uterine contraction and facilitating milk production in lactating mothers. It is used to induce labor and toaid milk production in breast-feeding mothers. Due to its structural similarity to vasopressin, oxytocin can reduce the production of urine slightly and in some species it can stimulate sodium excretion by the kidneys. Under certain circumstances, it can inhibit the release of adrenocorticotropic hormone and cortisol indirectly.

In the brain, oxytocin is associated with sexual arousal and pair bonding with her sexual mate in females. Oxytocin crosses the placenta and enters the brain of the fetus where it induces a switch in the effect of GABA from excitatory to inhibitory. This silences the fetal brain and reduces its vulnerability to hypoxic damage during birth. After birth, oxytocin appears to be responsible for maternal behavior. Oxytocin is also plays a role in the social behaviors of many species; in humans it appears to increase trust, reduce fear and increase empathy. Autistic child have been found to have significantly lower levels of oxytocin in their blood. Administered intravenously, oxytocin decreases repetitive behaviors and helps autistic adults to evaluate the emotional significance of speech intonation.

Oxytocin for research applications is available from AAPPTec.

The Apex 396 HT is unsurpassed for highthroughput synthesis of peptide libraries. It synthesizes up to 192 different sequences at the same time using standard 1 mL well titer plates. The Apex 396 HT is ideal for SAR studies. It can prepare complete alanine scan libraries, deletion libraries and truncation libraries to identify critical segments and residues. The library peptides are cleaved into standard 96 well titer plates.

Xhindoli D, Morgera F, Zinth U, Rizzo R, Pacor S, Tossi A., Biochem. J., 2015, 465, 443-57. doi: 10.1042/BJ20141016

The human cathelicidin peptide LL-37 is an important effector of our innate immune system and contributes to host defense with direct antimicrobialactivity and immunomodulatory properties, and by stimulating wound healing. Its sequence has evolved to confer specific structural characteristics that strongly affect these biological activities, and differentiate it from orthologues of other primate species. In the present paper we report a detailed study of the folding and self-assembly of this peptide in comparison with rhesus monkey peptide RL-37, taking into account the different stages of its trajectory from bulk solution to contact with, and insertion into, biological membranes. Phenylalanineresidues in different positions throughout the native sequences of LL-37 and RL-37 were systematically replaced with the non-invasive fluorescent and IR probe p-cyanophenylalanine. Steady-state and time-resolved fluorescence studies showed that LL- 37, in contrast to RL-37, forms oligomers with a loose hydrophobic core in physiological solutions, which persist in the presence of biological membranes. Fourier transform IR and surface plasmon resonance studies also indicated different modes of interaction for LL-37 and RL- 37 with anionic and neutral membranes. This correlated with a distinctly different mode of bacterial membrane permeabilization, as determined using a flow cytometric method involving impermeant fluorescent dyes linked to polymers of defined sizes.