The pain regulation of endokinin A/B or endokinin C/D on chimeric peptide MCRT in mice

The present study focused on the interactive pain regulation of endokinin A/B (EKA/B, the common C-terminal decapeptide in EKA and EKB) or endokinin C/D (EKC/D, the common C-terminal duodecapeptide in EKC and EKD) on chimeric peptide MCRT (YPFPFRTic-NH2, based on YPFP-NH2 and PFRTic-NH2) at supraspinal level in mice. Results demonstrated that the co-injection of nanomolar EKA/B and MCRT showed moderate regulation, while 30 pmol EKA/B had no effect on MCRT. The combination of EKC/D and MCRT produced enhancive antinociception, which was nearly equal to the sum of mathematical value of single EKC/D and MCRT. Mechanism studies revealed that pre-injected naloxone attenuated the combination significantly with the equivalent analgesic effects of EKC/D alone, suggesting that EKC/D and MCRT might act on two totally independent pathways. Moreover, based on the above results and previous reports, we made two reasonable hypotheses to explain the cocktail-induced analgesia, which potentially pave the way to explore the respective regulatory mechanisms of EKA/B, EKC/D and MCRT and better understand the complicated pain regulation of NK1 and Îź opioid receptors, as follows: (1) MCRT and endomorphin-1 possibly activated different Îź subtypes; (2) Picomolar EKA/B might motivate the endogenous NPFF system after NK1 activation.The accepted manuscript in pdf format is listed with the files at the bottom of this page. The presentation of the authors' names and (or) special characters in the title of the manuscript may differ slightly between what is listed on this page and what is listed in the pdf file of the accepted manuscript; that in the pdf file of the accepted manuscript is what was submitted by the author

Tetrazine-Containing Amino Acid for Peptide Modification and Live Cell Labeling

<div><p>A novel amino acid derivative 3-(4-(1, 2, 4, 5-tetrazine-3-yl) phenyl)-2-aminopropanoic acid was synthesized in this study. The compound possessed better water-solubility and was synthesized more easily compared with the well-known and commercially available 3-(p-benzylamino)-1, 2, 4, 5-tetrazine. Tetrazine-containing amino acid showed excellent stability in biological media and might be used for cancer cell labeling. Moreover, the compound remained relatively stable in 50% TFA/DCM with little decomposition after prolonged exposure at room temperature. The compound could be utilized as phenylalanine or tyrosine analogue in peptide modification, and the tetrazine-containing peptide demonstrated more significant biological activity than that of the parent peptide. The combination of tetrazine group and amino acid offered broad development prospects of the bioorthogonal labeling and peptide synthesis.</p></div

The compound 2 reacted with norbornene to label cancer cells.

<p>Cancer cells A549, which overexpressed EGFR, were exposed to the Cetuximab antibodies modified with norbornene and 5-carboxyfluorescein (green). In the next step, the pretargeted cells were labeled with a tetrazine bearing a fluorophore such as VT680 (red).</p

Fluorescent microscope images of A549 lung cancer cells after pretargeting Cetuximab antibodies modified with 5-carboxyfluorescein and norbornene and subsequent labeling with tetrazine-VT680.

<p>(A) Rhodamine channel. (B) Near-IR channel (tetrazine-VT680). Images of control experiments were also taken in near infrared channel. (C) Control experiment with unlabeled cetuximab and tetrazine-VT680. (D) Control experiment with norbornene-cetuximab and unlabeled VT680.</p

Peptides 7 and 8 (both with 10 μM) for small intestine contraction with acetylcholine (10 μM) as positive control.

<p>Peptides 7 and 8 (both with 10 μM) for small intestine contraction with acetylcholine (10 μM) as positive control.</p

Synthetic route of tetrazine-containing amino acid (S)-3-(4-(1, 2, 4, 5-tetrazin-3-yl) phenyl)-2-aminopropanoic acid 2 and protected tetrazine-containing amino acid 5 and 6, and the IED-DA reaction of 2 with 5-norbornene-2-carboxylic acid 3 give 4.

<p>a) (i) Formamidine acetate (4 eq), N₂H₄ (40 eq), S (1 eq), r.t, 22 h; (ii) CH₃COOH, NaNO₂ (5 eq), 0°C, 30 min, 64.9%. b) Phosphate buffered solution (PBS), r.t, 30 min. c) Fmoc-OSu (1.5 eq), 1: 1 Dioxane/H₂O (V/V), NaHCO₃ (2.5 eq), 0°C, overnight, 64.5%. d) Boc₂O (2 eq), 1: 1 Dioxane/H₂O (V/V), NaHCO₃ (2.5 eq), 0°C, 8h, 72.3%.</p