ABSTRACT
italic>α-Conotoxin ArIB[V11L,V16D] is currently the most optimal selective inhibitor of α7 nicotinic acetylcholine receptor (nAChR) known. In order to explore chemical modification methods and enrich its application in targeting nAChR, this study utilized the linker to covalently connect camptothecin and 7-amino-4-methylcoumarin to the [2,4] disulfide bond of ArIB[V11L,V16D]. Therefore, two peptide-drug conjugates (PDCs), ArIB[V11L,V16D]-5 and ArIB[V11L,V16D]-6, and one fluorescent-labeled peptide, ArIB[V11L,V16D]-7 were constructed. Cytotoxicity evaluation showed that the IC50 values against non-small cell lung cancer cell line A549 of the two PDCs were respectively 1.3 and 4.1 times of camptothecin, indicating slight reduction in activity at the cellular level which was related to the linker structure. Fluorescence spectrum scanning revealed that the excitation and emission wavelength of the fluorescent-labeled peptide were 340 nm and 403 nm respectively, and the fluorescence features of 7-amino-4-methylcoumarin as a marker were retained without fluorescence quenching. This modification strategy laid a solid foundation for the further application of α-conotoxin ArIB[V11L,V16D] in PDCs and fluorescent probes.
ABSTRACT
The α-conotoxins are peptide toxins that are identified from the venom of marine cone snails and they hold outstanding potency on various subtypes of nicotinic acetylcholine receptors (nAChRs). nAChRs have an important role in regulating transmitter release, cell excitability, and neuronal integration, so nAChR dysfunctions have been involved in a variety of severe pathologies. Four types of α-3/5 conotoxins MI, MIA, MIB and MIC have been found from Conus magus. Among them, the activity and selectivity of MIA and MIB have not been well studied. In this study, four α-3/5 conotoxins MI, MIA, MIB and MIC were synthesized by solid peptide synthesis method, and the bioactivities of them were screened by double electrode voltage clamp electrophysiology. The results showed that MIA and MIB selectively inhibited muscle type acetylcholine receptors with IC50 values of 14.45 and 72.78 nmol·L-1, respectively, which are slightly weaker than MI and MIC. Molecular docking results have shown MIA and MIB interact with muscle-type nAChRs with similar mechanism. The reasons for activity differences may relate to the size of the N-terminal amino acids. Together, the conotoxins MIA and MIB may have the potential to develop as a tool for detect the function of muscle type nAChRs, as well as the diagnosis or treat of related diseases.
ABSTRACT
italic>α7 nicotinic acetylcholine receptor (nAChR) is widely distributed in the central and peripheral nervous systems, and is closely related to a variety of neurological diseases and inflammation response. α-Conotoxin [A10L]PnIA, as an antagonist targeting α7 nAChR, plays an important role in studying the physiological and pathological processes involved in α7 nAChR. [A10L]PnIA was labeled with fluorescein 5-carboxytetramethylrhodamine, and the active peptide ([A10L]PnIA-F) was obtained by a two-step oxidative folding procedure in vitro. The Xenopus oocyte expression system and the two-electrode voltage clamp technique were used to identify the potency of [A10L]PnIA-F fluorescent peptide, and its cytotoxicity was detected by mouse macrophages and CCK8 method. The molecular weight of [A10L]PnIA-F fluorescent peptide was identified by mass spectrometry as 2 077.28 Da, which was consistent with the theoretical value. Electrophysiological determination of its half-maximal inhibitory concentration (IC50) for α7 nAChR is 17.32 nmol·L-1, which is consistent with [A10L]PnIA (IC50, 13.84 nmol·L-1). The cytotoxicity test results showed that within the concentration range of 5 nmol·L-1 to 10 μmol·L-1, there was no significant inhibition on the growth of mouse macrophages. The results showed that the α-conotoxin fluorescent probe [A10L]PnIA could provide pharmacological tools for the research of α7 nAChR-related neurophysiological and pathological mechanisms.
ABSTRACT
The cyanuric chloride linkers have been used for cyclizing polypeptide, but not used for α-conotoxin, the peptides with rich disulfide bonds and more amino acid residues. In this study, cyclic peptides c[A10L]PnIA-1-4 were synthesized efficiently by lysine assisted cyanuric chloride linkers with 28.92%-52.00% yields. The activity evaluation showed that the IC50 values of c[A10L]PnIA-1 against α7 and α3β2 nAChR subtypes were 5 and 7 times higher than [A10L]PnIA respectively, and the subtype selectivity was maintained. The results of circular dichroism show that this cyclization method had no significant effect on its secondary structure. Compared with the commonly used head-to-tail cyclization in conotoxin cyclization, this method has the advantages of rapid reaction and high yield, which is expected to be further applied to the cyclization study of various α-conotoxins.
ABSTRACT
GeXIVA[1,2] is a new type of conotoxin recently discovered in the transcriptome of Conus generalis and it is expected to be used clinically as a new type of analgesic. This study established and verified a sandwich enzyme-linked immunosorbent assay method for the marine drug GeXIVA[1,2] in the plasma of rats and Beagle dogs. The mouse monoclonal antibody 4B2 and biotin-labeled rabbit polyclonal antibody 2# were developed. The checkerboard method was used to optimize the antibody pairing concentration, minimum dilution ratio, incubation temperature, and incubation time to establish an antibody sandwich ELISA detection method. Verify the established testing methods. The established ELISA method has a quantitative range of 1.25-80 ng·mL-1 in rat and Beagle plasma. The precision, accuracy, selectivity, specificity, stability, dilution linearity, and hook effect all meet the requirements for biological sample analysis. All the procedures for the animal experiments were approved by the Animal Ethics Committee of the Institute (Permit Number: IACUC-DWZX-2020-698). This method can support the preclinical pharmacokinetic study of the marine drug GeXIVA.