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Conotoxin GIIIA in the Inhibition of Nav1.4

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Conotoxin GIIIA in the Inhibition of Nav1.4 marine drugs Article The Role of Individual Disulfide Bonds of µ-Conotoxin GIIIA in the Inhibition of NaV1.4 Penggang Han 1,2, Kang Wang 2, Xiandong Dai 2, Ying Cao 2, Shangyi Liu 2, Hui Jiang 2, Chongxu Fan 2,*, Wenjian Wu 1 and Jisheng Chen 1,2 1 College of Science, National University of Defense Technology, Changsha 410073, Hunan, China; [email protected] (P.H.); [email protected] (W.W.); [email protected] (J.C.) 2 Beijing Institute of Pharmaceutical Chemistry, Beijing 102205, China; [email protected] (K.W.); [email protected] (X.D.); [email protected] (Y.C.); [email protected] (S.L.); [email protected] (H.J.) * Correspondence: [email protected] Academic Editor: Peer B. Jacobson Received: 28 September 2016; Accepted: 11 November 2016; Published: 18 November 2016 Abstract: µ-Conotoxin GIIIA, a peptide toxin isolated from Conus geographus, preferentially blocks the skeletal muscle sodium channel NaV1.4. GIIIA folds compactly to a pyramidal structure stabilized by three disulfide bonds. To assess the contributions of individual disulfide bonds of GIIIA to the blockade of NaV1.4, seven disulfide-deficient analogues were prepared and characterized, each with one, two, or three pairs of disulfide-bonded Cys residues replaced with Ala. The inhibitory potency of the analogues against NaV1.4 was assayed by whole cell patch-clamp on rNaV1.4, heterologously expressed in HEK293 cells. The corresponding IC50 values were 0.069 ± 0.005 µM for GIIIA, 2.1 ± 0.3 µM for GIIIA-1, 3.3 ± 0.2 µM for GIIIA-2, and 15.8 ± 0.8 µM for GIIIA-3 (-1, -2 and -3 represent the removal of disulfide bridges Cys3–Cys15, Cys4–Cys20 and Cys10–Cys21, respectively). Other analogues were not active enough for IC50 measurement. Our results indicate that all three disulfide bonds of GIIIA are required to produce effective inhibition of NaV1.4, and the removal of any one significantly lowers its sodium channel binding affinity. Cys10–Cys21 is the most important for the NaV1.4 potency. Keywords: µ-conotoxin; GIIIA; disulfide bond; NaV1.4; electrophysiology 1. Introduction Conotoxins are small, cysteine-rich bioactive peptides derived from the venom of tropical marine cone snails, genus Conus. Conotoxins have a wide range of pharmacological targets, including different isoforms of ion channels and membrane receptors [1]. Thus, conotoxins are useful research tools and promising drug leads for diseases related to these targets. Ziconotide, !-conotoxin MVIIA, has been approved by the US Food and Drug administration as a local analgesic for severe chronic pain treatment [2], and several other pain relief drugs based on conotoxins are under preclinical or clinical testing [3,4]. µ-Conotoxins belong to the M superfamily of conopeptides and comprise 16 to 26 residues with six Cys residues arranged in a class III framework (–C1C2–C3–C4–C5C6–) [5]. µ-Conotoxins are the only venom peptides that selectively inhibit voltage-gated sodium channels by sterically and electrostatically blocking the ion flux via binding to the outer vestibule of the channel [6]. The µ-conotoxin GIIIA isolated from Conus geographus was among the first to be characterized. GIIIA consists of 22 amino acids (see Figure1) packed in a pyramidal structure, with three disulfide bonds and three hydroxyproline residues [7]. GIIIA preferentially blocks skeletal muscle sodium channel subtype NaV1.4 by a rather complex toxin–pore interaction, with several residues in the toxin contributing to the high-affinity binding [8]. Mar. Drugs 2016, 14, 213; doi:10.3390/md14110213 www.mdpi.com/journal/marinedrugs Mar. Drugs 2016, 14, 213 2 of 9 Mar.subtype Drugs 2016 Na, V141.4, 213 by a rather complex toxin–pore interaction, with several residues in the 2toxin of 9 contributing to the high‐affinity binding [8]. FigureFigure 1. 1.Amino Amino acid acid sequence sequence andand disulfidedisulfide bonds of of μ‐µ-conotoxinconotoxin GIIIA. GIIIA. *: *: amidated amidated CC‐terminus;-terminus; O: O:trans trans-4-hydroxyproline.‐4‐hydroxyproline. The most challenging step in the chemical synthesis of disulfide‐rich peptides, such as μ‐ The most challenging step in the chemical synthesis of disulfide-rich peptides, such as µ-conotoxin, conotoxin, is oxidative folding, which is often time‐consuming, low in efficiency and poor in yield is oxidative folding, which is often time-consuming, low in efficiency and poor in yield [9]. [9]. The disulfide bonds in the peptide sequences give rise to a well‐defined and constrained The disulfide bonds in the peptide sequences give rise to a well-defined and constrained framework framework and generally play an important role in their biological action [10]. However, growing and generally play an important role in their biological action [10]. However, growing evidence evidence suggests that many disulfide‐rich peptides do not need all their native disulfide bonds to suggests that many disulfide-rich peptides do not need all their native disulfide bonds to retain retain biological activity [11–13]. For example, the removal of the Cys1–Cys16 bridge in ω‐conotoxin biological activity [11–13]. For example, the removal of the Cys1–Cys16 bridge in !-conotoxin MVIIA MVIIA did not abolish its calcium channel binding affinity [14]. The deletion of a disulfide bond can did not abolish its calcium channel binding affinity [14]. The deletion of a disulfide bond can no doubt no doubt accelerate the chemical synthesis of the small neuropeptide and benefit the structure‐ accelerate the chemical synthesis of the small neuropeptide and benefit the structure-function studies. function studies. As a valuable probe for sodium channel research, GIIIA has attracted considerable attention. As a valuable probe for sodium channel research, GIIIA has attracted considerable attention. Previous studies have mainly focused on the key residues in the toxin-channel interaction, but the role Previous studies have mainly focused on the key residues in the toxin‐channel interaction, but the of individual disulfide bonds in the inhibition of NaV1.4 remains ambiguous. In this study, we assessed role of individual disulfide bonds in the inhibition of NaV1.4 remains ambiguous. In this study, we the contributions of the three individual disulfide bonds to the inhibitory activity against Na 1.4. GIIIA assessed the contributions of the three individual disulfide bonds to the inhibitory activityV against and seven analogues, each with one, two, or three pairs of disulfide-bonded Cys residues replaced NaV1.4. GIIIA and seven analogues, each with one, two, or three pairs of disulfide‐bonded Cys with Ala, were prepared, and their blocking potency was assayed using the whole cell patch-clamp residues replaced with Ala, were prepared, and their blocking potency was assayed using the whole technique on HEK293 cells transiently expressing sodium channel subtype rNaV1.4. cell patch‐clamp technique on HEK293 cells transiently expressing sodium channel subtype rNaV1.4. 2. Materials and Methods 2. Materials and Methods 2.1. Materials and Apparatus 2.1. Materials and Apparatus Rink amide resins, Fmoc-protected amino acids, and other reagents for peptide synthesis were purchasedRink from amide GL resins, Biochem, Fmoc Shanghai,‐protected China. amino HPLC-grade acids, and other ACN reagents was obtained for peptide from Sigma-Aldrich, synthesis were St.purchased Louis, MO, from USA. GL LipofectamineBiochem, Shanghai, 2000 China. was purchased HPLC‐grade from ACN Life Technologies,was obtained from Chagrin Sigma Falls,‐Aldrich, OH, USA.St. Louis, All reagents MO, USA. used Lipofectamine were analytical 2000 grade. was Thepurchased HEK293 from cell strainLife Technologies, was offered byChagrin the Cell Falls, Center OH, ofUSA. Chinese All reagents Academy used of Sciences, were analytical Shanghai, grade. China. The Standard HEK293 µcell-conotoxin strain was GIIIA offered was by purchased the Cell Center from Bachem,of Chinese Bubendorf, Academy Switzerland. of Sciences, Shanghai, China. Standard μ‐conotoxin GIIIA was purchased from Bachem,Solid-phase Bubendorf, peptide Switzerland. synthesis was performed on a CEM Liberty peptide synthesizer (CEM, Matthews,Solid‐phase NC, USA). peptide MALDI-TOF-MS synthesis was performed was measured on a onCEM a BrukerLiberty ultraflexpeptide synthesizer TOF/TOF mass(CEM, spectrometerMatthews, NC, (Bruker USA). Daltonics, MALDI Billerica,‐TOF‐MS MA, was USA) measured with α on-cyano-4-hydroxycinnamic a Bruker ultraflex TOF/TOF acid as mass the matrix.spectrometer Reversed-phase (Bruker Daltonics, HPLC was Billerica, performed MA, on USA) an Agilent with α‐ 1100cyano system‐4‐hydroxycinnamic with a dual wavelength acid as UV the detectormatrix. (Agilent,Reversed Santa‐phase Clara, HPLC CA, was USA). performed C18 Vydac on an columns Agilent 1100 were system purchased with from a dual Grace, wavelength Deerfield, UV IL,detector USA. Whole (Agilent, cell Santa patch-clamp Clara, CA, recordings USA). C18 were Vydac conducted columns on were an Axon700Bpurchased amplifierfrom Grace, (Molecular Deerfield, Devices,IL, USA. Sunnyvale, Whole cell CA, patch USA).‐clamp The recordings MF-900 Microforge were conducted and Shutter on an P-97 Axon700B Micropipette amplifier puller (Molecular were the productsDevices, of Sunnyvale, Narishige Group,CA, USA). Tokyo, The Japan MF‐900 and Microforge Sutter Instrument, and Shutter Novato, P‐97 CA, Micropipette USA, respectively. puller were the products of Narishige Group, Tokyo, Japan and Sutter Instrument, Novato, CA, USA, respectively. 2.2. Chemical Synthesis of Peptides 2.2. Chemical Synthesis of Peptides Peptides were synthesized on Rink amide resin using Fmoc chemistry and standard side chain protectionPeptides except were on the synthesized cysteine residues. on Rink Toamide prepare resin GIIIA using analogues Fmoc chemistry containing and two standard disulfide side bonds, chain theprotection peptides except were synthesized on the cysteine using residues. a selective To Cys prepare protection GIIIA scheme. analogues One containing pair of connected two disulfide Cys residuesbonds, the was peptides protected were by tritylsynthesized groups, using and thea selective other pair Cys was protection protected scheme. by Acm One groups.
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