From Synthetic Fragments of Endogenous Three-Finger Proteins to Potential Drugs.

Elena V. Kryukova,Yuri N. Utkin,Igor E. Kasheverov,Victor I. Tsetlin,Ekaterina N. Spirova,Aleksandra I. Garifulina,Maxim N. Zhmak,Natalia S. Egorova,Dmitry S. Lebedev,Denis S. Kudryavtsev

doi:10.3389/fphar.2019.00748

Abstract

The proteins of the Ly6 family have a three-finger folding as snake venom α-neurotoxins, targeting nicotinic acetylcholine receptors (nAChRs), and some of them, like mammalian secreted Ly6/uPAR protein (SLURP1) and membrane-attached Ly-6/neurotoxin (Lynx1), also interact with distinct nAChR subtypes. We believed that synthetic fragments of these endogenous proteins might open new ways for drug design because nAChRs are well-known targets for developing analgesics and drugs against neurodegenerative diseases. Since interaction with nAChRs was earlier shown for synthetic fragments of the α-neurotoxin central loop II, we synthesized a 15-membered fragment of human Lynx1, its form with two Cys residues added at the N- and C-termini and forming a disulfide, as well as similar forms of human SLURP1, SLURP2, and of Drosophila sleepless protein (SSS). The IC50 values measured in competition with radioiodinated α-bungarotoxin for binding to the membrane-bound Torpedo californica nAChR were 4.9 and 7.4 µM for Lynx1 and SSS fragments, but over 300 µM for SLURP1 or SLURP2 fragments. The affinity of these compounds for the α7 nAChR in the rat pituitary tumor-derived cell line GH4C1 was different: 13.1 and 147 µM for SSS and Lynx1 fragments, respectively. In competition for the ligand-binding domain of the α9 nAChR subunit, SSS and Lynx1 fragments had IC50 values of about 40 µM, which correlates with the value found for the latter with the rat α9α10 nAChR expressed in the Xenopus oocytes. Thus, the activity of these synthetic peptides against muscle-type and α9α10 nAChRs indicates that they may be useful in design of novel myorelaxants and analgesics.

Highlights

According to proteomic and transcriptomic studies, a dominant family in the venoms of Elapidae snakes are proteins containing about 60–80 amino acid residues, 4–5 disulfide bonds, and having a common type of the compact spatial structure built of three β-structural disulfide-confined loops (Kini and Doley, 2010; Utkin, 2013; Tsetlin, 2015), explaining theirAbbreviations: Ly6, lymphocyte antigen 6; Lynx1, Ly-6/neurotoxin; SLURP, secreted Ly6/uPAR protein; SSS, Drosophila sleepless protein; nAChR, nicotinic acetylcholine receptors; AChBP, acetylcholine-binding protein; ligand-binding domain (LBD), ligand binding domain; α‐Bgtx, α‐bungarotoxin.Synthetic Fragments of Ly6 proteins common name of “three-finger proteins” (TFPs)
They are basic to the respective fragment of a short neurotoxin II [which like Ly6 proteins does not have an additional disulfide in the central loop (Grishin et al, 1973)] and are much closer to pI 10.46 for a weak toxin Naja kaouthia respective fragment [which contains no additional disulfide in the central loop, but to Ly6 proteins possesses additional disulfide in the N-terminal loop (Utkin et al, 2001)]
Since high-affinity binding of radioiodinated α-bungarotoxin to muscle-type, α7 and α9α10 nAChRs occurs at their orthosteric sites, competition with this radioligand allows to detect binding to such sites for the compounds of interest

Summary

Introduction

According to proteomic and transcriptomic studies, a dominant family in the venoms of Elapidae snakes (cobras, kraits, coral snakes, and some others) are proteins containing about 60–80 amino acid residues, 4–5 disulfide bonds, and having a common type of the compact spatial structure built of three β-structural disulfide-confined loops (Kini and Doley, 2010; Utkin, 2013; Tsetlin, 2015), explaining theirSynthetic Fragments of Ly6 proteins common name of “three-finger proteins” (TFPs). The targets of the snake venom TFPs are not limited by receptors and ion channels: for example, fasciculins from the Dendroaspis angusticeps mamba venom inhibit acetylcholinesterase (Karlsson et al, 1984), while cytotoxins, known as cardiotoxins, do not have a selective target and penetrate the cell membrane (Dubovskii et al, 2013). It is a long history of using venoms as such for medical purposes (Chan et al, 2016; Estevão-Costa et al, 2018). As α-cobratoxin from the cobra venom and batroxobin from jararaca venom, are tested as possible remedies against pain (Gong et al, 2015) and as defibrinogenating agents (Ding et al, 2018), respectively

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Conclusion