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

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.

Keywords: Ly6 family; nAChRs; nicotinic acetylcholine receptors; peptide fragments; three-finger proteins.

Figure 1

Loop II fragment selection and design. (A) Loop II amino acid sequences alignment of Ly6 proteins. The respective synthetic peptide fragments of loop II prepared and studied in present work are labeled with numbers from 1 to 7 (see the Results section) and highlighted with the appropriate color. Cys residues added for the stabilization purposes are highlighted grey. (B) Design of artificial disulfide-stabilized loop II mimetics. To achieve hairpin conformation close to native, water-soluble Lynx1 structure (PDB 2L03) has been analyzed, Met28 and Ser44 (in case of peptide 1) or Thr29 and Lys43 (in case of peptide 3) residues were chosen because their radicals are situated on the same side of the beta-sheet. Selected residues were interchanged to Cys residues in loop II mimetic peptides. The same logic was applied to SLURP1 and SLURP2 structures. SSS loop II fragment was designed based on the sequence alignment.

Figure 2

Competition of [¹²⁵I]-labeled αBgt with Lynx1 peptides and SSS fragment for binding to (A) Torpedo californica nAChR, (B) human α7 nAChR expressed in the GH4C1 cell line, and (C) ligand-binding domain (LBD) of the human nAChR α9 subunit. IC₅₀ values and 95% confidence interval (CI 95%) for these data are presented in Table 2 . Each data point represents the mean ± SEM of three to four independent experiments.

Figure 3

Activity of synthetic Ly6 peptides on human and rat α9α10 nAChRs expressed in X. laevis oocytes. (A) Inhibition of ion currents induced by 30 μM acetylcholine by Lynx1 (1) fragment in the rat (▪, solid line) α9α10 nAChRs or by Lynx1 linear fragment (2) in the human (○, dashed line) α9α10 nAChRs. The oocytes were pre-incubated with peptides for 5 min. For Lynx1 (1) the calculated IC₅₀ was 27.00 μM (CI 95% 14.55–41.26 μM), n = 3. For Lynx1 linear fragment (2) the calculated IC₅₀ was 32.30 μM (CI 95% 14.70–70.99 μM), n = 3. (B) Bar graph of 100 μM SSS fragment (5) inhibition of ion currents induced by 25 μM acetylcholine (ACh) in the human α9α10 nAChR. Data are presented as mean ± SEM, n = 3. Paired Student’s t-test, p < 0.05 (black asterisks, normalized current evoked by acetylcholine in the presence of SSS vs normalized current induced by acetylcholine in the absence of SSS).

Figure 4

Bar graph of 50 μM Ly6 fragments of Lynx1 (1), SLURP1 (6), SLURP2 (7), Lynx1 linear (2), and SSS (5) inhibition of ion currents induced by 30 μM ACh in the human α3β2 nAChR expressed in X. laevis oocytes. The current recorded in response to 30 μM ACh application (ACh) is accepted as 1. The current recorded in response to application of acetylcholine in the presence of peptides was compared with the mean between previous and afterwards acetylcholine-induced current. Normalized currents are presented as mean ± SEM, n = 3 [for SLURP2 (7), Lynx1 linear (2), and SSS (5)] or n = 5 [for Lynx1 (1) and SLURP1 (6)]. Ion current values for Lynx1 (1) and SLURP1 (6) are 0.77 ± 0.08 and 0.74 ± 0.08, respectively.