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 their common name of “three-finger proteins” (TFPs). The snake venom TFPs greatly differ in their activity: short- and long-chain α-neurotoxins, as well as certain non-conventional neurotoxins are blocking nicotinic acetylcholine receptors (nAChRs) (Tsetlin, 2015), while another group of TFPs is attacking muscarinic acetylcholine receptors (Karlsson et al., 2000). There are toxins inhibiting β-adrenergic and other G-protein coupled receptors (GPCRs) (Blanchet et al., 2017). Among the recently found snake venom neurotoxins are mambalgins blocking the acid-sensitive ion channels (ASICs) (Brzezicki and Zakowicz, 2018). 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, also 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). Some individual components, 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.

There is no X-ray structure for a snake venom neurotoxin in complex with any whole-size nAChR subtype. However, high-resolution X-ray structures of toxins with the nAChR models are available: α-cobratoxin complex (Bourne et al., 2005) with the acetylcholine-binding protein (AChBP), which is an excellent model for the nAChR ligand-binding domain (LBD) (Smit et al., 2001; Ulens et al., 2006), α-bungarotoxin bound to the chimera of AChBP with the LBD of the α7 nAChR (Huang et al., 2013), as well as to the monomeric LBDs of the α1 and α9 nAChR subunits (Dellisanti et al., 2007; Zouridakis et al., 2014). All these structures, as well as earlier data on chemical modification and mutagenesis of α-neurotoxins, show the important role of the neurotoxin central loop II. Indeed, synthetic fragments of the central loop retained partial activity of the starting toxin and one such peptide was used for production of antisera for native toxin neutralization (de la Rosa et al., 2017).

Thus, synthetic fragments of snake venom toxins look promising for drug design. However, our paper is focusing not on the synthetic fragments of α-neurotoxins, but on the Ly6 proteins. Some of these proteins interact with nAChRs [see reviews (Ibañez-Tallon and Nitabach, 2012; Tsetlin, 2015)] and are considered as potential drugs (Miwa and Walz, 2012; Narumoto et al., 2013; Swamynathan et al., 2017; Lyukmanova et al., 2016a). Among them are mammalian proteins Lynx1 attached to the membrane in the vicinity of nAChRs by the glycosyl phosphoinositide anchor, and a secreted water-soluble protein SLURP1 (Durek et al., 2017). In view of the perspectives shown by synthetic fragments of α-neurotoxins, we synthesized the loop II fragments of these proteins and tested their activity against several nAChR subtypes. In case of positive results, the advantage of such peptides would be their origin from the endogenous proteins, allowing to expect from them only minimal toxicity. We synthesized the fragments of human Lynx1, SLURP1, and SLURP2, as well as a fragment of the a central loop of the Drosophila protein SSS, because for this protein [promoting sleep in Drosophila by inhibiting its neuronal nAChRs and activating a potassium channel (Wu et al., 2014)] the essential role of its whole loop II was demonstrated by heterologous expression of the protein lacking the N- and C-terminal loops (Wu et al., 2016).

Figure 1 and Table 1 show that the fragments of several Ly6 proteins were obtained as homogeneous peptides and their structure has been confirmed by mass spectrometry. Table 1 shows that the synthesized loops of human Lynx1 and Drosophila SSS have very close calculated pI values (9.7 and 10.0). They are basic similarly to the respective fragment (with the pI values of 7.9) 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 also contains no additional disulfide in the central loop, but similarly to Ly6 proteins possesses additional disulfide in the N-terminal loop (Utkin et al., 2001)]. However, the pI values for SPURP1 and SLURP2 fragments are much lower (3.80 and 4.37, respectively), which might explain a big difference from the Lynx1 and SSS fragments in our biological tests (see below).

Historically, Lynx-1 was the first protein of the Ly6 family found co-localized in the mouse brain with the α7 and α4β2 nAChRs and shown to inhibit their activity (Miwa et al., 1999). This protein is of special interest to us because several years ago at the Shemyakin-Ovchinnikov Institute a water-soluble analog of Lynx1, devoid of the GPI anchor, was heterologously expressed in Escherichia coli. It inhibited ion currents in several nAChR subtypes and its three-dimensional structure was established by ¹H-NMR (Lyukmanova et al., 2011). Moreover, several mutations in its central loop II were shown to affect the activity (Lyukmanova et al., 2013). However, a water-soluble ws-Lynx1 is only a model of the membrane-bound Lynx1 and the differences in behavior were demonstrated in mouse overexpressing Lynx 1 genes with or without the sequence coding for the GPI anchor (Miwa and Walz, 2012). The advantage of SLURP1 and SLURP2, the solution structures of which are also known (Lyukmanova et al., 2016b; Vasilyeva et al., 2017), is that they are naturally occurring water-soluble proteins and the research can be done on them as such, rather than on their models. Much information is available about the SLURP1/SLURP2 involvement in several diseases, predominantly in the skin disease Mal de Meleda (Perez and Khachemoune, 2016). However, until recently the work was done not on SLURP1 as such, but on various fusion proteins incorporating SLURP1. One publication revealed potentiation of ion currents in α7 nAChR at nanomolar concentration of the used fusion SLURP1 protein (Chimienti et al., 2003). However, on the human SLURP1, differing from the naturally occurring protein only by the additional N-terminal Met residue, the effect was the opposite: inhibition rather than enhancement of ion current in α7 nAChR and at micromolar, rather than at nanomolar concentration (Lyukmanova et al., 2016b). Moreover, with the SLURP1 prepared by peptide synthesis and having virtually the same primary structure as the native protein, no inhibition of the α7 nAChR took place, but inhibition of several heterooligomeric nAChRs was registered (Durek et al., 2017). Most interestingly, with this SLURP-1 for the first time for an Ly6 protein inhibition of the α9α10 nAChR was observed and binding was proved to occur at an allosteric site (Durek et al., 2017). This finding to a considerable degree stimulated the present work because α9α10 nAChRs are targets for developing new analgesics, as illustrated most convincingly by research on those α-conotoxins, which are selective for this nAChR subtype (Dutertre et al., 2017; Hone and McIntosh, 2018).

As was described in the Introduction, a certain success was achieved in research using mainly the central loop II of three-finger neurotoxins where the structure was additionally stabilized by connecting the N- and C-termini of peptide by the grafted disulfide. We decided to synthesize the appropriate loops of Lynx1, SLURP1, and SLURP 2 and test their possible activities against several nAChR subtypes. We also included the Drosophila protein SSS, which affects the sleep processes interacting both with the nAChRs and potassium channels (Wu et al., 2014). Additional importance of this protein for our work, focusing on the central loops II from the TFPs of the Ly6 family, was due to the recently demonstrated functional activity for its heterologically expressed loop II (Wu et al., 2016).

The results in this work show that the synthetic fragments of Lynx1 and SSS demonstrated a low micromolаr binding to the muscle-type Torpedo nAChR, as detected in competition with radioactive α-Bgt. Similar binding was registered with the SSS fragment (5) in case of the α7 nAChR, while Lynx1 fragment (1) was over 10-fold less active. A comparison of the cyclized and linear forms of Lynx1 fragment (1 and 2) shows that, contrary to expectations, stabilizing the spatial structure due to connecting the N- and C-termini did not cause a marked increase in the activity against certain targets. Surprisingly, shortening of the peptide (4) did not diminish its affinity, as compared to peptide (2), indicating that both peptides apparently retain the required conformation. Since peptides still should be relatively flexible it is difficult why cyclization of the short peptide (3) is weakening its interaction with the muscle-type and α9/α10 nAChRs.

Both for the SSS and Lynx1 fragments, their association with the α9 LBD was relatively weak (IC₅₀ on the average of 40 µM); inhibition (IC₅₀) of ion currents in the α9α10 nAChR expressed in Xenopus oocytes measured for the Lynx fragment (1) was about 30 µM and a slightly less activity was found for the SSS fragment (5).

Unfortunately, our hope that the synthetic fragment of SLURP1 will possess at least a part of the activity against α9α10 nAChR found in SLURP1 itself (Durek et al., 2017) was not realized. Neither SLURP1 fragment (6) nor that (7) of SLURP2 revealed noticeable inhibition of ¹²⁵I-αBgt binding to Torpedo and human α7 nAChRs or to the α9 domain. They also did not compete with ¹²⁵I-αBgt for binding to A. californica AChBP, but none other synthetic fragment (active against several nAChRs) also manifested a competition. This result is not easy to explain because usually AChBPs are not very discriminative and with low affinity can bind diverse compounds capable of interacting with one or another nAChR subtype or with a different Cys-loop receptor (Brejc et al., 2001; Rucktooa et al., 2009; Brams et al., 2011).

Although our work is focusing on the muscle-type, α7, and α9α10 nAChRs, all of which efficiently bind αBgt (which at nanomolar concentrations also blocks ion currents in them), we checked possible effects of our synthetic fragments on the heterooligomeric α3β2 nAChR. The reason for this is that Lynx1 in the very first publication (Miwa et al., 1999) was shown to inhibit α4β2 nAChRs, while its effects on the function of other heteromeric nAChRs (Ibañez-Tallon and Nitabach, 2012) and on the process of their assembly (Parker et al., 2017) were demonstrated later. In addition, SLURP1 not only inhibited α9α10 nAChR, but also (at low micromolar level) diminished the currents in the α3β2 and α3β4 nAChRs (Durek et al., 2017). The SSS protein was earlier shown to inhibit a heteromeric nAChR of Drosophila (Wu et al., 2014). As Figure 4 shows at least with the α3β2 nAChR, the SLURP1 fragment (6) had the activity comparable to that shown by other Ly6 synthetic fragments to one or another target.

It should be mentioned that there are many publications on the activity of the synthetic three-finger toxins and of their fragments: synthetic fragments of the central loop II of α-neurotoxins have been already mentioned (Kasheverov and Tsetlin, 2017), there are also full-size synthetic α-neurotoxins (Rey-Suárez et al., 2012), non-conventional neurotoxins (Poh et al., 2002), TFP toxins inhibiting acetylcholinesterase (fasciculins) (Falkenstein and Peña, 1997), synthetic mambalgins, and their analogs attacking ASICs (Schroeder et al., 2014). Not always in the work on TFP toxins the interest was focused on the central loop: for the synthetic N-terminal loop of cardiotoxin I from the Naja atra cobra venom the authors reported antimicrobial activity against Gram-positive and Gram-negative bacteria (Sala et al., 2018). Interestingly, molecular modeling indicates that binding of another cytotoxin I (Naja mossambica mossambica) to 20S proteasome (resulting in the inhibition of its chymotryptic activity) is due to the toxin N-terminal loop (Munawar et al., 2015). It is worth to mention that the N-terminal sequence of SIRS (soluble immune response suppressor) has a homology to short α-neurotoxin and its synthetic 21-membered N-terminal fragment was shown to inhibit the development of experimental allergic encephalitis (Webb, 2016).

On the contrary, there is not much work on the synthetic fragments of the Ly6 proteins, and we could not find any publications in scientific literature on the synthetic fragments of those Ly6 proteins which interact with nAChRs or affect their assembly. In the patent database we found one (Walz, 2014) where a series of peptides homologous to Lynx1 fragment (1) has been synthesized homologous to Lynx1 fragment (1) but devoid of the N- and C-terminal Cys residues. However, the peptides were designed to facilitate penetration of the blood–brain barrier, but their interaction with the nAChRs or any other receptor was not tested. Thus, to best of our knowledge, here are the first data on the interaction of synthetic fragments of the central loop of Ly6 proteins with nAChRs.

In the case of the SLURP1 fragment, we did not manage to register the activity against α9α10 nAChR, which would be comparable to that of the synthetic SLURP1 and thus might prove useful for design of new analgesics. The aforementioned examples of the snake-venom TFPs show that, in the future, a success might come with the fragments not only of the central loop II (see Figure 1 ) but of other loops as well. In particular, mutagenesis of ws-Lynx1 demonstrated the functional role of several residues both in the central loop and in the N-terminal loop (Lyukmanova et al., 2013).

The activity of Lynx1 and SSS synthetic fragments, although being in low µM range, should not be called low. The affinity of α-neurotoxins against the muscle-type and α7 nAChRs is in the nanomolar range, but earlier in radioligand and electrophysiology experiments, it was shown that both ws-Lynx1 and recombinant SLURP1 (bearing an additional N-terminal Met residue) bind to the Torpedo, α7, and several heteromeric nAChRs and inhibit their currents at low µM concentrations (Lyukmanova et al., 2016b). The concentration of Lynx1 in the brain was also shown to be in the µM range (Thomsen et al., 2014). With a similar µM affinity synthetic SLURP1 (identical in the amino acid sequence to the naturally occurring protein) inhibits α9α10 and α3β2 nAChRs (but does not inhibit either rat or human α7 nAChRs). The synthetic fragment of Lynx1, at least toward muscle-type Torpedo nAChR, reproduces the activity of the full-size ws-Lynx1. This activity might find practical applications—for example, for design of efficient myorelaxants. In this respect, about 30-fold lower affinity of Lynx1 fragment (1) toward α7 nAChR can be an advantage: it resembles the case of azemiopsin, a linear peptide from the viper venom, which is considerably more selective to muscle nAChRs than to α7 nAChRs and for which preclinical studies as a myorelaxant have been recently completed (Shelukhina et al., 2018).

VT contributed to the conception and planning of the study. EK contributed to the leadership of the project, planning of experiments, and radioligand assay. NE and MZ contributed to the synthesis and characterization of peptides. IK contributed to the preparation of radioligand. DK, DL, ES, and AG contributed to the analysis of biological activities of the peptides. EK, YU, and VT contributed to the drafting of the manuscript and its editing.

The major support of this work was by RSF grant 16-14-00215. VT was additionally supported by the RFBR grants 18-04-00844 and 17-00-00063 komfi. IK was additionally supported by the RFBR grant 18-04-01366.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.