A Single Amino Acid Substitution in Î±-Conotoxin TxID Reveals a

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A Single Amino Acid Substitution in #-Conotoxin TxID Reveals a Specific #3#4 Nicotinic Acetylcholine Receptor Antagonist Jinpeng Yu, Xiaopeng Zhu, Peta J. Harvey, Quentin Kaas, Dongting Zhangsun, David J Craik, and Sulan Luo J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00967 • Publication Date (Web): 25 Sep 2018 Downloaded from http://pubs.acs.org on September 26, 2018

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Journal of Medicinal Chemistry

A Single Amino Acid Substitution in α-Conotoxin TxID Reveals a Specific α3β4 Nicotinic Acetylcholine Receptor Antagonist Jinpeng Yu†, Xiaopeng Zhu†, Peta J. Harvey‡, Quentin Kaas‡, Dongting Zhangsun†, *, David J. Craik‡, Sulan Luo†, ** †

Key Laboratory of Tropical Biological Resources, Ministry of Education; Key Laboratory for

Marine Drugs of Haikou, Hainan University, Haikou 570228, China ‡

Institute for Molecular Bioscience, The University of Queensland, Brisbane, Queensland, 4072,

Australia

ABSTRACT The α3β4 nAChR is as an important target implicated in various disease states. α-Conotoxin TxID (1) is the most potent antagonist of the α3β4 nAChR, but also exhibits inhibition of the α6/α3β4 nAChR. The results of alanine scanning of (1) suggested a vital role of Ser9 on the selectivity of the peptide. In this study Ser9 was substituted with a series of 14 amino acids, including some non-natural amino acids, displaying different physico-chemical characteristics to further improve the selectivity of (1) on the α3β4 nAChR. Pharmacological activities of mutants were evaluated using an electrophysiological approach. The best selectivity was obtained with [S9K]TxID, (12), which inhibited the α3β4 nAChR with an IC50 of 6.9 nM, and had no effect on other nAChRs. Molecular modeling suggested a possible explanation for the high selectivity of (12) towards the α3β4 nAChR, providing a deeper insight into the interaction between α-conotoxins and nAChRs, as well as potential treatments for nAChR-related diseases.

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INTRODUCTION Nicotinic acetylcholine receptors (nAChRs) are transmembrane ligand-gated ion channels that are distributed throughout the central and peripheral nervous systems.1 All nAChR subtypes function as pentamers composed of either homologous α subunits (e.g. α7 and α9α10 nAChRs) or heterogenous complexes containing at least α and β subunits (e.g. α3β4, α6β4 and muscle type α1β1δε nAChRs).2 Neuronal nAChR subtypes are involved in a range of disease states, including pain, Parkinson’s disease, schizophrenia, depression, Alzheimer’s disease, nicotine addiction and lung cancer.3 The α3β4 nAChR has a vital role in the modulation of the synaptic release of some neurotransmitters. It is widely distributed in the peripheral nervous system and some regions of the central nervous system, including the medial habenula, interpeduncular nucleus and pineal gland.4, 5 It was recently reported that the selective α3β4 nAChR antagonists AT-1001, 18-methoxycoronaridine (18-MC) and α-conotoxin AuIB, (2), can reduce nicotine self-administration in rats, demonstrating the role of the α3β4 nAChR in nicotine dependence and drug-seeking behavior.6-8 α-Conotoxins (α-CTxs) are a family of natural 12−20 amino acid disulfide-rich peptides that selectively target nAChR subtypes and also bind to ACh binding proteins (AChBPs).9 Because of their high potency and selectivity for nAChR subtypes, α-CTxs are used as molecular probes or drug leads for nAChR-related diseases.10 α-CTx TxID, (1), is a novel α4/6-CTx identified from Conus textile, and is the most potent antagonist of α3β4 nAChR, with an IC50 of 3.6 nM. Since (1) also exhibits a slightly lower activity against the α6/α3β4 nAChR subtype with an IC50 of 34 nM, modification of this peptide is necessary to increase its selectivity and improve its potential as a medication for nicotine addiction or drug abuse.11, 12 Positional scanning is a successful approach for structure-function studies and modification of α-CTxs. For example, the double mutation V11L and V16D increased the selectivity of α-conotoxin


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ArIB at α7 versus α6/α3β2β3 nAChR from 36-fold to 760-fold.13 In another application of positional scanning, positions 4 and 9 of α-CTx Vc1.1 were identified as important for the selective inhibition of the α9α10 nAChR, a potential pain target, resulting in improved molecules targeting the human receptor.14, 15 Alanine scanning of (1) recently revealed that Ala substitution of Ser9 increased the selectivity for α3β4 relative to α6/α3β4 by 5-fold whereas [S9A]TxID, (3), had negligible activity on other nAChRs.16 In this study we generated a new series of Ser9 substituted mutants and analyzed their inhibitory activity by two-electrode voltage clamp electrophysiology on 10 nAChR subtypes with the aim of further improving the selectivity of TxID-based compounds for the α3β4 nAChR subtype.

RESULTS Chemical synthesis of peptide 1 analogs Since the globular isomer (Cys I-III, Cys II-IV) is the bioactive form of TxID11, all analogs were synthesized with this regioselective disulfide-bond arrangement by a two-step oxidation process (Scheme 1, Figure 1). The purity (>95%) and identity of each folded peptide was confirmed by analytical RP-HPLC and HRMS (Figure S1-S15).

Side chain diversity of residues at position 9 shows significant impacts on the selectivity of (1) Since the affinity preference for α3β4 over the α6/α3β4 nAChR subtype was notably increased to 46-fold by the S9A substitution of TxID, a suite of Ser9-substituted analogs were designed as the first generation mutants in the course of improving the selectivity towards α3β4 relative to α6/α3β4 nAChRs. The Ser at position 9 was substituted by various residues with differing characteristics,



including hydrophilic Thr and 2-aminobutyric acid (Abu) residues, hydrophobic Leu residues, bulky aromatic Phe and Tyr residues, positively charged Arg and negatively charged Asp residues (Figure 1). The potency of each synthetic peptide was evaluated using rα3β4 and rα6/α3β4 nAChRs expressed on the cell membranes of Xenopus oocytes (Table 1). The potency towards rα3β4 and rα6/α3β4 nAChR subtypes remained high for most of the analogs with the exception of [S9D]TxID (10). The mutants [S9T]TxID (4), [S9L]TxID (6), and [S9Y]TxID (8) showed similar activity relative to the native (1). The S9F substitution led to a 3-fold decrease in the potency on rα3β4 nAChR but a 3-fold increase in the potency on rα6/α3β4 nAChR, resulting in decreased selectivity towards rα3β4 relative to rα6/α3β4 nAChRs. Remarkably, replacing Ser9 with an Arg residue generated a 65-fold increase in selectivity compared with (1) and (3) by significantly reducing the potency on rα6/α3β4 nAChR while having no impact on the inhibition of rα3β4 nAChR. Although (10) was the only analog that significantly decreased the activity by greater than 100-fold on both receptors, its selectivity was also increased to >26-fold (Table 1).

Mutation of Ser9 to a charged amino acid residue reveals [S9K]TxID, (12), as a selective α3β4 nAChR antagonist The mutation of Ser9 with charged amino acid residues (S9R and S9D) resulted in increased selectivity for α3β4 over α6/α3β4 nAChR. To further improve the specificity of TxID for the α3β4 nAChR subtype, we synthesized second generation mutants that included other charged residues (S9H, S9E, S9K) and

D-amino

acid residues (S9D-Ser, S9D-Arg, S9D-Asp) (Figure 1). D-Amino acid

substitution led to a substantial decrease in activity; for example, [S9D-Asp]TxID (16) was ineffective at either receptor. The Cβ of a D-amino acid at position 9 is sterically incompatible with the


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Cys3-Cys15 disulfide bond in the context of the conformation of the native toxin, suggesting that the D-amino

acid variants display a slightly different conformation compared to the parent peptide. The

potency of [S9E]TxID (13) was comparable to that of (10), whereas the S9H mutation showed negligible difference in activity to TxID (Table 1). It is noteworthy that [S9K]TxID (12) showed an IC50 of 6.9 nM on rα3β4 nAChR, which was 2-fold less than the native (1). However, its inhibition on the rα6/α3β4 nAChR was negligible at a concentration of 10 µM, hence revealing (12) as a specific antagonist with high potency targeting only the rα3β4 nAChR (Figures 2 and 3, Table 2). When tested on other nAChR subtypes, including mα1β1δε, rα2β2, rα2β4, rα3β2, rα4β2, rα4β4, rα7 and rα9α10, the inhibition of (12) was also negligible at 10 µM.

NMR Spectroscopy and Structural Analysis of (12) The three-dimensional solution structure of (12) was determined using NMR methods to compare the effect of mutation at Ser-9 upon the structure relative to the native (1). NMR analysis revealed that (12) behaves similarly to both native TxID11 and its S9A mutant12, existing as two isomers due to the cis-trans isomerization of Pro-6. In aqueous solution at 308 K, the trans isomer dominates in a ratio of 70:30

but

this

ratio

increases

to

approximately 90:10

upon

addition

of

30%

TFE

(2,2,2-trifluoroethanol). Full assignment of the major isomer was possible apart from the N-terminal glycine residue and the major species was confirmed as having both prolines in the trans configuration by the observation of NOEs between the Hαi-1 – HδPi protons of the Pro-6 and Pro-13 and their preceding residues. No attempt was made to fully assign the minor isomer due to the low concentration of this species and spectral overlap. Secondary shift analysis was used to assess any changes in secondary structure elements of (12). Helicity was confirmed across the middle of the peptide which appears to be slightly strengthened in



this mutant compared to native TxID (Figure 4A). Structure calculations were performed with CYANA using distance restraints derived from NOESY spectra acquired in 30% TFE at 308 K. Dihedral restraints were generated from chemical shifts using TALOS-N.17 A set of 20 lowest energy structures, consistent with experimental data, were calculated (Figure 4B, Table 3). An alpha helix was identified by PROMOTIF18 as spanning residues 6-11 and the root-mean-square deviation (rmsd) was calculated as 0.81 ± 0.29 Å across backbone residues 2-15 of (12). The overall fold of this mutant is highly similar to that of native (1) with a rmsd of 1.14 Å across backbone residues 2-15.

Molecular modeling Molecular models of the complexes between (1) and the ligand binding domain of α3β4 or α6β4 nAChRs were generated using a molecular dynamics. The backbone root-mean-square deviations of the nAChR and toxins from their initial conformation stabilized at around 2 Å after 20–60 ns simulation time (Figure S16), suggesting that the systems reached equilibrium. The molecular dynamics was carried out with a simulation time of 200 ns for each system. The molecular models of the two complexes generated by this approach are similar to those previously reported, bringing an additional level of confidence in the molecular interactions that they describe. Specifically, we made use of the recent crystal structure of the α4β2 nAChR19, which provides detailed information on the interface between nAChR subunits that is targeted by agonist and antagonists such as α-CTxs. These toxins indeed compete with agonists in the orthosteric binding site, which is located at the interface between two subunits in the ligand-binding domain. In the case of α3β4 or α6β4 nAChRs, this binding site is contributed by either an α3 or α6 subunit and by a β4 subunit.

DISCUSSION


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In this study we synthesized a suite of Ser9-substituted mutants of TxID and determined their potency on nAChRs expressed in Xenopus oocytes. A strategy of single amino acid substitution at residue 9 was utilized to further investigate the structure-activity relationship of TxID. Ser9 was substituted by amino acid residues with different physico-chemical characteristics, including hydrophilic, hydrophobic, bulky aromatic, positively charged and negatively charged residues, as well as some non-natural amino acids. The results show that substitution of Ser9 with charged residues can lead to increased selectivity for α3β4 versus α6/α3β4 nAChRs compared with native (1). Notably, (12) was 2-fold less potent on rα3β4 nAChR compared with native (1), yet had no obvious effect on other tested nAChR subtypes including α6/α3β4 nAChR. We generated molecular models of the interaction between (1) and rα3β4 (Figure 5A) or rα6β4 (Figure 5C) nAChRs to rationalize our activity data. The molecular models suggest that the binding sites of the two nAChR subtypes are very similar; the positions that constitute the binding site of (1) at α3β4 or α6β4 nAChRs are occupied by the same amino acid residues with the exception of the α subunit positions 148 and 196 (Figure 5B). Nevertheless, the binding modes suggested by the two models are subtly different, resulting in a shift of the location of S9 of TxID in the binding site. This shift seems to stem from the α subunit position 196, which is occupied by Gln or Thr for the α3 or α6 subunit, respectively. Q196 is bulkier than T196 and interacts with the β8 strand of the α3 subunit, “pushing” the C-loop and (1) further towards the bottom of the orthosteric binding site than in the TxID/α6β4 model. The shift in the binding mode of (1) for α3β4 and α6β4 nAChRs results in different locations and interactions of S9, explaining the differential inhibitory activity of an Ala or a charged amino acid substitution at this position. In the TxID/α3β4 nAChR model, the side chain of S9 does not contact the β-strands of the β4 subunit, and is located in a cavity that is partly solvated (Figure 5D). This model suggests that the substitution of this position into an Ala or the long side chain amino acids Lys or Arg



should therefore be well accepted. Our mutational data indeed show that the corresponding substitutions are innocuous. The negatively-charged amino acids Asp or Glu have shorter side chains than Lys and Arg, and the variants (10) and (13) would bury the negative charge of D9 or E9 side chains in a negatively charged environment (Figure 5E). The D9 and E9 residues would cause a charge-charge repulsion at the interface, which is detrimental to affinity, as supported by the ca. 100-fold decrease in activity of the two mutants compared to the parent peptide. The S9 side chain of TxID in the TxID/α6β4 nAChR model faces the positively-charged β4 K81 and is in close proximity to the negatively-charged β4 E58 (Figure 5F). These two residues of the β4 subunit are only partly solvated due to the presence of the toxin in the binding site, and substituting S9 with a charged residue should result in charge repulsion with either β4 E58 or K81, as evidenced by the >300-fold decrease in activity of the variants (10), (12) and (13) compared to native (1). By contrast, the S9R substitution only caused a 10-fold decrease in activity. Arg residues have a positive charge located at the end of the longest side chain among natural amino acids, and the R9 charge of (9) could therefore position itself at a longer distance from K81 than K9 of (12), decreasing the energy penalty caused by the charge repulsion. In agreement with our previous study of the TxID/α6β4 nAChR system12, the S9 side chain of (1) establishes a hydrogen bond with the K81 side chain of β4. Hence, substitution of S9 into Ala, Arg, Lys, Glu or Asp would prevent the formation of this hydrogen bond with the receptor, contributing to a decrease in activity compared to the parent peptide of only ca. 5-fold according to the relative activity of (3) (Table 1). A substitution of position 9 in (1) allowed the creation of a more selective inhibitor of α3β4 but we also discovered that the substitution by Phe resulted in a less selective inhibitor (Figure 6, Table 4). Peptide (7) indeed inhibited all β4-containing nAChR subtypes (rα2β4, rα3β4, rα4β4 and rα6/α3β4)


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whereas (1) was inactive at rα2β4 and rα4β4. The substitution S9F resulted in a negligible 3-fold decrease or increase in activity for rα3β4 or rα6/α3β4 nAChRs compared to the parent peptide, respectively. In contrast, (7) had at least 30-fold increase inhibition of rα2β4 compared to (1). Interestingly, (2) is another α4/6-CTx targeting the α3β4 nAChR, albeit with a lower potency than (1), and it displays a Phe at position 9.10, 20 According to the modeling and mutagenesis experiments on (2) done by Grishin and co-workers, the aromatic ring of F9 interacts with β4 W79 or K81 of α3β4 nAChR (K61 and W59 in their numbering system).21 The proposed binding interaction of S9 (1) with α3β4 nAChR suggests that a similar van der Waals interaction between peptide 7 F9 and β4 W79 and/or K81 could happen. Van der Waals interactions are typically less specific than hydrogen bonds or charge-charge interactions. A small shift in the binding mode caused by a different α subunit might therefore not compromise these van der Waals interactions, which is consistent with the broader range of subtypes targeted by the S9F variant than the wild-type toxin. Introducing D-amino acid residues into peptides is known to increase their proteolytic stability while retaining bioactivity.22, 23 We consequently synthesized three variants (14) [S9D-Ser]TxID, (14), [S9D-Arg]TxID (15) and [S9D-Asp]TxID (16) incorporating D-amino acid residues at position 9 and evaluated their pharmacological activity at rα3β4 and rα6/α3β4 nAChR subtypes. Unfortunately, their potencies at rα3β4 were markedly decreased compared to the parent peptide.

CONCLUSIONS We have characterized a novel mutant of TxID, (12), as the most selective antagonist of α3β4 nAChR identified so far. This peptide exhibits inhibition of rα3β4 nAChR with an IC50 of 6.9 nM with no obvious effect on several other nAChR subtypes, especially for the closest subtype rα6/α3β4 nAChR. This peptide might find applications as a molecular probe or drug-lead for the development of



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compounds active at α3β4 nAChR, an important target for the treatment of nicotine addiction and drug abuse. The latter application would first require testing of the in vivo safety and efficacy of the compound, which will be the subject of future studies.

EXPERIMENTAL SECTION Strains and reagents The E. coli strain DH5α for storage of recombinant plasmid was purchased from Sangon (Shanghai, China). All restriction enzymes, DNA and RNA ladders were purchased from TaKaRa (Dalian, China). Reversed-phase C18 Vydac columns (5 µm, 4.6 mm × 250 mm; 10 µm, 22 mm × 250 mm) were purchased from Grace Vydac (Hesperia, CA, USA). Acetylcholine chloride, atropine, collagenase A, and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO, USA). Acetonitrile (ACN, HPLC grade) was purchased from Fisher Scientific (Pittsburgh, PA, USA). Trifluoroacetic acid (TFA) was purchased from Tedia Company (Fairfield, OH, USA). cRNA mMESSAGE mMACHINE In Vitro Transcription Kit and RNA MEGA Clear Kit were purchased from Ambion (Austin, TX, USA). Other chemical reagents were all of analytical grade. cDNA clones encoding rat (r) α2, α3, α4, α7, β2, β3, β4, and mouse (m) α1, β1, ε, δ subunits were kindly provided by S. Heinemann (Salk Institute, San Diego, CA). Since the expression of the native α6 subunit is poor, the α6/α3 chimera containing the N-terminal extracellular ligand-binding portion of the α6 subunit with the remainder as α3 subunit is utilized as a substitute, as previously reported.24 The rα6/α3 chimera clone was generously provided by J. E. Garrett (Cognetix, Inc., Salt Lake City, Utah). The rβ4 subunit clone in the high expressing pGEMHE vector was generously provided by C. W. Luetje (University of Miami, Miami, FL). Clones of rα9 and rα10 were generously provided by A. B. Elgoyen (Instituto de Investigaciones en Ingeniería Genética y Biología Molecular, Buenos Aires,


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Argentina). Female Xenopus laevis used for experiments were obtained from Nasco (USA), which were housed at 17 °C in SPF laboratory animal room and fed twice a week.

Peptide Synthesis The synthesis of the linear peptide analogs was performed using the solid-phase peptide synthesis (SPPS) method by GL Biochem (Shanghai, China). The Cys residues of all analogs were protected in pairs with S-trityl (Trt) on the first and third Cys while S-acetamidomethyl (Acm) were incorporated on the second and fourth Cys. Following addition of 60% ACN and purification by reversed-phase high-performance liquid chromatography (RP-HPLC), the linear peptides were oxidised by a two-step oxidation protocol as previously described (Scheme 1).25, 26 The HPLC separation program for all linear, monocyclic and bicyclic peptides purification included a linear increase of 5%–50% solvent B in 45 min at a flow rate of 12 mL/min on a reversed-phase C18 Vydac column (solvent A was ddH2O with 0.075% TFA, solvent B was 90% ACN and 10% ddH2O with 0.05% TFA). UV absorption wavelength was monitored at 214 nm. The purity (>95%) of all mature peptides was confirmed by analytical RP-HPLC performed with a linear gradient of 5%–40% solvent B in 20 min at a flow rate of 1 mL/min on an analytical reversed-phase C18 Vydac column. The identity of the mature peptides was confirmed by HRMS spectra, which was run on a LCMS-IT-TOF mass spectrometer (SHIMADZU) using electrospray ionization in positive ion mode.

cRNA preparation and injection Plasmids with cDNA clones encoding various nAChR subunits were transformed into the DH5α strain for storage and amplification. After digestion with the corresponding restriction enzyme, the



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linearized plasmids were used as templates for cRNA preparation. The reaction was performed using the mMESSAGE mMACHINE In Vitro Transcription Kit, and the generated capped cRNAs were purified using the RNA MEGA Clear Kit after overnight reaction. The purity and concentrations of cRNAs for various nAChR subunits were assessed by their absorbance at 260 and 280 nm. Oocytes were isolated from a female Xenopus laevis as described previously.27 All animal related procedures were performed in accordance with the guidelines of the Animal Ethics Committee of Hainan University. Then cRNA for each subtype were combined as equimolar ratios, and subsequently injected into Xenopus oocytes with a minimum of 10 ng for each subunit within 24 h of oocytes harvest. The oocytes were incubated at 17 °C and humidity of 35% in ND96 buffer (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mM MgCl2, and 5 mM HEPES, pH 7.1–7.5) with antibiotic (10 mg/L penicillin, 10 mg/L streptomycin, and 100 mg/L gentamicin), as previously described.28

Voltage clamp recording The membrane currents of the injected Xenopus oocytes were recorded by a two-electrode voltage clamp 2-5 days post-injection, as previously described.27, 29 The electrodes used for voltage clamping and current recording were pulled from borosilicate glass, whose resistance was 5-50 MΩ when filled with 3 M KCl. The oocyte chamber, consisting of a cylindrical groove with a volume of approximately 50 µl, was gravity perfused at a flowrate of 2–4 mL/min with ND-96 buffer containing 0.1 mg/mL BSA and 1 µM atropine for all nAChR subtypes except for mα1β1δε, rα7, and rα9α10. During the electrophysiological data recording, the oocytes were voltage-clamped at a potential of -70 mV, applied as a 1 s ACh pulse per min. Once a stable baseline was obtained, the oocytes expressing each kind of nAChR were pre-incubated with either ND96 alone or containing CTxs of different concentrations for 5 min prior to the ACh pulse. The ACh concentrations for nAChRs were 10 µM for


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rα1β1δγ and rα9α10, 200 µM for rα7, and 100 µM for all other subtypes.28, 30 Data were recorded at 50 Hz and filtered with 2 Hz. All recordings were generated at room temperature and repeated as 3-6 independent experiments.

Statistical analysis of data The average of three responses produced by ND-96 application was used as the normalized test response to calculate “% response” for different concentrations of CTxs. All IC50 values and dose– response curves were obtained by fitting to the non-linear regression equation using Graphpad Prism 6.0 (Graphpad Software, CA, USA), response% = 100/[1+([toxin]/IC50)nH], in which nH is the Hill coefficient.31 Each point of the dose–response curve was presented as average ± standard error of mean (SEM) for at least three panel data of % response recording.11

NMR Spectroscopy and Structure Calculations NMR spectra of (12) were recorded as described previously for (1)11 on a 1 mM sample in either 10% D2O/90% H2O or 30% d3-TFE/70% H2O (pH 3.5). Two-dimensional NMR experiments (TOCSY, NOESY,

15

N-HSQC, and

13

C- HSQC) were acquired using the 30% d3-TFE/70% H2O

sample at 308K on a Bruker Avance III 600 MHz equipped with a cryoprobe. Spectra were processed with Topspin (Bruker) and assigned using the program CcpNMR Analysis.32 Spectra were referenced to internal 2,2-dimethyl-2-silapentane-5-sulfonate (DSS) at 0 ppm. Structures were generated using CYANA33, and the 20 structures with the lowest energy and best quality were analysed using Molprobity.34



Molecular modeling Molecular models of the complexes between (1) and the ligand binding domain of the α3β4 nAChR or α6β4 nAChR were built by homology and refined using molecular dynamics (MD) simulation, following an approach similar but not identical to one of our previous studies.12 The initial models were built by homology modeling using Modeller 9v1535 using as templates the crystal structure of Aplysia californica AChBP bound to α-CTx PeIA (PDB: 2uz6)36 and the structure the human α4β2 nAChR bound to nicotine (PDB: 5kxi).19 A number of water molecules present in the PeIA/AChBP crystal structure were transferred to the homology models. The models were solvated by 18,000 water molecules in an octahedron box and thoroughly minimized. Sodium and chloride ions were introduced in the system to reach 150 mM sodium chloride concentration as well as neutralize the system. The systems were heated up over 100 ps to 300 K during which time the protein and peptide heavy atoms were restrained to their position using a force constant of 100 kcal/mol/Å2. The restraints were progressively removed over 20 ns apart from the backbone atoms of the loops that are in contact with the transmembrane domain in the crystal structure of α4β2 nAChR (PDB 5kxi). The position restraints on these loops were maintained during the following 200 ns production run. The Langevin thermostat (collision frequency of 2.0 ps-1)37 and isotropic Berendsen barostat (time constant of 1.0 ps)38 were used to maintain temperature and pressure to 300 K and 1 bar, respectively. Long-range electrostatics were treated using the Particle Mesh Ewald method.39 All bonds were constrained using the SHAKE algorithm and water molecules were modeled as rigid molecules using the SETTLE algorithm. The ff14SB force field was used for all simulations.40 The molecular dynamics simulations were carried on a GPU workstation using the pmemd engine from the AMBER16 molecular simulation package.41 Electrostatic potentials were computed using APBS.42


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■ ASSOCIATED CONTENT *Supporting Information

Additional figures illustrating analytical RP-HPLC and HRMS analysis of peptide (1) and its analogs (Figure S1-S15), as well as backbone root-mean-square deviations of the nAChR and toxins from their starting conformation for the 200 ns molecular dynamics simulations of the α3β4/TxID and α6β4/TxID systems (Figure S16). Table S1 providing the molecular masses of peptide (1) and its analogs observed by HRMS compared with the theoretical values. Table S2 providing the NMR chemical shift data of peptide (12). The two coordinate files correspond to the molecular dynamics simulations of the complexes between (1) and the binding site of α3β4 nAChR or α6β4 nAChR.

■ AUTHOR INFORMATION Corresponding Author ** E-mail: [email protected] (S.L.) * E-mail: [email protected] (D.Z.) Author Contributions The manuscript was written through the contributions of all authors. All authors have given approval to the final version of the manuscript. Notes The authors declare no competing financial interest.

■ ACKNOWLEDGMENTS This work was supported in part by Major International Joint Research Project of National



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Natural Science Foundation of China (81420108028), National Natural Science Foundation of China (31760249), Changjiang Scholars and Innovative Research Team in University Grant IRT_15R15, Hainan Provincial Department of Education (Hnky2017-16) and the Australian Research Council (DP150103990). DJC is an ARC Australian Laureate Fellow (FL150100146).

■ ABBREVIATIONS USED ACh, Acetylcholine; nAChRs ,

Nicotinic

acetylcholine

receptors;

18-MC,

18-Methoxycoronaridine; α-CTxs, α-Conotoxins; AChBPs, ACh binding proteins; BSA, Bovine serum albumin; SPPS, Solid-phase peptide synthesis; Trt, S-trityl; Acm, S-acetamidomethyl; RP-HPLC, Reversed-phase high-performance liquid chromatography; ESI-MS, Electrospray ionization-mass spectrometry; SEM, Standard error of mean; Abu (B); 2-Aminobutyric acid.


Page 17 of 37 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


REFERENCES 1.

Albuquerque, E. X.; Pereira, E. F.; Alkondon, M.; Rogers, S. W. Mammalian nicotinic

acetylcholine receptors: from structure to function. Physiol. Rev. 2009, 89, 73-120. 2.

Sine, S. M.; Bren, N.; Quiram, P. A. Molecular dissection of subunit interfaces in the nicotinic

acetylcholine receptor. J. Physiol. Paris 1998, 92, 101-105. 3.

Taly, A.; Corringer, P. J.; Guedin, D.; Lestage, P.; Changeux, J. P. Nicotinic receptors: allosteric

transitions and therapeutic targets in the nervous system. Nat. Rev. Drug Discov. 2009, 8, 733-750. 4.

Jensen, A. A.; Frolund, B.; Liljefors, T.; Krogsgaard-Larsen, P. Neuronal nicotinic acetylcholine

receptors: structural revelations, target identifications, and therapeutic inspirations. J. Med. Chem. 2005, 48, 4705-4745. 5.

Perry, D. C.; Xiao, Y.; Nguyen, H. N.; Musachio, J. L.; Davila-Garcia, M. I.; Kellar, K. J.

Measuring nicotinic receptors with characteristics of alpha4beta2, alpha3beta2 and alpha3beta4 subtypes in rat tissues by autoradiography. J. Neurochem. 2002, 82, 468-481. 6.

Glick, S. D.; Maisonneuve, I. M.; Kitchen, B. A. Modulation of nicotine self-administration in

rats by combination therapy with agents blocking alpha 3 beta 4 nicotinic receptors. Eur. J. Pharmacol. 2002, 448, 185-191. 7.

Toll, L.; Zaveri, N. T.; Polgar, W. E.; Jiang, F.; Khroyan, T. V.; Zhou, W.; Xie, X. S.; Stauber, G.

B.; Costello, M. R.; Leslie, F. M. AT-1001: a high affinity and selective alpha3beta4 nicotinic acetylcholine

receptor

antagonist

blocks

nicotine

self-administration

in

rats.

Neuropsychopharmacology 2012, 37, 1367-1376. 8.

Glick, S. D.; Sell, E. M.; McCallum, S. E.; Maisonneuve, I. M. Brain regions mediating

alpha3beta4 nicotinic antagonist effects of 18-MC on nicotine self-administration. Eur. J. Pharmacol. 2011, 669, 71-75.



9.

Ulens, C.; Hogg, R. C.; Celie, P. H.; Bertrand, D.; Tsetlin, V.; Smit, A. B.; Sixma, T. K. Structural

determinants of selective alpha-conotoxin binding to a nicotinic acetylcholine receptor homolog AChBP. Proc. Natl. Acad. Sci. U. S. A. 2006, 103, 3615-3620. 10. Chang, Y. P.; Banerjee, J.; Dowell, C.; Wu, J.; Gyanda, R.; Houghten, R. A.; Toll, L.; McIntosh, J. M.; Armishaw, C. J. Discovery of a potent and selective alpha3beta4 nicotinic acetylcholine receptor antagonist from an alpha-conotoxin synthetic combinatorial library. J. Med. Chem. 2014, 57, 3511-3521. 11. Luo, S.; Zhangsun, D.; Zhu, X.; Wu, Y.; Hu, Y.; Christensen, S.; Harvey, P. J.; Akcan, M.; Craik, D. J.; McIntosh, J. M. Characterization of a novel alpha-conotoxin TxID from Conus textile that potently blocks rat alpha3beta4 nicotinic acetylcholine receptors. J. Med. Chem. 2013, 56, 9655-9663. 12. Wu, Y.; Zhangsun, D.; Zhu, X.; Kaas, Q.; Zhangsun, M.; Harvey, P. J.; Craik, D. J.; McIntosh, J. M.; Luo, S. Alpha-conotoxin [S9A]TxID potently discriminates between alpha3beta4 and alpha6/alpha3beta4 nicotinic acetylcholine receptors. J. Med. Chem. 2017, 60, 5826-5833. 13. Whiteaker, P.; Christensen, S.; Yoshikami, D.; Dowell, C.; Watkins, M.; Gulyas, J.; Rivier, J.; Olivera, B. M.; McIntosh, J. M. Discovery, synthesis, and structure activity of a highly selective alpha7 nicotinic acetylcholine receptor antagonist. Biochemistry 2007, 46, 6628-6638. 14. Yu, R.; Kompella, S. N.; Adams, D. J.; Craik, D. J.; Kaas, Q. Determination of the alpha-conotoxin Vc1.1 binding site on the alpha9alpha10 nicotinic acetylcholine receptor. J. Med. Chem. 2013, 56, 3557-3567. 15. Halai, R.; Clark, R. J.; Nevin, S. T.; Jensen, J. E.; Adams, D. J.; Craik, D. J. Scanning mutagenesis of alpha-conotoxin Vc1.1 reveals residues crucial for activity at the alpha9alpha10 nicotinic acetylcholine receptor. J. Biol. Chem. 2009, 284, 20275-20284. 16. Wasko, M. J.; Pellegrene, K. A.; Madura, J. D.; Surratt, C. K. A role for fragment-based drug


Page 18 of 37

Page 19 of 37


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


design in developing novel lead compounds for central nervous system targets. Front. Neurol. 2015, 6, 197. 17. Shen, Y.; Bax, A. Protein backbone and sidechain torsion angles predicted from NMR chemical shifts using artificial neural networks. J. Biomol. NMR 2013, 56, 227-241. 18. Hutchinson, E. G.; Thornton, J. M. PROMOTIF--a program to identify and analyze structural motifs in proteins. Protein Sci. 1996, 5, 212-220. 19. Morales-Perez, C. L.; Noviello, C. M.; Hibbs, R. E. X-ray structure of the human alpha4beta2 nicotinic receptor. Nature 2016, 538, 411-415. 20. Grishin, A. A.; Wang, C. I.; Muttenthaler, M.; Alewood, P. F.; Lewis, R. J.; Adams, D. J. Alpha-conotoxin AuIB isomers exhibit distinct inhibitory mechanisms and differential sensitivity to stoichiometry of alpha3beta4 nicotinic acetylcholine receptors. J. Biol. Chem. 2010, 285, 22254-22263. 21. Grishin, A. A.; Cuny, H.; Hung, A.; Clark, R. J.; Brust, A.; Akondi, K.; Alewood, P. F.; Craik, D. J.; Adams, D. J. Identifying key amino acid residues that affect alpha-conotoxin AuIB inhibition of alpha3beta4 nicotinic acetylcholine receptors. J. Biol. Chem. 2013, 288, 34428-34442. 22. Najjar, K.; Erazo-Oliveras, A.; Brock, D. J.; Wang, T. Y.; Pellois, J. P. An l- to d-amino acid conversion in an endosomolytic analog of the cell-penetrating peptide TAT influences proteolytic stability, endocytic uptake, and endosomal escape. J. Biol. Chem. 2017, 292, 847-861. 23. Chen, S.; Gfeller, D.; Buth, S. A.; Michielin, O.; Leiman, P. G.; Heinis, C. Improving binding affinity and stability of peptide ligands by substituting glycines with D-amino acids. Chembiochem 2013, 14, 1316-1322. 24. Kuryatov, A.; Lindstrom, J. Expression of functional human alpha6beta2beta3* acetylcholine receptors in Xenopus laevis oocytes achieved through subunit chimeras and concatamers. Mol


Page 20 of 37

Pharmacol 2011, 79, 126-140. 25. Dowell, C.; Olivera, B. M.; Garrett, J. E.; Staheli, S. T.; Watkins, M.; Kuryatov, A.; Yoshikami, D.; Lindstrom, J. M.; McIntosh, J. M. Alpha-conotoxin PIA is selective for alpha6 subunit-containing nicotinic acetylcholine receptors. J. Neurosci. 2003, 23, 8445-8452. 26. Wu, X.; Wu, Y.; Zhu, F.; Yang, Q.; Wu, Q.; Zhangsun, D.; Luo, S. Optimal cleavage and oxidative folding of alpha-conotoxin TxIB as a therapeutic candidate peptide. Mar. Drugs 2013, 11, 3537-3553. 27. Cartier, G. E.; Yoshikami, D.; Gray, W. R.; Luo, S.; Olivera, B. M.; McIntosh, J. M. A new alpha-conotoxin which targets alpha3beta2 nicotinic acetylcholine receptors. J. Biol. Chem. 1996, 271, 7522-7528. 28. Luo, S.; Zhangsun, D.; Wu, Y.; Zhu, X.; Hu, Y.; McIntyre, M.; Christensen, S.; Akcan, M.; Craik, D. J.; McIntosh, J. M. Characterization of a novel alpha-conotoxin from conus textile that selectively targets alpha6/alpha3beta2beta3 nicotinic acetylcholine receptors. J. Biol. Chem. 2013, 288, 894-902. 29. Luo, S.; Zhangsun, D.; Harvey, P. J.; Kaas, Q.; Wu, Y.; Zhu, X.; Hu, Y.; Li, X.; Tsetlin, V. I.; Christensen, S.; Romero, H. K.; McIntyre, M.; Dowell, C.; Baxter, J. C.; Elmslie, K. S.; Craik, D. J.; McIntosh, J. M. Cloning, synthesis, and characterization of alphaO-conotoxin GeXIVA, a potent alpha9alpha10 nicotinic acetylcholine receptor antagonist. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, E4026-E4035. 30. Zhangsun, D.; Zhu, X.; Wu, Y.; Hu, Y.; Kaas, Q.; Craik, D. J.; McIntosh, J. M.; Luo, S. Key residues in the nicotinic acetylcholine receptor beta2 subunit contribute to alpha-conotoxin LvIA binding. J. Biol. Chem. 2015, 290, 9855-9862. 31. Luo, S.; Zhangsun, D.; Schroeder, C. I.; Zhu, X.; Hu, Y.; Wu, Y.; Weltzin, M. M.; Eberhard, S.; Kaas, Q.; Craik, D. J.; McIntosh, J. M.; Whiteaker, P. A novel alpha4/7-conotoxin LvIA from Conus lividus that selectively blocks alpha3beta2 vs. alpha6/alpha3beta2beta3 nicotinic acetylcholine


Page 21 of 37


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


receptors. FASEB J. 2014, 28, 1842-1853. 32. Vranken, W. F.; Boucher, W.; Stevens, T. J.; Fogh, R. H.; Pajon, A.; Llinas, M.; Ulrich, E. L.; Markley, J. L.; Ionides, J.; Laue, E. D. The CCPN data model for NMR spectroscopy: development of a software pipeline. Proteins 2005, 59, 687-696. 33. Guntert, P.; Mumenthaler, C.; Wuthrich, K. Torsion angle dynamics for NMR structure calculation with the new program DYANA. J. Mol. Biol. 1997, 273, 283-298. 34. Chen, V. B.; Arendall, W. B., 3rd; Headd, J. J.; Keedy, D. A.; Immormino, R. M.; Kapral, G. J.; Murray, L. W.; Richardson, J. S.; Richardson, D. C. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 12-21. 35. Sali, A.; Blundell, T. L. Comparative protein modelling by satisfaction of spatial restraints. J. Mol. Biol. 1993, 234, 779-815. 36. Dutertre, S.; Ulens, C.; Buttner, R.; Fish, A.; van Elk, R.; Kendel, Y.; Hopping, G.; Alewood, P. F.; Schroeder, C.; Nicke, A.; Smit, A. B.; Sixma, T. K.; Lewis, R. J. AChBP-targeted alpha-conotoxin correlates distinct binding orientations with nAChR subtype selectivity. EMBO J. 2007, 26, 3858-3867. 37. Izaguirre, J. A.; Catarello, D. P.; Wozniak, J. M.; Skeel, R. D. Langevin stabilization of molecular dynamics. J. Chem. Phys. 2001, 114, 2090-2098. 38. Berendsen, H. J. C.; Postma, J. P. M.; Gunsteren, W. F. V.; Dinola, A.; Haak, J. R. Molecular dynamics with coupling to an external bath. J. Chem. Phys. 1984, 81, 3684-3690. 39. Salomon-Ferrer, R.; Gotz, A. W.; Poole, D.; Le Grand, S.; Walker, R. C. Routine microsecond molecular dynamics simulations with AMBER on GPUs. 2. explicit solvent particle mesh ewald. J. Chem. Theory Comput. 2013, 9, 3878-3888. 40. Maier, J. A.; Martinez, C.; Kasavajhala, K.; Wickstrom, L.; Hauser, K. E.; Simmerling, C. ff14SB:


improving the accuracy of protein side chain and backbone parameters from ff99SB. J. Chem. Theory Comput. 2015, 11, 3696-3713. 41. Case, D. A.; Cerutti, D. S.; Cheatham III, T. E.; Darden, T. A.; Duke, R. E.; Giese, T.; Gohlke, H.; Goetz, A. W.; Greene, D.; Homeyer, N.; Izadi, S.; Kovalenko, A.; Lee, T.; LeGrand, S.; Li, P.; Lin, C.; Liu, T.; Luchko, T.; Luo, R.; Mermelstein, D.; Merz, K. M.; Monard, G.; Nguyen, H.; Omelyan, I.; Onufriev, A.; Pan, F.; Qi, R.; Roe, D.; Roitberg, A.; Sagui, C.; Simmerling, C. L.; Botello-Smith, W.; Swails, J.; Walker, R.; Wang, J.; Wolf, R.; Wu, X.; Xiao, L.; York, D.; Kollman, P. A. Amber 2017; University of California, San Francisco, 2017. 42. Baker, N. A.; Sept, D.; Joseph, S.; Holst, M. J.; McCammon, J. A. Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl. Acad. Sci. U. S. A. 2001, 98, 10037-10041.


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Figure Legends

Figure 1. Sequence of the native (1) and its mutants. All mutants of (1) were synthesized in the globular form with Cys I–III and Cys II–IV connectivities, which is the native isomer of (1). The amino acid residues substituted at position 9 are marked in red. Another α4/6-CTx AuIB, (2), which has been previously used for structure-function studies on the α3β4 nAChR is shown for comparison (not synthesized). (*, C-terminal amide; Abu, 2-aminobutyric acid).

Figure 2. Peptides 1 (A), 3 (B) and 12 (C) differentially block the currents mediated by rα3β4 and rα6/α3β4 nAChR subtypes expressed on Xenopus oocytes. The concentration for toxins was 100 nM except for (12) on rα6/α3β4 nAChR, which had no obvious effect. The ACh-evoked current amplitude of rα6/α3β4 nAChR was reduced to 75% by the application of 10 µM (12). “C” indicates control responses to ACh. Once a stable control response was acquired, the oocytes were incubated with 100 nM or 10 µM toxin prior to the application of ACh.

Figure 3. Dose response curves for the inhibition of (12) on rα3β4 and rα6/α3β4 nAChR subtypes. Each point was presented as the average ± SEM of 3-6 separate oocytes. Compared with the native (1) and peptide (3) which potentially block rα3β4 nAChR over rα6/α3β4 nAChR by 9.4-fold and 46-fold individually, the selectivity of (12) on rα3β4 nAChR is significantly improved, confirming (12) as a specific antagonist with high potency targeting only rα3β4 nAChR.

Figure 4. Structural analysis of (12). (A) Secondary chemical shift analysis of (12) compared to


Page 24 of 37

(1).11 Amino acid sequence of (1) is displayed, with substituted residue for the mutant shown in brackets. Grey bars represent (1); blue bars represent (12). (B) Ribbon representation of the lowest energy structure of (12) (blue backbone) superimposed with that of (1) (grey backbone).11 The N- and C-termini are labeled with N-ter and C-ter, respectively. The helical regions are shown in red and yellow. Cysteine resides are labelled with Roman numerals, and disulfide bonds are shown in yellow.

Figure 5. Molecular model of the interactions between (1) and rα3β4 or rα6β4 nAChRs. (A) Overview of binding mode of (1) in the rα3β4 nAChR orthosteric binding site. The α3 and β4 subunits are in magenta and yellow, respectively; (1) is in blue. (B) Overlay of the two binding modes, with nAChR subunits and (1) colored similarly as in panels A and B. A double-headed arrow indicates the shift in conformation of the nAChR binding sites and of the binding modes of (1). (C) Overview of binding mode of (1) inside the rα6β4 nAChR orthosteric binding site. The α6 and β4 subunits are in orange and yellow, respectively; (1) is in cyan. (D) Binding site of S9 at the interface with rα3β4 nAChR. The molecules are colored as in panel A. (E) Electrostatic potential on the solvent-accessible surface of the binding site of rα3β4 surrounding S9. The surface is colored according to the electrostatic potential with a color scale ranging from red (-5 kT/e) to white (0 kt/e) to blue (5 kt/e). (F) Binding site of S9 at the interface with rα6β4 nAChR. A dashed line indicates a hydrogen bond. The molecules are colored as in panel C. In all panels, the side chains of selected residues are in stick representation, with oxygen, nitrogen and sulfur atoms in red, blue and yellow, respectively. The crystal structure of Aplysia californica AChBP bound to α-CTx PeIA (PDB: 2uz6)36 and the structure of the human α4β2 nAChR bound to nicotine (PDB: 5kxi)19 were used as the starting points for molecular modeling.


Page 25 of 37


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Figure 6. The inhibition of (7) on β4-containing nAChR subtypes. (A-B) The inhibition of 100 nM (7) on rα3β4 and rα6/α3β4 nAChR subtypes. The block of (7) on these receptors was difficult to be reversed, which indicated the increase of affinity on main receptors by the mutation S9F. (C-D) The inhibition of 10 µM peptide (7) on rα2β4 and rα4β4 nAChR subtypes. (E) Dose response curves of (7) on all β4-containing nAChR subtypes. Each point was presented as the average ± SEM of 3-6 separate oocytes.


Scheme 1. Two-step oxidation protocol for chemical synthesis of peptide 1 analogs

a

Trt groups on the first and third Cys residues were removed while resin-bounded

prior to peptide cleavage and deprotection (done by GL Biochem). b

Mixed with an equal volume of 20 mM potassium ferricyanide, 0.1 M Tris base, pH

7.5, rt, 45 min. c

Added dropwise into an equal volume of 10 mM iodine dissolved in ACN:H2O:TFA

(24:71:5 by volume), rt, 5 min under N2 protection.25


Page 26 of 37

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Tables Table 1. Potencies of the native peptides (1) and its mutants on rα3β4 and rα6/α3β4 nAChR subtypes rα3β4

rα6/α3β4

Selectivity

Peptides IC50 a (nM)

Hillslope a

IC50 a (nM)

Hillslope a

( rα3β4: rα6/α3β4)

1

3.6 (1.8-7.3)

0.7 (0.4-1.1)

34 (24-49)

1.1 (0.8-1.5)

1:9.4 12

3

3.9 (2.5-5.9)

0.7 (0.5-1)

178 (137-232)

0.9 (0.7-1)

1:46 12

Mutants of first generation 4

7.4 (5.9-9.2)

1.5 (1.1-1.9)

50 (41-61)

1.2 (0.9-1.5)

1:6.8

5

1.9 (1.5-2.5)

1.3 (0.9-1.7)

6.2 (4.6-8.4)

1.3 (0.7-2)

1:3.3

6

9.9 (8.1-12)

1.7 (1.3-2)

21 (14-29)

1.1 (0.7-1.5)

1:2.1

7

11 (8.4-14)

1.8 (1.3-2.3)

10 (8.5-12)

1.9 (1.2-2.6)

1:0.9

8

7.9 (6.3-10)

1.6 (1.1-2.1)

23 (16-32)

1 (0.7-1.3)

1:2.9

9

5.4 (4-7.3)

1.1 (0.7-1.4)

350 (230-530)

0.6 (0.4-0.7)

1:65

10

380 (240-590)

0.7 (0.5-1)

> 10,000 b

/

/

Mutants of second generation 11

2 (1.5-2.3)

1.9 (1.4-2.3)

19 (13-26)

0.7 (0.6-0.9)

1:9.5

12

6.9 (5.3-8.9)

0.9 (0.7-1.2)

> 10,000 b

/

/

13

320 (240-420)

0.9 (0.7-1.1)

> 10,000 b

/

/

14

120 (90-170)

0.9 (0.7-1.1)

500 (360-700)

0.7 (0.5-0.8)

1:4.2

15

52 (39-71)

0.8 (0.6-0.9)

430 (320-580)

0.8 (0.6-1)

1:8.3

16

> 10,000 b

/

> 10,000 b

/

/

a

Numbers in parentheses are 95% confidence intervals.

b

The inhibition of mutants on nAChRs is < 50% at the concentration of 10-5 M.



Page 28 of 37

Table 2. IC50 and Hill slope values for peptides (1), (3) and (12) on nAChR subtypes Peptide

nAChR Subtypes

IC50 a (nM)

Hill Slope a

1

α3β4

3.6 (1.8-7.3)

0.7 (0.4-1.1)

α6/α3β4

34 (24-49)

1.1 (0.8-1.5) 12

α3β4

3.9 (2.5-5.9)

0.7 (0.5-1)

α6/α3β4

178 (137-232)

0.9 (0.7-1) 12

α3β4

6.9 (5.3-8.9)

0.9 (0.7-1.2)

α6/α3β4

>10,000 b

/

Mα1β1δε

>10,000 b

/

α2β2

>10,000 b

/

α2β4

>10,000 b

/

α3β2

>10,000 b

/

α4β2

>10,000 b

/

α4β4

>10,000 b

/

α7

>10,000 b

/

α9α10

>10,000 b

/

3

12

a

Numbers in parentheses are 95% confidence intervals.

b

The inhibition of mutants on nAChRs is < 50% at the concentration of 10-5 M. All

receptors are rat except for α1β1δε, which is mouse.


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Table 3. Statistical analysis of peptide (12) structures a Experimental restraints total no. distance restraints

72

intraresidue

24

sequential

32

medium range, i-j

A Single Amino Acid Substitution in Î±-Conotoxin TxID Reveals a

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