Single Amino Acid Substitution in α-Conotoxin TxID Reveals a Specific

Article Cite This: J. Med. Chem. 2018, 61, 9256−9265

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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,‡ and Sulan Luo*,† †

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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 S Supporting Information *

ABSTRACT: The α3β4 nicotinic acetylcholine receptor (nAChR) is an important target implicated in various disease states. α-Conotoxin TxID (1) is the most potent antagonist of α3β4 nAChR, but it also exhibits inhibition of α6/α3β4 nAChR. The results of alanine scanning of 1 suggested a vital role for Ser9 in 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 physicochemical characteristics to further improve the selectivity of 1 toward α3β4 nAChR. The pharmacological activities of the mutants were evaluated using an electrophysiological approach. The best selectivity was obtained with [S9K]TxID, 12, which inhibited α3β4 nAChR with an IC50 of 6.9 nM and had no effects on other nAChRs. Molecular modeling suggested a possible explanation for the high selectivity of 12 toward α3β4 nAChR, providing deeper insight into the interaction between α-conotoxins and nAChRs as well as potential treatments for nAChR-related diseases.

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α-Conotoxins 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, αconotoxins are used as molecular probes or drug leads for nAChR-related diseases.10 α-Conotoxin TxID, 1, is a novel α4/6-conotoxin identified from Conus textile, and it is the most potent antagonist of α3β4 nAChR, with an IC50 of 3.6 nM. Because 1 also exhibits 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-conotoxins. For example, the double mutation V11L−V16D increased the selectivity of α-conotoxin ArIB for α7 nAChR versus α6/ α3β2β3 nAChR from 36- to 760-fold.13 In another application of positional scanning, positions 4 and 9 of α-conotoxin Vc1.1

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 heterogeneous 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 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 α3β4 nAChR in nicotine dependence and drugseeking behavior.6−8 © 2018 American Chemical Society

Received: June 17, 2018 Published: September 25, 2018 9256

DOI: 10.1021/acs.jmedchem.8b00967 J. Med. Chem. 2018, 61, 9256−9265

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were identified as important for the selective inhibition of 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 that for 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 Ser9substituted mutants and analyzed their inhibitory activities by two-electrode-voltage-clamp electrophysiology experiments on 10 nAChR subtypes with the aim of further improving the selectivity of TxID-based compounds for the α3β4 nAChR subtype.

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RESULTS Chemical Synthesis of Peptide 1 Analogues. Because the globular isomer (Cys I−III, Cys II−IV) is the bioactive form of TxID,11 all analogues were synthesized with this regioselective disulfide-bond arrangement by a two-stepoxidation process (Scheme 1 and Figure 1). The purity (>95%) and identity of each folded peptide were confirmed by analytical RP-HPLC and HRMS (Figures S1−S15). Scheme 1. Two-Step-Oxidation Protocol for the Chemical Synthesis of Peptide 1 Analogues.

Figure 1. Sequence of the native peptide, 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-conotoxin AuIB, 2 (not synthesized), which has been previously used for structure−function studies on α3β4 nAChR, is shown for comparison. An asterisk (*) indicates a C-terminal amide. Abu, 2-aminobutyric acid.

a Trt groups on the first and third Cys residues were removed while the peptide was resin-bound prior to cleavage and deprotection. This was performed by GL Biochem. bMixed 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/H 2 O/TFA (24:71:5, by volume), rt, 5 min under N 2 protection.25

Xenopus oocytes (Table 1). The potency toward the rα3β4 and rα6/α3β4 nAChR subtypes remained high for most of the analogues, with the exception of [S9D]TxID (10). The mutants [S9T]TxID (4), [S9L]TxID (6), and [S9Y]TxID (8) showed similar activities relative to that of the native peptide, 1. The S9F substitution led to a 3-fold decrease in the potency toward rα3β4 nAChR but a 3-fold increase in the potency toward rα6/α3β4 nAChR, resulting in decreased selectivity toward rα3β4 nAChR relative to that toward rα6/ α3β4 nAChR. Remarkably, replacing Ser9 with an Arg residue generated a 65-fold increase in selectivity compared with those of 1 and 3 by significantly reducing the potency toward rα6/α3β4 nAChR while having no impact on the inhibition of rα3β4 nAChR. Although 10 was the only analogue that significantly decreased the activities of both receptors by >100-fold, its selectivity was also increased by >26-fold (Table 1). [S9K]TxID, 12, Revealed as a Selective α3β4 nAChR Antagonist by Mutation of Ser9 to a Charged Amino Acid Residue. Replacing Ser9 with charged amino acid residues (S9R and S9D) resulted in increased selectivity for

Side-Chain Diversity of Residues at Position 9 Showing Significant Impacts on the Selectivity of 1. Because the affinity preference for the α3β4 nAChR subtype over the α6/α3β4 nAChR subtype was notably increased to 46-fold by the S9A substitution of TxID, a suite of Ser9substituted analogues were designed as the first-generation mutants in the course of improving the selectivity toward α3β4 nAChR relative to that toward α6/α3β4 nAChR. The Ser at position 9 was substituted with various residues with differing characteristics, including hydrophilic Thr and 2-aminobutyric acid (Abu) residues, a hydrophobic Leu residue, bulky aromatic Phe and Tyr residues, a positively charged Arg residue, and a negatively charged Asp residue (Figure 1). The potency of each synthetic peptide was evaluated using rα3β4 and rα6/α3β4 nAChRs expressed on the cell membranes of 9257

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Table 1. Potencies of the Native Peptide, 1, and Its Mutants on rα3β4 and rα6/α3β4 nAChR Subtypesa rα3β4

rα6/α3β4

peptide

IC50 (nM)

Hill slope

1 3

3.6 (1.8−7.3) 3.9 (2.5−5.9)

0.7 (0.4−1.1) 0.7 (0.5−1)

4 5 6 7 8 9 10

7.4 (5.9−9.2) 1.9 (1.5−2.5) 9.9 (8.1−12) 11 (8.4−14) 7.9 (6.3−10) 5.4 (4−7.3) 380 (240−590)

1.5 (1.1−1.9) 1.3 (0.9−1.7) 1.7 (1.3−2) 1.8 (1.3−2.3) 1.6 (1.1−2.1) 1.1 (0.7−1.4) 0.7 (0.5−1)

11 12 13 14 15 16

2 (1.5−2.3) 6.9 (5.3−8.9) 320 (240−420) 120 (90−170) 52 (39−71) >10 000b

1.9 0.9 0.9 0.9 0.8

(1.4−2.3) (0.7−1.2) (0.7−1.1) (0.7−1.1) (0.6−0.9) 

IC50 (nM) 34 (24−49) 178 (137−232) First-Generation Mutants 50 (41−61) 6.2 (4.6−8.4) 21 (14−29) 10 (8.5−12) 23 (16−32) 350 (230−530) >10 000b Second-Generation Mutants 19 (13−26) >10 000b >10 000b 500 (360−700) 430 (320−580) >10 000b

Hill slope

selectivity (rα3β4 to rα6/α3β4)

1.1 (0.8−1.5) 0.9 (0.7−1)

1:9.412 1:4612

1.2 (0.9−1.5) 1.3 (0.7−2) 1.1 (0.7−1.5) 1.9 (1.2−2.6) 1 (0.7−1.3) 0.6 (0.4−0.7) 

1:6.8 1:3.3 1:2.1 1:0.9 1:2.9 1:65 

0.7 (0.6−0.9)   0.7 (0.5−0.8) 0.8 (0.6−1) 

1:9.5   1:4.2 1:8.3 

Numbers in parentheses are 95% confidence intervals. bThe inhibition of nAChRs by the mutants was 10 000b >10 000b >10 000b >10 000b >10 000b >10 000b >10 000b >10 000b >10 000b

0.7 (0.4−1.1) 1.1 (0.8−1.5)12 0.7 (0.5−1) 0.9 (0.7−1)12 0.9 (0.7−1.2)         

3 12

a Numbers in parentheses are 95% confidence intervals. All receptors are rat except for α1β1δε, which is mouse. bThe inhibition of nAChRs by the mutants was 300-fold decrease in activity of variants 10, 12, and 13 compared with that of the native peptide, 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 the natural amino acids; the R9 charge of 9 could therefore position itself at a longer distance from K81 than the K9 of 12, decreasing the energy penalty caused by the charge repulsion. In agreement with our previous study of the TxID−α6β4 nAChR system,12 the S9 side chain of 1 establishes a hydrogen bond with the K81 side chain of β4. Hence, substitution of S9 9260

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Table 3. Statistical Analysis of Peptide 12 Structuresa

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 by the wildtype 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, [S9DSer]TxID, 14; [S9D-Arg]TxID, 15; and [S9D-Asp]TxID, 16, incorporating D-amino acid residues at position 9 and evaluated their pharmacological activity against rα3β4 and rα6/α3β4 nAChR subtypes. Unfortunately, their potencies against rα3β4 were markedly decreased compared with that of the parent peptide.

experimental restraints Total No. of Distance Restraints intraresidue sequential medium range, i − j < 5 long range, i − j ≥ 5 Dihedral-Angle Restraints φ ψ violations

72 24 32 13 3 9 8

NOE violations exceeding 0.3 Å dihedral violations exceeding 2.0 Å average pairwise rmsd backbone atoms (residues 2−15) all heavy atoms (residues 2−15) stereochemical qualityb residues in most-favored Ramachandran region Ramachandran outliers unfavorable side-chain rotamers clashscore (all atoms) overall MolProbity score

0 0

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0.81 ± 0.29 Å 1.29 ± 0.25 Å 88.8 0.0 15.0 0.5 1.9

± ± ± ± ±

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 and no obvious effects on several other nAChR subtypes, even on the closest subtype, rα6/α3β4 nAChR. This peptide might find applications as a molecular probe or drug lead for the development of compounds active against 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.

5.1% 0.0% 7.5% 1.5 0.3

All statistics are given as means ± SD. bAccording to MolProbity.

a

interacts with β4 W79 or K81 of α3β4 nAChR (K61 and W59 in their numbering system). 21 The proposed binding interaction of S9 of 1 with α3β4 nAChR suggests that similar van der Waals interactions between F9 of peptide 7 and β4 W79 or K81 could happen. Van der Waals interactions are typically less specific than hydrogen bonds or charge−charge

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EXPERIMENTAL SECTION

Strains and Reagents. The Escherichia coli strain DH5α, for storage of recombinant plasmids, was purchased from Sangon

Figure 5. Molecular models of the interactions between 1 and rα3β4 nAChR and between 1 and rα6β4 nAChR. (A) Overview of the 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 the nAChR subunits and 1 colored as in panels A and B. The double-headed arrow indicates the shift in conformation of the nAChR binding sites and of the binding modes of 1. (C) Overview of the 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 (−5kT/e) to white (0kT/e) to blue (5kT/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-conotoxin PeIA (PDB: 2uz6)36 and the structure of human α4β2 nAChR bound to nicotine (PDB: 5kxi)19 were used as the starting points for molecular modeling. 9261

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previously reported.24 The rα6/α3 chimera clone was generously provided by J. E. Garrett (Cognetix, Inc., Salt Lake City, UT). 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 Ingenieriá Genética y Biologiá Molecular, Buenos Aires, Argentina). Female Xenopus laevis used for experiments were obtained from Nasco (Fort Atkinson, WI), which were housed at 17 °C in an SPF laboratory animal room and fed twice a week. Peptide Synthesis. The synthesis of the linear peptide analogues was performed by GL Biochem (Shanghai, China) using the solidphase-peptide-synthesis (SPPS) method. The Cys residues of all analogues were protected in pairs with S-trityl (Trt) on the first and third Cys residues, whereas S-acetamidomethyl (Acm) was incorporated on the second and fourth Cys residues. Following addition of 60% ACN and purification by reversed-phase high-performance liquid chromatography (RP-HPLC), the linear peptides were oxidized by a two-step-oxidation protocol as previously described (Scheme 1).25,26 The HPLC separation program for the purification of all linear, monocyclic, and bicyclic peptides 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, and solvent B was 90% ACN and 10% ddH2O with 0.05% TFA). The UVabsorption wavelength was monitored at 214 nm. The purities (>95%) of all the mature peptides were confirmed by analytical RPHPLC 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 identities of the mature peptides were confirmed by HRMS spectra, which were run on an LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, Japan) 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 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 purities and concentrations of the cRNAs for the various nAChR subunits were assessed by their absorbances 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. The cRNA for each subtype was combined in equimolar ratios and subsequently injected into Xenopus oocytes with a minimum of 10 ng for each subunit within 24 h of oocyte harvest. The oocytes were incubated at 17 °C with 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 postinjection, 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. While recording the electrophysiological data, the oocytes were voltage-clamped at a potential of −70 mV, applied as a 1 s ACh pulse per minute. Once a stable baseline was obtained, the oocytes expressing each kind of nAChR were preincubated for 5 min with either ND96 alone or conotoxins of different concentrations prior to the ACh pulse. The ACh concentrations for the nAChRs were 10 μM for rα1β1δγ and rα9α10, 200 μM for rα7, and 100 μM for all other subtypes.28,30 Data were recorded at 50 Hz and filtered

Figure 6. Inhibition of β4-containing nAChR subtypes by 7. (A,B) Inhibition by 100 nM 7 of rα3β4 and rα6/α3β4 nAChR subtypes. The block by 7 of these receptors was difficult to reverse, which indicated the increase of affinity to main receptors as a result of the S9F mutation. (C,D) Inhibition by 10 μM peptide 7 of rα2β4 and rα4β4 nAChR subtypes. (E) Dose−response curves of 7 with all β4containing nAChR subtypes. Each point is presented as the average ± SEM of 3−6 separate oocytes.

Table 4. IC50 and Hill-Slope Values for 7 on β4-Containing nAChR Subtypesa nAChR subtypes

IC50 (nM)

Hill slope

α2β4 α3β4b α4β4 α6/α3β4b

320 (250−410) 11 (8.4−14) 1730 (1410−2130) 10 (8.5−12)

0.9 (0.7−1.1) 1.8 (1.3−2.3) 1.3 (1−1.6) 1.9 (1.2−2.6)

Numbers in parentheses are 95% confidence intervals. All β4containing receptors are rat. bThe blocks of 7 on rα3β4 and rα6/ α3β4 nAChRs were difficult to wash out. a

(Shanghai, China). All restriction enzymes and DNA and RNA ladders were purchased from TaKaRa (Dalian, China). Reversedphase C18 Vydac columns (5 μm, 4.6 × 250 mm; 10 μm, 22 × 250 mm) were purchased from Grace Vydac (Hesperia, CA). Acetylcholine chloride, atropine, collagenase A, and bovine serum albumin (BSA) were obtained from Sigma (St. Louis, MO). Acetonitrile (ACN, HPLC grade) was purchased from Fisher Scientific (Pittsburgh, PA). Trifluoroacetic acid (TFA) was purchased from Tedia Company (Fairfield, OH). A cRNA mMESSAGE mMACHINE In Vitro Transcription Kit and an RNA MEGA Clear Kit were purchased from Ambion (Austin, TX). Other chemical reagents were all of analytical grade. cDNA clones encoding rat (r) α2, α3, α4, α7, β2, β3, and β4 and mouse (m) α1, β1, ε, and δ subunits were kindly provided by S. Heinemann (Salk Institute, San Diego, CA). Because the expression of the native α6 subunit is poor, an α6/α3 chimera containing the Nterminal, extracellular, ligand-binding portion of the α6 subunit with the remainder of the α3 subunit was utilized as a substitute, as 9262

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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 the percent response for different concentrations of conotoxins. All IC50 values and dose−response curves were obtained by fitting to the following nonlinear-regression equation using Graphpad Prism 6.0 (Graphpad Software, San Diego, CA): response (%) = 100/[1 + ([toxin]/IC50)nH], in which nH is the Hill coefficient.31 Each point of the dose−response curve was presented as the average ± standard error of the mean (SEM) for at least three panel data of percent-response recordings.11 NMR Spectroscopy and Structure Calculations. The NMR spectra of 12 were recorded as described previously for 111 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 13C- HSQC) were acquired using the 30% d3-TFE/ 70% H2O sample at 308 K on a Bruker Avance III 600 MHz (Billerica, MA) 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-2silapentane-5-sulfonate (DSS) at 0 ppm. Structures were generated using CYANA,33 and the 20 structures with the lowest energies and best qualities were analyzed using MolProbity.34 Molecular Modeling. Molecular models of the complexes between 1 and the ligand binding domains of α3β4 nAChR and α6β4 nAChR were built by homology and refined using moleculardynamics (MD) simulations, following an approach similar but not identical to that used in one of our previous studies.12 The initial models were built by homology modeling using Modeler 9v1535 and the crystal structures of Aplysia californica AChBP bound to αconotoxin PeIA (PDB: 2uz6)36 and human α4β2 nAChR bound to nicotine (PDB: 5kxi) as templates.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 into the system to reach a 150 mM sodium chloride concentration as well as to neutralize the system. The systems were heated over 100 ps to 300 K, during which time the protein and peptide heavy atoms were restrained to their positions using a force constant of 100 kcal/mol/Å2. The restraints were progressively removed over 20 ns, except for 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 the temperature and pressure at 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 out 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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as observed by HRMS and compared to the theoretical values, and NMR chemical-shift data of peptide 12 (PDF) Molecular-dynamics simulation of the complex between 1 and the binding site of α6β4 nAChR (PDB) Molecular-dynamics simulation of the complex between 1 and the binding site of α3β4 nAChR (PDB)

AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected] (S.L.) *E-mail: [email protected] (D.Z.) ORCID

Quentin Kaas: 0000-0001-9988-6152 David J. Craik: 0000-0003-0007-6796 Sulan Luo: 0000-0002-3568-5797 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.

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ACKNOWLEDGMENTS This work was supported in part by the Major International Joint Research Project of the National Natural Science Foundation of China (81420108028), the National Natural Science Foundation of China (31760249), the Changjiang Scholars and Innovative Research Team in University Grant IRT_15R15, the Hainan Provincial Department of Education (Hnky2017-16), and the Australian Research Council (ARC, DP150103990). D.J.C. is an ARC Australian Laureate Fellow (FL150100146).

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ABBREVIATIONS USED ACh, acetylcholine; nAChRs, nicotinic acetylcholine receptors; 18-MC, 18-methoxycoronaridine; AChBPs, ACh-binding proteins; BSA, bovine serum albumin; SPPS, solid-phase peptide synthesis; Trt, S-trityl; Acm, S-acetamidomethyl; RPHPLC, reversed-phase high-performance liquid chromatography; ESI-MS, electrospray-ionization mass spectrometry; SEM, standard error of the mean; Abu (B), 2-aminobutyric acid

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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. 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 Discovery 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.; DavilaGarcia, 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.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.8b00967. Analytical RP-HPLC and HRMS analysis of peptide 1 and its analogues, backbone root-mean-square deviations of nAChR and the toxins from their starting conformations for the 200 ns molecular dynamics simulations of the α3β4−TxID and α6β4−TxID systems, molecular masses of peptide 1 and its analogues 9263

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DOI: 10.1021/acs.jmedchem.8b00967 J. Med. Chem. 2018, 61, 9256−9265

Journal of Medicinal Chemistry

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DOI: 10.1021/acs.jmedchem.8b00967 J. Med. Chem. 2018, 61, 9256−9265