Rational Design of Potent α-Conotoxin PeIA Analogues with Non-Natural Amino Acids for the Inhibition of Human α9α10 Nicotinic Acetylcholine Receptors

α-Conotoxins (α-CTxs) are structurally related peptides that antagonize nicotinic acetylcholine receptors (nAChRs), which may serve as new alternatives to opioid-based treatment for pain-related conditions. The non-natural amino acid analogues of α-CTxs have been demonstrated with improved potency compared to the native peptide. In this study, we chemically synthesized Dab/Dap-substituted analogues of α-CTx PeIA and evaluated their activity at heterologously expressed human α9α10 nAChRs. PeIA[S4Dap, S9Dap] had the most potent half-maximal inhibitory concentration (IC50) of 0.93 nM. Molecular dynamic simulations suggested that the side chain amino group of Dap4 formed additional hydrogen bonds with S168 and D169 of the receptor and Dap9 formed an extra hydrogen bond interaction with Q34, which is distinctive to PeIA. Overall, our findings provide new insights into further development of more potent analogues of α-CTxs, and PeIA[S4Dap, S9Dap] has potential as a drug candidate for the treatment of chronic neuropathic pain.


Introduction
Chronic pain is a significant problem that threatens human health worldwide, but opioid-based medications can lead to drug tolerance and addiction.Thus, non-opioid therapeutics are urgently needed [1,2].Previous studies have shown that several subtypes of nAChRs are associated with pain and may serve as new treatment targets [3].
nAChRs belong to the Cys-loop superfamily and are prototypical characterized members of pentameric ligand-gated ion channels (LGICs) assembled from homologous subunits (α1-α10, β1-β4, δ, ε, and γ,) [4].The muscle-type nAChRs consist of α1, β1, δ, and ε/γ subunits, whereas other subunits form distinct homomeric or heteromeric nAChRs, such as α7, α3β2, α3β4, and α9α10 [5].The diverse subunit compositions of nAChRs give rise to many subtypes with differences in ion channel function and pharmacology.The two adjacent cysteines in loop C of the α subunit are essential for ACh to activate nAChRs, and the binding sites of nAChRs are located at the interface of two adjacent α and α/β subunits [5].There are several nAChR subtypes associated with pain, including α3-containing nAChRs such as α3β2 and α3β4 that are expressed in primary afferent nerve terminals or the spinal cord, which may mediate anti-allodynic actions [6].Additionally, α6β4containing nAChRs may be involved in the processing of pain-associated information and activation of these nAChRs could produce analgesic effects via purinergic (PX2) receptor inhibition [7].The α7 subtype is widely distributed in the nervous system and expressed by many non-neuronal cells (microglial, lymphocytes, and macrophage cells), and targeting α7 nAChRs has proven to be effective in attenuating neuropathic and inflammatory pain in animal models [8].The α9α10 nAChRs (Figure 1A,B) have been studied extensively and the analgesic effects of several α9α10 nAChR antagonists have been demonstrated in animal models for chronic constriction injury [9,10], oxaliplatin-induced neuropathy [11], and partial nerve ligation [10].The vital functions of α9α10 nAChRs in modulating the pathophysiology changes associated with traumatic and nerve injury [12] provide an initial basis for the development of non-opioid analgesic drugs that target these receptors.
nAChRs may be involved in the processing of pain-associated information and activation of these nAChRs could produce analgesic effects via purinergic (PX2) receptor inhibition [7].The α7 subtype is widely distributed in the nervous system and expressed by many non-neuronal cells (microglial, lymphocytes, and macrophage cells), and targeting α7 nA-ChRs has proven to be effective in attenuating neuropathic and inflammatory pain in animal models [8].The α9α10 nAChRs (Figure 1A,B) have been studied extensively and the analgesic effects of several α9α10 nAChR antagonists have been demonstrated in animal models for chronic constriction injury [9,10], oxaliplatin-induced neuropathy [11], and partial nerve ligation [10].The vital functions of α9α10 nAChRs in modulating the pathophysiology changes associated with traumatic and nerve injury [12] provide an initial basis for the development of non-opioid analgesic drugs that target these receptors.
Conotoxins (CTxs) derived from the venom of Conus marine snails are disulfide-rich peptides for rapid prey immobilization [13].Among them, α-CTxs are the most characterized and studied family, which are structurally related peptides that target nAChRs [14].The α-CTxs consist of 12~20 residues with four cysteines that form two disulfide bridges to provide conformation stability as well as interactions with receptors [15,16].The high affinity and selectivity of α-CTxs towards particular nAChR subtypes, coupled with low toxicity, give them an advantage as potential therapeutic candidates [17].(A,B) The extracellular ligand binding domain of the hα9α10 nAChR, based on the crystal structure of Aplysia californica acetylcholine binding protein (AChBP) in complex with PeIA (PDB Code: 5JME), was built using Modeler and AlphaFold2 as described in our previous study [9].(C,D) The (A,B) The extracellular ligand binding domain of the hα9α10 nAChR, based on the crystal structure of Aplysia californica acetylcholine binding protein (AChBP) in complex with PeIA (PDB Code: 5JME), was built using Modeler and AlphaFold2 as described in our previous study [9].(C,D) The backbone and side chains of PeIA are shown as a ribbon and sticks, respectively.α-Conotoxin PeIA is a 16amino-acid peptide that has two disulfide bonds and an amidated C-terminus (represented by "*" in the sequence).
Conotoxins (CTxs) derived from the venom of Conus marine snails are disulfide-rich peptides for rapid prey immobilization [13].Among them, α-CTxs are the most characterized and studied family, which are structurally related peptides that target nAChRs [14].The α-CTxs consist of 12~20 residues with four cysteines that form two disulfide bridges to provide conformation stability as well as interactions with receptors [15,16].The high affinity and selectivity of α-CTxs towards particular nAChR subtypes, coupled with low toxicity, give them an advantage as potential therapeutic candidates [17].

Design and Synthesis of PeIA Analogues
Based on the same structural subclass and the sequence similarity of PeIA, Vc1.1, and Mr1.1 (Figure 2A), in addition to similarities between the sequence in Loop 1 and the charge distribution in Loop 2 of PeIA and Mr1.1, we attempted to apply this strategy to PeIA by the substitution of Ser4 with Dab or Dap, Ser9 with Dap, or both Ser4 and Ser9 with Dap (Figure 2B).The peptides were synthesized by solid-phase peptide synthesis (SPPS) on Rink-Amide-MBHA resin, followed by peptide cleavage from the resin (Figure 2C).Regioselective oxidation was performed at CysI-CysIII and CysII-CysIV, and the Cys was introduced in pairs with Fmoc-Cys(Acm)-OH and Fmoc-Cys(Trt)-OH.Finally, the peptide was purified by preparative high-performance liquid chromatography (HPLC) with C 18 columns, followed by mass spectrometry (MS) and analytical HPLC to verify the molecular mass and purity of the peptides (Figures S7-S11).

Structural Analysis of the Designed PeIA Analogues
Circular dichroism (CD) spectroscopy was used to characterize the secondary structures of the designed analogues and PeIA (Figure 3).The four analogues exhibited significant negative absorption at 208 nM, similar to PeIA.The results indicated that the Dab/Dap-substituted analogues had minimal impact on the secondary structure of PeIA.

Structural Analysis of the Designed PeIA Analogues
Circular dichroism (CD) spectroscopy was used to characterize the secondary structures of the designed analogues and PeIA (Figure 3).The four analogues exhibited significant negative absorption at 208 nM, similar to PeIA.The results indicated that the Dab/Dap-substituted analogues had minimal impact on the secondary structure of PeIA.

Structural Analysis of the Designed PeIA Analogues
Circular dichroism (CD) spectroscopy was used to characterize the secondary structures of the designed analogues and PeIA (Figure 3).The four analogues exhibited significant negative absorption at 208 nM, similar to PeIA.The results indicated that the Dab/Dap-substituted analogues had minimal impact on the secondary structure of PeIA.
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Molecular Dynamic (MD) Simulations of PeIA and Its Analogue in Complex with Human α9α10 nAChRs
To explain the significantly improved potencies of the most potent analogu PeIA[S4Dap, S9Dap], we established the complex structure model of the peptide an hα9α10 nAChR using MD simulations (Figures 6 and S3-S6, Table S1) based on the mod els of Mr1.1 and Vc1.1 [9,20].The Ser residue at position 4 in PeIA possesses a hydroxy side chain, whereas the substituted Dap residue contains an amino side chain.The amin

Molecular Dynamic (MD) Simulations of PeIA and Its Analogue in Complex with Human α9α10 nAChRs
To explain the significantly improved potencies of the most potent analogue PeIA[S4Dap, S9Dap], we established the complex structure model of the peptide and hα9α10 nAChR using MD simulations (Figure 6 and Figure S3-S6, Table S1) based on the models of Mr1.1 and Vc1.1 [9,20].The Ser residue at position 4 in PeIA possesses a hydroxyl side chain, whereas the substituted Dap residue contains an amino side chain.The amino group formed three hydrogen bonds with D166, S168, and D169 at the α9(+)-α9(−) interface, whereas S4 of PeIA only had one hydrogen bond with D169.Moreover, Dap9 formed a hydrogen bond interaction with Q34 in the α9(-) subunit.The formation of extra hydrogen bonds as well as the electrostatic interactions between Dap and residues at the α9(+)-α9(−) are postulated to contribute to the significantly enhanced activity of PeIA[S4Dap, S9Dap] compared to the native PeIA.The α9(+)α9(−) binding site was generated using the crystal structure of A. califo nica AChBP in complex with PeIA as a template (PDB Code: 5JME), as described in our previo study [9].The parameters of non-natural amino acids were built in AMBER22.The complex mode were refined using MD stimulations in AMBER 22.
At the heterologous human nAChR subtypes tested, PeIA is most selective f hα9α10 followed by the hα3β2 subtype, and PeIA[S4Dap, S9Dap] retains the α9α10-sele tivity of PeIA.Remarkably, compared to the absence of inhibition by PeIA, the analogu has enhanced potency at hα7 nAChRs.Despite this, it has been proposed that activatio of the α7 subtype, as opposed to inhibition, mediates the attenuation of neuropathic pa in animal models [23][24][25].For hα9α10 nAChRs, the ~24-fold increase in potency f PeIA[S4Dap, S9Dap] compared to the native PeIA resulted in one of the most pote hα9α10 antagonists.To date, only two analogues of RgIA, RgIA-5524 and RgIA-5474, hav been reported to antagonize the hα9α10 subtype with IC50 values below 1 nM (0.9 and 0.0 nM, respectively) [26,27].Previously, substantial efforts have been made to optimize th structure of PeIA.Liang et al. [19] engineered dimeric PeIA with an 11-fold enhanced a tivity and an IC50 of 1.9 nM, presumably via simultaneous interactions with neighborin ] at the hα9α10 nAChR.The α9(+)α9(−) binding site was generated using the crystal structure of A. californica AChBP in complex with PeIA as a template (PDB Code: 5JME), as described in our previous study [9].The parameters of non-natural amino acids were built in AMBER22.The complex models were refined using MD stimulations in AMBER 22.
At the heterologous human nAChR subtypes tested, PeIA is most selective for hα9α10 followed by the hα3β2 subtype, and PeIA[S4Dap, S9Dap] retains the α9α10-selectivity of PeIA.Remarkably, compared to the absence of inhibition by PeIA, the analogue has enhanced potency at hα7 nAChRs.Despite this, it has been proposed that activation of the α7 subtype, as opposed to inhibition, mediates the attenuation of neuropathic pain in animal models [23][24][25].For hα9α10 nAChRs, the ~24-fold increase in potency for PeIA[S4Dap, S9Dap] compared to the native PeIA resulted in one of the most potent hα9α10 antagonists.To date, only two analogues of RgIA, RgIA-5524 and RgIA-5474, have been reported to antagonize the hα9α10 subtype with IC 50 values below 1 nM (0.9 and 0.05 nM, respectively) [26,27].Previously, substantial efforts have been made to optimize the structure of PeIA.Liang et al. [19] engineered dimeric PeIA with an 11-fold enhanced activity and an IC 50 of 1.9 nM, presumably via simultaneous interactions with neighboring binding sites on the receptor.Taken together, the potency of PeIA[S4Dap, S9Dap] is approximately two-fold higher than dimeric PeIA, and the synthesis procedure for the former is more facile.Thus, PeIA[S4Dap, S9Dap] possesses an advantage as a potential drug candidate in the treatment of pain-related conditions.

Peptide Synthesis
Peptides were synthesized by solid-phase peptide synthesis (SPPS).Rink amide MBHA resin (0.1 mmol; SV = 0.6 mmol/g) was used as a carrier for amino acid coupling for the amidated C-terminus of the peptide.There were 4 cysteine residues in the peptide, and an acid-labile trityl (Trt) protecting group was introduced in Cys2 and Cys8, while Cys3 and Cys16 were protected by acid-stable acetamidomethyl (Acm) to realize orthogonal oxidation.The specific experimental steps were described as follows: 166 mg of resin was put in a reaction vessel and swelled using a mixture solution of DMF/DCM (1:1).Before coupling, a 20% piperidine solution in DMF was used to deprotect the Fmoc (9fluorenylmethyloxycarbonyl) protecting group.The carboxyl group of the newly introduced N-Fmoc-protected residue was coupled with the amino group of the resin using HCTU (4.0 equiv) and DIPEA (8.0 equiv) at room temperature for an hour to realize peptide synthesis.The deprotection and coupling procedures were repeated until the full sequence was synthesized, and the terminal Fmoc group was removed.Then, a cleavage cocktail (TFA/TIPS/H 2 O, 90/5/5, v/v/v) was added to cleave the peptide for 3 h.After sufficient reaction, the solution was filtrated and then concentrated, precipitated with cold ether, and centrifuged to obtain the synthesized peptide.The molecular weight of the crude peptide was detected by Electrospray Ionization Mass Spectrometry (ESI-MS) (Figures S7-S9).

Disulfide Bond Formation between Cys2 and Cys8
The Trt group was removed along with peptide cleavage.The crude peptide was dissolved in a mixture of H 2 O/MeCN, and 1 equiv of 2,2 ′ -dithiodipyridine (DTDP) in MeOH was added at a slow rate.The solvent was then stirred at room temperature for 1 h.

Disulfide Bond Formation between Cys3 and Cys16
I 2 -mediated oxidation was used to form the second disulfide bond.The purified peptide was dissolved in H 2 O/MeCN (0.5 mg/mL), and superfluous I 2 in MeCN was added until the solution turned yellowish brown.After stirring at room temperature for 3 h, the reaction was quenched by ascorbic acid.

Peptide Purification
Semipreparative reverse-phase high-performance liquid chromatography (RP-HPLC) with a C 18 column was carried out in the purification assay.Deionized H 2 O and chromatographic grade MeCN were the components of the moving phase.The H 2 O/MeCN gradient at a flow rate of 6 mL/min containing 0.05% trifluoroacetic acid (TFA) is shown in Table 1.The analytical RP-HPLC used to determine the purity of the peptide and the H 2 O/MeCN gradient are shown in Table 1.

Circular Dichroism
Circular dichroism (CD) spectra were performed on Jasco J-810 spectropolarimeter over a wavelength range of 250-190 nm using a 1.0 mm path length cell, a bandwidth of 1.0 nm, a response time of 2 s, and averaging over three scans.Spectra were recorded at alone-evoked current amplitude was used to assess the activity of the peptides at nAChRs.Electrophysiological data were combined (n = 5-15) and represent the means ± standard deviations (SD).Data analysis was performed by GraphPad Prism 9 (GraphPad Software, version 9.1.0,La Jolla, CA, USA).α-CTx IC 50 values were determined from concentrationresponse relationships fitted to a non-linear regression function and reported with 95% confidence intervals.

Construction of Non-Natural Amino Acid Parameters
The three-dimensional spatial structure of α-CTx PeIA and its non-natural amino acid-substituted analogues were constructed using Mr1.1 as a template.Based on the structural similarity between the two α-CTxs, the molecular and non-natural amino acids were constructed using the protein builder and the molecule builder module of MOE followed by molecular optimization.Due to only the coordinate space of 20 natural amino acids being identified, the parameters of Dap needed preparation.Here, the Antechamber module of AMBER22 was used to conduct this experiment as described previously [19].

Molecular Dynamics Simulation
The structure of α-CTx/α9α10nAChR was obtained by modification of the Ser4/Ser9 to Dap from our previous MD refined model between Vc1.1/α9α10nAChR [20].Parameters for the non-natural amino acids were prepared in the Antechamber module of AM-BER22.R.E.D Tools were used to produce the atom partial charges for pyroglutamic acid.The PeIA/α9α10nAChR complexes were solvated at a size distance of a 10 Å truncated regular octahedron TIP3P water box in AMBER22.Sodium ion was added to neutralize the whole system, and then the energy of the system was optimized.Firstly, a harmonic force with an elastic constant of 100 kcal mol −1 •Å −2 was used to constrain the solute, and 3000 steps of the steepest descent method and the 3000-step conjugate gradient method were used to optimize the system.Secondly, the second round of optimization was carried out with all position restrictions withdrawn.The temperature of the whole system was gradually heated from 50 K to 300 K over 100 ps in the NVT complex, and over 100 ps and a 5 kcal mol −1 •Å −2 harmonic force were used to restrain the position of the solute.In the isothermal-isobaric (NPT) ensemble, MD simulations were performed with the position restrictions gradually removed.In this work, MD simulations were performed over 50 ns with the pressure and the temperature maintained at 1 atmosphere and 300 K, respectively.Visual Molecular Dynamics (VMD) was used to analyze the trajectories and the root mean square deviation (RMSD) of the backbone was calculated.

Conclusions
The introduction of non-natural amino acids to peptides or proteins can provide unique three-dimensional spatial structures with unique functionalities.In recent years, many conotoxins containing non-natural amino acids have been engineered to improve the physicochemical properties of peptides, including activity, selectivity, and serum stability.The replacement of Asn11 with non-natural amino acids with a negatively charged side chain and α-aminopimelic acid (Api) produced the PeIA-5466 analogue with ~300-fold greater selectivity for α3β2 than α6/α3β2β3 subtypes [28].In addition, the RgIA4 analogue was designed by substituting Arg9 and Tyr10 residues with Cit and 3-I-Try, respectively, which exhibited high potency at both human and rodent α9α10 nAChRs while retaining selectivity for the α9α10 subtype [29].
In this study, the non-natural amino acids Dab and Dap were used to substitute the Ser residue to optimize the activity of PeIA at inhibiting the hα9α10 nAChR.The IC 50 values of the PeIA[S4Dab], PeIA[S4Dap], PeIA[S9Dap], and PeIA[S4Dap, S9Dap] were improved by ~4-, 13-, 5-, and 24-fold, respectively, compared to PeIA.To date, PeIA[S4Dap, S9Dap] is one of the most potent peptide antagonists of hα9α10 nAChRs.Additionally, we performed an MD simulation to reveal additional interactions that may explain the enhanced potencies of the designed analogues.Overall, we provided a simple and effective way to improve the potency of α-CTx PeIA, and the PeIA[S4Dap, S9Dap] analogue has therapeutic potential to be explored in the future.

Figure 1 .
Figure 1.The extracellular domain of the hα9α10 nAChR and the structure of α-conotoxin PeIA.(A,B)The extracellular ligand binding domain of the hα9α10 nAChR, based on the crystal structure of Aplysia californica acetylcholine binding protein (AChBP) in complex with PeIA (PDB Code: 5JME), was built using Modeler and AlphaFold2 as described in our previous study[9].(C,D) The

Figure 1 .
Figure 1.The extracellular domain of the hα9α10 nAChR and the structure of α-conotoxin PeIA.(A,B)The extracellular ligand binding domain of the hα9α10 nAChR, based on the crystal structure of Aplysia californica acetylcholine binding protein (AChBP) in complex with PeIA (PDB Code: 5JME), was built using Modeler and AlphaFold2 as described in our previous study[9].(C,D) The backbone and side chains of PeIA are shown as a ribbon and sticks, respectively.α-Conotoxin PeIA is a 16amino-acid peptide that has two disulfide bonds and an amidated C-terminus (represented by "*" in the sequence).

Figure 2 .
Figure 2. Sequences of α-conotoxins and the synthesis of the PeIA analogues.(A) Sequences of αconotoxins containing two disulfide bonds (* represents the amidated C-terminus).Vc1.1, PeIA, and Mr1.1 belong to the 4/7 α-CTx subtype.(B) The Dab/Dap-substituted analogues of PeIA.The residue Dab/Dap is shown in red.(C) The synthesis of Dab/Dap-substituted analogues of PeIA using PeIA[S4Dab] as an example.The chemical structure was drawn using ChemBioDraw Ultra 14.0.
Mar. Drugs 2024,22,  x FOR PEER REVIEW 7 of group formed three hydrogen bonds with D166, S168, and D169 at the α9(+)-α9(−) inte face, whereas S4 of PeIA only had one hydrogen bond with D169.Moreover, Dap9 forme a hydrogen bond interaction with Q34 in the α9(-) subunit.The formation of extra hydr gen bonds as well as the electrostatic interactions between Dap and residues at the α9(+ α9(−) are postulated to contribute to the significantly enhanced activity of PeIA[S4Da S9Dap] compared to the native PeIA.

Figure 6 .
Figure 6.Binding modes of PeIA and PeIA[S4Dap,S9Dap] at the α9(+)α9(−) binding site.(A) T binding modes of PeIA at the hα9α10 nAChR.(B)The binding modes of PeIA[S4Dap, S9Dap] at t hα9α10 nAChR.The α9(+)α9(−) binding site was generated using the crystal structure of A. califo nica AChBP in complex with PeIA as a template (PDB Code: 5JME), as described in our previo study[9].The parameters of non-natural amino acids were built in AMBER22.The complex mode were refined using MD stimulations inAMBER 22.

Figure 6 .
Figure 6.Binding modes of PeIA and PeIA[S4Dap,S9Dap] at the α9(+)α9(−) binding site.(A) The binding modes of PeIA at the hα9α10 nAChR.(B) The binding modes of PeIA[S4Dap, S9Dap] at the hα9α10 nAChR.The α9(+)α9(−) binding site was generated using the crystal structure of A. californica AChBP in complex with PeIA as a template (PDB Code: 5JME), as described in our previous study[9].The parameters of non-natural amino acids were built in AMBER22.The complex models were refined using MD stimulations inAMBER 22.