A Single Amino Acid Replacement Boosts the Analgesic Activity of α-Conotoxin AuIB through the Inhibition of the GABABR-Coupled N-Type Calcium Channel

α-conotoxin AuIB is the only one of the 4/6 type α-conotoxins (α-CTxs) that inhibits the γ-aminobutyric acid receptor B (GABABR)-coupled N-type calcium channel (CaV2.2). To improve its inhibitory activity, a series of variants were synthesized and evaluated according to the structure–activity relationships of 4/7 type α-CTxs targeting GABABR-coupled CaV2.2. Surprisingly, only the substitution of Pro7 with Arg results in a 2–3-fold increase in the inhibition of GABABR-coupled CaV2.2 (IC50 is 0.74 nM); substitutions of position 9–12 with basic or hydrophobic amino acid and the addition of hydrophobic amino acid Leu or Ile at the second loop to mimic 4/7 type α-CTxs all failed to improve the inhibitory activity of AuIB against GABABR-coupled CaV2.2. Interestingly, the most potent form of AuIB[P7R] has disulfide bridges of “1–4, 2–3” (ribbon), which differs from the “1–3, 2–4” (globular) in the isoforms of wildtype AuIB. In addition, AuIB[P7R](globular) displays potent analgesic activity in the acetic acid writhing model and the partial sciatic nerve injury (PNL) model. Our study demonstrated that 4/6 type α-CTxs, with the disulfide bridge connectivity “1–4, 2–3,” are also potent inhibitors for GABABR-coupled CaV2.2, exhibiting potent analgesic activity.

To improve the potency of 4/6-type α-conotoxins against GABA B R-coupled Ca V 2.2, in the present study, a series of AuIB variants were designed and synthesized according to the structure-activity of 4/7 type α-CTxs (Table 2) targeting GABA B R-coupled Ca V 2.2 [11,12,14]. The inhibitory activity of variants against GABA B R-coupled Cav2.2 or Ca V 2.2 alone, expressed on HEK293, was evaluated using electrophysiological methods. The analgesic activities of AuIB and the most potent variant were also determined using the acetic acid writhing model, the hot plate model, and the partial sciatic nerve injury (PNL) model. We were surprised to find that only the substitution of Pro7 with Arg was able to increase the inhibitory activity of AuIB against GABA B R-coupled Ca V 2.2 (IC 50 is 0.74 nM). The AuIB variants with substitutions of amino acid at position 9-12 with basic or hydrophobic amino acid, as well as the addition of hydrophobic amino acid Leu or Ile at the second loop as 4/7 type α-CTxs, did not improve the inhibitory activity. The most potent isoform, AuIB[P7R], displays no significant inhibitory activity against nAChRs, including the α3β4 subtype. In addition, AuIB[P7R] is the ribbon isomer with the disulfide bridges "1-4, 2-3," but not "1-3, 2-4"(globular), as in wildtype AuIB. Finally, AuIB[P7R](ribbon) displays potent analgesic activity in the acetic acid writhing model and the partial sciatic nerve injury (PNL) model. Our study provides a new potent GABA B R-coupled Ca V 2.2 inhibitor, demonstrating that 4/6 type α-CTx, with the disulfide bridge connectivity "1-4, 2-3," is also a potent motif for designing potent inhibitors of GABA B R-coupled Ca V 2.2.

Synthesis and Characterization of AuIB and Its Variants
Most linear 4/6-type AuIB and its variants fold into two main products in 0.1 M NH 4 HCO 3 buffer (pH 8.0-8.2). The analyses of the folding products of AuIB[P7R] is shown in Figure 1A. All folding products (Table 1) were purified and assessed with analytical reversed-phase HPLC, and the purity of the peptides was greater than 95%. All peptides exhibited the expected molecular weights, as ascertained by mass spectrometry.
The disulfide bond connectivity of AuIB was confirmed, according to the methods of previous studies [21]. The disulfide bond connectivity of the AuIB mutant AuIB[P7R] was determined using the two-step synthesis method. The HPLC results of the one-step and two-step folding of the acetamidomethyl (Acm)-protected linear peptides are shown in Figure 1B,C. The disulfide bond connectivity of the AuIB[P7R]-I peak is "C1-C3, C2-C4" (globular, Figure 1B), and the disulfide bond connectivity of AuIB[P7R]-II is "C1-C4, C2-C3" (ribbon, Figure 1C). The elution time is different from that of wild type AuIB; the isomer AuIB(ribbon) was eluted first, but AuIB(globular) was eluted later [29].
dues, as well as 2 disulfide bonds [24]. Its analgesic activity has been recognized to originate from the inhibition of GABABR-coupled CaV2.2, since it only displays modest inhibition of the α3β4 nAChR subtype [27,28]. To date, no study of its structure-activity relationship has been reported regarding its inhibition of GABABR-coupled CaV2.2.
To improve the potency of 4/6-type α-conotoxins against GABABR-coupled CaV2.2, in the present study, a series of AuIB variants were designed and synthesized according to the structure-activity of 4/7 type α-CTxs (Table 2) targeting GABABR-coupled CaV2.2 [11,12,14]. The inhibitory activity of variants against GABABR-coupled Cav2.2 or CaV2.2 alone, expressed on HEK293, was evaluated using electrophysiological methods. The analgesic activities of AuIB and the most potent variant were also determined using the acetic acid writhing model, the hot plate model, and the partial sciatic nerve injury (PNL) model. We were surprised to find that only the substitution of Pro7 with Arg was able to increase the inhibitory activity of AuIB against GABABR-coupled CaV2.2 (IC50 is 0.74 nM). The AuIB variants with substitutions of amino acid at position 9-12 with basic or hydrophobic amino acid, as well as the addition of hydrophobic amino acid Leu or Ile at the second loop as 4/7 type α-CTxs, did not improve the inhibitory activity. The most potent isoform, AuIB[P7R], displays no significant inhibitory activity against nAChRs, including the α3β4 subtype. In addition, AuIB[P7R] is the ribbon isomer with the disulfide bridges "1-4, 2-3," but not "1-3, 2-4"(globular), as in wildtype AuIB. Finally, AuIB[P7R](ribbon) displays potent analgesic activity in the acetic acid writhing model and the partial sciatic nerve injury (PNL) model. Our study provides a new potent GABABR-coupled CaV2.2 inhibitor, demonstrating that 4/6 type α-CTx, with the disulfide bridge connectivity "1-4, 2-3," is also a potent motif for designing potent inhibitors of GABABR-coupled CaV2.2.

Synthesis and Characterization of AuIB and Its Variants
Most linear 4/6-type AuIB and its variants fold into two main products in 0.1 M NH4HCO3 buffer (pH 8.0-8.2). The analyses of the folding products of AuIB[P7R] is shown in Figure 1A. All folding products (Table 1) were purified and assessed with analytical reversed-phase HPLC, and the purity of the peptides was greater than 95%. All peptides exhibited the expected molecular weights, as ascertained by mass spectrometry.
The disulfide bond connectivity of AuIB was confirmed, according to the methods of previous studies [21]. The disulfide bond connectivity of the AuIB mutant AuIB[P7R] was determined using the two-step synthesis method. The HPLC results of the one-step and two-step folding of the acetamidomethyl (Acm)-protected linear peptides are shown in Figure 1B,C. The disulfide bond connectivity of the AuIB[P7R]-I peak is "C1-C3, C2-C4" (globular, Figure 1B), and the disulfide bond connectivity of AuIB[P7R]-II is "C1-C4, C2-C3" (ribbon, Figure 1C). The elution time is different from that of wild type AuIB; the isomer AuIB(ribbon) was eluted first, but AuIB(globular) was eluted later [29].  To improve the potency of the AuIB variants, a series of 4/6 type AuIB variants 2-18 (Table 1) were synthesized according to the structure-activity of 4/7 type α-CTxs targeting GABA B R-coupled Ca V 2.2 (Table 2), in which a basic acid at position 4, 7, 9, and 10, or an acidic amino acid at position 4 and 11, or the hydrophobic amino acid benzoyl group introduced at the N-terminus of Vc1.1 could enhance the potency [12,14,27]. In addition, 4/7 type AuIB variants 19-26 were also synthesized by the addition of a hydrophobic acid Leu at position 15.  Compared to 4/6 type AuIB variants, 4/7-type AuIB variants mainly formed a single primary product (data not shown), several AuIB variants, such as AuIB[A10R,+15L] and AuIB[P7R,+15L], also form two products [29].

Circular Dichroism of α-Conotoxin AuIB Variants
To determine if the difference in the potency of the AuIB variants resulted from their structures, we acquired the typical CD spectra of AuIB and its typical variants, shown in Figure 2. The results show that AuIB(globular) with the disulfide bond connectivity "C1-C3, C2-C4" and the 4/7-type AuIB variants have a low α-helix content, while others, including AuIB[P7R](globular) and AuIB[P7R](ribbon) possess no apparent α-helical structure.

Inhibitory Effects on GABABR-Coupled Cav2.2 Currents Induced by AuIB and Its Variants
The inhibitory activity of 1 μM of AuIB and its variants at the GABABR-coupled CaV2.2 channels was determined in the HEK293T cells (

Inhibitory Effects on GABA B R-Coupled Ca v 2.2 Currents Induced by AuIB and Its Variants
The inhibitory activity of 1 µM of AuIB and its variants at the GABA B R-coupled Ca V 2.2 channels was determined in the HEK293T cells ( In addition, the direct inhibitory effect of 10 µM AuIB[P7R](ribbon) on Ca V 2.2 was determined to be 14.87 ± 2.42%, much lower than that of GABA B R-coupled Ca v 2.2.

AuIB[P7R](ribbon) Significantly Reduces the Writhing Number in the Acetic Acid Writhing Model
The analgesic activity was first determined using the acetic acid writhing model. The Vc1.1 variant Benzoyl-Vc1.1[N9R] was selected as the positive control group because it possesses high analgesic activity and acts on GABABR-coupled CaV2.2 [12]. The results show that AuIB[P7R](ribbon) significantly reduces the writhing number ( Figure 5) compared to the saline group. Its analgesic activity is similar to Benzoyl-Vc1.  . Data were expressed as mean ± SEM (n = 10). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the saline group. All data are presented as mean ± SEM.

AuIB[P7R](ribbon) Also Displays Potent Analgesic Activity in the Rat Partial Sciatic Nerve Injury Model
The analgesic activity of AuIB(globular) and AuIB[P7R](ribbon) were also evaluated by the partial sciatic nerve injury (PNL) model. The results show that AuIB(globular) and AuIB[P7R](ribbon) have potent analgesic activity compared to the saline group. The PTEPs (mean pain threshold elevation percentages) values of each peptide group started to rise and reached the peak 2 h after ipsilateral muscular injection, then decreased to baseline levels around 6 h. Compared to Benzoyl-Vc1.  . Data were expressed as mean ± SEM (n = 10). * p < 0.05, ** p < 0.01, and *** p < 0.001 vs. the saline group. All data are presented as mean ± SEM.

AuIB[P7R](ribbon) Also Displays Potent Analgesic Activity in the Rat Partial Sciatic Nerve Injury Model
The analgesic activity of AuIB(globular) and AuIB[P7R](ribbon) were also evaluated by the partial sciatic nerve injury (PNL) model. The results show that AuIB(globular) and AuIB[P7R](ribbon) have potent analgesic activity compared to the saline group. The PTEPs (mean pain threshold elevation percentages) values of each peptide group started to rise and reached the peak 2 h after ipsilateral muscular injection, then decreased to baseline levels around 6 h. Compared to Benzoyl-Vc1.

Discussion
According to the structure-activity of 4/7-type α-CTxs, a series of AuIB variants at position 4, 5, 9, 10, 11, 12, and 15 were synthesized and evaluated. The results show that the substitutions or additions of one or two residues at the above position with basic or hydrophobic amino acid do not significantly enhance AuIB's potency to GAB-ABR-coupled CaV2.2. However, the substitution of Pro7 with Arg in AuIB[P7R](ribbon) with the disulfide bridges "1-4, 2-3" results in a 2-3-fold increase in the potency (IC50 is 0.74 nM). To our knowledge, AuIB[P7R](ribbon) is the most potent 4/6 type α-CTx targeting GABABR-coupled CaV2.2, and its potency is slightly lower than that of Benzoyl-Vc1.1[N9R], a known inhibitor of GABABR-coupled CaV2.2 [12]. The activity change trend may be related to the rigid structure of AuIB that has several Pro residues in loop1 and shorter amino acid residues in loop2. Interestingly, the further addition of a benzoyl group at N-terminus of AuIB[P7R] did not result in the increase in potency (Table 1), which is different from the results for Vc1. 1[N9R], where the addition of benzoyl group at its N-terminus significantly increased the inhibitory activity [12]. The benzoyl group may impair AuIB[P7R] binding to GABABR.

Discussion
According to the structure-activity of 4/7-type α-CTxs, a series of AuIB variants at position 4, 5, 9, 10, 11, 12, and 15 were synthesized and evaluated. The results show that the substitutions or additions of one or two residues at the above position with basic or hydrophobic amino acid do not significantly enhance AuIB's potency to GABA B R-coupled Ca V 2.2. However, the substitution of Pro7 with Arg in AuIB[P7R](ribbon) with the disulfide bridges "1-4, 2-3" results in a 2-3-fold increase in the potency (IC 50 is 0.74 nM). To our knowledge, AuIB[P7R](ribbon) is the most potent 4/6 type α-CTx targeting GABA B Rcoupled Ca V 2.2, and its potency is slightly lower than that of Benzoyl-Vc1.1[N9R], a known inhibitor of GABA B R-coupled Ca V 2.2 [12]. The activity change trend may be related to the rigid structure of AuIB that has several Pro residues in loop1 and shorter amino acid residues in loop2. Interestingly, the further addition of a benzoyl group at N-terminus of AuIB[P7R] did not result in the increase in potency (Table 1), which is different from the results for Vc1. 1[N9R], where the addition of benzoyl group at its N-terminus significantly increased the inhibitory activity [12]. The benzoyl group may impair AuIB[P7R] binding to GABA B R.
Interestingly, AuIB[P7R](ribbon) displays the greatest potency to GABA B R-coupled Ca V 2.2, but not the globular isomer of AuIB[P7R]. This character is also different from the isoforms of AuIB, in which the globular isomer is more potent than that of the ribbon isomer. In addition, AuIB[P7R](ribbon) displays no apparent inhibitory activity against the nAChRs subtypes at 10 µM. On the contrary, AuIB(globular) exhibits enhanced activity against α3β4 nAChRs compared to the ribbon isomer [29,30], but in the parasympathetic neurons, AuIB(ribbon) shows higher potency against whole nAchRs [31]. Furthermore, our experiments demonstrate that AuIB(globular) exhibits potent inhibitory activity against the GABA B R-coupled Ca V 2.2 ( Table 2), but not against its ribbon isomer. To date, only one 4/7-type α-CTx Lt1.3 with disulfide bridges (1-4, 2-3) has been found to target the GABA B R-coupled Ca V 2.2 [21], and AuIB[P7R](ribbon) is the first 4/6 type α-CTx with disulfide bridges (1-4, 2-3) inhibiting this target.
Finally, AuIB[P7R](ribbon) exhibits potent analgesic activity in the acetic acid writhing model and the rat partial sciatic nerve injury model (Figures 5 and 6). Compared to AuIB(globular), AuIB[P7R](ribbon) did not show higher analgesic activity in the acetic acid writhing model at the same dose (40 nmol/kg). This observation could potentially be explained by the compensatory action of AuIB(globular) on α3β4 nAChRs, which is a target for analgesic activity [10]. In addition, 40 nmol/kg AuIB(globular) and AuIB[P7R](ribbon) did not show apparent analgesic activity in the mice hot plate pain model (data not shown).
In summary, we found that a new 4/6 type α-conotoxin AuIB variant AuIB[P7R](ribbon) potently inhibits the GABA B R-coupled Ca V 2.2 with an IC 50 less than 1 nM, and the most potent isomer is the ribbon, but not the globular isomer. AuIB[P7R](ribbon) exhibits potent analgesic activity in the acetic acid writhing model and the rat partial sciatic nerve injury model. These finding demonstrate that 4/6 type α-CTxs, with the disulfide bridge connectivity "1-4, 2-3," are also potent inhibitors for GABA B R-coupled Ca V 2.2 and possess potent analgesic activity.

Chemicals and Biological Reagents
The N-Fmoc-amino acids, HOBt and HBTU, were purchased from GL Biochem Ltd.

Animals
Kunming mice (18-20 g, 3-4-week-old, quality license No. 110324220102142031) and Sprague-Dawley rats (200-220 g, 6-7-week-old, quality license No. 11032420102254558) were obtained from SPF (Beijing) Biotechnology Co., Ltd. (Beijing, China), and housed at a relative humidity of 50% under a 12 h light/dark cycle. Food pellets and water were available ad libitum. All experiments were conducted in accordance with the guidelines of the Animal Research Advisory Committee of the Beijing Institutes for Biological Science and conformed to the European Community directives for the care and use of laboratory animals.

Peptide Synthesis
The protected AuIB and its variants were synthesized on Rink resin using the coupling agents HOBt/HBTU and DIEA, as described previously [12]. The peptide-resin (0.05 mmol) is cleaved in the cleavage solution (4.4 mL TFA, 0.25 mL H 2 O, 0.25 g DTT, 0.1 mL TIPS) at room temperature for 3 h to remove the resin and side chain protecting groups. The released linear peptide was filtered, precipitated with cold anhydrous ether at 4 • C for 30 min, and folded in 0.1 M NH 4 HCO 3 buffer (pH 8.0-8.2) at a concentration of 0.2 mg/mL for 24-48 h at room temperature. After the completion of folding, the folded solution was acidified with acetic acid to a pH of 4.0-5.0. The mixture was then filtered and directly loaded onto a 25 × 250 mm preparative C18 column using a preparative HPLC pump (Waters Delta Prep 4000). The column was washed with buffer A (0.1% TFA in water) at a flow rate of 2.5 mL/min and then with buffer B (0.1% TFA in acetonitrile) at a flow rate of spectrometry on a ProFLEXTM-III MALDI-TOF spectrometer. The primary sequences of AuIB and its variants are listed in Table 1.

Disulfide Bond Connectivity Analysis
According to the potency, the disulfide bond connectivity of several AuIB variants folded by one-step oxidation was determined by comparison with peptide folding products of known disulfide connectivity, as described previously [32,33]. Briefly, linear AuIB variants containing an acetamidomethyl (Acm)-protecting group at the C1-C3 or C1-C4 positions were synthesized and then folded by incubation in 0.1 M NH 4 HCO 3 (pH 8.0) at room temperature for 24-72 h. The folded products were further oxidized with an iodine mixture (30% CH 3 CN, 2% TFA, 68% H 2 O) for 10 min to form disulfide bond connectivity "C1-C3, C2-C4" or "C1-C4, C2-C3," and then co-analyzed with a one-step folding product using HPLC.

Circular Dichroism (CD) Spectra
CD spectra were measured using a BioLogic Mos 450 spectropolarimeter (Grenoble, France), as described previously [32]. Briefly, 35 µM of peptide in 10 mM of phosphate buffer (pH 7.2) was added into a 0.1 cm path length quartz cell, and three individual scans (190-260 nm) were performed at 1.0 mm intervals with a bandwidth of 1.0 nm.

HEK293T Cell Electrophysiology
The inhibitory activities of AuIB and its variants against GABA B R-coupled Ca v 2.2 in vitro were determined, as previously described [12,21]. Briefly, HEK293 cells were cotransfected with human GABA B R plasmids (0.5 µg GABA B1 and 0.5 µg GABA B2 ), rat Ca v 2.2 plasmids (0.5 µg α1B, 0.5 µg α2δ and 0.5 µg β3), and 0.2 µg of the reporter gene EGFP for 10 min. After incubation at 37 • C in 5% CO 2 for 48 h, the trypsinized cells were added dropwise to a poly-L-lysine-coated glass coverslip for 30 min, and then 2 mL of culture medium was added and left to incubate for 3 h. Whole-cell voltage clamp recordings were completed using borosilicate glass pipettes at a resistance of~3 MΩ after being filled with intracellular solution. The intracellular solution was composed of 120 mM K-gluconate, 5 mM MgCl 2 ·6H 2 O, 2 mM CsCl, 2 mM MgATP, 0.6 mM Na 2 GTP, 10 mM HEPES, and 5 mM EGTA (pH adjusted to 7.2 with CsOH). The extracellular solution consisted of 90 mM NaCl, 5 mM CsCl, 30 mM TEA-Cl, 10 mM D-glucose, 10 mM BaCl 2 ·H 2 O, 1 mM MgCl 2 ·6H 2 O, and 10 mM HEPES (pH 7.4). AuIB and its variants were diluted to the appropriate final concentration and applied via perfusion; 10 µM baclofen was used as a positive control. All currents were elicited by a voltage step from a holding potential of −90 mV to a test pulse of +10 mV for 200 ms, with a 10 s interval per sweep at room temperature (20~25 • C) with the Axoclamp 700B patch-clamp. When the peak current of baclofen was inhibited by more than 50%, the drug tested was considered effective, and each current data was repeated for more than 4 cells. The concentration-response data were fitted to the equation: Y = Y min + (Y min − Y max )/(1 + 10 (Log IC 50 − X) × h)), where Y (response rate) is I/I 0 , h is the Hill coefficient (slope), and IC 50 is the half-maximal inhibitory concentration. The nonlinear regression analysis was performed using GraphPad Prism 8.0 (GraphPad Software, San Diego, CA, USA).
The inhibition of AuIB and its variants against Ca v 2.2 expressed in HEK293 was also determined using whole-cell voltage clamps [22,34]. The experimental conditions are similar to the tests of GABA B R-coupled Ca V 2.2, except for the lack of GABA B R plasmids. In addition, different external bath solutions (20 mM BaCl 2. H 2 O, 135 mM NMDG, 2 mM MgCl 2 .6H 2 O, 10 mM HEPES, pH 7.2) and intracellular solutions (140 mM CsCl, 10 mM NaCl, 10 mM HEPES, and 1 mM EGTA, pH 7.3) were used.

Two-Electrode Voltage-Clamp Recording on Oocytes Expressing nAChRs
Since AuIB[P7R](ribbon) has a significant inhibitory effect on GABA B R-coupled Ca V 2.2, its inhibitory activities on the nAChRs subtypes (α2β4, α3β2, α3β4, α4β2, α4β4, α7, and α9α10) were determined by the two-electrode voltage-clamp technique, as described previously [12,21]; the molar ratio of the cRNA of the subunits per injection was 1:1.

Acetic Acid Writhing Test
Male Kunming mice were randomly divided into seven groups, ten animals per group. Each group received saline, Benzoyl-Vc1.1[N9R] (10, 20, 40 nmol/kg), and AuIB[[P7R](ribbon) (10, 20 and 40 nmol/kg). All peptides and saline were intraperitoneal injected (i.p.) into the animals; the injection volume was 100 µL. A total of 30 min after the peptides or saline injection, acetic acid (1% in saline, 400 µL) was intraperitoneal administrated to each group. After 5 min, the number of abdominal contortions (writhes) was recorded for 15 min. Data are expressed as the total number of writhes. One-way ANOVA was performed, followed by Dunnett's multiple comparisons test using GraphGad prism 8 (GraphPad Software, La Jolla, CA, USA). All data are presented as mean ± SEM. A p-value less than 0.05 was considered significant.

Statistical Analysis
The results of analgesic tests were analyzed using repeated measures of ANOVA, followed by the unpaired t-test through GraphGad Prism 8. All data were presented as means ± SEM. p < 0.05 was considered as statistically significant.