SUPPORTING INFORMATION

Dicarba Analogues of -Conotoxin RgIA. Structure,

Stability and Activity at Potential Pain Targets

Sandeep Chhabra, Alessia Belgi, Peter Bartels, Bianca J. van Lierop, Samuel D.
Robinson, Shiva N. Kompella, Andrew Hung, Brid P. Callaghan, David J. Adams,
Andrea J. Robinson and Raymond S. Norton*,

Medicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University,

Parkville 3052, Victoria, Australia

School of Chemistry, Monash University, Clayton 3800, Victoria, Australia

Health Innovations Research Institute, RMIT University, Bundoora 3083, Victoria, Australia

These authors contributed equally to this study

Contents:
RP-HPLC and MALDI-TOF MS profiles
One-dimensional 1H NMR spectra
RP-HPLC profiles in human serum
1
H NMR spectra over a range of temperatures
Dicarba analogues effects on Ba2+ currents (IBa)
Effect of dicarba analogues on Cav2.2 channels alone
Homology modelling and receptor contacts
ESI-MS spectrum of Rg1A in human serum
Overlay of NMR structures
Chemical shifts for cis-[2,8]dicarba RgIA
Chemical shifts for trans-[3,12]-dicarba RgIA
Chemical shifts for cis-[3,12]-dicarba RgIA
NMR structural statistics
IC50 of RgIA and dicarba RgIA analogues

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Figure S1. RP-HPLC (Agilent: Vydac C18 analytical column, 0 30% buffer B over 30
min; left hand panels) and MALDI-TOF MS analysis (right hand panels) of dicarba-Rg1A
analogues. (A) cis-[2,8]-dicarba Rg1A RP-HPLC trace (13.9 min) and (B) MALDI-TOF MS;
(C) trans-[2,8]-dicarba Rg1A RP-HPLC trace (15.5 min) and (D) MALDI-TOF MS; (E) cis[3,12]-dicarba Rg1A (RP-HPLC trace (13.8 min) and (F) MALDI-TOF MS; (G) trans-[3,12]dicarba Rg1A RP-HPLC trace (14.1 min) and (H) MALDI-TOF MS. All mass spectra show
[M + H]+ions.

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Figure S2. Amide/aromatic region of one-dimensional 1H NMR spectra each of the dicarba
RgIA analogues at pH 3, 25C, acquired on a Bruker 600 MHz spectrometer.

S3

Figure S3. RP-HPLC profiles of (A) native Rg1A and (B) cis-[2,8]-dicarba Rg1A incubated
in human serum at 37 C for 24 h. The peaks for native Rg1A (15.5 min) and cis-[2,8]-dicarba
Rg1A (14.35 min), respectively, are marked with an arrow in each panel. Emerging peaks in
the profiles of native Rg1A at 14.2 and 14.8 min have lower molecular masses than native
Rg1A. Peaks at 14.0 and 14.7 min in the t=1h and t=2h RP-HPLC profiles of cis-[2,8]-dicarba
Rg1A also have lower molecular masses than cis-[2,8]-dicarba Rg1A. The RP-HPLC profiles
represent the region 12-22 min of the chromatographic runs, the peak of the internal standard
diazepam at about 27 min has been excluded from this figure. The peak at 13.05 min is
arising from the serum.

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Figure S4. Amide/aromatic region of one-dimensional 1H NMR spectra as a function of

temperature for cis-[2,8] (top) and cis-[3,12] (bottom) dicarba RgIA at pH 3, acquired on a
Bruker 600 MHz spectrometer.

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A.

B.

C.

D.
cis-[2,8]

+10
-80

Figure S5. Time course of peak Ba2+ current amplitude recorded from HEK293 cells
expressing Cav2.2 and GABABR as a function of time during bath application of either 1 M
cis-[2,8]-dicarba RgIA (A) or 1 M cis-[3,12]-dicarba RgIA (B) and subsequent bath
application of 50 M baclofen. Recovery of IBa amplitude was observed upon washout of
baclofen. (C) Representative superimposed depolarization-activated Ba2+ currents obtained in
the absence (control) and presence of a maximally effective concentration of cis-[2,8]-dicarba
RgIA (10 M) . (D) Concentration-response relationship for cis-[2,8]-dicarba RgIA inhibition
of IBa was determined from 3 different concentrations for a minimum of 3 cells and gave an
IC50 of ~300 nM (n = 3-5 cells).

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A.

B.

+10
-80

cis-[2,8]
Ctrl

Figure S6. (A) Representative depolarization-activated Ba2+ currents recorded from HEK293
cells expressing Cav2.2 channels alone in the absence (control) and presence of 1 M cis[2,8]-dicarba RgIA. Cells were depolarized from a holding potential of 80 mV to +10 mV
for 200 ms at a frequency of 0.2 Hz. (B) Bar graph of relative peak current amplitude
(I/Icontrol) for the dicarba RgIA analogues. Data are represented as mean S.E.M (n = 3) and
were not statistically significant difference from control (p > 0.05, unpaired t-test).

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Molecular Dynamics Simulations

In order to compare the overall structural fit of RgIA and the cis-[2,8]-dicarba analogue at the
two 910 interfaces, we calculated the change in interactions between toxin and nAChR at
9(+)10(-) and 10(+)9(-) in terms of total atomic contacts, number of H-bonds and toxinreceptor contact surface areas. Both atomic contact numbers and contact surface decrease by
~2014% and ~3015%, respectively, at both interfaces as a consequence of the cis-[2,8]dicarba modification. Similarly, simulations suggest that the total number of toxin-receptor Hbonds decrease by ~77% at 9(+)10(-), but increase by ~125% at 10(+)9(-). Thus, all
three measures indicate an overall marginal reduction in toxin-receptor contact at the
9(+)10(-) interface as a consequence of the cis-[2,8]-dicarba modification.

Figure S7. Homology modelling and receptor contacts (A) Surface representation of the
910 homology model based on AChBP bound to -conotoxin ImI (PDB ID 2BYP), with 9
in white, 10 in blue, and cis-[2,8]-dicarba RgIA in red. (B) Change in number of toxin atoms
(N) that lie within 4.5 of receptor residues (x-axis) due to cis-[2,8]-dicarba modification
of RgIA for 9 and (C) 10. Asterisks indicate 910 residues with significant gain (positive
values) or loss (negative) in toxin contacts. Error bars are standard errors of means calculated
over both (non-auxiliary) 9 and 10 subunits for 10 independent simulations (20 data points
for each 910 residue).

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Figure S8. A ESI-MS spectrum of Rg1A in serum at t=0 corresponding to the peak at 15.5
min in Figure S3; B ESI-MS spectrum of Rg1A in serum at t=2hr corresponding to the peak
at 14.2 min in Figure S3: the observed [M+2H]2+ of 708.0 and [M+3H]3+ of 472.5 converge to
a molecular ion M of 1414 which is consistent with the loss of one Arg residue from the
native sequence of Rg1A. The calculated mass for [Rg1A-Arg] is 1413.52; C ESI-MS
spectrum of Rg1A in serum at t=2hr corresponding to the peak at 14.8 min in Figure S3: the
observed [M+2H]2+ of 639.0 and [M+3H]3+ of 426.4 converge to a molecular ion M of 1276
which is consistent with the loss of a second Arg residue from native Rg1A. The calculated
mass for [Rg1A-2Arg] is 1275.43.

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Figure S9. Closest-to-average structures of RgIA (PDB ID 2JUT) (green) superimposed over
backbone heavy atoms of cis-[3,12]- (red), trans-[3,12]- (cyan) and cis-[2,8]-dicarba RgIA
(blue). The dicarba bridges are shown in green, disulde bridges are in yellow, and N- and Ctermini are labeled.

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NMR Chemical Shifts

Sequential amide NOEs were observed for the peptides with the obvious exception of Pro6,
which lacks amide protons. In all analogues, d NOEs and 13C values for Pro6 were
indicative of trans conformations about the X-Pro bonds. H resonances for Gly1 were
identified by strong 4Sub2/Cys2 HNGly1 H NOESY cross-peaks and subsequent
identification of H-H cross-peaks. The amide peak of 4Sub2 of cis-[2,8]-dicarba RgIA was
broadened by exchange with solvent and not observable at pH 4.8, as observed with native
RgIA.1 Dioxane (chemical shift of 3.75) was used as a reference.
1

H, 13C and 15N chemical shift assignments are tabulated below.

Table S1. Chemical shifts for cis-[2,8]-dicarba RgIA at pH 3, 25C.

Chemical Shift (ppm)
Residue
Gly1
Aba2
Cys3
Ser4
Asp5
Pro6
Arg7
Aba8
Arg9
Tyr10
Arg11
Cys12
Arg13

8.77
8.16
8.55
7.94

119.0
115.7
114.6
121.4

8.11
7.78
8.29
7.41
8.27
8.51
8.18

115.7
121.0
122.2
118.2
120.3
121.1
126.0

3.93,3.96
4.67
4.45
4.37
5.10
4.32
4.22
4.27
4.13
4.62
4.26
4.61
4.27

H
2.60,2.73
3.38,2.99
3.98
3.08,2.79
1.96,2.40
1.89,1.78
2.59
1.72,1.67
3.11,2.93
1.81,1.78
3.11,3.20
1.89,1.77

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3.92
3.21

7.42

1.56

3.11
7.12
3.18

7.08
6.87
7.16

1.62

3.2

7.18

43.4
56.3
57.0
58.6
50.8
64.9
56.6
56.1
57.1
56.3
56.2
56.5
57.4

5.63

2.06,2.03
1.64
5.63
1.48

Table S2. Chemical shifts for trans-[3,12]-dicarba RgIA (major conformation) at pH 3, 25C.
Chemical Shift (ppm)
Residue
Gly1
Cys2
Aba3
Ser4
Asp5
Pro6
Arg7
Cys8
Arg9
Tyr10
Arg11
Aba12
Arg13

8.76
8.30
8.35
8.21

116.5
118.1
113.2
122.7

8.51
7.96
8.62
7.43
8.22
8.20
8.05

115.4
119.4
125
117.8
122.1
122
123.5

H
3.97
4.90
4.04
4.36
5.08
4.32
4.23
4.50
4.11
4.73
4.42
4.41
4.27

3.66,3.02
2.56,2.59
3.98,4.05
3.08,2.74
1.96,2.42
1.81,1.97
3.46,3.05
1.71
2.95,3.06
1.72,1.80
2.59,2.46
1.77,1.89

5.58

2.07
1.63

3.90,3.96
3.22

7.40

1.47

3.14
7.11
3.18

7.12
6.85
7.17

3.21

7.19

1.60
5.58
1.64

C
43.3
55.2
59.8
51.1
64.9
56.5
55.3
57.6
56.5
58.7
55.2
56.2

Table S3. Chemical shifts for trans-[3,12]-dicarba RgIA (minor conformation) at pH 3, 25C.
Chemical Shift (ppm)
Residue
Gly1
Cys2
Aba3
Ser4
Asp5
Pro6
Arg7
Cys8
Arg9
Tyr10
Arg11
Aba12
Arg13

8.81
8.94
8.41
7.98

118.6
121.8
112.7
121.5

8.03
8.32
8.45
8.01
8.48
8.13
8.28

115.2
118.7
119.0
118.1
124.2
123.2
125.4

H
3.94
4.90
4.13
4.34
5.16
4.38
4.32
4.43
4.03
4.72
4.36
4.38
4.19

H
3.58,3.04
2.65,2.72
4.04
2.81,3.06
1.95,2.41
1.97,1.81
2.82,3.83
1.53
3.06,2.87
2.68,2.44

5.51

2.07
1.65

4.06
3.20

1.40

3.07

C
43.3
55.2
57.0
50.9
64.8

7.36
7.53
6.80

56.5

5.51
56.7

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Table S4. Chemical shifts for cis-[3,12]-dicarba RgIA (major conformation) at pH 3, 22C.

Chemical Shift (ppm)

Residue
Gly1
Cys2
Aba3
Ser4
Asp5
Pro6
Arg7
Cys8
Arg9
Tyr10
Arg11
Aba12
Arg13

8.82
8.50
8.23
8.29

116.5
115.8
112.9
124.6

8.53
7.96
8.55
7.34
8.21
8.37
8.08

116.0
120.4
125.5
117.8
119.8
122.3
125.1

H
4.05,3.96
4.97
3.93
4.45
5.09
4.36
4.26
4.37
4.08
4.77
4.38
4.63
4.25

H
3.68,2.99
2.77,2.48
4.03,3.94
2.67,3.19
2.41,1.98
1.96,1.77
2.92,3.60
1.72
3.03,2.95
1.84,1.74
2.62,2.65
1.88,1.76

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2.05
1.64

3.97
3.19,3.22

7.46

1.48

3.14
7.03
3.19

7.14
6.84
7.16

3.21

7.20

5.75

1.60
5.52
1.61

C
43.5
55.5
60.5
59.1
50.8
64.7
56.6
55.6
57.7
55.8
55.0
55.5
56.1

Table S5. Summary of experimental constraints and structural statistics for dicarba RgIA
analogues

[3,12]-dicarba RgIA
cis
trans
Conformational Restraints
total no. of distance restraints
intraresidue (i = j)
short/sequential (|i - j| = 1)
medium-range (1 < |i - j| < 5)
long-range (|i - j| > 5)
hydrogen bond
dihedral

[2,8]-dicarba RgIA
cis

185
106
56
17
6
1
14

92
46
41
10
4
0
16

179
107
50
16
6
1
7

ENOE (kcal mol-1)

1.79 0.60

0.65 0.28

7.18 1.51

EVdW (kcal mol-1)

rmsd from experimental data
NOEs ()
dihedrals (deg)

1.39 0.17

1.30 0.19

1.65 0.33

0.02 0.003
0.08 0.1

0.01 0.003
0.60 0.2

0.04 0.004
0.04 0.07

Deviations from idealb

angles (deg)
bonds ()
impropers (deg)

0.5 0.03
0.003 0
0.3 0.02

0.5 0.02
0.030 0
0.3 0.02

0.5 0.03
0.003 0
0.3 0.03

Atomic RMSDc
all heavy atoms
backbone heavy atoms

1.27 (2-12)
0.43 (2-12)

1.27 (2-12)
0.64 (2-12)

1.74 (2-12)
0.78 (2-12)

91.5
8.5
0.0
0.0

83.0
16.5
0.5
0.0

69.0
27.5
3.0
0.5

Energiesa

Ramachandran Plotd
most favoured (%)
allowed (%)
additionally allowed (%)
disallowed (%)

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Table S6. Inhibition of nAChR subtypes and GABABR/Cav2.2 channel by RgIA and dicarba
RgIA analogues.

Peptide

910

GABAB/Cav2.2

IC50 (95% CI)

nH

IC50 (95% CI)

nH

IC50 (95% CI)

RgIA

22 nM 2

0.9

4.66 M 3

1.2

22.4 nM 4

trans-[3,12]

1.15 M
(0.84 1.55)

1.9

3.95 M
(3.15 4.97)

1.5

ND

cis-[3,12]

1.47 M
(1.01 2.15)

1.2

ND

ND

trans-[2,8]

ND

ND

ND

cis-[2,8]

ND

ND

295 nM (2-5)
(104 309)

ND Not determined

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2.

3.

4.

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