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ARTICLE IN PRESS Model
EP 69015 1–7
Peptides xxx (2013) xxx– xxx
Contents lists available at SciVerse ScienceDirect
Peptides
jo ur nal homep age: www.elsev ier .com/ locate /pept ides
la-7, His-10 and Arg-12 are crucial amino acids for activity of aynthetically engineered �-conotoxin
line K.M. Lebbea, Steve Peigneura, Ward Brullotb, Thierry Verbiestb, Jan Tytgata,∗
Toxicology and Pharmacology, University of Leuven (KU Leuven), Campus Gasthuisberg O&N2, Herestraat 49, P.O. Box 922, 3000 Leuven, BelgiumLaboratory for Molecular Electronics and Photonics, Division Molecular Imaging and Photonics, Department of Chemistry, University of Leuven,elestijnenlaan 200D, P.O. Box 2425, 3001 Heverlee, Belgium
a r t i c l e i n f o
rticle history:eceived 13 May 2013eceived in revised form 1 July 2013ccepted 1 July 2013vailable online xxx
eywords:�-)Conotoxinoltage-gated sodium channelidi R2
ircular dichroismone snaileptidomimetics
a b s t r a c t
Cone snail toxins or conotoxins are often small cysteine-rich peptides which have shown to be highlyselective ligands for a wide range of ion channels such as voltage-gated sodium channels (NaVs). NaVsparticipate in a wide range of electrophysiological processes. Consequently, their malfunction has beenassociated with numerous diseases. The development of subtype-selective modulators of NaVs remainshighly important in the treatment of such disorders. In order to expand our knowledge in the searchfor novel therapeutics to treat NaV-related diseases, we explored the field of peptide engineering. In thecurrent study, the impact of well considered point mutations into a bioactive peptide that was foundto be a very potent and selective inhibitor of NaVs (i.e. Midi R2) was examined. We designed two pep-tides, named Midi R2[A7G] and Midi R2[H10A, R12A] which have mutations at position 7, and both 10and 12, respectively. Electrophysiological recordings indicated that an Ala to Gly mutation at position 7increased IC50-values from the nanomolar range to the micromolar range. For Midi R2[H10A, R12A] at
a concentration of 10 �M, activity is even reduced to 0–10% for all of the tested NaV-channels. Circulardichroism measurements proved that overall structural conformations did not change. These findingssuggest that the minimal space between the second and the third intercysteine loop of Midi R2 is thesequence RRWARDHSR and that His at position 10 and Arg at position 12 are crucial amino acids for thepotency and specificity of Midi R2. In this way, new insights into the structure–activity relationships of�-conotoxins were found.33
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. Introduction
Venoms from cone snails (genus Conus) can be seen as anntapped cocktail of biologically active compounds, being increas-
ngly recognized as a new emerging source of peptide-basedherapeutics [21]. These creatures (family Conidae) are uniqueecause of their possibility to use an incredibly diverse arse-al of small (<5 kDa), cysteine-rich peptides (called conopeptidesr conotoxins) to hunt for preys [1]. So far, about 500 differ-
Please cite this article in press as: Lebbe EKM, et al. Ala-7, His-10 and Arg�-conotoxin. Peptides (2013), http://dx.doi.org/10.1016/j.peptides.2013.07
nt cone snail species are known and each species produces annprecedented molecular diversity of pharmacologically activeomponents. Up to date, only a few hundred venom components
Abbreviations: NaV-channel, voltage-gated sodium channel; CD, circular dichro-sm.∗ Corresponding author. Tel.: +32 16 32 34 03; fax: +32 16 32 34 05.
E-mail addresses: [email protected], eline [email protected]. Lebbe), [email protected] (S. Peigneur),[email protected] (W. Brullot), [email protected]
T. Verbiest), [email protected] (J. Tytgat).
196-9781/$ – see front matter © 2013 Published by Elsevier Inc.ttp://dx.doi.org/10.1016/j.peptides.2013.07.005
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© 2013 Published by Elsevier Inc.
of over potentially 100,000 have been functionally and structurallyinvestigated [26]. Furthermore, the consideration of Conus venomsas gold mines for the discovery of new therapeutics is validated bythe knowledge that, out of the limited number studied conopep-tides, already six peptides have reached human clinical trials, andone was approved as analgesic in 2004 [2,14,27].
Conotoxins display a great molecular diversity which is mir-rored by the impressive diversity of their targets and even more inthe specific recognition of different binding sites within one tar-get [3]. Exactly this unparalleled specificity enables conotoxins tobe highly selective and extremely potent ligands of several sub-types of voltage-gated ion channels, while others are considered asimpressive selective and potent blockers of neuronal and musclesubtypes of nicotine-type acetylcholine receptors (nAChRs) [2,14].
Voltage-gated sodium channels (NaVs) are important trans-membrane proteins regarding generation as well as propagation ofaction potentials in excitable cells such as neuronal and muscular
-12 are crucial amino acids for activity of a synthetically engineered.005
cells [7,16,20]. Nine channel isoforms of the NaV1 subfamily havebeen identified up to now, having similar functional properties butdifferent distribution in muscle and nerve cells [8]. Consequently,defective NaVs cause several diseases or channelopathies like
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pileptic disorders [12], neuromuscular diseases, and cardiomy-pathies [6]. Blocking the aberrant Na+ current can be effective inreating these disorders [23].
To date, four families of conotoxins which target NaV-channelsave been described: �-conotoxins [31] and �-conotoxins [4,13]aving agonistic effects, whereas �O-conotoxins [25] and �-onotoxins [10,33] have antagonistic effects. The small size as wells their high potency and selectivity turn �-conotoxins into verynteresting compounds [20,29].
In previous research from our lab, miniaturized peptides thatotently and selectively block NaVs, were designed based on twonown �-conotoxins (KIIIA from Conus kinoshitai [5,24] and BuIIICrom Conus bullatus [17,36]) in order to create potent and selectiveeptides that can be synthesized cost-effectively [34]. In a studyf structure-activity relationships in KIIIA, key residues that aremportant for activity on mammalian neuronal NaV1.2 and skeletal
uscle NaV1.4 subtypes were identified. The authors demonstratedhat the engineering of KIIIA could provide subtype-selective ther-peutics against NaVs for the potential treatment of pain [24].imilarly, the design of two new Midi-analogous was investigatedn this study. Based on the most promising compound from pre-ious studies, namely Midi R2, we explored the effect of wellonsidered mutations into the pharmacophore of this miniaturizedeptide.
. Materials and methods
In this study, the following materials and methods were used.
.1. Chemical synthesis and analysis of synthetic peptides
The following peptides were synthesized by ChinaPeptides Co.,td. (Shanghai, China): Midi R2[A7G] and Midi R2[H10A,R12A]ith molecular masses of 1752.0 Da and 1614.9 Da, respectively.
urity was confirmed to be >98% by reversed-phase HPLC on annalytical Vydac C18 column (218MS54, 4.6 × 250 mm, 5-�m par-icle size; Grace, Deerfield, IL) with a flow rate of 1 ml min−1. UVbsorbance was monitored at 214 and 280 nm with a dual wave-ength absorbance detector. A linear gradient of 0–40% acetonitrilen 40 min was used at a flow rate of 1 ml min−1. The gradient waset by means of a mixture of solvent A (0.085% (v/v) trifluoroaceticcid (TFA) in acetonitrile) and solvent B (0.1% TFA (v/v) in water)ith an initial concentration of 0% solvent A.
.2. Folding of the peptides
Peptides were dissolved in the physiological buffer solution ND-6. Folding mixtures were retained at room temperature for twoeeks. At different time points following dissolution, aliquots wereithdrawn. Aliquots were analyzed with reversed-phase HPLC byeans of an analytical Vydac C18 column as described in the previ-
us section. The concentration of the folded synthetic peptides waseasured using a Nanodrop ND-1000 spectrophotometer (Isogen
ife Science, De Meern, the Netherlands).
.3. Heterologous expression in Xenopus oocytes
Complementary DNA encoding the NaV-channels wasubcloned into the corresponding vector: the �-subunitsNaV1.1/pLCT1(NotI), rNaV1.2/pLCT1(NotI), rNaV1.3/pNa3T(NotI),NaV1.4/pUI-2(NotI), hNaV1.5/pcDNA3.1(XbaI),NaV1.6/pLCT(NotI), rNaV1.7/pBSTA.rPN1(SacII), cockroach
Please cite this article in press as: Lebbe EKM, et al. Ala-7, His-10 and Arg�-conotoxin. Peptides (2013), http://dx.doi.org/10.1016/j.peptides.2013.0
latella germanica BgNaV1.1/pGH19(NotI), and the correspond-ng �-subunits r�1/pSP64T(EcoRI) and Drosophila melanogasteripE/pGH19(NotI). The linearized plasmids – respective restrictionnzymes are indicated in parentheses – were transcribed using
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the T7 (for rNaV1.1, rNaV1.2, rNaV1.3, rNaV1.4, mNaV1.6, rNaV1.7,BgNaV1.1, TipE) or the SP6 (for hNaV1.5 and r�1) mMESSAGE-mMACHINE transcription kit (Ambion, Austin, TX). The harvestingof stage V–VI oocytes from anaesthetized female Xenopus lae-vis frogs was previously described [35]. Oocytes were injectedwith 50–70 nl of cRNA at a concentration of 1–3 ng/nl using amicro-injector (Drummond Scientific, Broomall, PA). The oocyteswere incubated in a ND-96 solution containing: NaCl, 96 mM;KCl, 2 mM; CaCl2, 1.8 mM; MgCl2, 2 mM and HEPES, 5 mM (pH7.4), supplemented with 50 mg/l gentamycin sulfate and 0.5 mMtheophylline. Oocytes were stored for 1–5 days at 16 ◦C untilsufficient expression of NaVs was achieved.
2.4. Electrophysiology
Whole-cell currents from oocytes were recorded at room tem-perature (18–22 ◦C) by the two-electrode voltage clamp techniqueusing a GeneClamp 500 amplifier (Molecular Devices, Sunnyvale,CA) controlled by a pClamp data acquisition system (MolecularDevices). Oocytes were placed in a bath containing ND-96 solu-tion. Voltage and current electrodes were filled with 3 M KCl, andthe resistances of both electrodes were maintained as low as possi-ble (between 0.5 and 1.5 M�). The elicited currents were sampledat 20 kHz and filtered at 2 kHz using a four-pole, low pass Besselfilter. To eliminate the effect of the voltage drop across the bathgrounding electrode, the bath potential was actively controlled bya two-electrode bath clamp. Leak subtraction was performed usinga – P/4 protocol.
Whole-cell current traces were evoked every 5 s by a 100-msdepolarization to the voltage corresponding to the maximal acti-vation of the NaV-subtype in control conditions, starting from aholding potential of −90 mV. Concentration-response curves wereconstructed by adding different toxin concentrations directly to thebath solution. The percentage of NaV blockade was plotted againstthe logarithm of the applied concentrations and fitted with the Hillequation: y = 100/[1 + (EC50/[toxin])h], where y is the amplitude ofthe toxin-induced effect, EC50 is the toxin concentration at halfmaximal efficacy, [toxin] is the toxin concentration and h is the Hillcoefficient. To investigate the effects on the voltage dependence ofactivation, current traces were induced by 100-ms depolarizationsfrom a holding potential of −90 to 65 mV with 5-mV increments.The Na+ conductance was calculated from the currents using Ohm’slaw: gNa = INa/(V − Vrev), where INa represents the Na+ current peakamplitude at a given test potential V, and Vrev is the reversal poten-tial. The values of gNa were plotted as a function of voltage and fittedusing the Boltzmann equation: gNa/gmax = [1 + (exp(Vg − V)/k)]−1,where gmax represents maximal gNa, Vg is the voltage correspond-ing to half-maximal conductance and k is the slope factor. Toinvestigate the effects on the steady-state inactivation process,oocytes were depolarized using a standard two-step protocol.From a holding potential of −90 mV, 100-ms prepulses weregenerated, ranging from −90 to 65 mV with 5-mV increments,immediately followed by a 100-ms test pulse to −10 mV. Datawere normalized to the maximal Na+ current amplitude, plottedagainst prepulse potential and fitted using the Boltzmann equa-tion: INa/Imax = [(1 − C)/(1 + exp((V − Vh)/k))] + C, where Imax is themaximal INa, Vh is the voltage corresponding to half-maximal inac-tivation, V is the test voltage, k is the slope factor, and C is a constantrepresenting a non-inactivating persistent fraction (close to zero incontrol). Comparison of two sample means was made using a pairedStudent’s t test (p < 0.05). All data are presented as mean ± standarderror (SEM) of at least 3 independent experiments (n ≥ 3). All data
-12 are crucial amino acids for activity of a synthetically engineered7.005
was analyzed using pClamp Clampfit 10.0 (Molecular Devices®,Downingtown, PA) and Origin 7.5 software (Originlab®, Northamp-ton, MA).
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Fig. 1. Sequences of naturally occurring �-conotoxins (KIIIA en BuIIIC) and synthetically designed peptides.Shown are the sequences of two known, naturally occurring �-conotoxins, KIIIA from Conus kinoshitai (23, 24) and BuIIIC from Conus bullatus (25, 26) from which the Minipeptide and analogs Midi and Midi R2 were derived by Stevens et al. (27). Midi R2[A7G] and Midi R2[H10A, R12A] are investigated in this study. Dashes are put in thes k lines above the sequences represent the disulfide bridges. Cysteine amino acids are redc
2
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Fig. 2. CD spectrum of Midi, Midi R2 and its analogous.
and BgNa 1.1). From these Na -subtypes, Midi R2[H10A, R12A]
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equences to make all sequences and intercysteine loops of comparable length. Blacolored and mutated amino acids are indicated in bold.
.5. CD measurements
CD spectra were recorded on a JASCO J-820 spectropolarimetern H2O solution (MQ-water, pH 7.0) at 20 ◦C, with a quartz cell of-mm path length. The results are expressed in deg/(cm mol).
. Results
.1.1. �-Conotoxin engineering
Synthetic analogs of Midi and Midi R2, namely Midi R2[A7G]nd Midi R2[H10A, R12A] were engineered based on the studyf Stevens et al., 2012 [34] and McArthur et al., 2011 [24] Wettempted to design with Midi R2[A7G] a shorter peptide than theotent inhibitor Midi R2. The rationale for the double mutant, Midi2[H10A, R12A], was to strive for a more selective NaV1.7 inhibitor
unded on the study from [24] with KIIIA. Peptides were synthe-ized and folded in ND-96 solution. Peptide sequences are shownn Fig. 1.
.2. Folding of the peptides
To investigate the folding of the Midi analogs, changes in HPLCetention time were investigated and compared with the reten-ion time of the reduced peptide (data are not shown). As was alsoescribed in Stevens et al., 2012 [34], the Midi R2-analogs, dissolved
n ND-96 solution, were maximally folded after one week.
.3. CD measurements
The CD spectra of Midi R2, Midi R2[A7G] and Midi R2[H10A,12A] were strongly superimposable to each other with a mini-um around 202 nm (Fig. 2). A difference in signal strength can be
bserved between Midi and Midi R2 since Midi R2 seems to give atronger signal.
.4. Electrophysiological characterization
Using the two electrode voltage clamp technique our two
Please cite this article in press as: Lebbe EKM, et al. Ala-7, His-10 and Arg�-conotoxin. Peptides (2013), http://dx.doi.org/10.1016/j.peptides.2013.07
utants, Midi R2 [A7G] and Midi R2[H10A, R12A], were charac-erized by electrophysiological recordings in order to determineubtype selectivity profiles.
The peptides Midi, Midi R2, Midi R2[A7G] and Midi R2[H10A, R12A] were examinedby circular dichroism spectroscopy. This figure shows that the overall structure ofthe mutated peptides did not change.
3.4.1. Midi R2[A7G]Midi R2[A7G] was screened against a panel of seven mam-
malian NaV-channel isoforms at a concentration of 10 �M(Fig. 3). Among these isoforms Midi R2[A7G] inhibited all ofthe NaV-subtypes (NaV1.1 to NaV1.7) with inhibition percent-ages of 8.5 ± 3.3%, 25.3 ± 7.4%, 29.1 ± 4.2%, 23.8 ± 4.5%, 22.7 ± 6.2%,31.1 ± 8.2%, 12.4 ± 2.6% respectively. The IC50 values of MidiR2[A7G] on NaV1.2 and NaV1.4 are 14.7 ± 3.2 �M and 15.3 ± 3.9 �Mrespectively. Dose-response curves from NaV1.2 and NaV1.4 canbe found in Fig. 4 The NaV-subtypes NaV1.3 and NaV1.6 are mostsensitive for Midi R2[A7G].
Midi R2[A7G] was also tested on BgNaV1.1, an isoform from B.germanica, cockroach, representative for an insect NaV-channel, butno remarkable activity was observed at a concentration of 10 �M.
3.4.2. Midi R2[H10A, R12A]Midi R2[H10A, R12A] was submitted to a screening on the
same panel of voltage-gated sodium channels (NaV1.1 to NaV1.7
-12 are crucial amino acids for activity of a synthetically engineered.005
V V(10 �M) does not target any of them (Fig. 5). Remarkably, thecombination of the point mutations H10A and R12A completelyabolishes the activity of this peptide on the tested NaV-channels.
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Fig. 3. Electrophysiological measurements of Midi R2[A7G].Current traces were obtained by two-electrode voltage clamp on X. laevis oocytes heterologously expressing the NaV-isoforms as described under “Materials and Methods”.Currents were evoked by a depolarizing pulse starting from a holding potential of −90 mV to the voltage corresponding to the maximal activation in control conditions.T . The
p d NaV
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races shown are representative of at least three independent experiments (n ≥ 3)eak amplitude of the currents after exposure to 10 �M of Midi R2[A7G]. NaV1.3 an
comparison of the blocking percentages of the peptides Midi R2,idi R2[A7G] and Midi R2[H10A, R12A] is made in Fig. 6.
. Discussion
In the current study, we investigated the impact of point muta-ions into the pharmacophore of a bioactive peptide onto the
Please cite this article in press as: Lebbe EKM, et al. Ala-7, His-10 and Arg�-conotoxin. Peptides (2013), http://dx.doi.org/10.1016/j.peptides.2013.0
ctivity and potency of blocking NaV-channels. We integrated theesults of Stevens et al. [34] and those of McArthur et al. [24] intour design strategy. Stevens et al. [34] designed several bioactiveeptides (Mini and Midi) derived from two naturally occurring
ig. 4. Concentration-response curves on NaV1.2 and NaV1.4.. Midi R2 (�) and Midi R2[A7G] (©) were tested on NaV1.2, which was shown to be most
idi R2[A7G] are 34.1 ± 2.4 nM and 14.7 ± 3.2 �M respectively. B. Midi R2 (�) and Midi R2[5.3 ± 3.9 �M. Currents were obtained as described under “Materials and Methods”. The por both NaV-isoforms, results were fit with the Hill equation.
dotted line indicates the zero-current level. Asterisks (*) indicate the steady-state1.6 are most sensitive to Midi R2[A7G].
�-conotoxins, namely KIIIA from Conus kinoshitai, and BuIIIC fromConus bullatus. They illustrated that an extra Ala residue added tothe sequence of the Mini peptide restores the spacing (in compar-ison with the original �-conotoxins) which led to a peptide thatis active in the range of nanomolar concentrations onto NaV1.2,NaV1.4 and NaV1.6. They called this peptide Midi (sequence isshown in Fig. 1). The difference in activity between Mini (in a micro-molar range) and Midi (in a nanomolar range) was attributed to
-12 are crucial amino acids for activity of a synthetically engineered7.005
a difference in spacing whereupon they concluded that at leastsix residues are required to set up a fully functional bioactivescaffold that can interact properly with a binding area on the NaV-channel (see also [30]). Interestingly, the NMR structure of the
potently inactivated by Midi R2 (27). The corresponding IC50 values for Midi R2 andA7G] (©) were tested on NaV1.4 with corresponding IC50 values of 330 ± 570 nM andercentage of block was plotted against the logarithm of the tested concentrations.
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Fig. 5. Electrophysiological measurements of Midi R2[H10A, R12A]T ytes hr icatesa
Mtmla
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he current traces were obtained by two-electrode voltage clamp on X. laevis oocepresentative of at least three independent experiments (n ≥ 3). The dotted line indpplication of 10 �M Midi R2[H10A, R12A].
idi peptide does not show a complete alpha-helix which was
Please cite this article in press as: Lebbe EKM, et al. Ala-7, His-10 and Arg�-conotoxin. Peptides (2013), http://dx.doi.org/10.1016/j.peptides.2013.07
hought to be crucial for �-conotoxin biological activity [19,30]. Theutant Midi R2 in which an extra positive charge was integrated,
ed to lower IC50 values compared to Midi. The mutant exhibits very potent and selective block on NaV1.2 [34]. The overall
ig. 6. Overview of block by Midi R2, Midi R2[A7G] and Midi R2[H10A, R12A]he three peptides, Midi R2, Midi R2[A7G] and Midi R2[H10A, R12A], were tested on NaV1as seen at that concentration, this is represented by an arbitrary value of 1% for the clar
alues are discussed under “Results”. Data are represented as the means ± SE (indicated b
eterologously expressing a single type of cloned NaV-isoforms. Traces shown are the zero-current level. The asterisk (*) distinguishes the steady-state current after
higher potency is attributed to the larger electrostatic repulsion,
-12 are crucial amino acids for activity of a synthetically engineered.005
which was also previously proposed for BuIIIC [18]. Consequently,we decided to continue our research with this Midi R2-mutantin order to create smaller and/or more NaV-isoform selectivepeptides.
.1-NaV1.7 and BgNaV1.1 at a concentration of 10 �M. When no blocking activity (%)ity of the figure (this is the case for Midi R2[H10A, R12A] on NaV1.4). Other specificy error bars); each experiment was performed at least three times (n ≥ 3).
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The first mutant we created was Midi R2[A7G]. The purpose ofhis mutation was to investigate the minimal spacing of the sec-nd intercysteine loop. As Gly is an amino acid comparable to Ala,ut without a –CH3 group, we wanted to see if a smaller peptideolds or loses its activity. Our observations suggest that the smallerutant, Midi R2[A7G], is not only less active and potent, but it
oses also its selectivity. The IC50 value for NaV1.2 increased fromhe nanomolar to the micromolar range (34.1 ± 2.4 nM for Midi R2ersus 14.7 ± 3.2 �M for Midi R2[A7G]) as was also observed foraV1.4 (330 ± 570 nM for Midi R2 versus 15.2 ± 1.2 �M for Midi2[A7G]). Presumably, key residues are in a less favorable posi-ion because of the smaller spacing and thus interactions with theaV-channels are partially hindered. Further structural researchith site-directed mutagenesis or NMR or ideally co-crystallization
hould be conducted to verify if this is correct.From our observations, we can conclude that the minimal spac-
ng between the second and the third cysteine residues for aotent and selective inhibitor analog of Midi is not the sequenceRWGRDHSR, but RRWARDHSR, as CD measurements proved thatverall structural conformations did not change.
The second mutant, called Midi R2[H10A, R12A], has a dou-le mutation on positions 10 and 12. The idea behind these pointutations is based on the results from the work of McArthur et al.
24]. In their study, they described that the substitution H12A inIIIA severely reduced its affinity for NaV1.2 and NaV1.4 as IC50alues increased >2000-fold. In contrast, the increase in IC50 foraV1.7 was only 133-fold, which indicated that selectivity shifted
o NaV1.7. The substitution R14A in KIIIA also changed target speci-city for NaV1.7 over both NaV1.2 and NaV1.4. Unfortunately, bothubstitutions were found to substantially reduce KIIIA affinity. Inur study, we wanted to test if double substitutions [R10A, H12A]ould give an explicit shift toward NaV1.7 selectivity. The results
re dissimilar with what was suggested by McArthur et al., butonetheless very interesting. With the double mutant as toxin pep-ide we could not block any of the tested NaV-subtypes whereas CD
easurements proved that overall structural conformations stayedntact. Thus, compared to Midi R2, potency and activity decreasedramatically rendering Midi R2[H10A, R12A] inactive to the testedaV-subtypes.
Other studies investigating these mutation sites in several pep-ides, like for instance the �-conotoxin SIIIA from Conus striatus,ndicated that His-16 replacement caused large reductions in SIIIAffinity. NMR measurements pointed out that this altered affinityccurred from structural changes in the C-terminal half of the pep-ide which potentially disrupted the alpha-helix. Consequently, therientation of the major binding determinants required for highffinity interactions at NaVs was disturbed. It was suggested that anrginine and tyrosine (but not Ala) can replace His-16 as a structuraltabilizer [30].
Replacement of Arg-18 was found to be important for the block-ng effect of SIIIA. This mutation contributes significantly to theffinity at NaV1.4, and to a lesser extent at NaV1.2. Hereupon theyoncluded that the pharmacophoric residues important for the highffinity of SIIIA at NaVs are located along the alpha-helical motif plusrg-18 [30]. Li et al. (2001) investigated Arg-19 substitution into
he �-conotoxin GIIIA from Conus geographus. They suggested thatositively charged toxin residues (like for instance Arg-1, Lys-11,ys-16 and Arg-19) are critical for its biological activity. In this way,he charges, as well as their precise location within the pore, wereound to be critical for toxin-channel interaction [22]. Choudharyt al. showed that Arg-19 of GIIIA strongly interacts with domainV of the NaV-channels [9].
Please cite this article in press as: Lebbe EKM, et al. Ala-7, His-10 and Arg�-conotoxin. Peptides (2013), http://dx.doi.org/10.1016/j.peptides.2013.0
These and our observations suggest that the C-terminus of Midi2 and other smaller �-conotoxins like KIIIA or BuIIIC consist of
crucial epitope where charged residues cannot be changed. Ourtudy on Midi R2[H10A, R12A] is new in the fact that the double
[
PRESSs xxx (2013) xxx– xxx
mutant has never been analyzed before. We expected that potencywould be less, but we hoped that we could find a significant increasein affinity to NaV1.7. This NaV-subtype is namely an important anal-gesic drug target because of its role in human as well as animalpain perception [11,28]. However, our findings support the devel-opment of peptides with novel subtype specificity and analgesicpotential.
The importance of testing cockroach B. germanica BgNaV1.1 canbe seen in the world of the agro industry and the application ofinsecticides because voltage gated sodium channels are well rec-ognized targets in the development of novel insecticides [32].
5. Conclusion
In this study we investigated the influence of point mutationsto the potency and activity – i.e. the blocking effect on NaVs – ofa �-conotoxin, named Midi R2. As stated in previous research, anArg on position 2 (peptide Midi R2), leads to lower IC50 values andexhibits a very potent and selective block on NaV1.2 [34]. Whenthe Ala on position 7 in Midi R2 is mutated to a Gly, the IC50 valueincreases from the nanomolar range to the micromolar range. Thisindicates that for a potent and specific peptide, the minimal spacebetween the second and the third cysteine residues is the sequenceRRWARDHSR. For the double mutant, Midi R2[H10A, R12A], whereHis (position 10) and Arg (position 12) are mutated to Ala, the activ-ity of the peptide (measured at 10 �M) is even reduced to 0–10% forall NaV-subtypes. Circular dichroism measurements indicated thatstructural conformations of the mutants did not change. The muta-tions we made are thus critical for the function of the peptide. Thesefindings are important in the future engineering of �-conotoxin-based lead compounds in the development of novel therapeuticsthat can be used to treat NaV-related diseases such as epilepsy [38]and pain [15,37].
Acknowledgements
The authors would like to thank Beatrice Garcia Mille forher work in molecular biology. This work was supported by thefollowing grants: G.0433.12, G.A071.10N and G.0257.08 (F.W.O.Vlaanderen), EU-FP7-MAREX, IUAP 7/10 (Inter-University Attrac-tion Poles Program, Belgian State, Belgian Science Policy) andOT/12/081 (KU Leuven). W. Brullot received funding from theAgency for Innovation by Science and Technology (IWT) Flanders.
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