The Role of Disulfide Bond Replacements in Analogues of the Tarantula Toxin ProTx-II and Their Effects on Inhibition of the Voltage-Gated Sodium Ion Channel Nav1.7

Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Nav1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate.


Supporting Information
Synthesis of (2R, 6R)(Aloc, Allyl/Fmoc) lanthionine 1: General Experimental 1 H NMR spectra of protected lanthionine intermediates and 1 S10 Peptide synthesis: general methods S13 Synthesis of S-S bridged single ring peptides S14 Mass spec and HPLC traces for all peptides S16 X-ray crystallography methods S25 Figure S1 HPLC traces of ProTx-II samples (commercial and synthesised) S27 Patch clamp data: Study report from Essen Biosciences S27 Study report from B'SYS GmbH S32

S 3
Infrared spectra were recorded using a Perkin Elmer 100 FT-IR spectrometer and Optical Rotations were recorded using a Perkin Elmer 343 polarimeter and are reported in 10 −1 deg cm 2 g −1 . Melting points were recorded on a Gallenkamp Hot Stage apparatus and are uncorrected.
Flash column chromatography was carried out using silica gel with particle size <60 µm and reverse phase column chromatography was carried out using silica gel 60 silanized (53-200 µm). Thin layer chromatography (TLC) was performed on aluminium backed Sigma-Aldrich TLC plates with F254 fluorescent indicator. Developed plates were air dried and analysed under a UV light or by staining with the appropriate indicator.
Large scale column chromatography was carried out using a Biotage Isolera 4 with either Biotage 340 g KP-Sil Snap Cartridges filled with 50 µm silica or Biotage 375 g KP-NH Snap Cartridges filled with amine-bonded silica.
The following nomenclature is used throughout when assigning NMRs:

Safety Note
The large scale synthesis was carried out at the European Knowledge Centre of Eisai Ltd. in Hatfield, UK. When scaling up reactions, a number of problems can be encountered, 4,5 including localised exotherms and higher concentrations of reactants. This was particularly important for the Mitsunobu step to form (R)-Allyl 3-iodo-2-(trimethylphenylamino)propanoate, because the use of DEAD carries with it a risk of explosion. Although the replacement of DEAD with DIAD was investigated, none of the desired product formed, possibly due to the increased size of the ligand S 4 preventing reaction occurring with the starting material. During optimization of this reaction it was observed that large temperature spikes could occur during the addition of DEAD and again during the addition of MeI, and in the large scale procedure both of these reagents were slowly added in CH 2 Cl 2 solution with careful monitoring of the temperature. All other steps in the reaction sequence could be carried out in a similar manner to the small-scale synthesis previously reported. 1,2 Dithiothreitol (8.71 g, 56.5 mmol, 1.5 eq.) was added to a stirred solution of (Fmoc-Cys-O t Bu) 2 2 (30.0 g, 37.6 mmol, 1 eq.)
A solution of trifluoroacetic acid (20.4 mL, 265 mmol, 10 eq.) in CH 2 Cl 2 (50 mL) was added to a stirred solution of triethylsilane (42.2 mL, 265 mmol, 10 eq.) and (2R, 6R)-(Trt, Allyl/Fmoc, t Bu) lanthionine (20.3 g, 26.5 mmol, 1 eq.) in CH 2 Cl 2 (150 mL) on ice. The temperature inside the reaction vessel was maintained at 20 °C throughout the addition. The temperature was monitored throughout the reaction by the presence of a thermometer within the reaction vessel. Once addition was complete, the ice bath was removed and the reaction was left to stir at room temperature for 4 h. After this time, excess CH 2 Cl 2 (200 mL) was added and the organic layer was washed with sodium bicarbonate (2 x 200 mL) and brine (200 mL [α] D 20 -11.0 (c 1.0, CH 3 OH).

Peptide Synthesis
General Experimental Details: Protected amino acids and coupling reagents were purchased from Novabiochem, coupling reagents, bases and solvents were purchased from Sigma-Aldrich. All water used was either distilled using an Elga Purelab Option R 7 water purifier or used directly from a bottle of HPLC-grade water. The peptides were synthesised on a MultiSynTech Syro Peptide Synthesiser (Model MP-60). Amino acid and reagent concentrations were calculated based on the quantity and loading of the resin. The total volume of all reagents in each step was 1.5 mL. All reagents were dissolved in HPLC grade DMF. Peptides were centrifuged using an Eppendorf Centrifuge, model 5810R and were lyophilised using a Thermo Scientific Heto PowerDry LL1500 freeze-dryer. Amino Acid Coupling: Fmoc-protected amino acid (4 eq.) was added to the reaction syringe with HBTU (4 eq.) and DIPEA (8 eq.). The mixture was agitated for 20 sec every 3 min for a total of 40 min. The reagents were removed by filtration under vacuum and the resin washed with DMF (4 x 1.5 mL).
Cleavage from the Resin: Peptides were first washed with DMF (4 x 1.5 mL), CH 2 Cl 2 (4 x 1.5 mL), methanol (4 x 1.5 mL) and diethyl ether (4 x 1.5 mL) before drying in a desiccator for 45 min. A solution of 94% TFA, 2.5% water, 2.5% EDT and 1% TIPS (2.5 mL) was then added to the resin and left to agitate for 30 min on the platform shaker. After this time, the entire solution was transferred to a Falcon tube and 12 mL of diethyl ether was added to precipitate the peptide.
The cleavage procedure was then repeated with fresh cleavage solution (2.5 mL containing 94% TFA, 2.5% water, 2.5% EDT and 1% TIPS) and left to agitate for a further 40 min. The entire solution was again transferred to a Falcon tube before addition of 12 mL of diethyl ether to precipitate the peptide.
The Falcon tubes were then centrifuged for 10 min at 4000 rpm and 4 °C before decanting off the diethyl ether solution.
This process was performed 3 times in total before re-dissolving the peptide in water and lyophilising.
HPLC Purification: The peptides were analysed and purified via reverse phase HPLC using either a Varian ProStar system with a Model 210 solvent delivery module and a Model 320 UV detector or a Dionex system with a PDA-100 photodiode array detector and a model ASI-100 automated sample injector. The preparative purification was performed using an ACE C8-300 semi-preparative column (150 x 10 mm, flow rate of 8.0 mL/min), with UV detection at 215 and 254 nm, loaded with 200 -1850 µL aliquots of a 10-20 mg/mL solution of peptide dissolved in water. Gradient conditions are reported for each peptide. The fractions containing the correct peak were pooled, the solvent removed under reduced pressure to approximately 2 mL and the solution freeze-dried.
HPLC Analysis: All peptides were analysed using UV detection at 215 and 254 nm, using the conditions shown below.
Retention times are reported for each peptide.

Synthesis of S-S bridged single ring peptides
Single ring analog 2b: The synthesis was carried out on a 100 mg scale using pre-loaded H-Lys(Boc)-2-Cl-Trt resin

S 25
Crystallization A sample of ProTx-II was obtained from Smartox Biotechnology (570 rue de la chimie, 38400 Saint Martin d'Hères, France). The sample was dissolved in water to a concentration of 10 mg/mL, and the solution was passed by centrifuge down a pre-washed desalting column (Spin-OUT GT100, 2 min at 3750 rpm). Hanging-drop vapour-diffusion crystallisation trials were performed using 96-well plates with a well volume of 100 µL and drop volume of 100 nL. Trays were made using a Labtech Mosquito nanodrop robot, and incubated at room temperature in a Rock Imager (Formulatrix Inc). Crystal growth was monitored daily using Rock Maker software (Formulatrix Inc).
Hits were scaled up in 24-well plates with a well volume of 500 µL and drop volume of 2 µL. Crystals were grown in a buffer containing 2M Li 2 SO 4 , 100 mM Tris/HCl (pH 8.5), and 2% v/v PEG 400, and appeared within 48 h.

Diffraction
Crystals were flash cooled in liquid nitrogen without additional cryoprotection. A heavy atom derivative crystal was prepared by adding 2 µL of 500 mM potassium iodide to the crystallization drop and incubating at room temperature for 30 minutes. The native crystal was obtained from the same drop prior to heavy atom derivatization. Both datasets were collected collected using a Rigaku Micromax-007HF source equipped with Varimax HF optics, a Saturn 944 HG CCD detector, and a 4-circle AFC11 goniometer. High-resolution diffraction data on a native crystal was collected at beamline 5.0.2. at the Advanced Light Source (Berkeley, CA, USA).

Processing and Experimental Phasing
All diffraction data was indexed, integrated, merged and scaled using autoPROC. 7 Key statistics from the data processing are listed in Table S1.  S1 Data collection and refinement statistics for the three crystal diffraction datasets collected in this study. Initial phases were solved using single isomorphous replacement with anomalous signal (SIRAS) using the native and derivative datasets collected on the home source, followed by solvent flattening and electron density modification, all implemented in autoSHARP. 8 Manual building of the structure was carried out in Coot. A higher resolution native dataset was collected using synchrotron radiation. The model built using SIRAS phases was rigid body refined into the new dataset and least-squares restrained refinement continued within SHELXL 9 and Refmac. 10 Inspection of the electron density maps allowed solvent molecules and some alternative sidechain conformations to be modelled. Some sidechain conformations could not be determined from the electron density maps and were truncated in the final model. Refinement statistics are shown in Table 1. Patch clamp data Peptides were tested against stably transfected cell lines expressing the hNav1.7 receptor, using the hNav1.7-HEK cell IonWorks Population Patch Clamp Assay (Essen) and the QPatch™ Patch Clamp Assay (B'SYS GmbH). The Study Reports from the two companies are included below. As the testing was carried out blind, without knowledge of the structure and sequence of the peptides, Table S2 shows the codes used by B'Sys and correlates these to the numbering used in the present paper.