Single Residue Substitutions That Confer Voltage-Gated Sodium Ion Channel Subtype Selectivity in the Na_V1.7 Inhibitory Peptide GpTx‑1

There is interest in the identification and optimization of new molecular entities selectively targeting ion channels of therapeutic relevance. Peptide toxins represent a rich source of pharmacology for ion channels, and we recently reported GpTx-1 analogs that inhibit Na_V1.7, a voltage-gated sodium ion channel that is a compelling target for improved treatment of pain. Here we utilize multi-attribute positional scan (MAPS) analoging, combining high-throughput synthesis and electrophysiology, to interrogate the interaction of GpTx-1 with Na_V1.7 and related Na_V subtypes. After one round of MAPS analoging, we found novel substitutions at multiple residue positions not previously identified, specifically glutamic acid at positions 10 or 11 or lysine at position 18, that produce peptides with single digit nanomolar potency on Na_V1.7 and 500-fold selectivity against off-target sodium channels. Docking studies with a Na_V1.7 homology model and peptide NMR structure generated a model consistent with the key potency and selectivity modifications mapped in this work

Selectivity of AM-8145 for human Na_V channels in heterologous cells and native Na_V currents in mDRG neurons.

<p>A. Concentration response curves for TTX-S channels including Na_V1.1-Na_V1.4, Na_V1.6-Na_V1.7 and mDRG TTX-S. IC₅₀ values with fold-selectivity compared to Na_V1.7 are listed in parenthesis. AM-8145 was 30- to 120-fold selective for Na_V1.7 over Na_V1.1-Na_V1.4 and Na_V1.6. B. Concentration response curves for TTX-R channels including Na_V1.5, Na_V1.8 and mDRG TTX-R. AM-8145 was over 1,700-fold selective for Na_V1.7 over Na_V1.5, Na_V1.8 and mDRG TTX-R. Curves in A and B were derived from whole cell patch clamp experiments. Data are presented as mean ± SEM with n = 2–4 cells per data point. C. Representative current traces for TTX-S channels before (black) and after (red) 10 nM AM-8145 addition. Cells were held at a voltage yielding 20% channel inactivation and stepped to -10 mV for 14 msec. D. Representative current traces for TTX-R channels before (black) and after (red) 100 nM AM-8145 addition. Cells were held at a voltage yielding 20% channel inactivation and stepped to -10 mV for 14 msec (Na_V1.5), or 0 mV for 15msec (Na_V1.8 and mDRG TTX-R). Studies with mDRG TTX-R included 0.5 uM TTX to block endogenous TTX-S currents. 10 nM AM-8145 preferentially inhibited Na_V1.7.</p

Potency and selectivity of JzTx-V and peptide analogs.

<p>Potency and selectivity of JzTx-V and peptide analogs.</p

JzTx-V peptides displace ¹²⁵I-ProTx-II binding to hNa_V1.7 HEK293 cells.

<p>A. Binding of ¹²⁵I-ProTx-II (1.5 nM) to Na_V1.7 HEK293 but not parental cells. Specific binding counts were displaced by cold AM-8145 (1 μM). B. Saturation curves for ¹²⁵I-ProTx-II binding. Closed circles (blue) represent total binding, filled squares (black) represent non-specific binding, and open circles (red) represent specific binding, defined as the difference between total and non-specific binding. C. Competition binding curves with 0.5 nM ¹²⁵I-ProTx-II in the presence of increasing concentrations of the indicated peptides. ¹²⁵I-ProTx-II binding is competed by AM-8145, AM-0422, GpTx-1 and ProTx-II peptides. D. Correlation of Na_V1.7 IC₅₀ values by PX electrophysiology (x-axis) with K_i values for binding (y-axis) for a panel of reference and JzTx-V peptides (R² of 0.74).</p

Inhibition of mechanically-evoked action potential firing in TTX-sensitive C-fibers by active peptide AM-0422 but not inactive peptide AM-8374.

<p>Time- and concentration-dependent inhibition of action potential firing by AM-0422. *** P < 0.001, **** P = 0.0001 by repeated measure two-way ANOVA with Dunnett’s multiple comparison test compared to 0.1% BSA vehicle. Representative traces for each group are shown for the 25 min time point on the right. 150 mN mechanical stimuli were applied for 10 sec every 5 min. Data are mean ± SEM; n = 13–27 fibers per group.</p

Inhibition of capsaicin-induced action potential firing in MEA recordings in rat DRG neurons by active peptide AM-0422 but not inactive peptide AM-8374.

<p>Heat maps of active electrodes detecting action potential firing 30 seconds following vehicle, 25 nM capsaicin, 25 nM capsaicin plus 1–100 nM of AM-0422 (A) or 25 nM capsaicin plus 1–100 nM of AM-8394 (B). The colors white/red, yellow/green, and blue/black correspond to high, medium, and low action potential firing frequency respectively. Whole well raster plots for buffer (C), 25 nM capsaicin (D), 25 nM capsaicin plus 100 nM AM-0422 (E), or 25 nM capsaicin plus 100 nM AM-8394 (F) added at the time point indicated by the vertical black arrows. Each row in the raster plots represents a single electrode with 16 electrodes per well and all recordings were performed at 37°C. G. Summary of total spikes from active wells following 2 minute treatment with 25 nM capsaicin (Cap), vehicle, 25 nM capsaicin plus 100 nM AM-8394 or 25 nM capsaicin plus 100 nM AM-0422. *** p<0.001, * p<0.05 by two-tailed unpaired t-test compared to capsaicin group.</p

JzTx-V sequence and inhibition of Na_V1.7 currents in HEK293 cells.

<p>A. Amino acid sequence and disulfide connectivity of JzTx-V. B. Manual patch clamp traces for control (black) and JzTx-V (0.3 nM; red) channel block at a holding potential of -140 mV (left) or -82 mV (right). Voltage protocols are depicted below the traces. C. JzTx-V (0.3 nM) channel block is partially reversed by high-frequency strong depolarizations following peptide washout. Cells were held at -140 mV and stepped to -10 mV to record Na_V1.7 current. Downward arrows indicate time points during which a high frequency protocol (depicted to right of time course; step to +100 mV for 14msec at 10 Hz for 20 sec) was applied.</p

Pharmacological characterization of potent and selective Na_V1.7 inhibitors engineered from <i>Chilobrachys jingzhao</i> tarantula venom peptide JzTx-V - Fig 6

<p><b>Representative whole cell patch clamp recordings for active peptide AM-0422 (left) and inactive peptide AM-8394 (right) showing reversible block of TTX-S Na</b>_<b>V</b><b>channels in mDRG neurons.</b> TTX was used as a positive control. AM-0422 IC₅₀ 27.6 ± 7.7 nM (n = 4), and AM-8394 was inactive up to 1 uM (3.5±1.1% inhibition, n = 2). Voltage protocol is depicted below the traces.</p

Ala/Glu scan heat map and NMR structure of JzTx-V peptides.

<p>A. Heat map showing single residue scan IC₅₀ data of Ala- and Glu-mutants against Na_V1.7, Na_V1.5 and Na_V1.4 using the IWQ platform. Black rectangles indicated wild-type JzTx-V sequences and the yellow rectangle indicates the Ile28 mutation that confers selectivity to Na_V1.4. Cys residues were not mutated. B. NMR structural ensemble of peptide 3 containing the Ile28 residue. Disulfides are in yellow, the N-terminal region in blue and the C-terminal region in red. C. Average of Ala- and Glu-mutant Na_V1.7 IC₅₀’s mapped onto a space-filling model of peptide 3 based on the NMR structure, showing key residues that lose Na_V1.7 activity upon modification cluster on one face of the peptide. Colors correspond to IC₅₀ ranges in panel A. Residues tolerant to modification are in green and located on the reverse side of the putative binding face. The Ile28 residue is colored magenta. D. NMR structural ensemble of peptide AM-8145 containing the Glu28 residue that imparts selectivity over Na_V1.4. Colors are as in panel B. E. Comparison of key amino acid side chains from peptide 3 and AM-8145 NMR structures. The Glu28 modification induced a change in overall conformation, with the acidic Glu28 side chain pointing down towards the basic Arg20 in AM-8145.</p