Mechanism-specific assay design facilitates the discovery of Nav1.7-selective inhibitors

Abstract

Many ion channels, including Nav1.7, Cav1.3, and Kv1.3, are linked to human pathologies and are important therapeutic targets. To develop efficacious and safe drugs, subtype-selective modulation is essential, but has been extremely difficult to achieve. We postulate that this challenge is caused by the poor assay design, and investigate the Nav1.7 membrane potential assay, one of the most extensively employed screening assays in modern drug discovery. The assay uses veratridine to activate channels, and compounds are identified based on the inhibition of veratridine-evoked activities. We show that this assay is biased toward nonselective pore blockers and fails to detect the most potent, selective voltage-sensing domain 4 (VSD4) blockers, including PF-05089771 (PF-771) and GX-936. By eliminating a key binding site for pore blockers and replacing veratridine with a VSD-4 binding activator, we directed the assay toward non-pore-blocking mechanisms and discovered Nav1.7-selective chemical scaffolds. Hence, we address a major hurdle in Nav1.7 drug discovery, and this mechanistic approach to assay design is applicable to Cav3.1, Kv1.3, and many other ion channels to facilitate drug discovery.

Keywords: 1KαPMTX; N1742K; Nav1.7; VSD4; veratridine.

Fig. 1.

A conventional Nav1.7 membrane potential assay detects robust channel inhibition by TTX and tetracaine but fails to detect PF-771 and GX-936. (A–D) Representative membrane potential traces for TTX, tetracaine, PF-771, and GX-936. A total of 100 μM veratridine was added (indicated by arrows) to evoke channel activity, as represented by an increase in fluorescence signals. Cells were incubated with compounds for 3 min before veratridine application. (E) Inhibition concentration–response for TTX and tetracaine. The IC₅₀ was 0.034 ± 0.005 μM for TTX, and 3.6 ± 0.4 μM for tetracaine (n = 6). (F) Inhibition dose–response for PF-771 and GX-936. Only marginal inhibition was observed (n = 4). (G) Activities of 64,000 compounds from a membrane potential assay screen. Each dot represents one compound, numbered from 1 to 64,000. Yellow, 0.1% DMSO as negative control; blue, 1 μM TTX as positive control; red, four potent VSD4 blockers; and gray, library compounds. (H) Fold difference in IC₅₀ between Nav1.5 and Nav1.7 for 21 most potent Nav1.7 hits. No compounds showed greater than twofold selectivity (dotted line).

Fig. 2.

Biophysical and pharmacological characterization of Nav1.7 N1742K mutant channel. (A) Representative current traces for WT and N1742K channels. To assess voltage-dependent activation, currents were elicited by 20-ms test pulses (−80–15 mV in 5-mV increments) from a holding potential at −120 mV. Traces from 0-mV test pulses were highlighted. To determine voltage-dependent inactivation, peak currents at 0 mV were obtained after 500-ms conditioning prepulses varying from −120 to 15 mV. Traces obtained from test pulses (from −120 to 0 mV) were highlighted. Only selected voltage steps were shown in protocols. (B) Activation and inactivation curves for WT and N1742K mutant channels. Activation V_1/2: −29.58 ± 0.14 mV (n = 105, WT); −9.80 ± 0.09 mV (n = 155, N1742K); inactivation V_1/2: −62.85 ± 0.15 mV (n = 105, WT) and −48.85 ± 0.07 mV (n = 155, N1742K). (C) Pharmacology protocol and representative current traces. Cells were held at −50 mV, pulsed to 0 mV for 10 ms, followed by a 20-ms pulse at −150 mV and 10-ms pulse to 0 mV. Currents elicited at the second 0-mV pulse were used to derive inhibition at inactivated state. Current traces showed 20 nM TTX block of N1742K during a 10-min time period (from gray to black traces). (D) Fold change in IC₅₀ between N1742K and WT channel. Compounds were grouped based on binding sites. Tetracaine, CNV-802, and AMG-52 bind to the inner pore; TTX binds to outer vestibule (OV); ProTx-II binds to VSD2; PF-771 and GX-936 bind to VSD4.

Fig. 3.

Development of a N1742K-based membrane potential assay using 1KαPMTX as an activator. (A) Activities of 12 Nav activators on N1742K mutant channels tested at 100 µM in the presence and absence of 5 µM TTX (n = 4). The fluorescence signals were normalized to peak fluorescence obtained with 1KαPMTX. (B) Time-course of fluorescence responses to 100 µM 1KαPMTX. Signals were normalized by peak response of 1KαPMTX. (C) 1KαPMTX did not evoked robust response in WT channels. Signals were normalized by peak response of veratridine. Arrows indicate addition of 1KαPMTX.

Fig. 4.

Characterization of Nav1.7 blockers in N1742K-based membrane potential assay. A total of 100 μM 1KαPMTX and 100 μM veratridine were used to activate N1742K and WT Nav1.7, respectively. (A) Concentration–response relationships of TTX in N1742K (black line) and in WT (dotted line) membrane potential assays. TTX IC₅₀ was 0.048 ± 0.010 μM for N1742K, 0.034 ± 0.005 μM for WT (n = 6). (B) Concentration–response relationships for tetracaine for N1742K (black line) and for WT (dotted line) membrane potential assays. IC₅₀ was 488 ± 153 μM (n = 4) for N1742K and 3.6 ± 0.4 μM for WT (n = 6). (C) Representative kinetic traces for varying concentrations of ProTx-II in N1742K membrane potential assay. (D) Dose–response of ProTx-II in N1742K and WT membrane potential assays. IC₅₀ was 0.762 ± 0.066 (n = 4) for N1742K and 0.794 ± 0.037 μM for WT (n = 4). (E) Representative kinetic traces for PF-771 in N1742K membrane potential assay. (F) Concentration dose–responses of PF-771. IC₅₀ for PF-771 was 0.205 ± 0.005 μM for N1742K (n = 6); PF-771 only had marginal effect on WT (n = 4). (G) Representative kinetic traces for GX-936 in N1742K membrane potential assay. (H) Concentration–response relationship for GX-936. IC₅₀ for GX-936 was 0.040 ± 0.007 μM for N1742K (n = 6); GX-936 only had marginal effect on WT (n = 4). Dose–responses for WT (dotted lines in A, B, F, and H) appeared in Fig. 1 but were included for comparison purpose. Arrows indicate addition of 1KαPMTX.

Fig. 5.

A high-throughput screen using N1742K membrane potential assay. (A) Separation of activity between 0.1% DMSO (yellow, 9,476 wells) and 1 µM GX-936 treated group (blue, 3,708 wells). Activity was plotted as percent effect on 100 µM 1KαPMTX-evoked responses. Data were obtained from 103 plates as described in Materials and Methods. (B) Distribution of signal-to-background ratios for 103 plates. The signal was obtained as fluorescence responses to 100 µM 1KαPMTX, and the background was defined as responses to 1KαPMTX with 1 µM GX-936 treatment; 103 plates were included. (C) Distribution of Z′ values, reflecting robustness of the assay. Z′ > 0.5 is desired for high throughput screening (48). (D) Electrophysiological characterization of 384 randomly selected compounds from Roche library. Compounds were tested at 6.6 µM on N1742K mutant channel using patch-clamp protocols shown in Fig. 2C. Compounds were binned based on percentage of inhibition. (E) Electrophysiological characterization of 9,975 membrane potential assay hits. Compounds were binned based on percentage of inhibition.

Fig. 6.

The identification of GNE-0439 as a selective inhibitor of Nav1.7. (A) Chemical structure of GNE-0439. (B) GNE-0439 (5 μM) inhibited 1KαPMTX response in N1742K-based assay but had no effect on WT–veratridine assay. Fluorescence signals were normalized against peak response; faint dotted lines indicate baseline. Arrows indicate 1KαPMTX or veratridine addition. (C) Inhibition dose–response of GNE-0439 on Nav1.7, Nav1.5, N1742K, and R1608A channels. IC₅₀ value was 0.34 μM for Nav1.7 (95% confidence: 0.29–0.73 μM); 38.3 μM for Nav1.5 (95% confidence: 21.43–70.92 μM); and 0.37 μM for N1742K (95% confidence: 0.46–0.76 μM). GNE-0439 (66 μM) inhibited R1608A by 41.0 ± 12.8% (n = 6). The dotted line indicates 50% inhibition.

Fig. 7.

Drug binding sites for Nav1.7 activators and inhibitors underlie membrane potential assay performance. Veratridine and tetracaine bind to the inner pore, in the vicinity of residue N1742. In WT membrane potential assay, tetracaine affects veratridine binding and therefore inhibits response to veratridine; in N1742K mutant assay, tetracaine binding to N1742K channel is reduced. GX-936 and 1KαPMTX bind to the vicinity of VSD4 domain. In the N1742K mutant assay, GX-936 binding inhibits N1742K response to 1KαPMTX.