Discovery of a selective, state-independent inhibitor of NaV1.7 by modification of guanidinium toxins

Abstract

The voltage-gated sodium channel isoform Na_V1.7 is highly expressed in dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain-of-function and loss-of-function Na_V1.7 mutations have been identified in select individuals, and are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate Na_V1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting Na_V1.7 have been advanced to clinical development, no Na_V1.7-selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a Na_V1.7 inhibitor that blocks the extracellular vestibule of the channel with an IC₅₀ of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, Na_V1.1-1.6 and Na_V1.8. In contrast to other Na_V1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent in a protocol that favors the resting state of the channel, a protocol that favors the inactivated state, and a high frequency protocol. In a non-human primate study, animals treated with ST-2262 exhibited reduced sensitivity to noxious heat. These findings establish the extracellular vestibule of the sodium channel as a viable receptor site for the design of selective ligands targeting Na_V1.7.

Figure 1

(A) Cryo-EM structure of STX bound to human Na_V1.7-β1-β2 complex (PDB: 6j8g) with GX-936 positioned approximately based on PDB: 5ek0 in Pymol version 2.0.4 (Schrodinger, New York, NY). (B) Representative Na_V1.7 inhibitors that bind VSD IV. (C) Natural Na_V inhibitors that bind to the extracellular vestibule^–.

Figure 2

(A) The consensus pose for binding of STX in the extracellular vestibule of Na_V oriented C11 in proximity to the DIII pore loop prior to 2016. A revised pose based on mutant cycle analysis and recent cryo-EM structures orients the C13 carbamate near DIII^,. (B) ST-2262 was discovered by a rational design strategy aimed at identifying functional groups that interact with the DIII T1398/I1399 sequence motif unique to primate Na_V1.7. Values are mean (95% CI).

Figure 3

(A) Dose–response curves for the inhibitory effect of ST-2262 on Na_V1.1–Na_V1.8 stably expressed in CHO or HEK293 cells using a single-pulse (resting state) protocol with a 10 ms pulse from a holding potential of – 110 mV to voltage at peak activation (– 20 to + 10 mV). Na_V1.X IC₅₀ (in µM, mean, 95% CI). Na_V1.1: > 100; Na_V1.2: > 100; Na_V1.3: 65.3, 62.7–68.1; Na_V1.4: 80.7, 71.1–93.3; Na_V1.5: > 100; Na_V1.6: 17.9, 14.8–22.1; Na_V1.7: 0.072, 0.064–0.082; Na_V1.8: > 100. (B) Comparison of dose–response relationship of ST-2262 inhibition against Na_V1.7 using different stimulation protocols: resting state; two-pulse protocol contained an 8 s conditioning step to the voltage at half-inactivation, followed by a 20 ms step to voltage at full activation (half-inactivation protocol); high frequency single-pulse protocol stimulated at 30 Hz. Na_V1.7 IC₅₀ (in µM, mean, 95% CI). Resting state: 0.123, 0.104–0.145; half-inactivation: 0.087, 0.056–0.120; high frequency: 0.112, 0.015–0.357. (C) Comparison of dose–response relationship of Na_V1.7 inhibition against WT mNa_V1.7 and M1407T/D1408I mNa_V1.7 on a resting state protocol. mNa_V1.7 IC₅₀ (in µM, 95% CI). WT: 2.57, 2.30–2.87; M1407T/D1408I: 0.130, 0.055–0.307. (D) Comparison of dose–response of ST-2262 against transiently expressed hNa_V1.7 WT, hNa_V1.7 D1690N, and hNav1.7 T1398M/I1399D. IC₅₀ (in µM, mean, 95% CI). WT: 0.039, 0.032–0.047; D1690N: > 100; T1398M/I1399D: 1.87, 1.47–2.39.

Figure 4

ST-2262 increases withdrawal latency and reduces thermal evoked heart rate increase in a non-human primate noxious heat model. (A,B) Individual subject data points showing changes in withdrawal latency (A) and transient change in heart rate (ΔHR) (B) following thermal stimuli. Bar graphs are expressed as mean ± SEM. **Dunnett’s multiple comparison test, compared to baseline, p < 0.01. (C) Plasma level concentration of ST-2262 in plasma at different doses. (D,E) A lower heating rate thermal stimulus was presented for a maximum of 20 s, which selectively activates C fibers. In two subjects, the C-fiber-induced hand withdrawal response was replicable for testing. The efficacy endpoints measured were withdrawal latency (A) and heart rate change (B).