bioRxiv preprint doi: https://doi.org/10.1101/869206; this version posted December 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Discovery of a Selective, State-Independent Inhibitor of NaV1.7 by Modification of Guanidinium Toxins Authors: Pajouhesh H1, Beckley JT2, Delwig A1, Hajare HS3, Luu G1, Monteleone D1, Zhou X1, 5 Ligutti J4, Amagasu S4, Moyer BD4, Yeomans D5, Du Bois J4, Mulcahy JV1* Affiliations: 1SiteOne Therapeutics, South San Francisco, CA 94080 2SiteOne Therapeutics, Bozeman, MT 59718 3Department of Chemistry, Stanford University, Stanford, CA 94305 10 4Neuroscience Department, Amgen Research, Thousand Oaks, CA 91320 5Department of Anesthesiology, Perioperative, and Pain Medicine, Stanford University, Stanford, CA 94305 *Correspondence to: [email protected] 15 Abstract The voltage-gated sodium channel isoform NaV1.7 is highly expressed in small diameter dorsal root ganglion neurons and is obligatory for nociceptive signal transmission. Genetic gain- of-function and loss-of-function NaV1.7 mutations have been identified in select individuals, and 20 are associated with episodic extreme pain disorders and insensitivity to pain, respectively. These findings implicate NaV1.7 as a key pharmacotherapeutic target for the treatment of pain. While several small molecules targeting NaV1.7 have been advanced to clinical development, no NaV1.7- selective compound has shown convincing efficacy in clinical pain applications. Here we describe the discovery and characterization of ST-2262, a NaV1.7 inhibitor that blocks the extracellular 25 vestibule of the channel with an IC50 of 72 nM and greater than 200-fold selectivity over off-target sodium channel isoforms, NaV1.1–1.6 and NaV1.8. In contrast to other NaV1.7 inhibitors that preferentially inhibit the inactivated state of the channel, ST-2262 is equipotent against resting and inactivated protein conformers. In a non-human primate model, animals treated with ST-2262 exhibit markedly reduced sensitivity to noxious heat. These findings establish the extracellular 30 vestibule of the sodium channel as a viable receptor site for selective ligand design and provide insight into the pharmacology of state-independent inhibition of NaV1.7. Significance Statement Pain is among the most common reasons for seeking medical care, yet many frequently 35 prescribed drugs, particularly the opioids, cause problematic side effects and carry a risk of addiction. Voltage-gated sodium ion channels (NaVs) have emerged as promising targets for the development of non-opioid pain medicines. NaVs are involved in the propagation of electrical signals along neurons throughout the body. Humans born without a functional copy of one sodium channel subtype, NaV1.7, are unable to experience most types of pain. In the present work, we 40 disclose the discovery and characterization of a selective inhibitor of NaV1.7 that reduces sensitivity to a painful thermal stimulus in non-human primates. Findings from this work may help guide the development of novel, non-addictive drug candidates as alternatives to opioids. 1 bioRxiv preprint doi: https://doi.org/10.1101/869206; this version posted December 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. Introduction + The voltage-gated Na ion channel (NaV) isoform 1.7 has emerged as a high-interest target for the discovery of non-opioid pain therapeutics based on its preferential expression in small diameter peripheral sensory neurons and compelling Validation from human genetics.(1) NaV1.7 5 loss-of-function mutations result in whole-body insensitivity to pain; conversely, gain-of-function variants are associated with episodic extreme pain disorders and small fiber neuropathies.(2–5) Discovery of selective inhibitors of NaV1.7 has been challenging due to the structural conservation of off-target NaV isoforms (NaV1.1–1.6, NaV1.8 and NaV1.9), inhibition of which is likely to result in safety liabilities.(6–8) 10 NaVs are integral membrane proteins expressed in excitable cells that comprise a ~260 kD pore-forming a-subunit and two accessory b-subunits (Figure 1).(9) The central pore of the a- subunit is encircled by four voltage-sensing domains (VSD I–IV). Channel gating occurs through protein conformational changes in response to membrane depolarization. At least nine discrete binding sites on the NaV a-subunit have been identified for peptides and small molecules that 15 influence ion conductance.(10) The large majority of molecules that engage NaVs bind preferentially to a specific conformational state of the channel and show use-dependent activity. Clinical NaV inhibitors (e.g., bupivacaine, lidocaine, carbamazepine) are both state- and frequency- dependent agents that lodge in the intracellular pore of the a-subunit, a site that is highly conserved between isoforms. These drugs rely on local administration or frequency dependent inhibition to 20 achieve a margin between the desired pharmacodynamic effect and dose-limiting side effects. Investigational NaV inhibitors, such as peptide toxins isolated from venomous species, interact with VSDs, trapping the S4 helix in either an activated or deactivated conformation.(11, 12) Similarly, a class of small molecule aryl and acyl sulfonamide compounds bind to the activated conformation of VSD IV (Figure 1b).(13–16) By contrast, cationic guanidinium toxins and peptide 25 cone snail toxins inhibit ion conduction by sterically occluding the extracellular vestibule of the channel pore (Site 1). The former are a unique collection of natural products exemplified by saxitoxin and tetrodotoxin – high affinity, state independent blockers against six of nine NaV subtypes (Figure 1c).(17) N O O N A. B. O O S S CF S Cl Cl S O O O STX 3 N N N H S (extracellular H N N O O F H vestibule) H3CH2C CN H N O F N 2 N N NH GX-936 GX-936 PF-05089771 GDC-0276 (VSD IV) Genentech/Xenon Pfizer Genentech/Xenon hNaV1.7 IC50 0.001 µM hNaV1.7 IC50 0.011 µM hNaV1.7 IC50 0.0004 µM hNaV1.2 IC50 0.11 µM hNaV1.1 IC50 0.011 µM hNaV1.5 IC50 25 µM hNaV1.2 IC50 0.020 µM hNaV1.6 IC50 0.16 µM hNaV1.5 IC50 0.049 µM O OH HO O C. N HO NH2 O NH2 N NH O O O HO OH NH2 – O3 SO N N H HOHN HO OH HO NH HN NH H OH HOHN N O N OH H2N NH2 H2N O STX TTX ZTX (+)-saxitoxin (–)-tetrodotoxin (–)-zetekitoxin AB 30 Figure 1. (a) Cryo-EM structure of STX bound to human NaV1.7-b1-b2 complex (PDB: 6j8g) with GX-936 positioned approximately based on PDB: 5ek0. (b) RepresentatiVe NaV1.7 inhibitors that bind VSD IV. (c) Natural NaV inhibitors that bind to the extracellular vestibule.(18–21) In the pursuit of isoform-selective inhibitors of NaV1.7, two binding sites, the cystine knot 35 toxin site at VSD II and the sulfonamide site at VSD IV, have been heavily interrogated. Certain 2 bioRxiv preprint doi: https://doi.org/10.1101/869206; this version posted December 10, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. cystine knot toxins that engage VSD II such as HwTx-IV, Pn3a and ProTx-II exhibit 6–1000x selectivity for NaV1.7 over other channel isoforms. Potency and selectivity for this target have been improved with synthetic toxin derivatives.(22–26) Among small, Lipinski-type molecules, only the aryl and acyl sulfonamides pioneered by Icagen/Pfizer and subsequently investigated by 5 Amgen, Chromocell, Genentech/Xenon, Merck and others have shown evidence of significant NaV1.7 isoform selectivity.(16, 7) Within the sulfonamide series, selectivity levels are >1000x over certain off-target isoforms including the cardiac isoform, NaV1.5, but generally 10–50x against NaV1.2 and NaV1.6. Many but not all sulfonamide NaV1.7 inhibitors suffer from undesirable pharmaceutical properties, including high plasma protein binding (e.g. >99.8%), 10 cytochrome p450 inhibition, in vitro hepatotoxicity and high unbound clearance,(27, 28) which have hindered clinical development. Although a number of candidates have been advanced to human testing, one compound has been discontinued after a Phase 2 study likely due to limited efficacy (PF-05089771); others have been discontinued after Phase 1 trials for reasons that may be related to safety liabilities such as elevated expression of liver transaminases and hypotension 15 (GDC-0276).(29, 30) Electrophysiology studies with naturally occurring Site 1 ligands against different wild- type and mutant NaV isoforms have identified the extracellular vestibule of NaV1.7 as a promising locus for selective inhibitor design.(31–33) The outer mouth of the channel is formed from residues that link the S5–S6 helices (referred to as pore loops) from each of the four domains. The domain 20 III pore loop of human NaV1.7 contains a sequence variation, 1398T/1399I, that is not present in other human NaV subtypes (which contain MD at equivalent position, Suppl Table 1).(31) Comparison of the amino acid sequence of the domain III pore loop across species indicates that this variation is unique to primates. The half maximal inhibitory concentration (IC50) value for saxitoxin (STX) is markedly altered (250-fold change) depending on the presence or absence of 25 the 1398T/1399I variant. Against rNaV1.4, the IC50 of STX is 2.8 ± 0.1 nM compared to hNaV1.7 for which the IC50 = 702 ± 53 nM.(31) Introduction of the alternative domain III pore loop sequence by mutagenesis restores potency (hNaV1.7 T1398M/I1399D IC50 = 2.3 ± 0.2 nM).