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

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.

Nav1.7 inhibitors for the treatment of chronic pain.

The voltage gated sodium channel Nav1.7 plays an essential role in the transmission of pain signals. Strong human genetic validation has motivated extensive efforts to discover potent, selective, and efficacious Nav1.7 inhibitors for the treatment of chronic pain. This digest will introduce the structure and function of Nav1.7 and highlight the wealth of recent developments on a diverse array of Nav1.7 inhibitors, including optimization of their potency, selectivity, and PK/PD relationships.

Development of a concise synthesis of (+)-ingenol.

The complex diterpenoid (+)-ingenol possesses a uniquely challenging scaffold and constitutes the core of a recently approved anti-cancer drug. This full account details the development of a short synthesis of 1 that takes place in two separate phases (cyclase and oxidase) as loosely modeled after terpene biosynthesis. Initial model studies establishing the viability of a Pauson-Khand approach to building up the carbon framework are recounted. Extensive studies that led to the development of a 7-step cyclase phase to transform (+)-3-carene into a suitable tigliane-type core are also presented. A variety of competitive pinacol rearrangements and cyclization reactions were overcome to develop a 7-step oxidase phase producing (+)-ingenol. The pivotal pinacol rearrangement is further examined through DFT calculations, and implications for the biosynthesis of (+)-ingenol are discussed.

Cryo-EM reveals an unprecedented binding site for NaV1.7 inhibitors enabling rational design of potent hybrid inhibitors.

The voltage-gated sodium (NaV) channel NaV1.7 has been identified as a potential novel analgesic target due to its involvement in human pain syndromes. However, clinically available NaV channel-blocking drugs are not selective among the nine NaV channel subtypes, NaV1.1-NaV1.9. Moreover, the two currently known classes of NaV1.7 subtype-selective inhibitors (aryl- and acylsulfonamides) have undesirable characteristics that may limit their development. To this point understanding of the structure-activity relationships of the acylsulfonamide class of NaV1.7 inhibitors, exemplified by the clinical development candidate GDC-0310, has been based solely on a single co-crystal structure of an arylsulfonamide inhibitor bound to voltage-sensing domain 4 (VSD4). To advance inhibitor design targeting the NaV1.7 channel, we pursued high-resolution ligand-bound NaV1.7-VSD4 structures using cryogenic electron microscopy (cryo-EM). Here, we report that GDC-0310 engages the NaV1.7-VSD4 through an unexpected binding mode orthogonal to the arylsulfonamide inhibitor class binding pose, which identifies a previously unknown ligand binding site in NaV channels. This finding enabled the design of a novel hybrid inhibitor series that bridges the aryl- and acylsulfonamide binding pockets and allows for the generation of molecules with substantially differentiated structures and properties. Overall, our study highlights the power of cryo-EM methods to pursue challenging drug targets using iterative and high-resolution structure-guided inhibitor design. This work also underscores an important role of the membrane bilayer in the optimization of selective NaV channel modulators targeting VSD4.

Discovery of Acyl-sulfonamide Nav1.7 Inhibitors GDC-0276 and GDC-0310.

Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.

Structure- and Ligand-Based Discovery of Chromane Arylsulfonamide Nav1.7 Inhibitors for the Treatment of Chronic Pain.

Using structure- and ligand-based design principles, a novel series of piperidyl chromane arylsulfonamide Nav1.7 inhibitors was discovered. Early optimization focused on improvement of potency through refinement of the low energy ligand conformation and mitigation of high in vivo clearance. An in vitro hepatotoxicity hazard was identified and resolved through optimization of lipophilicity and lipophilic ligand efficiency to arrive at GNE-616 (24), a highly potent, metabolically stable, subtype selective inhibitor of Nav1.7. Compound 24 showed a robust PK/PD response in a Nav1.7-dependent mouse model, and site-directed mutagenesis was used to identify residues critical for the isoform selectivity profile of 24.

Chemical Proteomics Identifies SLC25A20 as a Functional Target of the Ingenol Class of Actinic Keratosis Drugs.

The diterpenoid ester ingenol mebutate (IngMeb) is the active ingredient in the topical drug Picato, a first-in-class treatment for the precancerous skin condition actinic keratosis. IngMeb is proposed to exert its therapeutic effects through a dual mode of action involving (i) induction of cell death that is associated with mitochondrial dysfunction followed by (ii) stimulation of a local inflammatory response, at least partially driven by protein kinase C (PKC) activation. Although this therapeutic model has been well characterized, the complete set of molecular targets responsible for mediating IngMeb activity remains ill-defined. Here, we have synthesized a photoreactive, clickable analogue of IngMeb and used this probe in quantitative proteomic experiments to map several protein targets of IngMeb in human cancer cell lines and primary human keratinocytes. Prominent among these targets was the mitochondrial carnitine-acylcarnitine translocase SLC25A20, which we show is inhibited in cells by IngMeb and the more stable analogue ingenol disoxate (IngDsx), but not by the canonical PKC agonist 12-O-tetradecanoylphorbol-13-acetate (TPA). SLC25A20 blockade by IngMeb and IngDsx leads to a buildup of cellular acylcarnitines and blockade of fatty acid oxidation (FAO), pointing to a possible mechanism for IngMeb-mediated perturbations in mitochondrial function.

14-step synthesis of (+)-ingenol from (+)-3-carene.

Ingenol is a diterpenoid with unique architecture and has derivatives possessing important anticancer activity, including the recently Food and Drug Administration-approved Picato, a first-in-class drug for the treatment of the precancerous skin condition actinic keratosis. Currently, that compound is sourced inefficiently from Euphorbia peplus. Here, we detail an efficient, highly stereocontrolled synthesis of (+)-ingenol proceeding in only 14 steps from inexpensive (+)-3-carene and using a two-phase design. This synthesis will allow for the creation of fully synthetic analogs of bioactive ingenanes to address pharmacological limitations and provides a strategic blueprint for chemical production. These results validate two-phase terpene total synthesis as not only an academic curiosity but also a viable alternative to isolation or bioengineering for the efficient preparation of polyoxygenated terpenoids at the limits of chemical complexity.