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

Abstract

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.

Carnitine Traffic in Cells. Link With Cancer

“…Among others, CAC results as the most prominent target of IngMeb. This drug impairs FAO through the inhibition of CAC transport activity and this can explain, at least in part, the IngMeb pharmacological mechanism of action (Parker et al, 2017). Another intriguing observation is that a relationship between the composition of the cardiolipin, the most abundant phospholipid of the inner mitochondrial membrane, and cancer cell proliferation has been found.…”

Chemical Biology Gateways to Mapping Location, Association, and Pathway Responsivity

“…To enable target ID, an alkyne-functionalized variant of native RES is used in G-REX. For lipid-derived electrophiles (LDEs), alkyne tagging is minimally (if at all) invasive, although alkynylated versions of many drugs have been successfully deployed for successful target ID (Wright and Sieber, 2016; Parker et al, 2017). Radioisotope tagging or antibody affinity methods present alternatives to alkyne.…”

Covalent Small Molecules as Enabling Platforms for Drug Discovery

“…In a similar vein, natural products and natural product‐derived compounds have been subjected to PAL approaches to identify their targets in live cells. Parker et al . used a diazirine‐tagged analogue of ingenol mebutate (Ing‐DAyne Figure C, a bioactive component of the Euphorbia peplus extract approved by the FDA in 2012 to treat actinic keratosis), to identify the solute carrier protein SLC25A20 as a target that may contribute to the clinical effects of this drug.…”

“…Interestingly, this study does not identify the primary known target of ingenol mebutate (protein kinase C), even in cells overexpressing this target. This important observation suggests that incorporation of PAL groups in a manner that does not interfere with protein‐ligand binding (i.e., a solvent‐exposed region) may prevent efficient photo‐crosslinking due to poor diazirine‐protein contacts . To counteract this issue, researchers could incorporate PAL groups at two different growth vectors from the parent compound.…”