Pharmacologic ATF6 activating compounds are metabolically activated to selectively modify endoplasmic reticulum proteins

Abstract

Pharmacologic arm-selective unfolded protein response (UPR) signaling pathway activation is emerging as a promising strategy to ameliorate imbalances in endoplasmic reticulum (ER) proteostasis implicated in diverse diseases. The small molecule N-(2-hydroxy-5-methylphenyl)-3-phenylpropanamide (147) was previously identified (Plate et al., 2016) to preferentially activate the ATF6 arm of the UPR, promoting protective remodeling of the ER proteostasis network. Here we show that 147-dependent ATF6 activation requires metabolic oxidation to form an electrophile that preferentially reacts with ER proteins. Proteins covalently modified by 147 include protein disulfide isomerases (PDIs), known to regulate ATF6 activation. Genetic depletion of PDIs perturbs 147-dependent induction of the ATF6-target gene, BiP, implicating covalent modifications of PDIs in the preferential activation of ATF6 afforded by treatment with 147. Thus, 147 is a pro-drug that preferentially activates ATF6 signaling through a mechanism involving localized metabolic activation and selective covalent modification of ER resident proteins that regulate ATF6 activity.

Dengue and Zika virus infection are impaired by small molecule ER proteostasis regulator 147 in an ATF6-independent manner

“…Prior work showed that 147 is a prodrug that is metabolically oxidized to an iminoquinone or quinone methide, which covalently targets nucleophilic cysteine residues on cellular protein (Palmer et al, 2020;Paxman et al, 2018;Plate et al, 2016). We showed that the antiviral activity of 147 could be attenuated with BME treatment, demonstrating that the metabolic activation mechanism and covalent targeting of reactive thiols is similarly required to reduce virus infection.…”

“…Previous studies showed that 147 is a prodrug and requires metabolic activation in cells to generate a reactive p-quinone methide that can then form protein adducts with reactive cysteine residues ( Fig. 4A) (Palmer et al, 2020;Paxman et al, 2018;Plate et al, 2016). In several cell types, ER-resident protein disulfide isomerases (PDIs) were identified as the common protein targets of 147, and this modulation of PDIs was linked to the activation of ATF6 .…”

Designing Novel Therapies to Mend Broken Hearts: ATF6 and Cardiac Proteostasis

“…An inhibitor of S1P, the Golgi protease that cleaves and activates ATF6, inhibited 147-mediated ATF6 target gene induction. Using "click" chemistry and forms of 147 with chemical handles, the 147 interactome was defined in an attempt to demonstrate the mechanism by which it activates ATF6 [129]. It was discovered that 147 binds PDIs, which regulate disulfide bond formation in the ER.…”

ER Proteostasis Regulators Reduce Amyloidogenic Light Chain Secretion Through an On-Target, ATF6-Independent Mechanism

“…The activation of ATF6 by 147 can be inhibited by co-administration of resveratrol or β-mercaptoethanol 99 (BME), which block different steps of the compound activation mechanism ( Figure 4A) (Paxman et al, 2018). 00…”

“…One of the most promising small 85 molecules emerging from this screen was compound 147. Using a combination of medicinal chemistry and 86 chemical biology, we showed that 147 activates ATF6 through a mechanism involving compound metabolic 87 activation and covalent labeling of multiple PDIs (Paxman et al, 2018). Since PDIs are involved in regulating 88 the proper folding and disulfide bond formation for ER-targeted proteins (Appenzeller-Herzog & Ellgaard, 2008, 89…”