Discovery of Fluoromethylketone-Based Peptidomimetics as Covalent ATG4B (Autophagin-1) Inhibitors

Abstract

ATG4B or autophagin-1 is a cysteine protease that cleaves ATG8 family proteins. ATG4B plays essential roles in the autophagosome formation and the autophagy pathway. Herein we disclose the design and structural modifications of a series of fluoromethylketone (FMK)-based peptidomimetics as highly potent ATG4B inhibitors. Their structure−activity relationship (SAR) and protease selectivity are also discussed.KEYWORDS: ATG4B, autophagy, covalent inhibitor, fluoromethylketone, peptidomimetics A utophagy is an evolutionarily conserved process essential for cell homeostasis and housekeeping by catabolizing aggregated proteins and damaged cellular components. 1 The hallmark of autophagy is the formation of autophagosomes and subsequent fusion with lysosomes to achieve degradation of their contents. Dysregulation of autophagy has been recently described in the pathogenesis of a variety of diseases such as cancer, neurodegenerative and metabolic disorders, and viral infections. 2,3 Modulation of autophagy has become a very active area of preclinical and clinical research, and particularly, there is high interest to identify potent and specific autophagy inhibitors. 4 ATG4 or autophagins are a class of cytosolic cysteine proteases that cleave ATG8 family proteins, such as light chain 3 (LC3). ATG4 plays essential roles in the formation and maturation of autophagosomes. 5,6 Among all four human ATG4 orthologues, ATG4B is functionally dominant, and it is the sole enzyme reported to efficiently cleave LC3 precursors and to deconjugate lipid from membrane bound LC3-phosphatidylethanolamine (LC3-PE). 7 ATG4B has been considered as a potential therapeutic target in the development of chemosensitizer for the treatment of certain cancer types. Recently, a number of small molecule ATG4B inhibitors such as Z-L-Phe-chloromethylketone and NSC185058 were reported to have modulatory effects on the autophagy process. 8−10 However, because of its high chemical reactivity, the chloromethylketone moiety is usually associated with significant cytotoxicity. NSC185058, however, is only a weak ATG4B inhibitor with an IC 50 of 51 μM in an assay detecting the cleavage of LC3-GST. 10 Previously, we reported the identification of Z-FA-FMK (1, Figure 1) as a covalent active-site directed ATG4B inhibitor from a TR-FRET based focused library screening. 11 The hit expansion of 1 led to the discovery of Z-FG-FMK (2), which was 10-times more potent than 1 with an IC 50 of 1.2 μM in the biochemical assay. 11 Herein we disclose the structure-guided optimization of 2 toward highly potent fluoromethylketone (FMK)-based ATG4B inhibitors.As an early step in the autophagosome formation, ATG4B cleaves proLC3 to expose the C-terminal Gly120 for subsequent PE-conjugation and autophagosome membrane insertion. 5 The cocrystal structures of catalytically inert C74S or

Autophagy-Independent Functions of the Autophagy Machinery

“…Indeed, considerable attention has been given so far to the structural characterization of single proteins and protein complexes linked to degradative autophagy, including ATG3 (Yamada et al, 2007), ATG4B (Sugawara et al, 2005), BECN1 (Li et al, 2012b), VPS34 (Miller et al, 2010;Rostislavleva et al, 2015), the ATG12-ATG5:ATG16L1 complex (Noda et al, 2008), LC3B (Sugawara et al, 2004), and several others. Although these studies provided profound mechanistic insights into bona fide autophagic responses (Li et al, 2012b;Satoo et al, 2009) and fostered the development of targeted inhibitors (Miller et al, 2010;Qiu et al, 2016), experimental design was often, if not always, biased toward autophagy-relevant domains and interactions. That said, it may not always be feasible to molecularly dissociate the autophagic and non-autophagic functions of a specific protein, even in the presence of detailed data on structure and physical interactions.…”

Stacking Interactions of Heterocyclic Drug Fragments with Protein Amide Backbones

“…Optimizing amide stacking has also been used to improve inhibition of a number of targets, including the aspartic protease endothiapepsin, protein kinase A (PKA), and the cysteine protease autophagin‐1 . Most recently, Diederich et al .…”

“…Optimizing amide stacking has also been used to improve inhibition of an umber of targets, includingt he aspartic protease endothiapepsin, [14] protein kinase A( PKA), [15] and the cysteine proteasea utophagin-1. [16] Most recently,D iederich et al [17] tried to design an inhibitor for the cysteine protease cathepsin Lb ym aximizing amide stackingi nteractions. Variations of ah eterocyclic core led to K i values spanning two orders of magnitude (from 1450 nm for quinolone to 4nm for benzothiophene).…”

A Systematic Analysis of Atomic Protein-Ligand Interactions in the PDB

“…83 These interactions were also shown to sometimes play an important role in ligand binding. [84][85][86] For example, the selective permeation of urea is facilitated by amide!! !π stacking interactions in the urea transporter.…”