Affinity-Based Selectivity Profiling of an In-Class Selective Competitive Inhibitor of Acyl Protein Thioesterase 2

Abstract

Activity-based protein profiling (ABPP) has revolutionized the discovery and optimization of active-site ligands across distinct enzyme families, providing a robust platform for in-class selectivity profiling. Nonetheless, this approach is less straightforward for profiling reversible inhibitors and does not access proteins outside the ABPP probe's target profile. While the active-site competitive acyl protein thioesterase 2 inhibitor ML349 (K i = 120 nM) is highly selective within the serine hydrolase enzyme family, it could still interact with other cellular targets. Here we present a chemoproteomic workflow to enrich and profile candidate ML349-binding proteins. In human cell lysates, biotinylated-ML349 enriches a recurring set of proteins, including metabolite kinases and flavin-dependent oxidoreductases that are potentially enhanced by avidity-driven multimeric interactions. Confirmatory assays by native mass spectrometry and fluorescence polarization quickly rank-ordered these weak off-targets, providing justification to explore ligand interactions and stoichiometry beyond ABPP.

Protein depalmitoylases

“…These isoform-selective inhibitors are active in vivo following intraperitoneal injection, and engage their respective targets across all major tissues (Adibekian et al 2012). Furthermore, chemical proteomics analysis of biotin-conjugated ML349 confirmed largely selective inhibition across the proteome at low micromolar concentrations (Won et al 2017). Other covalent APT1/APT2 dual inhibitors have been reported, including the commercially available triazole urea ML211 (Adibekian, Martin, Speers, et al 2010; Adibekian et al 2011) and the N -hydroxyhydantoin carbamate ML378 (Hulce et al 2010; Cognetta et al 2015).…”

Recent advances in identifying protein targets in drug discovery

“…Proteins enriched by both positive and negative probes can be indicators of nonspecific binders. In the off-target study of an acyl protein thioesterase 2 inhibitor (ML349), both positive and negative biotinylated target ID probes were synthesized by changing their substitution sites at the phenyl ring (Won et al, 2017). Besides the already known target protein of ML349, other binding kinases, including ADK, DCK, and PDXK, were identified to elucidate the off-target effects of this compound (Table 1, 12).…”

Characterisation of the ghrelinergic system in human models of neurodegenerative diseases

“…For instance, since APT1 has been shown to be involved in some cancer types (Wang et al 2015b), small inhibitory compounds have been extensively used in cancer research. Moreover, with the availability of the APT1 and APT2 crystal structures (Devedjiev et al 2000;Won et al 2017), different studies have been focusing on synthetising more effective and welltolerated small molecules that could effectively reduce APT1 de-acylation activity in the brain. One of this compounds, Palmostatin B, that inhibits both APT1 and 2, has been tested for neuroprotective effects in rodent models of neurodegenerative disease (Dekker et al 2010), confirming the potential therapeutic relevance of repristinating AG:UAG ratio by inhibiting its de-acylation.…”

“…APT1 has been long been considered the primary depalmitoylase enzyme in cells (Won et al 2017); indeed, it typically catalyses the removal of long acyl chains, like palmitic acid, but the high adaptability of its channel would potentially allow the enzyme to interact with much shorter or longer carbon chains alike. Consistently, APT1 has been shown to have a very broad range of targets, from lysophospholipids to a variety of esters and thioester metabolites (Lin and Conibear 2015).…”

“…Whilst ghrelin acylation has been confirmed to occur in the brain, and possibly in the circulation, hardly any information is known about its deacylation. APT1 mRNA transcript has been almost ubiquitously detected in rodents (Allen Cell Types Database -RNA-Seq; Wang et al 1999;Won et al 2017), whilst APT1 protein has been found in various cell types, including neurones (Duncan and Gilman 1998;Toyoda et al 1999). Notably, APT1 has also been shown to be expressed on circulating macrophages, hinting to the possibility that acyl-ghrelin may be deacylated in the circulation.…”