Click-generated triazole ureas as ultrapotent, in vivo-active serine hydrolase inhibitors

The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.
Nature Chemical Biology (Impact Factor: 13). 05/2011; 7(7):469-78. DOI: 10.1038/nchembio.579
Source: PubMed


Serine hydrolases are a diverse enzyme class representing ∼1% of all human proteins. The biological functions of most serine hydrolases remain poorly characterized owing to a lack of selective inhibitors to probe their activity in living systems. Here we show that a substantial number of serine hydrolases can be irreversibly inactivated by 1,2,3-triazole ureas, which show negligible cross-reactivity with other protein classes. Rapid lead optimization by click chemistry-enabled synthesis and competitive activity-based profiling identified 1,2,3-triazole ureas that selectively inhibit enzymes from diverse branches of the serine hydrolase class, including peptidases (acyl-peptide hydrolase, or APEH), lipases (platelet-activating factor acetylhydrolase-2, or PAFAH2) and uncharacterized hydrolases (α,β-hydrolase-11, or ABHD11), with exceptional potency in cells (sub-nanomolar) and mice (<1 mg kg(-1)). We show that APEH inhibition leads to accumulation of N-acetylated proteins and promotes proliferation in T cells. These data indicate 1,2,3-triazole ureas are a pharmacologically privileged chemotype for serine hydrolase inhibition, combining broad activity across the serine hydrolase class with tunable selectivity for individual enzymes.

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Available from: Ku-Lung Hsu, Oct 27, 2014
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    • "We selected one of the more promiscuous compounds, JJH221 (4), for analysis by mass spectrometry (MS)-based ABPP methods to assess the range of SHs targeted by NHH carbamates . JJH221-sensitive SHs were identified using the quantitative MS method ABPP-SILAC (stable isotope labeling by amino acids in cell culture; Mann, 2006) (Adibekian et al., 2011). In brief, proteomes from isotopically heavy-and lightamino acid-labeled human PC3 cells were treated with JJH221 (20 mM) or DMSO, respectively, for 4 hr followed by the biotinylated FP probe FP-biotin (Liu et al., 1999) (2.5 mM, 1 hr). "
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    ABSTRACT: Serine hydrolase inhibitors, which facilitate enzyme function assignment and are used to treat a range of human disorders, often act by an irreversible mechanism that involves covalent modification of the serine hydrolase catalytic nucleophile. The portion of mammalian serine hydrolases for which selective inhibitors have been developed, however, remains small. Here, we show that N-hydroxyhydantoin (NHH) carbamates are a versatile class of irreversible serine hydrolase inhibitors that can be modified on both the staying (carbamylating) and leaving (NHH) groups to optimize potency and selectivity. Synthesis of a small library of NHH carbamates and screening by competitive activity-based protein profiling furnished selective, in vivo-active inhibitors and tailored activity-based probes for multiple mammalian serine hydrolases, including palmitoyl protein thioesterase 1, mutations of which cause the human disease infantile neuronal ceroid lipofuscinosis. Copyright © 2015 Elsevier Ltd. All rights reserved.
    Chemistry & biology 06/2015; 22(7). DOI:10.1016/j.chembiol.2015.05.018 · 6.65 Impact Factor
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    • "The analysis below uses mice, wholly labeled with 15N, generated by raising postweaned mice for 10 weeks on a diet in which the nitrogen component is exclusively derived from 15N-labeled Spirulina. Greater than 95% of brain proteins within these reference mice are 15N labeled (Adibekian et al., 2011). "
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    ABSTRACT: Protein palmitoylation, a reversible lipid modification of proteins, is widely used in the nervous system, with dysregulated palmitoylation being implicated in a variety of neurological disorders. Described below is ABE/SILAM, a proteomic strategy that couples acyl-biotinyl exchange (ABE) purification of palmitoyl-proteins to whole animal stable isotope labeling (SILAM) to provide an accurate tracking of palmitoylation change within rodent disease models. As a first application, we have used ABE/SILAM to look at Huntington's disease (HD), profiling palmitoylation change in two HD-relevant mouse mutants: the transgenic HD model mouse YAC128 and the hypomorphic Hip14-gt mouse, which has sharply reduced expression for HIP14 (Zdhhc17), a palmitoyl-transferase implicated in the HD disease process. Rather than mapping to the degenerating neurons themselves, the biggest disease changes instead map to astrocytes and oligodendrocytes (i.e., the supporting glial cells).
    Chemistry & biology 11/2013; 20(11). DOI:10.1016/j.chembiol.2013.09.018 · 6.65 Impact Factor
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    • "To maximize our chances of success, we targeted the serine hydrolase superfamily because of its large size, estimated to represent 1% of the proteome, and its sensitivity to well-characterized carbamate probes. We identified a chlorinated phenyl carbamate (WWL91) that blocks fibrinogen binding to activated platelets and identified its endogenous enzyme targets as arylacetamide deacetylase-like 1 (AADACL1, also known as KIAA1363 or NCEH1), a microsomal lipid hydrolase and acylpeptide hydrolase (APEH), a recently described cytosolic hydrolase that cleaves acetylated peptides in T cells (Adibekian et al., 2012; Jessani et al., 2002; Sekiya et al., 2009). The inhibitory effects of WWL91 in platelets could not be attributed to APEH, but instead depended on AADACL1. "
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    ABSTRACT: A comprehensive knowledge of the platelet proteome is necessary for understanding thrombosis and for envisioning antiplatelet therapies. To discover other biochemical pathways in human platelets, we screened platelets with a carbamate library designed to interrogate the serine hydrolase subproteome and used competitive activity-based protein profiling to map the targets of active carbamates. We identified an inhibitor that targets arylacetamide deacetylase-like 1 (AADACL1), a lipid deacetylase originally identified in invasive cancers. Using this compound, along with highly selective second-generation inhibitors of AADACL1, metabolomics, and RNA interference, we show that AADACL1 regulates platelet aggregation, thrombus growth, RAP1 and PKC activation, lipid metabolism, and fibrinogen binding to platelets and megakaryocytes. These data provide evidence that AADACL1 regulates platelet and megakaryocyte activation and highlight the value of this chemoproteomic strategy for target discovery in platelets.
    Chemistry & biology 08/2013; 20(9). DOI:10.1016/j.chembiol.2013.07.011 · 6.65 Impact Factor
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