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

Adibekian A, Martin BR, Wang C, Hsu KL, Bachovchin DA, Niessen S, Hoover H, Cravatt BF - Nat. Chem. Biol. (2011)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
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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Competitive ABPP with clickable N-heterocyclic urea (NHU) activity-based probes AA6-AA10. (a) Structures of previously reported serine hydrolase inhibitors from the NHU class, including the endocannabinoid hydrolase inhibitor tetrazole urea LY218324015 (top) and the HSL inhibitors isoxazolonyl urea17 (middle) and 1,2,4-triazole urea18 (bottom). (b) Structures of carbamate- and NHU-alkyne probes with various leaving groups. (c) Competitive ABPP of AA6 - AA10 in the mouse brain membrane proteome. Brain membranes were incubated with 20 μM of AA6 - AA10 or DMSO for 30 min at 37 oC. Proteomes were then labeled with the SH-directed ABPP probe FP-Rh (2 μM, 30 min, 25 °C), separated by SDS-PAGE, and FP-Rh-labeled proteins detected by in-gel fluorescence scanning. This fluorescent gel and all gels in subsequent figures, are shown in grayscale. (d) Profiling the direct targets of AA6 - AA10 (20 μM, 30 min at 37 °C) in brain membranes in the presence or absence of the SH-directed probe FP-biotin (20 μM, 30 min at 37 °C). AA6 - AA10-labeled proteins were detected by reaction with an azide-Rh tag under click chemistry conditions following described protocols19,51. Targets of AA10 that are not competed by FP-biotin are highlighted with red boxes.
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Figure 1: Competitive ABPP with clickable N-heterocyclic urea (NHU) activity-based probes AA6-AA10. (a) Structures of previously reported serine hydrolase inhibitors from the NHU class, including the endocannabinoid hydrolase inhibitor tetrazole urea LY218324015 (top) and the HSL inhibitors isoxazolonyl urea17 (middle) and 1,2,4-triazole urea18 (bottom). (b) Structures of carbamate- and NHU-alkyne probes with various leaving groups. (c) Competitive ABPP of AA6 - AA10 in the mouse brain membrane proteome. Brain membranes were incubated with 20 μM of AA6 - AA10 or DMSO for 30 min at 37 oC. Proteomes were then labeled with the SH-directed ABPP probe FP-Rh (2 μM, 30 min, 25 °C), separated by SDS-PAGE, and FP-Rh-labeled proteins detected by in-gel fluorescence scanning. This fluorescent gel and all gels in subsequent figures, are shown in grayscale. (d) Profiling the direct targets of AA6 - AA10 (20 μM, 30 min at 37 °C) in brain membranes in the presence or absence of the SH-directed probe FP-biotin (20 μM, 30 min at 37 °C). AA6 - AA10-labeled proteins were detected by reaction with an azide-Rh tag under click chemistry conditions following described protocols19,51. Targets of AA10 that are not competed by FP-biotin are highlighted with red boxes.

Mentions: In the course of characterizing agents that perturb endocannabinoid uptake and metabolism, we discovered that the tetrazole urea LY218324015 (top, Fig. 1a) was a potent inhibitor of numerous SHs16. We confirmed that 1 inhibits the SH FAAH by covalent carbamoylation of the enzyme’s serine nucleophile16. There are a handful of other reports of N-heterocyclic ureas (NHUs) as SH inhibitors, including an isoxazolonyl urea17 (middle, Fig. 1a) and a 1,2,4-triazole urea18 (bottom, Fig. 1a), which are potent inhibitors of hormone-sensitive lipase (LIPE), but very limited selectivity data are available on these compounds. These initial findings suggested that the NHU might represent a useful scaffold for SH inhibitor design; however, several important questions remained unanswered. First, how does the electrophilicity of the NHU affect reactivity with SH enzymes, as well as potential cross-reactivity with other classes of proteins? Do NHUs inhibit SHs that are not susceptible to inactivation by other inhibitory scaffolds such as carbamates? Can one efficiently introduce structural diversity into the NHU scaffold by chemical synthesis? And, finally, can the properties of NHUs be optimized to achieve potent and selective inhibition of individual SHs in living systems?


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

Adibekian A, Martin BR, Wang C, Hsu KL, Bachovchin DA, Niessen S, Hoover H, Cravatt BF - Nat. Chem. Biol. (2011)

Competitive ABPP with clickable N-heterocyclic urea (NHU) activity-based probes AA6-AA10. (a) Structures of previously reported serine hydrolase inhibitors from the NHU class, including the endocannabinoid hydrolase inhibitor tetrazole urea LY218324015 (top) and the HSL inhibitors isoxazolonyl urea17 (middle) and 1,2,4-triazole urea18 (bottom). (b) Structures of carbamate- and NHU-alkyne probes with various leaving groups. (c) Competitive ABPP of AA6 - AA10 in the mouse brain membrane proteome. Brain membranes were incubated with 20 μM of AA6 - AA10 or DMSO for 30 min at 37 oC. Proteomes were then labeled with the SH-directed ABPP probe FP-Rh (2 μM, 30 min, 25 °C), separated by SDS-PAGE, and FP-Rh-labeled proteins detected by in-gel fluorescence scanning. This fluorescent gel and all gels in subsequent figures, are shown in grayscale. (d) Profiling the direct targets of AA6 - AA10 (20 μM, 30 min at 37 °C) in brain membranes in the presence or absence of the SH-directed probe FP-biotin (20 μM, 30 min at 37 °C). AA6 - AA10-labeled proteins were detected by reaction with an azide-Rh tag under click chemistry conditions following described protocols19,51. Targets of AA10 that are not competed by FP-biotin are highlighted with red boxes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3118922&req=5

Figure 1: Competitive ABPP with clickable N-heterocyclic urea (NHU) activity-based probes AA6-AA10. (a) Structures of previously reported serine hydrolase inhibitors from the NHU class, including the endocannabinoid hydrolase inhibitor tetrazole urea LY218324015 (top) and the HSL inhibitors isoxazolonyl urea17 (middle) and 1,2,4-triazole urea18 (bottom). (b) Structures of carbamate- and NHU-alkyne probes with various leaving groups. (c) Competitive ABPP of AA6 - AA10 in the mouse brain membrane proteome. Brain membranes were incubated with 20 μM of AA6 - AA10 or DMSO for 30 min at 37 oC. Proteomes were then labeled with the SH-directed ABPP probe FP-Rh (2 μM, 30 min, 25 °C), separated by SDS-PAGE, and FP-Rh-labeled proteins detected by in-gel fluorescence scanning. This fluorescent gel and all gels in subsequent figures, are shown in grayscale. (d) Profiling the direct targets of AA6 - AA10 (20 μM, 30 min at 37 °C) in brain membranes in the presence or absence of the SH-directed probe FP-biotin (20 μM, 30 min at 37 °C). AA6 - AA10-labeled proteins were detected by reaction with an azide-Rh tag under click chemistry conditions following described protocols19,51. Targets of AA10 that are not competed by FP-biotin are highlighted with red boxes.
Mentions: In the course of characterizing agents that perturb endocannabinoid uptake and metabolism, we discovered that the tetrazole urea LY218324015 (top, Fig. 1a) was a potent inhibitor of numerous SHs16. We confirmed that 1 inhibits the SH FAAH by covalent carbamoylation of the enzyme’s serine nucleophile16. There are a handful of other reports of N-heterocyclic ureas (NHUs) as SH inhibitors, including an isoxazolonyl urea17 (middle, Fig. 1a) and a 1,2,4-triazole urea18 (bottom, Fig. 1a), which are potent inhibitors of hormone-sensitive lipase (LIPE), but very limited selectivity data are available on these compounds. These initial findings suggested that the NHU might represent a useful scaffold for SH inhibitor design; however, several important questions remained unanswered. First, how does the electrophilicity of the NHU affect reactivity with SH enzymes, as well as potential cross-reactivity with other classes of proteins? Do NHUs inhibit SHs that are not susceptible to inactivation by other inhibitory scaffolds such as carbamates? Can one efficiently introduce structural diversity into the NHU scaffold by chemical synthesis? And, finally, can the properties of NHUs be optimized to achieve potent and selective inhibition of individual SHs in living systems?

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, California, USA.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus