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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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Proteomic characterization of endogenous APEH substrates using N-terminal labeling and enrichment. (a) Measured SILAC ratios for N-terminally enriched and unenriched peptides from the soluble proteome of mouse T-cells treated in situ with AA74-1 or AA39-2 (1 nM, 6 h). Green line designates the two-fold signal change cut-off used to define candidate APEH substrates in AA74-1-treated cells. (b) In vitro APEH exopeptidase activity assay with synthetic N-acetylated hexapeptides. APEH was recombinantly expressed in HEK-293 cells. Whole cell lysates were pre-treated with DMSO or AA74-1 (3 nM, 30 min), incubated with peptides for 10 h, and release of the N-terminal N-acetylated amino acid was measured by LC-MS. Data are presented as means ± s.d. (n = 3). Mock corresponds to control cells transfected with an empty vector. (c) Stimulation of mouse T-cell proliferation by APEH inhibition. Mouse T-cells were treated in situ with the indicated inhibitors (1 nM) or DMSO for 12 h. Cell proliferation was measured using the colorimetric agent WST-1 (*p < 0.05 for AA74-1-versus AA39-2-treated cells; **p < 0.01 for AA74-1- versus AA44-2-treated cells). Data are presented as means ± s.d. (n = 4).
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Figure 6: Proteomic characterization of endogenous APEH substrates using N-terminal labeling and enrichment. (a) Measured SILAC ratios for N-terminally enriched and unenriched peptides from the soluble proteome of mouse T-cells treated in situ with AA74-1 or AA39-2 (1 nM, 6 h). Green line designates the two-fold signal change cut-off used to define candidate APEH substrates in AA74-1-treated cells. (b) In vitro APEH exopeptidase activity assay with synthetic N-acetylated hexapeptides. APEH was recombinantly expressed in HEK-293 cells. Whole cell lysates were pre-treated with DMSO or AA74-1 (3 nM, 30 min), incubated with peptides for 10 h, and release of the N-terminal N-acetylated amino acid was measured by LC-MS. Data are presented as means ± s.d. (n = 3). Mock corresponds to control cells transfected with an empty vector. (c) Stimulation of mouse T-cell proliferation by APEH inhibition. Mouse T-cells were treated in situ with the indicated inhibitors (1 nM) or DMSO for 12 h. Cell proliferation was measured using the colorimetric agent WST-1 (*p < 0.05 for AA74-1-versus AA39-2-treated cells; **p < 0.01 for AA74-1- versus AA44-2-treated cells). Data are presented as means ± s.d. (n = 4).

Mentions: Despite having been postulated to serve as a key regulator of N-terminally acetylated proteins for many years25, very few endogenous substrates have been identified for APEH, nor have the biological effects of disrupting this enzyme been examined. The selective APEH inhibitor AA74-1 provided a pharmacological tool to investigate these questions. We measured changes in N-terminally modified proteins in AA74-1 (1 nM) versus DMSO-treated T-cells by SILAC combined with an established method for selective biotinylation of N-terminal amines26 (Supplementary Fig. 11a). Biotinylated proteins were enriched by avidin chromatography, digested on-bead with trypsin, and the resulting peptides analyzed by LC-MS on an LTQ-Orbitrap instrument. Data sets were filtered to identify proteins with multiple peptides that showed consistent two-fold or greater reductions in signals in AA74-1-treated cells. The resulting collection of ~25 proteins represented candidate APEH substrates (Fig. 6a, Table 1, and Supplementary Table 1). Importantly, none of these proteins showed altered N-terminal labeling profiles in T-cells treated with the PAFAH2 inhibitor AA39-2 (Table 1 and Supplementary Table 1), which produced a negligible number of total changes in the N-terminally modified protein profile (Fig. 6a). Furthermore, no changes were observed in total protein abundance for the subset of APEH substrates that could also be detected in unenriched proteomic profiles from AA74-1-treated cells (Supplementary Fig. 11b and Table 1), indicating that APEH inhibition affected the N-terminal modification state, but not overall stability of these proteins.


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)

Proteomic characterization of endogenous APEH substrates using N-terminal labeling and enrichment. (a) Measured SILAC ratios for N-terminally enriched and unenriched peptides from the soluble proteome of mouse T-cells treated in situ with AA74-1 or AA39-2 (1 nM, 6 h). Green line designates the two-fold signal change cut-off used to define candidate APEH substrates in AA74-1-treated cells. (b) In vitro APEH exopeptidase activity assay with synthetic N-acetylated hexapeptides. APEH was recombinantly expressed in HEK-293 cells. Whole cell lysates were pre-treated with DMSO or AA74-1 (3 nM, 30 min), incubated with peptides for 10 h, and release of the N-terminal N-acetylated amino acid was measured by LC-MS. Data are presented as means ± s.d. (n = 3). Mock corresponds to control cells transfected with an empty vector. (c) Stimulation of mouse T-cell proliferation by APEH inhibition. Mouse T-cells were treated in situ with the indicated inhibitors (1 nM) or DMSO for 12 h. Cell proliferation was measured using the colorimetric agent WST-1 (*p < 0.05 for AA74-1-versus AA39-2-treated cells; **p < 0.01 for AA74-1- versus AA44-2-treated cells). Data are presented as means ± s.d. (n = 4).
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Figure 6: Proteomic characterization of endogenous APEH substrates using N-terminal labeling and enrichment. (a) Measured SILAC ratios for N-terminally enriched and unenriched peptides from the soluble proteome of mouse T-cells treated in situ with AA74-1 or AA39-2 (1 nM, 6 h). Green line designates the two-fold signal change cut-off used to define candidate APEH substrates in AA74-1-treated cells. (b) In vitro APEH exopeptidase activity assay with synthetic N-acetylated hexapeptides. APEH was recombinantly expressed in HEK-293 cells. Whole cell lysates were pre-treated with DMSO or AA74-1 (3 nM, 30 min), incubated with peptides for 10 h, and release of the N-terminal N-acetylated amino acid was measured by LC-MS. Data are presented as means ± s.d. (n = 3). Mock corresponds to control cells transfected with an empty vector. (c) Stimulation of mouse T-cell proliferation by APEH inhibition. Mouse T-cells were treated in situ with the indicated inhibitors (1 nM) or DMSO for 12 h. Cell proliferation was measured using the colorimetric agent WST-1 (*p < 0.05 for AA74-1-versus AA39-2-treated cells; **p < 0.01 for AA74-1- versus AA44-2-treated cells). Data are presented as means ± s.d. (n = 4).
Mentions: Despite having been postulated to serve as a key regulator of N-terminally acetylated proteins for many years25, very few endogenous substrates have been identified for APEH, nor have the biological effects of disrupting this enzyme been examined. The selective APEH inhibitor AA74-1 provided a pharmacological tool to investigate these questions. We measured changes in N-terminally modified proteins in AA74-1 (1 nM) versus DMSO-treated T-cells by SILAC combined with an established method for selective biotinylation of N-terminal amines26 (Supplementary Fig. 11a). Biotinylated proteins were enriched by avidin chromatography, digested on-bead with trypsin, and the resulting peptides analyzed by LC-MS on an LTQ-Orbitrap instrument. Data sets were filtered to identify proteins with multiple peptides that showed consistent two-fold or greater reductions in signals in AA74-1-treated cells. The resulting collection of ~25 proteins represented candidate APEH substrates (Fig. 6a, Table 1, and Supplementary Table 1). Importantly, none of these proteins showed altered N-terminal labeling profiles in T-cells treated with the PAFAH2 inhibitor AA39-2 (Table 1 and Supplementary Table 1), which produced a negligible number of total changes in the N-terminally modified protein profile (Fig. 6a). Furthermore, no changes were observed in total protein abundance for the subset of APEH substrates that could also be detected in unenriched proteomic profiles from AA74-1-treated cells (Supplementary Fig. 11b and Table 1), indicating that APEH inhibition affected the N-terminal modification state, but not overall stability of these proteins.

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