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A new class of rhomboid protease inhibitors discovered by activity-based fluorescence polarization.

Wolf EV, Zeißler A, Vosyka O, Zeiler E, Sieber S, Verhelst SH - PLoS ONE (2013)

Bottom Line: Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones.They form covalent and irreversible complexes with the active site serine of GlpG.The presence of alkyne handles on the β-lactones also allowed activity-based labeling.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Freising, Germany.

ABSTRACT
Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases.

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Development of a rhomboid FluoPol ABPP assay.(A) Chemical structures of the ABPs EK2 and FP-R/FP-PEG-R. (B) 45 nM wild-type (WT) GlpG or the inactive S201A mutant (M) were preincubated with 100 µM inhibitor S016 or 1% DMSO vehicle control (30 min) and subsequently incubated with 1 µM of probe EK2 or FP-R (30 min). (C) Fluorescent polarization measured over time using 75 nM FP-R and 500 nM GlpG WT, 500 nM S201A or plain buffer. The mean of quadruplicate measurements is depicted with standard error. (D) The development of the Z’ value over time during a 5 h run of rhomboid FluoPol ABPP.
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pone-0072307-g001: Development of a rhomboid FluoPol ABPP assay.(A) Chemical structures of the ABPs EK2 and FP-R/FP-PEG-R. (B) 45 nM wild-type (WT) GlpG or the inactive S201A mutant (M) were preincubated with 100 µM inhibitor S016 or 1% DMSO vehicle control (30 min) and subsequently incubated with 1 µM of probe EK2 or FP-R (30 min). (C) Fluorescent polarization measured over time using 75 nM FP-R and 500 nM GlpG WT, 500 nM S201A or plain buffer. The mean of quadruplicate measurements is depicted with standard error. (D) The development of the Z’ value over time during a 5 h run of rhomboid FluoPol ABPP.

Mentions: Recently, we and others reported the first fluorescent ABPs for bacterial rhomboids [18], [20]. One ABP is the fluorophosphonate FP-PEG-rhodamine (FP-PEG-R; Figure 1A), the other one is based on the 4-chloro-isocoumarin scaffold (EK2; Figure 1A). Both FP-PEG-R and EK2 have only been used in gel-based applications. In view of previous work of the Cravatt laboratory [25], we expected that fluorescent rhomboid ABPs would be suitable for the development of a gel-free FluoPol ABPP screening method. Hence, we took EK2 and the commercially available fluorophosphonate FP-rhodamine (FP-R; Figure 1A) and verified whether these probes label rhomboid in an activity-based manner. Gratifyingly, both FP-R and EK2 labeled wild-type (WT) GlpG from E. coli, but not the inactive S201A mutant (Figure 1B). Labeling was also prevented by pre-inhibition of GlpG WT with the isocoumarin inhibitor S016, which we have identidfied in a previous MALDI-based screen (Figure 1B; see for structure Figure 2D) [18]. FP-R gave rise to a more intense labeling, probably due to the higher reactivity of the fluorophosphonate electrophile compared to the isocoumarin. We therefore chose this probe for subsequent FluoPol ABPP experiments. Until now FluoPol ABPP has only been performed on soluble enzymes without the presence of detergents. Hence, our initial experiments were focused on the optimization of FluoPol ABPP for usage with intramembrane proteases, which require the presence of detergents during their solubilization and purification. We found that detergents can interfere with FluoPol ABPP and lead to an unstable polarization signal over time (Figure S1). We tested a variety of conditions including different concentrations of dodecyl maltoside and Triton X-100 and found that a low amount of Triton X-100 (0.0125%) together with 0.01% Pluronic F127 gave robust and reproducible signals (Figure 1C). For assay quality assessment, we determined the Z’ value over time by measuring the fluorescence polarization signal for ten positive controls of the wild-type GlpG and ten negative controls of the inactive S201A mutant (Figure 1D). After 4 h, the Z’ reaches a value larger than 0.9, which is excellent for screening compound libraries [31].


A new class of rhomboid protease inhibitors discovered by activity-based fluorescence polarization.

Wolf EV, Zeißler A, Vosyka O, Zeiler E, Sieber S, Verhelst SH - PLoS ONE (2013)

Development of a rhomboid FluoPol ABPP assay.(A) Chemical structures of the ABPs EK2 and FP-R/FP-PEG-R. (B) 45 nM wild-type (WT) GlpG or the inactive S201A mutant (M) were preincubated with 100 µM inhibitor S016 or 1% DMSO vehicle control (30 min) and subsequently incubated with 1 µM of probe EK2 or FP-R (30 min). (C) Fluorescent polarization measured over time using 75 nM FP-R and 500 nM GlpG WT, 500 nM S201A or plain buffer. The mean of quadruplicate measurements is depicted with standard error. (D) The development of the Z’ value over time during a 5 h run of rhomboid FluoPol ABPP.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750051&req=5

pone-0072307-g001: Development of a rhomboid FluoPol ABPP assay.(A) Chemical structures of the ABPs EK2 and FP-R/FP-PEG-R. (B) 45 nM wild-type (WT) GlpG or the inactive S201A mutant (M) were preincubated with 100 µM inhibitor S016 or 1% DMSO vehicle control (30 min) and subsequently incubated with 1 µM of probe EK2 or FP-R (30 min). (C) Fluorescent polarization measured over time using 75 nM FP-R and 500 nM GlpG WT, 500 nM S201A or plain buffer. The mean of quadruplicate measurements is depicted with standard error. (D) The development of the Z’ value over time during a 5 h run of rhomboid FluoPol ABPP.
Mentions: Recently, we and others reported the first fluorescent ABPs for bacterial rhomboids [18], [20]. One ABP is the fluorophosphonate FP-PEG-rhodamine (FP-PEG-R; Figure 1A), the other one is based on the 4-chloro-isocoumarin scaffold (EK2; Figure 1A). Both FP-PEG-R and EK2 have only been used in gel-based applications. In view of previous work of the Cravatt laboratory [25], we expected that fluorescent rhomboid ABPs would be suitable for the development of a gel-free FluoPol ABPP screening method. Hence, we took EK2 and the commercially available fluorophosphonate FP-rhodamine (FP-R; Figure 1A) and verified whether these probes label rhomboid in an activity-based manner. Gratifyingly, both FP-R and EK2 labeled wild-type (WT) GlpG from E. coli, but not the inactive S201A mutant (Figure 1B). Labeling was also prevented by pre-inhibition of GlpG WT with the isocoumarin inhibitor S016, which we have identidfied in a previous MALDI-based screen (Figure 1B; see for structure Figure 2D) [18]. FP-R gave rise to a more intense labeling, probably due to the higher reactivity of the fluorophosphonate electrophile compared to the isocoumarin. We therefore chose this probe for subsequent FluoPol ABPP experiments. Until now FluoPol ABPP has only been performed on soluble enzymes without the presence of detergents. Hence, our initial experiments were focused on the optimization of FluoPol ABPP for usage with intramembrane proteases, which require the presence of detergents during their solubilization and purification. We found that detergents can interfere with FluoPol ABPP and lead to an unstable polarization signal over time (Figure S1). We tested a variety of conditions including different concentrations of dodecyl maltoside and Triton X-100 and found that a low amount of Triton X-100 (0.0125%) together with 0.01% Pluronic F127 gave robust and reproducible signals (Figure 1C). For assay quality assessment, we determined the Z’ value over time by measuring the fluorescence polarization signal for ten positive controls of the wild-type GlpG and ten negative controls of the inactive S201A mutant (Figure 1D). After 4 h, the Z’ reaches a value larger than 0.9, which is excellent for screening compound libraries [31].

Bottom Line: Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones.They form covalent and irreversible complexes with the active site serine of GlpG.The presence of alkyne handles on the β-lactones also allowed activity-based labeling.

View Article: PubMed Central - PubMed

Affiliation: Lehrstuhl für Chemie der Biopolymere, Technische Universität München, Freising, Germany.

ABSTRACT
Rhomboids are intramembrane serine proteases that play diverse biological roles, including some that are of potential therapeutical relevance. Up to date, rhomboid inhibitor assays are based on protein substrate cleavage. Although rhomboids have an overlapping substrate specificity, substrates cannot be used universally. To overcome the need for substrates, we developed a screening assay using fluorescence polarization activity-based protein profiling (FluoPol ABPP) that is compatible with membrane proteases. With FluoPol ABPP, we identified new inhibitors for the E. coli rhomboid GlpG. Among these was a structural class that has not yet been reported as rhomboid inhibitors: β-lactones. They form covalent and irreversible complexes with the active site serine of GlpG. The presence of alkyne handles on the β-lactones also allowed activity-based labeling. Overall, these molecules represent a new scaffold for future inhibitor and activity-based probe development, whereas the assay will allow inhibitor screening of ill-characterized membrane proteases.

Show MeSH