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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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Determination of the inhibition mechanism of the hit compounds.(A) Reversibility study by incubation of 500 nM GlpG WT with 100 µM of hit compounds, the two false positives, S016 or 1% DMSO vehicle control, followed by gel filtration and subsequent labeling with 1 µM EK2. (B) Tandem labeling of GlpG by the two hit compounds 31 and 43∶36 nM of GlpG wild-type (WT) or S201A mutant (M) was incubated with 100 µM the hit compounds or 1% DMSO vehicle control, followed by copper-mediated click reaction to attach a TAMRA-azide.
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pone-0072307-g003: Determination of the inhibition mechanism of the hit compounds.(A) Reversibility study by incubation of 500 nM GlpG WT with 100 µM of hit compounds, the two false positives, S016 or 1% DMSO vehicle control, followed by gel filtration and subsequent labeling with 1 µM EK2. (B) Tandem labeling of GlpG by the two hit compounds 31 and 43∶36 nM of GlpG wild-type (WT) or S201A mutant (M) was incubated with 100 µM the hit compounds or 1% DMSO vehicle control, followed by copper-mediated click reaction to attach a TAMRA-azide.

Mentions: Both 4-chloro-isocoumarins and β-lactones are electrophiles that can covalently and irreversibly react with active site serine residues of serine hydrolases [37]. To confirm whether the newly found 4-chloro-isocoumarins and β-lactones are indeed irreversible inhibitors, we pre-incubated GlpG with the hit compounds and the two false positives as negative controls, performed a gel filtration to remove non-covalently bound molecules and labeled the flow-through with the ABP EK2. As expected from their reported mechanism of action with soluble serine hydrolases, all verified hit compounds turned out to be irreversible inhibitors (Figure 3A).


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)

Determination of the inhibition mechanism of the hit compounds.(A) Reversibility study by incubation of 500 nM GlpG WT with 100 µM of hit compounds, the two false positives, S016 or 1% DMSO vehicle control, followed by gel filtration and subsequent labeling with 1 µM EK2. (B) Tandem labeling of GlpG by the two hit compounds 31 and 43∶36 nM of GlpG wild-type (WT) or S201A mutant (M) was incubated with 100 µM the hit compounds or 1% DMSO vehicle control, followed by copper-mediated click reaction to attach a TAMRA-azide.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3750051&req=5

pone-0072307-g003: Determination of the inhibition mechanism of the hit compounds.(A) Reversibility study by incubation of 500 nM GlpG WT with 100 µM of hit compounds, the two false positives, S016 or 1% DMSO vehicle control, followed by gel filtration and subsequent labeling with 1 µM EK2. (B) Tandem labeling of GlpG by the two hit compounds 31 and 43∶36 nM of GlpG wild-type (WT) or S201A mutant (M) was incubated with 100 µM the hit compounds or 1% DMSO vehicle control, followed by copper-mediated click reaction to attach a TAMRA-azide.
Mentions: Both 4-chloro-isocoumarins and β-lactones are electrophiles that can covalently and irreversibly react with active site serine residues of serine hydrolases [37]. To confirm whether the newly found 4-chloro-isocoumarins and β-lactones are indeed irreversible inhibitors, we pre-incubated GlpG with the hit compounds and the two false positives as negative controls, performed a gel filtration to remove non-covalently bound molecules and labeled the flow-through with the ABP EK2. As expected from their reported mechanism of action with soluble serine hydrolases, all verified hit compounds turned out to be irreversible inhibitors (Figure 3A).

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