Inhibitor Fingerprinting of Rhomboid Proteases by Activity-Based Protein Profiling Reveals Inhibitor Selectivity and Rhomboid Autoprocessing

Rhomboid proteases were discovered almost 15 years ago and are structurally the best characterized intramembrane proteases. Apart from the general serine protease inhibitor 3,4-dichloro-isocoumarin (DCI) and a few crystal structures of the <i>Escherichia coli</i> rhomboid GlpG with other inhibitors, there is surprisingly little information about inhibitors of rhomboids from other species, probably because of a lack of general methods to measure inhibition against different rhomboid species. We here present activity-based protein profiling (ABPP) as a general method to screen rhomboids for their activity and inhibition. Using ABPP, we compare the inhibitory capacity of 50 small molecules against 13 different rhomboids. We find one new pan rhomboid inhibitor and several inhibitors that display selectivity. We also demonstrate that inhibition profile and sequence similarity of rhomboids are not related, which suggests that related rhomboids may be selectively inhibited. Finally, by making use of the here discovered inhibitors, we were able to show that two bacterial rhomboids autoprocess themselves in their N-terminal part

A General Solid Phase Method for the Preparation of Diverse Azapeptide Probes Directed Against Cysteine Proteases

A solid phase approach is presented for the synthesis of azapeptide inhibitors and activity based probes (ABPs) for cysteine proteases. This synthetic method allows the incorporation of diverse reactive warheads linked to different peptide recognition elements. Application of this method to the synthesis of a series of caspase probes is described

Protease Specificity Profiling in a Pipet Tip Using “Charge-Synchronized” Proteome-Derived Peptide Libraries

About 2% of the genome of human and other organisms codes for proteases. An important step toward deciphering the biological function of a protease and designing inhibitors is the profiling of protease specificity. In this work we present a novel, label-free, proteomics-based protease specificity profiling method that only requires simple sample preparation steps. It uses proteome-derived peptide libraries and enriches the cleaved sequences using strong cation exchange chromatography (SCX) material in a pipet tip. As a demonstration of the method’s versatility, we successfully determined the specificity of GluC, caspase-3, chymotrypsin, MMP-1 and cathepsin G from several hundreds to almost 2000 cleavage events per protease. Interestingly, we also found a novel intrinsic preference of cathepsin G for Asn at the P1 subsite, which we confirmed using synthetic peptides. Overall, this method is straightforward and requires so far the lowest investment in material and equipment for protease specificity profiling. Therefore, we think it will be applicable in any biochemistry laboratory and promote an increased understanding of protease specificity

Protease Specificity Profiling in a Pipet Tip Using “Charge-Synchronized” Proteome-Derived Peptide Libraries

About 2% of the genome of human and other organisms codes for proteases. An important step toward deciphering the biological function of a protease and designing inhibitors is the profiling of protease specificity. In this work we present a novel, label-free, proteomics-based protease specificity profiling method that only requires simple sample preparation steps. It uses proteome-derived peptide libraries and enriches the cleaved sequences using strong cation exchange chromatography (SCX) material in a pipet tip. As a demonstration of the method’s versatility, we successfully determined the specificity of GluC, caspase-3, chymotrypsin, MMP-1 and cathepsin G from several hundreds to almost 2000 cleavage events per protease. Interestingly, we also found a novel intrinsic preference of cathepsin G for Asn at the P1 subsite, which we confirmed using synthetic peptides. Overall, this method is straightforward and requires so far the lowest investment in material and equipment for protease specificity profiling. Therefore, we think it will be applicable in any biochemistry laboratory and promote an increased understanding of protease specificity

Protease Specificity Profiling in a Pipet Tip Using “Charge-Synchronized” Proteome-Derived Peptide Libraries

About 2% of the genome of human and other organisms codes for proteases. An important step toward deciphering the biological function of a protease and designing inhibitors is the profiling of protease specificity. In this work we present a novel, label-free, proteomics-based protease specificity profiling method that only requires simple sample preparation steps. It uses proteome-derived peptide libraries and enriches the cleaved sequences using strong cation exchange chromatography (SCX) material in a pipet tip. As a demonstration of the method’s versatility, we successfully determined the specificity of GluC, caspase-3, chymotrypsin, MMP-1 and cathepsin G from several hundreds to almost 2000 cleavage events per protease. Interestingly, we also found a novel intrinsic preference of cathepsin G for Asn at the P1 subsite, which we confirmed using synthetic peptides. Overall, this method is straightforward and requires so far the lowest investment in material and equipment for protease specificity profiling. Therefore, we think it will be applicable in any biochemistry laboratory and promote an increased understanding of protease specificity

Protease Specificity Profiling in a Pipet Tip Using “Charge-Synchronized” Proteome-Derived Peptide Libraries

About 2% of the genome of human and other organisms codes for proteases. An important step toward deciphering the biological function of a protease and designing inhibitors is the profiling of protease specificity. In this work we present a novel, label-free, proteomics-based protease specificity profiling method that only requires simple sample preparation steps. It uses proteome-derived peptide libraries and enriches the cleaved sequences using strong cation exchange chromatography (SCX) material in a pipet tip. As a demonstration of the method’s versatility, we successfully determined the specificity of GluC, caspase-3, chymotrypsin, MMP-1 and cathepsin G from several hundreds to almost 2000 cleavage events per protease. Interestingly, we also found a novel intrinsic preference of cathepsin G for Asn at the P1 subsite, which we confirmed using synthetic peptides. Overall, this method is straightforward and requires so far the lowest investment in material and equipment for protease specificity profiling. Therefore, we think it will be applicable in any biochemistry laboratory and promote an increased understanding of protease specificity

A Selective Activity-Based Probe for the Papain Family Cysteine Protease Dipeptidyl Peptidase I/Cathepsin C

Dipeptidyl peptidase I is involved in the activation of a number of disease-related proteases by removal of N-terminal prodipeptides. We here report a selective activity-based probe for monitoring dipeptidyl peptidase I activity in whole proteomes as well as in intact cells, without labeling of closely related enzyme family members

Determination of the inhibition mechanism of the hit compounds.

<p>(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 <b>31</b> 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.</p

A New Class of Rhomboid Protease Inhibitors Discovered by Activity-Based Fluorescence Polarization

<div><p>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 <i>E. coli</i> 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.</p></div

Apparent IC₅₀ (µM) of the hit compounds and S016, determined in duplicate measurements by FluoPol ABPP.

<p>Apparent IC₅₀ (µM) of the hit compounds and S016, determined in duplicate measurements by FluoPol ABPP.</p