Counter Selection Substrate Library Strategy for Developing Specific Protease Substrates and Probes

Abstract

SUMMARY Legumain (AEP) is a lysosomal cysteine protease that is a lysosomal cysteine protease that was first characterized in leguminous seeds and later discovered in higher eukaryotes. AEP up-regulation is linked to a number of diseases including inflammation, arteriosclerosis and tumorigenesis. Thus legumain is an excellent molecular target for the development of new chemical markers. We deployed a hybrid combinatorial substrate library (HyCoSuL) approach to obtain P1-Asp fluorogenic substrates and biotin-labeled inhibitors that targeted legumain. Since this approach led to probes that were also recognized by caspases, we introduced a Counter Selection Substrate Library (CoSeSuL) approach that biases the peptidic scaffold against caspases, thus delivering highly selective legumain probes. The selectivity of these tools was validated using M38L and HEK293 cells. We also propose that the CoSeSuL methodology can be considered as a general principle in the design of selective probes for other protease families where selectivity is difficult to achieve by conventional sequence-based profiling.

Protease cleavage site fingerprinting by label‐free in‐gel degradomics reveals pH ‐dependent specificity switch of legumain

“…Advantages of DIPPS were demonstrated also at acidic pH where the profiling revealed that at low pH legumain is turned into an Asn/Asp‐specific protease capable of faster degradation of proteins. These results are in agreement with recent results obtained by combinatorial substrate libraries, which showed that recognition of small substrates by legumain depends solely on the P1 residue (Poreba et al , ). However, the total number of cleavages and the percentage of cleavages after Asp observed in large substrates at acidic pH were much higher as that in small fluorogenic substrates (z‐AAN‐AMC and z‐AAD‐AMC), suggesting differential binding of the substrates.…”

“…Human legumain was expressed in the baculovirus expression system (Poreba et al, 2016), whereas human cathepsins K, L, S, and V were expressed in the Pichia pastoris expression system (Invitrogen; Bromme et al, 2004;Mihelic et al, 2008). Active MMP-3 was purchased from Sigma (SRP7783).…”

Lansoprazole inhibits the cysteine protease legumain by binding to the active site

“…This indicated that lansoprazole directly formed S‐S bonds with the SH group of the cysteine in the active site of the proteases. This mechanism was confirmed by using the legumain‐selective activity‐based probe MP‐L01, which binds irreversibly to the active site of legumain . MP‐L01 competed with lansoprazole for binding to legumain and thus confirmed a direct binding of lansoprazole to the active site.…”

ABPP and Host–Virus Interactions

“…To allow specific activity-based labelling, a fluorogenic coumarin and a diphenyl phosphate warhead are added (Kasperkiewicz et al 2014). This library has previously been used to design probes targeting human proteases (Kasperkiewicz et al 2015;Poreba et al 2016Poreba et al , 2018. Recently, the HyCoSuL methodology was applied to design a probe, WRPK3, which rapidly labels Zika's NS3 protease at low nanomolar concentrations ( Fig.…”