Background

Protealysin-like proteases (PLPs) are a large group of zinc-containing metalloproteases belonging to the M4 family, according to the MEROPS database (www.ebi.ac.uk/merops) (Demidyuk et al., 2008, 2013). The interest in PLPs is primarily due to their possible involvement in bacterial pathogenesis, as they seem to be involved in bacterial invasion into eukaryotic cells, suppression of immune defense of some animals, and destruction of plant cell walls (Tsaplina et al., 2012, 2020; Demidyuk et al., 2013; Feng et al., 2014; Eshwar et al., 2018; Khaitlina et al., 2020).

We have previously developed a method for activity determination of protealysin (PLN), the prototype of PLP, from Serratia proteamaculans based on the hydrolysis of internally quenched fluorescent peptide substrate 2-aminobenzoyl-L-arginyl-L-seryl-L-valyl-L-isoleucyl-L-(ϵ-2,4-dinitrophenyl)lysine [Abz-RSVIK(Dnp)]. The proposed substrate was advantageous for quantitative activity analysis of PLN, as well as other M4 peptidases (Karaseva et al., 2019). Recently, we discovered emfourin (M4in) from S. proteamaculans, a novel potent slow-binding competitive proteinaceous inhibitor of PLN. To characterize the inhibition activity of M4in, we had to modify the PLN activity assay. Firstly, we significantly reduced the enzyme concentration and, in addition, introduced some minor modifications to decrease variation between replicates (Chukhontseva et al., 2021). Using the modernized protocol in the M4in study has shown that this approach is suitable for screening and characterizing PLP inhibitors.

Since Abz-RSVIK(Dnp) can be used to determine the activity of not only PLN but also other M4 family proteases (Karaseva et al., 2019), the proposed method can be adapted to determine the activity of inhibitors of various representatives of this family. Metalloproteases belonging to the M4 family are produced by significant human pathogens, such as Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis, Burkholderia cenocepacia, Enterococcus faecalis, Legionella pneumophila, Clostridium perfringens, and several pathogenic Vibrio species; their inhibitors, in the face of growing antibiotic resistance in bacteria, are considered as potential drug targets (Adekoya and Sylte, 2009). Thus, this method can be useful in the search for new antibacterial agents.