Evaluation of the preprotein translocation motor SecA as a target for the development of novel antibiotics

The fight against bacterial infections is one of the major concerns in modern medicine due to the rapid rise of antibiotic-resistant bacteria. Therefore, there is an urgent need for new antibacterials – preferably directed against alternative bacterial targets – to combat hardly treatable infections caused by these resistant bacteria. One such potential target is the preprotein translocation motor SecA. SecA is a peripheral membrane ATPase and a key component of the Sec-dependent protein translocation pathway, which is the major route for protein export across or into the bacterial cytoplasmic membrane. Since SecA is essential for bacterial viability, ubiquitous and highly conserved in bacteria, but not present in eukaryotic cells, it represents an attractive antibacterial target.  

In order to evaluate SecA as a target for the development of novel antibiotics with a new mode of action, first an in silico analysis of various SecA structures was performed to define potentially druggable pockets on the surface of SecA. Comparison of these potential binding sites for small-molecules in the different SecA structures showed that six of these pockets are highly conserved among SecA proteins of both Gram-negative and Gram-positive bacteria. To investigate whether three of these conserved pockets are essential for the function of SecA, various amino acid substitutions were introduced in each of the three predicted druggable pockets of both Escherichia coli SecA and Staphylococcus aureus SecA1, taken as representative SecAs for Gram-negative and Gram-positive bacteria, respectively. Subsequently the effect of the introduced mutations on the function of SecA was examined using an in vivo genetic complementation assay. This demonstrated that the three selected pockets are essential for the function of both E. coli SecA and S. aureus SecA1.

To examine whether these predicted druggable pockets may represent effective drug targets, structure-based virtual ligand screening was performed against one of the most attractive, essential pockets in E. coli SecA, the signal peptide-binding site. About 500,000 commercially available small-molecules from the ChemBridge compound library were virtually screened against this potentially druggable site in E. coli SecA using a multi-step virtual ligand screening protocol. The 1040 top-scoring molecules were tested in vitro for inhibition of the translocation ATPase activity of SecA. Five inhibitors of the translocation ATPase, and not of the basal or membrane ATPase activity, were identified with IC50 values <65 µM. The most potent inhibitor revealed an IC50 of 24 µM. For the 5 most potent SecA inhibitors, the antimicrobial activity was determined. Two compounds were found to possess weak antibacterial activity against E. coli (IC50 ~198 µM), while others showed moderate antibacterial activity against S. aureus (IC50 ~100 µM). These molecules may be used as lead compounds leading to the design of more potent SecA inhibitors which block the SecA-preprotein interaction and specifically inhibit Sec-dependent protein translocation.

As an alternative approach to discover SecA and/or Sec-dependent protein translocation inhibitors, we developed target-based whole-cell screening assays. Therefore, recombinant E. coli strains were constructed for which inhibition of protein translocation can be easily detected using either enzyme- or fluorescence-based approaches. These developed assays were validated using the known SecA inhibitor sodium azide and can be used in the future for the discovery of Sec-dependent protein translocation inhibitors.

In this study, we demonstrated that SecA is an attractive target for the development of novel antibiotics since the bacterial protein translocation can be inhibited by small-molecules targeting the signal peptide-binding site of SecA, indicating that SecA is druggable. The identified SecA inhibitors, which are the first reported SecA-preprotein interaction inhibitors, should be further optimized to improve their potency and antibacterial activity. Alternatively, additional in silico, in vitro and/or target-based whole-cell screenings could be performed to identify more potent SecA and/or Sec-dependent protein translocation inhibitors. Hopefully, these inhibitors may contribute to the generation of a novel class of antibiotics which can be used to combat hardly treatable infections caused by multidrug-resistant bacteria.