Development and Validation of a High‐Throughput Screening Assay for the Legionella ADP‐Ribosyl Transferase SdeA

Ubiquitination of proteins is one of the most crucial post-translational modifications in eukaryotic cells, typically involving conjugation of ubiquitin to a lysine residue in a substrate using a three-enzyme cascade that relies on ATP as energy source. The pathogen Legionella pneumophila, in contrast, employs a totally divergent pathway to ubiquitinate cellular host proteins in an unconventional manner that is crucial for bacterial proliferation. This multistep process is orchestrated by effector proteins from the SidE family which initially use NAD⁺ to adenosine diphosphate (ADP)-ribosylate ubiquitin in a mono-ADP-ribosyltransferase (mART) domain. The subsequent step relies on SidE phosphodiesterase activity to conjugate phosphoribosyl-Ubiquitin to serine residues in host substrates. Through these phosphoribosyl-ubiquitinating events, Legionella is able to gain local control over the host's ubiquitin system and simultaneously evades immune responses. Hence, pursuing new inhibitors which can disrupt these crucial steps in bacterial infection are essential towards further understanding and potentially blocking Legionella infection. Here, we present the application of an ε-NAD⁺ consumption-based fluorogenic assay to identify small molecule modulators of the SdeA effector enzyme in a High-Throughput Screening format, where over 600 compounds were screened. As a result, a potent inhibitor named cephalosporin C Zn²⁺ salt was discovered showing an IC₅₀ of 221 nM. To investigate the inhibitory properties more deeply, various cephalosporin analogs were synthesized where variations in charge and carbon length were introduced and their inhibitory efficiencies measured and compared. Our findings suggest that the inhibition is primarily attributed to the presence of the Zn²⁺ ion, rather than the cephalosporin core. We next compared the inhibitory potential of other bivalent metal ions, illustrating that the zinc ion causes the best inhibition of the Legionella effector.