Identification of selective inhibitors of S-adenosyl-L-homocysteine hydrolase from Pseudomonas aeruginosa via high-throughput screening and crystallographic approaches

M. Gawel¹, P. H. Malecki¹, M. Komorowska¹, J. Sadoch², M. Otrocka³,J. Kolanowski⁴, K. Brzezinski¹

¹Polish Academy of Sciences, Institute of Bioorganic Chemistry, Department of Structural Biology of Prokaryotic Organisms, 61-704, Poznan 

²Polish Academy of Sciences, Institute of Bioorganic Chemistry, High-throughput Screening Laboratory, 61-704, Poznan

³Polish Academy of Sciences, Institute of Bioorganic Chemistry, Laboratory of Molecular Assays, 61-704, Poznan

⁴Polish Academy of Sciences, Institute of Bioorganic Chemistry, Department of Molecular Probes and Prodrugs, 61-704, Poznan

mgawel@ibch.poznan.pl

S-adenosyl-L-methionine (SAM)-dependent methylation reactions are crucial for numerous vital processes in prokaryotic and eukaryotic organisms. In human cells, as well as its opportunistic pathogen, Pseudomonas aeruginosa, these reactions are controlled by only one enzyme, namely S-adenosyl-L-homocysteine hydrolase (SAHase). Therefore, SAHase is an essential element of cell metabolism. Thus, selective inhibition of the bacterial enzyme is an excellent possibility for drug intervention at the molecular level of cell metabolism. On the one hand, the goal is challenging because SAHases are conserved proteins, especially at the active site. However, our recent study indicates that the activity of SAHases is influenced by affecting the enzyme dynamics [1]. Therefore, targeting SAHase dynamics is an alternative strategy for developing new selective inhibitors of SAHase from Pseudomonas aeruginosa (PaSAHase) that do not bind in the highly-conserved active site but on the protein surface regions.

We started our research with a high-throughput inhibition study of PaSAHase, where we tested around 5000 compounds from the Pilot European Chemical Biology Library (EU-OPENSCREEN). This step identified 34 compounds that inhibited PaSAHase activity (hit rate 0.68%). However, after their validation, we limited the number of confirmed inhibitors to 22. To evaluate if these compounds exhibit any selectivity against the bacterial enzyme, we determined their IC₅₀ values, testing their influence on both P. aeruginosa and human SAHases. Parallelly, we determined crystal structures of PaSAHase-ligand complexes to characterize the binding modes of the analyzed inhibitors. It allowed us to distinguish between compounds that affect the enzyme activity through the binding in the active site or on the protein surface region involved in the enzyme dynamics. A combination of biochemical and structural methods allowed us to select several lead compounds that inhibit PaSAHase in a highly selective manner. Based on our structural studies, we characterized determinants that ensure the selectivity of these compounds against PaSAHase. These results will constitute the basis for further optimization of lead compounds.

1. Czyrko J. et al.,  Metal-cation regulation of enzyme dynamics is a key factor influencing the activity of S-adenosylL-homocysteine hydrolase from Pseudomonas aeruginosa, Sci Rep, 8, (2018), 11334.

This work has been funded by National Science Centre, Poland, grant number SONATA BIS 2018/30/E/NZ1/00729.