Synthesis and application of highly selective inhibitory probes for Fusobacterium nucleatum fusolisin
2Department of Chemical Biology and Bioimaging, Wroclaw University of Science and Technology, Poland
3Department of Microbiology, Faculty of Biochemistry, Biophysics and Biotechnology, Jagiellonian University, Poland
4Department of Oral Immunology and Infectious Diseases, University of Louisville School of Dentistry, USA
5The Concern foundation laboratories at the Lautenberg Center for General and Tumor Immunology, Department of Immunology and Cancer Research, Institute for Medical Research Israel Canada (IMRIC), Faculty of Medicine, The Hebrew University Medical School, Israel
Fusobacterium nucleatum is a common oral Gram-negative anaerobic bacterium that translocates to and colonizes breast cancer, colon adenocarcinoma, esophageal cancer, and other. When overabundant in tumors, F. nucleatum has been associated with the resistance to chemotherapy and poor prognosis. Therefore, it is critical to eliminate this bacterium in tumors.
Fusolisin, the only known functional F. nucleatum protease is essential for fusobacterial growth. Fusolisin inhibition with Phenyl-methane sulfonyl fluoride, impeded fusobacterial growth in culture. In addition, preliminary data indicate that fusolisin cleaves immune-stimulatory cytokines and abolished the killing activity of Natural killer (NK) cells by specifically targeting and degrading their activating receptors. Thus, fusolisin is hypothesized to play a major role in fusobacterial-mediated acceleration of the tumor progression.
The hybrid combinatorial substrate library (HyCoSuL) of tetrapeptides containing mixtures of 19 proteinogenic and <100 non-proteinogenic amino acids, amino acid mixtures at the P4–P2 positions, a fixed residue at the P1 position, and an ACC (7-amino-4-carbamoylmethylcoumarin) fluorescent tag occupying the P1′ position, was applied to screen specificity and identified sequential motives most efficiently cleaved by fusolisin. This approach allowed us to select optimal peptide substrates, which were converted to specific, small molecule inhibitory probes of fusolisin.
We expect fusolisin inhibition to reverse the immune-suppressing activity of F. nucleatum and to unleash an effective anti-tumor immunity.