Active site-directed probes targeting dipeptidyl peptidases 8 and 9.

Dipeptidyl peptidases (DPP) 8 and 9 are intracellular serine proteases that play key roles in various biological processes and recent findings highlight DPP8 and DPP9 as potential therapeutic targets for hematological and inflammasome-related diseases. Despite the substantial progress, the precise biological functions of these proteases remain elusive, and the lack of selective chemical tools hampers ongoing research. In this paper, we describe the synthesis and biochemical evaluation of the first active site-directed DPP8/9 probes which are derived from DPP8/9 inhibitors developed in-house. Specifically, we synthesized fluorescent inhibitors containing nitrobenzoxadiazole (NBD), dansyl (DNS) and cyanine-3 (Cy3) reporters to visualize intracellular DPP8/9. We demonstrate that the fluorescent inhibitors have high affinity and selectivity towards DPP8/9 over related S9 family members. The NBD-labeled DPP8/9 inhibitors were nominated as the best in class compounds to visualize DPP8/9 in human cells. Furthermore, a method has been developed for selective labeling and visualization of active DPP8/9 in vitro by fluorescence microscopy. A collection of potent and selective biotinylated DPP8/9-targeting probes was also prepared by replacing the fluorescent reporter with a biotin group. The present work provides the first DPP8/9-targeting fluorescent compounds as useful chemical tools for the study of DPP8 and DPP9's biological functions.

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Déclaration de conflit d'intérêts

Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Margarida Espadinha reports financial support was provided by European Commission Marie Sklodowska-Curie Actions. Joni De Loose reports financial support was provided by Research Foundation Flanders. Siham Benramdane reports financial support was provided by Research Foundation Flanders. Yentl Van Rymenant reports financial support was provided by Research Foundation Flanders. Sam Corthaut reports financial support was provided by Research Foundation Flanders. Ingrid De Meester reports financial support was provided by Research Foundation Flanders. Pieter Van der Veken reports financial support was provided by Research Foundation Flanders. Margarida Espadinha has patent #EP2023/064881 pending to University of Antwerp. Joni De Loose has patent #EP2023/064881 pending to University of Antwerp. Siham Benramdane has patent #EP2023/064881 pending to University of Antwerp. Ingrid De Meester has patent #EP2023/064881 pending to University of Antwerp. Pieter Van der Veken has patent #EP2023/064881 pending to University of Antwerp. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.