Dipeptidyl peptidases 8 and 9 (DPP8 and DPP9, respectively) are intracellular proteases widely expressed in mammalian tissues. They were discovered as DPP4 homologs having a DPP4-like peptidase activity [1,2]. DPP8 and DPP9 belong to the S9 family of serine proteases, together with DPP4, prolyl oligopeptidase (PREP) and fibroblast activation protein (FAP). Members of this family possess the rare ability to cleave post-proline bonds. More specifically, DPP4, DPP8 and DPP9 cleave N-terminal dipeptides from their substrates, primarily with proline or alanine at the penultimate position [3,4].

Emerging evidence suggests regulatory roles for DPP8 and DPP9 in human immunity. It was shown that DPP8/9 inhibition leads to pro-inflammatory cell death in various human cell types including acute myeloid leukemia (AML) cells, keratinocytes and primary blood mononuclear cells. More specifically, DPP8/9 inhibitors induce a lytic type of cell death that is suggestive of pyroptosis, characterized by NLRP1- and CARD8-inflammasome formation, pro-caspase-1 activation and gasdermin D cleavage [[5], [6], [7], [8], [9]]. Whilst DPP9 is identified as the primary inflammasome regulator, DPP8 can compensate for the absence or inhibiton of DPP9 activity [8,9]. The identification of DPP8 as substitute for DPP9 led to increased research focus on DPP9 and identified DPP9 as a putative therapeutic target for inflammasome modulation. Further, an in vivo study demonstrated that DPP8/9 inhibitors reduce tumor burden, and increase survival of immunodeficient mice injected with MV4-11 AML cells, showing that DPP8/9 inhibition may serve as an antitumor strategy [5]. On the other hand, targeted inactivation of DPP9 enzymatic activity caused mouse neonatal lethality [10]. In 2022, Bolgi et al. also revealed therapeutic possibilities of DPP9 inhibition in cancer cells by showing that DPP9 targets breast tumor-suppressor BRCA2 for degradation. Since BRCA2 is critical for DNA repair, DPP9-depleted cells were more sensitive to cancer therapies, which suggests that DPP9 inhibition might be of use in combination therapies for breast cancer patients [11]. Other recent findings show that DPP9 inhibition increases the potency of non-nucleoside reverse transcriptase inhibitors (NNRTI's) in killing HIV-1-infected cells via activation of the inflammasome [12,13]. Clearly, continued research efforts will further uncover the therapeutic potential of targeting DPP8/9-related pathways.

The high sequence identity between DPP8 and DPP9, especially around and in the active site, has hampered the development of selective inhibitors, substrates and other chemical tools [14]. Since their discovery at the beginning of this era, numerous non-selective small-molecule inhibitors have been used to study DPP8 and DPP9, such as ValboroPro (1), allo-Ile-5-fluoroisoindoline (2) and 1G244 (3) (Fig. 1). ValboroPro (1), also known as Talabostat or PT-100, is a pan-DPP inhibitor targeting DPP2, DPP4, DPP8, DPP9 and FAP with IC50 values in the nanomolar range [15]. Compared to ValboroPro (1), allo-Ile-5-fluoroisoindoline (2) has a higher selectivity for DPP8 and DPP9 over the other family members, but has comparable affinity for both proteases [16]. 1G244 (3) is frequently used in biochemical and cellular experiments and it has a 4-fold selectivity towards DPP8 compared to DPP9 [17]. Recently, advances have been made towards DPP8-selective inhibitors by Carvalho et al. who identified 4-Oxo-β-lactam variants as covalent DPP8/9 inhibitors with up to 21-fold selectivity towards DPP8 [18].

In 2022, our group reported a series of novel DPP8/9 inhibitors based on the commercial DPP4 inhibitor vildagliptin. From this collection of adamantyl derivatives, the most promising compounds (4–5, Fig. 1) had 7-fold preference to inhibit DPP9 over DPP8, with high nanomolar affinity towards DPP9 [19]. To further improve DPP9-over-DPP8 selectivity, we continued the optimization of these adamantyl-derivatives. We recently published this selection of non-covalent inhibitors that combine low nanomolar DPP9 affinity with unprecedented DPP9/DPP8 selectivity indices (SI > 100), together with an in vivo pharmacokinetic and toxicity study of the most promising derivative [20]. Given the urgent need for DPP8/9-targeting chemical tools, we designed probes based on the novel inhibitors 6–8 (Fig. 1) to target DPP8 and DPP9 in an activity-dependent manner.

Active site-directed chemical probes are powerful tools in protease research. In the presence of a warhead, the probe binds covalently to the active site of the target protease, which is the case for most activity-based probes (ABPs). Activity-based serine protease profiling with ABPs is excellently reviewed elsewhere [[21], [22], [23]]. Over the last years, various reactive electrophiles have been tuned to either target a group of serine proteases [24] or selectively react with the active site of a specific enzyme such as cathepsin G [25], neutrophil elastase [26,27] and neutrophil serine protease 4 [25]. As another approach, selective and potent covalent inhibitors have been used as ABPs, such as for FAP [28] and neutrophil elastase [29]. Given that activity rather than expression determines the biological functions of proteases, we also aimed to develop fluorescent- and biotin-labeled compounds based on in-house developed non-covalent small-molecule DPP8/9 inhibitors. Although the resulting probes do not comply with the strict definition of ABPs, they are active site-directed leading to an affinity- and not reactivity-driven inhibition mechanism, which may increase the selectivity in the proteome compared to classical, covalently-binding ABPs.

To the best of our knowledge, these are the first reported active site-directed probes that selectively target DPP8 and DPP9. In this study, we demonstrate the ability of the probes to inhibit the enzymatic activity of recombinant human DPP8 and DPP9 (rhDPP8 and rhDPP9 respectively). Moreover, we show that fluorescent probes are internalized in THP-1 and HEK293T cells and could be used for fluorescence microscopy to visualize intracellular DPP8/9 activity. In addition, we provide results of biophysical assays with biotinylated probes showing that the probes efficiently bind rhDPP9 but are unable to simultaneously bind rhDPP9 and avidin. We expect that the fluorescent probes will find application in the DPP8/9 research field and uncover novel research paths that can be translated to therapeutic possibilities.