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. Author manuscript; available in PMC: 2008 Jan 15.
Published in final edited form as: Bioorg Med Chem Lett. 2006 Oct 10;17(2):507–510. doi: 10.1016/j.bmcl.2006.10.012

Synthesis and activity of a potent, specific azabicyclo [3.3.0] octane-based DPP II inhibitor

Olga Danilova a, Bei Li b, Katrin Szardenings b, Brigitte T Huber a, Jonathan Rosenblum b
PMCID: PMC1828633  NIHMSID: NIHMS16749  PMID: 17055271

Abstract

A cell permeable DPP II (also known as DPP2, DPP7 and quiescent cell proline dipeptidase (QPP)) inhibitor has been synthesized. The azabicyclo[3.3.0]octane-based inhibitor is potent and selective and elicits very similar quiescent lymphocyte death to previously characterized inhibitors that are not as selective.

Keywords: Dipeptidyl peptidase

Recently, a family of serine proteases with the common ability to cleave after prolines has attracted attention as potential drug targets 1,2. The most widely studied enzyme in this family is dipeptidyl peptidase (DPP) IV (also known as the T-cell antigen CD26), which is a clinically validated target for type 2 diabetes 3. DPP IV is responsible for the rapid inactivation of the incretin GLP-1 by virtue of its ability to remove an amino-terminal dipeptide 4. It is possible that other members of the family function in analogous fashions, but determining the precise function of the other members has lagged for a number of reasons, including the unavailability of genetically modified animals, paucity of known biological substrates for the proteases and absence of specific, biologically active inhibitors.

In addition to DPP IV, the members include fibroblast activation protein α (FAP), DPP II (also known as DPP2, DPP7 and quiescent cell proline dipeptidase or QPP), prolylcarboxypeptidase, DPP 8, DPP9, acylpeptide hydrolase (APH), and prolyl oligopeptidase (POP). These enzymes share the ability to cleave peptides with proline (and sometimes alanine) at the P1 position, and some of the enzymes have specificity for P2 amino acids as well 5,6. In addition to demonstrating different levels of expression and tissue distribution, members of this family are found extracellularly (DPP IV), bound to the plasma membrane (DPP IV and FAPα), in the cytoplasm (DPP 8, DPP9, POP, and APH) and in specialized vesicles (DPP II) 7–12.

DPP II, a 58kDa glycoprotein, is localized to intracellular vesicles distinct from lysozymes and can be secreted in active form in response to calcium release13. Homodimerization via a leucine zipper motif is required for DPP II catalytic activity14. It is active within a broad range of pH with optimum between 5.5 and 7.0 5,8. It is believed that DPP II is essential for the G0 survival program of lymphocytes and neuronal cells. Inhibition of DPP II induces apoptosis of these quiescent cells15. DPP II may also be involved in pathogenesis of B cell chronic lymphocytic leukemia (B-CLL). B cells arrested in G0 accumulate in peripheral blood of CLL patients. Susceptibility to DPP II-induced apoptosis serves as a prognostic factor of CLL outcome16. Natural DPP II substrates remain unknown.

In order to gain further understanding of the biological role(s) of DPP II, we and others have synthesized small molecule inhibitors 17–19. A common starting point for the synthesis of DPP II inhibitors is the cationic P2 preference of the enzyme 5. For example, 2,4-diaminobutanoic acid (Dab) has been used as a P2 group in dipeptide inhibitors where P1 was piperidine or boronorleucine. Inhibitors of this type are typically potent and highly selective for DPP II over the other DPP enzymes (see Figure 1 for representative DPP II inhibitors). However, such hydrophilic inhibitors may suffer from low cell permeability, which could render them unable to target the intracellular compartment where DPP II is found.

Figure 1.

Figure 1

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Representative DPP II inhibitors 18, 17.

Here we report the discovery and biological characterization of a potent and selective azabicyclo[3.3.0]octane-based DPP II inhibitor.

In the course of exploring potential P1 groups for DPP II inhibitors, we focused on structures that could take advantage of DPP II’s larger S1 site relative to DPP IV, with a preference for lipophilic structures that would counterbalance the polar Dab group. Of the groups that were explored, the azabicyclo[3.3.0]octane shown in Figure 2 demonstrated the best combination of potency and specificity for DPP II.

Figure 2.

Figure 2

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Synthesis of AX8819. (a) Boc-N-2,4-diaminobutyric acid, DIEA, HOBt, EDC, DMF. (b) 4M HCl in dioxane, rt, 1h.

AX8819 was prepared by a standard coupling of amine 320 with Boc-N-2,4-diaminobutyric acid followed by HCl deprotection of the Boc groups.

AX8819 was tested for potency against members of the DPP family in cell-free extracts. As shown in Table 1, AX8819 is potent and selective for DPP II. Other compounds used in these studies were assayed side-by-side with AX8819 and their IC50 values are also shown in Table 1. We next tested the ability of the compounds to inhibit intracellular DPP II when added to intact cells. Testing the inhibitors in intact cells allows the determination of several factors, including cell permeability of the compound, the ability to target the DPP II-containing vesicles, compound potency and stability.

Table 1.

IC50 values of compounds tested in cell-free extractsa

Compound DPP IV IC50, nM DPP 2 IC50, nM FAP IC50, nM DPP8 IC50, nM DPP9 IC50, nM POP IC50, nM
AX8819 >33.000 0.88 >33,000 >20,000 >20,000 >20,000
2 >33,000 0.48 >33,000 4300 800 >20,000
VbP 0.10 30 24 17 2.0 7960
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a

IC50 values were determined as described previously 17,21

Three DPP II inhibitors were tested (Tables 1 and 2). The original cell-based potency testing was performed using DPP II-293T HEK and 293T HEK cell lines. DPP II-293T HEK, a cell line that overexpresses human DPP II14, was used to test the compounds’ permeability and selectivity. The 293T HEK cell line was employed as a control, since it does not express DPP II, but has other DPP activities (DPP8 and 9) and can be used to test the specificity of the compound. We used ValBoroPro (VbP1) as a standard non-specific DPP inhibitor (Table 1). As seen in Table 2, the compounds required between 1000 and 10,000 times their IC50 concentrations to inhibit approximately half of the DPP activity in DPP II overexpressing HEK cells (DPP II-293 T HEK column). As mentioned above, this level of compound reflects a combination of cell- and organelle-permeability, compound stability, and other factors 22. Taken together these results indicate that compounds 2 and AX8819 appear to bracket the intact cell DPP II inhibition of VbP, while being substantially more selective for DPP II over all of the other DPP enzymes.

Table 2.

Remaining Ala-Pro-AFC cleavage activity (%) upon treating intact cells with compounds21

Compound (concentration) DPP II-293T HEK 293T HEK Jurkat
VbP (10μM) 75.3 3.9 43.7
2 (4.8μM) 62.4 94.3
AX8819 (0.88μM) 65.48 97.3
AX8819 (4.4μM) 60.6 115.5 75.6
AX8819 (8.8μM) 57.7 65.8 68.4
AX8819 (22μM) 21.1 91.1 52.4
AX8819 (44μM) 28.9 96.1 46.8
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Compounds 2 and AX8819 were further studied for general toxicity. As observed previously, inhibition of DPP II does not cause cell death of proliferating Jurkat cells 15. Hence, if cell death is observed it is likely due to the toxicity of the studied compound. None of the compounds killed more than 5% of treated Jurkats cells at 5000 fold their DPPII IC50 after 16 hours of incubation. AX8819 did not cause any cell death in Jurkat cells at concentrations up to 44μM, 50,000-fold its DPP II IC50. By this assay AX8819 was therefore judged to have very low nonspecific toxicity. VbP and 2 also exhibit low nonspecific toxicity toward Jurkat cells.

We continued to examine functionality and specificity of AX8819 and to explore the consequences of DPP II inhibition on a cellular level. Also our goal was to determine the most efficient concentration of the inhibitor to use in intact cell applications. A range of doses of AX8819 was used to explore its ability to selectively inhibit DPP II when added to intact cells. AX8819 significantly inhibited the overexpressed DPP II in DPP II-293T HEK cells at all used concentrations and the endogenous DPP II in Jurkat cells at 22 and 44 μM doses. At the same time, at up to 44μM, it did not significantly inhibit the DPP activity of 293T HEK cells (Table 2). Thus, AX8819 demonstrated characteristics of a potent and specific inhibitor at a range of doses. At concentrations of up to 44μM, AX8819 did not kill proliferating Jurkat cells or inhibit other DPP enzymes in untransfected 293 cells.

As mentioned above, DPP II is involved in survival of resting cells. Inhibition of DPP II in these cells causes apoptosis. A series of cell death assays were performed on resting freshly isolated human peripheral blood mononuclear cells (PBMC) to demonstrate the results of specific DPP II inhibition. Apoptotic cell death was analyzed by annexin V (AnV) and propidium iodide (PI) staining. 23

Resting PBMCs were treated with various doses of AX8819 and 10μM VbP for 4, 8, and 16 h. Cells were washed and stained with AnV-allophycocyanin, propidium iodide was added immediately prior to analysis on FACScalibur. AX8819 caused cell death at 22 and 44μM. The kinetics and extent of cell death caused by 44μM AX8819 were identical to those of 10μM VbP, while 22μM AX8819 led to slightly less cell death and concentrations of AX8819 below 10μM were indistinguishable from DMSO (Figure 3). AX8819 caused apoptotic cell death (4 fold increase in the number AnV+/PI− and AnV+/PI+ cells) of resting PBMC comparable to VbP 24,25. The difference in the number of AnV+ cells between AX8819 (22 and 44μM) and VbP (10μM) treated cells compared to DMSO control is significant (t-test, p<0.001) at the 16 h time point .

Figure 3.

Figure 3

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Kinetics of apoptotic death of PBMC upon DPP II inhibition

To confirm the apoptotic nature of cell death, cells were treated with 100μM zVAD-fmk 1h prior to compound addition. For each compound, cell death was compared in the presence and absence of active caspases (with and without zVAD-fmk, Figure 4). A significant reduction (paired t-test, p<0.05) in the numbers of AnV+ cells was observed in the samples that were pre-treated with zVAD-fmk and incubated with AX8819 and VbP, thus confirming that cell death caused by DPP II inhibition is caspase-dependent.

Figure 4.

Figure 4

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Pretreatment of resting PBMC with 100μM zVAD-fmk reduces compound dependent cell death

A recent report 26 described studies with a DPP II inhibitor that apparently did not cause any form of cell death when PBMCs were treated with a cell permeable DPP II inhibitor. No cell death was seen with the DPP II inhibitor, but cells were killed with etoposide, a topoisomerase II inhibitor used in cancer chemotherapy. Etoposide triggers apoptosis through a mechanism distinct from that initiated by the DPP II inhibitors. For example, etoposide causes apoptosis in Jurkat cells 27 and DPP II inhibitors do not. Unfortunately, since Maes, et al. did not use a DPP II inhibitor shown previously to cause apoptosis in PBMC, for example VbP, as a positive control, it is possible that their PBMCs are resistant to apoptosis via this mechanism. It is important to note that we have observed that PBMCs derived from different individuals exhibit various degrees of susceptibility to DPPII inhibition-induced apoptosis (unpublished observation). Since some non-cycling cells that have DPP II activity are not susceptible to DPP II inhibition-induced apoptosis, it is clear that other factors are required to explain this phenotype.

In conclusion, AX8819 is a novel DPP II-specific inhibitor. Its functionality, specificity and potency were demonstrated in several cell-based systems. Though uniquely specific to DPP II among the DPP enzymes, AX8819 is able to kill PBMC in a similar fashion to VbP, a pan DPP inhibitor. As such, AX8819 further supports our hypothesis that DPP II is the relevant target in the apoptosis of quiescent cells and could be a useful tool in the further elucidation of the biological role of DPP II.

Acknowledgments

We thank John Kozarich for guidance and insightful suggestions and Kevin Shreder for supplying compound 2.

Footnotes

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