Crystal structure of Porphyromonas gingivalis dipeptidyl peptidase 4 and structure-activity relationships based on inhibitor profiling.

The Gram-negative anaerobe Porphyromonas gingivalis is associated with chronic periodontitis. Clinical isolates of P. gingivalis strains with high dipeptidyl peptidase 4 (DPP4) expression also had a high capacity for biofilm formation and were more infective. The X-ray crystal structure of P. gingivalis DPP4 was solved at 2.2 Å resolution. Despite a sequence identity of 32%, the overall structure of the dimer was conserved between P. gingivalis DPP4 and mammalian orthologues. The structures of the substrate binding sites were also conserved, except for the region called S2-extensive, which is exploited by specific human DPP4 inhibitors currently used as antidiabetic drugs. Screening of a collection of 450 compounds as inhibitors revealed a structure-activity relationship that mimics in part that of mammalian DPP9. The functional similarity between human and bacterial DPP4 was confirmed using 124 potential peptide substrates.

Dipeptidyl peptidases in atherosclerosis: expression and role in macrophage differentiation, activation and apoptosis.

Atherosclerosis is a chronic inflammatory disorder of the arterial wall leading to coronary artery disease, stroke, and peripheral arterial disease. Along with the discovery of dipeptidyl peptidase 4 (DPP4) as a therapeutic target in type 2 diabetes, a role for DPP4 in atherosclerosis is emerging. However, until now the expression and role of other DPPs such as DPP8 and DPP9 in atherosclerosis is completely unknown. In the present study, we first investigated DPP expression in human atherosclerotic plaques. DPP4 could only be observed in endothelial cells of plaque neovessels in half of the specimens. In contrast, DPP8 and DPP9 were abundantly present in macrophage-rich regions of plaques. We then focused on DPP expression and function in macrophage differentiation, activation and apoptosis. DPP8/9 was responsible for most of the DPP activity in macrophages. During monocyte to macrophage differentiation, DPP9 was upregulated both in pro-inflammatory M1 (3.7 ± 0.3-fold increase) and anti-inflammatory M2 macrophages (3.7 ± 0.4-fold increase) whereas DPP8 expression remained unchanged. Inhibition of DPP8/9 activity with compound 1G244 reduced activation of M1 macrophages (IL-6 88 ± 16 vs. 146 ± 19 pg/ml; TNFα 3.8 ± 1.0 vs. 6.6 ± 1.9 ng/ml in treated vs. untreated cells), but not of M2 macrophages. Likewise, DPP9 silencing reduced TNFα and IL-6 secretion, pointing to a DPP9-mediated effect of the inhibitor. DPP8/9 inhibition also enhanced macrophage apoptosis (15 ± 4 vs. 7 ± 3 % in untreated cells). Because pro-inflammatory macrophages play a key role in atherogenesis, plaque rupture and subsequent infarction, DPP9 inhibition might provide interesting therapeutic prospects in reducing atherosclerosis and/or in the prevention of plaque rupture.

Acylated Gly-(2-cyano)pyrrolidines as inhibitors of fibroblast activation protein (FAP) and the issue of FAP/prolyl oligopeptidase (PREP)-selectivity.

A series of N-acylated glycyl-(2-cyano)pyrrolidines were synthesized with the aim of generating structure-activity relationship (SAR) data for this class of compounds as inhibitors of fibroblast activation protein (FAP). Specifically, the influence of (1) the choice of the N-acyl group and (2) structural modification of the 2-cyanopyrrolidine residue were investigated. The inhibitors displayed inhibitory potency in the micromolar to nanomolar range and showed good to excellent selectivity with respect to the proline selective dipeptidyl peptidases (DPPs) DPP IV, DPP9 and DPP II. Additionally, selectivity for FAP with respect to prolyl oligopeptidase (PREP) is reported. Not unexpectedly, the latter data suggest significant overlap in the pharmacophoric features that define FAP or PREP-inhibitory activity and underscore the importance of systematically evaluating the FAP/PREP-selectivity index for inhibitors of either of these two enzymes. Finally, this study forwards several compounds that can serve as leads or prototypic structures for future FAP-selective-inhibitor discovery.

Novel diarylpyridinones, diarylpyridazinones and diarylphthalazinones as potential HIV-1 nonnucleoside reverse transcriptase inhibitors (NNRTIs).

In this Letter, we report on diarylpyridinone, diarylpyridazinone and diarylphthalazinone analogs as potential inhibitors of HIV-1 nonnucleoside reverse transcriptase (NNRTIs). The most promising compounds in these series are three diarylpyridazinones 25a, 25l and 25n which demonstrated submicromolar activity against wild-type HIV-1 and moderate activity against the single mutant strain Ba-L V106A.

Structure-activity relationship studies on isoindoline inhibitors of dipeptidyl peptidases 8 and 9 (DPP8, DPP9): is DPP8-selectivity an attainable goal?

This work represents the first directed study to identify modification points in the topology of a representative DPP8/9-inhibitor, capable of rendering selectivity for DPP8 over DPP9. The availability of a DPP8-selective compound would be highly instrumental for studying and untwining the biological roles of DPP8 and DPP9 and for the disambiguation of biological effects of nonselective DPP-inhibitors that have mainly been ascribed to blocking of DPPIV's action. The cell-permeable DPP8/9-inhibitor 7 was selected as a lead and dissected into several substructures that were modified separately for evaluating their potential to contribute to selectivity. The obtained results, together with earlier work from our group, clearly narrow down the most probable DPP8-selectivity imparting modification points in DPP8/9 inhibitors to parts of space that are topologically equivalent to the piperazine ring system in 7. This information can be considered of high value for future design of compounds with maximal DPP8 selectivity.

Measurement of carboxypeptidase U (active thrombin-activatable fibrinolysis inhibitor) in plasma: Challenges overcome by a novel selective assay.

This article introduces a novel assay for the measurement of carboxypeptidase U (CPU) in plasma using the selective CPU substrate Bz-o-cyano-Phe-Arg (N-benzoyl-ortho-cyano-phenylalanyl-arginine), thereby limiting the interference of plasma carboxypeptidase N (CPN) as well as the intrinsic activity of procarboxypeptidase U (proCPU). A limit of detection of 0.05 U/L (10 pM) was reached. In addition, the current assay has the advantage of being easy to perform and shows excellent linearity and variability, rendering it a useful tool in the screening of samples for the presence of CPU in several patient populations and encouraging in-depth exploration of the pathophysiological role of the proCPU/CPU system.

Development of a sensitive and selective assay for the determination of procarboxypeptidase U (thrombin-activatable fibrinolysis inhibitor) in plasma.

To date, several assays for procarboxypeptidase U (proCPU) determination exist, all having their own inherent disadvantages and advantages. A drawback of activity-based assays is the interference of the constitutively active carboxypeptidase N (CPN) in plasma. Recent screening of Bz-Xaa-Arg peptides with modified aromatic amino acids at the P1 position revealed a selective CPU substrate, N-benzoyl-ortho-cyano-phenylalanyl-arginine (Bz-o-cyano-Phe-Arg), which will allow straightforward determination of proCPU in plasma. Our assay shows an excellent linearity in the concentration range of 20-2600 U/L, with within- and between-run precision values of 2.7% and 4.6%, respectively. A good correlation with our high-performance liquid chromatography (HPLC)-assisted proCPU activity assay using hippuryl-l-arginine (HipArg) as substrate was found. Besides the major improvement regarding the selectivity, the assay is much easier to perform and far less time-consuming compared with the proCPU activity assay using HipArg as substrate.

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors.

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors' affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 1: identification of dipeptide derived leads.

Dipeptide derivatives bearing various P2 residues and pyrrolidine derivatives as P1 mimics were evaluated in order to identify lead structures for the development of DPP8 and DPP9 inhibitors. Structure-activity-relationship data obtained in this way led to the preparation of a series of alpha-aminoacyl ((2S, 4S)-4-azido-2-cyanopyrrolidines). These compounds were shown to be nanomolar DPP8/9 inhibitors with modest overall selectivity toward DPP IV and DPP II.