Identification of the reactive cysteine residues in yeast dipeptidyl peptidase III

Dipeptidyl peptidases III (DPPs III) form a distinct metallopeptidase family characterized by the unique HEXXGH motif. High susceptibility to inactivation by organomercurials suggests the presence of a reactive cysteine residue(s) in, or close to, their active site. Yeast DPP III contains five Cys, none of which is absolutely conserved within the family. In order to identify reactive residue(s), site-directed mutagenesis on yeast His₆-tagged DPP III was employed to substitute specifically all five cysteine residues to serine. The variant enzymes thus obtained were enzymatically active and showed an overall structure not greatly affected by the mutations as judged by circular dichroism. Analysis by native and SDS-PAGE under non-reducing conditions revealed the existence of a monomeric and dimeric form in all DPP III proteins except in the C130S, implying that dimerization of yeast DPP III is mediated by the surface-exposed cysteine 130.     

Aprotinin interacts with substrate-binding site of human dipeptidyl peptidase III

Abstract

Human dipeptidyl peptidase III (hDPP III) is a zinc-exopeptidase of the family M49 involved in final steps of intracellular protein degradation and in cytoprotective pathway Keap1-Nrf2. Biochemical and structural properties of this enzyme have been extensively investigated, but the knowledge on its contacts with other proteins is scarce. Previously, polypeptide aprotinin was shown to be a competitive inhibitor of hDPP III hydrolytic activity. In this study, aprotinin was first investigated as a potential substrate of hDPP III, but no degradation products were demonstrated by MALDI-TOF mass spectrometry. Subsequently, molecular details of the protein–protein interaction between aprotinin and hDPP III were studied by molecular modeling. Docking and long molecular dynamics (MD) simulations have shown that aprotinin interacts by its canonical binding epitope with the substrate binding cleft of hDPP III. Thereby, free N-terminus of aprotinin is distant from the active-site zinc. Enzyme-inhibitor complex is stabilized by intermolecular hydrogen bonding network, electrostatic and hydrophobic interactions which mostly involve constituent amino acid residues of the hDPP III substrate binding subsites S1, S1', S2, S2' and S3'. This is the first study that gives insight into aprotinin binding to a metallopeptidase.

Communicated by Ramaswamy H. Sarma     

Synthesis and biological evaluation of novel benzyl-substituted (S)-phenylalanine derivatives as potent dipeptidyl peptidase 4 inhibitors

A series of novel benzyl-substituted (S)-phenylalanine derivatives were synthesized and evaluated for their dipeptidyl peptidase 4 (DPP-4) inhibitory activity and selectivity. It was found that most synthesized target compounds were potent DPP-4 inhibitors with IC₅₀ values in 3.79–25.52 nM, which were significantly superior to that of the marketed drug sitagliptin. Furthermore, the 4-fluorobenzyl substituted phenylalanine derivative 6g not only displayed the potent DPP-4 inhibition with an IC₅₀ value of 3.79 nM, but also showed better selectivity against DPP-4 over other related enzymes including DPP-7, DPP-8, and DPP-9. In an oral glucose tolerance test (OGTT) in normal Sprague Dawley rats, compound 6g reduced blood glucose excursion in a dose-dependent manner.     

Human dipeptidyl peptidase III: insights into ligand binding from a combined experimental and computational approach

Human dipeptidyl peptidase III (DPP III) is a zinc-exopeptidase with implied roles in protein catabolism, pain modulation, and defense against oxidative stress. To understand the mode of ligand binding into its active site, we performed molecular modeling, site-directed mutagenesis, and biochemical analyses. Using the recently determined crystal structure of the human DPP III we built complexes between both, the wild-type (WT) protein and its mutant H568N with the preferred substrate Arg-Arg-2-naphthylamide (RRNA) and a competitive inhibitor Tyr-Phe-hydroxamate (Tyr-Phe-NHOH). The mutation of the conserved His568, structurally equivalent to catalytically important His231 in thermolysin, to Asn, resulted in a 1300-fold decrease of k(cat) for RRNA hydrolysis and in significantly lowered affinity for the inhibitor. Molecular dynamics simulations revealed the key protein-ligand interactions as well as the ligand-induced reorganization of the binding site and its partial closure. Simultaneously, the non-catalytic domain was observed to stretch and the opening at the wide side of the inter-domain cleft became enhanced. The driving force for these changes was the formation of the hydrogen bond between Asp372 and the bound ligand. The structural and dynamical differences, found for the ligand binding to the WT enzyme and the H568N mutant, and the calculated binding free energies, agree well with the measured affinities. On the basis of the obtained results we suggest a possible reaction mechanism. In addition, this work provides a foundation for further site-directed mutagenesis experiments, as well as for modeling the reaction itself.     

Design and evaluation of inhibitors for dipeptidyl peptidase I (Cathepsin C)

Dipeptidyl peptidase I (DPPI, cathepsin C) is a lysosomal cysteine protease that can activate zymogens of several different serine proteases by one step or sequential removal of dipeptides from the N-termini of the pro-protease protein substrates. To find DPPI inhibitors more suitable for cellular applications than diazomethyl ketones, we synthesized three types of inhibitors: dipeptide acyloxymethyl ketones, fluoromethyl ketones, and vinyl sulfones (VS). The acyloxymethyl ketones inhibited DPPI slowly and are moderate inhibitors of cellular DPPI. The fluoromethyl ketones were potent, but the inhibited DPPI regained activity quickly. The dipeptide vinyl sulfones were effective inhibitors for DPPI, but they also inhibited cathepsins B, H, and L weakly. The best inhibitor, Ala-Hph-VS-Ph, had a k2/K(I) of 2,000,000M(-1)s(-1). The vinyl sulfones also inhibited intracellular DPPI, and for this application the more stable inhibitors exhibit better potency. We conclude that vinyl sulfones are promising inhibitors to study the intracellular functions of DPPI.     

In silico approaches to predict the potential of milk protein-derived peptides as dipeptidyl peptidase IV (DPP-IV) inhibitors

Molecular docking of a library of all 8000 possible tripeptides to the active site of DPP-IV was used to determine their binding potential. A number of tripeptides were selected for experimental testing, however, there was no direct correlation between the Vina score and their in vitro DPP-IV inhibitory properties. While Trp-Trp-Trp, the peptide with the best docking score, was a moderate DPP-IV inhibitor (IC₅₀ 216 μM), Lineweaver and Burk analysis revealed its action to be non-competitive. This suggested that it may not bind to the active site of DPP-IV as assumed in the docking prediction. Furthermore, there was no significant link between DPP-IV inhibition and the physicochemical properties of the peptides (molecular mass, hydrophobicity, hydrophobic moment (μH), isoelectric point (pI) and charge). LIGPLOTs indicated that competitive inhibitory peptides were predicted to have both hydrophobic and hydrogen bond interactions with the active site of DPP-IV. DPP-IV inhibitory peptides generally had a hydrophobic or aromatic amino acid at the N-terminus, preferentially a Trp for non-competitive inhibitors and a broader range of residues for competitive inhibitors (Ile, Leu, Val, Phe, Trp or Tyr). Two of the potent DPP-IV inhibitors, Ile-Pro-Ile and Trp-Pro (IC₅₀ values of 3.5 and 44.2 μM, respectively), were predicted to be gastrointestinally/intestinally stable. This work highlights the needs to test the assumptions (i.e. competitive binding) of any integrated strategy of computational and experimental screening, in optimizing screening. Future strategies targeting allosteric mechanisms may need to rely more on structure–activity relationship modeling, rather than on docking, in computationally selecting peptides for screening.     

Design of potent dipeptidyl peptidase IV (DPP-4) inhibitors by employing a strategy to form a salt bridge with Lys554

We report a design strategy to obtain potent DPP-4 inhibitors by incorporating salt bridge formation with Lys554 in the S1′ pocket. By applying the strategy to the previously identified templates, quinoline 4 and pyridines 16a, 16b, and 17 have been identified as subnanomolar or nanomolar inhibitors of human DPP-4. Docking studies suggested that a hydrophobic interaction with Tyr547 as well as the salt bridge interaction is important for the extremely high potency. The design strategy would be useful to explore a novel design for DPP-4 inhibitors having a distinct structure with a unique binding mode.     

Design and synthesis of dipeptidyl nitriles as potent, selective, and reversible inhibitors of cathepsin C

A series of dipeptide nitriles with a thienyl alanine in P2 were identified as potent and selective cathepsin C inhibitors. Incorporation of a substituted cyclopropyl moiety in P1 effectively protects these derivatives against hydrolase activity in whole blood.     

3D-QSAR studies on fluoropyrrolidine amides as dipeptidyl peptidase IV inhibitors by CoMFA and CoMSIA

Three-dimensional quantitative structure-activity relationship (3D-QSAR) analyses using CoMFA and CoMSIA methods were conducted on a series of fluoropyrrolidine amides as dipeptidyl peptidase IV (DP-IV) inhibitors. The selected ligands were docked into the binding site of the 3D model of DP-IV using the GOLD software, and the possible interaction models between DP-IV and the inhibitors were obtained. Based on the binding conformations of these fluoropyrrolidine amides and their alignment inside the binding pocket of DP-IV, predictive 3D-QSAR models were established by CoMFA and CoMSIA analyses, which had conventional r ² and cross-validated coefficient values () up to 0.982 and 0.555 for CoMFA and 0.953 and 0.613 for CoMSIA, respectively. The predictive ability of these models was validated by six compounds that were in the testing set. Structure-based investigations and the final 3D-QSAR results provide the guide for designing new potent inhibitors.     

Improvement of high fat-diet-induced insulin resistance in dipeptidyl peptidase IV-deficient Fischer rats

F344/DuCrj rats are genetically deficient in dipeptidyl peptidase IV (DPPIV). This enzyme degrades glucagon-like peptide-1 (GLP-1), which induces glucose-dependent insulin secretion. Glucose tolerance of F344/DuCrj rats is improved as a result of enhanced insulin release induced by high levels of plasma GLP-1. In this study, we fed F344/DuCrj rats and DPPIV-positive F344/Jcl rats, aged five weeks, on a high-fat (HF) diet to examine the effect of DPPIV deficiency on food intake and insulin resistance. F344/Jcl rats gained significantly more body weight and consumed significantly more food than F344/DuCrj rats from Week 4 on either control or HF diet. Glucose excursion in the oral glucose tolerance test (OGTT) was improved in F344/DuCrj rats fed on the control or HF diet at all times examined, compared with F344/Jcl rats. Homeostasis model assessment (HOMA) insulin resistance values of F344/DuCrj and F344/Jcl rats fed on HF diet were higher than those of animals fed on control diet up to Week 6. However, HOMA insulin resistance values of F344/DuCrj rats fed on HF diet became significantly lower than those of F344/Jcl rats on HF diet during Weeks 8-10. The area under the insulin curve in the OGTT at Week 10 showed that the insulin resistance of HF-diet-fed F344/DuCrj rats was greatly ameliorated. Plasma active GLP-1 concentrations of F344/DuCrj rats in the fed state were significantly higher than those of F344/Jcl rats. These observations suggest that DPPIV deficiency results in improved glucose tolerance and ameliorated insulin resistance owing to enhanced insulin release and inhibition of food intake as a result of high active GLP-1 levels.     

Structure activity relationship modelling of milk protein-derived peptides with dipeptidyl peptidase IV (DPP-IV) inhibitory activity

Quantitative structure activity type models were developed in an attempt to predict the key features of peptide sequences having dipeptidyl peptidase IV (DPP-IV) inhibitory activity. The models were then employed to help predict the potential of peptides, which are currently reported in the literature to be present in the intestinal tract of humans following milk/dairy product ingestion, to act as inhibitors of DPP-IV. Two models (z- and v-scale) for short (2–5 amino acid residues) bovine milk peptides, behaving as competitive inhibitors of DPP-IV, were developed. The z- and the v-scale models (p < 0.05, R² of 0.829 and 0.815, respectively) were then applied to 56 milk protein-derived peptides previously reported in the literature to be found in the intestinal tract of humans which possessed a structural feature of DPP-IV inhibitory peptides (P at the N₂ position). Ten of these peptides were synthetized and tested for their in vitro DPP-IV inhibitory properties. There was no agreement between the predicted and experimentally determined DPP-IV half maximal inhibitory concentrations (IC₅₀) for the competitive peptide inhibitors. However, the ranking for DPP-IV inhibitory potency of the competitive peptide inhibitors was conserved. Furthermore, potent in vitro DPP-IV inhibitory activity was observed with two peptides, LPVPQ (IC₅₀ = 43.8 ± 8.8 μM) and IPM (IC₅₀ = 69.5 ± 8.7 μM). Peptides present within the gastrointestinal tract of human may have promise for the development of natural DPP-IV inhibitors for the management of serum glucose.     

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-l-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-l-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.     

Snake venom dipeptidyl peptidase IV: taxonomic distribution and quantitative variation

The present study examined the taxonomic distribution of dipeptidyl peptidase IV (DPP IV) activity in venoms of 59 ophidian taxa, representing seven subfamilies of the Families Elapidae and Viperidae. DPP IV activity is extremely variable at all taxonomic levels. It ranged from essentially none in laticaudine, hydrophiine, and some bungarine and elapine venoms, to 10.72 μmol 4-methoxy-β-naphthylamine liberated per min per 200 μg venom, for Ophiophagus hannah. Intra- and interpopulational variation were examined among eight populations of prairie rattlesnakes (Crotalus viridis viridis), Great Basin rattlesnakes (Crotalus viridis lutosus) and southern Pacific rattlesnakes (Crotalus viridis helleri). Among these populations, the mean weighted range of variation was 4.9-fold, and even among litter mates of C. v. lutosus, DPP IV activity varied as much as 5.6-fold. The two most salient findings, the near ubiquity of DPP IV in snake venoms and its great quantitative variability, even among full siblings, are paradoxical. The widespread distribution of the enzyme suggests an important role in envenomation, while the variable activity levels suggest that DPP IV and by extension, other individual enzymatic constituents, may not be under much individual selective pressure.     

Dipeptidyl peptidase IV (DPP-IV) inhibition prevents fibrosis in adipose tissue of obese mice

Background

During the development of obesity the expansion of white adipose tissue (WAT) leads to a dysregulation and an excessive remodeling of extracellular matrix (ECM), leading to fibrosis formation. These ECM changes have high impact on WAT physiology and may change obesity progression. Blocking WAT fibrosis may have beneficial effects on the efficacy of diet regimen or therapeutical approaches in obesity. Since dipeptidyl peptidase IV (DPP-IV) inhibitors prevent fibrosis in tissues, such as heart, liver and kidney, the objective of this study was to assess whether vildagliptin, a DPP-IV inhibitor, prevents fibrosis in WAT in a mouse model of obesity, and to investigate the mechanisms underlying this effect.

3D-QSAR studies of Dipeptidyl peptidase IV inhibitors using a docking based alignment

Dipeptidyl peptidase IV (DPP-IV) deactivates the incretin hormones GLP-1 and GIP by cleaving the penultimate proline or alanine from the N-terminal (P1-position) of the peptide. Inhibition of this enzyme will prevent the degradation of the incretin hormones and maintain glucose homeostasis; this makes it an attractive target for the development of drugs for diabetes. This paper reports 3D-QSAR analysis of several DPP-IV inhibitors, which were aligned by the receptor-based technique. The conformation of the molecules in the active site was obtained through docking methods. The QSAR models were generated on two training sets composed of 74 and 25 molecules which included phenylalanine, thiazolidine, and fluorinated pyrrolidine analogs. The 3D-QSAR models are robust with statistically significant r², q², and values. The CoMFA and CoMSIA models were used to design some new inhibitors with several fold higher binding affinity. Figure The CoMFA contours around molecule D1T155 (a) steric contours - favored (green); disfavored (yellow) (b) electrostatic contours - electropositive (blue); electronegative (red)     

Application of robotics to steady state enzyme kinetics: analysis of tight-binding inhibitors of dipeptidyl peptidase IV

Using available commercial robotics and instrumentation, we developed a fully automated and rigorous steady state enzyme kinetic assay for dipeptidyl peptidase IV (DPP IV; E.C. 3.4.14.5). The automated assay was validated with isoleucyl thiazolidide, a potent inhibitor of DPP IV with K(is)=110nM. Signal window analysis indicated that the assay had a 98% probability of detecting an inhibitor yielding 15% inhibition, with a predicted false positive rate of 0.13%. A mechanistic inhibition version of the automated assay was validated with isoleucyl 4-cyanothiazolidide, a very potent inhibitor of DPP IV. Isoleucyl 4-cyanothiazolidide was a competitive inhibitor of purified porcine DPP IV with K(is)=1 nM. Similar K(is) values were obtained for purified rat DPP IV and for DPP IV activity in human plasma from normal and diabetic donors. The pH dependence of K(is) for isoleucyl 4-cyanothiazolidide yielded a bell-shaped profile, with pK(a)=5.0 and pK(b)=7.6. To date, over 100,000 data points have been generated in profiling targeted compound libraries and in the analysis of tight-binding inhibitors of DPP IV. The data also show that robotic analysis is capable of producing full mechanistic inhibition analysis in a timely fashion to support drug discovery.     

Synthesis, kinetic evaluation, and utilization of a biotinylated dipeptide proline diphenyl phosphonate for the disclosure of dipeptidyl peptidase IV-like serine proteases

In this study, we report on the synthesis, kinetic characterisation, and application of a novel biotinylated and active site-directed inactivator of dipeptidyl peptidase IV (DPP-IV). Thus, the dipeptide-derived proline diphenyl phosphonate NH(2)-Glu(biotinyl-PEG)-Pro(P)(OPh)(2) has been prepared by a combination of classical solution- and solid-phase methodologies and has been shown to be an irreversible inhibitor of porcine DPP-IV, exhibiting an over all second-order rate constant (k(i)/K(i)) for inhibition of 1.57 x 10(3) M(-1) min(-1). This value compares favourably with previously reported rates of inactivation of DPP-IV by dipeptides containing a P(1) proline diphenyl phosphonate grouping [B. Boduszek, J. Oleksyszyn, C.M. Kam, J. Selzler, R.E. Smith, J.C. Powers, Dipeptide phophonates as inhibitors of dipeptidyl peptidase IV, J. Med. Chem. 37 (1994) 3969-3976; B.F. Gilmore, J.F. Lynas, C.J. Scott, C. McGoohan, L. Martin, B. Walker, Dipeptide proline diphenyl phosphonates are potent, irreversible inhibitors of seprase (FAPalpha), Biochem, Biophys. Res. Commun. 346 (2006) 436-446.], thus demonstrating that the incorporation of the side-chain modified (N-biotinyl-3-(2-(2-(3-aminopropyloxy)-ethoxy)-ethoxy)-propyl) glutamic acid residue at the P(2) position is compatible with inhibitor efficacy. The utilisation of this probe for the detection of both purified dipeptidyl peptidase IV and the disclosure of a dipeptidyl peptidase IV-like activity from a clinical isolate of Porphyromonas gingivalis, using established electrophoretic and Western blotting techniques previously developed by our group, is also demonstrated.     

Monitoring of the effects of transfection with baculovirus on Sf9 cell line and expression of human dipeptidyl peptidase IV

Human dipeptidylpeptidase IV (hDPPIV) is an enzyme that is in hydrolase class and has various roles in different parts of human body. Its deficiency may cause some disorders in the gastrointestinal, neurologic, endocrinological and immunological systems of humans. In the present study, hDPPIV enzyme was expressed on Spodoptera frugiperda (Sf9) cell lines as a host cell, and the expression of hDPPIV was obtained by a baculoviral expression system. The enzyme production, optimum multiplicity of infection, optimum transfection time, infected and uninfected cell size and cell behavior during transfection were also determined. For maximum hDPPIV (269 mU mL⁻¹) enzyme, optimum multiplicity of infection (MOI) and time were 0.1 and 72 h, respectively. The size of infected cells increased significantly (P < 0.001) after 24 h post infection. The results indicated that Sf9 cell line was applicable to the large scale for hDPPIV expression by using optimized parameters (infection time and MOI) because of its high productivity (4.03 mU m L⁻¹ h⁻¹).     

Synthesis,nitric oxide release,and dipeptidyl peptidase-4 inhibition of sitagliptin derivatives as new multifunctional antidiabetic agents

Nitric oxide (NO) dysfunction has been found to be an important factor in both the development and progression of diabetic complications due to its many roles in the vascular system. Multifunctional compounds with hypoglycemic and endothelial protective action will be promising agents for the treatment of diabetes and its complications. In this study, a series of novel NO-donating sitagliptin derivatives and relevant metabolites were synthesized and evaluated as potential multifunctional hypoglycemic agents. All of synthetic compounds shown remarkable inhibitory activity against dipeptidyl peptidase IV (DPP-IV) in vitro and demonstrated excellent hypoglycemic activities in diabetic mice, similar to the activity of sitagliptin, and compounds T1-T4 shown different extents of NO-releasing abilities and potent antioxidant abilities in vivo. By screening in DPP-4, compound T4 was recognized as a potent DPP-4 inhibitor with the IC₅₀ value of 0.060?μM. Docking study revealed compound T4 has a favorable binding mode. Furthermore, compounds T1-T4 exhibited different extents of NO-releasing abilities and excellent anti-platelet aggregation in vitro. The overall results suggested that T4 could help to the amelioration of endothelial dysfunction by reducing blood glucose, lessening oxidative stress and raising NO levels as well as inhibiting platelet aggregation. Based on this research, compound T4 deserves further investigation as potential new multifunctional anti-diabetic agent with antioxidant, anti-platelet aggregation and endothelial protective properties.     

Discovery of C-(1-aryl-cyclohexyl)-methylamines as selective,orally available inhibitors of dipeptidyl peptidase IV

The successful launches of dipeptidyl peptidase IV (DPP IV) inhibitors as oral anti-diabetics warrant and spur the further quest for additional chemical entities in this promising class of therapeutics. Numerous pharmaceutical companies have pursued their proprietary candidates towards the clinic, resulting in a large body of published chemical structures associated with DPP IV. Herein, we report the discovery of a novel chemotype for DPP IV inhibition based on the C-(1-aryl-cyclohexyl)-methylamine scaffold and its optimization to compounds which selectively inhibit DPP IV at low-nM potency and exhibit an excellent oral pharmacokinetic profile in the rat.