Ketopyrrolidines and ketoazetidines as potent dipeptidyl peptidase IV (DPP IV) inhibitors

In this paper, the synthesis and structure-activity relationships (SAR) of two classes of electrophile-based dipeptidyl peptidase IV (DPP IV) inhibitors, the ketopyrrolidines and ketoazetidines, is discussed. The SAR of these series demonstrate that the 2-thiazole, 2-benzothiazole, and 2-pyridylketones are optimal S1' binding groups for potency against DPP IV. In addition, both cyclohexyl glycine (CHG) and octahydroindole carboxylate (OIC) serve as the most potent S2 binding groups within each series. Stereochemistry at the alpha-position of the central ring is relevant to potency within the ketopyrrolidines series, but not in the ketoazetidine series. Finally, the ketoazetidines display enhanced stability over the corresponding ketopyrrolidines, while maintaining their potency. In fact, certain stabilized ketoazetidines can maintain their in vitro potency and inhibit DPP IV in the plasma for up to 6h.     

Selective fluorescence probes for dipeptidyl peptidase activity-fibroblast activation protein and dipeptidyl peptidase IV

Development of suitable tools to assess enzyme activity directly from their complex cellular environment has a dramatic impact on understanding the functional roles of proteins as well as on the discovery of new drugs. In this study, a novel fluorescence-based chemosensor strategy for the direct readout of dipeptidase activities within intact living cells is described. Selective activity-based probes were designed to sense two important type II transmembrane serine proteases, fibroblast activation protein (FAP) and dipeptidyl peptidase IV (DPP-IV). These serine proteases have been implicated in diverse cellular activities, including blood coagulation, digestion, immune responses, wound healing, tumor growth, tumor invasion, and metastasis. Here, we validated that Ac-GPGP-2SBPO and GPGP-2SBPO probes are excellent reporters of both proteolytic activities. Furthermore, the novel probes can differentiate between FAP and DPP-IV proteolytic activities in cellular assay. Potentially, this assay platform is immediately useful for novel drug discovery.     

Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8.

Dipeptidyl peptidase (DPP) IV has roles in T-cell costimulation, chemokine biology, type-II diabetes and tumor biology. Fibroblast activation protein (FAP) has been implicated in tumor growth and cirrhosis. Here we describe DPP8, a novel human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP. Northern-blot hybridization showed that the tissue expression of DPP8 mRNA is ubiquitous, similar to that of DPPIV. The DPP8 gene was localized to chromosome 15q22, distinct from a closely related gene at 19p13.3 which we named DPP9. The full-length DPP8 cDNA codes for an 882-amino-acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N-linked or O-linked glycosylation. Western blots and confocal microscopy of transfected COS-7 cells showed DPP8 to be a 100-kDa monomeric protein expressed in the cytoplasm. Purified recombinant DPP8 hydrolyzed the DPPIV substrates Ala-Pro, Arg-Pro and Gly-Pro. Thus recombinant DPP8 shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP. DPP8 enzyme activity had a neutral pH optimum consistent with it being nonlysosomal. The similarities between DPP8 and DPPIV in tissue expression pattern and substrates suggests a potential role for DPP8 in T-cell activation and immune function.     

Quantitative immunogold localization of dipeptidyl peptidase IV (DPP IV) in rat liver cells

We investigated ultrastructural localization of dipeptidyl peptidase IV (DPP IV) [EC3.4.14 5] in rat liver cells quantitatively by post embedding protein A-gold technique. In the hepatocyte, DPP IV was mainly localized on the bile canalicular surface and the lysosomal membranes, but were scarcely detectable on the sinusoid-lateral surface. A small number of DPP IV was also detected in the trans region of the Golgi apparatus, suggesting that this part may play important roles in intracellular transport or recycling of this enzyme. In the endothelial cell, DPP IV existed on the whole surface of the plasma membrane and the lysosomes. In the Kupffer cell DPP IV was mainly localized in lysosomes and a few were detected on the plasma membrane.     

Modulation of Kv4.2 channel expression and gating by dipeptidyl peptidase 10 (DPP10)

The dipeptidyl aminopeptidase-like protein DPPX (DPP6) associates with Kv4 potassium channels, increasing surface trafficking and reconstituting native neuronal ISA-like properties. Dipeptidyl peptidase 10 (DPP10) shares with DPP6 a high amino acid identity, lack of enzymatic activity, and expression predominantly in the brain. We used a two-electrode voltage-clamp and oocyte expression system to determine if DPP10 also interacts with Kv4 channels and modulates their expression and function. Kv4.2 coimmunoprecipitated with HA/DPP10 from extracts of oocytes heterologously expressing both proteins. Coexpression with DPP10 and HA/DPP10 enhanced Kv4.2 current by approximately fivefold without increasing protein level. DPP10 also remodeled Kv4.2 kinetic and steady-state properties by accelerating time courses of inactivation and recovery (taurec: WT = 200 ms, +DPP10 = 78 ms). Furthermore, DPP10 introduced hyperpolarizing shifts in the conductance-voltage relationship (approximately 19 mV) as well as steady-state inactivation (approximately 7 mV). The effects of DPP10 on Kv4.1 were similar to Kv4.2; however, distinct biophysical differences were observed. Additional experiments suggested that the cytoplasmic N-terminal domain of DPP10 determines the acceleration of inactivation. In summary, DPP10 is a potent modulator of Kv4 expression and biophysical properties and may be a critical component of somatodendritic ISA channels in the brain.     

Partial purification and characterization of dipeptidyl peptidase II (DPP II) from guinea pig testes

Depeptidyl peptidase (DPP II) was partially purified from guinea pig testes by (NH₄)₂SO₄ precipitation, Con A-Sepharose 4B chromatography, and Sephadex G-200 chromatography to a specific activity of 27.4 μmol Ala₃ hydrolyzed min^?1 mg^?1 protein. Chromatography on a calibrated G-200 column yielded a molecular weight of 135,000 daltons for the enzyme. Sodium dodecyl sulfate polyacrylamide electrophoresis showed an enrichment of a broad doublet at 64–66,000 daltons. The enzyme had optimal activity toward hydrolysis of L-alanyl-alanyl-alanine at pH 4.5 and showed sensitivity to cations of increasing size with Tris producing the most inhibition of those tested. The enzyme was moderately inhibited by serine proteinase inhibitors. Thin-layer chromatography revealed the dipeptidase nature of the enzyme's activity on tripeptides and dipeptidyl arylamides. A doublet of activity occurred when nitrocellulose electroblots of nondenaturing gel electrophoresis of the (NH₄)₂SO₄ fraction were reacted with the specific DPP II substrate, lysyl-alanyl-4-methoxy-2-napthylamide. Analytical isoelectric focusing of the G-200 fraction followed by fluorescent enzyme activity detection that used cellulose triacetate overlay membranes impregnated with the specific DPP II substrate, lysyl-alanyl-7-amino-4-trifluoromethylcou-marin, revealed multiple isoforms focusing at pI = 4.8–5.6. Two prominent bands focused at pI = 4.9 and pI = 5.1. The properties of guinea pig testicular DPP II are compared and contrasted with similar dipeptidyl peptidases from other sources.     

A time-resolved fluorescence resonance energy transfer-based assay for DEN1 peptidase activity

Neural precursor cell expressed, developmentally down-regulated gene 8 (NEDD8) is a recently discovered ubiquitin-like posttranslational modifier. NEDD8 acts predominantly as a regulator of ubiquitin-protein ligases and as a decoy for proteins targeted for proteasomal degradation. It thereby controls key events in cell cycle progression and embryogenesis. Deneddylase-1 (DEN1/NEDP1/SENP8) features a selective peptidase activity converting the proNEDD8 precursor to its mature form and an isopeptidase activity deconjugating NEDD8 from substrates such as cullins and p53. In this study, we describe a high-throughput screening (HTS)-compatible time-resolved fluorescent resonance energy transfer (TR-FRET) assay measuring the peptidase activity of DEN1.     

Inhibition of dipeptidyl peptidase IV (DPP IV) by 2-(2-amino-1-fluoro-propylidene)-cyclopentanecarbonitrile,a fluoroolefin containing peptidomimetic

Novel, potent inhibitors of dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5, CD26), containing the fluoroolefin peptide isostere psi [CFz.dbnd6;C], have been prepared via the intermediacy of the Peterson fluoroolefination reaction. The nitrile containing inhibitors were found to inhibit dipeptidyl peptidase IV competitively with K(i) values for the l-3 and u-3 inhibitors of 7.69 and 6.03 microM, respectively. In contrast to earlier reported fluoroolefin containing inhibitors, the nitriles underwent no detectable degradation at pH 7.6 under buffered conditions.     

Identification and characterization of human DPP9, a novel homologue of dipeptidyl peptidase IV

We used an in silico approach to identify new cDNAs with homology to dipeptidyl peptidase IV (DPP IV). DPP IV (EC 3.4.14.5) is a serine protease with a rare enzyme activity having an important role in the regulation of various processes, such as blood glucose control and immune responses. Here, we report the identification and characterization of a novel DPP IV-like molecule, termed dipeptidyl peptidase-like protein 9 (DPP9). The deduced amino acid sequence of DPP9 has a serine protease motif, GWSYG, identical to that found in DPP IV. The presence of this motif, together with a conserved order and spacing of the Ser, Asp, and His residues that form the catalytic triad in DPP IV, places DPP9 in the "DPP IV gene family". Northern blots showed that DPP9 is ubiquitously expressed, with the highest expression levels in skeletal muscle, heart, and liver, and the lowest in brain. In vitro translation of the cloned full-length DPP9 sequence resulted in a DPP9 protein product that migrated in sodium dodecyl sulfate-polyacrylamide gel electrophoresis at a position similar to the predicted protein size of 98 kDa. Consistent with the lack of predicted transmembrane domains and a signal sequence, DPP9 was found in a soluble, putative cytosolic form. A DPP9 orthologue in mice was identified by expressed sequence tag database searches and verified by cDNA cloning.     

Role of dipeptidyl peptidase IV (DPP4) in the development of dyslipidemia: DPP4 contributes to the steroid metabolism pathway

AimsWe previously reported that dipeptidyl peptidase IV (DPP4)-deficient rats were susceptible to dyslipidemia induced by streptozotocin (STZ). Hence, it is suggested that DPP4 is important for lipid metabolism.Main methodsIn this study, to verify the role of DPP4 in the development of dyslipidemia, we carried out a microarray analysis of the livers of STZ-treated wild-type and DPP4-deficient rats and showed that the expression levels of genes involved in metabolic processes (steroid metabolic processes and cellular lipid metabolic processes) were significantly altered by STZ treatment.Key findingsIn the wild-type rats, the expression of hydroxysteroid (17-beta) dehydrogenase 2 (Hsd7b2), which catalyzes sex steroid synthesis from cholesterol, was significantly increased by about 15-fold after STZ treatment; however, it did not change in the DPP4-deficient rats. In the STZ untreated group of DPP4-deficient rats, the expression levels of cytochrome P450, subfamily 51 (Cyp51) and sterol-C4-methyl oxidase-like (Sc4mol), which catalyze intermediate steps in cholesterol synthesis, were significantly elevated compared to those of other groups. Similar results were demonstrated in HuH7-cells after DPP4 overexpression or the addition of human sera containing DPP4.SignificanceDPP4 is crucial for regulating the expression of factors related to steroid metabolism such as Cyp51, Sc4mol, and Hsd17b2, and DPP4 deficiency or inhibition may cause dyslipidemia.     

Development and validation of a cell-based high-throughput screening assay for TRPM2 channel modulators

TRPM2 is a member of the transient receptor potential melastatin (TRPM)-related ion channel family. The activation of TRPM2 induced by oxidative/nitrosative stress leads to an increase in intracellular free Ca(2+). Although further progress in understanding TRPM2's role in cell and organism physiology would be facilitated by isolation of compounds able to specifically modulate its function in primary cells or animal models, no cell-based assays for TRPM2 function well suited for high-throughput screening have yet been described. Here, a novel suspension B lymphocyte cell line stably expressing TRPM2 was used to develop a cell-based assay. The assay uses the Ca(2+)-sensitive fluorescence dye, Fluo-4 NW (no wash), to measure TRPM2-dependent Ca(2+) transients induced by H(2)O(2) and N-methyl-N'-nitrosoguanidine in a 96-well plate format. Assay performance was evaluated by statistical analysis of the Z' factor value and was consistently greater than 0.5 under optimal conditions, suggesting that the assay is very robust. For assay validation, the effects of known inhibitors of TRPM2 and TRPM2 gating secondary messenger production were determined. Overall, the authors have developed a cell-based assay that may be used to identify TRPM2 ion channel modulators from large compound libraries.     

A new and highly sensitive fluorescence assay for collagenase-like peptidase activity.

A highly sensitive fluorescence assay for collagenase-like peptidase (CL-peptidase) has been developed using a newly synthesized substrate, (succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide (Suc-GPLGP-MCA). Suc-GPLGP-MCA was hydrolyzed at the Leu-Gly bond by CL-peptidase, (Gly-Pro)-4-methylcoumaryl-7-amide liberated by the enzyme was immediately hydrolyzed to Gly-Pro and 7-amino-4-methylcoumarin (AMC) by an excess of an auxiliary enzyme, X-prolyl dipeptidyl-aminopeptidase, and the fluorescence intensity of the AMC was measured at 460 nm with excitation at 380 nm. When assayed by this method, CL-peptidase partially purified from chick embryo showed a pH optimum at 8.0 and a K_m value of 4.0 × 10^?4m toward Suc-GPLGP-MCA. Under the optimum condition, the reaction proceeded linearly up to 4 h. The CL-peptidase activity was found in normal human sera by this method and the mean and standard deviation of the activity was 0.59 ± 0.10 nmol/min/ml of serum (n = 10). This assay was also applicable for the CL-peptidase in human liver and kidney. The results suggest that the CL-peptidase assayed by this new substrate may be different from the “PZ-peptidase” which cleaves a synthetic substrate for collagenase-like peptidase, 4-phenylazobenzyloxycarbonyl (PZ)-Pro-Leu-Gly-Pro-d-Arg (PZ-peptide). The new peptide, Suc-GPLGP-MCA, was found not to be a substrate for specific collagenase from tadpole.     

Dipeptidyl peptidase (DPP) IV in rat organs. Comparison of immunohistochemistry and activity histochemistry

Immunohistochemistry and activity histochemistry were used to study the localization of dipeptidyl peptidase (DPP) IV in rats. For immunohistochemistry, polyclonal as well as monoclonal anti-DPP IV antibodies were employed. The pattern of DPP IV immunoreactivity, determined with polyclonal anti-DPP IV antibody, corresponds to the histochemical pattern found for the enzymic activity of DPP IV. Immunoreactivity was present, in addition, in nerve cells, lateral membranes of certain surface epithelia, e.g., Fallopian tube, uterus and vesicular gland, in the luminal cytoplasm of e.g., vesicular gland epithelium, and in mucous cells of Brunner's gland. The monoclonal antibodies against DPP IV recognized four different epitopes (A-D) of the DPP IV molecule, and revealed that certain epitopes were not detectable by immunohistochemistry in some organs. Generally, the staining intensities for epitopes A, B, C and D decreased in that order. Usually, the monoclonal antibodies against epitopes A and B showed similar reaction patterns to those as obtained with the polyclonal antibody. Epitope D was recognized in the lumen of the duct system of exocrine glands and the intestine. Furthermore, high reactivity of this epitope was detected in goblet cells of the intestine, where no DPP IV activity was present.     

Molecular characterization of a novel dipeptidyl peptidase like 2-short form (DPL2-s) that is highly expressed in the brain and lacks dipeptidyl peptidase activity

DPL2 (DPP10) found at chromosome 2q14.1 is a member of the dipeptidyl peptidase IV (DPIV) gene family. Here we characterize a novel short DPL2 isoform (DPL2-s), a 789-amino acid protein, that differs from the previously described long DPL2 isoform (DPL2-l) at the N-terminal cytoplasmic domain by 13 amino acids. The two DPL2 isoforms use alternate first exons. DPL2 mRNA was expressed mainly in the brain and pancreas. Multiple forms of recombinant DPL2-s protein were observed in 293T cells, having mobilities 96 kDa, 100 kDa, and approximately 250 kDa which may represent soluble DPL2, transmembrane DPL2 and multimeric DPL2 respectively. DPL2 is glycosylated as a band shift is observed following PNGase F deglycosylation. DPL2-s was expressed primarily on the cell surface of transfected 293T and PC12 cells. DPL2-s exhibits high sequence homology with other DPIV peptidases, but lacks a catalytic serine residue and lacks dipeptidyl peptidase activity. Substitutions of Gly(644)-->Ser, Lys(643)Gly(644)-->TrpSer, or Asp(561)Lys(643)Gly(644)-->TyrTrpSer in the catalytic motif did not confer dipeptidyl peptidase activity upon DPL2-s. Thus, although DPL2 is similar in structure and sequence to the other dipeptidyl peptidases, it lacks vital residues required to confer dipeptidyl peptidase activity and has instead evolved features that enable it to act as an important component of voltage-gated potassium channels.     

A simple and sensitive fluorescence assay for tyrosine hydroxylase activity.

A simple and sensitive fluorescence assay for tyrosine hydroxylase (TH) activity wasdevised based on rapid isolation of enzymatically formed dopa by a double-column procedure fitted together sequentially (the top column of Amberlite CG-50 and the bottom column of aluminum oxide). Interfering substances were removed by the first Amberlite CG-50 column. Dopa was adsorbed on the second aluminum oxide column, then eluted with 0.5 m acetic acid, and assayed by the highly sensitive hydroxyindole method of Johnson et al. (1973, Anal. Biochem.54, 129–136). The standard incubation mixture (total volume, 0.5 ml) contained 0.3 mm l-tyrosine, 1.0 mm 6-methyl-5,6,7,8-tetrahydropterin, 100 mm mercaptoethanol, and an optimal concentration of ferrous ion. d-Tyrosine was used for the blank incubation. Recovery of dopa added to the standard incubation mixture as internal standard was about 70% and was reproducible. The fluorescence characteristics of the product were the same as those of authentic dopa. Blank fluorescence was very low even with crude enzyme preparations. The limit of sensitivity was 100 pmol of dopa formed, which is close to the sensitivity of radioassays. TH activity in homogenates of rat brain stem or human putamen could be assayed in the standard incubation system containing ferrous ion. The validity of this fluorescence assay has been shown by the agreement between the values obtained by this method and by radioassay using l-[U-¹⁴C]tyrosine as substrate. In the rapid assay procedure dopa in the eluate from aluminum oxide was assayed directly by native fluorescence. Although the sensitivity was about 1 nmol, this rapid assay procedure was found to be particularly useful for the purification of TH.     

Development of a high-throughput fluorescence polarization assay for Bcl-x(L)

Antiapoptotic protein Bcl-x(L) has been demonstrated to play a very important role in a variety of diseases such as cancer. Its biological function can be inhibited by proapoptotic proteins such Bak, Bad, and Bax by forming complexes mediated primarily by the Bcl-2 homology 3 (BH3) domain. To facilitate drug discovery for Bcl-x(L) inhibitors, we have developed and optimized a fluorescence polarization assay based on the interaction between Bcl-x(L) and BH3 domain peptides. We observed that the fluorescein-labeled Bad BH3 peptide [NLWAAQRYGRELRRMSDK(fluorescein)FVD or fluorescent Bad peptide] generates best overall results. Fluorescent Bad peptide interacts strongly with Bcl-x(L) with a K(d) of 21.48nM. The assay is stable over a 24-h period and can tolerate the presence of dimethyl sulfoxide up to 8%. By using a competition assay, several peptides derived from the BH3 region of Bak, Bad, Bax, and Bcl-2 were investigated. Bad and Bak BH3 peptides compete efficiently with IC(50) values of 0.048 and 1.14 microM, respectively, while the peptides from the BH3 region of Bcl-2 and Bax compete weakly. A mutated Bak peptide, which has been shown to be inactive for binding to Bcl-x(L), did not compete. The relative binding order of the peptides (Bad>Bak>Bcl-2>Bax>mutated Bak) correlates well with previously published results. When tested in high-throughput formats, the assay has a signal-to-noise ratio of 15.37 and a Z(') factor of at least 0.73. The plate-to-plate variability for free peptide control and bound peptide control is minimal. This validates the assay not only for investigating the nature of Bcl-x(L)-peptide interaction, but also for high-throughput screening of Bcl-x(L) inhibitors.     

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.     

Development and validation of a fluorescence HPLC-based screening assay for inhibition of human estrogen sulfotransferase

Human estrogen sulfotransferase (SULT1E1) is involved in the regulation of 17beta-estradiol responsiveness and is believed to protect peripheral tissues from excessive estrogenic effects. Several assays already have been developed to investigate the inhibitory effect of endocrine-disrupting compounds (EDCs) on SULT1E1. However, most of these assays make use of the radiolabeled cofactor [(35)S]3'-phosphoadenosine 5'-phosphosulfate (PAPS) or radiolabeled substrate [(3)H]estradiol. In this article, we describe the development and validation of an assay for the inhibition of human SULT1E1 that is rapid and simple and that uses the nonradioactive and noncarcinogenic 1-hydroxypyrene. A gradient HPLC separation of 15 min using a C18-RP column was developed to detect 1-hydroxypyrene and its metabolite pyrene 1-sulfate fluorescently. Time- and protein-dependent formation of pyrene 1-sulfate was investigated, and enzyme kinetics was determined (K(m)=6.4+/-0.8 nM and V(max)=158+/-19 pmol/min/microg SULT1E1). At higher 1-hydroxypyrene concentrations, the assay displayed non-Michaelis-Menten kinetics involving substrate inhibition. IC(50) values have been determined for eight known SULT1E1 inhibitors or competing substrates (17beta-estradiol, 17alpha-estradiol, genistein, 17alpha-ethynylestradiol, estrone, diethylstilbestrol, estriol, and hexestrol) and two previously unknown SULT1E1 inhibitors (zearalenone and dienestrol). The method was demonstrated to be easy, feasible, and highly reproducible for SULT1E1 screening assay inhibition studies.     

A homogeneous cell-based assay for measurement of endogenous paraoxonase 1 activity

Paraoxonase 1 (PON1) is a high-density lipoprotein-associated enzyme that plays an important role in organophosphate detoxification and prevention of atherosclerosis. Thus, there is significant interest in identifying nutritional and pharmacological enhancers of PON1 activity. To identify such compounds, we developed a rapid homogeneous assay to detect endogenous cell-associated PON1 activity. PON1 activity was measured by the simple addition of fluorigenic PON1 substrate DEPFMU to live Huh7 cells in medium and monitoring change in fluorescence. A specific PON1 inhibitor, 2-hydroxyquinoline, was used to confirm that the observed activity was due to PON1. The assay was optimized and characterized with regard to time course, substrate and sodium chloride concentration, number of cells, and tolerance to dimethyl sulfoxide and serum. Aspirin, quercetin, and simvastatin are compounds reported to increase PON1 expression. Consistent with the literature and Western blot data, these compounds enhanced PON1 activity in this assay with comparable efficacies and potencies. A known toxic compound did not increase assay signal. This assay method also detected PON1 activity in normal hepatocytes. Thus, a novel homogeneous assay for detection of endogenous PON1 expression has been developed and is amenable to high-throughput screening for the identification of small molecules that enhance PON1 expression.     

Development of a cell-based diabetic wound assay

Chronic wounds require prolonged healthcare and adversely affect the quality of life of patients. They are particularly prominent in patients with diabetes, and their relative numbers are set to increase with the rise of diabetes within our population. Research is still needed to understand the factors leading to such wounds, to understand why they persist for such long periods of time, and also to develop new and efficacious treatment strategies. One problem facing this research is a lack of adequate animal models, as the current models do not truly reflect the human condition and often lead to much animal suffering. Hence, over the past four years, our group has been trying to develop a human-based in vitro diabetic wound model, which could be used as a high-throughput screening system to pre-screen potential chronic diabetic wound healing agents and to reduce unnecessary animal pain and suffering. To this end, we have isolated healthy and diseased skin fibroblasts from patient tissue biopsies. Crucially, to create a cell reporter system that can be widely used in the future, the cells were immortalised in order to escape senescence. By using microarray analysis, gene expression pattern differences have been identified between healthy and diseased cells, and disease-specific 'reporter' genes have been selected for further studies. The promoters of these reporter genes have been coupled to fluorescent reporter constructs and inserted back into the diseased fibroblasts, so that we now have proof-of-concept for a real-time diabetic reporter system for future exploitation.