Apical uptake of choline and cationic drugs in epithelial cell lines derived from human placenta.

Many cationic drugs are administered during pregnancy and might enter the fetal circulation by transplacental passage. This study was performed to characterize the apical uptake of choline and several cationic drugs at cultured epithelial cells of the human placenta. Total uptake of [3H]choline in BeWo cells was H(+)-independent and to 65% Na(+)-independent. Uptake rates into both cell lines were saturable with Michaelis-Menten constants (Kt) of 108 microM (BeWo) and 206 microM (JEG-3), respectively. Cationic drugs such as etilefrine, clonidine, ranitidine, diphenhydramine, imipramine and butylscopolamine strongly inhibited the [3H]choline uptake in BeWo cells and in JEG-3 cells, with Ki values ranging from 0.18 to 3.3 mM. In contrast, tetraethylammonium had only little inhibitory effect on [3H]choline uptake. Using high-performance capillary electrophoresis for quantitative analyses, uptake of etilefrine and diphenhydramine into JEG-3 or BeWo cells was measured. Diphenhydramine was transported into JEG-3 cells in a saturable manner with a Kt value of 0.75 mM. In the presence of sodium, diphenhydramine uptake at BeWo cells was inhibited to 69% by the addition of 50 mM choline chloride. Like choline uptake, total diphenhydramine uptake was to 68% Na(+)-independent in BeWo cells. We conclude that in addition to choline, several cationic drugs, in particular diphenhydramine, are taken up by placental epithelial cells from the maternal blood by carrier-mediated processes. Etilefrine, clonidine, ranitidine, diphenhydramine and butylscopolamine interact with the Na(+)-independent placental choline transport system.

A novel inhibitor of the mammalian peptide transporter PEPT1.

This study was initiated to develop inhibitors of the intestinal H(+)/peptide symporter. We provide evidence that the dipeptide derivative Lys[Z(NO(2))]-Pro is an effective competitive inhibitor of mammalian PEPT1 with an apparent binding affinity of 5-10 microM. Characterization of the interaction of Lys[Z(NO(2))]-Pro with the substrate binding domain of PEPT1 has been performed in (a) monolayer cultures of human Caco-2 cells expressing PEPT1, (b) transgenic Pichia pastoris cells expressing PEPT1, and (c) Xenopus laevis oocytes expressing PEPT1. By competitive uptake studies with radiolabeled dipeptides, HPLC analysis of Lys[Z(NO(2))]-Pro in cells, and electrophysiological techniques, we unequivocally show that Lys[Z(NO(2))]-Pro binds with high affinity to PEPT1, competes competitively with various dipeptides for uptake into cells, but is not transported itself. Lack of transport was substantiated by the absence of Lys[Z(NO(2))]-Pro in Caco-2 cell extracts as determined by HPLC analysis, and by the absence of any positive inward currents in oocytes when exposed to the inhibitor. The fact that Lys[Z(NO(2))]-Pro can bind to PEPT1 from the extracellular as well as the intracellular site was shown in the oocyte expression system by a strong inhibition of dipeptide-induced currents under voltage clamp conditions. Our findings serve as a starting point for the identification of the substrate binding domain in the PEPT1 protein as well as for studies on the physiological and pharmacological role of PEPT1.

Targeting dipeptidyl peptidase IV (CD26) suppresses autoimmune encephalomyelitis and up-regulates TGF-beta 1 secretion in vivo.

CD26 or dipeptidyl peptidase IV (DP IV) is expressed on various cell types, including T cells. Although T cells can receive activating signals via CD26, the physiological role of CD26/DP IV is largely unknown. We used the reversible DP IV inhibitor Lys[Z(NO(2))]-pyrrolidide (I40) to dissect the role of DP IV in experimental autoimmune encephalomyelitis (EAE) and to explore the therapeutic potential of DP IV inhibition for autoimmunity. I40 administration in vivo decreased and delayed clinical and neuropathological signs of adoptive transfer EAE. I40 blocked DP IV activity in vivo and increased the secretion of the immunosuppressive cytokine TGF-beta1 in spinal cord tissue and plasma during acute EAE. In vitro, while suppressing autoreactive T cell proliferation and TNF-alpha production, I40 consistently up-regulated TGF-beta1 secretion. A neutralizing anti-TGF-beta1 Ab blocked the inhibitory effect of I40 on T cell proliferation to myelin Ag. DP IV inhibition in vivo was not generally immunosuppressive, neither eliminating encephalitogenic T cells nor inhibiting T cell priming. These data suggest that DP IV inhibition represents a novel and specific therapeutic approach protecting from autoimmune disease by a mechanism that includes an active TGF-beta1-mediated antiinflammatory effect at the site of pathology.

Potent inhibitors of dipeptidyl peptidase IV and their mechanisms of inhibition.

Dipeptidyl peptidase IV (DP IV) is a proline specific serine protease which cleaves Xaa-Pro-dipeptides from the N-terminus of longer peptides. A series of product analogous amino acid amides containing different structure modifications like substitution of a ring atom, variation of the ring size and/or the introduction of a thioxo amide bond, phosphono amide bond or reduced amide bond were done to characterize these compounds as inhibitors of DP IV. These compounds are mostly classical reversible inhibitors of DP IV. In contrast amino acyl-2-cyanopyrrolidides inhibit DP IV according to a slow-binding mechanism with inhibition constants in the nanomolare range. On the other hand, diaryl dipeptide phosphonates inhibit irreversibly. In conclusion, this work shows, that the mechanism of inhibition of DP IV depends on the structure of the investigated compounds.

Signal transduction events induced or affected by inhibition of the catalytic activity of dipeptidyl peptidase IV (DP IV, CD26).

DP IV (CD26) represents an accessory surface molecule playing an important role in the process of activation and proliferation of human lymphocytes. The molecular events mediated by this ectoenzyme are only partly established and the necessity of DP IV enzymatic activity for its signalling capacity has been discussed controversial. Focusing on the putative role of the catalytic domain of this peptidase, it could be shown that inhibition of the catalytic activity can provoke many cellular effects, including induction of tyrosine phosphorylations and p38 MAP kinase activation as well as suppression of DNA synthesis and reduced production of various cytokines. TGF-beta 1, the production and secretion of which is increased after DP IV inhibition, supposedly mediates the observed suppressive effects by maintaining p27kip expression levels which leads to a cell cycle arrest in G1. Moreover, anti-CD3-induced signalling pathways, including Ca2+ mobilisation, MEK1-, Erk1/2- and PKB-activation, can be strongly affected by DP IV inhibition. Thus, the enzymatic activity or at least the interaction of effectors with the catalytic domain of CD26 seems to be important for crucial functions of this cell surface antigen.

Dipeptidyl peptidase IV in inflammatory CNS disease.

Current pathogenic concepts of inflammatory demyelinating disorders such as multiple sclerosis (MS) are based on the hypothesis that a T cell-mediated autoimmune response is involved in the disease process. One of the primary goals in the in the development of immunotherapies for autoimmune diseases has been to achieve inactivation of disease-inducing lymphocytes either by direct inhibition or suppression through regulatory cells and/or cytokines. The CD26 antigen is identical with the cell surface ectopeptidase dipeptidyl peptidase IV (DP IV, EC 3.4.14.5) which is involved in regulating T cell activation and growth. Activated T cells, including those specific for myelin antigens, express high levels of CD26/DP IV. In vitro, reversible DP IV inhibitors suppress T cell proliferation and pro-inflammatory cytokine production in response to myelin antigens. Further studies will evaluate the role of DP IV inhibition in T cell-mediated inflammatory disease of the central nervous system.

Dipeptidyl peptidase IV (CD26): role in T cell activation and autoimmune disease.

The ectoenzyme dipeptidyl peptidase IV (DP IV; EC 3.4.14.5; CD26) has been shown to play a crucial role in T cell activation. In the present study, we show by flow cytometry and by enzymatic DP IV assay that myelin basic protein (MBP)-specific, CD4+ T cell clones (TCC) derived from patients with multiple sclerosis (MS) express high levels of DP IV/CD26. The enzymatic activity of resting TCC was found to be three to fourfold higher than on resting peripheral blood T cells and close to that of T cells 48 hours after PHA stimulation. The DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide suppress in a dose-dependent manner DNA synthesis and IFN-gamma, IL-4, and TNF-alpha production of the antigen-stimulated TCC. These data suggest that CD26 plays a role in regulating activation of autoreactive TCC. Further in vivo investigations will clarify, whether the inhibition of the enzymatic activity of DP IV could be a useful tool for therapeutic interventions in MS and/or other autoimmune diseases.

DNA synthesis in cultured human keratinocytes and HaCaT keratinocytes is reduced by specific inhibition of dipeptidyl peptidase IV (CD26) enzymatic activity.

The ectopeptidase dipeptidyl peptidase IV (DP IV, CD26, EC 3.4.14.5) is present on most mammalian cells. Using specific inhibitors of DP IV, it has been shown that this enzyme is involved in the regulation of DNA synthesis and in production of various cytokines in lymphocytes. The aim of the present work was to investigate the expression of DP IV/CD26 on human keratinocytes and to answer the question, whether the proliferation (DNA synthesis) of human keratinocytes is influenced by inhibition of the enzymatic activity of DP IV. Using flow cytometry, RT-PCR, and specific enzymatic activity assays, expression of DP IV-mRNA and CD26 antigen were shown on primary keratinocyte strains and on the HaCaT keratinocyte cell line. The synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and -pyrrolidide suppress the DNA-synthesis of these cells in a dose-dependent manner. These data demonstrate that CD26 is also involved in the regulation of DNA synthesis of keratinocytes and that the enzymatic activity is required for mediating these effects.

Decisive structural determinants for the interaction of proline derivatives with the intestinal H+/peptide symporter.

To elucidate the decisive structural factors relevant for dipeptide-carrier interaction, the affinity of short amide and imide derivatives for the intestinal H+/peptide symporter (PEPT1) was investigated by measuring their ability to inhibit Gly-Sar transport in Caco-2 cells. Dipeptides with proline or alanine in the C-terminal position displayed affinity constants (Ki) of 0.15-1.2 mM and 0.08-9.5 mM, respectively. There was no clear relationship between hydrophobicity, size or ionization status of the N-terminal amino acid and the affinity of the dipeptides. However, analyzing the individual peptide bond conformations of Xaa-Pro dipeptides, a striking correlation between the cis/trans ratios (trans contents 24-70%) and the affinity constants was observed. After correcting the Ki values for the incompetent cis isomers, the Ki corr values of most dipeptides were in a small range of 0.1-0.16 mM. This result revealed the decisive role of peptide bond conformation even for a transport protein that is quite promiscuous in substrate translocation. When measuring affinity constants of Xaa-Pro and Xaa-Sar dipeptides, the cis/trans ratios cannot be ignored. Lower affinities of Lys-Pro, Arg-Pro and Pro-Pro indicate that additional molecular factors affect their binding at PEPT1. The Ki values obtained for the corresponding Xaa-Ala dipeptides support this conclusion. Potential substrates or inhibitors of peptide transport were found among Xaa-piperidides and Xaa-thiazolidides. Dipeptides with N-terminal proline displayed a very diverse affinity profile. However, in contrast to current knowledge, several Pro-Xaa dipeptides such as Pro-Leu, Pro-Tyr and Pro-Pro are recognized by PEPT1 with appreciable affinities. Binding seems mainly determined by the hydrophobicity of the C-terminal amino acid and the rigidity of the structure.

[Can knee joint proprioception by reconstruction of the anterior cruciate ligament be restored? A prospective longitudinal study]. / Kann die Kniegelenkspropriozeption durch Rekonstruktion des vorderen Kreuzbandes wiederhergestellt werden? Eine prospektive Longitudinalstudie.

Knee proprioception was assessed in 20 healthy volunteers, 10 patients with acute anterior instability and in 20 patients with chronic anterior instability preoperatively as well as after 3 and 6 months postoperatively. To determine proprioception, an angle reproduction test was performed. There were no differences between the right and the left knee joint or between men and women. Best proprioception was measured near full extension. The acute trauma causes massive damage of the proprioception, which could be improved significantly by rehabilitation. However, rehabilitation could not restore proprioception. 3 months postoperatively, there was a slight decrease of proprioception as compared to the preoperative values, but 6 months after reconstruction, restoration of proprioception was documented near full extension and full flexion. In the mid range position, the proprioception could not be restored. There was no difference between open and arthroscopic techniques. The highest correlation was found between proprioception and patient's satisfaction.

Recurrent chromosomal imbalances detected in biopsy material from oral premalignant and malignant lesions by combined tissue microdissection, universal DNA amplification, and comparative genomic hybridization.

Biopsies routinely performed for the histopathological diagnosis of oral epithelial lesions before treatment were screened for chromosomal imbalances by comparative genomic hybridization. Comparative genomic hybridization was performed on 12 oral premalignant lesions (OPLs; dysplasias and carcinomas in situ) and 14 oral squamous cell carcinomas (OSCCs). Eight biopsies displayed areas of different histopathological appearance, so that OPLs and OSCCs from the same patient were analyzed. To avoid contamination with nonneoplastic cells, defined cell populations were isolated by micromanipulation with a glass needle. Before comparative genomic hybridization analysis, universal DNA amplification was performed using the DOP-polymerase chain reaction protocol. In the 14 OSCCs examined, the average number of chromosomal imbalances was significantly higher than in the 12 OPLs (mean +/- SEM: 11.9 +/- 1.9 versus 3.2 +/- 1.2; P = 0.003). The DNA copy number changes identified in more than one OPL were gains on 8q (3 of 12) and 16p (2 of 12), as well as losses on 3p (5 of 12); 5q (4 of 12); 13q (3 of 12); and 4q, 8p, and 9p (2 of 12 each). In more than 30% of OSCCs, gains of chromosomal material were identified on 20q (8 of 14); 8q, 11q, 22q (7 of 14 each); 3q, 15q, and 17p (6 of 14 each); and 14q, 17q, and 20p (5 of 14 each), and losses were identified on 3p and 4q (9 of 14 each), 5q (7 of 14), 13q (6 of 14), and 2q and 9p (5 of 14 each). These results were validated by positive and negative control comparative genomic hybridization experiments and microsatellite analysis for the detection of allelic loss. The vast majority of genomic alterations found in OPLs were again identified in OSCCs from the same biopsy, supporting the hypothesis that multiple lesions in the same patient are clonally related. In summary, we show that comprehensive information on the genomic alterations in oral epithelial lesions can be obtained from small biopsies. Such data may identify prognostic indicators that could eventually assist in designing therapeutic strategies.

Inhibitors of dipeptidyl peptidase IV/CD26 suppress activation of human MBP-specific CD4+ T cell clones.

The ectoenzyme dipeptidyl peptidase IV (DP IV, EC 3.4.14.5, CD26) has been shown to play a crucial role in T cell activation. Specific inhibitors of DP IV suppress DNA synthesis as well as cytokine production (IL-2, IL-10, IL-12, IFN-gamma) of stimulated human and mouse T cells suggesting a potential application of these effectors in transplantations and autoimmune diseases. In the present study, we have examined the expression of DP IV/CD26 on six myelin basic protein (MBP)(87-99)-specific, CD4+ T cell clones (TCC) derived from patients with multiple sclerosis (MS) as well as the biological effects of the two synthetic DP IV inhibitors Lys[Z(NO2)]-thiazolidide and Lys[Z(NO2)]-pyrrolidide on the function of these cells. All TCC expressed high levels of DP IV/CD26, as shown by flow cytometry and by enzymatic DP IV assay. Enzymatic activity of resting TCC was found to be three to fourfold higher than on resting peripheral blood T cells and close to that of T cells 48 h after PHA stimulation. The DP IV inhibitors suppress DNA synthesis and IFN-gamma, IL-4, and TNF-alpha production of the antigen-stimulated TCC. These data suggest that CD26 plays a role in regulation of activation of autoreactive TCC. Further in-vivo investigations, first in experimental models, will clarify, whether the inhibition of the enzymatic activity of DP IV could be a useful tool for therapeutic interventions in MS or other autoimmune diseases.

Dipeptidyl peptidase IV (DP IV, CD26) is involved in regulation of DNA synthesis in human keratinocytes.

Various studies have shown that the membrane ectoenzyme dipeptidyl peptidase IV (DP IV, CD26), expressed on T, NK, and B cells in the human immune system, is involved in the regulation of DNA synthesis and cytokine production. Here, we clearly demonstrate that this enzyme is highly expressed also on human epidermal foreskin and split-skin keratinocytes and that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-pyrrolidide inhibit the enzymatic activity as well as the DNA synthesis of these cells. These data demonstrate that CD26 plays a role also in regulation of DNA synthesis of epidermal keratinocytes and that the enzymatic activity is required for mediating these effects.

Inhibitors of dipeptidyl peptidase IV (DP IV, CD26) induces secretion of transforming growth factor-beta 1 (TGF-beta 1) in stimulated mouse splenocytes and thymocytes.

Various studies have shown that the ectoenzyme dipeptidyl peptidase IV (DP IV, CD26), expressed on T, NK and B cells in the human immune system, is involved in the regulation of DNA synthesis and cytokine production. The DP IV/CD26 was found also on mouse splenocytes and thymocytes. Here, we show that the specific DP IV inhibitors Lys[Z(NO2)]-thiazolidide, Lys[Z(NO2)]-pyrrolidide inhibit DNA synthesis as well as production of IL-2, IL-6 and IL-10 of PHA-stimulated mouse splenocytes and Con A-stimulated mouse thymocytes. Most importantly, these inhibitors induce a three to fourfold increased secretion of latent transforming growth factor beta 1 (TGF-beta 1) by mitogen-stimulated mouse immune cells, as measured with a specific TGF-beta 1 enzyme-linked immunosorbent assay (ELISA). These data demonstrate that CD26 plays a role also in regulation of DNA synthesis and cytokine production by murine immune cells, that the enzymatic activity is required for mediating these effects, and that TGF-beta 1 might have key functions in these processes.

Probing substrate backbone function in prolyl oligopeptidase catalysis--large positional effects of peptide bond monothioxylation.

Site-specific effects on the catalytic activity of prolyl oligopeptidase from human placenta were studied using oligopeptide substrates in which a peptide bond has been replaced by a thioxo peptide bond. Two series of tetrapeptide-4-nitroanilides, Ala-Gly-Pro-Phe-NH-Np and Ala-Ala-Pro-Phe-NH-Np, along with all possible monothioxylated derivatives, were synthesised and k(cat) and Km values were determined for proteolytic cleavage at the Pro-Phe bond. Regardless of either Gly or Ala in the P2 subsite, tetrapeptides were rendered uncleavable by thioxylation at the Pro-Phe linkage. As a result, Ala-Xaa-Pro-psi[CS-NH]-Phe-NH-Np (Xaa = Gly or Ala) displayed competitive inhibition with Ki-values of 12 microM and 44 microM, respectively. Furthermore, in controlling proteolytic susceptibility of the substrates, cooperation of the P3-P2 thioxylation site and the side chain at the P2 subsite was obtained. Thioxylation at this position enhanced k(cat)/Km fivefold in the Gly series, but led to a 1.7-fold decrease in the Ala series of substrates. With respect to the Xaa-Pro peptide bond, all of the substrates underwent cis/trans isomerisation, thus presenting two stable conformers to the protease. However, the magnitudes of the isomerisation constants suggested that neither isomerisation rates nor cis/trans equilibria can explain the effect of thioxylation on the steady-state constants of proteolysis.

Redifferentiation of oral dysplastic mucosa by the application of the antioxidants beta-carotene, alpha-tocopherol and vitamin C.

In a clinical trial the effect of chemoprevention with beta-carotene, vitamin E and C on dysplastic tissue was studied. The study included 24 patients with oral leukoplakia and 24 patients after radical resection of a primary oral cancer. There was a reduction of increased cell kinetic parameters like the S-phase portion or the average number of nuclear-organizer regions (NOR) per cell nucleus, a decrease of the micronuclei portion and a normalization of the cytokeratin gene-expression. The general response was 97.5%. Stopping the alcohol and tobacco abuse the effect of the antioxidative vitamins on redifferentiation of the oral mucosa was more intense than by persistance of the alcohol and tobacco abuse, but a long term prevention seems to be ineffective.

[Effects of chronic alcohol drinking on mouth mucosa. A morphometric study]. / Auswirkungen des chronischen Alkoholkonsums auf die Mundschleimhaut. Eine morphometrische Untersuchung.

BACKGROUND: Chronic alcohol consumption is a major risk factor for oral and pharyngeal cancer. Besides other mechanisms a toxic effect of ethanol and/or its metabolite acetaldehyde on the oral mucosa and a resulting increased cell regeneration seem to play an important role in tumor promotion. METHODS: In the present study the effect of chronic ethanol consumption on the morphology of the oral mucosa of 20 male wistar rats that had been fed nutritionally adequate liquid diets containing 36% of total calories either as ethanol or isocaloric carbohydrates for six months was investigated. RESULTS: Morphometric analysis showed that in the ethanol rats, the size of the basal cell nuclei of the oral mucosa from the floor of the mouth, the edge of the tongue, and the base of the tongue was significantly increased (p < 0.001). The size of the basal cell layer in these rats was increased, and the stratification of the cells was altered. Mean epithelial thickness of the mucosa from the floor of the mouth was significantly reduced in the ethanol rats (p < 0.01). CONCLUSIONS: In conclusion, our results indicate that chronic ethanol consumption causes oral mucosa atrophy associated with hyperregeneration, which may result in an enhanced susceptibility of the mucosal epithelium to chemical carcinogens.

[Ultrasound diagnosis in primary staging of head-neck tumors]. / Die Sonographie im Primärstaging von Kopf-Hals-Tumoren.

The radiologic imaging methods play an important role in the precise staging as basic requirement for an effective concept of tumor therapy. The accuracy of ultrasound in the primary staging according to the TNM-classification (UICC) was therefore prospectively investigated in 260 patients with head and neck tumors of the clinical stages T1N0 to T4N3. The clinically (C1) and sonographically (C2) evaluated pretherapeutic stages were compared to the postoperative histopathologic tumor classifications. The clinical staging was correct in 75.0%, high in 7.7%, low in 17.3%, the N-stages were correct in 59.2%, high in 17.7%, low in 23.1%. The sonographic staging was apparently superior with the T-classifications correct in 92.3%, high in 7.7%, low 0.0%. The N-stages were correct in 89.6%, high in 9.2%, low in 1.2%. The accuracy of combined TN classification rose from clinical 46.5% to 84.6% by sonography. Accompanying inflammations, foregoing biopsies and tooth extractions were the main reasons for incorrect staging. Therefore, the thorough sonographic investigation performed after the clinical examination and before invasive procedures, due to little patient discomfort, good availability and high accuracy, is an excellent sectional imaging method for staging, therapy-planning and follow up of tumors of the head and neck especially of the orofacial regions.

Effect of photodynamic therapy on the healing of a rat skin flap and its implication for head and neck reconstructive surgery.

BACKGROUND AND OBJECTIVE: Photodynamic therapy (PDT) may as adjuvant therapy by used to reduce tumor recurrence in the head and neck with surgery, given intraoperatively after resection. A concern with the use of intraoperative PDT is the possible effect on wound healing, especially on the healing of myocutaneous skin flaps, which are widely used to reconstruct defects following resections for head and neck cancer. STUDY DESIGN/ MATERIALS AND METHODS: A flap, based on the inferior epigastric artery, was prepared in thirty male Lewis rats. Group I did not receive any further treatment but the wound was left open for 20 minutes. Group II was injected with 5mg/kg Photofrin, 48 hours prior to the operation and also did not receive any further treatment. The wound bed and wound borders of group III were treated with 630nm light of different dosages, delivered by an argon dye laser. Animals in group IV received 5mg/kg Photofrin 48 hours prior to the operation and their wound beds were treated with the same light dosages as group III. After the treatment all flaps were replaced into the wound bed and the incisions were closed. Biopsies for histological analysis were taken at several time points; and on day 21, biopsies for wound tensile strength measurements were taken. RESULTS: The wound healing in group I, II, and III appeared normal and there were no differences seen between these groups. Also, the tensile strength did not differ significantly. The flaps of group IV showed serous effusion, epidermal necrosis, and weaker tensile strength (P = .04 and .02 for the light doses of 50 J/sq cm and 75 J/sq cm respectively) at a specific time point. CONCLUSION: The results of this study demonstrate that PDT given immediately before flap reconstruction will result in delayed wound healing. These results should be considered when contemplating the use of PDT as adjuvant intraoperative therapy for tumor surgery requiring flap reconstruction after ablative surgery.

A model of the active site of dipeptidyl peptidase IV predicted by comparative molecular field analysis and molecular modelling simulations.

A molecular model of the active site of the serine protease dipeptidyl peptidase IV (DPP IV or CD26) has been developed on the basis of comparative molecular field analysis (CoMFA) of competitive inhibitors and by force field calculations. By application of CoMFA experimentally obtained inhibition constants Ki have been successfully predicted. The resulting steric and electrostatic coefficients of CoMFA were used for the development of the molecular model. The main assumptions of the model are the recognition of substrates or inhibitors by the side chains of a tyrosine (S1-position) and a tryptophan residue (S2-position). The model helps us to understand a multitude of experimental data regarding the substrate specificity of this enzyme as well as results obtained by genetic engineering experiments by other authors. General conclusions concerning a new family of serine proteases are drawn and discussed.