Biomedical subjects

P W Swaan

Publications and source records attributed to P W Swaan.

16 recordsLinked to original sources

PubMed2004

Molecular requirements of the human nucleoside transporters hCNT1, hCNT2, and hENT1.

Concentrative nucleoside transporters (CNTs) and equilibrative nucleoside transporters (ENTs) are important in physiological and pharmacological activity and disposition of nucleosides and nucleoside drugs. A better understanding of the structural requirements of inhibitors for these transporters will aid in designing therapeutic agents. To define the relative and unified structural requirements of nucleoside analogs for interaction with hCNT1, hCNT2, and hENT1, we applied an array of structure-activity techniques. Unique pharmacophore models for each respective nucleoside transporter were generated. These models reveal that hCNT2 affinity is dominated by hydrogen bonding features, whereas hCNT1 and hENT1 displayed mainly electrostatic and steric features. Hydrogen bond formation over 3'-OH is essential for all nucleoside transporters. Inhibition of nucleoside transporters by a series of uridine and adenosine analogs and a variety of drugs was analyzed by comparative molecular field analysis. Cross-validated r2 (q2) values were 0.65, 0.52, and 0.74 for hCNT1, hCNT2, and hENT1, respectively. The predictive quality of the models was further validated by successful prediction of the inhibition of a set of test compounds. Addition of a hydroxyl group around the 2-position of purine (or 3-position of pyrimidine) may increase inhibition to hCNT2 transporter; addition of hydroxyl group around the 2,7-position of purine (or the 3,5-position of pyrimidine) would increase the inhibition to hENT1 transporter. Utilization of these models should assist the design of high-affinity nucleoside transporter inhibitors and substrates for both anticancer and antiviral therapy.

Biological Transport↗

PubMed2001

Downregulation of topoisomerase I in differentiating human intestinal epithelial cells.

To better understand the increased sensitivity of proliferating intestinal epithelial cells to topoisomerase I (topo I) poisons, we examined differentiation of a human intestinal cell line (Caco-2) in the presence of camptothecin (CPT) and its analogs irinotecan (CPT-11) and topotecan (TPT). The prodrug CPT-11 exerts its antitumor activity after transformation to SN-38. We show that cleavable complex formation in vivo (on genomic DNA) induced by CPT or SN-38 is 4- to 7-fold reduced in fully differentiated cells relative to undifferentiated cells. TPT-induced cleavable complexes, however, are reduced by 30-fold. In contrast, CPT-11-driven cleavable complexes did not change during cell differentiation. In general, cytotoxicity closely paralleled cleavable complex formation, as attested to by the four- to 6-fold decrease in cytotoxicity in fully differentiated cells treated with CPT and SN-38 compared with proliferating cells. Topo I activity and polypeptide levels decreased 4-fold over the course of differentiation. This reduction occurs as Caco-2 cells approach G(1) and simultaneously differentiate. In contrast, human diploid fibroblasts do not show a reduction in topo I when entering G(1); therefore, topo I downregulation is a differentiation-specific event in the Caco-2 cell line. Cleavable complex formation and cytotoxicity induced by CPT and SN-38 correlate with topo I level and activity in cells at different stages in their differentiation. Thus, high target levels correspond closely with drug sensitivity and since proliferating cells contain larger amounts of topo I, we conclude that epithelial crypt cells probably succumb to chemotherapy involving topo I poisons.

Caco-2 Cells↗

PubMed2001

Sequential processing of human ProIL-1beta by caspase-1 and subsequent folding determined by a combined in vitro and in silico approach.

PURPOSE: Interleukin-1beta is a multifunctional cytokine produced by activated monocytes and macrophages that requires caspase-1 (IL-1 converting enzyme/ICE) to process the 31kDa inactive precursor protein to the biologically active 17kDa peptide. This activation event involves ICE cleavage at Asp27 (site 1) and Asp116 (site 2). To address the sequential processing at ICE cut sites we combined in vitro analysis and molecular modeling to investigate the sequence of molecular events. METHODS: Pulse chase labeling followed by immunoprecipitation of IL-1beta in activated human monocyte lysates demonstrated sequential cutting by ICE at site 1 before site 2 in vitro. To corroborate these findings, we constructed a homology model of proIL-1beta after the crystal structure of another ICE substrate, human alpha1-antitrypsin (23% sequence identity). RESULTS: Comparative modeling revealed that site 1 on proIL-1beta is accessible to ICE but site 2 is not. Molecular dynamics simulations following ICE cleavage at site 1 and removal of the 3kDa amino-terminal fragment, rendered site 2 accessible to ICE. CONCLUSIONS: The close agreement between the in vitro and modeled behavior of IL-1beta support our contention that IL-1beta may be structurally related to alpha1-antitrypsin and also predicts that proIL-1beta requires sequential processing for activation. These findings may facilitate the development of novel pharmacological agents that control posttranslational proIL-1beta modification, thereby preventing excessive production of this potent inflammatory cytokine.

Amino Acid Sequence↗

PubMed2001

Riboflavin uptake in human trophoblast-derived BeWo cell monolayers: cellular translocation and regulatory mechanisms.

Riboflavin (vitamin B2) is essential for fetal development and must be acquired from maternal sources. The uptake mechanism of riboflavin and the major regulatory pathways involved were characterized in a model for the placental barrier, the human choriocarcinoma cell line, BeWo. Uptake of [3H]riboflavin was saturable (Kt = 1.32 +/- 0.68 nM, Jmax = 266.63 +/- 26.89 fmol/mg of protein/20 min), and was significantly reduced at low temperature and in the presence of metabolic inhibitors (azide, 2-deoxyglucose) or structural analogs. Ouabain, amiloride, sodium-free buffers, and medium with pH values ranging from 3 to 8 did not affect uptake of [3H]riboflavin. In contrast, substitution of chloride with other monovalent anions significantly inhibited its uptake. Induced differentiation of BeWo cells into syncytiotrophoblasts by forskolin or 8-bromo-cyclic adenosine monophosphate introduced a time-dependent decrease of riboflavin uptake. Preincubation with activators of cyclic nucleotide-dependent protein kinase pathways (3-isobutyl-1-methylxanthine and p-chlorophenylthio-cyclic guanosine monophosphate) and calmodulin antagonists (calmidazolium and W-13) resulted in a concentration-dependent reduction of [3H]riboflavin uptake, whereas specific modulators of protein kinase C pathways did not have significant effects. 3-Isobutyl-1-methylxanthine exerted its regulatory effect on riboflavin uptake via decreasing both Kt and Jmax of the riboflavin uptake process (Kt = 6.32 +/- 1.29 nM, Jmax = 135.57 +/- 10.42 fmol/mg of protein/20 min). In summary, we report the presence of high- affinity riboflavin transporter(s) on the microvillous membrane of BeWo cells that appears to be modulated by cellular cyclic nucleotide levels and calmodulin.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Involvement of a receptor-mediated component in cellular translocation of riboflavin.

This study addresses the transport mechanism of riboflavin (vitamin B(2)) across intestinal epithelium in the presence and absence of pharmacologically active compounds. A polarized transport process with a 6-fold higher basolateral (BL)-to-apical (AP) flux was observed in both a human intestinal cell model (Caco-2) and rat intestinal tissue. Riboflavin-specific translocation systems on both the AP and BL cell surfaces were saturable with affinity values close to most receptors (K(m): 9.72 +/- 0.85 and 4.06 +/- 0.03 nM, respectively). Pharmacological agents known to alter intracellular endocytic events were used to examine the potential involvement of receptor-mediated events. Nocodazole significantly inhibited AP uptake (58.4%), BL-to-AP riboflavin (56.7%) and fluorescein isothiocyanate-labeled transferrin (FITC-Tf) (31.8%) transport without affecting mannitol or cholic acid transport, whereas AP-to-BL riboflavin (252.8%) and FITC-Tf (145.1%) transport was increased. Brefeldin A significantly enhanced AP-to-BL riboflavin (37.1%) and bidirectional FITC-Tf transport (AP-to-BL: 13-fold; BL-to-AP: 5-fold). without affecting BL-to-AP riboflavin transport. Combined, these data suggest an essential role of microtubule-dependent movement and vesicular sorting component(s) in the bidirectional transport of riboflavin. Dissociation of riboflavin from the cell surface was pH-dependent with significantly higher substrate release at acidic pH, indicating the presence of riboflavin-specific cell surface receptors. In summary, our studies provide biochemical evidence of the involvement of a receptor-mediated mechanism in the cellular translocation of riboflavin.

Biological Transport↗

PubMed1999

Determination of membrane protein glycation in diabetic tissue.

Diabetes-associated hyperglycemia causes glycation of proteins at reactive amino groups, which can adversely affect protein function. Although the effects of glycation on soluble proteins are well characterized, there is no information regarding membrane-associated proteins, mainly because of the lack of reproducible methods to determine protein glycation in vivo. The current study was conducted to establish such a method and to compare the glycation levels of membrane-associated proteins derived from normal and diabetic tissue. We present a detailed sample preparation protocol based on the borohydride-periodate assay, modified to allow manipulation of animal tissue. Assay noise associated with extraction protocols and nonproteinaceous buffer components was eliminated by the using 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS) as a membrane detergent, applying desalting columns, and including a protein precipitation step. The glycation level of membrane proteins from diabetic rats is elevated to 4.89 nmol/mg protein (standard deviation [SD] 0.48) compared with normoglycemic control tissue (2.23 nmol/mg protein, SD 0.64). This result is consistent with and correlated to the total glycated hemoglobin levels in diabetic and normoglycemic rats. Using <100 microg protein, the described methods allow further study of protein glycation effects on the function of individual transporter proteins and the role of these modifications in diabetes.

Animals↗

PubMed1998

Recent advances in intestinal macromolecular drug delivery via receptor-mediated transport pathways.

Receptor-mediated transport mechanisms provide a pathway for the trafficking of extracellular macromolecules into (endocytosis) and across (transcytosis) the cell. This comprises the binding of a ligand to a specific cell-surface receptor, clustering of the ligand-receptor complexes in endocytotic vesicles and vesicular sorting. This review focuses on recent advances in cellular and molecular biology pertaining to receptor-mediated endocytosis. A concise overview is presented of current and potential future applications of targeting to RME mechanisms to improve oral macromolecular drug delivery.

Administration, Oral↗

PubMed1998

Mapping the binding site of the small intestinal peptide carrier (PepT1) using comparative molecular field analysis.

The present study was undertaken to examine the relationship between chemical structure (steric and electrostatic fields) and affinity for the small intestinal oligopeptide carrier (PepT1) using comparative molecular field analysis (CoMFA), a three-dimensional approach towards building quantitative structure-activity relationships. Various biological activity parameters (Kt, Jmax, Pc) and molecular descriptors (CoMFA fields, isobutylalcohol/water distribution coefficients) were examined. The resulting field map provides information on the geometry of the binding site cavity and the relative weights of various properties in different site pockets for each of the substrates considered. The results indicate that carrier permeability (Pc), calculated as the ratio of the half-maximal concentration (Kt) and the maximal carrier flux (Jmax), is sensitive to composition, size and hydrophobicity of the ligands. The best model obtained showed a high correlation between the carrier permeability (Pc) and the steric (76.3% contribution) and electrostatic (23.7% contribution) molecular fields with a cross-validated r2 (q2) of 0.754. The model fitted the experimental data with a correlation coefficient of 0.993 and a standard error of 0.041, while the regression line between experimental and calculated Pc had a slope of 0.994 with an intercept of 0.009. These results lead to a better understanding of the molecular requirements for optimal drug-carrier interactions with the intestinal peptide transporter and offers a useful visual aid for designing new potentially interesting structures with affinity for the oligopeptide transporter PepT1.

Binding Sites↗

PubMed1997

Molecular determinants of recognition for the intestinal peptide carrier.

Computer-aided conformational analysis was used to characterize the pharmacophore for the intestinal peptide carrier. The active analog approach to pharmacophore building was applied as implemented in the SYBYL software package. Conformational analysis and MOPAC calculations were used to determine the lowest energy conformation of carrier substrates, as well as the conformations of compounds that displayed a common pharmacophoric geometry (i.e., inhibitors and inactive structural analogs). A pharmacophore map was calculated, and based on structural mutualities and functional topology, three substrate groups were suggested: compounds that bind to the transporter and are transferred across the membrane; compounds that show affinity for the peptide carrier (i.e., known to inhibit transport of substrates) but are not transferred across the membrane; and compounds that contain the pharmacophoric geometry but show no affinity for the carrier. Affinity for the peptide transporter can be diminished or abolished in either of three ways: esterification of the free carboxylic acid moiety; introduction of a second negative group; and intramolecular steric hindrance of the free carboxylic acid by either side chains with a positively charged nitrogen function or groups capable of hydrogen bond formation.

Administration, Oral↗

PubMed1997

Molecular modeling of the intestinal bile acid carrier: a comparative molecular field analysis study.

A structure-binding activity relationship for the intestinal bile acid transporter has been developed using data from a series of bile acid analogs in a comparative molecular field analysis (CoMFA). The studied compounds consisted of a series of bile acid-peptide conjugates, with modifications at the 24 position of the cholic acid sterol nucleus, and compounds with slight modifications at the 3, 7, and 12 positions. For the CoMFA study, these compounds were divided into a training set and a test set, comprising 25 and 5 molecules, respectively. The best three-dimensional quantitative structure-activity relationship model found rationalizes the steric and electrostatic factors which modulate affinity to the bile acid carrier with a cross-validated, conventional and predictive r2 of 0.63, 0.96, and 0.69, respectively, indicating a good predictive model for carrier affinity. Binding is facilitated by positioning an electronegative moiety at the 24-27 position, and also by steric bulk at the end of the side chain. The model suggests substitutions at positions 3, 7, 12, and 24 that could lead to new substrates with reasonable affinity for the carrier.

Adenosine Triphosphatases↗

PubMed1997

Use of the intestinal bile acid transporter for the uptake of cholic acid conjugates with HIV-1 protease inhibitory activity.

PURPOSE: To investigate the ability of the human intestinal bile acid transporter to transport cholic acid conjugates with potential HIV-1 protease inhibitory activity. METHODS: Cholic acid was conjugated at the 24 position of the sterol nucleus with various amino acids and amino acid analogs. The CaCo-2 cell line was used as a model to investigate the interaction of these bile acid conjugates with the human intestinal bile acid transporter. Interaction between the carrier and the conjugates was quantified by inhibition of taurocholic acid transport and confirmed by transport of radiolabelled conjugates in this cell line. RESULTS: The highest interaction with the transporter, as quantified by inhibition of taurocholic acid transport, occurred when a single negative charge was present around the 24 to 29 region of the sterol nucleus. A second negative charge or a positive charge significantly reduced the interaction. Transport of radiolabelled cholyl-L-Lys-epsilon-tBOC ester and cholyl-D-Asp-beta-benzyl ester was inhibited by taurocholic acid. Of all tested compounds, only cholyl-D-Asp-beta-benzyl ester showed modest HIV-1 protease inhibitory activity with an IC50 of 125 microM. CONCLUSIONS: Cholic acid-amino acid conjugates with appropriate stereochemistry are recognized and transported by the human bile acid transporter and show modest HIV-1 protease inhibitory activity. Transport of these conjugates by the bile acid carrier is influenced by charge and hydrophobicity around the 24 position of the sterol nucleus.

Amino Acids↗

PubMed1995

Molecular mechanism for the relative binding affinity to the intestinal peptide carrier. Comparison of three ACE-inhibitors: enalapril, enalaprilat, and lisinopril.

The affinity of three substrates for the intestinal peptide carrier is explained based on their three-dimensional (3D) structural data. The kinetic transport parameters of three ACE-inhibitors, enalapril, enalaprilat, and lisinopril, have been determined in an in vivo system using rat intestine. The observed kinetic transport parameters (+/- asymptotic standard error) of enalapril are: 0.81 (+/- 0.23) mM, 0.58 (+/- 0.37) mumol/h per cm2, and 0.56 (+/- 0.04) cm/h for the half-maximal transport concentration (KT), the maximal transport flux (Jmax) and the passive permeability constant (Pm). Enalaprilat was transported by passive diffusional with a Pm of 0.51 (+/- 0.04) cm/h. For lisinopril the kinetic transport parameters were 0.38 (+/- 0.19) mM, 0.12 (+/- 0.07) mumol/h per cm2, and 0.18 (+/- 0.02) cm/h for KT, Jmax, and Pm, respectively. The affinity of the ACE-inhibitors for the intestinal peptide carrier has been evaluated based on their ability to inhibit the transport rate of cephalexin. The inhibition constants (Ki) of enalapril, enalaprilat and lisinopril were 0.15, 0.28 and 0.39 mM, respectively. 3D structural analysis of lisinopril using molecular modelling techniques reveals that intramolecular hydrogen bond formation is responsible for decreased carrier affinity.

Angiotensin-Converting Enzyme Inhibitors↗

PubMed1995

Carrier-mediated transport mechanism of foscarnet (trisodium phosphonoformate hexahydrate) in rat intestinal tissue.

New findings are presented on the specific transport mechanisms of foscarnet (trisodium phosphonoformate hexahydrate) in rat small intestinal tissue and proof for the partial participation of the Na(+)-phosphate co-transport system in foscarnet transport. The transport of the free acid form of foscarnet, phosphonoformic acid (PFA), was studied in rat small intestine by applying Ussing chambers. Transport studies in both mucosal (m)-to-serosal (s) and s-to-m directions revealed polarization of PFA transport. In m-to-s studies, nonlinear concentration-dependent transport was observed and described by the following transport parameters (estimate +/- asymptotic standard error): 0.84 +/- 0.13 mumol/h.cm2, 1.13 +/- 0.29 mM and 0.22 +/- 0.05 cm/h for the maximal transport rate (Jmax), the half-maximal transport concentration (Kt) and the passive membrane permeability constant (Pm), respectively. PFA transport (1.0 mM) was reduced to 72% and to 56% in the presence of the structural analogs phosphate and arsenate (10 mM), respectively. Bidirectional transport studies of PFA at 38 degrees C and 4 degrees C revealed a higher decrease in transport rate for the m-to-s studies than for the s-to-m studies. The combined results of the experiments described in this study demonstrate that PFA transport across rat small intestine is partly passive, using both the paracellular and transcellular pathways, and partly carrier-mediated, involving the phosphate co-transport system.

Animals↗

PubMed1994

Determination of transport rates for arginine and acetaminophen in rabbit intestinal tissues in vitro.

The in vitro Ussing technique was employed to examine transport rates for acetaminophen and arginine across rabbit intestinal tissues. Mannitol and transepithelial conductance were used to monitor the integrity of rabbit intestinal tissues and the basal and stimulated short-circuit current were measured to assess functional viability. Transepithelial transport of acetaminophen, arginine, and mannitol was determined in rabbit jejunum, ileum, and distal colon. Transepithelial transport of arginine in the ileum and jejunum was composed of both passive (nonsaturable) (Pm = 0.06) and saturable components (KT = 0.6-0.7 mM; Jmax = 0.3-0.4 mumol/hr.cm2). The saturable component of arginine fluxes was abolished by pretreatment of the tissue with serosal ouabain (0.1 mM). In the distal colon, both unidirectional arginine fluxes were nonsaturable. In the segments examined, both unidirectional fluxes of acetaminophen were nonsaturable over the concentration range from 0.1 to 30 mM. These results provide values for maximal permeabilities attained by molecules traversing both the cellular and the paracellular pathways and, by comparison to their in vivo bioavailabilities, provide selection criteria for evaluating drug candidates for oral activity.

Acetaminophen↗

PubMedDate not supplied

Progress in predicting human ADME parameters in silico.

Understanding the development of a scientific approach is a valuable exercise in gauging the potential directions the process could take in the future. The relatively short history of applying computational methods to absorption, distribution, metabolism and excretion (ADME) can be split into defined periods. The first began in the 1960s and continued through the 1970s with the work of Corwin Hansch et al. Their models utilized small sets of in vivo ADME data. The second era from the 1980s through 1990s witnessed the widespread incorporation of in vitro approaches as surrogates of in vivo ADME studies. These approaches fostered the initiation and increase in interpretable computational ADME models available in the literature. The third era is the present were there are many literature data sets derived from in vitro data for absorption, drug-drug interactions (DDI), drug transporters and efflux pumps [P-glycoprotein (P-gp), MRP], intrinsic clearance and brain penetration, which can theoretically be used to predict the situation in vivo in humans. Combinatorial synthesis, high throughput screening and computational approaches have emerged as a result of continual pressure on pharmaceutical companies to accelerate drug discovery while decreasing drug development costs. The goal has become to reduce the drop-out rate of drug candidates in the latter, most expensive stages of drug development. This is accomplished by increasing the failure rate of candidate compounds in the preclinical stages and increasing the speed of nomination of likely clinical candidates. The industry now understands the reasons for clinical failure other than efficacy are mainly related to pharmacokinetics and toxicity. The late 1990s saw significant company investment in ADME and drug safety departments to assess properties such as metabolic stability, cytochrome P-450 inhibition, absorption and genotoxicity earlier in the drug discovery paradigm. The next logical step in this process is the evaluation of higher throughput data to determine if computational (in silico) models can be constructed and validated from it. Such models would allow an exponential increase in the number of compounds screened virtually for ADME parameters. A number of researchers have started to utilize in silico, in vitro and in vivo approaches in parallel to address intestinal permeability and cytochrome P-450-mediated DDI. This review will assess how computational approaches for ADME parameters have evolved and how they are likely to progress.

Catalysis↗

PubMedDate not supplied

Enhanced transepithelial transport of peptides by conjugation to cholic acid.

The potential of the intestinal bile acid transporter to serve as a shuttle for small peptide molecules was investigated. Eleven peptides with a 2-6 amino acid backbone were conjugated to the 24-position of 3 alpha, 7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oic acid (cholic acid) via an amide bond using an automated peptide synthesizer. In a human intestinal cell line (CaCo-2), cholic acid-peptide conjugates were able to inhibit the transepithelial transport of [3H]taurocholic acid, a natural substrate for the bile acid carrier, at a 100:1 conjugate/substrate ratio. Affinity for the carrier decreased significantly when the conjugate in the 24-position increased from 1 to 2 amino acids. Further increase in the amino acid chain length caused only minor decrease in affinity. A tetrapeptide-bile acid conjugate, [3H]-ChEAAA (Ch = cholic acid), was transported by the bile acid transporter, showing markedly higher apical (AP)-to-basolateral (BL) compared to BL-to-AP transport and inhibition by a 100-fold excess taurocholic acid. Another conjugate with 6 amino acids (ChEASASA) was transported by a passive diffusion pathway but still showed higher transport rates than the passive permeability marker mannitol, suggesting the possibility that the cholic acid moiety aids the passive membrane transfer of peptide molecules by increasing its lipophilicity. Metabolism of bile acid-peptide conjugates in CaCo-2 cells was 3% over 3 h. In conclusion, these studies show that the coupling of peptides to the 24-position of the sterol nucleus in cholic acid results in a combination of decreased metabolism and increased intestinal absorption, either by a carrier-mediated pathway or by accelerated passive diffusion.

Biological Transport↗