ABSTRACT
Matrix metalloproteinase-7 (MMP-7) has been shown to play an important role in pathophysiological processes such as cancer and fibrosis. We previously discovered selective MMP-7 inhibitors by molecular hybridization and structure-based drug design. However, the systemic clearance (CLtot) of the biologically active lead compound was very high. Because our studies revealed that hepatic uptake by organic anion transporting polypeptide (OATP) was responsible for the high CLtot, we found a novel approach to reducing their uptake based on isoelectric point (IP) values as an indicator for substrate recognition by OATP1B1/1B3. Our "IP shift strategy" to adjust the IP values culminated in the discovery of TP0628103 (18), which is characterized by reduced in vitro OATP-mediated hepatic uptake and in vivo CLtot. Our in vitro-in vivo extrapolation of OATP-mediated clearance and the "IP shift strategy" provide crucial insights for a new medicinal chemistry approach to reducing the systemic clearance of OATP1B1/1B3 substrates.
Subject(s)
Matrix Metalloproteinase 7 , Organic Anion Transporters , Liver-Specific Organic Anion Transporter 1 , Isoelectric Point , Liver , Drug Interactions , HepatocytesABSTRACT
Matrix metalloproteinase-7 (MMP-7) has been shown to play important roles in pathophysiological processes involved in the development/progression of diseases such as cancer and fibrosis. We discovered selective MMP-7 inhibitors composed of arylsulfonamide, carboxylate, and short peptides by a molecular hybridization approach. These compounds interacted with MMP-7 via multiple hydrogen bonds in the cocrystal structures. To obtain compounds for in vivo evaluation, we attempted structural optimization, particularly targeting Tyr167 at the S3 subsite through structure-based drug design, and identified compound 15 as showing improved MMP-7 potency and MMP subtype selectivity. A novel π-π stacking interaction with Tyr167 was achieved when 4-pyridylalanine was introduced as the P3 residue. Compound 15 suppressed the progression of kidney fibrosis in a dose-dependent manner in a mouse model of unilateral ureteral obstruction. Thus, we demonstrated, for the first time, that potent and selective MMP-7 inhibitors could prevent the progression of kidney fibrosis.
Subject(s)
Matrix Metalloproteinase 7 , Matrix Metalloproteinase Inhibitors , Mice , Animals , Matrix Metalloproteinase Inhibitors/pharmacology , Matrix Metalloproteinase Inhibitors/therapeutic use , Drug Design , Fibrosis , KidneyABSTRACT
Hyperbilirubinemia (HB) is sometimes encountered following bile-duct cannulation in rats. It possibly originates from the reduced functioning of multidrug resistance-associated protein 2 (Mrp2) and subsequent adaptive alterations in the expression of Mrp3 and the organic anion transporting polypeptides (Oatps). Our aim was to clarify the importance of excluding bile-duct-cannulated (BDC) rats with HB for proper conduct of drug excretion studies. We detected HB [serum total bilirubin concentration (TBIL) ≥0.20 mg/dl] in 16% of all BDC rats prepared. The serum activities of aspartate aminotransferase, alanine aminotransferase, leucine aminopeptidase, and alkaline phosphatase were within the respective normal ranges in the BDC rats with mild HB (TBIL, 0.20-0.79 mg/dl), indicating the absence of hepatic failure. In the pharmacokinetics of pravastatin, an Oatps/Mrp2 probe drug in the BDC rats, the apparent volume of distribution and the clearance were smaller in the mild HB group as compared with the normal group, suggesting the reduction of apparent hepatic uptake and hepatobiliary elimination. The biliary excretion (percentage of dose) was significantly reduced by 54%, suggesting that the biliary efflux activity via Mrp2 was reduced to a greater extent relative to metabolic activity in hepatocytes. The serum γ-glutamyltransferase (GGT) activity correlated with TBIL and inversely correlated with biliary excretion of pravastatin, a finding which could serve as a clue to uncover the regulatory system involving cooperation between GGT and Mrp2. In conclusion, BDC rats with HB, however mild, should be excluded from drug excretion studies to avoid the risk of underestimation of the biliary excretion of drugs.
Subject(s)
Bile Ducts/metabolism , Bile/metabolism , Catheterization/adverse effects , Hepatobiliary Elimination , Hyperbilirubinemia/etiology , Pravastatin/pharmacokinetics , ATP-Binding Cassette Transporters/metabolism , Animals , Bilirubin/blood , Biomarkers/blood , Hyperbilirubinemia/blood , Hyperbilirubinemia/metabolism , Liver/metabolism , Male , Models, Biological , Organic Anion Transporters/metabolism , Pravastatin/administration & dosage , Rats , Rats, Sprague-Dawley , Reproducibility of ResultsABSTRACT
Organic cation transporters (OCT) encoded by members of the solute carrier (SLC) 22 family of genes are involved in the disposition of physiological substrates and xenobiotics, including drugs used in the treatment of chronic obstructive lung diseases and asthma. The aim of this work was to identify continuously growing epithelial cell lines that closely mimic the organic cation transport of freshly isolated human alveolar type I-like epithelial cells (ATI) in primary culture, and which consequently, can be utilised as in vitro models for the study of organic cation transport at the air-blood barrier. OCT activity was investigated by measuring [(14)C]-tetraethylammonium (TEA) uptake into monolayers of Calu-3, NCI-H441 and A549 lung epithelial cell lines in comparison to ATI-like cell monolayers in primary culture. Levels of time-dependent TEA uptake were highest in A549 and ATI-like cells. In A549 cells, TEA uptake had a saturable and a non-saturable component with Km=528.5±373.1µM, Vmax=0.3±0.1nmol/min/mg protein and Kd=0.02µl/min/mg protein. TEA uptake into Calu-3 and NCI-H441 cells did not reach saturation within the concentration range studied. RNAi experiments in A549 cells confirmed that TEA uptake was mainly facilitated by OCT1 and OCT2. Co-incubation studies using pharmacological OCT modulators suggested that organic cation uptake pathways share several similarities between ATI-like primary cells and the NCI-H441 cell line, whereas more pronounced differences exist between primary cells and the A549 and Calu-3 cell lines.
Subject(s)
Epithelial Cells/metabolism , Organic Cation Transport Proteins/metabolism , Pulmonary Alveoli/cytology , Cell Line , Humans , In Vitro Techniques , Models, Biological , Organic Cation Transport Proteins/physiology , Organic Cation Transporter 1/metabolism , Organic Cation Transporter 1/physiology , Organic Cation Transporter 2 , Pulmonary Alveoli/metabolism , Tetraethylammonium/metabolismABSTRACT
Taurine transporter (TauT/SLC6A6) is an "honorary" γ-aminobutyric acid (GABA) transporter because of its low affinity for GABA. The sequence analysis of TauT implied the role of Gly57, Phe58, Leu306 and Glu406 in the substrate recognition of TauT, and amino acid-substitutions were performed. Immunocytochemistry supported no marked effect of mutations on the expression of TauT. TauT-expressing oocytes showed a reduction in [(3)H]taurine uptake by G57E, F58I, L306Q and E406C, and change in [(3)H]GABA uptake by G57E and E406C, suggesting their significant roles in the function of TauT. G57E lost the activity of [(3)H]taurine and [(3)H]GABA uptake, suggesting that Gly57 is involved in the determination of substrate pocket volume and in the interaction with substrates. E406C exhibited a decrease and an increase in the affinity for taurine and GABA, respectively, suggesting the involvement of Glu406 in the substrate specificity of TauT. The inhibition study supported the role of Glu406 in the substrate specificity since [(3)H]taurine and [(3)H]GABA uptake by E406C was less sensitive to taurine and ß-alanine, and more sensitive to GABA and nipecotic acid than was the case with wild type of TauT. F58I had an increased affinity for GABA, suggesting the involvement of Phe58 in the substrate accessibility. The kinetic parameters showed the decreased and increased affinities of L306Q for taurine and GABA, respectively, supporting that substrate recognition of TauT is conformationally regulated by the branched-side chain of Leu306. In conclusion, the present results suggest that these residues play important roles in the transport function and substrate specificity of TauT.
Subject(s)
Amino Acids/metabolism , Biological Transport/genetics , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Membrane Transport Proteins/chemistry , Membrane Transport Proteins/metabolism , Taurine/metabolism , gamma-Aminobutyric Acid/metabolism , Animals , Membrane Glycoproteins/genetics , Membrane Transport Proteins/genetics , Mutagenesis, Site-Directed , Nipecotic Acids/metabolism , Oocytes/metabolism , Substrate Specificity/genetics , Tritium , Xenopus laevis , beta-Alanine/metabolismABSTRACT
We have reported previously that taurine transporter (TauT) mediates γ-aminobutyric acid (GABA) as a substrate in a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2 cells). This study investigates how TauT-mediated GABA transport is regulated in TR-iBRB2 cells under hypertonic conditions. [³H]GABA uptake by TR-iBRB2 cells exposed to 12 h- to 24 h-hypertonic culture medium was significantly greater than that of isotonic culture medium. [³H]GABA uptake by TR-iBRB2 cells was Na(+)-, Cl(-)-, and concentration-dependent with a Michaelis-Menten (K(m)) constant of 3.5 mM under isotonic conditions and K(m) of 0.324 and 5.48 mM under hypertonic conditions. Under hypertonic conditions, [³H]GABA uptake by TR-iBRB2 cells was more potently inhibited by substrates of TauT, such as taurine and ß-alanine, than those of GABA transporters such as GABA, nipecotic acid, and betaine. These results suggest that an unknown high-affinity GABA transport process and TauT-mediated GABA transport are enhanced under hypertonic conditions. In conclusion, hypertonicity enhances GABA uptake by cultured rat retinal capillary endothelial cells.