ABSTRACT
We optimized the structure of an active metabolite (1) of WAY-123783, which was obtained from mouse urine after oral administration, to improve selectivity for SGLT2 and oral bioavailability. O-glucoside derivative 24 (remogliflozin etabonate) was subsequently identified as a potent, highly selective, and orally available SGLT2 inhibitor.
Subject(s)
Glucosides/chemistry , Glucosides/pharmacology , Pyrazoles/chemistry , Pyrazoles/pharmacology , Sodium-Glucose Transporter 2 Inhibitors/chemistry , Sodium-Glucose Transporter 2 Inhibitors/pharmacology , Administration, Oral , Animals , Biological Availability , COS Cells , Chlorocebus aethiops , Drug Discovery , Glycosuria , Mice , Molecular Structure , Pyrazoles/metabolism , Sodium-Glucose Transporter 2/genetics , Sodium-Glucose Transporter 2/metabolism , Sodium-Glucose Transporter 2 Inhibitors/metabolismABSTRACT
To test the hypothesis that inhibitors of human concentrative nucleoside transporter 2 (hCNT2) suppress increases in serum urate levels derived from dietary purines, we previously identified adenosine derivative 1 as a potent hCNT2 inhibitor (IC50 = 0.64 µM), but further study was hampered due to its poor solubility. Here we describe the results of subsequent research to identify more soluble and more potent hCNT2 inhibitors, leading to the discovery of the benzimidazole nucleoside 22, which is the most potent hCNT2 inhibitor (IC50 = 0.062 µM) reported to date. Compound 22 significantly suppressed the increase in plasma uric acid levels after oral administration of purine nucleosides in rats. Because compound 22 was poorly absorbed orally in rats (F = 0.51%), its pharmacologic action was mostly limited to the gastrointestinal tract. These findings suggest that inhibition of hCNT2 in the gastrointestinal tract can be a promising approach for the treatment of hyperuricemia.
Subject(s)
Adenine/chemistry , Benzimidazoles/chemistry , Gout/drug therapy , Hyperuricemia/drug therapy , Membrane Transport Proteins/drug effects , Nucleosides/pharmacology , Animals , Humans , Male , Nucleosides/chemistry , Nucleosides/pharmacokinetics , Nucleosides/therapeutic use , Rats , Rats, Sprague-DawleyABSTRACT
Although 6-hydroxydopamine-induced (6-OHDA-induced) rats are a well-known Parkinson's disease model, the effects of dopamine D2 agonists in mice with 6-OHDA-induced lesions are not completely understood. We produced mice with 6-OHDA-induced lesions and measured their total locomotion counts following administration of several dopamine D2 agonists (pramipexole, ropinirole, cabergoline, rotigotine, apomorphine, talipexole, and quinelorane). Cabergoline showed the longest duration of drug action, which was in agreement with its long-lived anti-Parkinson effects in rats and humans. In contrast, pramipexole and ropinirole had notably short durations of drug action. We demonstrated that mice with 6-OHDA-induced lesions accompanied with significant lesions in the striatum may be reasonable models to predict the action duration of anti-Parkinson drug candidates in humans.
Subject(s)
Antiparkinson Agents/pharmacokinetics , Corpus Striatum/pathology , Dopamine Agonists/pharmacokinetics , Motor Activity/drug effects , Parkinsonian Disorders/chemically induced , Receptors, Dopamine D2/agonists , Animals , Apomorphine/pharmacokinetics , Azepines/pharmacokinetics , Benzothiazoles/pharmacokinetics , Cabergoline , Corpus Striatum/drug effects , Disease Models, Animal , Ergolines/pharmacokinetics , Indoles/pharmacokinetics , Injections, Intraventricular , Male , Mice , Oxidopamine/pharmacology , Parkinsonian Disorders/pathology , Parkinsonian Disorders/psychology , Pramipexole , Quinolines/pharmacokinetics , Tetrahydronaphthalenes/pharmacokinetics , Thiophenes/pharmacokineticsABSTRACT
Purine-rich foods have long been suspected as a major cause of hyperuricemia. We hypothesized that inhibition of human concentrative nucleoside transporter 2 (hCNT2) would suppress increases in serum urate levels derived from dietary purines. To test this hypothesis, the development of potent hCNT2 inhibitors was required. By modifying adenosine, an hCNT2 substrate, we successfully identified 8-aminoadenosine derivatives as a new class of hCNT2 inhibitors. Compound 12 moderately inhibited hCNT2 (IC50 = 52 ± 3.8 µM), and subsequent structure-activity relationship studies led to the discovery of compound 48 (IC50 = 0.64 ± 0.19 µM). Here we describe significant findings about structural requirements of 8-aminoadenosine derivatives for exhibiting potent hCNT2 inhibitory activity.
ABSTRACT
BACKGROUND AND AIM OF THE STUDY: The symptoms of Parkinson's disease are alleviated by dopamine D2 agonists, which are classified as ergot dopamine D2 agonists and non-ergot D2 agonists. Among the former, pergolide has been associated with valvular heart disease, since it has both potent D2 receptor and serotonin 5-HT(2B) receptor agonistic properties. Among the latter, pramipexole has few incidences of heart valve disease onset, since it has an absence of 5-HT(2B) receptor agonism. METHOD: A [3H]thymidine incorporation assay was performed to monitor function, and microarray global analysis to monitor gene expression, on porcine heart valve interstitial cells (VICs) treated with pergolide or pramipexole. RESULTS: The 5-HT(2B) receptor was abundantly expressed in porcine VICs. The 5-HT(2B) receptor agonist pergolide induced an increase in [3H]thymidine incorporation, accompanied by a decrease in 5-HT(2B) receptor mRNA expression. [3H]thymidine incorporation was blocked by lisuride, a 5-HT(2B) receptor antagonist, and also by LY-294002, a specific inhibitor of PI3K and Akt. Moreover, type 2 iodothyronine deiodinase (Dio2) expression in porcine VICs treated with pergolide was shown, by a global analysis of mRNA, to be markedly increased compared to that induced by pramipexole. Such changes in VICs may correlate with the mechanism of heart valve disease pathogenesis. CONCLUSION: There were substantial differences (increased [3H]thymidine incorporation, and Dio2 expression) between pergolide and pramipexole, which might correlate with the mechanism of heart valve disease onset.
Subject(s)
Benzothiazoles/toxicity , Dopamine Agonists/toxicity , Mitral Valve/drug effects , Pergolide/toxicity , Receptors, Dopamine D2/agonists , Animals , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Gene Expression Profiling/methods , Gene Expression Regulation/drug effects , Mitral Valve/metabolism , Mitral Valve/pathology , Oligonucleotide Array Sequence Analysis , Pramipexole , Protein Kinase Inhibitors/pharmacology , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction , Receptor, Serotonin, 5-HT2B/drug effects , Receptor, Serotonin, 5-HT2B/genetics , Receptor, Serotonin, 5-HT2B/metabolism , Receptors, Dopamine D2/metabolism , Reproducibility of Results , Serotonin 5-HT2 Receptor Agonists/toxicity , Serotonin 5-HT2 Receptor Antagonists/pharmacology , Signal Transduction/drug effectsABSTRACT
We have developed concentrative nucleoside transporter 2 (CNT2) inhibitors as a novel pharmacological approach for improving hyperuricemia by inhibiting intestinal absorption of purines. Dietary purine nucleosides are absorbed in the small intestines by CNTs expressed in the apical membrane. In humans, the absorbed purine nucleosides are rapidly degraded to their final end product, uric acid, by xanthine oxidase. Based on the expression profile of human CNTs in digestive tract tissues, we established a working hypothesis that mainly CNT2 contributes to the intestinal absorption of purine nucleosides. In order to confirm this possibility, we developed CNT2 inhibitors and found that (2R,3R,4S,5R)-2-(6-amino-8-{[3'-(3-aminopropoxy)-biphenyl-4-ylmethyl]-amino}-9H-purin-9-yl)-5-hydroxymethyl-tetrahydrofuran-3,4-diol (KGO-2142) and 1-[3-(5-{[1-((2R,3R,4S,5R)-3,4-dihydroxy-5-hydroxymethyl-tetrahydrofuran-2-yl)-1H-benzimidazol-2-ylamino]-methyl}-2-ethoxyphenoxy)-propyl]-piperidine-4-carboxylic acid amide (KGO-2173) were inhibitory. These CNT2 inhibitors had potent inhibitory activity against inosine uptake via human CNT2, but they did not potently interfere with nucleoside uptake via human CNT1, CNT3 or equilibrative nucleoside transporters (ENTs) in vitro. After oral administration of KGO-2173 along with [(14)C]-inosine, KGO-2173 significantly decreased the urinary excretion of radioactivity at 6 and 24h in rats. Since dietary purine nucleosides are not utilized in the body and are excreted into the urine rapidly, this decrease in radioactivity in the urine represented the inhibitory activity of KGO-2173 toward the absorption of [(14)C]-inosine in the small intestines. KGO-2142 almost completely inhibited dietary RNA-induced hyperuricemia and the increase in urinary excretion of uric acid in cebus monkeys. These novel CNT2 inhibitors, KGO-2142 and KGO-2173, could be useful therapeutic options for the treatment of hyperuricemia.
Subject(s)
Furans/pharmacology , Intestinal Absorption/drug effects , Membrane Transport Proteins/metabolism , Purine Nucleosides/metabolism , Renal Tubular Transport, Inborn Errors/drug therapy , Renal Tubular Transport, Inborn Errors/metabolism , Urinary Calculi/drug therapy , Urinary Calculi/metabolism , Animals , Biological Transport/drug effects , COS Cells , Cebus , Chlorocebus aethiops , Dose-Response Relationship, Drug , Furans/chemistry , Furans/therapeutic use , Gene Expression Regulation/drug effects , HeLa Cells , Humans , Inosine/metabolism , Male , RNA, Fungal/administration & dosage , RNA, Fungal/pharmacology , Rats , Rats, Sprague-Dawley , Renal Tubular Transport, Inborn Errors/blood , Uric Acid/blood , Urinary Calculi/bloodABSTRACT
In the canonical Notch signaling pathway, intramembrane cleavage by gamma-secretase serves to release an intracellular domain of Notch that has activity in the nucleus through binding to transcription factors. In addition, we showed that Notch also supplies signals to Delta, a major Notch ligand, to release the intracellular domain of Delta by gamma-secretase from the cell membrane, which then translocates to the nucleus, where it mediates the transcription of specific genes. Therefore, the Notch-Delta signaling pathway is bi-directional and similar mechanisms regulated by gamma-secretase are involved in both directions. Recently, it was demonstrated that many type 1 transmembrane proteins including Notch, Delta and amyloid precursor protein (APP) are substrates for gamma-secretase and release intracellular domains of these proteins from cell membranes. These observations that the common enzyme, gamma-secretase, modulates proteolysis and the turnover of possible signaling molecules have led to the attractive hypothesis that mechanisms similar to the Notch-Delta signaling pathway may widely contribute to gamma-secretase-regulated signaling pathways, including APP signaling which leads to Alzheimer's disease. Here, we review the molecular mechanisms of the Notch-Delta signaling pathway in a bi-directional manner, and discuss the recent progress in understanding the biology of gamma-secretase-regulated signaling with respect to neurodegeneration.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Cell Membrane/metabolism , Membrane Proteins/metabolism , Receptors, Notch/metabolism , Amino Acid Sequence , Animals , Humans , Intracellular Signaling Peptides and Proteins , Molecular Sequence Data , Sequence Alignment , Signal TransductionABSTRACT
Although amyloid precursor protein (APP) has central roles in Alzheimer's disease, the physiological functions of this protein have yet to be fully elucidated. APP homologues show significant sequence conservation in the intracellular domain through evolution, which may reflect the functional importance of the intracellular domain of APP (AICD). To examine this possibility, we established embryonic carcinoma P19 cell lines overexpressing AICD. Although neurons could be differentiated from these cell lines with retinoic acid treatment, overexpression of AICD gave rise to neuron-specific cell death. Furthermore, DNA fragmentation was detected and TUNEL-positive cells were also Tuj1-positive neurons. Taken together, we concluded that AICD can induce neuron-specific apoptosis.
Subject(s)
Amyloid beta-Protein Precursor/physiology , Apoptosis , Neurons/physiology , Amino Acid Sequence , Amyloid beta-Protein Precursor/genetics , Animals , Cell Differentiation , Cell Line, Tumor , Humans , In Situ Nick-End Labeling , Intracellular Space/metabolism , Mice , Molecular Sequence Data , Transfection , Tretinoin/pharmacologyABSTRACT
Delta is a major transmembrane ligand for Notch receptor that mediates numerous cell fate decisions. The Notch signaling pathway has long been thought to be mono-directional, because ligands for Notch were generally believed to be unable to transmit signals into the cells expressing them. However, we showed here that Notch also supplies signals to neighboring mouse neural stem cells (NSCs). To investigate the Notch-Delta signaling pathway in a bi-directional manner, we analyzed functional roles of the intracellular domain of mouse Delta like protein 1 (Dll1IC). In developing mouse NSCs, Dll1IC, which is released from cell membrane by proteolysis, is present in the nucleus. Furthermore, we screened for transcription factors that bind to Dll1IC and demonstrated that Dll1IC binds specifically to transcription factors involved in TGF-beta/Activin signaling--Smad2, Smad3 and Smad4--and enhances Smad-dependent transcription. In addition, the results of the present study indicated that over-expression of Dll1IC in embryonic carcinoma P19 cells induced neurons, and this induction was blocked by SB431542, which is a specific inhibitor of TGF-beta/Activin signaling. These observations strongly suggested that Dll1IC mediates TGF-beta/Activin signaling through binding to Smads and plays an important role for bi-directional Notch-Delta signaling pathway.
Subject(s)
Activins/physiology , Intercellular Signaling Peptides and Proteins/metabolism , Receptor, Notch1/metabolism , Smad Proteins/metabolism , Transforming Growth Factor beta/physiology , Amino Acid Sequence , Animals , Binding Sites , Calcium-Binding Proteins , Cell Differentiation , Cell Line, Tumor , Cell Nucleus/chemistry , Cells, Cultured , Evolution, Molecular , Intercellular Signaling Peptides and Proteins/analysis , Intercellular Signaling Peptides and Proteins/chemistry , Mice , Molecular Sequence Data , Neurons/cytology , Neurons/metabolism , Protein Structure, Tertiary , Sequence Homology, Amino Acid , Signal Transduction , Stem Cells/cytology , Stem Cells/metabolism , Transcription, GeneticABSTRACT
The low-affinity sodium glucose cotransporter (SGLT2), which is expressed specifically in the kidney, plays a major role in renal glucose reabsorption in the proximal tubule. We have discovered sergliflozin, a prodrug of a novel selective SGLT2 inhibitor, based on benzylphenol glucoside. In structure, it belongs to a new category of SGLT2 inhibitors and its skeleton differs from that of phlorizin, a nonselective SGLT inhibitor. We investigated its pharmacological properties and potencies in vitro and in vivo. By examining a Chinese hamster ovary-K1 cell line stably expressing either human SGLT2 or human high-affinity sodium glucose cotransporter (SGLT1), we found sergliflozin-A (active form) to be a highly selective and potent inhibitor of human SGLT2. At pharmacological doses, sergliflozin, sergliflozin-A, and its aglycon had no effects on facilitative glucose transporter 1 activity, which was inhibited by phloretin (the aglycon of phlorizin). The transport maximum for glucose in the kidney was reduced by sergliflozin-A in normal rats. As a result of this effect, orally administered sergliflozin increased urinary glucose excretion in mice, rats, and dogs in a dose-dependent manner. In an oral glucose tolerance test in diabetic rats, sergliflozin exhibited glucose-lowering effects independently of insulin secretion. Any glucose excretion induced by sergliflozin did not affect normoglycemia or electrolyte balance. These data indicate that selective inhibition of SGLT2 increases urinary glucose excretion by inhibiting renal glucose reabsorption. As a representative of a new category of antidiabetic drugs, sergliflozin may provide a new and unique approach to the treatment of diabetes mellitus.
Subject(s)
Blood Glucose/analysis , Glucose/metabolism , Glucosides/pharmacology , Hypoglycemic Agents/pharmacology , Kidney/metabolism , Sodium-Glucose Transporter 2 Inhibitors , Absorption , Animals , Dogs , Electrolytes/urine , Erythrocytes/metabolism , Female , Glucose Tolerance Test , Glycosuria/chemically induced , Male , Mice , Mice, Inbred C57BL , Rats , Rats, Sprague-Dawley , Sodium-Glucose Transporter 2/physiologyABSTRACT
We isolated a cDNA clone of SLC5A9/SGLT4 from human small intestinal full-length cDNA libraries, and functionally characterized it in vitro. The messenger RNA encoding SGLT4 was mainly expressed in the small intestine and kidney, among the human tissues tested. COS-7 cells transiently expressing SGLT4 exhibited Na(+)-dependent alpha-methyl-D-glucopyranoside (AMG) transport activity with an apparent K(m) of 2.6 mM, suggesting that SGLT4 is a low affinity-type transporter. The rank order of naturally occurring sugar analogs for the inhibition of AMG transport was: D-mannose (Man) >> D-glucose (Glc) > D-fructose (Fru) = 1,5-anhydro-D-glucitol (1,5AG) > D-galactose (Gal). Recognition of Man as a substrate was confirmed by direct uptake of Man into the cell. COS-7 cells expressing a putative murine SGLT4 ortholog showed similar Na(+)-dependent AMG transport activity and a similar deduced substrate specificity. These results suggest that SGLT4 would have unique physiological functions (i.e., absorption and/or reabsorption of Man, 1,5AG, and Fru, in addition to Glc).
Subject(s)
Deoxyglucose/metabolism , Fructose/metabolism , Mannose/metabolism , Monosaccharide Transport Proteins/physiology , Amino Acid Sequence , Animals , COS Cells , Humans , Molecular Sequence Data , Monosaccharide Transport Proteins/genetics , RNA, Messenger/analysis , Sodium-Glucose Transporter 2ABSTRACT
We cloned the 5'-flanking region of the mouse homolog of the Delta gene (Dll1) and demonstrated that the sequence between nucleotide position -514 and -484 in the 5'-flanking region of Dll1 played a critical role in the regulation of its tissue-specific expression in neural stem cells (NSCs). Further, we showed that multiple POU-binding motifs, located within this short sequence of 30bp, were essential for transcriptional activation of Dll1 and also that multiple tissue-specific nuclear factors recognized these POU-binding motifs in various combinations through differentiation of NSCs. Thus, POU-binding factors may play an important role in Dll1 expression in developing NSCs.