Izumi Nakagome

α-1-C-butyl-1,4-dideoxy-1,4-imino-l-arabinitol as a second-generation iminosugar-based oral α-glucosidase inhibitor for improving postprandial hyperglycemia.

We report on the synthesis and the biological evaluation of a series of α-1-C-alkylated 1,4-dideoxy-1,4-imino-l-arabinitol (LAB) derivatives. The asymmetric synthesis of the derivatives was achieved by asymmetric allylic alkylation, ring-closing metathesis, and Negishi cross-coupling as key reactions. α-1-C-Butyl-LAB is a potent inhibitor of intestinal maltase, isomaltase, and sucrase, with IC50 values of 0.13, 4.7, and 0.032 μM, respectively. Matrix-assisted laser desorption ionization time-of-flight mass spectrometric analysis revealed that this compound differs from miglitol in that it does not influence oligosaccharide processing and the maturation of glycoproteins. A molecular docking study of maltase-glucoamylase suggested that the interaction modes and the orientations of α-1-C-butyl-LAB and miglitol are clearly different. Furthermore, α-1-C-butyl-LAB strongly suppressed postprandial hyperglycemia at an early phase, similar to miglitol in vivo. It is noteworthy that the effective dose was about 10-fold lower than that for miglitol. α-1-C-Butyl-LAB therefore represents a new class of promising compounds that can improve postprandial hyperglycemia.

Estimation of the Three-Dimensional Pharmacophore of Ligands for Rat Multidrug-Resistance–Associated Protein 2 Using Ligand-Based Drug Design Techniques

No HeadingPurpose.Multidrug-resistance–associated protein 2 (Mrp2) shows a broad substrate specificity toward amphiphilic organic anions. This study identified key functional groups of ligand molecules for binding to rat Mrp2, determined their relative locations, and examined substrate specificity through receptor mapping using three-dimensional (3D) quantitative structure-activity relationship (3D-QSAR) analysis.Methods.Ligand-binding conformations were estimated using conformational analysis (CAMDAS) and molecular superposition (SUPERPOSE) methods to clarify the substrate specificity of rat Mrp2 in relation to 3D ligand structures.Results.Two types of binding conformations of ligands for rat Mrp2 were identified. 3D-QSAR comparative molecular-field analysis (CoMFA) revealed a statistically significant model for one type, in which the steric, electrostatic, and log P contributions to the binding affinity for rat Mrp2 were 63.0%, 33.4%, and 3.6%, respectively (n = 16, q2 = 0.59, n = 3, r2 = 0.99, and s = 0.08).Conclusions.The 3D pharmacophore of ligands for rat Mrp2, and the ligand-binding region of rat Mrp2, were estimated. Ligand recognition of rat Mrp2 is achieved through interactions in two hydrophobic and two electrostatically positive sites (primary binding sites). The broad substrate specificity of rat Mrp2 might result from the combination of secondary (two electrostatically positive and two electrostatically negative sites) and primary binding sites.

Design and Synthesis of Labystegines, Hybrid Iminosugars from LAB and Calystegine, as Inhibitors of Intestinal α-Glucosidases: Binding Conformation and Interaction for ntSI

This paper identifies the required configuration and orientation of α-glucosidase inhibitors, miglitol, α-1-C-butyl-DNJ, and α-1-C-butyl-LAB for binding to ntSI (isomaltase). Molecular dynamics (MD) calculations suggested that the flexibility around the keyhole of ntSI is lower than that of ctSI (sucrase). Furthermore, a molecular-docking study revealed that a specific binding orientation with a CH-π interaction (Trp370 and Phe648) is a requirement for achieving a strong affinity with ntSI. On the basis of these results, a new class of nortropane-type iminosugars, labystegines, hybrid iminosugars of LAB and calystegine, have been designed and synthesized efficiently from sugar-derived cyclic nitrones with intramolecular 1,3-dipolar cycloaddition or samarium iodide catalyzed reductive coupling reaction as the key step. Biological evaluation showed that our newly designed 3(S)-hydroxy labystegine (6a) inherited the selectivity against intestinal α-glucosidases from LAB, and its inhibition potency was 10 times better than that of miglitol. Labystegine, therefore, represents a promising new class of nortropane-type iminosugar for improving postprandial hyperglycemia.

Docking and SAR studies of D- and L-isofagomine isomers as human β-glucocerebrosidase inhibitors.

We report the structure-activity relationship of a series of D-, and L-isofagomine and fagomine isomers as glycosidase inhibitors. Our study revealed that a positive charge at the anomeric position of d-isofagomines enhanced the potency toward β-glycosidases, while the epimerization at the C3 OH group drastically reduced their inhibitory potency by over three orders of magnitude. Furthermore, d-3,4-di-epi-isofagomine abolished their inhibition activities against all enzymes. L-Isofagomine was also a fairly potent inhibitor of human β-glucocerebrosidase, with an IC₅₀ value of 8.7 μM. A molecular docking study revealed that the positions and orientations of the piperidine ring of D-3-epi-isofagomine in the binding site was similar to that of D-isofagomine, while D-3-epi-isofagomine missed the hydrogen bond interactions between Asp127 and the 3-OH group and between Trp179 and the 3-OH group. Furthermore, the top 10 docking models ranked by IFDscore suggested that D-3,4-di-epi-isofagomine can not bind to β-glucocerebrosidase at a stable interaction mode. These results provide an insight into the structural requirements of isofagomine isomers for developing a new type of pharmacological chaperone for Gaucher disease.

Genistein and daidzein, typical soy isoflavones, inhibit TNF‐α‐mediated downregulation of adiponectin expression via different mechanisms in 3T3‐L1 adipocytes

SCOPE Previous reports suggest that soy isoflavones have multiple biological functions and may help to restore adiponectin expression and insulin sensitivity. However, little is known about whether soy isoflavones can inhibit the downregulation of adiponectin and their molecular mechanisms. In the present study, we demonstrate that genistein (Gen) or daidzein (Dai) can significantly inhibit the downregulation of adiponectin expression via unique and different molecular mechanisms. METHODS AND RESULTS Pretreatment with Gen or Dai significantly inhibited the tumor necrosis factor-alpha (TNF-α)-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes. Gen inhibited the TNF-α-induced c-Jun-NH(2) -terminal kinase (JNK) signaling that is involved in adiponectin expression. Molecular docking studies based on JNK1 with Gen or Dai clearly supported our experimental results. However, Dai did not significantly inhibit JNK signaling. Dai did, however, inhibit the TNF-α-induced downregulation of forkhead box-containg protein O1, which is also involved in adiponectin expression. CONCLUSION These data demonstrate that: (i) both Gen and Dai significantly inhibit the TNF-α-mediated downregulation of adiponectin in adipocytes; (ii) Gen is an effective inhibitor of JNK activation, thus inhibiting the TNF-α-mediated downregulation of adiponectin; and (iii) Dai can inhibit the downregulation of adiponectin by restoring the TNF-α-mediated reduction of forkhead box-containg protein O1 protein expression.

Design, Synthesis, and Structural Analysis of Phenylpropanoic Acid-Type PPARγ-Selective Agonists: Discovery of Reversed Stereochemistry−Activity Relationship

Peroxisome proliferator-activated receptor gamma (PPARγ) is a ligand-mediated transcription factor with roles in glucose, lipid, and lipoprotein homeostasis, and PPARγ ligands are expected have therapeutic potential in these as well as other areas. We report here the design, synthesis, crystallographic analysis, and computational studies of α-benzylphenylpropanoic acid PPARγ agonists. Interestingly, these compounds show a reversal of the stereochemistry-transactivation activity relationship observed with other phenylpropanoic acid ligands.

Brazilian propolis-derived components inhibit TNF-α-mediated downregulation of adiponectin expression via different mechanisms in 3T3-L1 adipocytes.

BACKGROUND Previous reports suggest that Brazilian propolis has multiple biological functions and may help to restore adiponectin expression and insulin sensitivity. However, little is known about the molecular mechanisms by which these compounds inhibit the downregulation of adiponectin. METHODS The effect of various Brazilian propolis-derived components on inhibition of tumor necrosis factor-α (TNF-α)-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes and molecular mechanism was investigated. RESULTS AND CONCLUSIONS Pretreatment with either artepillin C (C3) or its derivative (C4) significantly inhibited TNF-α-mediated downregulation of adiponectin expression in 3T3-L1 adipocytes. Interestingly, C3 strongly activated peroxisome proliferator-activated receptor γ (PPARγ) transcriptional activity. Treatment of adipocytes with C3 resulted in the upregulation of adiponectin and fatty acid-binding protein 4 expression, but C4 did not significantly induce PPARγ transactivation. C4 did, however, inhibit the TNF-α-induced c-Jun-NH(2)-terminal kinase (JNK) signaling that is involved in adiponectin expression. Molecular docking studies based on hPPARγ with C3 and JNK1 with C4 clearly supported our experimental results. These data demonstrate that 1) both C3 and C4 significantly inhibit the TNF-α-mediated downregulation of adiponectin in adipocytes, 2) C3 functions as a PPARγ agonist, and its inhibition of the effect of TNF-α is due to this PPARγ transactivation, and 3) C4 is an effective inhibitor of JNK activation, thus inhibiting the TNF-α-mediated downregulation of adiponectin. GENERAL SIGNIFICANCE Brazilian propolis-derived components (C3 and C4) can significantly inhibit TNF-α-mediated downregulation of adiponectin in adipocytes, although they do so via different mechanisms.

Immunochemical characterization of developmental changes in rat hepatic hydroxysteroid sulfotransferase

A major isoenzyme of hepatic androsterone-sulfating sulfotransferase (AD-ST) was purified from adult female rats. The activity was purified 122-fold over that found in the cytosol and showed a single protein band with a subunit molecular mass of 30 kDa after sodium dodecyl sulfate polyacrylamide gel electrophoresis. The purified enzyme exhibited four isoelectric variants of subunits on denaturing isoelectrofocusing gels (pI = 5.8, 6.1, 6.7 and 7.2). Rabbit antiserum raised against the enzyme specifically detected AD-ST polypeptide in rat liver cytosol. Immunoblot analysis of liver cytosol from female and male rats at various ages showed good correlation between the levels of AD-ST activity and AD-ST polypeptide. Significant levels of AD-ST activity and polypeptide were detected in senescent male rats, though normal adult male rats have very low levels of AD-ST activity and protein. The relative content of the isoelectric variants of AD-ST were different in liver cytosol of weanling and adult females, indicating that age- and gender-related alterations of hepatic AD-ST activity are primarily determined by the levels of AD-ST polypeptide and the relative amounts of the four isoelectric variants of the enzyme.

Structure-based design, synthesis, and nonalcoholic steatohepatitis (NASH)-preventive effect of phenylpropanoic acid peroxisome proliferator-activated receptor (PPAR) α-selective agonists

A series of α-ethylphenylpropanoic acid derivatives was prepared as candidate peroxisome proliferator-activated receptor (PPAR) α-selective agonists, based on our PPARα/δ dual agonist 3 as a lead compound. Structure-activity relationship studies clearly indicated that the steric bulkiness and position of the distal hydrophobic tail part are critical for PPARα agonistic activity and PPARα selectivity, as had been predicted from a molecular-modeling study. A representative compound blocked the progression of nonalcoholic steatohepatitis (NASH) in an animal model.

Studies on rat hepatic hydroxysteroid sulfotransferase — immunochemistry, development and pI variants

Rat hepatic hydroxysteroid sulfotransferase with sulfoconjugates androsterone (androsterone-sulfating sulfotransferase) is an oligomer consisting of several subunits with distinct pI values but with the same molecular mass (pI variants). N-terminal amino acid sequences of the pI variants are all identical. The enzyme is exclusively present in the liver, in which its lobular localization is sex-dependent. The localization of the enzyme is markedly different from that of an isoenzyme of phenol sulfotransferase. In weanling and adult female rats, the relative abundance of the pI variants is different. During development from weanling stage to adulthood, the amounts of acidic variants increase, whereas the relative levels of alkaline variants remain constant.