Synthesis and Structure-Activity Relationships of 2-Aminoacetamide Derivatives as Peroxisome Proliferator-Activated Receptor α/γ Dual Agonists.

We describe the design, syntheses, and structure-activity relationships of novel zwitterionic compounds as nonthiazolidinedion-based peroxisome proliferator-activated receptor (PPAR) α/γ dual agonists. In our previous report, we obtained compound 1 showing potent PPARα/γ dual agonistic activities, together with a sufficient glucose-lowering effect in db/db mice. However, this compound possessed an issue, i.e., the 1,3,4-oxadiazole ring was not stable in acidic conditions. Thus, we carried out further optimization to improve the stability while maintaining the other favorable profile features including potent PPARα/γ dual agonistic activity. We addressed the issue by changing the oxadiazole ring to a bioisostere amide group. These amide derivatives were stable in acidic conditions and decreased plasma glucose and plasma triglyceride levels significantly without marked weight gain.

Synthesis and structure-activity relationships of novel zwitterionic compounds as peroxisome proliferator activated receptor α/γ dual agonists with improved physicochemical properties.

We describe herein the design, syntheses and structure-activity relationships (SAR) of novel zwitterionic compounds as non-thiazolidinedion (TZD) based peroxisome proliferator activated receptor (PPAR) α/γ dual agonists. In the previous report, we obtained compound 1 showing potent PPARα/γ dual agonistic activities, together with a great glucose lowering effect in the db/db mice. However, this compound possessed fatal issues such as potent cytochrome P450 (CYP)3A4 direct inhibitory activity. Thus, we carried out the medicinal optimization to improve these while maintaining the potent PPAR agonistic activity. As a result, the issues were addressed by changing the furan ring to a low lipophilic 1,3,4-oxadiazole ring. Additionally, these oxadiazole derivatives exhibited a significant decrease in plasma glucose and plasma triglyceride levels without marked weight gain.

Phosphatidylinositol-4-phosphate 5-kinase gamma is associated with cell-cell junction in A431 epithelial cells.

Cell to cell contact in epithelial cells is crucial for tissue integrity and is maintained by junctional complexes, such as the adherens junction (AJ). Actin polymerization has been shown to be important for AJ formation; however, the molecular mechanisms have yet to be clarified. It has been shown that increased phosphatidylinositol-4,5-bisphosphate (PIP2) induces actin polymerization. It is thus of interest to know more about the production of PIP2 during cell-cell adhesion formation in epithelial cells. The distribution of phosphatidylinositol-4-phosphate 5-kinase gamma635 (PIP5Kgamma635), an isoform of the PIP2 synthesizing enzymes, was examined in epithelial cell line A431. It was found that, in non-contact cells, PIP5Kgamma635 was not concentrated at the plasma membrane. However, in cells that were in contact, PIP5Kgamma635 localized to the intercellular contact sites and colocalized with E-cadherin and beta-catenin, two components of AJ, and with polymerized actin, but did not colocalize with focal adhesion, integrin-mediated cell-substratum complex. Decreasing calcium ion concentration induced both disruption of intercellular adhesion and the dissociation of both PIP5Kgamma635 and actin from the contact site. These results suggest that PIP5K has an important role in actin polymerization in epithelial cell-cell adhesion.

A combinatorial G protein-coupled receptor reconstitution system on budded baculovirus. Evidence for Galpha and Galphao coupling to a human leukotriene B4 receptor.

To investigate the coupling selectivity of G proteins and G protein-coupled receptors (GPCRs), we developed a reconstitution system made up of GPCR and heterotrimeric G proteins on extracellular baculovirus particles (budded virus (BV)). BV released from Sf9 cells infected with a recombinant baculovirus coding for human leukotriene B4 receptor (BLT1) cDNA exhibited a high level of BLT1 expression (27.3 pmol/mg of protein) and specific [3H]leukotriene B4 binding activity (Kd = 3.67 nm). The apparent low affinity of the expressed BLT1 is thought to be due to relative non-availability of the Galphai isoform, which couples to BLT1, in BV. Co-infection of heterotrimeric G protein recombinant viruses led to co-expression of BLT1 and G protein subunits on BV. A guanosine-5'-(beta,gamma-imido)triphosphate-sensitive, high affinity ligand binding was observed in the BLT1 BV co-expressing Galphai1beta1gamma2 (Kd = 0.17 nm). A relatively large amount of high affinity receptor protein was recovered in the co-expressing BV fraction (6.81 pmol/mg of protein). A combination of BLT1 and Galphai1 without Gbeta1gamma2 did not exhibit high affinity ligand binding on BV, indicating the low background environment for the GPCR-G protein coupling in this BV reconstitution system. To test other G proteins for coupling, various Galpha subunits were combinatorially expressed in BV with BLT1 and Gbeta1gamma2. The BLT1 BV co-expressing GalphaoAbeta1gamma2 exhibited a comparably high affinity ligand binding as well as ligand-stimulated guanosine 5'-3-O-(thio)triphosphate binding to Galphai1beta1gamma2. Co-expression of other Galpha isoforms such as Galphas, Galpha11, Galpha14, Galpha16, Galpha12, or Galpha13 did not exhibit any significant effects on ligand binding affinity in this system. These results reveal that BLT1 and coupled trimeric G proteins were functionally reconstituted on BV and that Galphao as well as Galphai couples to BLT1. This expression system should prove highly useful for pharmacological characterization, biosensor chip applications, and also drug discovery directed at highly important targets of the membrane receptor proteins.