Identification of sulfonylpyrimidines as novel selective aldosterone synthase (CYP11B2) inhibitors.

4-[(5-[2-Methyl-5-(methylsulfonyl)pentan-2-yl]sulfonylpyrimidin-4-yl)amino]benzonitrile 2 was identified as a novel potent aldosterone synthase inhibitor. Compound 2 was found to inhibit human CYP11B2 in the nanomolar range, and showed an aldosterone-lowering effect in a furosemide-treated cynomolgus monkey model. Although human CYP11B2 has the high homology sequence with human CYP11B1, compound 2 showed more than 80 times higher selectivity over human CYP11B1 in vitro.

A xanthene derivative, DS20060511, attenuates glucose intolerance by inducing skeletal muscle-specific GLUT4 translocation in mice.

Reduced glucose uptake into the skeletal muscle is an important pathophysiological abnormality in type 2 diabetes, and is caused by impaired translocation of glucose transporter 4 (GLUT4) to the skeletal muscle cell surface. Here, we show a xanthene derivative, DS20060511, induces GLUT4 translocation to the skeletal muscle cell surface, thereby stimulating glucose uptake into the tissue. DS20060511 induced GLUT4 translocation and stimulated glucose uptake into differentiated L6-myotubes and into the skeletal muscles in mice. These effects were completely abolished in GLUT4 knockout mice. Induction of GLUT4 translocation by DS20060511 was independent of the insulin signaling pathways including IRS1-Akt-AS160 phosphorylation and IRS1-Rac1-actin polymerization, eNOS pathway, and AMPK pathway. Acute and chronic DS20060511 treatment attenuated the glucose intolerance in obese diabetic mice. Taken together, DS20060511 acts as a skeletal muscle-specific GLUT4 translocation enhancer to facilitate glucose uptake. Further studies of DS20060511 may pave the way for the development of novel antidiabetic medicines.

Discovery of novel pyridazine derivatives as glucose transporter type 4 (GLUT4) translocation activators.

We report herein the synthesis and structure-activity relationships (SAR) of a series of pyridazine derivatives with the activation of glucose transporter type 4 (GLUT4) translocation. Through a cell-based phenotype screening in L6-GLUT4-myc myoblasts and functional glucose uptake assays, lead compound 1a was identified as a functional small molecule. After further derivatization, the thienopyridazine scaffold as the central ring (B-part) was revealed to have potent GLUT4 translocation activities. Consequently, we obtained promising compound 26b, which showed a significant blood glucose lowering effect in the severe diabetic mice model (10-week aged db/db mice) after oral dosing even at 10 mg/kg, implying that our pyridazine derivatives have potential to become novel therapeutic agents for diabetes mellitus.

4-Anilino-pyrimidine, novel aldosterone synthase (CYP11B2) inhibitors bearing pyrimidine structures.

2,2,2-Trifluoro-1-{4-[(4-fluorophenyl)amino]pyrimidin-5-yl}-1-[1-(methylsulfonyl)piperidin-4-yl]ethanol 1 was identified as a novel potent aldosterone synthase inhibitor. Despite large species differences, compound 1 inhibits both human and rodent CYP11B2 in a nano-molar range.

A novel aldosterone synthase inhibitor ameliorates mortality in pressure-overload mice with heart failure.

It has been elucidated that mineralocorticoid receptor antagonists reduce mortality in patients with congestive heart failure and post-acute myocardial infarction. A direct inhibition of aldosterone synthase (CYP11B2) is also expected to have therapeutic benefits equal in quality to mineralocorticoid receptor antagonists in terms of reducing mineralocorticoid receptor signaling. Therefore, we have screened our chemical libraries and identified a novel and potent aldosterone synthase inhibitor, 2,2,2-trifluoro-1-{4-[(4-fluorophenyl)amino]pyrimidin-5-y}-1-[1-(methylsulfonyl)piperidin-4-yl]ethanol (compound 1), by lead optimization. Pharmacological properties of compound 1 were examined in in vitro cell-based assays and an in vivo mouse model of pressure-overload hypertrophy by transverse aortic constriction (TAC). Compound 1 showed potent CYP11B2 inhibition against human and mouse enzymes (IC50; 0.003µM and 0.096µM, respectively) in a cell-based assay. The oral administration of 0.06% compound 1 in the food mixture of a mouse TAC model significantly reduced the plasma aldosterone level and ameliorated mortality rate. This study is the first to demonstrate that a CYP11B2 inhibitor improved survival rates of heart failure induced by pressure-overload in mice. The treatment of 0.06% compound 1 did not elevate plasma potassium level in this model, although further evaluation of hyperkalemia is needed. These results suggest that compound 1 can be developed as a promising oral CYP11B2 inhibitor for pharmaceutical applications. Compound 1 could also be a useful compound for clarifying the role of aldosterone in cardiac hypertrophy.

Inherent pacemaker function of duodenal GIST.

Gastrointestinal stromal tumours (GIST) are thought to derive from interstitial cells of Cajal (ICCs), which are putative pacemaker cells for gut motility. Isolated cells were obtained by enzymatic treatment of human duodenum GIST tissue having a frequent gain-of-function gene mutation. After cell culturing, c-Kit immunoreactivity was preserved and the cells developed long processes. Whole cell patch clamp recordings revealed voltage-dependent outward currents, without transient inward currents. Intracellular Ca(2+) measurements showed oscillation-like spontaneous activity in some GIST cells. RT-PCR revealed expression of ion channels (Kv1.1, Kv1.6 and KCNH2; IP3R1, and IP3R2; TRPC1, 3, 6 and 7; Cx43), which have been suggested to play important roles in pacemaker activity. However, SCN5A, a TTX-resistant Na(+) channel known to be expressed in human ICCs, was below detectable levels. These data suggest that GIST cells appear to preserve some, but not all ionic mechanisms underlying pacemaker activity in ICC.

Sulphonylurea receptors differently modulate ICC pacemaker Ca2+ activity and smooth muscle contractility.

Appropriate gastrointestinal motility is essential to properly control the body energy level. Intracellular Ca2+ ([Ca2+]i) oscillations in interstitial cells of Cajal (ICCs; identified with c-Kit immunoreactivity) are considered to be the primary mechanism for the pacemaker activity in gastrointestinal motility. In the present study, RT-PCR examinations revealed predominant expression of the type 1 isoform of sulphonylurea receptors (SUR1) in ICCs of the mouse ileum, but expression of SUR2 was predominant in smooth muscle. In cell clusters prepared from the same tissue, smooth muscle contractility and pacemaker [Ca2+]i activity in ICCs were found to be differentially modulated by K(ATP) channel openers and sulphonylurea compounds, in accordance with the expression of SUR isoforms. 1 microM cromakalim nearly fully suppressed the mechanical activity in smooth muscle, whereas ICC pacemaker [Ca2+]i oscillations persisted. Greater concentrations (approximately 10 microM) of cromakalim attenuated pacemaker [Ca2+]i oscillations. This effect was not reversed by changing the reversal potential of K+, but was prevented by glibenclamide. Diazoxide at 30 muM terminated ICC pacemaker [Ca2+]i oscillations, but again treatment with high extracellular K+ did not restore them. These results suggest that SUR can modulate pacemaker [Ca2+]i oscillations via voltage-independent mechanism(s), and also that intestinal pacemaking and glucose control are closely associated with SUR.

Purinergic modulation of pacemaker Ca2+ activity in interstitial cells of Cajal.

Purinoceptors are widely distributed throughout the body, and are thought to have important contributions to numerous functions. In this study, we characterised the contribution of purinoceptors to the mechanisms underlying spontaneous rhythmicity of the gastro-intestinal tracts. Using cell cluster preparations (100-200 microm diameter) obtained from murine ileum, we measured spontaneous intracellular Ca2+([Ca2+]i) oscillations in the presence of nifedipine, as an index of pacemaker [Ca2+]i activity in interstitial cells of Cajal (ICCs, c-Kit-immunopositive cells), the pacemaker cells for gastrointestinal motility. This small preparation also contained smooth muscle and enteric neurones. Using various purinoceptor agonists and an antagonist, we characterised both TTX-sensitive and insensitive modulations of pacemaker [Ca2+]i activity in ICCs. Continuous application of either ATP, ATPgammaS, suramin or alpha,beta-methylene ATP (alpha,beta-meATP) suppressed pacemaker [Ca2+]i activity. The inhibitory effect of alpha,beta-meATP was completely abolished by a prior application of TTX. On the other hand, even in the presence of TTX, continuous application of 2-methylthio ATP (2-MeSATP) at concentrations greater than 30 microM caused a prompt rise followed by a slow decline of the baseline [Ca2+]i, and pacemaker [Ca2+]i oscillations were gradually suppressed during the decline. Neither UTP nor alpha,beta-meATP at high concentrations (30-100 microM) produced a similar [Ca2+]i response. These results suggest that the TTX-resistant, direct purinergic modulation of pacemaker [Ca2+]i activity in ICCs is mediated via P2X purinoceptors distinct from those involved in TTX-sensitive modulation. The slow decline may be attributed to desensitisation of these purinoceptors. The possible involvement of other purinoceptors is also discussed.

Co-contribution of IP3R and Ca2+ influx pathways to pacemaker Ca2+ activity in stomach ICC.

Intracellular Ca2+ oscillations in interstitial cells of Cajal (ICCs) are thought to be the primary pacemaker activity in the gut. In the present study, the authors prepared small tissues of 100-to 300-microm diameter (cell cluster preparation) from the stomach smooth muscle (including the myenteric plexus) of mice by enzymatic and mechanical treatments. After 2 to 4 days of culture, the intracellular Ca2+ concentration ([Ca2+]i) was measured. In the presence of nifedipine, a dihydropyridine Ca2+ channel antagonist, spontaneous [Ca2+]i oscillations were observed within limited regions showing positive c-Kitimmunoreactivity, a maker for ICCs. In the majority of cell cluster preparations with multiple regions of [Ca2+]i oscillations, [Ca2+]i oscillated synchronously in the same phase. A small number of cell clusters (8 of 53) showed multiple regions of [Ca2+]i oscillations synchronized but with a considerable phase shift. Neither tetrodotoxin (250 nM) nor atropine (10 microM) significantly affected [Ca2+]i oscillations in the presence of nifedipine. Low concentrations (40 microM) of Ni2+ had little effect on the spontaneous [Ca2+]i oscillation, but SK&F96365 (40 microM) and Cd2+ (120 microM) terminated it. Applications of either 2-aminoethoxydiphenyl borate (10 microM) or xestosponginC(10 microM) completely and rather rapidly (approximately 2 min) abolished the spontaneous [Ca2+]i oscillations. The results suggest that pacemaker [Ca2+]i oscillations in ICCs are produced by close interaction of intracellular Ca2+ release channels, especially inositol 1,4,5-trisphosphate receptor (InsP3R) and Ca2+ influx pathways, presumably corresponding to store-operated type channels. Reverse transcription polymerase chain reaction examinations revealed expression of TRPC2, 4, and 6, as well as InsP3R1 and 2 in ICCs.

Requirement of ryanodine receptors for pacemaker Ca2+ activity in ICC and HEK293 cells.

Intracellular Ca(2+) ([Ca(2+)](i)) oscillations seen in interstitial cells of Cajal (ICCs) are considered to be the primary pacemaker activity in the gut. Here, we show evidence that periodic Ca(2+) release from intracellular Ca(2+) stores produces [Ca(2+)](i) oscillations in ICCs, using cell cluster preparations isolated from mouse ileum. The pacemaker [Ca(2+)](i) oscillations in ICCs are preserved in the presence of dihydropyridine Ca(2+) antagonists, which suppress Ca(2+) activity in smooth muscle cells. However, applications of drugs affecting either ryanodine receptors or inositol 1,4,5-trisphosphate receptors terminated [Ca(2+)](i) oscillations at relatively low concentrations. RT-PCR analyses revealed a predominant expression of type 3 RyR (RyR3) in isolated c-Kit-immunopositive cells (ICCs). Furthermore, we demonstrate that pacemaker-like global [Ca(2+)](i) oscillation activity is endowed by introducing RyR3 into HEK293 cells, which originally express only IP(3)Rs. The reconstituted [Ca(2+)](i) oscillations in HEK293 cells possess essentially the same pharmacological characteristics as seen in ICCs. The results support the functional role of RyR3 in ICCs.