Chi Hyun Kim

Discovery of cyclicsulfonamide derivatives as 11β-hydroxysteroid dehydrogenase 1 inhibitors

A new series of cyclic sulfonamide derivatives was synthesized and evaluated for their ability to inhibit 11beta-HSD1. Cyclic sulfonamides with phenylacetyl substituents at the 2-position showed nanomolar inhibitory activities. Among them, compound 4e exhibited a good in vitro inhibitory activity and selectivity toward human 11beta-HSD2.

Cell-based assay for screening 11β-hydroxysteroid dehydrogenase 1 inhibitors

11beta-Hydroxysteroid dehydrogenase 1 (11beta-HSD1) is primarily responsible for intracellular biosynthesis of active glucocorticoid, and its tissue-specific dysregulation has been implicated in the development of metabolic syndromes. We have developed a cell-based assay for measuring 11beta-HSD1 activities using murine skeletal muscle cell line C2C12. We found that the messenger RNA (mRNA) expression of 11beta-HSD1 increased on differentiation with enhanced enzyme activity as determined by homogeneous time-resolved fluorescence (HTRF) assay. Carbenoxolone, a well-known 11beta-HSD1 inhibitor, exhibited an IC(50) value similar to that in in vitro microsomal assay (IC(50) = 0.3 microM). Unlike in vitro microsomal assay, cosubstrate NADPH was not required in the cell-based assay, indicating that viable cells might provide a sufficient amount of endogenous NADPH to catalyze the enzymatic conversion of inactive cortisone to active cortisol. Treatment of C2C12 myotubes with cortisone concentration dependently transactivated and transrepressed glutamine synthase and interleukin-6, respectively, which were abrogated by carbenoxolone or RU-486 (mifepristone), a glucocorticoid receptor antagonist. Accordingly, a newly designed cell-based assay using differentiated skeletal muscle cells would be useful for high-throughput screening of 11beta-HSD1 inhibitors as well as for understanding the molecular mechanisms of glucocorticoid action.

Synthesis and 11β hydroxysteroid dehydrogenase 1 inhibition of thiazolidine derivatives with an adamantyl group.

A new series of thiazolidine derivatives with an adamantyl group was synthesized and evaluated for their ability to inhibit 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1). Our initial compound 5a showed a weak inhibitory activity. Significant improvements in potency were achieved by substituent modification. The potent compound 8g (E) showed good in vitro inhibitory activity toward human 11β-HSD1, selectivity toward 11β-HSD2, metabolic stability, pharmacokinetic, and safety profile. Furthermore, this compound significantly inhibited 11β-HSD1 activity in rat and monkey models, and showed improved glycemic control in KKAy mice.

Protective effects of arachidonic acid against palmitic acid-mediated lipotoxicity in HIT-T15 cells.

Saturated fatty acids have been considered major contributing factors in type 2 diabetes, whereas unsaturated fatty acids have beneficial effects for preventing the development of diabetes. However, the effects of polyunsaturated fatty acids in pancreatic β cells have not been reported. Here, we examined the effects of arachidonic acid (AA) on palmitic acid (PA)-mediated lipotoxicity in clonal HIT-T15 pancreatic β cells. AA prevented the PA-induced lipotoxicity as indicated by cell viability, DNA fragmentation and mitochondrial membrane potential, whereas eicosatetraynoic acid (ETYA), a non-metabolizable AA, had little effect on PA-induced lipotoxicity. In parallel with its protective effects against PA-induced lipotoxicity, AA restored impaired insulin expression and secretion induced by PA. AA but not ETYA increased intracellular triglyceride (TG) in the presence of PA compared with PA alone, and xanthohumol, a diacylglycerol acyltransferase (DGAT) inhibitor, reversed AA-induced protection from PA. Taken together, our results suggest that AA protects against PA-induced lipotoxicity in clonal HIT-T15 pancreatic β cells, and the protective effects may be associated with TG accumulation, possibly through sequestration of lipotoxic PA into TG.

Discovery of (2-Fluoro-Benzyl)-(2-Methyl-2-Phenethyl-2H-Chromen-6-yl)-Amine (KRH-102140) as an Orally Active 5-Lipoxygenase Inhibitor with Activity in Murine Inflammation Models

Objective and Design: We investigated anti-inflammatory properties of a novel 5-lipoxygenase (5-LO) inhibitor, KRH-102140, in vitro and in vivo. 5-LO enzyme activity was assayed using insect cell lysates overexpressing rat 5-LO. The leukotriene B4 (LTB4) level was assayed in rat basophilic leukemia (RBL-1) cell line. ICR (Institute of Cancer Research) mice were used for in vivo assays. Mouse ear edema was induced by topical application of arachidonic acid. An air pouch was induced by subcutaneous injection of sterile air into mice, followed by zymosan treatment. Sprague-Dawley rats were used for pharmacokinetic studies. Results: KRH-102140 inhibited 5-LO activity with an IC50 value of 160 ± 23 nmol/l in parallel with LTB4 inhibition in RBL-1 cells. Oral administration of KRH-102140 (10–100 mg/kg) reduced ear edema, myeloperoxidase activity and LTB4 production in murine inflammation models. Oral bioavailability as determined in rats was 66%. Conclusions: Our results show that KRH-102140, a new 5-LO inhibitor, exhibits potent anti-inflammatory activities in vitro as well as in vivo.

Rosiglitazone reduces a wide range of proinflammatory profiles in synovial fibroblast SW982 under spheroid culture

Rosiglitazone (RSG) has been known to play a role in the modulation of inflammatory responses. Therefore, we sought to elucidate the underlying molecular mechanism by which RSG regulates the development of rheumatoid arthritis. Firstly, we examined the preventive effect of RSG on the inflammatory mediators induced by spheroid culture of synovial sarcoma SW982. Expression of proinflammatory cytokines under spheroid culture was more elevated than that under monolayer culture while RSG abolished inflammatory responses. The upregulation of inflammation-related genes by spheroid culture was closely associated with NFkappaB (NFkappaB) activation. Also, activation of p38 and c-Jun N-terminal kinase (JNK) by spheroid culture was abrogated with RSG treatment. Lastly, it was demonstrated that RSG reduced the development of arthritis in mice immunized with collagen, improving the histology of inflamed joint. In summary, RSG reduces inflammatory responses of synovial fibroblast via not only inhibition of NFkappaB but also modulation of both p38 and JNK.

Identification of 4-[4-(4-Fluoro-Phenyl)-Thiazol-2-Ylamino]-2,6-Dimethyl-Phenol (KR-33749) as an Inhibitor of 5-Lipoxygenase with Potent Antiinflammatory Activity

Background/Aim: To discover new 5-lipoxygenase (5-LO) inhibitors applicable to inflammation-related skin disease, we identified and examined antiinflammatory properties of a novel 5-LO inhibitor, KR-33749, in vitro and in vivo. Methods: 5-LO enzyme activity was assayed using insect cell lysates overexpressing rat 5-LO. The leukotriene B4 (LTB4) level was assayed in rat basophilic leukemia (RBL-1) cell line. Mouse ear edema was induced by topical application of arachidonic acid. Atopic dermatitis-like skin lesion was induced by topical application of 1-chloro-2,4-dinitrobenzene (DNCB) to NC/Nga mice. Results: KR-33749 inhibited 5-LO activity with an IC50 value of 70.5 ± 6.0 nmol/l in parallel with LTB4 inhibition in RBL-1 cells. The compound exhibited a >1,000-fold selectivity against 12-LO and 15-LO. KR-33749 showed in vivo protective effects against arachidonic acid-induced ear edema and DNCB-induced atopic dermatitis-like symptoms in NC/Nga mice. Conclusion: Our results show that KR-33749, a new 5-LO inhibitor exhibits potent antiinflammatory activities in vitro as well as in vivo.

Selection and optimization of MCF‐7 cell line for screening selective inhibitors of 11β‐hydroxysteroid dehydrogenase 2

An 11β‐hydroxysteroid dehydrogenase type 1 (11β‐HSD1) produces glucocorticoid (GC) from 11‐keto metabolite, and its modulation has been suggested as a novel approach to treat metabolic diseases. In contrast, type 2 isozyme 11β‐HSD2 is involved in the inactivation of glucocorticoids (GCs), protecting the non‐selective mineralocorticoid receptor (MR) from GCs in kidney. Therefore, when 11β‐HSD1 inhibitors are pursued to treat the metabolic syndrome, preferential selectivity of inhibitors for type 1 over type 2 isozyme is rather important than inhibitory potency. Primarily, to search for cell lines with 11β‐HSD2 activity, we investigated the expression profiles of enzymes or receptors relevant to GC metabolism in breast, colon, and bone‐derived cell lines. We demonstrated that MCF‐7 cells had high expression for 11β‐HSD2, but not for 11β‐HSD1 with its cognate receptor. Next, for the determination of enzyme activity indirectly, we adopted homogeneous time resolved fluorescence (HTRF) cortisol assay. Obviously, the feasibility of HTRF to cellular 11β‐HSD2 was corroborated by constructing inhibitory response to an 11b‐HSD2 inhibitor glycyrrhetinic acid (GA). Taken together, MCF‐7 that overexpresses type 2 but not type 1 enzyme is chosen for cellular 11β‐HSD2 assay, and our results show that a nonradioactive HTRF assay is applicable for type 2 as well as type 1 isozyme. Copyright