Hyojin Ko

Suppression of innate immunity (natural killer cell/interferon-γ) in the advanced stages of liver fibrosis in mice

Activation of innate immunity (natural killer [NK] cell/interferon‐γ [IFN‐γ]) has been shown to play an important role in antiviral and antitumor defenses as well as antifibrogenesis. However, little is known about the regulation of innate immunity during chronic liver injury. Here, we compared the functions of NK cells in early and advanced liver fibrosis induced by a 2‐week or a 10‐week carbon tetrachloride (CCl4) challenge, respectively. Injection of polyinosinic‐polycytidylic acid (poly I:C) or IFN‐γ induced NK cell activation and NK cell killing of hepatic stellate cells (HSCs) in the 2‐week CCl4 model. Such activation was diminished in the 10‐week CCl4 model. Consistent with these findings, the inhibitory effect of poly I:C and IFN‐γ on liver fibrosis was markedly reduced in the 10‐week versus the 2‐week CCl4 model. In vitro coculture experiments demonstrated that 4‐day cultured (early activated) HSCs induce NK cell activation via an NK group 2 member D/retinoic acid–induced early gene 1–dependent mechanism. Such activation was reduced when cocultured with 8‐day cultured (intermediately activated) HSCs due to the production of transforming growth factor‐β (TGF‐β) by HSCs. Moreover, early activated HSCs were sensitive, whereas intermediately activated HSCs were resistant to IFN‐γ–mediated inhibition of cell proliferation, likely due to elevated expression of suppressor of cytokine signaling 1 (SOCS1). Disruption of the SOCS1 gene restored the IFN‐γ inhibition of cell proliferation in intermediately activated HSCs. Production of retinol metabolites by HSCs contributed to SOCS1 induction and subsequently inhibited IFN‐γ signaling and functioning, whereas production of TGF‐β by HSCs inhibited NK cell function and cytotoxicity against HSCs. Conclusion: The antifibrogenic effects of NK cell/IFN‐γ are suppressed during advanced liver injury, which is likely due to increased production of TGF‐β and expression of SOCS1 in intermediately activated HSCs. (HEPATOLOGY 2011;)

Design, synthesis and X-ray crystallographic study of NAmPRTase inhibitors as anti-cancer agents

NAmPRTase (PBEF/Visfatin) plays a pivotal role in the salvage pathway of NAD(+) biosynthesis. NAmPRTase has been an attractive target for anti-cancer agents that induce apoptosis of tumor cells via a declining plasma NAD(+) level. In this report, a series of structural analogs of FK866 (1), a known NAmPRTase inhibitor, was synthesized and tested for inhibitory activities against the proliferation of cancer cells and human NAmPRTase. Among them, compound 7 showed similar anti-cancer and enzyme inhibitory activities to compound 1. Further investigation of compound 7 with X-ray analysis revealed a co-crystal structure in complex with human NAmPRTase, suggesting that Asp219 in the active site of the enzyme could contribute to an additional interaction with the pyrrole nitrogen of compound 7.

Pheophorbide-a conjugates with cancer-targeting moieties for targeted photodynamic cancer therapy

Pheophorbide-a, a non-selective photosensitizer, was conjugated with cancer-targeting moieties, such as folic acid, the CRGDLASLC peptide, the cRGDfK peptide and leuprorelin, for the purpose of targeted photodynamic cancer therapy. The cellular uptake of pheophorbide-a conjugates in cancer cells overexpressing the corresponding receptors of the targeting moieties was largely enhanced compared with that in the receptor-negative cells. In the study of in vitro photodynamic activity and selectivity of pheophorbide-a conjugates in the receptor-positive and receptor-negative cells, a pheophorbide-a conjugate, (14) with an αvβ6 ligand (CRGDLASLC) exhibited the highest selectivity in the positive FaDu cells. Targeted PDT with 14 induced cell death through apoptosis and morphological apoptosis-like characteristics. These results suggest that pheophorbide-a conjugate 14 could be utilized in selective photodynamic therapy for oral cancers primarily expressing the αvβ6 receptor.

Synthesis of pheophorbide-a conjugates with anticancer drugs as potential cancer diagnostic and therapeutic agents.

Pheophorbide-a, a chlorine based photosensitizer known to be selectively accumulated in cancer cells, was conjugated with anticancer drugs, doxorubicin and paclitaxel in the purpose of selective cancer diagnosis and therapy. Pheophorbide-a was conjugated with anticancer drugs via directly and by the use of selective cleavage linkers in cancer cell. The fluorescence of pheophorbide-a and doxorubicin conjugate by excitation at 420 or 440 nm was greatly diminished possibly by the energy transfer mechanism between two fluorescent groups. However, upon treatment in cancer cells, the conjugate showed to be cleaved to restore each fluorescence of pheophorbide-a and doxorubicin after 48 h of incubation. Also, pheophorbide-a conjugates either with doxorubicin and paclitaxel inhibited the growth of various cancer cells more potently than pheophorbide-a, which displayed very weak inhibitory activity. The results indicated that the pheophorbide-a conjugates with anticancer drugs could be utilized for selective cancer therapy as well as for the fluorescence detection of cancer.

Synthesis and biological evaluation of α,β-unsaturated lactones as potent immunosuppressive agents

Compounds having α,β-unsaturated lactones display a variety of biological activities. Many research groups have tested both natural and unnatural α,β-unsaturated lactones for as-yet undiscovered biological properties. We synthesized α,β-unsaturated lactones with various substituents at the δ-position and studied their immunosuppressive effects, that is, the inhibition of Interleukin-2 (IL-2) production. Among the compounds synthesized, the benzofuran-substituted α,β-unsaturated lactone 4h showed the best inhibitory activity toward IL-2 production in Jurkat e6-1 T lymphocytes (IC(50)=66.9 nM) without cytotoxicity at 10 μM. The results indicated that 4h may be useful as a potent immunosuppressive agent, as well as in IL-2-related studies.

Structure-activity relationship studies of pyrimidine-2,4-dione derivatives as potent P2X7 receptor antagonists.

As an optimization strategy, the flexible structure of KN-62, a known P2X7 receptor antagonist, was converted into conformationally constrained derivatives using pyrimidine-2,4-dione as the core skeleton. Various modifications at the 4-position of the piperazine moiety of the new lead compound were performed to improve P2X7 receptor antagonistic activities, which were evaluated in HEK293 cells stably expressing the human P2X7 receptor (EtBr uptake assay) and in THP-1 cells (IL-1β ELISA assay). According to the results, polycycloalkyl acyl or di-halogenated benzoyl substituents were much more favorable than the original phenyl group of KN-62. Among these compounds, the trifluoromethyl-chloro benzoyl derivative 18 m and adamantyl carbonyl derivatives 19 g-19 i and 19k showed potent antagonistic effects, with IC50 values ranging from 10 to 30 nM. In addition, the in vitro adsorption, distribution, metabolism, excretion, and toxicity (ADMET) profile of 18 m was determined to be in acceptable ranges in terms of metabolic stability and cytotoxicity. These results suggest that pyrimidine-2,4-dione derivatives may be promising novel P2X7 receptor antagonists for the development of anti-inflammatory drugs.

Immunosuppressive effects of subglutinol derivatives.

Immunosuppressants are used to prevent organ rejection after transplantation, to treat autoimmune diseases such as rheumatoid arthritis and Crohn’s disease, and to treat non-autoimmune diseases such as long-term allergic asthma. Cyclosporine A, a representative immunosuppressant, is widely used following allogeneic organ transplantation to reduce the activity of the recipient’s immune system. However, cyclosporine A has undesirable side effects, including gingival hyperplasia, convulsions, peptic ulcers, pancreatitis, fever, and kidney and liver dysfunction. Therefore, efforts are underway to identify novel immunosuppressive agents with improved safety profiles. 8] Among the emerging potential agents are natural-productbased immunosuppressives that exert their activity without concomitant cytotoxicity or other undesirable side effects. These natural products include subglutinols A and B, diterpenoid pyrones isolated from Fusarium subglutinans, an endophytic fungus from the vine Tripterygium wilfordii, which has been used in Chinese traditional medicine for over 2000 years. The structure and relative stereochemistry of subglutinols A and B were determined by extensive NMR spectroscopy and X-ray diffraction analysis. Subglutinols A and B were shown to be equipotent in mixed lymphocyte reactions (MLR) and thymocyte proliferation (TP) assays (IC50 = 0.1 mm). Recently, a stereoselective total synthesis of subglutinols A and B was described, further confirming that subglutinols A and B are immunosuppressive agents with dose-dependent osteogenic activity. 12] Despite the availability of the synthetic compounds, the detailed mechanism underlying the immunosuppressive effects of subglutinols and their target molecules in lymphocyte activation and proliferation have not been fully elucidated. Their mechanism of action may be similar to that of similar members of the diterpenoid g-pyrone class of natural products, nalanthalide and candelalides A–C, which have been reported to inhibit the Kv1.3 voltage-gated potassium channels. Kv1.3 may be a target of immunosuppressant agents because blockade of these channels has been found to result in the depolarization of cells. Moreover, channel blockade limits further Ca + entry into the cytosol, which normally occurs upon activation of the T cell receptor. This inhibition of Ca entry results in decreased interleukin-2 (IL-2) production via a calcium-dependent pathway, thus suppressing the activation and proliferation of T cells. To further assess their functional mechanisms of immunomodulation, we evaluated the activity of subglutinol A and some of its intermediates at the molecular level, including their inhibition of IL-2 production by stimulated Jurkat T cells, and human and murine CD4 primary T cells. We also assayed the effects of these derivatives on the cellular expression of the T cell activation markers, CD25 and CD69, and their ability to inhibit Kv1.3 channel activity. IL-2, one of the major target cytokines in inflammatory diseases, is produced by activated T cells during immune responses. IL-2 binds to the IL-2 receptor then stimulates the growth, differentiation and survival of T cells via induction of the expression of specific genes. Initially, we tested the effects of subglutinol derivatives on IL-2 production by Jurkat T cells stimulated with phorbol 12-myristate 13-acetate (PMA)/ ionomycin (A23187), using an enzyme-linked immunosorbent assay (ELISA). As positive controls, we used the immunosuppressant cyclosporine A and a Kv1.3 blocker, Psora-4. A parallel MTT assay showed that the ability of subglutinol derivatives to inhibit IL-2 production by Jurkat T cells was not due to cytotoxicity (Table 1). We found that structural fragments of subglutinols, including the pyrone moieties 1 and 2 and the decalin skeletons 3 and 4, were completely inactive at inhibiting IL-2 production, suggesting that both of these groups are essential pharmacophores for immunosuppressive activity. Compounds 5 and 6 with hydroxy groups at the R position showed significant lower activity than compounds 7 and 8, indicating that steric or electronic effects around this position may be critical for immunosuppressive activity. Comparisons of compounds 7 and 8 with compounds 9 and 10 showed that isobutene provided greater activity than ethene in the diterpenoid structure and that the R configuration at the C-12 position was preferred in the series of g-pyrone subglutinol derivatives 5–10. However, a-pyrone derivatives 11–14 showed a totally different profile of effects on IL-2 production. For example, methyl-a-pyrone analogue 11, possessing the same configurations and substituents as the corresponding methyl-g-pyrone derivative (12R) 10, showed negligible inhibition of IL-2 production. In contrast, subglutinol A, the a-pyrone analogue with a 12S configuration, potent[a] W.-G. Lee, W.-S. Kim, Prof. Dr. S.-G. Park, Prof. Dr. Y.-C. Kim School of Life Sciences, Gwangju Institute of Science and Technology (GIST) 261 Cheomdan-gwagiro(Oryongdong), Buk-gu Gwangju 500-712 (Republic of Korea) E-mail : yongchul@gist.ac.kr [b] Prof. Dr. H. Kim College of Pharmacy, Ajou University San 5, Woncheon-dong, Yeongtong-gu Suwon, 443-749 (Republic of Korea) [c] Prof. Dr. J. Hong Department of Chemistry, Duke University 124 Science Dr. , Durham, NC 27708 (USA) [d] Dr. H. Ko Institute of Medical System Engineering Gwangju Institute of Science and Technology (GIST) 261 Cheomdan-gwagiro(Oryongdong), Buk-gu Gwangju 500-712 (Republic of Korea)

The discovery of 2,5-isomers of triazole-pyrrolopyrimidine as selective Janus kinase 2 (JAK2) inhibitors versus JAK1 and JAK3.

Members of the Janus kinase (JAK) family are potential therapeutic targets. Abnormal signaling by mutant JAK2 is related to hematological malignancy, such as myeloproliferative neoplasms (MPNs), and tyrosine kinase inhibitor (TKI)-resistance in non-small cell lung cancer (NSCLC). We discovered a potent and highly selective inhibitor of JAK2 over JAK1 and -3 based on the structure of 4-(2,5-triazole)-pyrrolopyrimidine. Among all triazole compounds tested, 2,5-triazole regioisomers more effectively inhibited JAK2 kinase activity than isomers with substitutions of various alkyl groups at the R2 position, except for methyl-substituted 1,5-triazole, which was more potent than the corresponding 1,4- and 2,5-triazoles. None of the synthesized 1,4-isomers inhibited all three JAK family members. Compounds with phenyl or tolyl group substituents at the R1 position were completely inactive compared with the corresponding analogues with a methyl substituted at the R1 position. As a result of this structure-activity relationship, 54, which is substituted with a cyclopropylmethyl moiety, exhibited significant inhibitory activity and selectivity (IC50=41.9nM, fold selectivity JAK1/2 10.6 and JAK3/2 58.1). Compound 54 also exhibited an equivalent inhibition of wild type JAK2 and the V617F mutant. Moreover, 54 inhibited the proliferation of HEL 92.1.7 cells, which carry JAK2 V617F, and gefitinib-resistant HCC827 cells. Compound 54 also suppressed STAT3 phosphorylation at Y705.

Synthesis and structure-activity relationships of quinolinone and quinoline-based P2X7 receptor antagonists and their anti-sphere formation activities in glioblastoma cells

Screening a compound library of quinolinone derivatives identified compound 11a as a new P2X7 receptor antagonist. To optimize its activity, we assessed structure-activity relationships (SAR) at three different positions, R1, R2 and R3, of the quinolinone scaffold. SAR analysis suggested that a carboxylic acid ethyl ester group at the R1 position, an adamantyl carboxamide group at R2 and a 4-methoxy substitution at the R3 position are the best substituents for the antagonism of P2X7R activity. However, because most of the quinolinone derivatives showed low inhibitory effects in an IL-1β ELISA assay, the core structure was further modified to a quinoline skeleton with chloride or substituted phenyl groups. The optimized antagonists with the quinoline scaffold included 2-chloro-5-adamantyl-quinoline derivative (16c) and 2-(4-hydroxymethylphenyl)-5-adamantyl-quinoline derivative (17k), with IC50 values of 4 and 3 nM, respectively. In contrast to the quinolinone derivatives, the antagonistic effects of the quinoline compounds (16c and 17k) were paralleled by their ability to inhibit the release of the pro-inflammatory cytokine, IL-1β, from LPS/IFN-γ/BzATP-stimulated THP-1 cells (IC50 of 7 and 12 nM, respectively). In addition, potent P2X7R antagonists significantly inhibited the sphere size of TS15-88 glioblastoma cells.

Discovery and structure-activity relationship studies of quinolinone derivatives as potent IL-2 suppressive agents.

The quinolinone skeleton has been utilized to develop various mechanism-based immune modulators. However, the effects of quinolinone derivatives on the release of T cell-associated interleukin-2 (IL-2) have not been established. In this study, a series of novel quinolinone derivatives was synthesized, and their immunosuppressive activity was evaluated by measuring suppression of IL-2 release from activated Jurkat T cells. Optimizing the three side chains around the quinolinone skeleton revealed the most active compound: 11l. This compound exhibits potent inhibitory activity toward IL-2 release in both 12-o-tetradecanoylphorbol-13-acetate (PMA)/A23187 (ionomycin) (IC50=80±10nM) and anti-CD3/CD28-stimulated Jurkat T cells (83% inhibition at 10μM) without cytotoxic effects. Further investigation into the underlying mechanism of 11l indicated the suppression of NF-κB and nuclear factor of activated T cells (NFAT) promoter activities in Jurkat T cells.