Pentagamavunone-1 inhibits aggressive breast cancer cell proliferation through mitotic catastrophe and ROS-mediated activities: in vitro and in vivo studies.

Pentagamavunone-1 (PGV-1), an analog of curcumin, has been studied for its cytotoxic effects in 4T1, MCF7, MCF7/HER2, and T47D breast cancer cells. Its antiproliferative effect is partly mediated through G2/M arrest; however, its molecular mechanism during cell cycle progression remains unknown. In this study, we aimed to determine whether PGV-1 has any anticancer effects on highly aggressive breast cancer cells, with a focus on cell cycle regulatory activity, reactive oxygen species (ROS) generation, and their mediated effects on cancer cells. MDA-MB-231 (triple-negative) and HCC1954 (overexpressed HER2) immortalized human breast cancer cells were used in the study. PGV-1 exhibited cytotoxic activity with an irreversible antiproliferative impact on treated cells and had good selectivity when tested in fibroblast cells. Oral PGV-1 administration suppressed tumor growth in a cell-derived xenograft mouse model. PGV-1 induced the phosphorylation of Aurora A kinase and PLK1 in MDA-MB-231 cells, while PLK1 and cyclin B1 phosphorylation were enhanced in the PGV-1-treated HCC1954 cells during prometaphase arrest. Intracellular ROS production was substantially higher upon PGV-1 treatment following mitotic arrest, and this activity caused impairment of mitochondrial respiration, induced senescence, and subsequently triggered early-to-late apoptosis. Collectively, these results suggest that the molecular mechanism of PGV-1 involves the regulation of mitotic kinases to cause cell cycle arrest and the enhancement of ROS production to impair mitochondrial activity and induce cellular senescence. The therapeutic activities demonstrated by PGV-1 in this study show its potential as an appealing candidate for chemotherapy in breast cancer treatment.

Tumour-suppressive effects of curcumin analogs CCA-1.1 and Pentagamavunone-1 in colon cancer: In vivo and in vitro studies.

This study aimed to evaluate the efficacy of Chemoprevention Curcumin Analog-1.1 (CCA-1.1) and Pentagamavunone-1 (PGV-1) in vivo and in vitro in colorectal cancer model. CCA-1.1 or PGV-1 was administered orally to 1,2-dimethylhydrazine (DMH)-induced rats for 16 weeks. The cytotoxicity of both compounds was tested on Caco-2, CT26, and NIH/3T3 cells using the MTT method. The cell cycle, apoptosis, and reactive oxygen species (ROS) levels were analyzed through flow cytometry. X-gal staining was used to examine the compound's effect on senescence. Oral co-administration of CCA-1.1 or PGV-1 significantly suppressed the carcinogenic characteristics and symptoms of premalignant colon cancer relative to DMH-only and untreated groups. CCA-1.1 and PGV-1 administration did not affect the blood profile. CCA-1.1 and PGV-1 demonstrated great cytotoxicity on Caco-2 and CT26 cells, with 50% inhibition concentration (IC50) values of 4.3 ± 0.2 and 3.1 ± 0.1 µM for CCA-1.1 and 11.2 ± 1.1 and 4.8 ± 0.1 µM for PGV-1, respectively, while not toxic against fibroblast cells. Both compounds instigated G2/M arrest and efficiently induced cell senescence and apoptosis. Moreover, these analogs selectively elevated oxidative stress in colon cancer cells without inducing noticeable changes in fibroblasts. In conclusion, PGV-1 and CCA-1.1 suppressed colorectal tumor formation and induced mitotic arrest.

Chemoprevention curcumin analog 1.1 promotes metaphase arrest and enhances intracellular reactive oxygen species levels on TNBC MDA-MB-231 and HER2-positive HCC1954 cells.

Background and purpose: Previous studies highlighted that chemoprevention curcumin analog-1.1 (CCA-1.1) demonstrated an antitumor effect on breast, leukemia, and colorectal cancer cells. By utilizing immortalized MDA-MB-231 and HCC1954 cells, we evaluated the anticancer properties of CCA-1.1 and its mediated activity to promote cellular death. Experimental approach: Cytotoxicity and anti-proliferation were assayed using trypan blue exclusion. The cell cycle profile after CCA-1.1 treatment was established through flow cytometry. May-Grünwald-Giemsa and Hoechst staining were performed to determine the cell cycle arrest upon CCA-1.1 treatment. The involvement of CCA-1.1 in mitotic kinases (aurora A, p-aurora A, p-PLK1, and p-cyclin B1) expression was investigated by immunoblotting. CCA-1.1-treated cells were stained with the X-gal solution to examine the effect on senescence. ROS level and mitochondrial respiration were assessed by DCFDA assay and mitochondrial oxygen consumption rate, respectively. Findings/Results: CCA-1.1 exerted cytotoxic activity and inhibited cell proliferation with an irreversible effect, and the flow cytometry analysis demonstrated that CCA-1.1 significantly halted during the G2/M phase, and further assessment revealed that CCA-1.1 caused metaphase arrest. Immunoblot assays confirmed CCA-1.1 suppressed aurora A kinase in MDA-MB-231 cells. The ROS level was elevated after treatment with CCA-1.1, which might promote cellular senescence and suppress basal mitochondrial respiration in MDA-MB-231 cells. Conclusion and implications: Our data suggested the in vitro proof-of-concept that supports the involvement in cell cycle regulation and ROS generation as contributors to the effectiveness of CCA-1.1 in suppressing breast cancer cell growth.

Discovery of a potent and subtype-selective TYK2 degrader based on an allosteric TYK2 inhibitor.

TYK2, a member of the JAK family of proximal membrane-bound tyrosine kinases, has emerged as an attractive target for the treatment of autoimmune diseases. Herein, we report the discovery of first-in-class potent and subtype-selective TYK2 degraders. By conjugating a TYK2 ligand from a known allosteric TYK2 inhibitor with a VHL ligand as the E3 ligase ligand via alkyl linkers of various lengths, we rapidly identified TYK2 degrader 5 with moderate TYK2 degradation activity. Degrader 5 induced TYK2 degradation without affecting the protein level of subtype kinases (JAK1, JAK2, and JAK3) in Jurkat cellular assays. Furthermore, modifying the TYK2 ligand moiety of degrader 5 yielded the more potent TYK2 degrader 37 with retained selectivity for JAKs. Our subtype-selective TYK2 degraders represent valuable chemical probes for investigating the biology of TYK2 degradation.

Preclinical evaluation of pentagamavunone-1 as monotherapy and combination therapy for pancreatic cancer in multiple xenograft models.

We previously reported that pentagamavunone-1 (PGV-1) effectively inhibited cell proliferation in many types of human tumors, including pancreatic cancer, by inducing M phase (prometaphase) arrest, senescence, and apoptosis with few side effects. However, a detailed evaluation of the effects of PGV-1 on pancreatic cancer cells in an in vivo setting has not yet been conducted. The present study investigated the potential efficacy of PGV-1 as both monotherapy and combination therapy for pancreatic cancer using multiple xenograft mouse assays. A cell-line derived xenograft model (CDX-M) with pancreatic cancer cell line and a patient-derived xenograft mouse model (PDX-M) using resected pancreatic cancer samples without neoadjuvant chemotherapy were established in both heterotopic and orthotopic manners. PGV-1 effectively suppressed tumor formation at the heterotopic and orthotopic sites in CDX-M than in untreated mice. Combination therapy with PGV-1 and gemcitabine more effectively suppressed tumor formation than monotherapy with PGV-1 or gemcitabine when administered after tumor formation. Monotherapy with PGV-1 or gemcitabine less effectively suppressed tumor formation in PDX-M than in CDX-M, whereas combination therapy with PGV-1 and gemcitabine more effectively suppressed tumor formation. PGV-1 as monotherapy and combination therapy with gemcitabine effectively inhibited tumor formation and has potential as an anticancer candidate for pancreatic cancer.

Preparation and Cytotoxic Evaluation of PGV-1 Derivative, CCA-1.1, as a New Curcumin Analog with Improved-Physicochemical and Pharmacological Properties.

Purpose: This study aimed to challenge the anticancer potency of pentagamavunone-1 (PGV- 1) and obtain a new compound (Chemoprevention-Curcumin Analog 1.1, CCA-1.1) with improved chemical and pharmacological properties. Methods: CCA-1.1 was prepared by changing the ketone group of PGV-1 into a hydroxyl group with NaBH4 as the reducing agent. The product was purified under preparative layer chromatography and confirmed with HPLC to show about 93% purity. It was tested for its solubility, stability, and cytotoxic activities on several cancer cells. The structure of the product was characterized using 1HNMR, 13C-NMR, FT-IR, and HR-mass spectroscopy. Results: Molecular docking analysis showed that CCA-1.1 performed similar or better interaction to NF-κB pathway-related signaling proteins (HER2, EGFR, IKK, ER-alpha, and ER-beta) and reactive oxygen species (ROS) metabolic enzymes (NQO1, NQO2, GSTP1, AKC1R1, and GLO1) compared with PGV-1, indicating that CCA-1.1 exhibits the same or better anticancer activity than PGV-1. CCA-1.1 also showed better solubility and stability than PGV-1 in aqueous solution at pH 1.0-7.4 under light exposure at room temperature. The cytotoxic activities of CCA-1.1 against several (10) cancer cell lines revealed the same or better potency than PGV-1. Conclusion: In conclusion, CCA-1.1 performs better chemical and anticancer properties than PGV-1 and shows promise as an anticancer agent with high selectivity.

The integrative bioinformatic analysis deciphers the predicted molecular target gene and pathway from curcumin derivative CCA-1.1 against triple-negative breast cancer (TNBC).

BACKGROUND: The poor outcomes from triple-negative breast cancer (TNBC) therapy are mainly because of TNBC cells' heterogeneity, and chemotherapy is the current approach in TNBC treatment. A previous study reported that CCA-1.1, the alcohol-derivative from monocarbonyl PGV-1, exhibits anticancer activities against several cancer cells, as well as in TNBC. This time, we utilized an integrative bioinformatics approach to identify potential biomarkers and molecular mechanisms of CCA-1.1 in inhibiting proliferation in TNBC cells. METHODS: Genomics data expression were collected through UALCAN, derived initially from TCGA-BRCA data, and selected for TNBC-only cases. We predict CCA-1.1 potential targets using SMILES-based similarity functions across six public web tools (BindingDB, DINIES, Swiss Target Prediction, Polypharmacology browser/PPB, Similarity Ensemble Approach/SEA, and TargetNet). The overlapping genes between the CCA-1.1 target and TNBC (CPTGs) were selected and used in further assessment. Gene ontology (GO) enrichment and the Kyoto Encyclopedia of Genes and Genomes (KEGG) network analysis were generated in WebGestalt. The protein-protein interaction (PPI) network was established in STRING-DB, and then the hub-genes were defined through Cytoscape. The hub-gene's survival analysis was processed via CTGS web tools using TCGA database. RESULTS: KEGG pathway analysis pointed to cell cycle process which enriched in CCA-1.1 potential targets. We also identified nine CPTGs that are responsible in mitosis, including AURKB, PLK1, CDK1, TPX2, AURKA, KIF11, CDC7, CHEK1, and CDC25B. CONCLUSION: We suggested CCA-1.1 possibly regulated cell cycle process during mitosis, which led to cell death. These findings needed to be investigated through experimental studies to reinforce scientific data of CCA-1.1 therapy against TNBC.

CCA-1.1, a Novel Curcumin Analog, Exerts Cytotoxic anti- Migratory Activity toward TNBC and HER2-Enriched Breast Cancer Cells.

OBJECTIVE: Chemoprevention curcumin Analog-1.1 (CCA-1.1) demonstrates antineoplastic effect toward cancer cells. By using triple-negative breast cancer (TNBC), 4T1, and human epidermal growth factor receptor 2 (HER2)-enriched metastatic cells (MCF-7/HER2), we evaluate the cytotoxic and antimigration activities from CCA-1.1. METHODS: The cytotoxic activities from a single treatment of CCA-1.1 and in combination with doxorubicin were determined through MTT assay. We also calculated the selectivity index and combination index of CCA-1.1 from the cytotoxic data. Migrating cells were evaluated using wound healing assay, and the MMP2 and MMP9 secretion levels were determined through gelatin zymography. RESULTS: As hypothesized, CCA-1.1 performed cytotoxic activity during treatment in 4T1 and MCF-7/HER2 cancer cells with good selectivity (Selectivity Index >2). In addition, CCA-1.1 demonstrated a synergistic effect in combinatorial treatment with a low dose of doxorubicin. A single treatment of CCA-1.1 repressed cell migration in 4T1 and MCF-7/HER2 cells. Under gelatin zymography, CCA-1.1 subsided the activities of MMP-9, thereby revealing the potency of CCA-1.1 as an anti-migratory agent. Moreover, MMP-9 was also eminently expressed in TNBC and HER2-enriched breast cancer cells when compared with that in other subtypes. CONCLUSION: Our preliminary study collectively reinforces the potential effect of CCA-1.1 through the inhibition of highly aggressive cell migration, particularly in breast cancer.

Stabilization of fatty acid synthesis enzyme acetyl-CoA carboxylase 1 suppresses acute myeloid leukemia development.

Cancer cells reprogram lipid metabolism during their malignant progression, but limited information is currently available on the involvement of alterations in fatty acid synthesis in cancer development. We herein demonstrate that acetyl-CoA carboxylase 1 (ACC1), a rate-limiting enzyme for fatty acid synthesis, plays a critical role in regulating the growth and differentiation of leukemia-initiating cells. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPA, a transcription factor regulating myeloid differentiation, for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). We identified ACC1 as a target of the Trib1-COP1 complex and found that an ACC1 mutant resistant to degradation because of the lack of a Trib1-binding site attenuated complex-driven leukemogenesis. Stable ACC1 protein expression suppressed the growth-promoting activity and increased ROS levels with the consumption of NADPH in a primary bone marrow culture, and delayed the onset of AML with increases in mature myeloid cells in mouse models. ACC1 promoted the terminal differentiation of Trib1-COP1-expressing cells and eradicated leukemia-initiating cells in the early phase of leukemic progression. These results indicate that ACC1 is a natural inhibitor of AML development. The upregulated expression of the ACC1 protein has potential as an effective strategy for cancer therapy.

The Target Differences of Anti-Tumorigenesis Potential of Curcumin and its Analogues Against HER-2 Positive and Triple-Negative Breast Cancer Cells.

Purpose: The current study aims to evaluate the in vitro cytotoxic and cell migration effects of synthetic curcumin and its analogues on HER2 and nuclear factor kappa B (NFκB) pathways, as well as the in vivo inhibitory effect on cancer growth of metastatic breast cancer. Methods: Cell viability, protein expression, and protein localization were determined in vitro using MTT assay, western blotting, and immunofluorescence, respectively. Meanwhile, scratch wound healing assay and gelatin zymography were conducted to investigate the metastasis inhibitory effect. The in vivo anti-tumor ability was evaluated in xenograft mouse model using triple-negative breast cancer (TNBC) cells. Results: Curcumin, PGV-0, and PGV-1 exhibited cytotoxic effect against HER2-overexpressing breast cancer cells. Although PGV-1 showed the best activity in the single cytotoxic assay, curcumin showed the strongest synergism with doxorubicin. Curcumin and PGV-0 inhibited membrane localization of HER2. In contrast, PGV-1 neither inhibited localization nor decreased the expression of HER2, nonetheless showed the most potent inhibition against nuclear localization of p65 indicating the different mechanisms of curcumin, PGV-0, and PGV-1. Regarding cancer metastasis, curcumin and PGV-1 showed inhibitory activities against cell migration and inhibited MMP-2 and MMP-9 protein expression. Lastly, PGV-1 was more potent compared to curcumin to suppress the tumor formation of metastatic breast cancer xenograft model in nude mice. Conclusion: Overall, our study strengthens the potency of curcumin analogue, PGV-1, for treating several types of cancer, including metastatic breast cancer.

Curcumin Derivatives Verify the Essentiality of ROS Upregulation in Tumor Suppression.

BACKGROUND: Curcumin has been shown to exert pleiotropic biological effects, including anti-tumorigenic activity. We previously showed that curcumin controls reactive oxygen species (ROS) levels through the ROS metabolic enzymes, to prevent tumor cell growth. In this study, we synthesized 39 novel curcumin derivatives and examined their anti-proliferative and anti-tumorigenic properties. METHODS AND RESULTS: Thirty-nine derivatives exhibited anti-proliferative activity toward human cancer cell lines, including CML-derived K562 leukemic cells, in a manner sensitive to an antioxidant, N-acetyl-cysteine (NAC). Some compounds exhibited lower GI50 values than curcumin, some efficiently induced cell senescence, and others markedly increased ROS levels, efficiently induced cell death and suppressed tumor formation in a xenograft mouse model, without any detectable side effects. A clustering analysis of the selected compounds and their measurement variables revealed that anti-tumorigenic activity was most well-correlated with an increase in ROS levels. Pulldown assays and a molecular docking analysis showed that curcumin derivatives competed with co-enzymes to bind to the respective ROS metabolic enzymes and inhibited their enzymatic activities. CONCLUSIONS: The analysis of novel curcumin derivatives established the importance of ROS upregulation in suppression of tumorigenesis, and these compounds are potentially useful for the development of an anti-cancer drug with few side effects.

Pentagamavunon-1 (PGV-1) inhibits ROS metabolic enzymes and suppresses tumor cell growth by inducing M phase (prometaphase) arrest and cell senescence.

We previously showed that curcumin, a phytopolyphenol found in turmeric (Curcuma longa), targets a series of enzymes in the ROS metabolic pathway, induces irreversible growth arrest, and causes apoptosis. In this study, we tested Pentagamavunon-1 (PGV-1), a molecule related to curcumin, for its inhibitory activity on tumor cells in vitro and in vivo. PGV-1 exhibited 60 times lower GI50 compared to that of curcumin in K562 cells, and inhibited the proliferation of cell lines derived from leukemia, breast adenocarcinoma, cervical cancer, uterine cancer, and pancreatic cancer. The inhibition of growth by PGV-1 remained after its removal from the medium, which suggests that PGV-1 irreversibly prevents proliferation. PGV-1 specifically induced prometaphase arrest in the M phase of the cell cycle, and efficiently induced cell senescence and cell death by increasing intracellular ROS levels through inhibition of ROS-metabolic enzymes. In a xenograft mouse model, PGV-1 had marked anti-tumor activity with little side effects by oral administration, whereas curcumin rarely inhibited tumor formation by this administration. Therefore, PGV-1 is a potential therapeutic to induce tumor cell apoptosis with few side effects and low risk of relapse.

Anti-proliferative and Anti-metastatic Potential of Curcumin Analogue, Pentagamavunon-1 (PGV-1), Toward Highly Metastatic Breast Cancer Cells in Correlation with ROS Generation.

Purpose: Pentagamavunon-1 (PGV-1) is a curcumin analogue that shows cytotoxic activity in various cancer cells. In this study, we evaluated the effect of PGV-1 on a highly metastatic breast cancer cell line, the 4T1 cells, as an anti-metastatic and anti-proliferative agent. Methods: Cell viability was evaluated using MTT assay; while cell cycle profile, apoptosis incidence, and ROS intracellular level were determined by flow cytometry. Cell senescence was observed under senescence-associated-ß-galactosidase (SA-ß-gal) staining assay. The expression of matrixmetalloproteinase-9 (MMP-9) was determined using immunoreaction based-ELISA, while other proteins expression were detected using immunoblotting. Results: Curcumin and PGV-1 showed cytotoxic effects on 4T1 cells with IC50 value of 50 and 4 µM, respectively. The cytotoxic activity of PGV-1 was correlated to the induction of G2/M cell cycle arrest and cell senescence. Furthermore, PGV-1 increased the accumulation of intracellular ROS level. We also revealed that PGV-1 bound to several ROS-metabolizing enzymes, including glyoxalase I (GLO1), peroxiredoxin 1 (PRDX1), N-ribosyldihydronicotinamide: quinone reductase 2 (NQO2), aldo-keto reductase family 1 member c1 (AKR1C1). As an antimetastatic agent, PGV-1 showed less inhibitory effect on cell migration compared to curcumin. However, PGV-1 significantly decreased MMP-9 protein expression in a dose-dependent manner suggesting it still potent to inhibit metastatic cells. Conclusion: Overall, our findings suggest that PGV-1 is potential to be developed as an antiproliferative and anti-metastatic agent.

Cell Cycle Modulation of CHO-K1 Cells Under Genistein Treatment Correlates with Cells Senescence, Apoptosis and ROS Level but in a Dose-Dependent Manner.

Purpose: Genistein, a soy isoflavone, exhibits a biphasic effect on cells proliferation with some different effects between ER-alpha and ER-beta. The objective of this present study is to determine the modulatory effect based on cell cycle progression under genistein treatment in combination with 17-ß estradiol (E2) on CHO-K1 cells. Methods: The effect of genistein 0.1-100 µM on cells proliferation was examined by MTT assay. The modulation of genistein and estradiol (E2) on cell cycle and apoptosis were observed by using flowcytometry with PI and PI/AnnexinV staining, respectively. Moreover, the effect of genistein and E2 on senescence cells, and ROS level were determined by senescence-associated ß-galactosidase (SA ß-gal) staining and by using flowcytometry with 2', 7'-dichlorofluorescin diacetate (DCFDA) staining, respectively. The expression level of the cell cycle and senescence protein markers were observed by immunoblotting. Results: Single treatment of genistein at physiologically achievable (low) concentration (<2 µM) induced proliferation of CHO-K1 cells while at a pharmacological (high) concentration (50 and 100 µM) suppressed cells proliferation. Interestingly, treatment of genistein at the physiological concentration in combination with E2 for 24, 48 and 72 h decreased cells viability on CHO-K1 cells compared to untreated cells. Further analysis of the cells showed that 50 µM genistein induced G2/M phase accumulation and induced apoptosis. Moreover, genistein induced cell senescence and increased ROS level. Immunoblotting analysis showed the decreasing of ERalpha, Bcl2, and ppRb protein level upon treatment of 1 µM Gen and 1 nM E2. Conclusion: Our results suggest that the cell proliferation inhibitory mechanism of genistein at pharmacological concentration involved the induction of cell senescence, and the elevation of ROS level. Moreover, the decreased of cells proliferation upon treatment of physiological concentration of genistein in combination with E2 may be correlated with the alteration of ER expression.

Curcumin targets multiple enzymes involved in the ROS metabolic pathway to suppress tumor cell growth.

Curcumin has been reported to exhibit anti-tumorigenic activity; however, since its precise actions remain unclear, its effects are considered to be deceptive. In the present study, we confirmed the anti-tumorigenic effects of curcumin on CML-derived leukemic cells in a xenograft model and in vitro culture system. In vitro pull-down and mass analyses revealed a series of enzymes (carbonyl reductase, glutathione-S-transferase, glyoxalase, etc.) that function in a reactive oxygen species (ROS) metabolic pathway as curcumin-binding targets, the expression of which was up-regulated in human leukemia. Curcumin increased ROS levels over the threshold in leukemic cells, and the antioxidant, glutathione (GSH) and overexpression of curcumin-binding enzymes partially mitigated the up-regulation of ROS and growth inhibition caused by curcumin. These results show that curcumin specifically inhibits tumor growth by increasing ROS levels over the threshold through the miscellaneous inhibition of ROS metabolic enzymes. Curcumin has potential in therapy to regulate ROS levels in tumor cells, thereby controlling tumor growth.

Myeloid leukemia factor 1 stabilizes tumor suppressor C/EBPα to prevent Trib1-driven acute myeloid leukemia.

C/EBPα is a key transcription factor regulating myeloid differentiation and leukemogenesis. The Trib1-COP1 complex is an E3 ubiquitin ligase that targets C/EBPα for degradation, and its overexpression specifically induces acute myeloid leukemia (AML). Here we show that myeloid leukemia factor 1 (MLF1) stabilizes C/EBPα protein levels by inhibiting the ligase activity of the Trib1-COP1 complex. MLF1 directly interacts with COP1 in the nucleus and interferes with the formation of the Trib1-COP1 complex, thereby blocking its ability to polyubiquitinate C/EBPα for degradation. MLF1 overexpression suppressed the Trib1-induced growth advantage in a murine bone marrow (BM) culture and Trib1-induced AML development in BM-transplanted mouse models. MLF1 was expressed in hematopoietic stem cells and myeloid progenitors (common myeloid progenitors and granulocyte-macrophage progenitors) in normal hematopoiesis, which is consistent with the distribution of C/EBPα. An MLF1 deficiency conferred a more immature phenotype on Trib1-induced AML development. A higher expression ratio of Trib1 to MLF1 was a key determinant for AML development in mouse models, which was also confirmed in human patient samples with acute leukemia. These results indicate that MLF1 is a positive regulator that is critical for C/EBPα stability in the early phases of hematopoiesis and leukemogenesis.

Discovery of non-competitive thrombin inhibitor derived from competitive tryptase inhibitor skeleton: Shift in molecular recognition resulted from skeletal conversion of carboxylate into phosphonate.

A novel series of terminal and internal phosphonate esters based on our previously developed aryl carboxylate-type tryptase selective inhibitor 1 was synthesized. The potency of these synthesized compounds was assessed in vitro with an enzyme inhibition assay using three available serine proteases, that is, tryptase, trypsin, and thrombin. The internal phosphonate derivative 6 showed potent thrombin inhibitory activity with an IC50 value of 1.0 µM, whereas it exhibited no or only weak tryptase and trypsin inhibition at 10 µM. The Lineweaver-Burk plot analysis indicates that the inhibition pattern of thrombin with 6 is non-competitive in spite of the fact that the lead carboxylate compound 1 is competitive inhibitor. Therefore, the skeletal conversion of the carboxylate into a phosphonate alters the mode of molecular recognition of these inhibitors by thrombin.

Design and synthesis of 5-[(2-chloro-6-fluorophenyl)acetylamino]-3-(4-fluorophenyl)-4-(4-pyrimidinyl)isoxazole (AKP-001), a novel inhibitor of p38 MAP kinase with reduced side effects based on the antedrug concept.

Inhibitors of p38 mitogen-activated protein (MAP) kinase, which are closely involved in the production of inflammatory cytokines, are considered promising curative drugs for chronic inflammatory disorders. However, there is also a growing concern regarding its systemic side effects. To reduce the occurrence of side effects, we have identified a novel p38 MAP kinase inhibitor that shows properties of an antedrug, which imparts its effect solely on the inflammatory site and is metabolically inactivated right after. We have designed isoxazole derivatives through the addition of a fresh interacting fourth site to the structure of the prototypical p38 MAP kinase inhibitor that harbors three point interactive sites. The derivative 26d (AKP-001) shows excellent p38 MAP kinase inhibitory activity and a high selectivity for various kinases. Its rapid metabolism has been confirmed in rats. Moreover, 26d has been shown to be effective in animal models of inflammatory bowel disease.

COP1 targets C/EBPα for degradation and induces acute myeloid leukemia via Trib1.

The ubiquitin ligase constitutively photomorphogenic 1 (COP1) is involved in many biological responses in mammalian cells, but its role in tumorigenesis remains unclear. Here we show that COP1 is a ubiquitin ligase for the tumor suppressor CCAAT/enhancer-binding protein (C/EBPα) and promotes its degradation in vivo, thereby blocking myeloid differentiation of hematopoietic cells for tumorigenesis. In this process, mammalian homolog of Tribbles, Trib1, which contains a COP1-binding motif, is essential for down-regulation of C/EBPα expression. Murine bone marrow transplantation experiments showed that coexpression of COP1 accelerates development of acute myeloid leukemia induced by Trib1, which pathologically resembles that of p42C/EBPα-deficient mice. Interestingly, coexpression of ligase activity-deficient COP1 mutant abrogated Trib1-induced leukemogenesis. These results indicate that COP1 and Trib1 act as an oncoprotein complex functioning upstream of C/EBPα, and its ligase activity is crucial for leukemogenesis.

Novel strategy for synthesis of substituted benzimidazo[1,2-a]quinolines.

An efficient method for the synthesis of benzimidazo[1,2-a]quinolines under transition-metal-free conditions has been developed through a cascade reaction involving sequential aromatic nucleophilic substitution and intramolecular Knoevenagel condensation reactions. This method is applicable for the synthesis of a wide range of benzimidazo[1,2-a]quinoline derivatives from readily available 2-fluoroarylaldehyde and benzimidazole substrates.