Discovery of vanoxerine dihydrochloride as a CDK2/4/6 triple-inhibitor for the treatment of human hepatocellular carcinoma.

BACKGROUND: Cyclin-dependent kinases 2/4/6 (CDK2/4/6) play critical roles in cell cycle progression, and their deregulations are hallmarks of hepatocellular carcinoma (HCC). METHODS: We used the combination of computational and experimental approaches to discover a CDK2/4/6 triple-inhibitor from FDA approved small-molecule drugs for the treatment of HCC. RESULTS: We identified vanoxerine dihydrochloride as a new CDK2/4/6 inhibitor, and a strong cytotoxicdrugin human HCC QGY7703 and Huh7 cells (IC50: 3.79 µM for QGY7703and 4.04 µM for Huh7 cells). In QGY7703 and Huh7 cells, vanoxerine dihydrochloride treatment caused G1-arrest, induced apoptosis, and reduced the expressions of CDK2/4/6, cyclin D/E, retinoblastoma protein (Rb), as well as the phosphorylation of CDK2/4/6 and Rb. Drug combination study indicated that vanoxerine dihydrochloride and 5-Fu produced synergistic cytotoxicity in vitro in Huh7 cells. Finally, in vivo study in BALB/C nude mice subcutaneously xenografted with Huh7 cells, vanoxerine dihydrochloride (40 mg/kg, i.p.) injection for 21 days produced significant anti-tumor activity (p < 0.05), which was comparable to that achieved by 5-Fu (10 mg/kg, i.p.), with the combination treatment resulted in synergistic effect. Immunohistochemistry staining of the tumor tissues also revealed significantly reduced expressions of Rb and CDK2/4/6in vanoxerinedihydrochloride treatment group. CONCLUSIONS: The present study isthe first report identifying a new CDK2/4/6 triple inhibitor vanoxerine dihydrochloride, and demonstrated that this drug represents a novel therapeutic strategy for HCC treatment.

Discovery of a New CDK4/6 and PI3K/AKT Multiple Kinase Inhibitor Aminoquinol for the Treatment of Hepatocellular Carcinoma.

Background: Hepatocellular carcinoma (HCC) is a lethal malignancy lacking effective treatment. The Cyclin-dependent kinases 4/6 (CDK4/6) and PI3K/AKT signal pathways play pivotal roles in carcinogenesis and are promising therapeutic targets for HCC. Here we identified a new CDK4/6 and PI3K/AKT multi-kinase inhibitor for the treatment of HCC. Methods: Using a repurposing and ensemble docking methodology, we screened a library of worldwide approved drugs to identify candidate CDK4/6 inhibitors. By MTT, apoptosis, and flow cytometry analysis, we investigated the effects of candidate drug in reducing cell-viability,inducing apoptosis, and causing cell-cycle arrest. The drug combination and thermal proteomic profiling (TPP) method were used to investigate whether the candidate drug produced antagonistic effect. The in vivo anti-cancer effect was performed in BALB/C nude mice subcutaneously xenografted with Huh7 cells. Results: We demonstrated for the first time that the anti-plasmodium drug aminoquinol is a new CDK4/6 and PI3K/AKT inhibitor. Aminoquinol significantly decreased cell viability, induced apoptosis, increased the percentage of cells in G1 phase. Drug combination screening indicated that aminoquinol could produce antagonistic effect with the PI3K inhibitor LY294002. TPP analysis confirmed that aminoquinol significantly stabilized CDK4, CDK6, PI3K and AKT proteins. Finally, in vivo study in Huh7 cells xenografted nude mice demonstrated that aminoquinol exhibited strong anti-tumor activity, comparable to that of the leading cancer drug 5-fluorouracil with the combination treatment showed the highest therapeutic effect. Conclusion: The present study indicates for the first time the discovery of a new CDK4/6 and PI3K/AKT multi-kinase inhibitor aminoquinol. It could be used alone or as a combination therapeutic strategy for the treatment of HCC.

The antipsychotic agent flupentixol is a new PI3K inhibitor and potential anticancer drug for lung cancer.

Background: The phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway is hyperactivated in lung cancer and regulates a broad range of cellular processes, including proliferation, survival, angiogenesis, and metastasis. Thus PI3K is considered a promising target for therapy. To date, PI3K inhibitors have not been approved for lung cancer. Recent studies showed that the antipsychotic agent flupentixol induced apoptosis of lung cancer cell, however the anti-tumor mechanism of flupentixol remains unclear. Methods: (1) The idock software simulated the molecular docking between the PI3Kα protein and flupentixol. (2) Inhibition of PI3Kα by the flupentixol was examined by in vitro kinase assays. (3) The cytotoxicity of flupentixol on the NSCLC cell lines was tested by MTT assays. (4) We treated A549 and H661 cells with flupentixol and then measured the percentage of apoptotic cells by the Annexin V/PI analysis. (5) We investigated the effect of flupentixol on the expression of critical PI3K/AKT signaling pathway proteins, further analyzed on the cleavage of PARP and caspase-3 by Western blotting. (6) BALB/C nude mice were subcutaneously injected with A549 cells to evaluate the effect of flupentixol on the growth of lung carcinoma. Results: Structural analysis of the predicted binding conformation suggested that flupentixol docks to the ATP binding pocket of PI3Kα. Kinase assays demonstrate that flupentixol indeed inhibited the PI3Kα kinase activity. Flupentixol exhibited cytotoxicity in lung cancer cell lines A549 and H661 in a dose- and time-dependent manner. Furthermore, flupentixol more strongly inhibited the phosphorylation of AKT (T308 and S473) and the expression of its downstream target gene Bcl-2 than two known PI3K inhibitors (BYL719 and BKM120). Flupentixol induced apoptosis as measured by PARP and caspase-3 cleavage. Finally, flupentixol significantly suppressed A549 xenograft growth in BALB/C nude mice. Conclusions: Flupentixol could be docked to the PI3Kα protein and specifically inhibit the PI3K/AKT pathway and survival of lung cancer cells in vitro and in vivo. As an old drug, flupentixol is a new PI3K inhibitor that may be used for the treatment of lung cancers.

In silico prediction and in vitro and in vivo validation of acaricide fluazuron as a potential inhibitor of FGFR3 and a candidate anticancer drug for bladder carcinoma.

Bladder carcinoma (BC) is the ninth most common cause of cancer worldwide. Surgical resection and conventional chemotherapy and radiotherapy will ultimately fail due to tumor recurrence and resistance. Thus, the development of novel treatment is urgently needed. Fibroblast growth factor receptor 3 (FGFR3) is an important and well-established target for BC treatment. In this study, we utilized the free and open-source protein-ligand docking software idock to prospectively identify potential inhibitors of FGFR3 from 3,167 worldwide approved small-molecule drugs using a repositioning strategy. Six high-scoring compounds were purchased and tested in vitro. Among them, the acaricide drug fluazuron exhibited the highest antiproliferative effect in human BC cell lines RT112 and RT4. We further demonstrated that fluazuron treatment significantly increased the percentage of apoptosis cells, and decreased the phosphorylation level of FGFR3 and its downstream proteins FRS2-α, AKT, and ERK. We also investigated the anticancer effect of fluazuron in vivo in BALB/C nude mice subcutaneously xenografted with RT112 cells. Our results showed that oral treatment with fluazuron (80 mg/kg) significantly inhibited tumor growth. These results suggested for the first time that fluazuron is a potential inhibitor of FGFR3 and a candidate anticancer drug for the treatment of BC.

Econazole nitrate inhibits PI3K activity and promotes apoptosis in lung cancer cells.

The phosphatidylinositol-3-kinase (PI3K)/AKT signaling pathway plays a pivotal role in many cellular processes, including the proliferation, survival and differentiation of lung cancer cells. Thus, PI3K is a promising therapeutic target for lung cancer treatment. In this study, we applied free and open-source protein-ligand docking software, screened 3167 FDA-approved small molecules, and identified putative PI3Kα inhibitors. Among them, econazole nitrate, an antifungal agent, exhibited the highest activity in decreasing cell viability in pathological types of NSCLC cell lines, including H661 (large cell lung cancer) and A549 (adenocarcinoma). Econazole decreased the protein levels of p-AKT and Bcl-2, but had no effect on the phosphorylation level of ERK. It inhibited cell growth and promote apoptosis in a dose-dependent manner. Furthermore, the combination of econazole and cisplatin exhibited additive and synergistic effects in the H661 and A549 lung cancer cell lines, respectively. Finally, we demonstrated that econazole significantly suppressed A549 tumor growth in nude mice. Our findings suggest that econazole is a new PI3K inhibitor and a potential drug that can be used in lung cancer treatment alone or in combination with cisplatin.

Adapalene inhibits the activity of cyclin-dependent kinase 2 in colorectal carcinoma.

Cyclin-dependent kinase 2 (CDK2) has been reported to be overexpressed in human colorectal cancer; it is responsible for the G1­to­S­phase transition in the cell cycle and its deregulation is a hallmark of cancer. The present study was the first to use idock, a free and open­source protein­ligand docking software developed by our group, to identify potential CDK2 inhibitors from 4,311 US Food and Drug Administration­approved small molecular drugs with a re­purposing strategy. Among the top compounds identified by idock score, nine were selected for further study. Among them, adapalene (ADA; CD271,6­[3­(1­adamantyl)­4­methoxyphenyl]­2­naphtoic acid) exhibited the highest anti­proliferative effects in LOVO and DLD1 human colon cancer cell lines. Consistent with the expected properties of CDK2 inhibitors, the present study demonstrated that ADA significantly increased the G1­phase population and decreased the expression of CDK2, cyclin E and retinoblastoma protein (Rb), as well as the phosphorylation of CDK2 (on Thr­160) and Rb (on Ser­795). Furthermore, the anti­cancer effects of ADA were examined in vivo on xenograft tumors derived from DLD1 human colorectal cancer cells subcutaneously inoculated in BALB/C nude mice. ADA (20 mg/kg orally) exhibited marked anti­tumor activity, comparable to that of oxaliplatin (40 mg/kg), and dose­dependently inhibited tumor growth (P<0.05), while combined administration of ADA and oxaliplatin produced the highest therapeutic effect. To the best of our knowledge, the present study was the first to indicate that ADA inhibits CDK2 and is a potential candidate drug for the treatment of human colorectal cancer.

In Silico Identification and In Vitro and In Vivo Validation of Anti-Psychotic Drug Fluspirilene as a Potential CDK2 Inhibitor and a Candidate Anti-Cancer Drug.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Surgical resection and conventional chemotherapy and radiotherapy ultimately fail due to tumor recurrence and HCC's resistance. The development of novel therapies against HCC is thus urgently required. The cyclin-dependent kinase (CDK) pathways are important and well-established targets for cancer treatment. In particular, CDK2 is a key factor regulating the cell cycle G1 to S transition and a hallmark for cancers. In this study, we utilized our free and open-source protein-ligand docking software, idock, prospectively to identify potential CDK2 inhibitors from 4,311 FDA-approved small molecule drugs using a repurposing strategy and an ensemble docking methodology. Sorted by average idock score, nine compounds were purchased and tested in vitro. Among them, the anti-psychotic drug fluspirilene exhibited the highest anti-proliferative effect in human hepatocellular carcinoma HepG2 and Huh7 cells. We demonstrated for the first time that fluspirilene treatment significantly increased the percentage of cells in G1 phase, and decreased the expressions of CDK2, cyclin E and Rb, as well as the phosphorylations of CDK2 on Thr160 and Rb on Ser795. We also examined the anti-cancer effect of fluspirilene in vivo in BALB/C nude mice subcutaneously xenografted with human hepatocellular carcinoma Huh7 cells. Our results showed that oral fluspirilene treatment significantly inhibited tumor growth. Fluspirilene (15 mg/kg) exhibited strong anti-tumor activity, comparable to that of the leading cancer drug 5-fluorouracil (10 mg/kg). Moreover, the cocktail treatment with fluspirilene and 5-fluorouracil exhibited the highest therapeutic effect. These results suggested for the first time that fluspirilene is a potential CDK2 inhibitor and a candidate anti-cancer drug for the treatment of human hepatocellular carcinoma. In view of the fact that fluspirilene has a long history of safe human use, our discovery of fluspirilene as a potential anti-HCC drug may present an immediately applicable clinical therapy.