Rebamipide and Derivatives are Potent, Selective Inhibitors of Histidine Phosphatase Activity of the Suppressor of T Cell Receptor Signaling Proteins.

The suppressor of T cell receptor signaling (Sts) proteins are negative regulators of immune signaling. Genetic inactivation of these proteins leads to significant resistance to infection. From a 590,000 compound high-throughput screen, we identified the 2-(1H)-quinolinone derivative, rebamipide, as a putative inhibitor of Sts phosphatase activity. Rebamipide, and a small library of derivatives, are competitive, selective inhibitors of Sts-1 with IC50 values from low to submicromolar. SAR analysis indicates that the quinolinone, the acid, and the amide moieties are all essential for activity. A crystal structure confirmed the SAR and reveals key interactions between this class of compound and the protein. Although rebamipide has poor cell permeability, we demonstrated that a liposomal preparation can inactivate the phosphatase activity of Sts-1 in cells. These studies demonstrate that Sts-1 enzyme activity can be pharmacologically inactivated and provide foundational tools and insights for the development of immune-enhancing therapies that target the Sts proteins.

Human uridine 5'-monophosphate synthase stores metabolic potential in inactive biomolecular condensates.

Human uridine 5'-monophosphate synthase (HsUMPS) is a bifunctional enzyme that catalyzes the final two steps in de novo pyrimidine biosynthesis. The individual orotate phosphoribosyl transferase and orotidine monophosphate domains have been well characterized, but little is known about the overall structure of the protein and how the organization of domains impacts function. Using a combination of chromatography, electron microscopy, and complementary biophysical methods, we report herein that HsUMPS can be observed in two structurally distinct states, an enzymatically active dimeric form and a nonactive multimeric form. These two states readily interconvert to reach an equilibrium that is sensitive to perturbations of the active site and the presence of substrate. We determined that the smaller molecular weight form of HsUMPS is an S-shaped dimer that can self-assemble into relatively well-ordered globular condensates. Our analysis suggests that the transition between dimer and multimer is driven primarily by oligomerization of the orotate phosphoribosyl transferase domain. While the cellular distribution of HsUMPS is unaffected, quantification by mass spectrometry revealed that de novo pyrimidine biosynthesis is dysregulated when this protein is unable to assemble into inactive condensates. Taken together, our data suggest that HsUMPS self-assembles into biomolecular condensates as a means to store metabolic potential for the regulation of metabolic rates.

HI-511 overcomes melanoma drug resistance via targeting AURKB and BRAF V600E.

Rationale: Melanoma is an aggressive tumor of the skin and drug resistance is still a major problem in melanoma therapy. Novel targets and effective agents to overcome drug resistant melanoma are urgently needed in clinical therapy. Methods: Gene Expression Omnibus (GEO) database analysis, pathway enrichment analysis, and survival rate analysis were utilized to identify a candidate target. An anchorage-independent cell growth assay, flow cytometry, Western blot, and a xenograft mouse model were used to study the function of Aurora kinase B (AURKB) in both drug-sensitive and drug-resistant melanoma. Next, HI-511, a novel dual-target inhibitor targeting both AURKB and BRAF V600E, was designed and examined by an in vitro kinase assay. Methods as indicated above in addition to a BRAF V600E/PTEN-loss melanoma mouse model were used to demonstrate the effect of HI-511 on melanoma development in vitro and in vivo. Results: AURKB is highly expressed in melanoma and especially in vemurafenib-resistant melanoma and the expression was correlated with patient survival rate. Knocking down AURKB inhibited cell growth and induced apoptosis in melanoma, which was associated with the BRAF/MEK/ERKs and PI3-K/AKT signaling pathways. Importantly, we found that HI-511, a novel dual-target inhibitor against AURKB and BRAF V600E, suppresses both vemurafenib-sensitive and vemurafenib-resistant melanoma growth in vitro and in vivo by inducing apoptosis and mediating the inhibition of the BRAF/MEK/ERKs and PI3K/AKT signaling pathways. Conclusion: AURKB is a potential target for melanoma treatment. HI-511, a novel dual-target inhibitor against both AURKB and BRAF V600E, could achieve durable suppression of melanoma growth, even drug-resistant melanoma growth.

Discovery of a novel dual-target inhibitor against RSK1 and MSK2 to suppress growth of human colon cancer.

Colon cancer is the most aggressive tumor in both men and women globally. As many the chemotherapeutic regimens have adverse side effects and contribute to the resistance and recurrence, therefore, finding novel therapeutic targets and developing effective agents are urgent. Based on the TCGA and GTEx database analysis, RSK1 and MSK2 were found abnormal expressed in colon cancer. RSK1 and MSK2 were overexpressed in colon cancer tissues confirmed by western blot and IHC. After knocking down RSK1 or MSK2, cell proliferation and anchorage-independent cell growth were markedly inhibited. Using a computer docking model, we identified a novel dual-target inhibitor, APIO-EE-07, that could block both RSK1 and MSK2 kinase activity in a dose-dependent manner. APIO-EE-07 inhibited cell growth and induced apoptosis and also increased expression of Bax as well as cleaved caspase-3 and -PARP in colon cancer cells by downregulating RSK1 and MSK2 downstream targets, including CREB and ATF1. Furthermore, APIO-EE-07 decreased tumor volume and weight in human patient-derived xenografts tumors implanted in SCID mice. In summary, our results demonstrate that RSK1 and MSK2 are the potential targets for the treatment of colon cancer. APIO-EE-07, a novel dual-target inhibitor of RSK1 and MSK2, can suppress the growth of colon cancer by attenuating RSK1 and MSK2 signaling.

Suppression of the solar ultraviolet-induced skin carcinogenesis by TOPK inhibitor HI-TOPK-032.

Nonmelanoma skin cancer (NMSC) such as cutaneous squamous cell carcinoma (cSCC) is caused by solar ultraviolet (SUV) exposure and is the most common cancer in the United States. T-LAK cell-originated protein kinase (TOPK), a serine-threonine kinase is activated by SUV irradiation and involved in skin carcinogenesis. Strategies with research focusing on the TOPK signaling pathway and targeted therapy in skin carcinogenesis may helpful for the discovery of additional treatments against skin cancer. In this study, we found that TOPK can directly bind to and phosphorylate c-Jun (as one of the core member of AP-1) at Ser63 and Ser73 after SSL exposure in a JNKs-independent manner. TOPK knocking down, or HI-TOPK-032 (TOPK specific inhibitor) attenuated colony formation and cell proliferation of skin cancer cells. Phosphorylated levels of c-Jun were overexpressed in human AK and cSCC compared with normal skin tissues, and HI-TOPK-032 inhibited the phosphorylation of c-Jun in SCC cell line in a dose-dependent manner. Furthermore, HI-TOPK-032 decreased SSL-induced AP-1 transactivation activity. Moreover, acute SSL-induced inflammation was attenuated by the topical application of HI-TOPK-032 in SKH1 hairless mice. Importantly, HI-TOPK-032 suppressed chronic SSL-induced skin carcinogenesis and c-Jun phosphorylation levels in SKH1 hairless mice. Our results demonstrate that TOPK can phosphorylate and activate c-Jun at Ser63 and Ser73 in the process of skin carcinogenesis and HI-TOPK-032 could be used as a potential chemopreventive drug against cSCC development.

Aurora B kinase as a novel molecular target for inhibition the growth of osteosarcoma.

Osteosarcoma is the primary human malignant tumor affecting bone. This cancer most frequently arises in children and adolescents, with a second peak in those over the age of 50. Currently, surgery followed by radiotherapy and chemotherapy are the main treatments, but long-term positive effects are very poor. Aurora B kinase is a serine/threonine kinase that is a key regulator of cell cycle and mitosis. Tissue array analysis revealed that Aurora B kinase is overexpressed in osteosarcoma compared with normal bone tissue. We developed a compound, HOI-07 (i.e., (E)-3-((E)-4-(benzo[d] [1,3]dioxol-5-yl)-2-oxobut-3-en-1-ylidene)indolin-2-one), as a specific Aurora B kinase inhibitor and examined its effectiveness against osteosarcoma cell growth in this study. This compound inhibited Aurora B kinase activity in osteosarcoma and induced apoptosis, caused G2-M phase arrest, and attenuated osteosarcoma anchorage-independent cell growth. Moreover, knocking down the expression of Aurora B effectively reduced the sensitivity of osteosarcoma to HOI-07. Results of a xenograft mouse study indicated that HOI-07 treatment effectively suppressed the growth of 143B and KHOS xenografts, without affecting the body weight of mice. The expression of phosphorylated histone H3 (Ser10) was reduced in mice treated with HOI-07. Overall, we identified HOI-07 as a specific Aurora B kinase inhibitor for osteosarcoma treatment and this compound warrants further investigation.

The Ashitaba (Angelica keiskei) Chalcones 4-hydroxyderricin and Xanthoangelol Suppress Melanomagenesis By Targeting BRAF and PI3K.

Malignant melanoma is an aggressive tumor of the skin and still lacks effective preventive and therapeutic treatments. In melanoma, both the BRAF/MEK/ERK and PI3-K/AKT signaling pathways are constitutively activated through multiple mechanisms, which result in cell-cycle progression and prevention of apoptosis. Therefore, the development of novel strategies for targeting BRAF and PI3K are of utmost importance. In this study, we found that Ashitaba (Angelica keiskei) chalcones, 4-hydroxyderricin (4HD) and xanthoangelol (XAG), suppressed melanoma development by directly targeting both BRAFV600E and PI3K, which blocked the activation of downstream signaling. This led to the induction of G1 phase cell-cycle arrest and apoptosis in melanoma cells. Importantly, 4HD or XAG dramatically attenuated tumor incidence and volume in the BRAF-activated Pten-deficient melanoma mouse model. Our findings suggest that 4HD and XAG are promising chemopreventive or potential therapeutic agents against melanomagenesis that act by targeting both BRAF and PI3K, providing hope for rapid clinical translation. Cancer Prev Res; 11(10); 607-20. ©2018 AACR.

A Small Molecule Inhibitor of the ß-Catenin-TCF4 Interaction Suppresses Colorectal Cancer Growth In Vitro and In Vivo.

Colorectal cancer is associated with aberrant activation of the Wnt pathway. ß-Catenin plays essential roles in the Wnt pathway by interacting with T-cell factor 4 (TCF4) to transcribe oncogenes. We synthesized a small molecule, referred to as HI-B1, and evaluated signaling changes and biological consequences induced by the compound. HI-B1 inhibited ß-catenin/TCF4 luciferase activity and preferentially caused apoptosis of cancer cells in which the survival is dependent on ß-catenin. The formation of the ß-catenin/TCF4 complex was disrupted by HI-B1 due to the direct interaction of HI-B1 with ß-catenin. Colon cancer patient-derived xenograft (PDX) studies showed that a tumor with higher levels of ß-catenin expression was more sensitive to HI-B1 treatment, compared to a tumor with lower expression levels of ß-catenin. The different sensitivities of PDX tumors to HI-B1 were dependent on the ß-catenin expression level and potentially could be further exploited for biomarker development and therapeutic applications against colon cancer.

APIO-EE-9 is a novel Aurora A and B antagonist that suppresses esophageal cancer growth in a PDX mouse model.

Esophageal cancer (EC) is one of the most aggressive malignancies of the upper aerodigestive tract. Over the past three decades, with advances in surgical techniques and treatment, the prognosis of esophageal cancer has only slowly improved. Thus identifying novel molecular targets and developing therapeutic agents are critical. Aurora kinases play a crucial role in mitosis and selective inhibitors might provide an effective therapeutic treatment for cancer. However, the role of Aurora kinases in EC is still inadequately studied. Here, we identified a novel compound, referred to as APIO-EE-9, which inhibits growth and colony formation and induces apoptosis of esophageal cancer cells. Using computer modeling, we found that APIO-EE-9 interacted with both Aurora A and B in the ATP-binding pocket. APIO-EE-9 inhibited both Aurora A and B kinase activities in a dose-dependent manner. Treatment with APIO-EE-9 substantially reduced the downstream Aurora kinase phosphorylation of histone H3 (Ser10), resulting in formation of multiple nuclei and centrosomes. Additionally, esophageal cancer cells expressing shAurora A or shAurora B kinase exhibited a dramatic reduction in proliferation and colony formation. Injection of these cells as xenografts in mice reduced tumor formation compared to wildtype cells. Importantly, APIO-EE-9 significantly decreased the size of esophageal patient-derived xenograft (PDX) tumors implanted in SCID mice. These results demonstrated that APIO-EE-9 is a specific Aurora kinase inhibitor that could be developed as a therapeutic agent against esophageal cancer.

ADA-07 Suppresses Solar Ultraviolet-Induced Skin Carcinogenesis by Directly Inhibiting TOPK.

Cumulative exposure to solar ultraviolet (SUV) irradiation is regarded as the major etiologic factor in the development of skin cancer. The activation of the MAPK cascades occurs rapidly and is vital in the regulation of SUV-induced cellular responses. The T-LAK cell-originated protein kinase (TOPK), an upstream activator of MAPKs, is heavily involved in inflammation, DNA damage, and tumor development. However, the chemopreventive and therapeutic effects of specific TOPK inhibitors in SUV-induced skin cancer have not yet been elucidated. In the current study, ADA-07, a novel TOPK inhibitor, was synthesized and characterized. Pull-down assay results, ATP competition, and in vitro kinase assay data revealed that ADA-07 interacted with TOPK at the ATP-binding pocket and inhibited its kinase activity. Western blot analysis showed that ADA-07 suppressed SUV-induced phosphorylation of ERK1/2, p38, and JNKs and subsequently inhibited AP-1 activity. Importantly, topical treatment with ADA-07 dramatically attenuated tumor incidence, multiplicity, and volume in SKH-1 hairless mice exposed to chronic SUV. Our findings suggest that ADA-07 is a promising chemopreventive or potential therapeutic agent against SUV-induced skin carcinogenesis that acts by specifically targeting TOPK. Mol Cancer Ther; 16(9); 1843-54. ©2017 AACR.

PPMP, a novel tubulin-depolymerizing agent against esophageal cancer in patient-derived tumor xenografts.

Esophageal cancer is one of the least studied and deadliest cancers worldwide with a poor prognosis due to limited options for treatment. Chemotherapy agents such as the microtubule-targeting compounds are the mainstay of palliation for advanced esophageal cancer treatment. However, the toxicity and side effects of tubulin-binding agents (TBAs) have promoted the development of novel, more potent but less toxic TBAs. Herein, we identified 2-[4-(3,4-dimethoxyphenyl)-3-methyl-1H-pyrazol-5-yl]-5-[(2-methylprop-2-en-1-yl)oxy] phenol (PPMP) as a novel TBA for esophageal cancer treatment. PPMP markedly inhibited tubulin polymerization, and decreased viability and anchorage-independent growth of esophageal cancer cell lines, effects that were accompanied by G2/M arrest and apoptosis. Importantly, we produced patient-derived esophageal cancer xenografts to evaluate the therapeutic effect of PPMP in a setting that best mimics the clinical context in patients with esophageal cancer. Overall, we identified PPMP as a novel microtubule-destabilizing compound and as a new therapeutic agent against esophageal carcinoma.

Herbacetin Is a Novel Allosteric Inhibitor of Ornithine Decarboxylase with Antitumor Activity.

Ornithine decarboxylase (ODC) is a rate-limiting enzyme in the first step of polyamine biosynthesis that is associated with cell growth and tumor formation. Existing catalytic inhibitors of ODC have lacked efficacy in clinical testing or displayed unacceptable toxicity. In this study, we report the identification of an effective and nontoxic allosteric inhibitor of ODC. Using computer docking simulation and an in vitro ODC enzyme assay, we identified herbacetin, a natural compound found in flax and other plants, as a novel ODC inhibitor. Mechanistic investigations defined aspartate 44 in ODC as critical for binding. Herbacetin exhibited potent anticancer activity in colon cancer cell lines expressing high levels of ODC. Intraperitoneal or oral administration of herbacetin effectively suppressed HCT116 xenograft tumor growth and also reduced the number and size of polyps in a mouse model of APC-driven colon cancer (ApcMin/+). Unlike the well-established ODC inhibitor DFMO, herbacetin treatment was not associated with hearing loss. Taken together, our findings defined the natural product herbacetin as an allosteric inhibitor of ODC with chemopreventive and antitumor activity in preclinical models of colon cancer, prompting its further investigation in clinical trials.

244-MPT overcomes gefitinib resistance in non-small cell lung cancer cells.

The epidermal growth factor receptor (EGFR) is known to play a critical role in non-small cell lung cancer(NSCLC). Several EGFR tyrosine kinase inhibitors(TKIs), such as gefitinib, have been used as effective clinical therapies for patients with NSCLC. Unfortunately, acquired resistance to gefitinib commonly occurs after 6-12 months of treatment. The resistance is associated with the appearance of the L858R/T790M double mutation of the EGFR. In our present study, we discovered a compound,referred to as 244-MPT, which could suppress either gefitinib-sensitive or -resistant lung cancer cell growth and colony formation, and also suppressed the kinase activity of both wildtype and double mutant (L858R/T790M) EGFR. The underlying mechanism reveals that 244-MPT could interact with either the wildtype or double-mutant EGFR in an ATP-competitive manner and inhibit activity. Treatment with 244-MPT could substantially reduce the phosphorylation of EGFR and its downstream signaling pathways, including Akt and ERK1/2 in gefitinib-sensitive and -resistant cell lines. It was equally effective in suppressing EGFR phosphorylation and downstream signaling in NL20 cells transfected with wildtype, single-mutant (L858R) or mutant (L858R/T790M) EGFR. 244-MPT could also induce apoptosis in a gefitinib-resistant cell line and strongly suppress gefitinib-resistant NSCLC tumor growth in a xenograft mouse model. In addition, 244-MPT could effectively reduce the size of tumors in a gefitinib-resistant NSCLC patient-derived xenograft (PDX) SCID mouse model. Overall, 244-MPT could overcome gefitinib-resistance by directly targeting the EGFR.

3,6,2',4',5'-Pentahydroxyflavone, an orally bioavailable multiple protein kinase inhibitor, overcomes gefitinib resistance in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) is the most lethal cancer, causing more than 150,000 deaths in the United States in 2013. The receptor tyrosine kinase inhibitors such as gefitinib are not perfect clinical therapeutic agents for NSCLC treatment due to primary or acquired tyrosine kinase inhibitor resistance. Herein, 3,6,2',4',5'-pentahydroxyflavone (36245-PHF) was identified as a multiple kinase inhibitor for NSCLC treatment based on the computational screening of a natural products database. 36245-PHF was shown to inhibit PI3K and Aurora A and B kinases and overcome gefitinib-resistant NSCLC growth. Our data clearly showed that 36245-PHF markedly inhibited anchorage-independent growth of gefitinib-resistant NSCLC cell lines and exerted a substantial chemotherapeutic effect following oral administration in a gefitinib-resistant NSCLC xenograft model. The evidence from three different subsequent methodological approaches, in vitro, ex vivo, and in vivo, all confirmed that 36245-PHF as a multiple protein kinase inhibitor. Overall, we identified 36245-PHF as a multiple protein kinase inhibitor and as a novel therapeutic agent to overcome gefitinib-resistant NSCLC growth, which could provide a new option for clinical NSCLC oral treatment.

[6]-shogaol inhibits growth and induces apoptosis of non-small cell lung cancer cells by directly regulating Akt1/2.

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality worldwide. Despite progress in developing chemotherapeutics for the treatment of NSCLC, primary and secondary resistance limits therapeutic success. NSCLC cells exhibit multiple mutations in the epidermal growth factor receptor (EGFR), which cause aberrant activation of diverse cell signaling pathways. Therefore, suppression of the inappropriate amplification of EGFR downstream signaling cascades is considered to be a rational therapeutic and preventive strategy for the management of NSCLC. Our initial molecular target-oriented virtual screening revealed that the ginger components, including [6]-shogaol, [6]-paradol and [6]-gingerol, seem to be potential candidates for the prevention and treatment of NSCLC. Among the compounds, [6]-shogaol showed the greatest inhibitory effects on the NSCLC cell proliferation and anchorage-independent growth. [6]-Shogaol induced cell cycle arrest (G1 or G2/M) and apoptosis. Furthermore, [6]-shogaol inhibited Akt kinase activity, a downstream mediator of EGFR signaling, by binding with an allosteric site of Akt. In NCI-H1650 lung cancer cells, [6]-shogaol reduced the constitutive phosphorylation of signal transducer and activator of transcription-3 (STAT3) and decreased the expression of cyclin D1/3, which are target proteins in the Akt signaling pathway. The induction of apoptosis in NCI-H1650 cells by [6]-shogaol corresponded with the cleavage of caspase-3 and caspase-7. Moreover, intraperitoneal administration of [6]-shogaol inhibited the growth of NCI-H1650 cells as tumor xenografts in nude mice. [6]-Shogaol suppressed the expression of Ki-67, cyclin D1 and phosphorylated Akt and STAT3 and increased terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling-positivity in xenograft tumors. The current study clearly indicates that [6]-shogaol can be exploited for the prevention and/or treatment of NSCLC.

The P110 subunit of PI3-K is a therapeutic target of acacetin in skin cancer.

The identification of primary molecular targets of cancer-preventive phytochemicals is essential for a comprehensive understanding of their mechanism of action. In the present study, we investigated the chemopreventive effects and molecular targets of acacetin, a flavonoid found in Robinia p seudoacacia, also known as black locust. Acacetin treatment significantly suppressed epidermal growth factor (EGF)-induced cell transformation. Immunoblot analysis revealed that acacetin attenuated EGF-induced phosphorylation of Akt and p70(S6K), which are downstream effectors of phosphatidylinositol 3-kinase (PI3-K). An immunoprecipitation kinase assay of PI3-K and pull-down assay results demonstrated that acacetin substantially inhibits PI3-K activity by direct physical binding. Acacetin exhibited stronger inhibitory effects against anchorage-dependent and -independent cell growth in cells expressing higher PI3-K activity compared with those exhibiting relatively low PI3-K activity. Binding assay data combined with computational modeling suggest that acacetin binds in an adenosine triphosphate (ATP)-competitive manner with the p110α subunit of PI3-K and interacts with Val828, Glu826, Asp911, Trp760, Ile777, Ile825, Tyr813, Ile910 and Met900 residues. Acacetin was also found to significantly reduce SK-MEL-28 tumor growth and Akt phosphorylation in vivo. Taken together, these results indicate that acacetin is an ATP-competitive PI3-K inhibitor and a promising agent for melanoma chemoprevention.

USP8 is a novel target for overcoming gefitinib resistance in lung cancer.

PURPOSE: Common treatment modalities for non-small cell lung cancer (NSCLC) involve the EGF receptor-tyrosine kinase inhibitors (EGFR-TKIs) like gefitinib and erlotinib. However, the vast majority of treated patients acquire resistance to EGFR-TKIs, due, in large part, to secondary mutations in EGFR or amplification of the MET gene. Our purpose was to test ubiquitin-specific peptidase 8 (USP8) as a potential therapeutic target for gefitinib-resistant and -sensitive non-small cell lung cancer (NSCLC). EXPERIMENTAL DESIGN: Testing the effect of knockdown of USP8 and use of a synthetic USP8 inhibitor to selectively kill gefitinib-resistant (or -sensitive) NSCLCs with little effect on normal cells in cell culture and a xenograft mouse model. RESULTS: Knockdown of ubiquitin-specific peptidase 8 (USP8) selectively kills gefitinib-resistant NSCLCs while having little toxicity toward normal cells. Genetic silencing of USP8 led to the downregulation of several receptor tyrosine kinases (RTK) including EGFR, ERBB2, ERBB3, and MET. We also determined that a synthetic USP8 inhibitor markedly decreased the viability of gefitinib-resistant and -sensitive NSCLC cells by decreasing RTK expression while having no effect on normal cells. Moreover, treatment with a USP8 inhibitor led to significant reductions in tumor size in a mouse xenograft model using gefitinib-resistant and -sensitive NSCLC cells. CONCLUSIONS: Our results show for the first time that the inhibition of USP8 activity or reduction in USP8 expression can selectively kill NSCLC cells. We propose USP8 as a potential therapeutic target for gefitinib-resistant and -sensitive NSCLC cells.

Discovery of the novel mTOR inhibitor and its antitumor activities in vitro and in vivo.

The phosphoinositide 3-kinase (PI3-K)/Akt and mTOR signaling pathway plays a critical role in cell survival and proliferation and is often aberrantly activated in many types of cancer. The mTOR kinase protein, one of the key molecules in this pathway, has been shown to be an important target for cancer therapy. In the present study, a ligand docking method was used to screen for novel scaffold mTOR inhibitors. Sixty thousand compounds in the Natural Product Database were screened against the mTOR homologous structure, and 13 commercially available compounds listed in the top-ranked 100 compounds were selected for further examination. Compound [(E)-3-(4-(benzo[d][1,3]dioxol-5-yl)-2-oxobut-3-en-1-yl)- 3-hydroxyindolin-2-one; designated herein as 3HOI-BA-01] was then selected for further study of its antitumor activity. An in vitro study has shown that 3HOI-BA-01 inhibited mTOR kinase activity in a dose-dependent manner by directly binding with mTOR. In a panel of non-small cell lung cancer cells, the compound also attenuated mTOR downstream signaling, including the phosphorylation of p70S6K, S6, and Akt, resulting in G1 cell-cycle arrest and growth inhibition. Results of an in vivo study have shown that intraperitoneal injection of 3HOI-BA-01 in A549 lung tumor-bearing mice effectively suppressed cancer growth without affecting the body weight of the mice. The expression of downstream signaling molecules in the mTOR pathway in tumor tissues was also reduced after 3HOI-BA-01 treatment. Taken together, we identified 3HOI-BA-01 as a novel and effective mTOR inhibitor.

CInQ-03, a novel allosteric MEK inhibitor, suppresses cancer growth in vitro and in vivo.

The mitogen-activated protein kinase kinase 1 and 2 signaling pathway is a major component of the RAS (Rat sarcoma)/RAF (Radpidly accelerated fibrosarcoma)/MEK (mitogen-activated protein kinase kinase)/ERKs (Extracellular signal-regulated kinases) signaling axis that regulates tumorigenesis and cancer cell growth. MEK is frequently activated in various cancers that have mutations in the KRAS and BRAF oncogenes. Therefore, MEK has been suggested as a therapeutic target for inhibitor development against tumors that are dependent on the activating mutations in mitogen-activated protein kinase signaling. Herein, we report the discovery of three novel MEK inhibitors, herein referred to as CInQ-01, CInQ-03 and CInQ-06. All three inhibitors were highly effective in suppressing MEK1 and MEK2 in vitro kinase activity as well as anchorage-dependent and anchorage-independent cell growth. The inhibitory activity was associated with markedly reduced phosphorylation of ERKs and ribosomal S6 kinases. Furthermore, administration of CInQ-03 inhibited colon cancer cell growth in an in vivo xenograft mouse model and showed no skin toxicity. Overall, these results suggest that these novel MEK inhibitors might be used for chemotherapy or prevention.

Identification of an Aurora kinase inhibitor specific for the Aurora B isoform.

Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. In the present study, we used a ligand docking method to explore the novel scaffold of potential Aurora B inhibitors. One thousand compounds from our in-house compound library were screened against the Aurora B structure and one compound, (E)-3-((E)-4-(benzo[d][1,3]dioxol-5-yl)-2-oxobut-3-en-1-ylidene)indolin-2-one (designated herein as HOI-07) was selected for further study. HOI-07 potently inhibited in vitro Aurora B kinase activity in a dose-dependent manner, without obvious inhibition of another 49 kinases, including Aurora A. This compound suppressed Aurora B kinase activity in lung cancer cells, evidenced by the inhibition of the phosphorylation of histone H3 on Ser10 in a dose- and time-dependent manner. This inhibition resulted in apoptosis induction, G(2)-M arrest, polyploidy cells, and attenuation of cancer cell anchorage-independent growth. Moreover, knocking down the expression of Aurora B effectively reduced the sensitivity of cancer cells to HOI-07. Results of an in vivo xenograft mouse study showed that HOI-07 treatment effectively suppressed the growth of A549 xenografts, without affecting the body weight of mice. The expression of phospho-histone H3, phospho-Aurora B, and Ki-67 was also suppressed in the HOI-07 treatment group. Taken together, we identified HOI-07 as a specific Aurora B inhibitor, which deserves further investigation.