Tumor Cell Growth Inhibitory Activity Research Articles

De Novo Class of Momordicoside with Potent and Selective Tumor Cell Growth Inhibitory Activity as Pyruvate Kinase Muscle Isozyme 2 and Anti-apoptotic Myeloid Leukemia 1 Inhibitors

The difficulty in treating cancer resides in targeting abnormal proliferation while protecting normal proliferation, necessitating a thorough comprehension of the normal and malignant mechanisms that promote cell growth and proliferation. Targeting cell death signaling pathways such as glycolytic and mitochondrial apoptosis is the hallmark of many cancers, the aim of which this research is ready to evaluate. Atomistic molecular dynamics simulation of top hits after molecular docking and ADMET profiling of the ligands were performed for main protease-hit complexes. Docking scores of ligands used against PKM2 range from –9.36 to –12.1 kcal/mol, wherein momordicoside-F2 had the highest score (2.1 kcal/mol), performing better than the FDA-approved drug benserazide. Likewise, the scores ranged between –8.51 and –12.05 kcal/mol for Anti-apoptotic Myeloid Leukemia 1 (MCL-1), with momordicoside-F1 being the highest-ranked compound. The RMSD plots depicted stable trajectories with consistent and minor fluctuations implying that the protein (PKM2 and MCL1) backbone underwent minor structural perturbations. In addition, several significant peaks of increased fluctuations were also observed, indicating their increased interaction potential, implying that the ligands could adapt well in the protein's binding pocket. The SASA analysis results show that the ligands retained inside their shallow binding pocket. The phylogenetic tree obtained implies the likelihood of recurring results of the in silico profiling. Conclusively, this research unveils that Mormordicoside F1 shows good stability with MCL-1, likewise, momordicoside-F2 against PKM2. These hits can be a better re-purposing option.

Design, synthesis, anticancer evaluation, molecular docking, and in silico ADME analysis of novel chalcones incorporated indole-pyrimidine derivatives as promising anticancer agents

A novel library of aryl rings attached chalcone derivatives of indole-pyrimidines ( 10a-j ) were designed, synthesized, and evaluated for anticancer activities against four human cancer cell lines such as PC3, DU-145 (prostate), A549 (lung), and (MCF-7 (breast) by employing MTT method. The results of the MTT assay showed that all the synthetic compounds had some tumor cell growth inhibitory activities with IC 50 values range from 0.01±0.005 µM to 14.6 ± 6.32 µM, and standard drug with values 1.97 ± 0.45 µM to 3.08 ± 0.135 µM, respectively. Among them, compounds ( 10a, 10b, 10c, 10d , and 10e ) displayed remarkable activities, and out of which compounds 10a and 10d were found to be the most promising with potential anti-cancer activity. Molecular docking studies of the compounds were carried out against SARM (PDB ID: 3V49) and the docking results exclusively proposed binding modes in the active site of SARM. Among all, 10a, 10b, 10d , 10 h and 10j showed comparable dock score, 10a and 10b (-142.89 and -145.13) displayed pronounced interactions when compared with co-crystal ligand (-143.40) . Finally, in silico predictions of ADME and pharmacokinetic parameters indicated that all these compounds could be considered as drug candidates with oral bioavailability.

Semi-Synthesis of Small Molecules of Aminocarbazoles: Tumor Growth Inhibition and Potential Impact on p53.

The tumor suppressor p53 is inactivated by mutation in approximately 50% of human cancers. Small molecules that bind and stabilize those mutants may represent effective anticancer drugs. Herein, we report the tumor cell growth inhibitory activity of carbazole alkaloids and amino derivatives, as well as their potential activation of p53. Twelve aminocarbazole alkaloids were semi-synthesized from heptaphylline (1), 7-methoxy heptaphylline (2), and 7-methoxymukonal (3), isolated from Clausena harmandiana, using a reductive amination protocol. Naturally-occurring carbazoles 1–3 and their amino derivatives were evaluated for their potential effect on wild-type and mutant p53 activity using a yeast screening assay and on human tumor cell lines. Naturally-occurring carbazoles 1–3 showed the most potent growth inhibitory effects on wild-type p53-expressing cells, being heptaphylline (1) the most promising in all the investigated cell lines. However, compound 1 also showed growth inhibition against non-tumor cells. Conversely, semi-synthetic aminocarbazole 1d showed an interesting growth inhibitory activity in tumor cells expressing both wild-type and mutant p53, exhibiting low growth inhibition on non-tumor cells. The yeast assay showed a potential reactivation of mutant p53 by heptaphylline derivatives, including compound 1d. The results obtained indicate that carbazole alkaloids may represent a promising starting point to search for new mutp53-reactivating agents with promising applications in cancer therapy.

Synthesis, DNA/RNA-interaction and biological activity of benzo[k,l]xanthene lignans

Interactions of two newly synthesized and six previously reported benzoxanthene lignans (BXLs), analogues of rare natural products, with DNA/RNA, G-quadruplex and HSA were evaluated by a set of spectrophotometric methods. Presence/absence of methoxy and hydroxy groups on the benzoxanthene core and minor modifications at C-1/C-2 side pendants – presence/absence of phenyl ring and presence/absence of methoxy and hydroxy groups on phenyl ring – influenced the fluorescence changes and the binding strength to double-stranded (ds-) and G-quadruplex structures. In general, compounds without phenyl ring showed stronger fluorescence changes upon binding than phenyl-substituted BXLs. On the other hand, BXLs with an unsubstituted phenyl ring showed the best stabilization effects of G-quadruplex. Circular dichroism spectroscopy results suggest mixed binding mode, groove binding and partial intercalation, to ds-DNA/RNA and end-stacking to top or bottom G-tetrads as the main binding modes of BXLs to those targets. All compounds exhibited micromolar binding affinities toward HSA and an increased protein thermal stability. Moderate to strong antiradical scavenging activity was observed for all BXLs with hydroxy groups at C-6, C-9 and C-10 positions of the benzoxanthene core, except for derivative bearing methoxy groups at these positions. BXLs with unsubstituted or low-substituted phenyl ring and one derivative without phenyl ring showed strong growth inhibition of Gram-positive Staphylococcus aureus. All compounds showed moderate to strong tumor cell growth-inhibitory activity and cytotoxicity.

Design, synthesis and biological evaluation of novel potent STAT3 inhibitors based on BBI608 for cancer therapy

Persistently activated signal transducer and activator of transcription 3 (STAT3) plays an important role in the development of multiple cancers, and therefore is a potential therapeutic target for cancer prevention. Herein, we report the rational design, synthesis, and biological evaluation of novel potent STAT3 inhibitors based on BBI608. Among them, compound A11 exhibited the most potent in vitro tumor cell growth inhibitory activities toward MDA-MB-231, MDA-MB-468 and HepG2 cells with IC50 values as low as 0.67 ± 0.02 μM, 0.77 ± 0.01 μM and 1.24 ± 0.16 μM, respectively. Fluorescence polarization (FP) assay validated the binding of compound A11 in STAT3 SH2 domain with the IC50 value of 5.18 μM. Further mechanistic studies indicated that A11 inhibited the activation of STAT3 (Y705), and thus reduced the expression of STAT3 downstream genes CyclinD1 and C-Myc. Simultaneously, it induced cancer cell S phase arrest and apoptosis in a concentration-dependent manner. An additional in vivo study revealed that A11 suppressed the MDA-MB-231 xenograft tumor growth in mice at the dose of 10 mg/kg (i.p.) without obvious body-weight loss. Finally, molecular docking study further elucidated the binding mode of A11 in STAT3 SH2 domain.

Abstract A106: A peptide based on transmembrane domain of S1PR4 as a tumor cell growth inhibitor

Abstract Background: Many anti-tumor agents are used in chemotherapy for cancers. However, they also have serious side effects because they express similar growth inhibitory activity in normal cells. Meanwhile, the development of molecular target drugs inhibiting biomolecules relating to tumor growth, invasion, and metastasis has been proceeding with the goal of selectively attacking cancer cells. It has been revealed that the proliferation of cancer stem cells involved in redevelopment, invasion, and metastasis is promoted by sphingosine-1-phosphate (S1P). We focused on the transmembrane domains of sphingosine 1 phosphate receptor 4 (S1PR4), and hypothesized that peptides which utilized these domains would exhibit a selective anti-tumor effect in cancer cells. We designed peptides based on the transmembrane domains of S1PR4 and evaluated whether the peptides showed tumor cell growth inhibitory activity against various kinds of human tumor cells. Material and methods: We chemically synthesized conjugation peptides of the human S1PR4 transmembrane domain sequences and a cell-penetrating peptide HIV TAT. The peptides were evaluated for growth inhibitory activity against human melanoma, non-small cell lung cancer (NSCLC),kidney cancer by WST assay. Results: The peptide based on the S1PR4 transmembrane domain 2 exhibited the strongest anti-tumor activity in the synthesized peptides based on the domain from 1 to 7. The result was that more than 95% of the tumor cells were inhibited in cells treated with a concentration of 25μM for 48 hours. Conclusions: We demonstrated a novel strategy for discovering an anti-tumor molecule by focusing on transmembrane domains of proteins involved in the proliferation of cancer cells. In particular, the peptide based on the transmembrane domain 2 of S1PR4 exhibits remarkable anti-tumor activity against human melanoma, NSCLC, kidney cancer which suggests that the peptide could be a candidate for a new anti-tumor agent. COI: I/we (in case of co-authors) have no potential conflict of interest to disclose Citation Format: Tetsuhiko Yoshida, Kenichi Tanaka, Shuichi Asakawa, Makoto Sawada, Yoshinori Hasegawa, Ikuhiko Nakase, Nahoko Bailey Kobayashi. A peptide based on transmembrane domain of S1PR4 as a tumor cell growth inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr A106. doi:10.1158/1535-7163.TARG-19-A106

Abstract 3864: A novel PDE10/β-catenin pathway inhibitor, MCI-030, for the treatment of colorectal cancer

Abstract Over 90% of colorectal cancers harbor mutations in β-catenin or pathway components (e.g. APC) that stabilize β-catenin, causing nuclear translocation and constitutive Tcf-mediated transcription of genes encoding proteins essential for the proliferation and survival of tumor cells. We recently reported that the cyclic nucleotide degrading phosphodiesterase (PDE) isozyme PDE10 is overexpressed in colorectal cancers relative to normal tissue. Its expression and enzymatic activity are essential for colon tumor cell growth, as evidenced by knockdown of PDE10 expression using siRNA or inhibition of enzyme activity using known inhibitors such as PF-2545920. PDE10 inhibition in tumor cells expressing high levels of PDE10 causes increased intracellular cGMP levels to activate PKG and phosphorylate β-catenin, which induces ubiquitination and proteasomal degradation to suppress nuclear translocation and Tcf transcriptional activity. Conversely, ectopic expression of PDE10 in normal colonocytes or precancerous adenoma cells causes increased levels of β-catenin and the expression of proteins (e.g. cyclin D and survivin) essential for the proliferation and survival of tumor cells. To identify novel antitumor PDE10 inhibitors, we screened a chemically diverse library of indenes for PDE10 and tumor cell growth inhibitory activity. Following extensive chemical optimization, MCI-030 emerged as a potent and selective inhibitor of tumor cell growth. Similar to PF-2545920, but with appreciably greater potency and tumor cell selectivity, MCI-030 inhibited colon tumor cell growth by activating cGMP/PKG signaling to phosphorylate and induce β-catenin degradation. MCI-030 also inhibited colon tumor cell spheroid formation and reduced spheroid size and growth at concentrations that inhibit PDE10. Oral administration of MCI-030 significantly inhibited colon tumor formation in the Apc+/min-FCCC mouse model without discernable toxicity. Importantly, unlike PDE10 inhibitors developed to cross the blood-brain barrier for the treatment of CNS disorders, MCI-030 lacks the sedation side effects. Together, these findings support preclinical development of MCI-030 for the treatment of colorectal cancer as a novel PDE10 inhibitor capable of selectively inhibiting the growth of tumors harboring β-catenin or APC mutations. Funding provided by NCI grants R01CA131378, R01CA148817, R01CA197147, and R01CA155638. Citation Format: Antonio B. Ward, Xi Chen, Jacob Valiyaveettil, Kevin Lee, Wen-Chi L. Chang, Yulia Maxuitenko, Veronica Ramirez-Alcantara, Kristy Berry, Luciana Madeira da Silva, Bing Zhu, Tyler Mattox, Michael R. Boyd, Adam B. Keeton, Margie L. Clapper, Harry S. Cooper, Gary A. Piazza. A novel PDE10/β-catenin pathway inhibitor, MCI-030, for the treatment of colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3864.

Synthesis of Novel Pyrazole Derivatives and Their Tumor Cell Growth Inhibitory Activity.

To find novel antitumor agents, a series of 1H-benzofuro[3,2-c]pyrazole derivatives 4a-e were designed and synthesized. The treatment of 6-methoxybenzofuran-3(2H)-one 3 with LiHMDS in anhydrous tetrahydrofuran (THF) followed by reaction with 3-substitued phenyl isothiocyanate gave the thioamide intermediates, which underwent condensation with hydrazine monohydrate in dioxane/EtOH (1:1) to provide the benzofuropyrazole derivatives 4a–e as well as the unexpected pyrazole derivatives 5a–e. In tumor cell growth inhibitory assay, all the benzofuropyrazole derivatives were not active against the breast tumor MCF-7 cell, only 4a was highly active and more potent than ABT-751 against the leukemia K562 (GI50 = 0.26 μM) and lung tumor A549 cells (GI50 = 0.19 μM), while other benzofuropyrazoles showed very weak inhibitory activity. In contrast, the pyrazoles 5a-e were in general more potent than the benzofuropyrazoles 4a–e. Compound 5a exhibited a similar tendency to that of 4a with high potency against K562 and A549 cells but weak effects on MCF-7 cell. Both pyrazoles 5b and 5e exhibited high inhibitory activities against K562, MCF-7 and A549 cells. The most active compound 5b was much more potent than ABT-751 against K562 and A549 cells with GI50 values of 0.021 and 0.69 μM, respectively. Moreover, 5b was identified as a novel tubulin polymerization inhibitor with an IC50 of 7.30 μM.

Design, synthesis, and anticancer properties of isocorydine derivatives

Isocorydine (ICD), an aporphine alkaloid, is widely distributed in nature. Its ability to target side population (SP) cells found in human hepatocellular carcinoma (HCC) makes it and its derivative 8-amino-isocorydine (NICD) promising chemotherapeutic agents for the treatment of HCC. To improve the anticancer activity of isocorydine derivatives, twenty derivatives of NICD were designed and synthesized through chemical structure modifications of the aromatic amino group at C-8. The anti-proliferative activities of all synthesized compounds against human hepatocellular (HepG2), cervical (HeLa), and gastric (MGC-803) cancer cell lines were evaluated using an MTT assay. The results showed that all the synthetic compounds had some tumor cell growth inhibitory activity. The compound COM33 (24) was the most active with IC50 values under 10 μM (IC50 for HepG2 = 7.51 µM; IC50 for HeLa = 6.32 μM). FICD (12) and COM33 (24) were selected for further investigation of their in vitro and in vivo activities due to their relatively good antiproliferative properties. These two compounds significantly downregulated the expression of four key proteins (C-Myc, β-Catenin, CylinD1, and Ki67) in HepG2 cells. The tumor inhibition rate of COM33 (24) in vivo was 73.8% after a dose 100 mg/kg via intraperitoneal injection and the combined inhibition rate of COM33 (24) (50 mg/kg) with sorafenib (50 mg/kg) was 66.5%. The results indicated that these isocorydine derivatives could potentially be used as targeted chemotherapy agents or could be further developed in combination with conventional chemotherapy drugs to target cancer stem cells (CSCs) and epithelial-to-mesenchymal transition (EMT), the main therapeutic targets in HCC.

Abstract 1140: Characterization of a novel PDE10 inhibitor in lung tumor cells and an orthotopic mouse model of lung cancer

Abstract BACKGROUND: Screening a focused library of indene derivatives for PDE10 inhibitory activity identified novel leads with potent and selective tumor cell growth inhibitory activity. ADT-030 emerged from lead optimization chemistry with excellent drug-like properties and oral bioavailability. Here we characterize the anti-tumor activity of ADT-030 in human lung tumor cells and an orthotopic mouse model of lung cancer. METHODS: Growth inhibitory activity of ADT-030 was measured in a panel of human lung tumor cell lines by ATP quantification following 72 h of treatment. The effect of ADT-030 on intracellular cGMP/cAMP was measured in whole cell lysates using a competitive ELISA assay. PDE inhibitory activity of ADT-030 was evaluated in lysates of human lung tumor cells and by recombinant PDE isozymes using the IMAP fluorescence polarization PDE assay. Activation of PKG signaling and suppression of β-catenin levels in response to ADT-030 treatment was evaluated by Western blot using whole cell lysates of human lung tumor cells. ADT-030 was orally administrated to C57BL/6 mice and free levels quantified in plasma and tissues by LC-MS. Anti-tumor activity of ADT-030 was evaluated in athymic nude-Foxn1nu mice after inoculating the left lung with 1x106 A549 lung tumor cells and treating once daily by oral administration at dosages ranging from 25 - 125 mg/kg. Tumor growth was monitored by in situ bioluminescence using IVIS as well as necropsy and pathological grading after 4 weeks of treatment. RESULTS: ADT-030 inhibited the growth of human lung tumor cell lines with IC50 values in the low micromolar range by inducing apoptosis, while appreciably higher concentrations were required to affect the growth of normal human airway epithelial cells. ADT-030 treatment of human lung tumor cells increased both intracellular cGMP and cAMP levels, activated PKG and suppressed β-catenin within the same concentration range as required for tumor cell growth inhibition. Pharmacokinetic studies in mice demonstrated a half-life suitable for once a day dosing. Tissue distribution studies revealed appreciably higher concentrations of ADT-030 in lungs relative to plasma and other tissues, with the highest accumulation measured in the parenchyma. ADT-030 was well tolerated in mice implanted with A549 tumor cells and displayed strong anti-tumor activity as evident by reduced luminescence, tumor grading, and double-blinded pathological evaluation. CONCLUSIONS: ADT-030 represents a prospective drug development candidate with favorable drug-like properties that concentrates in lung after oral administration exhibiting a strong anti-tumor activity in a pre-clinical mouse model. The mechanism of lung tumor cell growth inhibition involves PDE10 inhibition, elevation of cGMP, activation of PKG, and attenuation of β-catenin. Citation Format: Veronica Ramirez-Alcantara, Bing Zhu, Xi Chen, Rajkumar Savai, Prema Subbarayal, Michele A. Schuler, Kevin J. Lee, Ashley S. Lindsey, Kristy L. Berry, Dennis Otali, Joshua Canzoneri, Jacob Valiyaveettil, Adam Keeton, Lori Coward, Gregory Gorman, William Grizzle, Michael Boyd, Gary A. Piazza. Characterization of a novel PDE10 inhibitor in lung tumor cells and an orthotopic mouse model of lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1140. doi:10.1158/1538-7445.AM2017-1140

Abstract 5268: Nexrutine and palmatine mediated effects on the STAT3/EP4/IL-6 axis in pancreatic cancer

Abstract With a 5 year survival of less than 8%, the poor prognosis of pancreatic cancer (PanCA) underlines the importance of improving therapies for effective management of this disease. In previous studies, we identified the tumor cell growth inhibitory activities for Nexrutine® (Nx, a bark extract from Phellodendron amurense) and palmatine (PMT) which is a constituent of Nx. These studies revealed an important role for downregulation of KRAS downstream effectors including GLI, STAT3 and NF-κB in mediating growth inhibitory effects. Remarkably, both Nx and PMT exerted synergistic growth inhibitory effects in combination with the conventional chemotherapeutic agent, Gemcitabine (GEM). Despite such promising in vitro observations, the in vivo efficacy of Nx or PMT had not been established. In this investigation, we evaluated the ability of Nx and PMT to (i) prevent the development of pancreatic tumors; and (ii) inhibit the growth of patient derived pancreatic cancer tissue in short term ex vivo cultures. Studies were also conducted to investigate the underlying molecular mechanism using cell culture models. Our results show that both Nx and PMT are well tolerated in vivo as evidenced by a lack of significant changes in the body weights of athymic mice implanted with Capan-2 cells. Interestingly, both Nx and PMT showed a significant decrease in the circulating levels of inflammatory molecules including IL-6 and CXCL1. In this first proof-of-concept study, PMT treated animals demonstrated a trend towards decreased pancreatic tumor weight with associated histopathogical changes. Remarkably, both Nx and PMT inhibited the growth of patient derived pancreatic cancer cells. Mechanistic investigations identified downregulation of STAT3, TrkA, Src, and RPS6 activities as potential contributors to Nx or PMT-induced synergistic growth inhibitory effects with GEM. Further investigation of STAT3 signaling revealed the involvement of the prostaglandin receptor, EP4, in a potential feedback loop with STAT3 and in asserting STAT3-mediated inhibition of autophagy. Taken together these data support potential utility for Nx and/or PMT in the management of PanCA and suggest that these agents use multiple mechanisms to affect growth of pancreatic tumors. Supported by NCCIH (R01 AT007448; APK) and VA-MERIT Award (I01 BX 000766; APK). Citation Format: Amanda R. Munoz, Roble G. Bedolla, Paul Rivas, Divya Chakravarthy, Robert L. Reddick, Martha A. Hanes, Glenn A. Halff, Rita Ghosh, Addanki P. Kumar. Nexrutine and palmatine mediated effects on the STAT3/EP4/IL-6 axis in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5268. doi:10.1158/1538-7445.AM2017-5268

Synthesis, characterization and biological evaluation of some new indomethacin analogs with a colon tumor cell growth inhibitory activity

Focusing particularly on colorectal cancer, and suggesting research strategies that may help to accelerate the future clinical application of indomethacin for the treatment of cancer, the molecular structures of indomethacin was used as starting scaffold to design novel amide analogs, and the effects of those analogs on the proliferation of human cancer cells were evaluated against three colon cancer cell lines, namely, HCT-116, CACO-2, and HT-29. Compared to indomethacin, the new derivatives displayed significantly increased activities. Interestingly two of the indomethacin analogs 7a and 8c displayed high growth inhibitory activity in nano-molar to micro-molar range against all three human colon cancer cell lines with IC50 values ranging from 0.055 to 4.0 µg/ml compared to 0.7–5.45 µg/ml for 5-fluorouracil (5-FU). Moreover, the potential mechanisms of the cytotoxic activity of the promising compounds 7a and 8c on the HT-29 and HCT-116 cell lines respectively were studied. The results indicated that compounds 7a and 8c arrested the cell cycle at G1/S and G0/G1 phase in HT-29 and HCT-116 cells respectively and might induced apoptosis via caspase-3 dependent pathway.

Design, synthesis and tumor cell growth inhibitory activity of 3-nitro-2H-cheromene derivatives as histone deacetylaes inhibitors

As a continuous research for the discovery of coumarin-based targeted anticancer agents, we designed and synthesized a series of novel histone deacetylases (HDAC) inhibitors using the 8-ethoxy-3-nitro-2H-chromene as the surface binding or cap group, linear dicarboxylic acid or ω-amino acid moiety with different length as the linking motif, ortho-aminoanilides, amides or α-aminoamides as the zinc binding group and the internal cavity motifs. Most of these 3-nitro-2H-chromene derivatives exhibited good growth inhibitory activity against K562, A549, MCF-7, PC3 and Hela cells and were more potent than the reference drug SAHA and MS-275. At the concentration of 10µM, the ortho-aminoanilide series and the d-Phe derived α-aminoamide derivatives 16a and 16b displayed more potent activity toward HADC1 over HADC2, and only moderate to weak activity over HADC6. In contrast, the amide ZBG analogues, 12a and 12b, 14 and 15, were only moderate HDAC6 inhibitors, but more selective over HDAC1 and HDAC2. The ortho-aminoanilides 9b, 9c, 10b, 10c, 11b, and the α-aminoamides 16a and 16b were potent HADC1 inhibitors with the IC50 values in the nanomolar ranges. The ortho-aminoanilides 10b and10c with a phenyl internal cavity motif were more potent than MS-275 as HADC1 inhibitors and more selective over HADC2.

Abstract B23: DC070-547, a novel Ras inhibitor potently and selectivity inhibits colon tumor growth in vitro and in vivo

Abstract Introduction: Activating mutations in Ras oncogenes play a critical role in the development of colorectal cancer and are an indicator of poor prognosis. Constitutive activation of Ras proteins is also associated with resistance to chemotherapy and radiation in which treatment options are limited. Despite extensive efforts, no drugs have been successfully developed that target these aberrant gene products, in part because of the high affinity of Ras binding to GTP, which is essential for Ras activation. Using a phenotypic screening assay designed to select for Ras inhibitors and follow-up chemical optimization, a novel Ras inhibitor, DC070-547 was identified, which potently and selectively inhibits the growth of colon tumor cells with activated Ras in vitro and in vivo. Methods: Tumor cell growth inhibitory activity of DC070-547 was measured in a panel of human colon tumor cell lines using the CellTiter Glo assay following 72 h of treatment. Ras activation levels were measured by precipitating GTP-bound Ras from human colon tumor cell lysates with GST-Raf1-RBD/GSH Sepharose followed by western blotting using anti-Ras antibody. Isogenic cell lines were established by transfecting Ras wild-type HT-29 colon tumor cells with mutant H-Ras. Disruption of Ras-Raf binding was determined by pre-incubating cell lysates or intact cells in the presence of DC070-547 for 30 or 45 min followed by Ras pull-down with GST-Raf beads and western blotting using anti-Ras or an anti-GST antibody as a loading control. Cell cycle arrest and apoptosis were measured by DNA content and annexin V levels, respectively. Antitumor activity was determined in a mouse xenograft model subcutaneously implanted with mutant K-Ras HCT-116 colon tumor cells. Mice were treated with DC070-547 administered i.p. for 14 days at a dose of 2.5 mg/kg bid in a co-solvent formulation. Results: DC070-547 potently inhibits the growth of mutant K-Ras HCT-116 and other colon tumor cell lines having constitutively activated Ras with IC50 values as low as 2 nM and selectivity indices of approximately 100-fold for HT-29 and other colon tumor cells lacking activated Ras. Among a panel of six colon tumor cell lines, a strong correlation was measured between IC50 values for growth inhibition and the level of Ras activation, suggesting that activated Ras is the primary target. Isogenic cell line pairs involving transfecting Ras wild-type cells with mutant H-Ras confirmed that sensitivity to DC070-547 required activated Ras. DC070-547 also blocked Ras-Raf binding in cell lysates and intact cells at concentrations that inhibit the growth of tumor cells with activated Ras. The mechanism of growth inhibition by DC070-547 appears to involve mitotic arrest and apoptosis. Moreover, cells obtained from normal colon mucosa were essentially refractory to treatment with DC070-547. In a mouse xenograft model involving mutant K-Ras human HCT-116 colon tumors, DC070-547 was well tolerated and caused a sustained inhibition of tumor growth in which there was complete tumor regression in 3 of 7 mice. Conclusions: DC070-547 potently and selectively inhibits the growth of colon tumor cells with constitutively activated Ras by disrupting Ras-effector interactions and represents a first-in-class drug development candidate for the treatment of Ras-driven colorectal cancer. Citation Format: Veronica Ramirez-Alcantara, Adam B. Keeton, Bing Zhu, Kevin J. Lee, Joshua Canzoneri, Ashley S. Lindsey, Luciana Madeira da Silva Barnes, Kristy Berry, Jacob Valiyaveettil, Xi Chen, Michael R. Boyd, Gary Piazza. DC070-547, a novel Ras inhibitor potently and selectivity inhibits colon tumor growth in vitro and in vivo. [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer: From Initiation to Outcomes; 2016 Sep 17-20; Tampa, FL. Philadelphia (PA): AACR; Cancer Res 2017;77(3 Suppl):Abstract nr B23.

Structural modification of luteolin from Flos Chrysanthemi leads to increased tumor cell growth inhibitory activity

The luteolin from Flos Chrysanthemi was found to directly bind to the Bcl-2 protein and inhibit the tumor cell growth in our previous study. However, it has been shown to possess wide and week biological activities. In this study, a series of derivatives of luteolin were designed and synthesized, and their tumor cell growth inhibitory activities were evaluated against human leukemia cell line HL-60. The results showed that compounds 1B-2, 2A-3, and 2B-5, with hydrophobic substituted benzyl groups introduced to B ring and hydrogen or methyl introduced to 7-OH group of luteolin, exhibited the strongest inhibitory activity with the IC50 lower than 10μM, which were significantly more potent than luteolin. The studies presented here offer a good example for modifications of flavones to improve their tumor cell growth inhibitory activities.

Design, synthesis and cytotoxicity of pyrano[4,3-b]indol-1(5H)-ones: A hybrid pharmacophore approach via gold catalyzed cyclization

Reported herein is the gold(III)-catalyzed 6-endo-dig cycloisomerization of 2-alkynyl-indole-3-carboxylic acids to form pyrano[4,3-b]indol-1(5H)-ones, which are pharmaceutically important structural motifs. The hitherto unknown substrates required for this methodology were conveniently synthesized in five steps with good overall yields. The utility of this new cycloisomerization is demonstrated by the excellent regioselectivity obtained using a range of substrates. The mildness of the method allowed functional group compatibility towards hydroxyl tether, displaying exquisite chemoselectivity. All the synthesized compounds were screened for their tumor cell growth inhibitory activity against human cervix adenocarcinoma (HeLa). Compound 7d emerged as the most active (IC50=0.69μM) among the tested series compared to the standard cis-platin (IC50=0.08μM).

Flexible synthesis of isomeric pyranoindolones and evaluation of cytotoxicity towards HeLa cells

A hybrid pharmacophore approach for the synthesis of isomeric pyranoindolones was achieved by employing gold(III) chloride-catalyzed cycloisomerization of alkyne-tethered indole carboxylic acids in good to excellent yield. All the synthesized compounds were evaluated for their tumor cell growth inhibitory activity against human cervix adenocarcinoma (HeLa) which revealed that three compounds exhibited activity comparable with the standard cis-platin (IC50 = 0.08 μM). Molecular docking of all the compounds in Vaccinia H1-Related (VHR) Phosphatase receptor also supported that compound 7d as the most active with a free energy of binding as −8.27 kcal/mol.

Abstract 4360: Validation of phosphodiesterase 10A as a cancer target

Abstract Phosphodiesterase 10A (PDE10) is a cAMP and cGMP degrading PDE isozyme that is highly expressed in the brain striatum where it plays an important role in cognition and psychomotor activity. PDE10 inhibitors are being developed for the treatment of schizophrenia and Huntington's disease and are generally well tolerated, likely because of low expression levels in peripheral tissues. We recently reported high levels of PDE10 in tumors and that genetic silencing by siRNA inhibits tumor cell growth with a high degree of selectivity over normal cells (Li et al., Oncogene 2014). These observations suggest that PDE10 may have an unrecognized role in tumorigenesis and represents a novel cancer target. To further test this possibility, we studied the effects of a highly specific PDE10 inhibitor, Pf-2545920 (MP-10) on colon tumor cell growth. Here we show that Pf-2545920 selectively inhibits tumor cell growth, causes G1 cell cycle arrest, and induces apoptosis. The concentration range by which Pf-2545920 inhibits tumor cell growth parallels the concentration range required to increase intracellular cyclic nucleotide levels and activate PKA and PKG. Moreover, PDE10 knockdown by siRNA reduces the sensitivity of tumor cells to the growth inhibitory activity of Pf-2545920. Using the crystal structure of PDE10 to design novel inhibitors, a series of compounds were synthesized and screened for tumor cell growth inhibitory activity and PDE10 isozyme specificity. A lead compound, ADT-030 was found to inhibit tumor cell growth and PDE10 enzymatic activity with IC50 values in the nanomolar range, but did not significantly affect the growth of normal cells. Unlike Pf-2545920, ADT-030 exhibits high selectivity for activating PKG signaling without affecting PKA signaling. Inhibitors of PKA and PKG were used to confirm that the tumor cell growth inhibitory activity associated with PDE10 inhibition involves PKG activation, while PKA activation appears to be ancillary. These findings serve to validate PDE10 as a cancer target, whereby novel inhibitors can be designed to specifically activate cGMP/PKG signaling with a high degree of tumor cell selectivity. Supported by NIH grants 1R01CA155638 and 1R01CA131378 (Piazza). Citation Format: Kevin Lee, Nan Li, Xi Chen, Bing Zhu, Larry Yet, Luciana Madeira da Silva, Suzanne Russo, Adam B. Keeton, Michael R. Boyd, Gary A. Piazza. Validation of phosphodiesterase 10A as a cancer target. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4360. doi:10.1158/1538-7445.AM2015-4360

Abstract 1914: A novel celecoxib derivative that lacks COX-2 inhibition but displays potent colon tumor cell growth and PDE5 inhibitory activity

Abstract Background: The use of celecoxib for cancer chemoprevention is not recommended because of modest efficacy and potentially fatal cardiovascular toxicity due to COX-2 inhibition. There is evidence that celecoxib inhibits tumor growth by a COX-2 independent mechanism, suggesting that efficacy and safety could be improved by chemical optimization towards the underlying target. We synthesized a novel series of celecoxib derivatives that lack COX-2 inhibitory activity, yet display higher potency to inhibit colon tumor cell growth. Here we study a potential mechanism involving phosphodiesterase 5 (PDE5) inhibition, elevation of intracellular cGMP levels, PKG activation and suppression of β-catenin signaling. Methods: The IMAP fluorescence polarization PDE assay was used to measure PDE activity. Colon tumor cell lines (HT-29 and HCT-116) were used to measure growth inhibitory activity using the Cell Titer Glo Assay. COX inhibitory activity was measured using a fluorescent assay. Intracellular cGMP levels in live cells were measured using a cGMP biosensor assay, while cGMP levels in cell and tissues lysates were measured by EIA assay. PKG activity was measured by western blotting using a phosphospecific antibody to VASP. Levels of β-catenin, survivin, and cyclin D1 were determined by western blotting. TCF/LEF transcriptional activity was measured using a luciferase reporter assay. Cell cycle arrest was measured using an IXM imager and MetaXpress software. Oral bioavailability was determined based on drug concentrations in plasma and colon mucosal tissue after treatment by oral gavage in C57BL/6 mice. Results: Celecoxib inhibits HT-29 and HCT-115 colon tumor cell growth and PDE5 activity with IC50 values of 34.3, 35.3, and 37.0 μM, while a derivative, RF26, had values of 5.8, 6.6, and 0.001 μM, respectively. RF26 was highly selective for PDE5 compared with other PDE isozymes. Celecoxib displays an IC50 of 0.058 μM for COX-2 inhibition, while RF26 lacks COX-2 inhibitory activity. RF26 increases cGMP levels, activates PKG, and decreases β-catenin levels and activity demonstrated by decreased survivin and cyclin D1 protein levels and TCF/LEF transcriptional activity. The mechanism of growth inhibition involves G1 cell cycle arrest. In mice, RF26 displays promising pharmacokinetics and drug levels in the target tissue that increase cGMP levels. Conclusions: A novel series of celecoxib derivatives was identified that lack COX-2 inhibitory activity; yet display high potency to inhibit colon tumor cell growth. PDE5 inhibition appears to be an important mechanism for this class of compounds, which is consistent with previous observations from our group implicating cGMP/PKG activation as an anticancer target. These findings may lead to the development of novel celecoxib derivatives with improved anti-tumor efficacy and reduced risk of toxicity. Supported by NIH grants 1R01CA155638 and 1R01CA131378 (Piazza). Citation Format: Sara C. Sigler, Veronica Ramirez-Alcantara, Adam B. Keeton, Mohammad Abdel-Halim, Ashraf H. Abadi, Gary A. Piazza. A novel celecoxib derivative that lacks COX-2 inhibition but displays potent colon tumor cell growth and PDE5 inhibitory activity. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1914. doi:10.1158/1538-7445.AM2015-1914

Abstract 950: Antitumor activity of a novel phosphodiesterase 10 inhibitor in an orthotopic mouse model of lung cancer

Abstract BACKGROUND: We recently reported that phosphodiesterase 10A (PDE10) is elevated in certain tumor types and essential for tumor cell growth (Li et al., Oncogene 2014). Here we evaluate anti-tumor efficacy, pharmacokinetics, tissue distribution, and tolerance of a novel PDE10 inhibitor, ADT-048, in mice. METHODS: ADT-048 was orally administrated to C57BL/6 mice and free levels quantified in plasma and tissues by LC-MS. Anti-tumor activity was evaluated in athymic nude-Foxn1nu mice after inoculating the left lung with 1×106 A549 lung tumor cells transfected with luciferase. Tumor growth was monitored by in situ bioluminescence using the In Vivo Imaging System and efficacy was evaluated by necropsy grading. RESULTS: ADT-048 inhibited A549 lung tumor cell growth with an IC50 value of 0.7 μM, while appreciably higher concentrations were required to inhibit the growth of normal airway epithelial cells (IC50 = 35 μM). Pharmacokinetic studies demonstrated a half-life suitable for once a day dosing, while tissue distribution studies demonstrated a unique capacity to achieve high concentrations in lungs relative to plasma and other tissues. ADT-048, at a dose of 100 mg/Kg, displayed significant anti-tumor activity as evident by reduced luminescence and tumor grading following 28 days of treatment. CONCLUSIONS: A novel PDE10 inhibitor, ADT-048, was found to display highly potent and selective tumor cell growth inhibitory activity in vitro. ADT-048 was well tolerated in mice and had attractive pharmaceutical properties with a unique capacity to achieve high lung concentrations following oral administration. Efficacy studies demonstrate strong anti-tumor activity of ADT-048, which represents a candidate for clinical development. Supported by NIH grants 1R01CA155638 and 1R01CA131378 (Piazza). Citation Format: Veronica Ramirez-Alcantara, Michele Schuler, Bing Zhu, Nan Li, Evrim Gurpinar, Dennis Otali, Joshua Canzoneri, Adam Keeton, Bernard Gary, Suzanne Russo, Lori Coward, Gregory Gorman, William Grizzle, Xi Chen, Michael Boyd, Gary Piazza. Antitumor activity of a novel phosphodiesterase 10 inhibitor in an orthotopic mouse model of lung cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 950. doi:10.1158/1538-7445.AM2015-950