Mean GI50 Research Articles

N-Sulfonyl-aminobiaryls as Antitubulin Agents and Inhibitors of Signal Transducers and Activators of Transcription 3 (STAT3) Signaling.

A series of N-sulfonyl-aminobiaryl derivatives have been examined as novel antitubulin agents. Compound 21 [N-(4'-cyano-3'-fluoro-biphenyl-2-yl)-4-methoxy-benzenesulfonamide] exhibits remarkable antiproliferative activity against four cancer cell lines (pancreatic AsPC-1, lung A549, liver Hep3B, and prostate PC-3) with a mean GI50 value of 57.5 nM. Additional assays reveal that 21 inhibits not only tubulin polymerization but also the phosphorylation of STAT3 inhibition with an IC50 value of 0.2 μM. Four additional compounds (8, 10, 19, and 35) are also able to inhibit this phosphorylation. This study describes novel N-sulfonyl-aminobiaryl (biaryl-benzenesulfonamides) as potent anticancer agents targeting both STAT3 and tubulin.

Abstract 5136: The Flint Animal Cancer Center (FACC) canine tumor cell line panel: A resource for comparative and translational oncology

Abstract Purpose: Mammalian cell tissue culture has been a critical tool leading to our current understanding of cancer including many aspects of cellular transformation, growth, and response to therapies. The current use of large panels of human cancer cell lines (i.e. NCI60, GDSC) with associated phenotypic and genotypic information allows for informatics approaches and in silico screens to rapidly test hypotheses based on simple as well as complex relationships. We have assembled a panel of canine cancer cell lines to facilitate translational studies in canine cancer and report here the characteristics and some applications of this panel in comparative oncology. Methods: Canine cancer cell lines isolated from tumors have been accumulated from a number of sources over the years. Our current panel consists of 29 cell lines that have been validated as being of canine origin and “fingerprinted” by microsatellite analysis. The tumor types represented include 10 osteosarcomas, 5 melanomas, 2 mammary carcinomas, 1 hemangiosarcoma, 2 bladder carcinomas, 4 lymphoma/leukemias, 1 mast cell tumor, 2 histiocytic sarcomas, 1 thyroid carcinoma and 1 soft-tissue sarcoma. Drug sensitivity analysis was carried out using a resazurin-based bioreductive fluorometric assay. Gene expression microarray analysis was carried out using the Affymetrix GeneChip Canine 2.0 at the Genomics and Microarray Core (University of Colorado Cancer Center). Results: Drug sensitivity to the cytotoxic chemotherapeutic agents carboplatin, cisplatin, doxorubicin, lomustine, paclitaxel and vinblastine were assessed and compared to responses in the human NCI-60 panel. Results show that the human and canine cell lines show similar ranges of sensitivity to these agents. The mean GI50 values for the human and canine cell lines are significantly different for DOX, VBL, CARBO, CCNU as well as PTX. The variances in the data were also significantly different for CARBO and PTX. Overall, however, the drug sensitivity/resistance patterns in the canine and human tumor cell line panels shared general trends with regard to sensitivity and variance observed to the specific agents with the mean Log GI50 values for each agent showing a cross-species correlation (r = 0.88, p = 0.0194, Pearson). Cluster analysis of the gene expression data using the top 100 most variant genes as well as the 100 top most variant cancer genes largely separates the panel into groups with similar histiotypes. Cluster analysis of cancer genes also highlights alterations in gene expression that may contribute to pathogenesis in some cell lines such as loss of the PTEN tumor suppressor or elevated expression of receptor tyrosine kinase genes. Conclusions: The FACC canine tumor cell line panel represents a validated panel of representative cancers from dogs that can be utilized for comparative oncology studies as well as applications in drug development where use of a companion animal model is appropriate. Citation Format: Daniel L. Gustafson, Jared S. Fowles, Douglas H. Thamm, Rodney L. Page, Dawn L. Duval. The Flint Animal Cancer Center (FACC) canine tumor cell line panel: A resource for comparative and translational oncology. [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 5136. doi:10.1158/1538-7445.AM2015-5136

Synthesis and potential antitumor activity of 7-(4-substituted piperazin-1-yl)-4-oxoquinolines based on ciprofloxacin and norfloxacin scaffolds: in silico studies

The potential antitumor activities of a series of 7-(4-substituted piperazin-1-yl)fluoroquinolone derivatives (1–14a,b) using ciprofloxacin and norfloxacin as scaffolds are described. These compounds exhibit potent and broad spectrum antitumor activities using 60 human cell lines in addition to the inherent antibacterial activity. Compounds 1a, 2a, 3b, 6b and 7a were found to be the most potent, while 2b, 5b, and 6a were found to have an average activity. The results of this study demonstrated that compounds 1a, 2a, 3b, 6b and 7a (mean GI50; 2.63–3.09 µM) are nearly 7-fold more potent compared with the positive control 5-fluorouracil (mean GI50; 22.60 µM). More interestingly, compounds 1a, 2a, 3b, 6b and 7a have an almost antitumor activity similar to gefitinib (mean GI50; 3.24 µM) and are nearly 2-fold more potent compared to erlotinib (mean GI50; 7.29 µM). In silico study and ADME-Tox prediction methodology were used to study the antitumor activity of the most active compounds and to identify the structural features required for antitumor activity.

Steroidal alkaloids from the marine sponge Corticium niger that inhibit growth of human colon carcinoma cells.

Bioinformatic analysis of data from the NCI-60 cell cytotoxicity screen revealed a subset of extracts that showed selective cytotoxic activity toward human colon carcinoma cell lines. Bioassay-guided fractionation of a colon cancer selective extract from a Philippines collection of the marine sponge Corticium niger provided two new steroidal alkaloids, plakinamines N (1) and O (2), along with two known compounds of the plakinamine class (3, 4). The structures of these compounds were elucidated by interpretation of combined MS and NMR spectroscopic data. Plakinamines N (1), O (2), and J (4) were tested for antiproliferative activity in the NCI-60 screen, and they showed enhanced inhibitory effects against all of the colon cell lines with mean GI50 values of 11.5, 2.4, and 1.4 μM, respectively.

Abstract 4575: Structure-based design, synthesis and biological testing of highly potent semi-synthetic epipodophyllotoxin-derived hybrid etoposide analogs

Abstract Drugs that target DNA topoisomerase II and interact with DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Using a structure-based design approach a series of epipodophyllotoxin-derived hybrid etoposide analogs were designed, synthesized and biologically characterized. It was hypothesized that combining an epipodophyllotoxin moiety that could interact with specific protein residues on topoisomerase II and DNA-interacting moieties in the same molecule would produce much stronger binding and yield more potent and wide spectrum cytotoxic anticancer drugs. Because the hybrids have unique dual targeting mechanisms, cancer cells should be much less able to develop resistance. The most potent hybrid had a mean GI50 in the NCI-60 cell screen much lower than etoposide. The compounds were much less cross-resistant to a K562-derived etoposide-resistant cell line. All of the hybrids tested targeted topoisomerase II as evidenced by their ability to inhibit kDNA decatenation, to produce linear DNA in a DNA cleavage assay and to form covalent topoisomerase II-DNA covalent complexes in a cellular ICE assay. The DNA double strand breaks the hybrids produced in a cellular γH2AX assay and the G2/M cell cycle blocks were also characteristic of topoisomerase II-targeted drugs. Cell cycle analysis was also used to characterize the targets. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles of both etoposide and the DNA-interacting moieties from which they were derived. This data supports the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA. Support: CIHR and a Canada Research Chair in Drug Development. Citation Format: Brian B. Hasinoff. Structure-based design, synthesis and biological testing of highly potent semi-synthetic epipodophyllotoxin-derived hybrid etoposide analogs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4575. doi:10.1158/1538-7445.AM2014-4575

Structure-based design, synthesis and biological testing of etoposide analog epipodophyllotoxin–N-mustard hybrid compounds designed to covalently bind to topoisomerase II and DNA

Drugs that target DNA topoisomerase II isoforms and alkylate DNA represent two mechanistically distinct and clinically important classes of anticancer drugs. Guided by molecular modeling and docking a series of etoposide analog epipodophyllotoxin–N-mustard hybrid compounds were designed, synthesized and biologically characterized. These hybrids were designed to alkylate nucleophilic protein residues on topoisomerase II and thus produce inactive covalent adducts and to also alkylate DNA. The most potent hybrid had a mean GI50 in the NCI-60 cell screen 17-fold lower than etoposide. Using a variety of in vitro and cell-based assays all of the hybrids tested were shown to target topoisomerase II. A COMPARE analysis indicated that the hybrids had NCI 60-cell growth inhibition profiles matching both etoposide and the N-mustard compounds from which they were derived. These results supported the conclusion that the hybrids displayed characteristics that were consistent with having targeted both topoisomerase II and DNA.

Synthesis and antiproliferative activity of novobiocin analogues as potential hsp90 inhibitors

A series of substituted coumarins1–10 was designed and synthesized as a novel class of 4TCNA analogues. Compound 2a showed excellent antiproliferative activity with mean GI50 values at a micromolar level in a diverse set of human cancer cells (GI50 = 2–30 μM) and induced a high apoptosis level in MCF-7 breast cancer cell line. The molecular signature of hsp90 inhibition was assessed by depletion of the Erα hsp90 client protein.

Inhibition of the checkpoint kinase Chk1 induces DNA damage and cell death in human Leukemia and Lymphoma cells.

BackgroundChk1 forms a core component of the DNA damage response and small molecule inhibitors are currently being investigated in the clinic as cytotoxic chemotherapy potentiators. Recent evidence suggests that Chk1 inhibitors may demonstrate significant single agent activity in tumors with specific DNA repair defects, a constitutively activated DNA damage response or oncogene induced replicative stress.MethodsGrowth inhibition induced by the small molecule Chk1 inhibitor V158411 was assessed in a panel of human leukemia and lymphoma cell lines and compared to cancer cell lines derived from solid tumors. The effects on cell cycle and DNA damage response markers were further evaluated.ResultsLeukemia and lymphoma cell lines were identified as particularly sensitive to the Chk1 inhibitor V158411 (mean GI50 0.17 μM) compared to colon (2.8 μM) or lung (6.9 μM) cancer cell lines. Chk1 inhibition by V158411 in the leukemia and lymphoma cell lines induced DNA fragmentation and cell death that was both caspase dependent and independent, and prevented cells undergoing mitosis. An analysis of in vitro pharmacodynamic markers identified a dose dependent decrease in Chk1 and cyclin B1 protein levels and Cdc2 Thr15 phosphorylation along with a concomitant increase in H2AX phosphorylation at Ser139 following V158411 treatment.ConclusionsThese data support the further evaluation of Chk1 inhibitors in hematopoietic cancers as single agents as well as in combination with standard of care cytotoxic drugs.

Design, synthesis and biological evaluation of some novel substituted quinazolines as antitumor agents

A novel series of 6-chloro-2-p-tolylquinazolinone and substituted-(4-methylbenzamido)benzamide (1–20) were designed, synthesized and evaluated for their in-vitro antitumor activity. Compounds 3, 14 and 16 possessed remarkable broad-spectrum antitumor activity. Compound 16 was found to be a particularly active growth inhibitor of the renal cancer (GI50 = 4.07 μM), CNS cancer (GI50 = 7.41 μM), ovarian cancer (GI50 = 7.41 μM) and non-small cell lung cancer (GI50 = 7.94 μM). Compound 16 ranks as nearly 1.5-fold more potent (mean GI50 = 15.8 μM) compared to 5-FU (mean GI50 = 22.6 μM).

Discovery of azaisoerianin derivatives as potential antitumors agents

A series of N-methyl-diarylamines 2 was designed and synthesized as a novel class of CA-4 and isoCA-4 analogues. Compounds 2b and 2m showed excellent antiproliferative activity with mean GI50 values at a nanomolar level in a diverse set of human cancer cells. These compounds also inhibited tubulin assembly at a micromolar range, arrested the cellular cycle in the G2/M phase and induced apoptosis at very low concentrations. Preliminary in vitro results revealed that 2b and 2m displayed substantial efficacy as potent antivascular agents. Docking studies indicates that these lead compounds showed a binding mode similar to those observed with isoCA-4 at the colchicine binding site of tubulin.

Abstract B26: Design and synthesis of furoquinoline derivatives as potential drug candidates for the treatment of lung cancers.

Abstract Quinoline moiety is present in many classes of biologically active compounds. Recently, we have synthesized certain quinolone, 2-phenylquinoline, 2-furanylquinoline, and 4-anilinofuro[2,3-b]quinoline derivatives for biological evaluations. Among them, 1-[4-(furo[2,3-b]quinolin-4-ylamino)phenyl]ethanone (CIL-102) was the most potent with a mean GI50 value of 0.025 μM against the growth of nine types of cancer cells and therefore, was further evaluated on its effects of cell cycle distribution. Results indicated CIL-102 induces cell cycle arrest in G2/M followed by apoptosis. CIL-102 bounds to tubulin at a single site with a dissociation constant of 0.4 μM. An analysis of the modified Dixon plot suggested that CIL-102 competitively inhibited the binding of podophyllotoxin, a colchicine-binding site agent, to tubulin. However, some drawbacks such as lack of selective cytotoxicity, poor oral bioavailability, and poor water solubility exhibited by CIL-102 prompted us to search for newer derivatives. Through extensive structural optimization, the new lead compound named as CYW-1865 which possesses aminoalkoxyimino side chain, exhibited selective anticancer activity, higher oral bioavailability, and higher water solubility than that of CIL-102. Xenographic studies indicated the tumor size with CYW-1865 treated nude mice was significantly lower than control. Further evaluation in an orthotopic lung cancer model indicated that CYW-1865 can be absorbed readily through oral administration, distributed to lung tissue, and exhibited significant efficacy in inhibiting the growth of lung cancers. Strategies for design and synthesis of CYW-1865 and its further structural optimization will be presented. Citation Format: Cherng-Chyi Tzeng, Chih-Hua Tseng, Yeh-Long Chen. Design and synthesis of furoquinoline derivatives as potential drug candidates for the treatment of lung cancers. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B26.

Abstract A33: Therapeutic potential of sapacitabine in ovarian cancers defective in homologous recombination

Abstract Background: Sapacitabine is an oral nucleoside analogue that is metabolised in-vivo to CNDAC (2′-C-cyano-2′-deoxy-1-β-D-arabino-pentofuranosylcytosine). It has a novel mechanism of action by causing single-strand breaks and replication-associated double-strand breaks (DSBs) after its incorporation into DNA. DSB, if unrepaired, are cytotoxic and are predominantly repaired by homologous recombination (HR). Over 50% of high-grade serous ovarian cancers are estimated to be defective in HR, only a small proportion of which are accounted for by germline BRCA mutations. HR-defective ovarian cancers have been shown to be sensitive to PARP inhibitors ex-vivo and platinum in-vivo and we therefore hypothesised that HR defective ovarian cancers would be sensitive to CNDAC and aimed to explore the potential use of CNDAC in ovarian cancer stratified for HR function. Methods: Primary cultures (PCO) were generated from ascitic fluid collected from patients undergoing ovarian cancer surgery, characterised and confirmed to be ovarian and epithelial in origin based upon morphology, expression of immunofluorescent-labelled markers and cross-referencing with formal examination of FFPE tissue and cytology of ascites. The functional status of HR was determined for each cell line and PCO culture by quantification of nuclear Rad51 focus formation following induction of DNA DSB. SRB assays were used to calculate the GI50 for cisplatin and CNDAC, (Cyclacel, UK), in cell lines nad PCO cultures. Results: As hypothesised, HR defective cell lines were highly sensitive to CNDAC (VC8 BRCA2 mutant: GI50=366 nM) in comparison to their matched HR competent cell lines (VC8-BRCA2 reconstructed: GI50=18 μM; VC8-PARPi-resistant BRCA revertant: GI50=3 μM). Sensitivity to CNDAC in patient PCO cultures (n=39) was heterogeneous with a mean GI50 of 297 nM (95% CI 136-646 nM). Stratifying PCO cultures according to their HR status demonstrated greater sensitivity in HR defective cultures with a mean GI50 of 135 nM (46-394; n=12) in defective cultures, compared to 477 nM in HR competent cultures (164-1386; n=27). Of the 34 cisplatin resistant PCO cultures (GI50 >5 μM), 20 (59%) remained sensitive to CNDAC with a mean GI50 of 426 nM (148-1223; n=13) in HR competent cultures and a GI50 of 230 nM (70-759; n=7) in HR defective cultures. CNDAC sensitivity was evaluated in base excision repair (BER) defective EM9 cell line (GI50=374 nM) compared with parental wild type AA8 cells (GI50=6.9 μM), p<0.0001. Using detection of 8-hydroxy-2′-deoxyguanosine (8-OHdG) as an indicator of BER functional status in PCO cultures, a trend between BER dysfunction and sensitivity to CNDAC was seen with a mean GI50 of 42 nM (18-97; n=2) in BER defective PCO cultures in comparison to 187 nM (74-473; n=15) in BER competent cultures. Conclusions: Cell lines defective in HR or BER are highly sensitive to CNDAC suggesting a possible use of HR or BER status as biomarkers for the stratified use of CNDAC in the treatment of ovarian cancer. When applied to ex-vivo studies HR status as a single marker cannot reliably predict sensitivity and a panel of pathways may therefore be needed to enable reliable treatment stratification. Nevertheless, stratification based on these markers is likely to permit enrichment of patients more likely to respond to CNDAC. Additionally CNDAC may have a role in the treatment of platinum resistant disease as approximately 60% of patients with resistant disease may benefit. Further work is required to explore the possibility of CNDAC as a chemosensitiser in platinum sensitive disease and as a second line agent in platinum resistant disease. Citation Format: Rachel L. O'Donnell, James CC Murray, Adriana GB Buskin, Sheelagh Frame, David G. Blake, Michelle Dixon, Aiste McCormick, Angelika Kaufmann, E Ruth Plummer, Richard J. Edmondson, Nicola J. Curtin. Therapeutic potential of sapacitabine in ovarian cancers defective in homologous recombination. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A33.

Abstract 700: Gene expression profiling of cyclin-dependent kinase (CDK) inhibition in cancer cells.

Abstract Cyclin-dependent kinases (CDKs) form complexes with partner cyclins to regulate cell cycle progression and transcription. CDK1, CDK2, CDK4 and CDK6 all regulate cell cycle progression and CDK8 and CDK9 are regulators of transcription. CDK7 is a unique member of this family because it can regulate the cell cycle CDKs as part of the CDK-activating kinase (CAK) and also transcription in conjunction with the TFIIH transcription factor by phosphorylating the COOH-terminal domain of RNA polymerase II (Pol II CTD). A hallmark of cancer is deregulated cell cycle progression and many cancers have abnormal CDK activity which makes CDKs a good target for cancer therapy. Here we show BS-194, a selective and potent CDK inhibitor, which inhibits CDK2, CDK1, CDK5, CDK7 and CDK9 (IC50 = 3, 30, 30, 250 and 90nmol/L, respectively). In vitro cell based assays have shown that BS-194 inhibits the phosphorylation of CDK substrates, Rb and RNA Poll II, down regulation of cyclins A, E and D1 and cell cycle arrest in the S and G2/M phases. BS-194 also has potent anti-proliferative activity in 60 cancer cell lines tested (mean GI50 = 28nmol/L). BS-194 is orally bioavailable and shows potent in vivo tumour growth inhibition of xenograft models of MCF7 and HCT116 cells. We have used BS-194 as a potent inhibitor of CDK activity in HCT116 cells and performed gene microarray analysis to determine novel pathways and identify novel biomarkers associated with CDK inhibition. In summary, the gene microarray has identified genes differentially expressed in response to BS-194 treatment that are primarily involved in cell cycle, apoptosis and transcription pathways. Citation Format: Silvia Ottaviani, Sean Delaney, Hetal Patel, Manikandan Periyasamy, Alexander Bondke, Brian Slafer, Richard Starkey, Sebastian H.B. Kroll, Matthew J. Fuchter, Anthony G.M. Barrett, R. Charles Coombes, Simak Ali. Gene expression profiling of cyclin-dependent kinase (CDK) inhibition in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 700. doi:10.1158/1538-7445.AM2013-700

Design, synthesis and antiproliferative activity of indole analogues of indanocine

The design and synthesis of a novel series of indole-analogues of indanocine is reported, together with their antiproliferative activity in the NCI's panel of cancer cell lines. Indanocine displays potent activity against a wide range of cancer cell lines (mean GI50 < 20 nM), including drug-resistant cancer cell lines, and also inhibits the migration of metastatic cancer cells. A number of the described indole-analogues display a similar activity profile to indanocine, exhibiting potent antiproliferative activities in several cancer cell lines, and offer new leads for further development.

Serum Metabolomics Uncovers a New Therapeutic Target in Diffuse Large B Cell Lymphoma (DLBCL)

Abstract 1648DLBCL is a molecularly heterogeneous disease usually treated with chemoimmunotherapy ultimately curing ∼65% of pts. In order to improve therapy for these pts, the identification of broadly relevant therapeutic targets is critical. One such target is HSP90. Tumor cells are enriched for a fraction of HSP90 found in higher-order multi-chaperone complexes. Tumor-enriched HSP90 (teHSP90) displays higher affinity for HSP90 inhibitors than normal tissues, which contain latent, uncomplexed HSP90. Many client proteins are depleted upon exposure to teHSP90 inhibitors. PU-H71 is a highly teHSP90 selective inhibitor with an excellent toxicity profile currently being tested for DLBCL in clinical trials. Combination therapies involving teHSP90 inhibitors may synergize with drugs targeting client proteins of teHSP90 by more powerfully inhibit survival pathways. Cell replication involves substantial metabolic demands and PU-H71-induced degradation of enzymatic client proteins may affect critical metabolic pathways. We hypothesized that by identifying PU-H71-induced metabolic changes, metabolomics could point to potential new targets for combinatorial therapeutic intervention.We therefore analyzed the global metabolic consequence of teHSP90 inhibition in DLBCL by PU-H71. The metabolome was analyzed in the serum of LY7 DLBCL xenografted mice treated with 75 mg/m2 of PU-H71 for 24 h (n=5) or vehicle (n=5) by HPLC/MS. Bioinformatic analysis revealed significant changes in 122 metabolites in PUH-71- vs. vehicle-treated mice; including significantly lower levels of xanthine, hypoxanthine, adenosine, xanthosine monophosphate (XMP), depletion of the guanine nucleoside pool, together with higher levels of inosine and inosine monophosphate (IMP). These metabolic changes pointed towards possible PU-H71 mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). IMPDH catalyzes the NAD-dependent oxidation of IMP to XMP, which is the committed step in de novo guanosine nucleotide biosynthesis. This reaction is particularly important to lymphocytes, which depend on IMPDH activity to generate the guanosine nucleotide levels needed to initiate a proliferative response to antigen. Increased IMPDH activity has also been observed in leukemia and lymphoma, mostly as consequence of up-regulation of the IMPDH2 isoform. In order to determine whether IMPDH1/2 stability depends on teHSP90, we treated a panel of 6 DLBCL cell lines (including LY7) with the mean GI50 of PU-H71 (1 μM) for up to 24 h and checked for IMPDH1/2 abundance. We found a time-dependent decrease in IMPDH2 protein levels. Similar results were obtained with the chemically unrelated HSP90 inhibitor 17-DMAG. To confirm that IMPDH2 binds to teHSP90, we took advantage of an affinity-based PUH-71 pull-down method we recently developed. In this assay PU-H71-beads preferentially bind to teHSP90 complexes pecipitating cancer-related client proteins. By using this assay, we determined that IMPDH2 was indeed a teHSP90 client in DLBCL cells. We also found that PU-H71 (and 17-DMAG) inhibited the activity of IMPDH in DLBCL cells by shortening its half-live (IMPDH t1/2from 2 h to 45 min). To determine whether the combination of teHSP90 inhibitors will synergize with IMPDH inhibitors in killing DLBCL, we treated a panel of 10 DLBCL cell lines with the combination of PU-H71 and two IMPDH inhibitors in clinical use, mycophenolic acid (MPA, an uncompetitive inhibitor) and ribavirin (RIB, a competitive inhibitor). We found that most cell lines showed synergistic killing effect when treated with the combination of drugs compared to each drug alone (determined by isobologram method). This prompted us to test the combination in vivo. SCID mice were xenografted with LY7 and SUDHL6 and once tumor developed, they were treated with vehicle, PU-H71, mycophenolate mofetil (MMF, the pro-drug of MPA), RIB and the combination of PU-H71 and MMF or RIB. We found that mice treated with the combination of drugs exhibited greater effect that each drug alone (p=0.002 for SU-DHL6 and p>0.001 for LY7 for PU-H71+MMF, and p=0.01 for SU-DHL6 and p=0.02 for LY7 for PU-H71+RIB, all T-test day 10). There were no toxic effects.In sum, our work uses serum metabolomics to provide new insights into the pharmacological targets of a particular HSP90 inhibitor, and unveiled a critical survival pathway in DLBCL that was harnessed to develop a rationally combined targeted therapy. Disclosures:No relevant conflicts of interest to declare.

Molecular docking and synthesis of novel quinazoline analogues as inhibitors of transcription factors NF-κB activation and their anti-cancer activities

NF-kB is a transcription factor protein complex that can be found in almost all animal cell types and is a key player in some cancers and inflammatory responses. It can enhance the proliferation rate, reduce apoptosis, as well as creating more blood flow to ensure the survival of cancer, thus blocking the NF-kB pathway has potential therapeutic benefit. We designed a series of compounds based on a quinazoline scaffold pharmacophore model which may have high binding affinity with the p50 subunit of NF-kB. The compound series with phenyl substitution at the 2 position of the quinazoline proved to be more effective at inhibiting NF-kB function both theoretically and experimentally. These compounds also reduce the proliferation of numerous tumor cell lines and the mean GI50 for compound 2a is 2.88μM against the NCI-60 cell line. At the same time, compound 2a can induce significant apoptosis in EKVX cell line at the concentration of 1μM.

Synthesis of New 4-Thiazolidinone-, Pyrazoline-, and Isatin-Based Conjugates with Promising Antitumor Activity

The synthesis and antitumor activity screening of novel 3-[2-(3,5-diaryl-4,5-dihydropyrazol-1-yl)-4-oxo-4,5-dihydro-1,3-thiazol-5-ylidene]-2,3-dihydro-1H-indol-2-ones 1-23 and 3-(3,5-diarylpyrazol-1-yl)-2,3-dihydro-1H-indol-2-ones 24-39 are performed. In vitro anticancer activity of the synthesized compounds was tested by the National Cancer Institute. Most of them displayed anticancer activity on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate, and breast cancers cell lines. The structure-activity relationship is discussed. The most effective anticancer compound 10 was found to be active with mean GI₅₀ and TGI values of 0.071 μM and 0.76 μM, respectively. It demonstrated the highest antiproliferative influence on the non-small-cell lung cancer cell line HOP-92 (GI₅₀ < 0.01 μM), colon cancer line HCT-116 (GI₅₀ = 0.018 μM), CNS cancer cell line SNB-75 (GI₅₀ = 0.0159 μM), ovarian cancer cell line NCI/ADR-RES (GI₅₀ = 0.0169 μM), and renal cancer cell line RXF 393 (GI₅₀ = 0.0197 μM).

Structure-based design and synthesis of novel pseudosaccharine derivatives as antiproliferative agents and kinase inhibitors

This study is concerned with the implementation of structure-based techniques for the design of new heterocyclic compounds based on pseudosaccharine scaffold with protein kinase inhibition activity. This nucleus was exploited based on the well-known quinazoline core and its interactions with several protein kinases. Two series of compounds employing this new scaffold were synthesized and evaluated at enzymatic and cellular levels. Compound 9b displayed broad spectrum antiproliferative activity on NCI 60-cell lines panel with mean GI50 of 5.4 μM. Investigation of the molecular mechanism showed probable inhibitory activity against Src kinase.

Methylenedioxy- and Ethylenedioxy-Fused Indolocarbazoles: Potent Human Topoisomerase I Inhibitors and Antitumor Agents

The indolo[2,3-a]carbazole alkaloids constitute an important class of natural products with interesting and diverse biological activities. A series of novel ring-fused indolocarbazoles were synthesized and evaluated for inhibition of topoisomerase I-mediated relaxation of supercoiled DNA and in vitro antitumor activity. The derivatives bearing a methylenedioxy or an ethylenedioxy ring fused onto the nonglycosylated indole (1a, 1b) demonstrated more potent anti-topoisomerase I activity. The isopropylenedioxy analogue 1c was approximately half as active as 1a, while the O-dimethoxy analogue 1d and the regioisomers 2a and 2b were essentially devoid of measurable activity, implying that the stacking with the intact DNA strand has been impeded by these compounds due to steric hindrance. The newly synthesized indolocarbazoles were screened against the NCI's 60 tumor cell lines. The order of activity, based on the mean GI50 values, is as follows: 1a > 2a ~ 1d > 1b > MCR-47 > 2b. Though in general the analogues that showed potent activity against topoisomerase I (1a, 1b) also showed potent in vitro inhibition of tumor cell growth, the antitumor activity of the anti-topoisomerase I inactive 1d and 2a were intriguing. COMPARE analyses confirmed that the topoisomerase I is the primary target for 1a and 1b; however, other target(s) or pathway(s) may also be involved, with PLD1 and MERTK suggested. Further investigation of these molecular targets against these indolocarbazoles is warranted.

Synthesis and Antiproliferative Activity of the Ring System [1,2]Oxazolo[4,5‐g]indole

Brand new ring: A series of 27 derivatives of the new ring system [1,2]oxazolo[4,5-g]indole were conveniently prepared and tested at the NCI for antiproliferative studies. Several of them showed good inhibitory activity toward all tested cell lines, reaching GI50 values generally at the micromolar and sub-micromolar levels and in some cases at nanomolar concentrations. The mean GI50 values, calculated on the full panel, were in the range 0.25-7.08 μM.