NCI-60 Cell Line Screen Research Articles

Protein Structure Inspired Discovery of a Novel Inducer of Anoikis in Human Melanoma.

Drug discovery historically starts with an established function, either that of compounds or proteins. This can hamper discovery of novel therapeutics. As structure determines function, we hypothesized that unique 3D protein structures constitute primary data that can inform novel discovery. Using a computationally intensive physics-based analytical platform operating at supercomputing speeds, we probed a high-resolution protein X-ray crystallographic library developed by us. For each of the eight identified novel 3D structures, we analyzed binding of sixty million compounds. Top-ranking compounds were acquired and screened for efficacy against breast, prostate, colon, or lung cancer, and for toxicity on normal human bone marrow stem cells, both using eight-day colony formation assays. Effective and non-toxic compounds segregated to two pockets. One compound, Dxr2-017, exhibited selective anti-melanoma activity in the NCI-60 cell line screen. In eight-day assays, Dxr2-017 had an IC50 of 12 nM against melanoma cells, while concentrations over 2100-fold higher had minimal stem cell toxicity. Dxr2-017 induced anoikis, a unique form of programmed cell death in need of targeted therapeutics. Our findings demonstrate proof-of-concept that protein structures represent high-value primary data to support the discovery of novel acting therapeutics. This approach is widely applicable.

Modernizing the NCI60 Cell Line Screen for Phenotypic Drug Discovery in the 21st Century.

Over the past three decades, high-throughput phenotypic cancer cell line screens have revealed unanticipated small-molecule activities and illuminated connections between tumor genotypes and anticancer efficacy. Founded in 1984, the National Cancer Institute's "NCI60" screen laid the conceptual groundwork for the contemporary landscape of phenotypic drug discovery. NCI60 first operated as a primary bioactivity screen, but molecular characterization of the NCI60 cell line panel and development of a small-molecule sensitivity pattern recognition algorithm (called "COMPARE") have enabled subsequent studies into drug mechanisms of action and biomarker identification. In this issue of Cancer Research, Kunkel and colleagues report an updated version of the NCI60 screen, dubbed "HTS384" NCI60, that better aligns with current cell proliferation assay standards and has higher throughput. Changes include the use of a 384-well plate format, automated laboratory equipment, 3 days of compound exposure, and a CellTiter-Glo luminescent endpoint. To confirm that data from the HTS384 and classic NCI60 screen are comparable, the authors tested a library of 1,003 anticancer agents using both protocols and applied COMPARE to analyze patterns of cell line sensitivities. More than three dozen groups of targeted therapies showed high comparability between screens. Modernization of NCI60, and closer integration with other large-scale pharmacogenomic screens and molecular feature sets, will help this public screening service remain pertinent for cancer drug discovery efforts for years to come. See related article by Kunkel et al., p. 2403.

Design and Synthesis of New 4-(3,4,5-Trimethoxyphenyl)Thiazole-Pyrimidine Derivatives as Potential Antiproliferative Agents.

A new series of 3,4,5-trimethoxyphenyl thiazole pyrimidines has been synthesized and biologically evaluated for its in vitro anticancer activity. Compounds 4a, 4b, and 4h with substituted piperazine showed the best antiproliferative activity. In the NCI-60 cell line screening, compound 4b showed promising cytostatic activity against multiple cell lines. Notably, it elicited a GI value of 86.28% against the NSCL cancer cell line HOP-92 at a 10 μM dose. Compounds 4a and 4h at 10 μM showed promising GI values of 40.87% and 46.14% against HCT-116 colorectal carcinoma and SK-BR-3 breast cancer cell lines, respectively. ADME-Tox prediction of compounds 4a, 4b, and 4h revealed their acceptable drug-likeness properties. In addition, compounds 4a, 4b, and 4h showed a high probability of targeting kinase receptors via Molinspiration and Swiss TargetPrediction.

Abstract 3097: Prodrugs of a 1-hydroxy-2-oxopiperidin-3-yl phosphonate enolase inhibitor for the treatment of ENO1-deleted cancers

Abstract Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). We previously identified and characterized a competitive, small molecule phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX), and its lipophilic bis-ester prodrug (POMHEX) in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma. Treatment with either HEX (150 mg/kg IV/IP) or POMHEX (20 mg/kg IV/IP) yielded tumor regression even after drug discontinuation. However, due to the poor pharmacokinetics of esterase-labile POMHEX, we synthesized a library of novel phosphonate prodrugs with distinct mechanisms of bioactivation and assessed their potency in D423 (ENO1-/-) cells. By conducting a prodrug structure activity relationship (SAR) study, we found that phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, while canonical McGuigan (ProTide) prodrugs were not. Other strategies, including salicylic alcohol (cycloSal) and lipid prodrugs of HEX, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over POMHEX. En route, we developed a novel class of aliphatic amine/ester prodrugs that can be broadly applied to efficiently deliver phosph(on)ate pharmacophores in cells. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation. In sum, we have developed a novel class of phosph(on)ate prodrugs that is efficiently bioactivated in cells in vitro. Our prodrug SAR study disputes the common notion that ProTides are universally advantageous promoieties on phosph(on)ate pharmacophores and we provide mechanistic rationale for this observation with HEX. Finally, we show that the cycloSal prodrug yields efficient intracellular delivery of HEX in vitro, with a mechanism of bioactivation consistent with the GBM microenvironment, making this promoiety promising for further evaluation in vivo. Citation Format: Victoria C. Yan, Cong-Dat Pham, Elliot S. Ballato, Kristine L. Yang, Sunada Khadka, Yasaman Barektain, Prakriti Shrestha, Theresa Tran, Anton H. Poral, Mykia Washington, Sudhir Raghavan, Barbara Czako, Federica Pisaneschi, Yu-Hsi Lin, Nikunj Satani, Naima Hammoudi, Jeffrey J. Ackroyd, Dimitra K. Georgiou, Steven W. Millward, Florian L. Muller. Prodrugs of a 1-hydroxy-2-oxopiperidin-3-yl phosphonate enolase inhibitor for the treatment of ENO1-deleted cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3097.

Multifunctional metallochaperone modifications for targeting subsite cavities in mutant p53-Y220C

The p53 protein, known as the ‘guardian of the genome’, plays an important role in cancer prevention. Unfortunately, p53 mutations result in compromised activity with over 50% of cancers resulting from point mutations to p53. There is considerable interest in mutant p53 reactivation, with the development of small-molecule reactivators showing promise. We have focused our efforts on the common p53 mutation Y220C, which causes protein unfolding, aggregation, and can result in the loss of a structural Zn from the DNA-binding domain. In addition, the Y220C mutant creates a surface pocket that can be stabilized using small molecules. We previously reported the bifunctional ligand L5 as a Zn metallochaperone and reactivator of the p53-Y220C mutant. Herein we report two new ligands L5-P and L5-O that are designed to act as Zn metallochaperones and non-covalent binders in the Y220C mutant pocket. For L5-P the distance between the Zn-binding di-(2-picolyl)amine function and the pocket-binding diiodophenol was extended in comparison to L5, while for L5-O we extended the pocket-binding moiety via attachment of an alkyne function. While both new ligands displayed similar Zn-binding affinity to L5, neither acted as efficient Zn-metallochaperones. However, the new ligands exhibited significant cytotoxicity in the NCI-60 cell line screen as well as in the NUGC3 Y220C mutant cell line. We identified that the primary mode of cytotoxicity is likely reactive oxygen species (ROS) generation for L5-P and L5-O, in comparison to mutant p53 reactivation for L5, demonstrating that subtle changes to the ligand scaffold can change the toxicity pathway.

Prodrugs of a 1-Hydroxy-2-oxopiperidin-3-yl Phosphonate Enolase Inhibitor for the Treatment of ENO1-Deleted Cancers.

Cancers harboring homozygous deletion of the glycolytic enzyme enolase 1 (ENO1) are selectively vulnerable to inhibition of the paralogous isoform, enolase 2 (ENO2). A previous work described the sustained tumor regression activities of a substrate-competitive phosphonate inhibitor of ENO2, 1-hydroxy-2-oxopiperidin-3-yl phosphonate (HEX) (5), and its bis-pivaloyoxymethyl prodrug, POMHEX (6), in an ENO1-deleted intracranial orthotopic xenograft model of glioblastoma [Nature Metabolism 2020, 2, 1423-1426]. Due to poor pharmacokinetics of bis-ester prodrugs, this study was undertaken to identify potential non-esterase prodrugs for further development. Whereas phosphonoamidate esters were efficiently bioactivated in ENO1-deleted glioma cells, McGuigan prodrugs were not. Other strategies, including cycloSal and lipid prodrugs of 5, exhibited low micromolar IC50 values in ENO1-deleted glioma cells and improved stability in human serum over 6. The activity of select prodrugs was also probed using the NCI-60 cell line screen, supporting its use to examine the relationship between prodrugs and cell line-dependent bioactivation.

Dimers of isatin derived α-methylene-γ-butyrolactone as potent anti-cancer agents

The IKK-NFκB complex is a key signaling node that facilitates activation of gene expression in response to extracellular signals. The kinase IKKβ and the transcription factor RELA have been targeted by covalent modifiers that bind to surface exposed cysteine residues. A common feature in well characterized covalent modifiers of RELA and IKKβ is the Michael acceptor containing α-methylene-γ-butyrolactone functionality. Through synthesis and evaluation of a focused set of α-methylene-γ-butyrolactone containing spirocyclic dimers (SpiDs) we identified SpiD3 as an anticancer agent with low nanomolar potency. Using cell-free and cell-based studies we show that SpiD3 is a covalent modifier that generates stable RELA containing high molecular weight complexes. SpiD3 inhibits TNFα-induced IκBα phosphorylation resulting in the blockade of RELA nuclear translocation. SpiD3 induces apoptosis, inhibits colony formation and migration of cancer cells. The NCI-60 cell line screen revealed that SpiD3 potently inhibits growth of leukemia cell lines, making it a suitable pre-therapeutic lead for hematological malignancies.

Indolin-2-one derivatives as selective Aurora B kinase inhibitors targeting breast cancer

Aurora B is a pivotal cell cycle regulator where errors in its function results in polyploidy, genetic instability, and tumorigenesis. It is overexpressed in many cancers, consequently, targeting Aurora B with small molecule inhibitors constitutes a promising approach for anticancer therapy. Guided by structure-based design and molecular hybridization approach we developed a series of fifteen indolin-2-one derivatives based on a previously reported indolin-2-one-based multikinase inhibitor (1). Seven derivatives, 5g, 6a, 6c-e, 7, and 8a showed preferential antiproliferative activity in NCI-60 cell line screening and out of these, carbamate 6e and cyclopropylurea 8a derivatives showed optimum activity against Aurora B (IC50 = 16.2 and 10.5 nM respectively) and MDA-MB-468 cells (IC50 = 32.6 ± 9.9 and 29.1 ± 7.3 nM respectively). Furthermore, 6e and 8a impaired the clonogenic potential of MDA-MB-468 cells. Mechanistic investigations indicated that 6e and 8a induced G2/M cell cycle arrest, apoptosis, and necrosis of MDA-MB-468 cells and western blot analysis of 8a effect on MDA-MB-468 cells revealed 8a‘s ability to reduce Aurora B and its downstream target, Histone H3 phosphorylation. 6e and 8a displayed better safety profiles than multikinase inhibitors such as sunitinib, showing no cytotoxic effects on normal rat cardiomyoblasts and murine hepatocytes. Finally, 8a demonstrated a more selective profile than 1 when screened against ten related kinases. Based on these findings, 8a represents a promising candidate for further development to target breast cancer via Aurora B selective inhibition.

Synthesis, Characterization, and Biological Activity of Anthraquinone-Substituted Imidazolium Salts for the Treatment of Bladder Cancer

BACKGROUND: Bladder cancer is one of the most common types of cancer diagnosed each year, and more than half of patients have non-muscle invasive bladder cancer (NMIBC). The standard of care for patients with high-grade NMIBC is Bacillus Calmette-Guerin (BCG). Unfortunately, multiple BCG shortages have limited access to this treatment. Available alternatives using intravesical administration of chemotherapy have some efficacy, but lack prospective validation and long-term outcomes. Development of novel intravesical therapies may provide more active alternatives to BCG for patients with high-grade NMIBC. OBJECTIVE: To develop an optimal imidazolium salt for the intravesical treatment of NMIBC and determine preliminary in vitro activity of anthraquinone-substituted imidazolium salts. METHODS: The development of the anthraquinone-substituted imidazolium salts was undertaken in an attempt to increase the potency of this class of compounds by incorporating the quinone functional group observed in the chemotherapeutics doxorubicin, valrubicin, and mitomycin. All compounds were characterized by 1H and 13C NMR spectroscopy and infrared spectroscopy. Furthermore, these imidazolium salts were tested for in vitro cytotoxicity by the Developmental Therapeutics Program (DTP) on the NCI-60 human tumor cell line screening. Additional in vitro testing was performed against diverse bladder cancer cell lines (RT112, TCCSUP, J82, and UMUC13) using CellTiter-Glo® assays and colony-forming assays. RESULTS: The NCI-60 cell line screening indicated that compound 7 had the highest activity and was concluded to be the optimal compound for further study. Using CellTiter-Glo® assays on bladder cancer cell lines, 50% growth inhibitory concentration (IC50) values were determined to range from 32–50μM after an exposure of 1 h, for compound 7. Further evaluation of the compound by colony-forming assays showed the complete inhibition of growth at 10 days post a 100μM dose of compound 7 for 1 h. CONCLUSIONS: The most active lipophilic anthraquinone imidazolium salt, compound 7, could be a viable treatment for non-muscle invasive bladder cancer as it exhibits a cell-killing effect at a 1 h time period and completely inhibits cancer regrowth in colony-forming assays.

Specific RITA Modification Produces Hyperselective Cytotoxicity While Maintaining In Vivo Antitumor Efficacy.

The preclinical antitumor agent RITA (2,5-bis[5-hydroxymethyl-2-thienyl] furan, NSC 652287), an analog of the natural product α-terthiophene, failed during the development phase due to acute pulmonary toxicity in animal models. A series of synthetic modifications to RITA's heterocyclic scaffold resulted in activity ranging from broadly cytotoxic to highly selective. In the NCI 60-cell line screen, these "hyperselective" agents (e.g., imatinib) are rare. A selectivity index (SI) was developed to quantify this desirable feature, which is 20 for imatinib, whereas RITA's SI is only 0.10. One of the described hyperselective RITA analogs (SI = 7.9) completely lost activity in the presence of a known SULT1A1 inhibitor. These results, coupled with previous evidence that RITA is a SULT1A1 substrate, suggest that carbinol modification by a sulfate leaving group and subsequent formation of a reactive carbocation may explain RITA's broad cytotoxicity. Although SULT1A1 expression is required for susceptibility, hyperselective analogs exhibited reduced association of activity with SULT1A1 mRNA expression compared with RITA, apparently requiring some additional target(s). In support of this hypothesis, there is a strong correlation (P < 0.01, r = 0.95) between quantum mechanically calculated energy barriers for carbocation formation from sulfonated analogs and SI, indicating that hyperselective RITA analogs generate reactive carbocations less readily after sulfate activation. Importantly, narrowing the cytotoxicity profile of RITA did not eliminate its analogs' in vivo antitumor activity, as several new hyperselective agents, NSC 773097 (1), 773392 (2), and 782846 (6), displayed impressive activity against A498 xenografts in mice.

Cytotoxic Triterpenes from Salacia crassifolia and Metabolite Profiling of Celastraceae Species.

The new pentacyclic triterpene 11β-hydroxypristimerin (1), along with the known metabolites pristimerin (2), 6-oxopristimerol (3) and vitideasin (4), were isolated from a Salacia crassifolia root wood extract, following a bioassay-guided fractionation approach. Both the extract and the purified triterpenes displayed pronounced cytotoxic activity against human cancer cell lines. The NCI-60 cell line screen revealed that compound 2 was the most active, with a mean GI50 of 0.17 μM, while compound 1 had a mean GI50 of 8.7 μM. A COMPARE analysis of the screening results showed that pristimerin is likely to be the main compound responsible for the cytotoxic activity of the extract (mean GI50 of 0.3 μg·mL−1). A targeted search for pristimerin and related derivatives using LC-MS/MS revealed the presence of pristimerin (2) and 6-oxopristimerol (3) in all Celastraceae species examined and in all plant parts tested, while vitideasin (4) was only detected in the genus Salacia.

Discovery and Characterization of R/S-N-3-Cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea, a New Histone Deacetylase Class III Inhibitor Exerting Antiproliferative Activity against Cancer Cell Lines.

A new series of N-aryl-N'-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)ureas bearing an alkoxycarbonylamino group at the 6-position were synthesized and examined as putative anticancer agents targeting sirtuins in glioma cells. On the basis of computational docking combined to in vitro sirtuin 1/2 inhibition assays, we selected compound 18 [R/S-N-3-cyanophenyl-N'-(6-tert-butoxycarbonylamino-3,4-dihydro-2,2-dimethyl-2H-1-benzopyran-4-yl)urea] which displays a potent antiproliferative activity on various glioma cell types, assessed by quantitative videomicroscopy, eventually triggering senescence. The impact on normal glial cells was lower with a selectivity index of >10. Furthermore, human U373 and Hs683 glioblastoma cell lines served to demonstrate the inhibitory activity of 18 against histone deacetylase (HDAC) class III sirtuins 1 and 2 (SIRT1/2) by quantifying acetylation levels of histone and non-histone proteins. The translational potential of 18 was validated by an NCI-60 cell line screen and validation of growth inhibition of drug resistant cancer cell models. Eventually, the anticancer potential of 18 was validated in 3D glioblastoma spheroids and in vivo by zebrafish xenografts. In summary, compound 18 is the first representative of a new class of SIRT inhibitors opening new perspectives in the medicinal chemistry of HDAC inhibitors.

Design, Synthesis and Biochemical Evaluation of Novel Selective Estrogen Receptor Ligand Conjugates Incorporating an Endoxifen-Combretastatin Hybrid Scaffold.

Nuclear-receptors are often overexpressed in tumours and can thereby be used as targets when designing novel selective chemotherapeutic agents. To date, many conjugates incorporating an estrogen receptor (ER) ligand have been synthesised in order to direct chemical agents to tissue sites containing ERs. A series of ER ligand conjugates were synthesised incorporating an antagonistic ER ligand scaffold based on endoxifen, covalently-bound via an amide linkage to a variety of combretastatin-based analogues, which may act as antimitotic agents. These novel endoxifen-combretastatin hybrid scaffold analogues were biochemically evaluated in order to determine their antiproliferative and cytotoxicity effects in both the ER-positive MCF-7 and the ER-negative MDA-MB-231 human breast cancer cell lines. ER competitive binding assays were carried out to assess the binding affinity of the lead conjugate 28 towards both the ERα and ERβ isoforms. In results from the NCI 60-cell line screen, the lead conjugate 28 displayed potent and highly selective antiproliferative activity towards the MCF-7 human cancer cell line (IC50 = 5 nM). In the ER-binding assays, the lead conjugate 28 demonstrated potent ER competitive binding in ERα (IC50 value: 0.9 nM) and ERβ (IC50 value: 4.7 nM). Preliminary biochemical results also demonstrate that the lead conjugate 28 may exhibit pure antagonism. This series makes an important addition to the class of ER antagonists and may have potential applications in anticancer therapy.

NCI-60 Cell Line Screening: A Radical Departure in its Time.

The year 2015 marked the 30th anniversary of National Cancer Institute's (NCI's) initial effort to establish a human cell line panel as the basis for discovering new cancer drugs. At its inception, the NCI-60 panel was a controversial departure, born of frustration with previous efforts that employed murine tumors and grounded in the hope that the biology of human tumors was diverse and somehow quite different than the murine leukemia used for the previous 30 years. And while the NCI-60 has not revolutionized cancer drug discovery in terms of the new drugs that resulted, it represents a turning point in the philosophy and practice of cancer drug research.

Synthesis and Biochemical Evaluation of 3-Phenoxy-1,4-diarylazetidin-2-ones as Tubulin-Targeting Antitumor Agents.

Structure-activity relationships for a series of 3-phenoxy-1,4-diarylazetidin-2-ones were investigated, leading to the discovery of a number of potent antiproliferative compounds, including trans-4-(3-hydroxy-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (78b) and trans-4-(3-amino-4-methoxyphenyl)-3-phenoxy-1-(3,4,5-trimethoxyphenyl)azetidin-2-one (90b). X-ray crystallography studies indicate the potential importance of the torsional angle between the 1-phenyl "A" ring and 4-phenyl "B" ring for potent antiproliferative activity and that a trans configuration between the 3-phenoxy and 4-phenyl rings is generally optimal. These compounds displayed IC50 values of 38 and 19 nM, respectively, in MCF-7 breast cancer cells, inhibited the polymerization of isolated tubulin in vitro, disrupted the microtubular structure in MCF-7 cells as visualized by confocal microscopy, and caused G2/M arrest and apoptosis. Compound 90b possessed a mean GI50 value of 22 nM in the NCI60 cell line screen, displayed minimal cytotoxicity, and was shown to interact at the colchicine-binding site on β-tubulin. Phosphate and amino acid prodrugs of both 78b and 90b were synthesized, of which the alanine amide 102b retained potency and is a promising candidate for further clinical development.

Abstract 3659: Target validation and structural optimization of selective small molecule survivin inhibitors as potential anti-cancer agents

Abstract The anti-apoptotic protein survivin is highly expressed in most human cancer cells, but has very low expression in normal differentiated cells. Thus survivin is widely considered as an attractive cancer drug target. Herein we report the in vitro and in vivo study on a series of novel survivin inhibitors modified from our recently identified hit compound UC-112. NCI 60-cell line screening assay showed that three new analogs with oxyquinoline scaffold had GI50 values within the nanomolar range (513 nM, 646 nM and 891 nM). These new compounds can overcome the P-gp mediated multidrug-resistance in cancer cell lines. Western blot analyses demonstrated the high selectivity for survivin inhibition among other members in the inhibitor of apoptosis protein family. Mechanistic studies such as isothermal calorimetry measurements, survivin protein pull-down assay and fluorescence polarization assay confirm that these novel compounds indeed target survivin and inhibit its function. The most active compound showed excellent aqueous solubility of 148.7 μM (PBS pH7.4), reasonable metabolic stability (half-life) of 51 minutes as tested in human liver microsomes, and no significant inhibitions to major CYP enzymes except one of CYPs. Rat pharmacokinetic studies indicated good pharmacokinetic profiles for this novel scaffold. When tested in an A375 human melanoma xenograft model, this compound effectively suppressed tumor growth and strongly induced cancer cell apoptosis in tumor tissues. Overall, our study has demonstrated that these cell-permeable small molecular survivin inhibitors possessing a novel scaffold could be developed as potent anti-tumor therapeutic agents. Citation Format: Jin Wang, Min Xiao, Duane D. Miller, Wei Li. Target validation and structural optimization of selective small molecule survivin inhibitors as potential anti-cancer agents. [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 3659. doi:10.1158/1538-7445.AM2015-3659

Organometallic Iridium(III) Anticancer Complexes with New Mechanisms of Action: NCI-60 Screening, Mitochondrial Targeting, and Apoptosis

Platinum complexes related to cisplatin, cis-[PtCl2(NH3)2], are successful anticancer drugs; however, other transition metal complexes offer potential for combating cisplatin resistance, decreasing side effects, and widening the spectrum of activity. Organometallic half-sandwich iridium (IrIII) complexes [Ir(Cpx)(XY)Cl]+/0 (Cpx = biphenyltetramethylcyclopentadienyl and XY = phenanthroline (1), bipyridine (2), or phenylpyridine (3)) all hydrolyze rapidly, forming monofunctional G adducts on DNA with additional intercalation of the phenyl substituents on the Cpx ring. In comparison, highly potent complex 4 (Cpx = phenyltetramethylcyclopentadienyl and XY = N,N-dimethylphenylazopyridine) does not hydrolyze. All show higher potency toward A2780 human ovarian cancer cells compared to cisplatin, with 1, 3, and 4 also demonstrating higher potency in the National Cancer Institute (NCI) NCI-60 cell-line screen. Use of the NCI COMPARE algorithm (which predicts mechanisms of action (MoAs) for emerging anticancer compounds by correlating NCI-60 patterns of sensitivity) shows that the MoA of these IrIII complexes has no correlation to cisplatin (or oxaliplatin), with 3 and 4 emerging as particularly novel compounds. Those findings by COMPARE were experimentally probed by transmission electron microscopy (TEM) of A2780 cells exposed to 1, showing mitochondrial swelling and activation of apoptosis after 24 h. Significant changes in mitochondrial membrane polarization were detected by flow cytometry, and the potency of the complexes was enhanced ca. 5× by co-administration with a low concentration (5 μM) of the γ-glutamyl cysteine synthetase inhibitor L-buthionine sulfoximine (L-BSO). These studies reveal potential polypharmacology of organometallic IrIII complexes, with MoA and cell selectivity governed by structural changes in the chelating ligands.

Abstract C176: Activity of novel indolequinone inhibitors of thioredoxin reductase 1 in pancreatic and colon cancers and in melanomas. Signaling mechanisms underlying antiproliferative activity.

Abstract Novel indolequinones (including QG001, 5-methoxy-1-methyl-3-[(2,4,6-trifluorophenoxy)methyl]-1H-indole-4,7-dione and NJ824, 2-(hydroxymethyl)-5-methoxy-1-methyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione) demonstrated marked anti-proliferative activity in MIAPaCa-2 and BxPC-3 pancreatic cancer cells as well as in colon, melanoma and renal cell lines as indicated by testing in the NCI-60 cell line screen. In studies using MIAPaCa-2 and BxPC-3 cell lines we examined the mechanism of action of lead IQs and results demonstrated inhibition of the selenoprotein thioredoxin reductase 1 (TR1) and induction of apoptosis in pancreatic cell lines at doses approximating their IC50 values obtained from MTT growth inhibition assays. Other selenoproteins such as glutathione peroxidase were not inhibited and the depletion of cellular thiols was not observed following treatment with lead IQs indicating target selectivity. The mechanisms underlying IQ-induced apoptosis were investigated and lead IQs were found to induce oxidation of cellular thioredoxin subsequent to inhibition of thioredoxin reductase 1 and downstream activation of p38 and JNK leading to apoptosis. IQ-induced apoptosis could be inhibited by the use of p38 and JNK inhibitors in MIAPaCa-2 cells confirming the importance of this pathway. The role of ASK1 as an intermediary MAPKKK modulating signaling from oxidation of thioredoxin to activation of p38 and JNK was investigated in MIAPaCa-2 cells overexpressing ASK1 and lead IQs were found to induce ASK1 phosphorylation. Studies were extended to panels of eighteen human colorectal cancers and nineteen pancreatic cancer cell lines and lead IQs were found to exhibit marked growth inhibitory activity with IC50 values ranging from 0.05 to 0.5 micromolar. In vivo studies were performed in xenograft systems and lead IQs demonstrated significant growth inhibitory activity in both pancreatic and melanoma xenografts. These data demonstrate that this class of IQ has marked growth inhibitory activity in pancreatic, colon and melanoma tumors and are worthy of further translational evaluation as potential therapeutics. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C176.

In vitro and in vivo evaluation of select kahalalide F analogs with antitumor and antifungal activities

Kahalalide F (KF) and the regioisomer isoKF are novel anticancer drugs of marine origin and currently under clinical investigation. Here we report the synthesis of two new KF analogs with significant in vitro and in vivo antifungal and antitumor activities. The primary amine hydrogen of ornithine in KF has been replaced with 4-fluoro-3-methylbenzyl and morpholin-4-yl-benzyl via reductive N-alkylation. The TGI of these analogs using the NCI-60 cell line screening revealed promising results when compared to paclitaxel. The result of in vivo hollow fiber and animal toxicity assays are presented.

Induced Production ofN-Formyl Alkaloids fromAspergillus fumigatusby Co-culture withStreptomyces peucetius

Co-culture of the fungus Aspergillus fumigatus with the bacteria Streptomyces peucetius led to the induction of production of formyl xanthocillin analogues. This mixed fermentation yielded two new metabolites, fumiformamide (1) and N,N'-((1Z,3Z)-1,4-bis(4-methoxyphenyl)buta-1,3-diene-2,3-diyl)diformamide (2), together with two known N-formyl derivatives and the xanthocillin analogue BU-4704. The structures were determined by spectroscopic methods and by comparison with literature. Cytotoxic activity of all the analogues was tested on the NCI-60 cell line screen, and compound 2 exhibited significant activity against several cell lines. The analogues did not show antimicrobial activity.