Proteolytic inactivation of MAP-kinase-kinase by anthrax lethal factor.

Anthrax lethal toxin, produced by the bacterium Bacillus anthracis, is the major cause of death in animals infected with anthrax. One component of this toxin, lethal factor (LF), is suspected to be a metalloprotease, but no physiological substrates have been identified. Here it is shown that LF is a protease that cleaves the amino terminus of mitogen-activated protein kinase kinases 1 and 2 (MAPKK1 and MAPKK2) and that this cleavage inactivates MAPKK1 and inhibits the MAPK signal transduction pathway. The identification of a cleavage site for LF may facilitate the development of LF inhibitors.

An information-intensive approach to the molecular pharmacology of cancer.

Since 1990, the National Cancer Institute (NCI) has screened more than 60,000 compounds against a panel of 60 human cancer cell lines. The 50-percent growth-inhibitory concentration (GI50) for any single cell line is simply an index of cytotoxicity or cytostasis, but the patterns of 60 such GI50 values encode unexpectedly rich, detailed information on mechanisms of drug action and drug resistance. Each compound's pattern is like a fingerprint, essentially unique among the many billions of distinguishable possibilities. These activity patterns are being used in conjunction with molecular structural features of the tested agents to explore the NCI's database of more than 460,000 compounds, and they are providing insight into potential target molecules and modulators of activity in the 60 cell lines. For example, the information is being used to search for candidate anticancer drugs that are not dependent on intact p53 suppressor gene function for their activity. It remains to be seen how effective this information-intensive strategy will be at generating new clinically active agents.

Topoisomerase II inhibition and cytotoxicity of the anthrapyrazoles DuP 937 and DuP 941 (Losoxantrone) in the National Cancer Institute preclinical antitumor drug discovery screen.

BACKGROUND: The cumulative cardiotoxicity of anthracyclines is thought to result from the generation of free radicals. New DNA topoisomerase II inhibitors less prone to redox reactions, such as mitoxantrone and more recently the anthrapyrazoles, were developed to circumvent this toxicity. PURPOSE: Two anthrapyrazoles currently in clinical evaluation, DuP 941 (Losoxantrone) and DuP 937, were compared to other topoisomerase II inhibitors with respect to their cytotoxic potency and selectivity and with respect to topoisomerase II inhibition. METHODS: Cytotoxicity was tested in the 60 cell lines of the National Cancer Institute preclinical antitumor drug discovery screen (NCI screen). The potency of anthrapyrazoles to inhibit purified topoisomerase II was determined. The specificity of drug-induced topoisomerase II pattern of cleavage, one of the cellular determinants of cytotoxicity, was investigated in human c-myc DNA. RESULTS: Using the COMPARE analysis, we found that the most closely related cytotoxic profiles in the NCI screen were between the anthrapyrazoles and mitoxantrone. Among topoisomerase II inhibitors, the cytostatic potency was by decreasing order: mitoxantrone; doxorubicin, which was slightly greater than DuP 941, azatoxin; DuP 937; and amsacrine, which was much greater than VP-16. The potency of mitoxantrone and anthrapyrazoles to generate DNA double-strand breaks, by induction of the topoisomerase II cleavable complexes in nuclear extracts, was in agreement with cytotoxicity. Sequencing of drug-induced topoisomerase II cleavages in c-myc DNA showed a common cleavage pattern for anthrapyrazoles and mitoxantrone. This pattern was different from the patterns obtained with other topoisomerase II inhibitors. CONCLUSION: At the molecular and cellular levels, anthrapyrazoles are potent topoisomerase II inhibitors closely related to mitoxantrone. IMPLICATIONS: These results validate the COMPARE analysis using the NCI screen to predict molecular mechanisms of drug action. Anthrapyrazoles, which are unlikely to produce free radicals, might be useful in the same indications as mitoxantrone, especially for patients with cardiac risks, for pediatric patients, and for patients treated with intensified protocols.

Use of the Kohonen self-organizing map to study the mechanisms of action of chemotherapeutic agents.

BACKGROUND: Many natural and synthetic compounds might prove to be effective in cancer chemotherapy. To identify potentially useful agents, the National Cancer Institute screens over 10,000 compounds annually against a panel of 60 distinct human tumor cell lines in vitro. This screening program generates large amounts of data that are organized into relational databases. Important questions concern the information content of the data and ways to extract that information. Previously, statistical techniques have revealed that compounds with similar patterns of activity against the 60 cell lines are often similar in structure and mechanism of action. Feed-forward, back-propagation neural networks have been trained on this type of data to predict broadly defined mechanisms of action of chemotherapeutic agents. PURPOSE AND METHOD: In this report, we examine the information that can be extracted from the screening data by means of another type of neural network paradigm, the Kohonen self-organizing map. This is a topology-preserving function, obtained by unsupervised learning, that nonlinearly projects the high-dimensional activity patterns into two dimensions. Our dataset is almost identical to that used in the earlier neural network study. RESULTS: The self-organizing maps we constructed have several important characteristics. 1) They partition the two-dimensional array into distinct regions, each of which is principally occupied by agents having the same broadly defined mechanism of action. 2) These regions can be resolved into distinct subregions that conform to plausible submechanisms and chemically defined subgroups of submechanism. 3) These results (and exceptions to them) are consistent with those obtained with the use of such deterministic measures of similarity among activity patterns as the Euclidean distance or Pearson correlation coefficient. CONCLUSIONS: Our results indicate that the activity patterns obtained from the screen contain detailed information about mechanism of action and its basis in chemical structure. The self-organizing map can be used to suggest the mechanism of action of compounds identified by the screen as potentially useful chemotherapeutic agents and to probe the biology of the cell lines in the cancer screen. Kohonen self-organizing maps, unlike the previously applied neural networks, preserve and reveal the relationships among compounds acting by similar mechanisms and therefore have the potential to identify compounds that act by novel cytotoxic mechanisms.

Display and analysis of patterns of differential activity of drugs against human tumor cell lines: development of mean graph and COMPARE algorithm.

The objective of this study was to develop and investigate an approach to optimally detect, rank, display, and analyze patterns of differential growth inhibition among cultured cell lines. Such patterns of cellular responsiveness are produced by substances tested in vitro against disease-oriented panels of human tumor cell lines in a new anticancer screening model under development by the National Cancer Institute. In the first phase of the study, we developed a key methodological tool, the mean graph, which allowed the transformation of the numerical cell line response data into graphic patterns. These patterns were particularly expressive of differential cell growth inhibition and were conveniently amenable to further analyses by an algorithm we devised and implemented in the COMPARE computer program.

Identification of epidermal growth factor receptor and c-erbB2 pathway inhibitors by correlation with gene expression patterns.

BACKGROUND: Growth factor receptor-signaling pathways are potentially important targets for anticancer therapy. The interaction of anticancer agents with specific molecular targets can be identified by correlating target expression patterns with cytotoxicity patterns. We sought to identify new agents that target and inhibit the activity of the epidermal growth factor (EGF) receptor and of c-erbB2 (also called HER2 or neu), by correlating EGF receptor, transforming growth factor (TGF)-alpha (a ligand for EGF receptor), and c-erbB2 messenger RNA (mRNA) expression levels with the results of cytotoxicity assays of the 49000 compounds in the National Cancer Institute (NCI) drug screen database. METHODS: The levels of mRNAs were measured and used to generate a molecular target database for the 60 cell lines of the NCI anticancer drug screen. The computer analysis program, COMPARE, was used to search for cytotoxicity patterns in the NCI drug screen database that were highly correlated with EGF receptor, TGF-alpha, or c-erbB2 mRNA expression patterns. The putative EGF receptor-inhibiting compounds were tested for effects on basal tyrosine phosphorylation, in vitro EGF receptor tyrosine kinase activity, and EGF-dependent growth. Putative ErbB2-inhibiting compounds were tested for effects on antibody-induced ErbB2 tyrosine kinase activity. RESULTS: EGF receptor mRNA and TGF-alpha mRNA levels were highest in cell lines derived from renal cancers, and c-erbB2 mRNA levels were highest in cells derived from breast, ovarian, and colon cancers. Twenty-five compounds with high correlation coefficients (for cytotoxicity and levels of the measured mRNAs) were tested as inhibitors of the EGF receptor or c-erbB2 signaling pathways; 14 compounds were identified as inhibitors of these pathways. The most potent compound, B4, inhibited autophosphorylation (which occurs following activation) of ErbB2 by 50% in whole cells at 7.7 microM. CONCLUSIONS: Novel EGF receptor or c-erbB2 pathway inhibitors can be identified in the NCI drug screen by correlation of cytotoxicity patterns with EGF receptor or c-erbB2 mRNA expression levels.

Comparison of in vitro anticancer-drug-screening data generated with a tetrazolium assay versus a protein assay against a diverse panel of human tumor cell lines.

The National Cancer Institute (NCI) is implementing a large-scale in vitro drug-screening program that requires a very efficient automated assay of drug effects on tumor cell viability or growth. Many laboratories worldwide have adopted a microculture assay based on metabolic reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). However, because of certain technical advantages to use of the protein-binding dye sulforhodamine B (SRB) in a large-scale screening application, a detailed comparison of data generated by each type of assay was undertaken. The MTT and SRB assays were each used to test 197 compounds, on simultaneous days, against up to 38 human tumor cell lines representing seven major tumor categories. On subsequent days, 38 compounds were retested with the SRB assay and 25 compounds were retested with the MTT assay. For each of these three comparisons, we tabulated the differences between the two assays in the ratios of test group values to control values (T/C) for cell survival; calculated correlation coefficients for various T/C ratios; and estimated the bivariate distribution of the values for IC50 (concentration of drug resulting in T/C values of 50%, or 50% growth inhibition) for the two assays. The results indicate that under the experimental conditions used and within the limits of the data analyses, the assays perform similarly. Because the SRB assay has practical advantages for large-scale screening, however, it has been adopted for routine use in the NCI in vitro antitumor screen.

The ras oncogene-mediated sensitization of human cells to topoisomerase II inhibitor-induced apoptosis.

BACKGROUND: Among the inhibitors of the enzyme topoisomerase II (an important target for chemotherapeutic drugs) tested in the National Cancer Institute's In Vitro Antineoplastic Drug Screen, NSC 284682 (3'-hydroxydaunorubicin) and NSC 659687 [9-hydroxy-5,6-dimethyl-1-(N-[2(dimethylamino)ethyl]carbamoyl)-6H-pyrido -(4,3-b)carbazole] were the only compounds that were more cytotoxic to tumor cells harboring an activated ras oncogene than to tumor cells bearing wild-type ras alleles. Expression of the multidrug resistance proteins P-glycoprotein and MRP (multidrug resistance-associated protein) facilitates tumor cell resistance to topoisomerase II inhibitors. We investigated whether tumor cells with activated ras oncogenes showed enhanced sensitivity to other topoisomerase II inhibitors in the absence of the multidrug-resistant phenotype. METHODS: We studied 20 topoisomerase II inhibitors and individual cell lines with or without activated ras oncogenes and with varying degrees of multidrug resistance. RESULTS: In the absence of multidrug resistance, human tumor cell lines with activated ras oncogenes were uniformly more sensitive to most topoisomerase II inhibitors than were cell lines containing wild-type ras alleles. The compounds NSC 284682 and NSC 659687 were especially effective irrespective of the multidrug resistant phenotype. The ras oncogene-mediated sensitization to topoisomerase II inhibitors was far more prominent with the non-DNA-intercalating epipodophyllotoxins than with the DNA-intercalating inhibitors. This difference in sensitization appears to be related to a difference in apoptotic sensitivity, since the level of DNA damage generated by etoposide (an epipodophyllotoxin derivative) in immortalized human kidney epithelial cells expressing an activated ras oncogene was similar to that in the parental cells, but apoptosis was enhanced only in the former cells. CONCLUSIONS: Activated ras oncogenes appear to enhance the sensitivity of human tumor cells to topoisomerase II inhibitors by potentiating an apoptotic response. Epipodophyllotoxin-derived topoisomerase II inhibitors should be more effective than the DNA-intercalating inhibitors against tumor cells with activated ras oncogenes.

Identification of novel antimitotic agents acting at the tubulin level by computer-assisted evaluation of differential cytotoxicity data.

Data generated in the new National Cancer Institute drug evaluation program, which are based on inhibition of cell growth in 60 human tumor cell lines, were probed with nine known antimitotic agents using the COMPARE algorithm. Cytotoxicity data were available on approximately 7000 compounds at the time of the analysis, and, based on the criteria used, 82 compounds were selected as positive by the computer search. Nine were the probe compounds themselves, and 41 were analogues of known antimitotic agents. Among the remaining 32 compounds there were 19 distinct chemical species. Agents in ten of these groups (containing 20 compounds) were effective inhibitors of in vitro tubulin polymerization and caused the mitotic arrest of cells grown in culture. Two compounds were related natural products binding in the Vinca domain of tubulin, and the others were synthetic agents which interfered with colchicine binding. The remaining 12 agents (one natural product, the remainder synthetic) fell into several groups: two compounds were weak inhibitors of tubulin polymerization, inhibited colchicine binding, and caused mitotic arrest; one compound weakly inhibited tubulin polymerization but did not cause an increase in the number of cells arrested in mitosis; two compounds caused mitotic arrest at micromolar concentrations, but thus far no in vitro interaction with tubulin has been observed; the remainder neither inhibited tubulin polymerization nor caused a rise in the number of cultured cells arrested in mitosis. Tubulin-dependent GTP hydrolysis was stimulated or inhibited by all agents which inhibited tubulin polymerization with the exception of one compound. The analysis of differential cytotoxicity data thus appears to have great promise for the identification of new antimitotic agents with antineoplastic potential.

Evaluation of a soluble tetrazolium/formazan assay for cell growth and drug sensitivity in culture using human and other tumor cell lines.

We have previously described the application of an automated microculture tetrazolium assay (MTA) involving dimethyl sulfoxide solubilization of cellular-generated 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-formazan to the in vitro assessment of drug effects on cell growth (M.C. Alley et al., Proc. Am. Assoc. Cancer Res., 27:389, 1986; M.C. Alley et al., Cancer Res. 48:589-601, 1988). There are several inherent disadvantages of this assay, including the safety hazard of personnel exposure to large quantities of dimethyl sulfoxide, the deleterious effects of this solvent on laboratory equipment, and the inefficient metabolism of MTT by some human cell lines. Recognition of these limitations prompted development of possible alternative MTAs utilizing a different tetrazolium reagent, 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl] -2H- tetrazolium hydroxide (XTT), which is metabolically reduced in viable cells to a water-soluble formazan product. This reagent allows direct absorbance readings, therefore eliminating a solubilization step and shortening the microculture growth assay procedure. Most human tumor cell lines examined metabolized XTT less efficiently than MTT; however, the addition of phenazine methosulfate (PMS) markedly enhanced cellular reduction of XTT. In the presence of PMS, the XTT reagent yielded usable absorbance values for growth and drug sensitivity evaluations with a variety of cell lines. Depending on the metabolic reductive capacity of a given cell line, the optimal conditions for a 4-h XTT incubation assay were 50 micrograms of XTT and 0.15 to 0.4 microgram of PMS per well. Drug profiles obtained with representative human tumor cell lines for several standard compounds utilizing the XTT-PMS methodology were similar to the profiles obtained with MTT. Addition of PMS appeared to have little effect on the metabolism of MTT. The new XTT reagent thus provides for a simplified, in vitro cell growth assay with possible applicability to a variety of problems in cellular pharmacology and biology. However, the MTA using the XTT reagent still shares many of the limitations and potential pitfalls of MTT or other tetrazolium-based assays.

Preclinical antitumor activity of an alpha-picoline derivative, penclomedine (NSC 338720), on human and murine tumors.

Penclomedine, a synthetic alpha-picoline derivative, was identified as a potential antitumor agent in the P388 leukemia prescreen of the National Cancer Institute. Upon further evaluation in the National Cancer Institute in vivo tumor panel, the compound demonstrated good activity against two breast tumors. A single i.p. dose or five daily doses caused partial regressions of advanced-stage s.c. implanted mouse CD8F1 mammary adenocarcinomas. Also, penclomedine administered i.p. on Days 1,5, and 9 caused regression of the human MX-1 mammary carcinoma implanted under the renal capsule of athymic mice. In contrast, penclomedine demonstrated only marginal to moderate activity against the i.p. implanted L1210 leukemia and M5076 sarcoma and was inactive in three additional non-breast tumor models (i.p. B16 melanoma, i.v. Lewis lung carcinoma, and s.c. colon adenocarcinoma 38). Penclomedine administered p.o. and i.p. was equally effective against the subrenal capsule MX-1. Doses given p.o. every fourth day caused complete regression of 39 of 40 advanced-stage s.c. implanted MX-1 tumors but were much less effective against human H82 small cell lung carcinomas (13 of 80 complete regressions). Penclomedine p.o. also inhibited growth of the human MCF-7 and mouse 16/C breast adenocarcinomas. Further studies to support the development of penclomedine to clinical trial are in progress.

Multidrug-resistant phenotype of disease-oriented panels of human tumor cell lines used for anticancer drug screening.

Disease-oriented panels of human tumor cell lines used by the National Cancer Institute for large-scale in vitro anticancer drug screening were evaluated for multidrug-resistant phenotype at the functional (in vitro drug sensitivity) and molecular levels. The cell line panels manifested a broad range of sensitivities to drugs typically associated with multidrug resistance (MDR) as well as to drugs not associated with MDR. Individual cell lines displayed unique and characteristic profiles of response. Patterns of correlated response were observed among, but not between, MDR and non-MDR drugs. Strong evidence of correlated response was limited to drugs sharing an intracellular mechanism of action. Several tumor cell lines exhibited a high degree of resistance to MDR drugs and relative sensitivity to non-MDR drugs, contained high levels of MDR-1 mRNA, and expressed cell surface P-glycoprotein detectable with one or more monoclonal antibodies. Parallel expression of all of these features representing the classic MDR phenotype was observed among members of the colon and renal tumor panels. Certain individual cell lines among other panels (lung, ovarian, melanoma, and central nervous system) also manifested some aspects of the MDR phenotype to various extents. Identification of MDR cell lines used for large-scale in vitro anticancer drug screening will facilitate interpretation of data in a way which may allow identification of new drug leads of potential value in treatment of MDR tumor cell populations.

Enhanced sensitivity to 1-beta-D-arabinofuranosylcytosine and topoisomerase II inhibitors in tumor cell lines harboring activated ras oncogenes.

We used human tumor cell lines from the National Cancer Institute's In Vitro Antineoplastic Drug Screen to assess whether sensitivity to any of the approximately 45,000 compounds tested previously correlated with the presence of a ras oncogene. Among these cell lines, the mutations in Ki-ras2 clustered in non-small cell lung and colon carcinoma subpanels, and five of the six leukemia lines contained mutations in either N-ras or Ki-ras2. These analyses revealed a striking correlation with 1-beta-D-arabinofuranosylcytosine (Ara-C) and 2,2'-O-cyclocytidine sensitivity in the cell lines harboring ras mutations compared to the tumor lines with wild-type ras alleles. Strong correlations were also found with topoisomerase (topo) II inhibitors, especially 3'-hydroxydaunorubicin and an olivacine derivative. These differential sensitivities persisted in an additional 22 non-small cell lung carcinoma lines (ras mutations, n = 12 and wild-type ras, n = 10). Thus, the association with Ara-C sensitivity was greatest while topo II inhibitors showed a lower, but significant, correlation. These results suggest that the ras oncogene may play a determinant role in rendering tumor cells sensitive to deoxycytidine analogues and topo II inhibitors.

Activity of a novel 4-quinolinecarboxylic acid, NSC 368390 [6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinolinecarb oxylic acid sodium salt], against experimental tumors.

A novel, substituted 4-quinolinecarboxylic acid (NSC 339768) demonstrated antitumor activity against L1210 leukemia and B16 melanoma in the National Cancer Institute's Developmental Therapeutics Program. An extensive analogue synthesis program was initiated; over 200 derivatives were synthesized and tested for anticancer activity. One of these compounds, 6-fluoro-2-(2'-fluoro-1,1'-biphenyl-4-yl)-3-methyl-4-quinolinecarboxylic acid sodium salt, NSC 368390 (DuP-785), was selected for further investigation because of its efficacy against a spectrum of human solid tumors and its water solubility. In initial studies with L1210 leukemia, the compound caused an increase in life span of greater than 80%. The activity was schedule dependent, and the compound was equally efficacious when administered i.p., i.v., s.c., or p.o. In tests against human tumors xenografted under the renal capsule of nude mice, NSC 368390 when injected i.p. in doses of 20-40 mg/kg daily for 9 days inhibited the growth of the MX-1 breast, LX-1 lung, BL/STX-1 stomach, and CX-1 colon carcinomas by greater than 90%. NSC 368390 also inhibited the growth of three distinct human colon carcinomas, the HCT-15, clone A, and DLD-2 tumors, growing s.c. in nude mice. An i.p. dose of 25 mg/kg given daily for 9 days inhibited the growth of the DLD-2 colon cancer by 98%. 1-beta-D-Arabinofuranosylcytosine and Adriamycin were ineffective, and fluorouracil was only moderately effective against these colon tumors. Because of its good activity against human colon tumors and other human carcinomas and its water solubility, NSC 368390 (DuP-785) is being developed as a Phase 1 anticancer agent.

In vivo antitumor activity of 6-benzyl-1,3-benzodioxole derivatives against the P388, L1210, B16, and M5076 murine models.

A series of 6-benzyl-1,3-benzodioxoles have been synthesized and evaluated against the in vivo ip P388 murine lymphocytic leukemia. Selected activities against this system were tested against the additional in vivo systems L1210, B16, M5076, and MX1. The most active of the 6-benzyl-1,3-benzodioxoles tested were as effective as podophyllotoxin as experimental antitumor agents in vivo, but larger doses were required. Three of the P388-active series members were active against the in vitro astrocytoma assay, which detects compounds that interfere with or bind to tubulin.

Some substituted naphthazarins as potential anticancer agents.

Some 2,3-bis(substituted methyl)naphthazarins and related compounds were synthesized by the Diels-Alder reaction of benzoquinone and 2,3-dimethylbutadiene followed by oxidation and substitution reactions. These compounds were prepared as potential biological alkylating agents. Screening results indicated that 1,4-diacetyl-6,7-dimethyl-4a,5,8,8a-tetrahydronaphthalene and 5,8-bis(benzoyloxy)-2,3-dimethyl-1,4-naphthoquinone possessed borderline activity against leukemia P388 and that naphthazarin diacetate possessed confirmed cytotoxicity against the cell culture of human epidermoid carcinoma of the nasopharynx.

Design and synthesis of ellipticinium salts and 1,2-dihydroellipticines with high selectivities against human CNS cancers in vitro.

9-Methoxy-2-methylellipticinium acetate (6), along with the 9-methyl and 9-chloro derivatives (7, and 8, respectively) have shown remarkable selectivities in vitro against the NCI human CNS cancer subpanel. In order to target these types of compounds to the CNS in vivo, a series of 1,2-dihydroellipticines was synthesized. 9-Methoxy-2-methyl-1,2-dihydroellipticine (9) retained the potency and selectivity of the parent compound 6 but was unstable toward oxidation to 6. In order to improve the stability of 9, it was converted to the vinylogous amide 33 by introduction of a formyl group in the 4-position. Compound 33 proved to be much more stable than 9, but it was also less potent than 9 by about 1 order of magnitude, and it was less selective for the CNS subpanel than 9. To overcome the limited water solubilities of the ellipticines and dihydroellipticines, several ellipticine analogues incorporating polar groups on the N-2 nitrogen were prepared. The 2-(methoxymethyl)ellipticinium salts 24 and 25, as well as the (methylthio)methyl congener 26, were relatively potent anticancer agents which displayed cytotoxicity selectivity profiles similar to compound 6. The cytotoxic dihydroellipticines 9 and 10 exhibited potencies approaching that of ellipticine itself in facilitating the formation of a "cleavable complex", while the least cytotoxic ellipticine derivatives exhibited no cleavage above background.

Synthesis and cytotoxicity of 1,6,7,8-substituted 2-(4'-substituted phenyl)-4-quinolones and related compounds: identification as antimitotic agents interacting with tubulin.

A series of 1,6,7,8-substituted 2-(4'-substituted phenyl)-4-quinolones and related compounds have been synthesized and evaluated as cytotoxic compounds and as antimitotic agents interacting with tubulin. The 2-phenyl-4-quinolones (22-30) with substituents (e.g. F, Cl, and OCH3) at C-6, C-7, and C-8 show, in general, potent cytotoxicity against human lung carcinoma (A-549), ileocecal carcinoma (HCT-8), melanoma (RPMI-7951), and epidermoid carcinoma of the nasopharynx (KB) and two murine leukemia lines (P-388 and L1210). Introduction of alkyl groups at N-1 or C-4 oxygen led to inactive compounds (35-43 and 50). In addition, compounds 24, 26, and 27 were evaluated in the National Cancer Institute's 60 human tumor cell line in vitro screen. These compounds demonstrated the most marked effects in the screen on two colon carcinoma cell lines (COLO-205 and KM-20L2) and on a central nervous system tumor cell line (SF-539) with compound 26 the most potent of the three agents. Compounds 24, 26, and 27 were potent inhibitors of tubulin polymerization, with activity nearly comparable to that of the potent antimitotic natural products colchicine, podophyllotoxin, and combretastatin A-4. The three agents also inhibited the binding of radiolabeled colchicine to tubulin, but this inhibition was less potent than that obtained with the natural products.

Antitumor 2,3-dihydro-2-(aryl)-4(1H)-quinazolinone derivatives. Interactions with tubulin.

A series of derivatives of 2,3-dihydro-2-(aryl)-4(1H)-quinazolinone (DHQZ) with known antitumor activity was re-evaluated in the National Cancer Institute cancer cell line screen. Analysis by the COMPARE algorithm suggested that their cytotoxicity derived from interactions with tubulin. Significant inhibition of tubulin assembly and of the binding of radiolabeled colchicine to tubulin was demonstrated with several of the compounds, particularly NSC 145669, 175635, and 175636. The DHQZ derivatives are structurally analogous to a number of antimitotic agents, flavonols and derivatives of 2-styrylquinazolin-4(3H)-one and of 2-phenyl-4-quinolone. Structure-activity analogies between these agents, the combretastatins, and the colchicinoids were analyzed and summarized.

Dual inhibition of topoisomerase II and tubulin polymerization by azatoxin, a novel cytotoxic agent.

Azatoxin (NSC 640737) is a synthetic molecule that was rationally designed as a topoisomerase II inhibitor (Leteurtre et al., Cancer Res 52: 4478-4483, 1992). The present study was undertaken in order to investigate the molecular pharmacology and the cytotoxic activity of azatoxin in human tumor cells. Alkaline elution experiments performed in HL-60 cells demonstrated that: (1) azatoxin induces DNA-protein cross-links and protein-linked DNA single- and double-strand breaks characteristics of topoisomerase II inhibition in HL-60 cells; and (2) the potency of azatoxin is comparable to that of etoposide (VP-16). Testing of azatoxin in 45 human cell lines in the National Cancer Institute (NCI) in vitro Drug Screening Program indicated that azatoxin was potent (mean IC50 = 0.13 microM), but that its cell line sensitivity profile was correlated with that of tubule inhibitors rather than that of topoisomerase II inhibitors. These data led us to investigate the anti-tubulin activity of azatoxin. We found that azatoxin inhibited tubulin polymerization in vitro and was a mitotic inhibitor at 1 microM and above in the human colon cancer cell line KM20L2. In these cells topoisomerase II inhibition, as detected by the induction of protein-linked DNA strand breaks, required azatoxin concentrations of at least 10 microM. In summary, azatoxin is a potent cytotoxic agent that inhibited both tubulin and topoisomerase II. At lower azatoxin concentrations the former activity prevailed whereas at higher concentrations topoisomerase II inhibition became prominent.