Kenneth D. Paull

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.

Mining the NCI Anticancer Drug Discovery Databases: Genetic Function Approximation for the QSAR Study of Anticancer Ellipticine Analogues

The U.S. National Cancer Institute (NCI) conducts a drug discovery program in which approximately 10,000 compounds are screened every year in vitro against a panel of 60 human cancer cell lines from different organs of origin. Since 1990, approximately 63,000 compounds have been tested, and their patterns of activity profiled. Recently, we analyzed the antitumor activity patterns of 112 ellipticine analogues using a hierarchical clustering algorithm. Dramatic coherence between molecular structures and activity patterns was observed qualitatively from the cluster tree. In the present study, we further investigate the quantitative structure-activity relationships (QSAR) of these compounds, in particular with respect to the influence of p53-status and the CNS cell selectivity of the activity patterns. Independent variables (i.e., chemical structural descriptors of the ellipticine analogues) were calculated from the Cerius2 molecular modeling package. Important structural descriptors, including partial atomic charges on the ellipticine ring-forming atoms, were identified by the recently developed genetic function approximation (GFA) method. For our data set, the GFA method gave better correlation and cross-validation results (R2 and CVR2 were usually approximately 0.3 higher) than did classical stepwise linear regression. A procedure for improving the performance of GFA is proposed, and the relative advantages and disadvantages of using GFA for QSAR studies are discussed.

Reduced folate carrier gene (RFC1) expression and anti-folate resistance in transfected and non-selected cell lines

Methotrexate transport deficiency due to decreased reduced folate carrier (RFC) activity has been observed in several cell lines selected for resistance to methotrexate (MTX). Since MTX resistance is multifactorial, however, it is difficult to quantify the relative importance of changes in RFC activity in selected cell lines and even more so to determine the relative contribution of naturally occurring RFC activity in the MTX sensitivity of non‐selected cell lines. We examined the role of RFC in MTX resistance by studying a transport‐deficient cell line transfected with the gene for human RFC, RFC1, and by correlating relative RFC1 expression with MTX and trimetrexate (TMTX) growth inhibition (GI50) in a panel of cell lines used in the NCI Anticancer Drug Screen. Clones of transport‐deficient, MTX‐resistant ZR‐75‐1 human breast cancer cells (MTXR ZR‐75‐1) transfected with RFC1 were 250‐fold more sensitive to MTX and 300‐fold more resistant to TMTX than control cell clones, showing that restoration of RFC activity has a significant impact on MTX and TMTX cytotoxicity. We also surveyed 40 of the 60 cell lines in the NCI drug screen panel for RFC1 RNA levels by a quantitative RT‐PCR assay. RFC1 RNA levels varied over a range of 15‐fold, with only 1 cell line found to be null in expression. Using data from the 6‐day drug exposure assay, RFC1 correlated positively with MTX and negatively with TMTX cytotoxicity. As predicted by transfection studies, the calculated difference between MTX and TMTX potency was even more strongly correlated with RFC1 RNA levels of the cell lines. In addition, compounds in the NCI Anticancer Drug Screen database with cytotoxicity profiles which correlated with RFC1 RNA levels or with the calculated difference in MTX‐TMTX potency were examined for MTX uptake inhibition and cytotoxicity in the RFC1‐transfected MTXR ZR‐75‐1 cell line. Overall, our data demonstrate the importance of RFC1 in MTX resistance both as a transgene and as a constitutively expressed gene in non‐selected cell lines. Int. J. Cancer 72:184–190, 1997.

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.

Cytotoxicity of a new IMP dehydrogenase inhibitor, benzamide riboside, to human myelogenous leukemia K562 cells

COMPARE computer program suggested that benzamide riboside, BR, 3-(1-deoxy-beta-D-ribofuranosyl)benzamide, should have a similar mechanism of action as that of tiazofurin, an inhibitor of IMP dehydrogenase (IMPDH). This hypothesis was tested in K562 cells in culture. BR was cytotoxic to K562 cells with an IC50 of 2 microM. Incubation of K562 cells with BR resulted in a significant decrease in GMP and GTP levels with a concurrent increase in IMP pools, and with a significant inhibition of IMPDH activity. However, 290-fold higher BR concentration was needed to demonstrate in vitro inhibition of IMPDH activity, suggesting that the agent may require metabolism to exert its action. These results provide evidence that BR is a new inhibitor of IMPDH. This investigation should be helpful to design new analogues having activity against IMPDH.

Preclinical antitumor activity of batracylin (NSC 320846)

SummaryBatracylin (NSC 320846, BAY H 2049), given ip on days 2 and 9 at a dose of 400 mg/kg, inhibited tumor growth completely in 80–100% of mice with early-stage colon adenocarcinoma 38. Therapeutic efficacy against this subcutaneously implanted tumor was retained upon oral administration of Batracylin although, compared to ip treatment, larger doses were required. Batracylin also caused regression of advanced (400 mg) colon 38 tumors. Only modest activity was observed for this compound against P388 leukemia, but P388 sublines with acquired resistance to either adriamycin or cisplatin demonstrated collateral sensitivity. Batracylin currently is undergoing toxicological evaluation by NCI prior to clinical trials.

Comparison of biochemical parameters of benzamide riboside, a new inhibitor of IMP dehydrogenase, with tiazofurin and selenazofurin

The biochemical and cytotoxic activities of the IMP dehydrogenase (IMPDH) inhibitors benzamide riboside, tiazofurin, and selenazofurin were compared. These three C-nucleosides exert their cytotoxicity by forming an analogue of NAD, wherein nicotinamide is replaced by the C-nucleoside base. The antiproliferative activities of these three agents were compared in a panel of 60 human cancer cell lines. To examine the relationship of benzamide riboside and selenazofurin to tiazofurin, COMPARE computer analysis was performed, and correlation coefficients of 0.761 and 0.815 were obtained for benzamide riboside and selenazofurin, respectively. The biochemical activities of these agents were examined in human myelogenous leukemia K562 cells. Incubation of K562 cells for 4 hr with 10 microM each of benzamide riboside, selenazofurin and tiazofurin resulted in a 49, 71, and 26% decrease in IMPDH activity with a concurrent increase in intracellular IMP pools. As a consequence of IMPDH inhibition, GTP and dGTP concentrations were curtailed. These studies demonstrated that selenazofurin was the most potent of the three agents. To compare the cellular synthesis of NAD analogues of these agents, K562 cells were incubated with 10 microM each of benzamide riboside, tiazofurin and selenazofurin after prelabeling the cells with [2,8-3H]adenosine. The results demonstrated that benzamide riboside produced 2- and 3-fold more of NAD analogue (BAD) than tiazofurin and selenazofurin did. To elucidate the effects of the three compounds on other NAD-utilizing enzymes, the inhibitory activities of purified benzamide adenine dinucleotide (BAD), thiazole-4-carboxamide adenine dinucleotide (TAD) and selenazole-4-carboxamide adenine dinucleotide (SAD) were studied in commercially available purified preparations of lactate dehydrogenase, glutamate dehydrogenase and malate dehydrogenase. TAD and SAD did not inhibit these three dehydrogenases. Although BAD did not influence lactate and glutamate dehydrogenases, it selectively inhibited 50% of malate dehydrogenase activity at a 3.2 microM concentration. These studies demonstrate similarities and differences in the biochemical actions of the three C-nucleosides, even though they share similar mechanisms of action.

Computer Assisted Structure-Antileukemic Activity Correlations of Organotin Compounds and Initial Exploration of their Potential Anti-HIV Activity

The National Cancer Institute (NCI) and others have a continuing interest in exploring the anticancer and recently the anti-HIV potential of metal and metalloid compounds (Cleare et al 1980; Lippard 1983; Crowe et al 1980). The number of metal-containing compounds in the NCI collection is given in Table 1. Organotin compounds are the largest class among metals as represented by more than 2,000 compounds. This emphasis is the natural consequence of the wide biological use (Cardarelli 1972; Arakawa et al 1988) of tin compounds and their subsequent availability for screening by the NCI. A good deal of work has been done on the chemistry (Davis et al 1980; Wardell 1967), toxicology (Cardarelli 1986; Smith et al 1975), and metabolism (Thayer 1974; Kimmel 1977) of tin compounds. This study focuses on the structure-anticancer activity of tin-containing compounds evaluated by the NCI.

Exploratory data analytic techniques to evaluate anticancer agents screened in a cell culture panel

Information theory is used to provide a measure of selectivity, i.e., the degree to which a drug has preferential toxicity or growth inhibition for one or a few cell lines from a large panel. The selectivity measure is intended to complement a measure of differential growth inhibition in evaluating the drug development potential of a new compound. Also, a similarity measure obtained from information theory is used to classify drugs according to their pattern of responses on the panel. Some structure-activity relations emerge. This work is applied to 176 agents selected to be tested by the National Cancer Institute in about 50 cell lines.

Computer-Assisted Structure-Activity Correlations

Several types of Michael acceptors, including alpha,beta-unsaturated ketones, lactones, and lactams, have been extensively studied as potential anticancer agents. A concerted effort was made to explore the relationship between the structures of these compounds and their antitumor activity against P388 and L1210 leukemias. This article describes the computer-assisted structure-activity evaluation of more than 14,000 compounds, representing different classes of Michael acceptors, in the NCI file. In this study, advantage has been taken of the computers ability to search substructures according to precise definitions and to manipulate these substructures utilizing Boolean logic. Olefinic conjugated Michael-type acceptors, e.g., styrenes with different activating groups, cinnamic acid derivatives, and alpha,beta-unsaturated nitro, cyano, sulfone, sulfoxide, and acetylenic compounds, have shown appreciable activity against P388 lymphocytic leukemia. The analysis of lactones and lactams includes substructures representing a wide variety of exocyclic and endocyclic alpha,beta-unsaturated compounds. The analysis describes the effect of certain groups, such as an --OH, --OR, alpha,beta-unsaturated ester, or epoxide, adjacent to the alpha,beta-unsaturated center, on the antitumor activity of these compounds. A combination of one or more of these activating groups with an alpha-methylene-gamma-lactone moiety significantly enhanced the activity against P388. In general, many of the classes of compounds studied have shown poor activity against the more stringent L1210 lymphoid leukemia.