NSC-3852

We searched the National Cancer Institute (NCI) compound library for structures related to the antitumor quinoline NSC3852 (5-nitroso-8-quinolinol) and used a computer algorithm to predict the antiprotozoan activity for each of 13 structures. Half of these compounds inhibited Toxoplasma gondii tachyzoite propagation in human fibroblasts at < or =1 microM. The active compounds comprise a series of low-molecular-weight quinolines bearing nitrogen substituents in the ring-5 position. NSC3852 (EC(50) 80 nM) and NSC74949 (EC(50) 646 nM) were the most potent. NSC3852 also inhibited Plasmodium falciparum growth in human red blood cells (EC(50) 1.3 microM). To investigate the mechanism for NSC3852's anti-T. gondii activity, we used chemiluminescence assays to detect reactive oxygen species (ROS) formation in freshly isolated tachyzoites and in infected host cells; the absence of ROS generation by NSC3852 in these assays indicated NSC3852 does not redox cycle in T. gondii. Inhibitors of enzyme sources of free radicals such as superoxide anion, nitric oxide (NO), and their reaction product peroxynitrite did not interfere with the anti-T. gondii activity of NSC3852. However, inhibition of T. gondii tachyzoite propagation by NSC3852 involved redox reactions because tachyzoites were protected from NSC3852 by inclusion of the cell permeant superoxide dismutase mimetic, MnTMPyP, or N-acetylcysteine in the culture medium. We conclude that the Prediction of Activity Spectra for Substances (PASS) computer program is useful in finding new compounds that inhibit T. gondii tachyzoites in vitro and that NSC3852 is a potent T. gondii inhibitor that acts by indirect generation of oxidative stress in T. gondii.

NSC3852 (5-nitroso-8-quinolinol) has cell differentiation and antiproliferative activity in human breast cancer cells in tissue culture and antitumor activity in mice bearing P388 and L1210 leukemic cells. We investigated the mechanism of NSC3852 action in MCF-7 human breast cancer cells using electron spin resonance (ESR). Reactive oxygen species (ROS) were detected in MCF-7 cell suspensions incubated with NSC3852 using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). Formation of the DMPO-OH adduct was quenched by the addition of superoxide dismutase but not by catalase, and we concluded that superoxide was generated in the NSC3852-treated cells. The flavoprotein inhibitor diphenylene iodonium suppressed ROS production, providing evidence for the involvement of a flavin-dependent enzyme system in the ROS response to NSC3852. A biologically significant oxidative response to NSC3852 occurred in MCF-7 cells. An early marker of oxidative stress was a decrease in the [glutathione]/[glutathione disulfide] ratio 1 h after NSC3852 addition. Oxidative DNA damage, marked by the presence of 8-oxoguanine, and DNA-strand breakage occurred in cells exposed to NSC3852 for 24 h. Apoptosis peaked 48 h after exposure to NSC3852. Pretreatment with the glutathione precursor N-acetyl-l-cysteine (NAC) prevented DNA-strand breakage and apoptosis. Pretreatment with NAC also reversed NSC3852 decreases in E2F1, Myc, and phosphorylated retinoblastoma protein, indicative of redox-sensitive pathway(s) in MCF-7 cells during G(1) phase of the cell cycle. We conclude that ROS formation is involved in the apoptotic and cell differentiation responses to NSC3852 in MCF-7 cells.