Volume 12, Issue 12, Pages 1317-1324 (December 2005) The β-Carboline Analog Mana-Hox Causes Mitotic Aberration by Interacting with DNA  Lan Chun Tu, Chien-Shu Chen, I-Ching Hsiao, Ji-Wang Chern, Chi-Hung Lin, Ya-Ching Shen, Sheau Farn Yeh  Chemistry & Biology  Volume 12, Issue 12, Pages 1317-1324 (December 2005) DOI: 10.1016/j.chembiol.2005.09.014 Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 1 Structure of Mana-Hox and Analogs Chemistry & Biology 2005 12, 1317-1324DOI: (10.1016/j.chembiol.2005.09.014) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 2 Cellular Localization of Mana-Hox in HeLa Cells (A) HeLa cells were exposed to 4 μM 1 for 1 min, 10 min, 60 min, or 5 hr. After drug removal, cells were observed with a fluorescence microscope, and the presence and distribution of 1 were determined by its fluorescence. (B–D) HeLa cells were transfected with organelle markers as described in Experimental Procedures. Cells were treated with 1 for the indicated time at 12 hr posttransfection. The colocalization of 1 and the subcellular organelles were examined with a fluorescence microscopy. (E) HeLa cells were exposed to 4 μM Mana-Hox for 13 hr. After drug removal, cells were observed with a fluorescence microscope. Note the accumulation of 1 in the aberrant metaphase chromosomes. (F) Drug treatments were as described in (E). The presence of Mana-Hox (stained green) in the aberrant metaphase chromosomes was confirmed by the staining with the DNA dye Hoechst 33258 (stained blue). Chemistry & Biology 2005 12, 1317-1324DOI: (10.1016/j.chembiol.2005.09.014) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 3 Fluorescence Spectrum of Mana-Hox and Its Alteration upon the Addition of DNA Solutions were excited at 287 nm, and emission spectra were recorded. Solutions consist of 1 μM 1 (D) and calf thymus DNA (P) at 0.25, 0.5, 1, 2.5, 5, 7.5, and 10 μM. Chemistry & Biology 2005 12, 1317-1324DOI: (10.1016/j.chembiol.2005.09.014) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 4 Mana-Hox Can Intercalate DNA and Bind to the DNA Minor Groove (A) Quenching of fluorescence intensity of the ethidium bromide-DNA complex by Mana-Hox and analogs. Calf thymus DNA (20 μM) was preincubated with 2 μM ethidium bromide in 0.01 M ionic strength buffer, and was then incubated separately with 1, 2, and 3 in various concentrations for another 10 min. The samples were excited at 538 nm, and the fluorescence was measured at 590 nm (mean ± SEM; n = 3). (B) Effect of Mana-Hox on the electrophoretic mobility of supercoiled DNA. pC86 (2 μg) was preincubated with various concentrations of 1 for 30 min, loading buffer was then added, and the samples were electrophoresed through 1% agarose gels. Abbreviations: S, plasmid DNA; EB, ethidium bromide; Tx, taxol; OC, nicked, open circular DNA; SC, supercoiled DNA. (C) Quenching of fluorescence intensity of the Hoechst 33258-DNA complex by Mana-Hox and analogs. Calf thymus DNA (1 μg) was preincubated with 6.1 μM Hoechst 33258 in PBS for 10 min at room temperature, and was then incubated separately with 1, 2, and 3 in various concentrations for another 10 min. The samples were excited at 355 nm, and the fluorescence was measured at 460 nm (mean ± SEM; n = 3). Chemistry & Biology 2005 12, 1317-1324DOI: (10.1016/j.chembiol.2005.09.014) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 5 The Energy-Minimized Docked Model of the Mana-Hox-DNA Complex (A) The lowest-energy docked conformations proposed the energetically favorable binding of 1, with the β-carboline moiety being intercalated into d(CCGGCGGT)2. a, line plot; b, space-filling plot. c, hydrogen bonding interactions in the intercalation site of the energy-minimized docked model of the 1-DNA complex. The water molecule was from the solvation calculations. (B) The lowest-energy docked conformations proposed the energetically favorable binding of 1 to d(CGCAAATTTGCG)2 through the minor groove. a, line plot; b, space-filling plot. The following color scheme is used: 1: carbon, green; nitrogen, blue; hydrogen, white; hydrogen bond, white dotted line; oxygen, red; DNA duplex, purple. Chemistry & Biology 2005 12, 1317-1324DOI: (10.1016/j.chembiol.2005.09.014) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 5 The Energy-Minimized Docked Model of the Mana-Hox-DNA Complex (A) The lowest-energy docked conformations proposed the energetically favorable binding of 1, with the β-carboline moiety being intercalated into d(CCGGCGGT)2. a, line plot; b, space-filling plot. c, hydrogen bonding interactions in the intercalation site of the energy-minimized docked model of the 1-DNA complex. The water molecule was from the solvation calculations. (B) The lowest-energy docked conformations proposed the energetically favorable binding of 1 to d(CGCAAATTTGCG)2 through the minor groove. a, line plot; b, space-filling plot. The following color scheme is used: 1: carbon, green; nitrogen, blue; hydrogen, white; hydrogen bond, white dotted line; oxygen, red; DNA duplex, purple. Chemistry & Biology 2005 12, 1317-1324DOI: (10.1016/j.chembiol.2005.09.014) Copyright © 2005 Elsevier Ltd Terms and Conditions

Figure 6 Mana-Hox Inhibits Topoisomerase II Relaxation Plasmid DNA (1 μg) was incubated with various concentrations of 1, and was then treated with 8 U topoisomerase II for 30 min at 37°C. The reaction was stopped by adding EDTA/SDS. The samples were electrophoresed through 1% agarose gel. S, supercoiled DNA; T, topoisomerase II; E, etoposide (85 μM). Chemistry & Biology 2005 12, 1317-1324DOI: (10.1016/j.chembiol.2005.09.014) Copyright © 2005 Elsevier Ltd Terms and Conditions