Imipramine

ICZ (cis‐4[4‐4‐4[[2‐(2‐4‐dichlorophenyl)‐2‐(1H‐1,2,4,triazol‐1‐methyl)‐1,3‐dioxolan‐4‐yl]‐1‐piperazinyl]phenyl]‐2,4‐dihydro‐2‐(1‐methyl‐propyl)‐3H‐1,2,4‐triazol‐3‐one; MW: 705.64) and ketoconazole were purchased from Sigma‐Aldrich (catalogue numbers #I6657 and #K1003, respectively, St‐Louis, MO, USA), while OSW‐1 ((3β,16β)‐3,17‐dihydroxy‐22‐oxocholest‐5‐en‐16‐yl‐2‐O‐acetyl‐3‐O‐[2‐O‐(4‐methoxybenzoyl)‐β‐D‐xylopyranosyl]‐α‐L‐arabinopyranoside) was from Alfa Chemistry (#ACM145075816, Ronkonkoma, NY, USA). The hydroxy‐ICZ (H‐ICZ), thapsigargin, imipramine and 25‐HC were all purchased from Cayman Chemical (#22576, #10522, #15890 and #11097, respectively, Ann Arbor, MI, USA). Stock solutions were prepared at 10 mM in dimethyl sulfoxide (DMSO) (VWR International, Radnor, PA, USA) for ICZ, H‐ICZ, ketoconazole and thapsigargin; 100 mM for imipramine; 20 mM for 25‐HC; and 1 mM for OSW‐1. Filipin complex from Streptomyces filipinensis was obtained from Sigma‐Aldrich (#F9765), and the stock solution prepared at 25 mg/ml in DMSO. λ‐phosphatase was also purchased from Sigma‐Aldrich (#P9614) at a stock solution of 400,000 units (U) per ml.

DNA relaxation assay was carried out using negatively supercoiled pBluescript plasmid and purified LdTOPIA, LdTOPIA^-Y357A, LdTOPIA^-E135A, LdTOPIAΔNLS, LdTOPIA^-1-684, LdTOPIA^-1-640 in relaxation assay buffer (50 mM Tris-HCl, pH 7.5, 0.5 mM DTT, 100 mM NaCl, 10 mM MgCl₂ and 50 μg/ml BSA, 0.5 mM EDTA, 5% glycerol) (25 (link)). Magnesium and salt dependency assays were carried out with assay buffers not containing MgCl₂ and NaCl respectively. DNA and protein were incubated at various time points at 37 °C in a thermostat and electrophoresed in 1% TAE agarose gel without EtBr in 1× TAE running buffer and later visualized using 1 μg/ml EtBr staining. Inhibition assays were performed using drugs, 10, 20 and 40 μM of Imipramine (TCI) and Norclomipramine (Cayman Chemicals), 20 μM and 50 μM of Camptothecin (TCI) and Etoposide (Sigma) with LdTOPIA, and 10 μM of Camptothecin and Etoposide with Human TOPIB (Sigma) and Human TOPII (Sigma).