The S. cerevisiae strain EKY3 (MAT$\alpha$, ura3–52, his3$\mathrm{\Delta }$200, leu2$\mathrm{\Delta }$1, trp1$\mathrm{\Delta }$63, top1$\mathrm{\Delta }$::TRP1) previously described [28, 29, 30] was kindly provided by Dr. Leroy Liu at UMDNJ. The human TOP1 cDNA sequence (Accession No. NM_003286.3) with its starting ATG and termination codon was cloned into plasmid pYES2.1/TOPO (Invitrogen). The resulting clone pYES2-TOP1 expresses recombinant human TOP1 under the control of the GAL1 promoter. The T718A substitution was created in pYES2-TOP1 using the Q5 site-directed mutagenesis kit (New England BioLabs) with primers 5${}^{\prime }$-TCGCCTGGGAGCCTCCAAACTCA-3${}^{\prime }$ and 5${}^{\prime }$- ATCTGTTTATTTTCCTCTCGGTCTGTG-3${}^{\prime }$.

Recombinant human TOP1 enzyme was purchased from TopoGEN, and also purified from the lysate of S. cerevisiae EKY3/pYES2-TOP1 induced for expression of wild-type human TOP1 with the PrepEase Histidine-Tagged Protein Purification Midi Kit-High Specificity (from Affymetrix) according to the manufacturer’s procedures. Camptothecin, myricetin and other flavonoids were purchased from Adooq Bioscience. NSC65860 (3-hydroxy-4-[[4-[[4-[4-[(2-hydroxy-3, 6-disulfonaphthalen-1-yl) diazenyl] phenyl] sulfanylphenyl] carbamoyl] phenyl] diazenyl]naphthalene-2, 7-disulfonic acid) was provided by NCI/DTP Open Chemical Repository.

Human TOP1 was assayed in reaction buffer containing 10 mM Tris-HCl pH 7.9, 1 mM EDTA, 0.15 M NaCl, 0.1% BSA, 0.1 mM spermidine, 5% glycerol [31]. The compounds were added to the enzyme suspended in the buffer, followed by addition of 200 ng of negatively supercoiled DNA in the same buffer for a final volume of 20 $\mu$L. The amount of enzyme added was first determined to be the minimal amount required for complete relaxation of the input DNA following 30 min incubation at 37 ${}^{\circ }$C in the presence of 0.5 $\mu$L DMSO as control. The reactions were terminated with 4 $\mu$L of 6x SDS stop buffer (6% SDS, 6 mg/mL bromophenol blue, 40% glycerol) and was analyzed via electrophoresis in 1% agarose gel with TAE buffer [32]. The gel was stained in a 1 $\mu$g/mL solution of ethidium bromide for 30–45 minutes and rinsed for 10–15 minutes in deionized water before photographed over UV light using the ProteinSimple AlphaImager Mini system. The percent inhibition was determined with the AlphaImager software based on intensity of supercoiled DNA remaining. The average and standard error of results from three experiments was plotted with GraphPad Prism to obtain the IC${}_{50}$ for inhibition.

For spot test of growth inhibition from expression of human TOP1-T718A, EKY3/pYES-TOP1 and EKY3/pYES2-TOP1-T718A were grown from single colonies in 3 mL of Synthetic Complete-Uracil (SC-U) media supplemented with 2% dextrose and incubated overnight at 30 ${}^{\circ }$C. On the second day, the cultures were diluted with SC-U + 2% raffinose 1:50 for a final volume of 3 mL and grown overnight at 30 ${}^{\circ }$C. On the third day, the cultures were diluted into 2 sets with SC-U media + 2% raffinose with OD600 adjusted to 0.1 and 0.5 respectively. Five-fold serial dilutions were then made with SC-U media + 2% raffinose. Five microliters of these serial dilutions were spotted on SC-U agar plates supplemented with either 2% dextrose (expression repressor) or 2% galactose (expression inducer). Agar plates were incubated at 30 ${}^{\circ }$C for 4–5 days.

To monitor growth in 96 well microplates, EKY3/pYES2-TOP1 and EKY3/pYES2-TOP1-T718A cultures were grown from single colonies in SC-U media supplemented with 1% dextrose for 24 hours at 30 ${}^{\circ }$C. The culture was then diluted 1:50 into SC-U supplemented with 1% raffinose, and allowed to grow for a second day at 30 ${}^{\circ }$C. On the day of the experiment, OD600 values of the growing cultures were adjusted to 0.05 with SC-U supplemented with either 1% dextrose or 1% galactose. The compounds tested were diluted serially in the same media in the successive rows of a sterile CELLTREAT polystyrene 96 well round bottom non-treated plate before the addition of an equal volume of the diluted cultures, achieving a final volume of 100 $\mu$L of OD600 = 0.025 starting culture in each well. The plate was then incubated at 30 ${}^{\circ }$C for at least 66 hours. OD600 measurements were taken to monitor growth using a Synergy HT plate reader at each time point after 2 min of shaking. Statistical significance of observed differences in OD600 from compound treatment was determined by using the two-way t-test for two independent means.

EKY3/pYES2-TOP1-T718A was grown from single colonies to saturation first in SC-U media supplemented with 1% dextrose for 24 hours at 30 ${}^{\circ }$C. The culture was then diluted 1:50 into SC-U media supplemented with 1% raffinose, and grown for a second day at 30 ${}^{\circ }$C. This saturated culture was used to prepare in 96-well plates 200 $\mu$L volume of diluted cultures (final OD600 = 0.025) containing DMSO (1% v/v) or 100 $\mu$M NSC65860. Following incubation at 30 ${}^{\circ }$C for the indicated amount of time, 20 $\mu$L of cultures were removed for serial dilutions in Phosphate Buffered Saline (PBS) and plated on YPD plates. Viable colonies were counted after incubation at 30 ${}^{\circ }$C for 3 days.

To confirm recombinant human TOP1 expression under the control of the GAL1 promoter in EKY3 cells, transformants were grown from colonies on plates in Synthetic Complete-Uracil (SC-U) media supplemented with 1% dextrose for 24 hours at 30 ${}^{\circ }$C. The culture was then diluted 1:50 into SC-U supplemented with 1% raffinose, and allowed to grow for a second day at 30 ${}^{\circ }$C. Finally, the OD600 values of the cultures were adjusted to 0.05 with SC-U media supplemented with either 1% dextrose (expression repressor) or 1% galactose (expression inducer) and allowed to grow for 66 hours at 30 ${}^{\circ }$C. Cell pellets were collected by centrifugation at 16,000 $\times$ g for 5 min.

For relaxation activity assay, cells were resuspended in a cell breaking buffer consisting of 50 mM sodium phosphate pH 7.4, 5% glycerol, 1 mM EDTA and 1 mM PMSF. OD600 values of the cell pellets in breaking buffer were adjusted to 25, then acid-washed glass beads were added to the buffer and vortexed with the resuspended cell pellets for four cycles of 30 sec agitation followed by 30 sec of cooling on ice. Soluble lysates were obtained by centrifugation at 16,000 $\times$ g for 10 min. Supercoiled plasmid DNA was used as substrate to assay the DNA-relaxing activity. Two microliters of soluble lysates were added to 200 ng of negatively supercoiled pBAD/Thio DNA in a reaction buffer that composed of 10 mM Tris HCl, pH 7.9, 1 mM EDTA, 0.15 M NaCl, 0.1% BSA, 0.1 mM spermidine, and 5% glycerol [31]. All reactants were incubated for 30 minutes at 37 ${}^{\circ }$C, then each reaction was stopped by adding 6 $\mu$L of 4X SDS stop buffer (6% SDS, 0.3% bromophenol blue, 30% glycerol) and the supercoiled DNA and relaxed DNA were then separated by electrophoresis in 1% agarose gel. Purified recombinant human TOP1 (TopoGEN) was used as positive control.

For Western blot analysis of the human TOP1 expression, cell pellets were washed with 500 $\mu$L deionized water, then re-suspended in a volume of 0.1 M NaOH that resulted in OD600 readings of 2–3. Following 10 minutes at room temperature, the cell pellets were collected by centrifugation again and boiled for 5 minutes in the same volume of a sample buffer consisting of 60 mM Tris-HCl, pH 6.8, 4% 2-mercaptoethanol, 5% glycerol, 2% SDS, and 0.0025% bromophenol blue [33]. Samples were loaded and run in 7.5% SDS-PAGE. After transferring separated proteins to a nitrocellulose membrane, the membrane was blocked with 5% Bovine Serum Albumin (BSA) at room temperature for 1 hour, then incubated with a 1:1000 (v/v) solution of mouse anti-human TOP1 antibodies (Developmental Studies Hybridoma Bank, Cat. No. CPTC-Top1-2) and mouse anti-Actin antibodies (Developmental Studies Hybridoma Bank, Cat. No. JLA-20) in 1X TBST buffer at 4 ${}^{\circ }$C overnight. The membrane was washed three times for 5 min each in 1X TBST buffer, and then a 1:5000 (v/v) solution of horse-radish peroxidase (HRP) conjugated mouse IgG kappa binding protein (Santa Cruz Biotechnology Cat. No. sc-516102) in 1% BSA solution in 1X TBST buffer was incubated with the membrane at room temperature for one hour. After thorough washing and rinsing of the membrane in 1X TBST buffer, SuperSignal West Pico PLUS Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA) was used to visualize the protein bands.

We performed docking of NSC65860 against a human TOP1 structure following previously published procedures [34]. Briefly, the 2D structure of NSC65860 (obtained from PubChem) was converted to a 3D structure. A random 3D ligand conformer was then obtained and converted to a pdbqt file by adding polar hydrogens using Open Babel [35]. PDB 1K4T [36] was used to extract human TOP1 and DNA coordinates only and I-TASSER [37] was used to model missing residues to generate the full length structure of human TOP1. A 10 ns molecular dynamics (MD) simulation was conducted for this newly generated full length human TOP1-DNA structure using the NAMD simulation package [38]. The topology and parameter files used to generate and simulate the structure are as described previously [34]. Protein only coordinates (full length human TOP1) from the covalent complex after 10 ns MD simulation was then selected as the target structure for docking. AutoDockTools [39] was used to generate a pdbqt file of the human TOP1 structure. Finally, AutoDock vina [40] was used to dock the NSC65860 against human TOP1 target structure. The dimensions of the search box was 90 $\times$ 120 $\times$ 130 Å${}^3$ that covered the entire human TOP1. Five independent dockings were performed against the same human TOP1 target using the same NSC65860 pdbqt file as mentioned above. The docked conformation with the highest affinity among the affinities of the top predicted model from the results of each docked result was then selected as the best conformation. VMD [41] was used to generate the docked position of the best docked NSC65860 structure against human TOP1 and to analyze hydrogen bonds. An atomic distance of 3.5 Å and angle cut off of 30 degrees were used to predict hydrogen bonding.

To validate the catalytic function of recombinant human TOP1 induced in EKY3 yeast cells, EKY3 cell lysates were prepared and assayed with supercoiled plasmid DNA as substrate. The relaxation activity of the human TOP1 in the lysates were compared with purified recombinant human TOP1 (from TopoGEN) as control. As shown in Fig. 2, EKY3/pYES2-TOP1 cells expressed active human TOP1 in the presence of galactose as inducer of the GAL1 promoter (lane 3). The human TOP1 activity was present at lower level when the GAL1 promoter was repressed by dextrose (lane 5). Under our assay conditions, we could not detect any relaxation activity in the soluble lysates of EKY3/pYES2-TOP1-T718A cells growth in presence of either dextrose or galactose (Fig. 2, lanes 4 and 6).

Fig. 2.

Activity of recombinant human TOP1 expressed in EKY3. Lane 1. Supercoiled plasmid DNA substrate. Lane 2. 1 U of human TOP1 from TopoGEN added. Lane 3–6. Equal amounts of lysates of EKY3 transformed with wild-type pYES2-TOP1 plasmid (lanes 3, 5) or mutant pYES2-TOP1-T718A plasmid (lanes 4, 6) were compared. Expression of recombinant human TOP1 was induced by galactose (lanes 3, 4) or repressed by dextrose (lanes 5, 6). S, supercoiled DNA; N, nicked DNA; FR, fully relaxed DNA; PR, partially relaxed DNA. The lanes shown here are from the same gel.

We confirmed that the accumulation of the covalent intermediate formed by recombinant TOP1-T718A in EKY3 led to galactose-dependent growth inhibition. Growth observed from serial dilutions spotted on agar plates (Fig. 3A) showed equal growth of wild-type and mutant transformants on agar plate with 2% dextrose but no growth for transformant of mutant TOP1-T718A on agar plate with 2% galactose. We tested lower galactose concentrations for induction of TOP1-T718A expression in 96 well microplates so that the rescue of growth by TOP1 suppressors could be more readily observed in screening assays. Growth inhibition of EKY3 from induction of mutant TOP1-T718A could be readily detected in 96 well microplates when the OD600 readings of cultures in 1% galactose or dextrose were monitored (Fig. 3B). Galactose induction of wild-type TOP1 is well tolerated at in both assays.

Fig. 3.

Galactose-dependent growth inhibition of EKY3 from expression of TOP1-T718A mutant. (A) EKY3/pYES2-TOP1 and EKY3/pYES2-TOP1-T718A saturated cultures in SC-U media with 2% raffinose were diluted to OD600 = 0.1 (top 2 rows) or 0.5 (bottom two rows) before 5-fold serial dilutions were spotted on SC-U agar plates supplemented with either 2% dextrose or galactose. (B) Growth curves of EKY3/pYES2-TOP1 and EKY3/pYES2-TOP1-T718A in SC-U media supplemented with 1% dextrose or galactose.

Camptothecin is an extensively characterized human TOP1 poison with synthetic analogs that are used clinically in anticancer treatment [14, 17]. Addition of camptothecin to culture of EKY3/pYES2-TOP1 resulted in growth inhibition to a much greater extent in the presence of galactose versus dextrose, as expected from the toxic effect from the trapping of the covalent complex formed by induced recombinant TOP1 (Fig. 4). In contrast, camptothecin had relatively little effect on growth of EKY3/pYES2-TOP1-T718A with expression of the mutant TOP1-T718A induced by galactose, since this mutant protein already forms a stabilized TOP1cc. These results demonstrate that utility of the yeast screening system for studying the TOP1 poisons. The slight increase in OD values observed for camptothecin treatment of EKY3/pYES2-TOP1-T718A is not statistically significant, but could be due to other unknown effects of camptothecin.

Fig. 4.

Effect of camptothecin on growth of EKY3/pYES2-TOP1 and EKY3/pYES-TOP1-T718A in media with dextrose or galactose. (A) Representative growth curves with increasing concentrations of camptothecin present. (B) Effect of camptothecin on relative OD600 of cultures after 66 hours in dextrose media with non-induced versus galactose media with induced expression of wild-type (WT) or T178A mutant recombinant human TOP1. ****: p 0.0001, **: p 0.01, ns, not significant.

In addition to camptothecin, natural products have been reported in the literature as human TOP1 inhibitors [42, 43, 44, 45]. It is not always clear if these natural products act as poisons or suppressors in their TOP1 inhibition mechanism [45]. A set of nine flavonoids and similar compounds (Supplementary Fig. 1) were first tested for inhibition of TOP1 relaxation activity at up to 250 $\mu$M concentration. Myricetin was the only compound found in this preliminary study to inhibit the relaxation activity of recombinant human TOP1 expressed in yeast completely. Follow-up assay of serial dilution of myricetin showed that the IC${}_{50}$ for inhibition of more highly purified human TOP1 from TopoGEN is ~10 $\mu$M (Fig. 5A). Testing of myricetin in the yeast-based assay (Fig. 5B) showed that it inhibited growth of EKY3/pYES2-TOP1 with either galactose or dextrose in the media, with a greater degree of growth inhibition in galactose containing medium. There is also no increase in the growth of EKY3/pYES2-TOP1-T718A. These results suggested that in addition to inhibiting TOP1 activity as a poison, myricetin can inhibit yeast cell growth with TOP1-independent mechanism. Myricetin was found previously to inhibit both human TOP1 and TOP2$\alpha$ [42, 44]. The yeast-based screening assay described here is thus useful for identifying TOP1 inhibitors that may have other cellular targets.

Fig. 5.

Testing of myricetin in human TOP1 relaxation and yeast-based growth assay. (A) Human TOP1 (0.3 U from TopoGEN) was assayed for relaxation of supercoiled plasmid DNA in the presence of serial dilutions of myricetin. Lane 1: no enzyme; lane 2: DMSO control; lane 3: 25 $\mu$M CPT; lanes 4–9: 50, 25, 12.5, 6.2, 3.1, 1.6 $\mu$M myricetin. S, supercoiled DNA; N, nicked DNA; FR, fully relaxed DNA; PR, partially relaxed DNA. Results from three replicates were plotted to obtain the IC${}_{50}$. (B) Effect of myricetin on relative OD600 of cultures after 66 hours in 1% dextrose media with non-induced or 1% galactose media with induced expression of wild-type (WT) or T178A mutant recombinant human TOP1. ****: p 0.0001, *: p 0.05, ns, not significant.

Compounds with functional groups that may compete for nucleic acid binding to topoisomerases were obtained from NCI Developmental Therapeutics and tested for preventing human TOP1 from relaxing supercoiled plasmid DNA in gel-based assay as well as the yeast-based screening assay described here. NSC65860 was found to prevent the relaxation of supercoiled plasmid DNA by human TOP1, with IC${}_{50}$ of 0.89 $\mu$M (Fig. 6A). When incubated with supercoiled plasmid DNA in the absence of human TOP1, NSC65860 had no effect on the DNA electrophoretic mobility (Supplementary Fig. 2). When NSC65860 was added to relaxed DNA and incubated further with human TOP1, there was no significant change in plasmid supercoiling as seen with ethidium bromide as a positive control for DNA intercalator in the same experiment (Supplementary Fig. 3), ruling out the possibility that the effect of NSC65860 on plasmid DNA relaxation by human TOP1 shown in Fig. 6A was due to DNA intercalation.

Fig. 6.

Testing of NSC65860 in human TOP1 relaxation and yeast-based growth assay. (A) Human TOP1 (0.3 U from TopoGEN) was assayed for relaxation of supercoiled plasmid DNA in the presence of serial dilutions of NSC65860. Lane 1: no enzyme; lane 2: DMSO control; lane 3: 25 $\mu$M CPT; lanes 4–9: 5, 2.5, 1.2, 0.62, 0.31, 0.16 $\mu$M NSC65860. S, supercoiled DNA; N, nicked DNA; FR, fully relaxed DNA; PR, partially relaxed DNA. Results from three replicates were plotted to obtain the IC${}_{50}$. (B) Effect of NSC65860 on relative OD600 of cultures after 66 hours in 1% dextrose media with non-induced or 1% galactose media with induced expression of wild-type (WT) or T178A mutant recombinant human TOP1. *: p 0.05; **: p 0.01; ***: p 0.001.

NSC65860 had no significant effect on the growth of EKY3/pYES2-TOP1 in the presence of either dextrose or galactose (Fig. 6B). In the presence of dextrose to repress the expression of the toxic mutant TOP1-T718A protein, NSC65860 had also little effect on growth of EKY3/pYES2-TOP1-T718A. However the growth of EKY3/pYES2-TOP1-T718A in the presence of galactose was enhanced significantly by the addition of NSC65860, with up to 3.5-fold increase in OD600 at 66 hours (Fig. 6B). This is consistent with NSC65860 acting as TOP1 suppressor to prevent the formation of intracellular TOP1-T718A covalent complex from the induced toxic mutant TOP1. The protective effect of NSC65860 on EKY3/pYES2-TOP1-T718A has been confirmed with measurement of CFU/mL following culture in SC-U media with 1% galactose (Supplementary Table 1 and Supplementary Fig. 4).

NSC65860 does not affect growth of EKY3/pYES2-TOP1 in media with dextrose (Supplementary Fig. 5), so it is unlikely to have significant effect on any possible targets in yeast that may result in growth inhibition. It needs to be confirmed that treatment of EKY3 with NSC65860 did not impair the induction of recombinant protein expression from the GAL1 promoter. Western blot with antibodies against human TOP1 was used to show that treatment with as much as 100 $\mu$M NSC65850 had little or no effect on the production of recombinant human TOP1 from the GAL1 promoter following induction with 1% galactose (Fig. 7). The induction of TOP1-T718A has also been examined by western blot to confirm that NSC65860 did not affect the induction of the mutant TOP1 by galactose (Supplementary Fig. 6).

Fig. 7.

Western blot analysis of effect of NSC65860 on induction of GAL1 promoter. Total lysates of equal number of EKY3/pYES2-TOP1 cells grown in media with 1% galactose or dextrose, with and without treatment of 100 $\mu$M NSC65860 were analyzed in duplicates by SDS-PAGE followed by western blots with antibodies against human TOP1 and actin.

Molecular docking is a widely used computational technique to predict ligand-receptor complexes. In this work, we used AutoDock vina to predict a possible binding site for NSC65860 when human TOP1 was used as a target. The structure of NSC65860 is shown in Fig. 8A. Notably, the search box used in our docking procedure covered the entire TOP1 structure allowing NSC65860 to dock randomly in the target. The human TOP1 target structure is taken from its covalent complex with DNA. Fig. 8B shows the best-predicted docked position of NSC65860 in human TOP1. Our result predicts that the region near the active site tyrosine (TYR732) is a possible binding site for NSC65860. Analysis of the docked NSC65860 predicted that the three amino acid residues ARG364, ARG488, and TYR501 establish hydrogen bindings with NSC65860. The hydrogen bonds are shown as dashed lines in magenta color in Fig. 8B.

Fig. 8.

Docking of NSC65860 near the active site of human TOP1. (A) Structure of NSC65860. (B) Docked position of NSC65860 against human TOP1 target. The structure in grey color represents human TOP1. CPK structure represents TYR723 in the active site, and licorice structure represents NSC65860. Left panel shows the entire human TOP1 with the docked NSC65860. Right panel shows the TOP1-NSC65860 complex zoomed near the active site TYR723. The three human TOPOI residues that were predicted to form hydrogen bonds (magenta colors dashed lines) with NSC65860 are shown in stick representations with their backbone atoms enclosed inside wired surfaces.