Academic Editor: Graham Pawelec
Background: Inhibition of human topoisomerase I (TOP1) by camptothecin and topotecan has been shown to reduce excessive transcription of PAMP (Pathogen-Associated Molecular Pattern)-induced genes in prior studies, preventing death from sepsis in animal models of bacterial and SARS-CoV-2 infections. The TOP1 catalytic activity likely resolves the topological constraints on DNA that encodes these genes to facilitate the transcription induction that leads to excess inflammation. The increased accumulation of TOP1-DNA covalent complex (TOP1cc) following DNA cleavage is the basis for the anticancer efficacy of the TOP1 poisons developed for anticancer treatment. The potential cytotoxicity and mutagenicity of TOP1 targeting cancer drugs pose serious concerns for employing them as therapies in sepsis prevention. Methods: In this study we set up a novel yeast-based screening system that employs yeast strains expressing wild-type or a dominant lethal mutant recombinant human TOP1. The effect of test compounds on growth is monitored with and without overexpression of the recombinant human TOP1. Results: This yeast-based screening system can identify human TOP1 poisons for anticancer efficacy as well as TOP1 suppressors that can inhibit TOP1 DNA binding or cleavage activity in steps prior to the formation of the TOP1cc. Conclusions: This yeast-based screening system can distinguish between TOP1 suppressors and TOP1 poisons. The assay can also identify compounds that are likely to be cytotoxic based on their effect on yeast cell growth that is independent of recombinant human TOP1 overexpression.
Life-threatening complications and fatalities attributable to infections might not be directly caused by the infectious agent. Instead, they might happen as consequences of the host immune responses that are triggered by the infection process. Acute respiratory distress syndrome (ARDS) and pulmonary fibrosis (PF) are two deadly complications of coronavirus infection that are caused by the host immune response to the viral infection [1]. Bacterial infections can also lead to severe immune-related complications, such as ARDS, acute lung injury [2, 3], and TB sepsis which has an 80% mortality rate [4]. When microorganisms infect the host, pathogen-associated molecular patterns (PAMPs), which are distinct microbial structures and macromolecules, and damage-associated molecular patterns (DAMPs) that result from damaged host tissues, are identified by pattern recognition receptors (PRRs) that belong to the host immune system. The expression of PRRs through PAMP response genes activates the production of innate immune cells that target and kill the invading pathogens [5, 6]. However, excess immune response can cause systemic tissue injuries and sepsis [7]. Sepsis is defined as life-threatening organ dysfunction caused by a dysregulated host response [8] to infection caused by viruses, bacteria or other infectious agents. During sepsis, the overexpression of PAMP response genes leads to the uncontrollable release of inflammatory mediators, such as cytokines and chemokines leading to cytokine storm [9, 10].
Topoisomerases play a major role in resolving the topological problems encountered in transcription and replication [11, 12, 13]. Human topoisomerase I (TOP1) has been a target for clinically approved cancer treatment [14, 15]. FDA-approved drugs that target TOP1 derived from camptothecin are TOP1 poisons that stabilize the enzyme-DNA covalent complex [16, 17]. These poisons cause accumulation of topoisomerase I-DNA covalent complex (TOP1cc) and upon collision with replication or transcription proteins forms double-stranded breaks (DSBs) in DNA. Unrepaired DNA DSBs lead to cell death, which is useful in cancer treatment [15, 16, 18]. An earlier study showed that the activation of PAMP response genes requires human TOP1, and that the depletion and reversible inhibition of TOP1 selectively suppress PAMP response genes [19]. Treatment with camptothecin suppressed PAMP response genes, and rescued mice infected with Staphylococcus aureus from death by sepsis [19]. Topoisomerase activities can modulate chromatin structure and influence the gene expression patterns relevant for both cancer and inflammation [20]. A recent study also showed that therapy with the FDA-approved TOP1 poison topotecan suppresses lethal inflammation induced by SARS-CoV-2 and protects hamsters against SARS-CoV-2-induced lethal inflammation [21]. However, cytotoxicity associated with topoisomerase poisons is not desirable in sepsis management. Suppressors of human TOP1 that inhibits TOP1 catalytic activity by preventing DNA binding and cleavage by TOP1 may be preferable because they are not expected to cause DNA breaks and cell death. The TOP1 inhibitors that act as such suppressors of TOP1 DNA binding and cleavage can potentially repress PAMP response gene expression and alleviate the excess immune response caused by infections.
Currently, use of radio-labeled DNA substrate is generally needed to conduct the
TOP1 DNA cleavage-religation inhibition assays to determine if a TOP1 inhibitor
acts as a topoisomerase poison, or if the inhibitor acts as a suppressor to
prevent DNA cleavage [22, 23]. Yeast-based assays for identification of
topoisomerase poisons are easier to perform and do not require handling of
radioactive material. The budding yeast Saccharomyces cerevisiae has
been used for several decades as a model to study human topoisomerases-targeting
drugs [24, 25]. S. cerevisiae strains with
Scheme of yeast screening assay for human TOP1 inhibitors that act as suppressors. Wild-type human TOP1 does not accumulate TOP1cc and DNA breaks. Increased DNA breaks from TOP1cc because of T718A mutation leads to cell death. A TOP1 suppressor can reduce DNA breaks from mutant TOP1cc to rescue cell growth.
The S. cerevisiae strain EKY3 (MAT
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
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
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
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
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
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
For Western blot analysis of the human TOP1 expression, cell
pellets were washed with 500
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
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).
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.
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.
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
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
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
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
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
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
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
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.
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.
The lethal mutant human TOP1-T718A enzyme forms a stabilized covalent complex
following DNA cleavage, leading to accumulation of DNA breaks and eventually
cellular death when overexpressed from the GAL1 promoter in yeast cells cultured
in media containing galactose [27]. S. cerevisiae cells overexpressing
TOP1-T718A can be used as a model to screen for suppressors of human TOP1 that
inhibit DNA binding and cleavage by TOP1. Such suppressors can be expected to
reduce the lethal effect of TOP1-T718A expression. This would increase the
viability of these cells when grown in media with galactose inducer. The TOP1
catalytic activity is not required for yeast cell growth. As discussed previously
[46], cyotoxicity from action on other cellular targets is an important concern
for identifying specific human TOP1 suppressors or poisons that can be addressed
through yeast-based assay. A specific human TOP1 suppressor should not negatively
impact the growth rates of yeast cells expressing WT human TOP1. In this study,
we used yeast EKY3 transformed with WT or T718A mutant TOP1 clones to demonstrate
that compound NSC65860 is likely to be a human TOP1 suppressor based on results
from this yeast-based assay. The growth of EKY3/pYES-TOP1-T718A in the presence
of galactose was enhanced by
The results reported here provide validation that this yeast-based screening system could be used as a rapid and economical screening approach for identifying specific human TOP1 suppressors. Compared to enzyme-based gel electrophoresis assay, the yeast-based assay would require less resources to scale up for screening of a large library of molecules for TOP1 inhibitors. Candidates for either TOP1 suppressors or poisons can be identified based on the differential effects of the compound on growth of EKY3/pYES-TOP1 and EKY3/pYES-TOP1-718A in media containing dextrose versus galactose. A specific human TOP1 poison would diminish growth only when WT TOP1 expression is induced by galactose. Myricetin resulted in most significant growth suppression of EKY3 with galactose induction of WT TOP1 to indicate that it can act as a TOP1 poison, as reported in previous research [42, 44]. There was also some degree of suppression of growth of EKY3/pYES-TOP1 in the presence of dextrose, suggesting that myricetin can affect yeast cell viability in mode of action that does not involve TOP1 inhibition. Myricetin is likely to act on an essential target in yeast, with TOP2 being just one of the possibilities. Significant growth suppression of EKY3/pYES-TOP1 in dextrose containing media can thus be an indication that the molecule is not likely to be a selective human TOP1 inhibitor and may not be a good candidate for consideration in further investigation of TOP1 poisons or suppressors for potential clinical applications in cancer or infectious disease treatment. This useful information on selectivity at the cellular level can be obtained readily from this yeast-based screening system to facilitate planning of development of human TOP1 suppressors into drug leads for reducing harmful inflammatory response during bacterial and viral infections.
The yeast-based screening system described in this study can be used to identify human TOP1 inhibitors acting as poisons that trap TOP1cc, or as suppressors that prevent DNA cleavage by TOP1. The yeast growth inhibition assays can complement the relaxation inhibition assay to provide information on the mode of action and selectivity of human TOP1 inhibitors. Compounds that are likely to be cytotoxic can also be identified based on their effect on yeast cell growth that is independent of recombinant human TOP1 overexpression.
PAMP, pathogen-associated molecular pattern; ARDS, acute respiratory distress syndrome; PRR, pattern recognition receptor; TOP1, Topoisomerase I; TOP1cc, TOP1 covalent complex; CPT, camptothecin; WT, wild-type.
YCDT, TA and AW conceived and designed the experiments; AS, CM (Christian Madeira), TA, PT performed the experiments; AS, CM (Christian Madeira), TA, PT and YCDT analyzed the data; CM (Christopher Medros) and AW contributed reagents. AS and YCDT prepared the manuscript. All authors have read and approved the manuscript.
Not applicable.
We thank Norhan Mohammed for technical assistance. We acknowledge the NCI/DTP Open Chemical Repository (http://dtp.cancer.gov) for supply of NSC65860.
This research was supported by the Florida International University Food Science Initiative with funding from the College of Arts, Sciences, Education and the Chaplin School of Hospitality and Tourism Management. Ahmed Seddek was supported by a FIU University Graduate School Dissertation Year Fellowship.
The authors declare no conflict of interest.