The PDB structures were obtained for the target proteins - p53 (PDB ID: 1OLG), mortalin (PDB ID: 4KBO), and PARP1 (PDB ID: 4ZZZ) (Clore et al., 1994; Amick et al., 2014; Papeo et al., 2015). The 3D structure of Mortaparib^Mild was downloaded from the PubChem repository (CID: 91464252) (Figure 1A). The protein structures were preprocessed using the Protein Preparation module, and the ligand structure was prepared using the Ligprep module in the Schrodinger Maestro suite. Docking grids were generated around the mortalin binding region of p53 (312–352), the p53 binding region of mortalin (253–282), and the Olaparib-binding site in the catalytic domain of PARP1 (862–880) (Kaul et al., 2001; Papeo et al., 2015). Flexible docking was done using the Extra-precision algorithm of the Glide docking program (Friesner et al., 2006).

Desmond module of Schrodinger software was used for performing the simulations (Schrödinger 2020). The docked protein-ligand complexes were solvated using the “system builder” program of Desmond using a predefined TIP3P water model. In the boundary conditions option, an orthorhombic periodic boundary was set up to give the shape and size of the box buffered at a distance of 10 Å, and then ions (Na+/Cl−) was added to every system depending on the complex for balancing the charge. After building the solvated protein-ligand complex systems, the energy of the prepared systems was minimized through small steps Brownian dynamics for 20 ps at low-temperature (10 K) in NVT ensemble to remove steric clashes (Harder et al., 2016). Further, the minimized systems were equilibrated in seven steps in NVT and NPT ensembles using the relaxation protocol defined in the Desmond Schrodinger suite. Finally, the production simulations were performed for 100 ns in the NPT ensemble with a time step of 2 fs. The pressure and temperature of the systems were kept at 1 atmospheric pressure (using Martyna–Tobias–Kelin barostat) with a relaxation time of 2 ps and 300 K temperature (using Nose–Hoover chain thermostat) with a relaxation time of 1 ps, respectively. The cutoff radius for short-range Coulombic interactions was set to 9 Å. No restraints were added to any molecule and all other options were set to default.

The MD trajectories were analyzed using the “Simulation Event Analysis” module in the Schrodinger suite (Bowers et al., 2006). Root Mean Square Deviation (RMSD) of protein–Mortaparib^Mild structures were analyzed as a function of time to investigate the stability of the protein-ligand complexes. The number of hydrogen bonds between the ligand and proteins was calculated throughout the simulation time. The RMSD of ligands was also calculated and compared to investigate their flexibility, binding inside the active pocket of protein, and their stability throughout the simulation (Liu and Kokubo, 2017). The average structure was finally visualized using the PyMOL suite of the Schrodinger package.

The average representative structure was chosen from the simulation trajectories using the “Average Structure” module of the Schrodinger suite. Prime MM/GBSA was used for binding free energy calculation (Li et al., 2011).

The equation used for the calculation was: MM/GBSA  Δ G bind =Δ G complex − ( Δ G receptor +Δ G ligand ) where, ∆G _complex, ∆G _receptor, and ∆G _ligand represent the free energies of the complex, receptor, and ligand, respectively. MM/GBSA refers to the binding affinity of the ligand towards the target protein; a more negative value represents stronger affinity (Genheden and Ryde, 2015). The calculated binding free energy values are not absolute due to the inherent limitations of end-point free energy calculation methods.

Human cancer cells, colorectal (HCT116 and DLD1), breast (T47D, MCF7, MDA-MB231, and MDA-MB453), cervical (HeLa, ME-180, SKG-II, SKG-IIIb, and CASKI), hepatic (HuH7), lung (A549), bone (U2OS), and the normal fibroblasts (TIG3 and MRC5) (JCRB, Tokyo, Japan) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) low glucose with L-glutamine and phenol red (Fujifilm WAKO Pure Chemical Corporation, Osaka, Japan) supplemented with 5% fetal bovine serum (Thermo Fisher Scientific, Japan), 1% penicillin-streptomycin (Invitrogen, Carlsbad, CA, United States) at 37°C in an atmosphere of 5% CO₂.

Mortaparib^Mild (triazole derivative 4-[(4-amino-5-thiophen-2-yl-1,2,4-triazol-3-yl)sulfanylmethyl]-N-(4-methoxyphenyl)-1,3-thiazol-2-amine) (NAMIKI SHOJI Co., Ltd.; Shinjuku, Japan) was dissolved in dimethyl sulfoxide (DMSO) (WAKO, Osaka, Japan) to prepare 50 mM stock. The stock was diluted in a complete cell culture media to obtain working concentrations (1–80 μM). The cells were treated with Mortaparib^Mild at 60–70% of confluency for 24–48 h.

Screening was performed from the library consisting of 12,000 synthetic and natural compounds to obtain candidates abrogating mortalin-p53 interactions, as previously described (Putri et al., 2019).

The short- and long-term cytotoxicity of Mortaparib^Mild was determined by MTT-based cell viability and colony formation assays in 96-well and 6-well plates, respectively, as described earlier (Putri et al., 2019; Sari et al., 2021). Morphology of control and treated cells was also captured under phase contrast light microscope (Nikon TS100-F, Tokyo, Japan).

Wild type p53 dependent luciferase reporter assay was performed in HCT116 and T47D cells with/without the treatment with Mortaparib^Mild. Cells (2 × 10⁵/well of 6-well plates) were transfected with PG13-luc (the wild type p53 responsive luciferase-reporter plasmid) using X-tremeGENE HP DNA transfection reagent (Roche, Basel, Switzerland) as described earlier (Sari et al., 2021). Cells, treated with either DMSO or Mortaparib^Mild for 24 h, were collected by trypsinization, lysed using the passive lysis buffer (PLB) (Promega, WI, United States), and subjected to luciferase activity assay using the Luciferase Reporter Assay System (Promega, Madison, WI, United States) as described previously (Sari et al., 2021).

HCT116 cells were seeded in the 6-well plates (2 × 10⁵ cells/well). After 24 h, control and Mortaparib^Mild-treated cells were harvested along with the floating cells by centrifugation at 3,000 rpm at 4°C for 5 min. The cell pellet was resuspended in 100 µl fresh media and stained with Guava Nexin Reagent (EMD Millipore Corporation, Berlington, MA, United States). Apoptotic cells were quantified and analyzed by Guava PCA-96 System (Luminex Corporation, Austin TX, United States) and FlowJo software (Version 7.6, Flow Jo, LLC, Ashland, OR, United States).

Cells were seeded in 6-well plates (2 × 10⁵ cells/well). After 24 h, control and Mortaparib^Mild-treated cells were harvested, cold (4°C)-centrifuged at 2,000 rpm for 5 min, washed with cold PBS, fixed with 70% ethanol on slow vortex, and kept at −20°C for up to 72 h. The fixed cells were cold (4°C)-centrifuged at 3,000 rpm for 10 min followed by two cycles of cold PBS washing. The cells were stained with Guava Cell Cycle Reagent (4500-0220) (Luminex Corporation, Austin, TX, United States) in the dark for 30 min. RNase-A (1 mg/ml at 37°C for 30 min) treatment was performed to eliminate RNA in the samples. Cell cycle progression analysis was done using Guava PCA-96 System (Luminex Corporation, Austin, TX United States). FlowJo software (Version 7.6, Flow Jo, LLC, Ashland, OR, United States) was used to analyze the flow cytometry data.

Control and Mortaparib^Mild-treated cells were harvested by trypsinization. Cell pellets were incubated with RIPA Lysis Buffer (Thermo Fisher Scientific, Waltham, MA, United States) supplemented with complete protease inhibitor cocktail (Roche Applied Science, Mannheim, Germany) for 30 min with slow vortex at 4°C. Lysates were centrifuged at 15,000 rpm for 15 min. Protein concentrations in lysates was determined by Bicinchoninic acid (BCA) protein assay (Thermo Fisher Scientific, Waltham, MA, United States). The cell lysates containing 10–20 µg protein were separated in 8–12% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, United States) using a wet transfer [Mini-PROTEAN Tetra Cell (BIO-RAD, California, United States)] for 75 min or a semi dry transfer blotter (ATTO Corporation, Tokyo, Japan) for 65 min. Membrane blocking was done using 3% bovine serum albumin at room temperature for ∼1 h. Blocked membranes were probed with the target protein-specific primary antibodies overnight at 4°C. Primary antibodies included anti-mortalin (37-6) raised in our laboratory; p21^WAF11 (12D1) (Cell Signaling Technology, Danvers, MA, United States); anti-cleaved PARP1, anti-Poly (ADP-Ribose) Polymer [10H] (Abcam); MMP 3/10 (F-10), PARP-1 (F-2), p53 (DO-1) (Santa Cruz Biotechnology, Paso Robles, CA, United States). The blots were then incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies [anti-rabbit IgG and anti-mouse IgG (Santa Cruz Biotechnology, CA, United States)] and developed using the enhanced chemiluminescence system (GE Healthcare, Buckinghamshire, United Kingdom). Direct-blot™ anti-β-actin antibody (BioLegend) was used as an internal control. The protein band images were analyzed by ImageJ (National Institutes of Health, Bethesda, MD, United States) software.

Cells (4 × 10⁴/well) were plated on 18-mm glass coverslips placed in 12-well plates and allowed to settle overnight followed by treatment with Mortaparib^Mild for 24 h and then fixed in methanol:acetone (1:1) at 4°C for 5 min. Cells were washed with phosphate buffered saline (PBS) and permeabilized using PBST (PBS with 0.1% Triton X-100) for 10 min, followed by blocking with 2% bovine serum albumin in PBST at room temperature for 1 h. Fixed cells were incubated with primary antibodies as described previously in Western blotting section and others including, phospho-Histone H2A.X (Ser139) (20E3) (Cell Signaling Technology, Danvers, MA, United States), PUMAα/β (H-136) (Santa Cruz Biotechnology, Paso Robles, CA, United States), Bax (B-9) (Santa Cruz Biotechnology, Paso Robles, CA, United States) and anti-Cytochrome C [EPR1327] (Abcam). Immunostaining was visualized by secondary antibody conjugated with fluorochromes including either FITC or Alexa-488 or Alexa-594 (Molecular Probes, Eugene, OR, United States). Hoechst 33,342 (Invitrogen, Molecular Probes, Eugene, OR, United States) was used for nuclear staining. The coverslips were mounted on glass slides and examined under Zeiss Axiovert 200 M microscope with AxioVision 4.6 software (Carl Zeiss, Tokyo, Japan). Several images were captured from each of the three independent experiments. Protein levels represented by the fluorescence signals obtained using ImageJ software (National Institutes of Health, Bethesda, MD, United States). In each image, the mean intensity of the targeted protein per cell was calculated from 10 to 15 cells. The average of mean intensity per cell was determined along with the standard deviation.

The indirect co-immunoprecipitation was performed to investigate the effect of Mortaparib^Mild on mortalin-p53 interaction. Control and Mortaparib^Mild-treated cells were harvested, PBS-washed, and lysed using NP-40 lysis buffer. The protein concentrations were measured by BCA assay (Thermo Fisher Scientific, Rockford, IL). Cell lysates containing 400 μg total protein were precleared with normal rabbit IgG (2729; Cell Signaling Technology, MA, United States) and Protein A/G PLUS-Agarose beads (sc-2003) (Santa Cruz Biotechnology, United States) at 4°C with slow rotation for 1 h, followed by centrifugation at 2,500 rpm at 4°C for 5 min. The supernatant was collected and incubated with either anti-mortalin polyclonal antibody raised in our lab, anti-p53 polyclonal antibody (FL-393; Santa Cruz Biotech., United States) or control normal rabbit IgG at 4°C overnight with slow rotation. Fifty microliters of Protein A/G PLUS-Agarose beads was added to the mixture and incubated for 4 h. Immunoprecipitants were collected by centrifugation at 2,500 rpm at 4°C for 5 min. Pellets were washed with NP-40 lysis buffer followed by centrifugation at 2,500 rpm at 4°C for 5 min (4 times). Immunoprecipitants were boiled at 99°C in SDS sample buffer for 10 min, resolved on SDS-PAGE and subjected to Western blotting with anti-p53 mouse monoclonal antibody (DO-1) from Santa Cruz Biotech. (United States) or anti-mortalin monoclonal antibody raised in our lab. Furthermore, to segregate the effect of Mortaparib^Mild on mortalin-p53 interaction from its effect on transcription, translation, post-translation and molecular signaling networks in living cells, we treated cell lysates (prepared in NP-40 lysis buffer) with Mortaparib^Mild. Cell lysate containing 700 μg protein was precleared with normal rabbit IgG and Protein A/G PLUS-Agarose beads as described above. The supernatant was divided equally into two parts and incubated with either 0.1% DMSO (control) or 50 μM Mortaparib^Mild at room temperature for 3 h with slow rotation, respectively. The control and Mortaparib^Mild-treated lysates were then incubated with either anti-mortalin or anti-p53 antibodies at 4°C overnight, as described above. Immunocomplexes were separated with Protein A/G PLUS-Agarose beads, resolved by SDS-PAGE and Western blotting analysis, as described above.

Total RNA from DMSO (control) and Mortaparib^Mild-treated cells was collected by RNeasy mini kit (Qiagen, Stanford Valencia, CA, United States) following the manufacturer’s protocol. Equal amounts of RNA (1 µg) from samples were reverse transcribed into cDNA following the protocol from QuantiTect Reverse Transcription kit (Qiagen, Tokyo, Japan). Real Time quantitative Polymerase Chain Reaction (RT-qPCR) was performed using the protocols for SYBR Select Master Mix (Applied Biosystem, Life Technologies, Foster City, CA, United States). The conditions of RT-qPCR using specific primers (Table 1) were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles (denaturation at 95°C for 15 s, annealing at 60°C for 1 min, and extension at 72°C for 15 s). The relative expression level of target genes was normalized against 18S gene as an internal control.

Trapping assay was aimed to fractionate PARP-DNA complexes in different stringency hypotonic buffers (A, B, C, and/or D; Table 2), wherein the tight complex is characterized by fractionation of PARP in higher stringency buffer as described earlier (Murai et al., 2012). Mortaparib^Mild-treated and control cells were collected by trypsinization and centrifugation (2,500 rpm) at 4°C for 3 min. Supernatants were removed, and pellets were mixed with hypotonic buffers (Table 2), vortexed for 10 min followed by centrifugation at 16,000 rpm at 4°C for 10 min. The collected supernatant was labelled as P1 and pellet was re-suspended with Buffer A. This step was serially repeated in the sequence of buffer A-D. Supernatants from each centrifugation step were collected and labelled as A, B, C, and D. Western blotting analysis was perform using anti-PARP1/2 and anti-histone H3 antibodies.

Cells (2 × 10⁵/well) were plated in 6-well plates and allowed to make monolayers overnight. The monolayer of cells was manually scraped using a p200 pipette tip to create a linear wound. The cells were washed with PBS and cultured in complete culture medium-supplemented with DMSO (control) or non-toxic concentration (5 µM for HCT116 and T47D) of Mortaparib^Mild. The migration of cancer cells into the gap was imaged over 0–96 h under a microscope (Nikon TS100-F, Tokyo, Japan).

Data obtained from three or more independent experiments were statistically expressed as mean ± standard deviation. Unpaired t test (GraphPad Prism, GraphPad Software, San Diego, CA) was performed to determine statistical significance between the control and experimental samples. Values of p > 0.05 (^ns), p ≤ 0.05 (*)‬, p ≤ 0.01 (**), p ‬≤ 0.001 (***) and p‬ ≤ 0.0001 (****) were considered non-significant, statistically significant‬, very significant, highly significant, and extremely significant, respectively.

Molecular docking was performed to analyze the interaction of Mortaparib^Mild in the mortalin-binding region of p53, p53-binding region of mortalin, and catalytic site of PARP1. Mortaparib^Mild was found to be interacting with 1) the mortalin-binding domain (amino acid residue number: 323–337) of p53 with a docking score of −3.348 kcal/mol, 2) within the p53 binding domain (amino acid residue number: 260–280) of mortalin with a docking score of -2.873 kcal/mol, and 3) with the catalytic binding domain of PARP1 with a docking score of −8.202 kcal/mol, where known drug Olaparib (inhibitor of PARP1) binds. The docking score was calculated for determining the strength of ligand binding with the target protein based on the hydrogen bonding, hydrophobic interactions, ionic interactions, and aromatic ring stacking (Bohm, 1998). Docking scores represent empirical values for relative assessment of ligand binding; higher docking score signifies stronger interaction. Furthermore, each of the protein-drug complexes was subjected to molecular dynamics (MD) simulation for 100 ns that determined the stability of ligand-target protein interaction. RMSD of all the protein-drug complexes converged within 40 ns indicating a stable interaction (Figure 1B). The hydrogen bond plot obtained throughout the simulation revealed that Mortaparib^Mild stably interacted with p53, mortalin and PARP1 at the docked site, and formed continuous hydrogen bonds throughout the simulation (Figure 1C). After every 10 ns simulation, frames of all three complexes were extracted and the stable binding of the Mortaparib^Mild with p53, mortalin, (Supplementary Figures S1A,B), and PARP1 (Supplementary Figure S2) was visualized.

Mortaparib^Mild formed hydrogen bonds with residues Phe328 and Leu330 and showed hydrophobic interactions with residues Thr329 and Tyr327 in the mortalin binding site of p53 (Figure 2A). All the residues that interacted with Mortaparib^Mild were found to be in the mortalin-binding region of p53. From the hydrogen bond occupancy plot of the simulation, it was found that residues Thr329, Phe328, Leu330, and Arg337 were involved for more than 40% of the time during the entire simulation period (Supplementary Figure S3A). Mortaparib^Mild interacted with mortalin by forming hydrogen bonds with residues Arg85, Gly247, and Glu313, and had hydrophobic interactions with residues Gly275 and Glu276 in the p53-binding region of mortalin (Figure 2B). Most of the residues were in common with the known p53 binding region involving residues from 253 to 282 in the N-terminal of mortalin (Kaul et al., 2001; Iosefson and Azem, 2010). Moreover, from the hydrogen bond occupancy plot of the simulation, it was found that residues Arg85, Gly247, Glu276, and Glu313 were involved for more than 20% of the time of the entire simulation period (Supplementary Figure S3B). Interestingly, Mortaparib^Mild was found to interact with the catalytic domain of PARP1 in a way similar to Olaparib (an established inhibitor of PARP1). It inhibited the catalytic site by forming firm interactions with residue Gly863 through a hydrogen bond, with residue Tyr896 through pi-pi interaction of aromatic ring, and with residues Lys903, Phe988, and Tyr 889 by hydrophobic interactions (Figure 2C). From the hydrogen bond occupancy plot of the simulation, it was found that residues Gly863, Tyr889, Tyr896, and Lys903 were making interactions for more than 25% of the simulation time (Supplementary Figure S3C). The interactions of Olaparib at Gly863, Lys903, and Tyr896 of PARP1 were observed at similar residues and found to be comparable with Mortaparib^Mild. This confirmed that Mortaparib^Mild and Olaparib bind to the same catalytic region of PARP1 (Figure 2D).

The average structures from the simulation trajectories were used for MM/GBSA binding energy calculation. The binding energy of Mortaparib^Mild with the mortalin binding site of p53, the p53 binding site of mortalin, and the catalytic site of PARP1 was −55.43 kcal/mol, −55.96 kcal/mol, and −53.64 kcal/mol respectively. Based on this, it was concluded that Mortaparib^Mild possesses similar affinity towards the three tested targets (p53, mortalin, and PARP1).

We next examined if Mortaparib^Mild could function by independent mechanism(s) as well and therefore first determined the level of mortalin expression in control and Mortaparib^Mild-treated cells. The doses were selected, based on independent cell viability assays that showed Mortaparib^Mild inhibited cell proliferation with IC₅₀ of 50–80 μM for most cancer cell types (Supplementary Figure S4A). Based on the dose-dependent cytotoxicity assays, HCT116 (p53 wild type) and T47D (p53-mutant) cells were selected for the present study; cell viability assays for both cells (24–48 h) are shown in Supplementary Figure S4B,C. By multiple dose- and time-dependent cytotoxicity assays, we determined IC₅₀, IC₃₀, and IC₁₀ concentrations for HCT116 and T47D cells. Of note, Mortaparib^Mild showed weaker cytotoxicity as compared to Mortaparib^Plus (Sari et al., 2021), and HCT116 cells were more responsive to Mortaparib^Mild than T47D cells (Supplementary Figure S4D,E). Based on these data, 50 μM (∼48 h; IC₅₀) was selected for further analyses (Figure 3A).

First, to validate the in-silico results showing the ability of Mortaparib^Mild to interact with mortalin and p53, co-immunoprecipitation analyses were performed. As shown in Figure 3B, by Western blotting, decrease in mortalin and increase in p53 protein in Mortaparib^Mild-treated cells was recorded. Mortalin-immunocomplexes probed with anti-p53 antibody revealed decrease in the amount of p53 in mortalin-complexes from Mortaparib^Mild treated HCT116 cells. In order to rule out the effect of unspecific immunoprecipitation of mortalin with IgG, we also obtained p53-immunocomplexes and probed them with anti-mortalin antibody. The data showed decrease in mortalin in p53-immunocomplexes in Mortaparib^Mild treated cells. Quantitation of the data from more than three experiments (Figure 3B) endorsed that Mortaparib^Mild caused disruption of mortalin-p53 interaction. To further support the ability of Mortaparib^Mild to disrupt mortalin-p53 interactions, and to exclude the effect of transcription, translation, post-translation as well as molecular signaling networks that operate in live cells, we treated cell lysates (prepared in NP-40 lysis buffer) with Mortaparib^Mild. As shown in Supplementary Figure S5A–C, cell lysates treated with Mortaparib^Mild showed disruption of mortalin-p53 complexes as evidenced by decreased amount of mortalin in p53-immunocomplexes (Supplementary Figure S5A) as well as decrease amount of p53 in mortalin-immunocomplexes (Supplementary Figure S5B). Of note, the treated cell lysates showed higher amount of mortalin and p53 in supernatant after the precipitation of p53 and mortalin, respectively (Supplementary Figure S5C). These data reaffirmed that Mortaparib^Mild abrogated mortalin-p53 interaction. We also used T47D cells that harbor mutant p53 (Supplementary Figure S6A). Of note, Mortaparib^Mild− treated cells did not show increase in p53 level due to the stable nature and excessive accumulation of mutant p53 in these cells. However, decrease in mortalin (mRNA and protein; Supplementary Figure S6A,B) and mortalin-p53 complexes (Supplementary Figure S6A) was similar to HCT116 cells.

Next, further to the decrease in mortalin protein in Mortaparib^Mild-treated cells as detected by Western blotting (Figure 3C), we performed immunostaining. As shown in Figure 3D, decrease in mortalin and increase in nuclear p53 in Mortaparib^Mild-treated cells was clearly observed. Since the reduction in mortalin was anticipated to be independent of the binding of Mortaparib^Mild to either mortalin or p53, we performed RT-qPCR and found significant decrease in mortalin mRNA in Mortaparib^Mild-treated cells (Figure 3E; Supplementary Figure S6B). We also examined the transcriptional activation function of p53 in control and Mortaparib^Mild-treated cells by wild type p53-responsive luciferase reporter (PG13-luc) assay. As shown in Figure 3F, Mortaparib^Mild-treated HCT116 (wild type p53) cells showed remarkable increase in luciferase reporter activity, indicating the transcriptional activation of p53, also supported by increase in the level of p21 protein (Figure 3C) and decrease in cell viability. Similar reporter assays performed in T47D (mutant p53) cells did not show any increase in reporter activity in Mortaparib^Mild-treated cells (Supplementary Figure S6C). Of note, Mortaparib^Mild disrupted the p53 did not increase in T47D cells and caused relocation of mutant p53 in the nucleus (Supplementary Figure S6A,D). These data demonstrated that Mortaparib^Mild-mediated reactivation of transcriptional activation of p53 was specific to wild type p53. However, T47D cells were responsive to Mortaparib^Mild and showed growth arrest in short- and long-term dose-dependent viability assays (Supplementary Figure S6E,F, respectively). Taken together, these data suggested that Mortaparib^Mild has capability to disrupt mortalin-p53 interaction and activate wild type p53 function. It may also work through mechanisms independent to its effects on mortalin-p53 binding and wild p53 activity.

Based on our earlier findings on the effect of Mortaparib (Putri et al., 2019) and Mortaparib^Plus (Elwakeel et al., 2021; Sari et al., 2021) on PARP1 activity, we next investigated the expression levels of PARP1 and cleaved PARP1 in the Mortaparib^Mild-treated HCT116 cells by Western blotting. Decrease in full-length PARP1 protein and mRNA was observed in Mortaparib^Mild-treated cells (Figures 4A,B). Furthermore, the 89-kDa cleaved fragments of PARP1 and PAR were seen to be accumulated in Mortaparib^Mild-treated cells (Figure 4A). Trapping assay in control and treated cells was performed to fractionate PARP1-DNA complexes in hypotonic buffers (A-D; Table 2) of variable stringency, wherein the tight complex is characterized by fractionation of PARP1 in higher stringency buffer as described earlier (Murai et al., 2012). The data showed fractionation of PARP1 from control cells in lower stringency (P1) and from treated cells in higher stringency buffer (D) suggesting that PARP1 was trapped into the DNA in the treated cells (Figure 4C) and hence may cause anomalous DNA repair. Although the direct experimental evidence to show the physical interaction of the compound with PARP1 and binding domain analyses remain to be clarified, these data demonstrated the potential of Mortaparib^Mild to impair DNA damage repair signaling in cancer cells.

Based on the above computational and experimental evidence, it was predicted that Mortaparib^Mild would inhibit cancer cells proliferation in p53-dependent and independent mechanisms. As described above, mutant p53 harboring T47D cells showed growth arrest (Supplementary Figure S6E,F). Consistently, as shown in Figure 5A, HCT116 cells treated with low (10 µM) and high (50 µM) concentration of Mortaparib^Mild for 24 h showed growth arrest and apoptotic phenotypes, respectively. In long-term clonogenic assays, HCT116 cells showed dose-dependent decrease in colony forming efficiency (Figure 5B). Whereas 10 μM Mortaparib^Mild-treated (∼10 days) cells showed insignificant effect on colony forming efficiency, 50 μM Mortaparib^Mild caused about 50% reduction in the colony number. Cell cycle analysis in control and Mortaparib^Mild-treated cells showed a dose-dependent increase in the percentage of cell population in G2 phase (Figure 5C), and apoptosis assay revealed increase in percentage of apoptotic cells in response to high dose (50 μM) of Mortaparib^Mild (Figure 5D). The effect was further confirmed by immunostaining of proteins involved in apoptosis (PUMA, Bax and Cytochrome C) (Figure 5E). Furthermore, consistent with the PARP-1 trapping data that suggested anomalous DNA repair in Mortaparib^Mild-treated cells, γH2AX staining showed remarkable increase in the latter (Figure 5E).

Overexpression of mortalin and PARP1 has been shown to promote migration of cancer cells. Knock-down of these proteins has been connected to compromised migration ability of cancer cells (Schiewer and Knudsen, 2014; Na et al., 2016) by p53-independent mechanism. In light of this information and downregulation of mortalin and PARP1 in Mortaparib^Mild-treated cells, we next examined the migration of cells by Scratch-wound assay in HCT116 as well as T47D cells. In order to rule out the effect on cell proliferation, extremely low non-toxic concentration of Mortaparib^Mild (5 μM) was used. As shown in Figure 6A and Supplementary Figure S7, Mortaparib^Mild-treated both HCT116 and T47D cells showed delay in migration endorsing that the effect on cell migration involves p53-independent mechanism(s). Cancer cell migration is largely influenced by matrix metalloproteinases (MMPs) that are enriched in most cancers and function in the degradation of the extracellular matrix and non-matrix proteins (Jabłońska-Trypuć, et al., 2016). Earlier studies have shown upregulation of MMP3 in mortalin-enriched metastatic cancer cells (Wadhwa et al., 2006.) In light of this information, we examined the expression of MMP 3/10 in control and Mortaparib^Mild-treated cells and found decrease in the latter (Figure 6B) suggesting it as one of the p53-independent mechanisms involved in Mortaparib^Mild-mediated decrease in cancer cell migration.