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. 2023 Jan 12;6(2):281–289. doi: 10.1021/acsptsci.2c00214

Repurposing Ponatinib as a PD-L1 Inhibitor Revealed by Drug Repurposing Screening and Validation by In Vitro and In Vivo Experiments

Anjali Barnwal †,‡, Sanjeev Das §, Jayanta Bhattacharyya †,‡,*
PMCID: PMC9926522  PMID: 36798474

Abstract

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Cancer treatment by inhibiting the PD-1/PD-L1 pathway using monoclonal antibodies has made great advances as it showed long-lasting antitumor responses in a wide range of cancers. However, antibodies exhibit several disadvantages, which include low permeability, immune-related adverse effects, complex synthetic procedures, and high treatment costs. Hence, small-molecule inhibitors can be used as alternatives; however, no small molecule with in vivo activity has been reported. In addition, there are many challenges in developing a new drug, including the timeline and escalating cost. Therefore, repurposing an approved drug offers advantages over the development of an entirely new drug. Herein, we identify an FDA-approved small-molecule drug, Ponatinib, as a PD-L1 inhibitor via virtual drug screening of the ZINC database. Ponatinib showed stable binding with PD-L1, with the highest binding energy among all of the screened FDA-approved drugs. The binding of Ponatinib with PD-L1 was supported by a fluorescence quenching assay and immunofluorescence study. Further, we compared the in vivo antitumor efficacy of Ponatinib with a commercially available anti-PD-L1 antibody in the murine melanoma model. Ponatinib was found to be more efficient in delaying tumor growth than the anti-PD-L1 antibody. Furthermore, Ponatinib also reduced the expression of PD-L1 in tumors and increased the T-cell population. Interestingly, splenocytes isolated from Ponatinib-treated mice showed enhanced cytotoxic T-cell (CTL) activity against B16-F10 cells. However, Ponatinib itself did not have any direct toxic effect on cancer cells in vitro. These findings suggest that Ponatinib can be used as a potent small-molecule inhibitor of PD-L1 to overcome the disadvantages associated with antibodies.

Keywords: drug repurposing, cancer immunotherapy, immune checkpoint inhibitor, PD-1/PD-L1 interaction, small-molecule inhibitor, melanoma

Research on the development of immune checkpoint inhibitors has been motivated by the clinical success of the use of antibodies to target immune inhibitory pathways, specifically the programmed cell death protein 1 (PD-1) and its ligand 1 (PD-L1).1 In clinics, several studies showed that the inhibition of the PD-1/PD-L1 interaction enhances the T-cell response and tumor regression.2−4 Currently, more than 1000 clinical trials are evaluating the efficacy of antibodies targeting the PD-1/PD-L1 pathway and some have been approved for the treatment of many tumors such as non-small-cell lung cancer (NSCLC), renal cell carcinoma (RCC), melanoma, Hodgkin’s lymphoma, bladder cancer, etc.5−7 However, antibodies are associated with several disadvantages, which include a high production cost, lower stability, poor tissue penetration, and immunogenicity.8 Moreover, the PD-L1 antibody induces immune-related adverse effects (irAEs) due to long-lasting systemic immune activation, whereas small molecules could reduce this problem because of their shorter half-life in a systemic environment.9 To this end, the development of small-molecule inhibitors appears to be the most effective alternative as it will overcome the problems associated with antibodies.

At present, the research progress on small-molecule inhibitors targeting the PD-1/PD-L1 pathway lags behind antibody development. Although recent studies reported a few series of small molecules, peptides, macrocyclic peptides, and peptidomimetics that target the PD-1/PD-L1 interaction, publicly disclosed validation in the animal model is rare.10−12 Conversely, the development of a new drug is associated with several bottlenecks, such as toxicity, cost, and the time required to bring it to the market. Hence, an existing drug used for a different target can be repurposed to minimize the time required for the development and to reduce the risk of failure as the drug has already been evaluated for its toxicity, formulation, and safety assessment.13 Here, we sought to repurpose an FDA-approved small-molecule drug, Ponatinib, as an inhibitor of PD-L1. Ponatinib is a tyrosine kinase inhibitor (TKI) used for the treatment of Philadelphia chromosome–positive (Ph+) acute lymphoblastic leukemia (ALL) and chronic myeloid leukemia (CML).14 Small molecules available in the ZINC database were screened in silico for the binding of the PD-L1 protein. Among all of the molecules screened from the database, Ponatinib showed the highest binding energy and the most stable interaction with PD-L1 throughout the molecular dynamics (MD) run. Fluorescence quenching data demonstrated that Ponatinib exhibits a dose-dependent binding to PD-L1 protein in solution. Ponatinib also showed efficient binding to surface PD-L1 expressed by B16-F10 cells. When administered to mice bearing the B16-F10 melanoma tumor, Ponatinib delayed the tumor growth more efficiently than the commercially available anti-PD-L1 antibody (aPD-L1). As aPD-L1 inhibits tumor growth via the modulation of the PD-1/PD-L1 pathway by promoting cytotoxic T-lymphocyte (CTL) activity and T-cell proliferation, we analyzed the same with Ponatinib treatment, and it was found to be superior to aPD-L1 in promoting the CTL activity and T-cell population. However, earlier studies have reported that at low doses, TKIs, such as imatinib, inhibited the T-regulatory cell population in tumors and modulated the immune response indirectly. In addition, these TKI inhibitors also inhibited T-cell receptor (TCR)-mediated signal transduction and T-cell responses.15,16 Thus, in this study, for the first time, we have shown that Ponatinib can bind to PD-L1, inhibit PD-1/PD-L1 interaction, and delay tumor growth by enhancing CTL activity and T-cell proliferation. Thus, Ponatinib can be potentially repurposed as a small-molecule inhibitor of PD-L1 and hence can overcome the disadvantages associated with anti-PD-L1 antibodies.

Results

In Silico Screening of Small-Molecule Inhibitors

Drugs from the ZINC database were screened to identify potential small molecules that can bind to PD-L1. These molecules were then subjected to atomic-level docking and scoring using ParDOCK. Eleven molecules with the best dock scores (binding affinity in kcal mol–1) were subjected to MD simulation with the docked PD-L1 protein to determine the dynamic stability of the interaction (Table S1). The interaction stability of compounds with PD-L1-binding residues was analyzed by root-mean-square fluctuation per residue (RMSF). Ponatinib showed the most stable interaction with PD-L1 throughout the MD simulation run (Figure 1A). Figure 1B shows that Ponatinib forms a hydrophobic bond with residues Glu-54, Glu-55, Asp-56, Gln-49, Val-51, Tyr-39, Ser-100, Ile-99, Met-98, Ala-104, and Asp-105. Based on the root-mean-square deviation (RMSD) fluctuations and binding interaction, Ponatinib was selected for further experiments.

Figure 1.

Figure 1

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In silico interaction of Ponatinib with the PD-L1 protein. (A) RMSF plots of the protein–small molecule complex. The plot obtained from MD simulations during 50 ns for Ponatinib bound with PD-L1 shows a stable interaction throughout the simulation run. (B) LigPlot of the protein–small molecule complex. During molecular dynamics simulations, one of the frames where the ligand was found to be stable most of the time was used to plot the protein–small molecule drug interaction diagram. The complex shows the hydrophobic bonds (in red color) between Ponatinib and amino acid residues of PD-L1.

Ponatinib Binds to the PD-L1 Protein in Solution and on B16-F10 Cells

To evaluate the cell-free binding efficacy of Ponatinib to the PD-L1 protein, a fluorescence-quenching assay was performed. The addition of Ponatinib to the protein drastically alters the intrinsic fluorescence properties of the protein. The emission spectrum of the PD-L1 protein and the increase in fluorescence quenching of the protein with an increase in the concentration of Ponatinib is shown in Figure S1. The addition of Ponatinib results in the quenching of the fluorescence emission with the maximum effect at an emission wavelength of 334 nm. The half-maximum quenching was observed at a concentration of 1.912 μM (Figure 2A).

Figure 2.

Figure 2

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Binding of Ponatinib to PD-L1 in solution and on B16-F10 cells. (A) Fluorescence spectra of PD-L1 (5 μM) with different concentrations of Ponatinib (0.5–10 μM) were measured using a spectrofluorometer. A significant decrease in the fluorescence of the PD-L1 protein was observed with an increasing concentration of Ponatinib; 50% quenching in fluorescence was observed at a Ponatinib concentration of 1.912 μM. (B) B16-F10 cells were preincubated with 0.5 and 1 μM Ponatinib at 37 °C for 2 h. Cells were then fixed with 4% paraformaldehyde and incubated with the anti-PD-L1 antibody. The Alexa fluor 598 secondary antibody was used for detection (red). Cells were counterstained with DAPI (blue) and imaged by a cell-imaging multimode reader. Scale bar, 100 μm.

As Ponatinib binds with high affinity and specificity to the purified PD-L1 protein, next, we tried to evaluate whether Ponatinib can bind to the PD-L1 protein expressed on the cell surface. We used B16-F10 and A549 cells because B16-F10 shows abundant PD-L1 expression, whereas A549 cells show low or no expression of the PD-L1 membrane protein (Figure S2). After 2 h of treatment with 0.5 or 1 μM Ponatinib, the presence of free PD-L1 protein was determined by immunofluorescence. As shown in Figure 2B, the amount of free PD-L1 significantly decreased with Ponatinib treatment in a dose-dependent manner, which indicated the efficient binding of Ponatinib to PD-L1.

In Vivo Efficacy in a Tumor Isograft Model

The in vivo antitumor efficacy was evaluated in mice bearing B16-F10 melanoma treated with Ponatinib (15 mg kg–1 bodyweight (BW) i.p. daily for 10 days) or aPD-L1, a commercially available anti-PD-L1 antibody (clone 10F.9G2, BioXcell) (200 μg i.p. on days 0, 3, 6, and 9) (Figure 3A). Mice treated with Ponatinib or aPD-L1 showed a significant delay in tumor growth compared with vehicle-treated mice (Figure 3B). As shown in Figure 3C, on day 22, the mice treated with Ponatinib and aPD-L1 had a mean volume of 470.8 ± 55.89 and 1242 ± 37.45 mm3, respectively, versus 2174 ± 80.66 mm3 for mice that received the vehicle only (Tukey’s multiple comparisons test, P < 0.0001; each treatment group compared with the vehicle-treated group, P < 0.0001; Ponatinib vs the aPD-L1 treatment group, n = 5). A significant increase in tumor size was observed in all vehicle-treated and aPD-L1-treated mice compared with the Ponatinib-treated mice (Figure 3D). On day 22, all of the mice were sacrificed, and the tumor weights were measured. The mean tumor weights were found to be 1.191 ± 0.06772, 2.182 ± 0.05083, and 3.324 ± 0.1414 g, respectively, for the mice treated with Ponatinib, aPD-L1, and the vehicle (Tukey’s multiple comparisons test, P < 0.0001; each treatment group compared with the control group, P < 0.0001; Ponatinib vs aPD-L1-treated group, n = 5) (Figure S3). No significant change in the body weight of the mice was observed during the course of the experiment (Figure S4). The above data clearly indicated that Ponatinib treatment is more potent in delaying tumor growth and in reducing tumor weights than aPD-L1 treatment.

Figure 3.

Figure 3

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Antitumor efficacy of Ponatinib in the B16-F10 tumor isograft model. (A) Scheme of the experimental design. Tumor cells (B16-F10) were subcutaneously inoculated in the right flank of the mice on day 1. When the tumor volume reached 50–100 mm3, mice received i.p. doses of Ponatinib (15 mg kg–1 BW, n = 5), aPD-L1 (200 μg/mouse, n = 5), or vehicle only (n = 5). (B) Change in the tumor volume up to 22 days. Tumor growth was significantly delayed with Ponatinib treatment compared with the control. (C) Tumor volume at day 22 (mean ± 95%) CI. Tukey’s multiple comparisons test: P values for the Ponatinib- and aPD-L1-treated groups relative to vehicle groups are ****P < 0.0001; P value for the Ponatinib-treated group in comparison with the aPD-L1-treated group is ####P < 0.0001. (D) Individual tumor growth curves of mice treated with control, Ponatinib, and aPD-L1.

Ponatinib Inhibited the Expression of PD-L1 in Tumors

Next, to analyze the inhibition of PD-L1 expression by Ponatinib, we determined the expression of PD-L1 in melanoma tumor tissues. Immunohistochemistry staining of tumor sections showed that PD-L1 expression was significantly lowered in the Ponatinib-treated group compared with the vehicle- and aPD-L1-treated mice (Figure 4).

Figure 4.

Figure 4

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Ponatinib treatment decreases in vivo PD-L1 expression. At the end of the experiment, tumors were excised from all of the groups, fixed in 4% paraformaldehyde for 24 h, and then embedded in paraffin. Tumor sections (4 μm) were prepared. All immunostained tumor sections were examined by light microscopy at a magnification of 400×.

Ponatinib Mediated the Antitumor Effect via Cytotoxic T-Cell (CTL) Activity and not via Direct Toxicity

To assess the possibility of the direct toxicity of Ponatinib, B16-F10 cells were treated with increasing concentrations of Ponatinib, whereas doxorubicin (Dox), an anti-cancer drug, was used as a control. Up to a concentration of 1 μM, Ponatinib treatment showed no significant toxicity toward B16-F10 cells (Figure 5A). Furthermore, the IC50 for Ponatinib was found to be 1000-fold higher than that of doxorubicin (P < 0.0001, n = 3) (Table S2). This result clearly demonstrated that the delay in tumor growth with Ponatinib treatment might not be due to the direct killing of tumor cells. Next, to prove that the antitumor efficacy of Ponatinib is due to immune activation, we evaluated the cytotoxic activity of splenocytes (E-Effector cells) against B16-F10 cells (T-target cells). The CTL activity of splenocytes at E/T ratios of 20:1 and 40:1 increased in both treatment groups in B16-F10-bearing mice compared with the mice that received the vehicle only (Sidak’s multiple comparison test; each treatment, P < 0.0001 at every ratio, n = 3) (Figure 5B). Interestingly, splenocytes isolated from mice treated with Ponatinib showed better cytotoxicity toward B16-F10 cells compared with the mice treated with aPD-L1 (Sidak’s multiple comparisons test, P < 0.0001 at every ratio, n = 3).

Figure 5.

Figure 5

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Ponatinib exerts antitumor activity via CTL activation. (A) Direct toxicity of Ponatinib toward B16-F10 cells was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Doxorubicin was used as the positive control. (B) Spleens were collected from each group (n = 3) on day 22, and single-cell suspensions were prepared. The cytotoxic activities of splenocytes were measured by co-culturing splenocytes with B16-F10 cells at E/T ratios of 20:1 and 40:1. Sidak’s multiple comparison test: P values for Ponatinib- and aPD-L1-treated groups relative to the vehicle groups for both ratios are ****P < 0.0001. P values for the Ponatinib-treated group in comparison with the aPD-L1-treated group for both ratios are ####P < 0.0001.

T-Cell Population in the Spleen

As the blockade of the PD-1/PD-L1 signaling pathway induces T-cell proliferation and activation, we evaluated T-cell subpopulations in the spleen.17 As shown in Figure 6A–C, respectively, an increase in the percentages of CD3+, CD4+, and CD8+ T cells was observed in the spleen of mice treated with Ponatinib and aPD-L1 compared with the control group. Interestingly, the population of T cells (CD3+, CD4+, and CD8+) was higher in the spleen of Ponatinib-treated mice (35.6, 17, and 11%, respectively) than in those of aPD-L1-treated mice (32.4, 14.8, and 10.3%, respectively). Taken together, the above data suggest that Ponatinib binds to PD-L1 and delays tumor growth by inducing antitumor immunity.

Figure 6.

Figure 6

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T-cell subsets in the spleen of B16-F10-bearing mice. At the end of the experiment, mouse spleens were harvested, and single-cell suspensions were prepared. CD3+ (A), CD4+ (B), and CD8+ (C) T-cell populations in spleens were determined by flow cytometry.

Discussion

PD-L1 overexpression, which helps tumors evade the immune system, has been an important marker for tumors.18 There are several antibodies available that target PD-1/PD-L1 pathways, such as atezolizumab, nivolumab, and pembrolizumab, and they can achieve long-lasting tumor regression.10 However, the response rate is still low, which could be due to their lower tissue penetration and larger size. In recent years, several classes of small molecules and peptidomimetics have been patented.10,11 Aurigene Discovery Technologies Limited19 and Bristol-Myers Squibb (BMS)20 have reported a series of small molecules that can target the PD-1/PD-L1 pathway.19 However, reports on the in vivo efficacy of such molecules is rare. Again, the time required to bring new drugs to the market and other associated risks altogether contribute to a higher cost.13 Hence, repurposing approved drugs for new targets provides numerous advantages over the development of new ones.

The present study has demonstrated the repurposing of an FDA-approved small-molecule drug, Ponatinib, as an inhibitor of the PD-L1 protein. In silico screening of the small-molecule database showed that Ponatinib displayed stable binding to the active site of human PD-L1 (hPD-L1_protein). The binding of Ponatinib was further confirmed with recombinant PD-L1 by a cell-free fluorescence-quenching study, which shows that Ponatinib can efficiently bind to the PD-L1 protein and quench its fluorescence. A recent report showed that the structure of mouse PD-L1 (mPD-L1) is similar to that of hPD-L1, and mPD-L1 interacts with hPD1 in a manner similar to hPD-L1.21 Interestingly, they found major differences in the druggability of hPD-L1 and mPD-L1 using therapeutic antibodies and small molecules. However, among antibodies approved as PD-L1 inhibitors (atezolizumab and durvalumab), durvalumab did not show any binding with mPD-L1, but atezolizumab showed a binding similar to that with hPD-L1. Hence, in our study, we also confirmed the binding of Ponatinib to mPD-L1 present on B16-F10 cells, and immunofluorescence data showed that Ponatinib binds to surface PD-L1. Moreover, the free PD-L1 on the surface of B16-F10 also decreased with increasing concentration of Ponatinib, indicating the binding of Ponatinib with the mPD-L1 protein. Altogether, these data suggest that Ponatinib can efficiently bind to both hPD-L1 and mPD-L1.

PD-L1 has become a significant target for cancer immunotherapy as an increased PD-L1 level is related to poor prognosis in melanoma and many other tumors.19 Hence, we used melanoma-bearing mice to evaluate and compare the in vivo efficacy of Ponatinib with a commercially available anti-PD-L1 antibody. In this study, the antitumor efficacy of aPD-L1 at the maximum tolerable dose (MTD) is compared with a low dose of Ponatinib.22,23 In addition, it is also reported that repetitive doses of aPD-L1 (10 mg kg–1) induced fatal hypersensitivity reactions and organ-specific toxicities in a murine breast cancer model.23 Previously, Ponatinib has not been injected i.p. in mice; however, in murine tumor models, Ponatinib has been administered orally for 3 weeks with a considerably higher dose than the one used in this study.24,25 Strikingly, Ponatinib treatment showed a significant delay in tumor growth compared with the vehicle control as well as the aPD-L1-treated group. PD-1/PD-L1 interaction diminishes T-cell proliferation and activation and hence inhibits antitumor immunity.26 CD4+ T cells induce antitumor immunity by recruiting macrophages and eosinophils, whereas CD8+ T cells exert a direct cytotoxic effect on tumor cells.27 Splenic immune cells play a critical role in the antitumor immune response as T cell priming occurs in lymphoid tissues.28 In previous reports, inhibition of the PD-1/PD-L1 interaction by anti-PD-L1 antibody treatment showed increased T-cell proliferation in the spleen and CTL activity.17,29−31 We found that Ponatinib also increased the CD4+ and CD8+ T-cell populations in the spleen and enhanced the CTL activity of splenocytes against B16-F10 cells. Moreover, Ponatinib was found to be more efficient in delaying tumor growth and enhancing T-cell immunity compared with aPD-L1. Interestingly, unlike other chemo drugs, Ponatinib did not show any direct toxicity toward B16-F10 cells; however, it induced antitumor immunity by enhancing T-cell proliferation and CTL activity in the spleen. These data indicate that Ponatinib delays the growth of melanoma by inducing antitumor immunity via the inhibition of PD-1/PD-L1 interaction.

Conclusions

Here, we report the repurposing of an FDA-approved small-molecule drug, Ponatinib, as a PD-L1 inhibitor, which might overcome the challenges associated with antibodies, such as higher cost and poor outcomes due to immune resistance. Our cell-free and in vitro cell-based assays suggest that it can efficiently bind to PD-L1. Similar to aPD-L1, Ponatinib also delayed tumor growth in melanoma mice by enhancing the proliferation of T cells and CTL activity. However, it was more efficient than aPD-L1. Furthermore, Ponatinib can directly be used in clinical trials as the toxicity associated with it is known. In conclusion, the findings demonstrate a new role of Ponatinib as it binds to PD-L1 and inhibits its expression in vivo. It elicits antitumor immunity in melanoma-bearing mice more efficiently than the antibody. Although the in vivo activity has been demonstrated here in a murine melanoma model, Ponatinib can also be used for the treatment of other PD-L1-positive tumors.

Materials and Methods

Drugs and Reagents

Ponatinib was purchased from Selleckchem (Cat #S1490). Anti-mouse PD-L1 (clone 10F.9G2) was purchased from BioXcell (Cat #BE0101), West Lebanon. FITC anti-mouse CD3 Antibody (Cat #100204), FITC anti-mouse CD4 Antibody (Cat #100405), Alexa fluor 647 anti-mouse CD8 (Cat #100727), and PD-L1 antibody were purchased from Biolegend (Cat #124302).

Cell Line and Cell Cultures

The mouse melanoma cell line B16-F10 was purchased from the National Centre for Cell Sciences (NCCS), India. Cells were maintained in complete media consisting of Dulbecco’s modified Eagle medium (DMEM) and 10% (v/v) heat-inactivated fetal bovine serum in a humidified 5% CO2 incubator at 37 °C.

Animals

Male C57BL/6J mice (6–8 weeks old) were used for in vivo experiments. Animal experiments were performed according to the institutional guide under protocols approved by the NII Institutional Animal Ethical Committee.

In Silico Screening of the Small-Molecule Inhibitor

The X-ray crystal structure of human PD-1/PD-L1 was retrieved from the Protein Data Bank (PDB) database. The protein–ligand three-dimensional (3D) structure was then used to screen small molecules from the ZINC database using the RASPD tool to select potential molecules with affinity toward human PD-L1. Small molecules with an affinity score of −8 kcal mol–1 and more were subjected to docking with PD-L1 using ParDOCK.32,33 Compounds showing significantly high binding were then subjected to short MD simulations. All MD simulations were carried out using the AMBER suite.34,35 MM/GBSA binding energy (ΔG) calculations were performed using the MM/GBSA method implemented in Amber14.36,37

Cell-Free Binding Efficacy by the Fluorescence-Quenching Assay

Purified PD-L1 was purchased from Sinobiologicals and diluted to 5 μM in a phosphate-buffered saline solution. The fluorescence emission spectra of protein solutions were measured with increasing concentrations of Ponatinib (0.5–10 μM) at room temperature (298 K) on a PC-based spectrofluorometer (Edinburgh Instruments FLS920) equipped with a Xenon lamp and a 1 cm quartz cell. Excitation and emission slit widths were fixed at 5 nm. Samples were excited at 280 nm as the fluorescence is exclusively due to the intrinsic tryptophan (Trp). For all experiments, the emission intensity was recorded between the wavelengths of 305 and 580 nm with an increment of 1 nm. The data were plotted as the change in the fluorescence intensity against wavelength.

Binding to Surface PD-L1 on B16-F10 Cells by Immunofluorescence

Cells were seeded at a density of 104 cells per well in 96-well plates and incubated at 37 °C for 16 h. Cells were then incubated with different concentrations of Ponatinib at 37 °C for 2 h and fixed with 4% paraformaldehyde. Cells were incubated with the primary anti-PD-L1 antibody for 1 h and detected using the Alexa fluor 594 anti-rat antibody. Nuclei were counterstained with DAPI. Images were obtained by a cell-imaging multi-mode reader, Cytation 3, Biotek.

In Vivo Tumor Isograft Model and Dosing Regimen

For the establishment of the B16-F10 melanoma model, 2 × 105 cells resuspended in phosphate-buffered saline (PBS) and an equal volume of matrigel were injected subcutaneously into the right flank of C57BL/6J male mice. When the tumor volume reached 50–100 mm3, the mice were randomly divided into three groups (5 in each group). Mice were injected intraperitoneally (i.p.) with Ponatinib (15 mg kg–1 BW) dissolved in dimethylsulfoxide (DMSO), DMSO alone (control) every day, or aPD-L1 (200 μg on days 0, 3, 6, and 9).

The BW and tumor volume were measured every 1 or 2 days. Tumor volumes were determined with a caliper and calculated by the formula mentioned below:

graphic file with name pt2c00214_m001.jpg

In Vitro Cytotoxicity Assay

To determine the toxicity of Ponatinib on B16-F10 cells, cell survival was measured by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cells were plated in 96-well plates with a density of 5 × 103 cells/well. After overnight incubation, the cells were treated with different concentrations (1 nM-100 μM) of Ponatinib and doxorubicin (Dox) and cultured at 37 °C in a humidified atmosphere for 48 h. Then, 20 μL of MTT solution (5 mg mL–1) was added to each well and incubated for 3 h at 37 °C. The medium was discarded, and 100 μL of 100% DMSO was added to each well to dissolve the formazan crystals. The absorbance was measured at 570 nm using a BioTek SynergyH1 multiplate reader.

T-Cell Population in Spleens of Melanoma-Bearing Mice

At the end of the experiment, spleens were harvested and mechanically dissociated into single-cell suspensions with the back of the syringe. Cells were washed 2–3 times with PBS and resuspended in the staining buffer (PBS containing 4% FBS). Thereafter, 106 cells were used for antibody staining using FITC-conjugated anti-CD3 and -CD4 antibodies and the Alexa Fluor-conjugated anti-CD8 Antibody. The cells were acquired on a BD FACSVerse flow cytometer (BD Biosciences), and data were analyzed using FlowJo LLC software, Oregon.

Cytotoxic T-Lymphocyte (CTL) Activity in Melanoma-Bearing Mice

The CTL activity was analyzed by an MTT assay, where B16-F10 cells were used as target cells (T) and splenocytes isolated from mice bearing B16-F10 tumors were used as effector cells (E). In brief, at the end of the in vivo experiment (day 22), the spleens were collected from each group (n = 3), gently homogenized, and passed through a 70 μm mesh sieve to obtain single-cell suspensions. The suspension was treated with RBC lysis buffer; the splenocytes were washed with PBS three times and resuspended in DMEM complete medium.

B16-F10 cells were seeded in a 96-well microtiter plate at a density of 1 × 104 cells/well in DMEM complete medium. Splenocytes (E) were added to B16-F10 cells (T) at ratios of 20:1 and 40:1 (effector cells/target cells) and then incubated at 37 °C for 20 h. MTT solution (20 μL, 5 mg mL–1) was added to each well, and the plates were incubated for 3 h. The medium was discarded, and 100 μL of 100% DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a BioTek SynergyH1 multiplate reader. The percentage of killed cancer cells was calculated using following equation:

graphic file with name pt2c00214_m002.jpg

where ODT, ODE, and ODS, represent the optical densities (absorbances) of control target cells, control effector cells, and co-cultured cells, respectively.

Immunohistochemistry

At the end of the experiment, excised tumors were fixed and paraffinized. For IHC analysis, serial microsections were prepared, and antigens were retrieved after deparaffinization. The sections were then blocked with serum for 1 h and then incubated with the anti-PD-L1 antibody overnight at 4 °C. The slides were washed with PBS and incubated with the HRP-conjugated secondary antibody for 3 h, followed by washing with PBS. For color development, 3, 3′-diaminobenzidine (DAB) was placed on the tissue sections and the slides were immediately washed with water for 10–15 min. Nuclei were counterstained with hematoxylin. Dehydration was performed by washing the sections with ethanol and xylene. Images were obtained by light microscopy (Olympus, Japan).

Statistical Analysis

All experiments were performed in triplicate. The results are presented as means of at least three experiments ±95% CI. Statistical analyses were performed by GraphPad Prism 6, and P values were calculated by one-way and two-way analyses of variance (ANOVA). The significance of the difference between different treatment groups was analyzed by Sidak’s multiple comparisons test and Tukey’s multiple comparison test.

Acknowledgments

This research was supported by a grant from the Indian Institute of Technology Delhi (to J.B.). The authors thank the Supercomputing Facility, IIT Delhi, for providing the software used for computational studies. They also acknowledge the animal facility at NII, Delhi, for allowing them to perform the animal studies. The authors acknowledge Mr. Amit Kumar Rathore, (Institute of Liver and Biliary Sciences, New Delhi) for preparing IHC slides.

Data Availability Statement

All materials used to obtain the data in this study are available from the corresponding authors upon reasonable request.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsptsci.2c00214.

  • Computational parameters for the protein–small molecule interaction (Table S1); IC50 of Ponatinib and doxorubicin in B16-F10 cells (Table S2); cell-free binding assay (Figure S1); expression of PD-L1 on A549 cells (Figure S2); tumor weight on day 23 (Figure S3); change in BW (Figure S4) (PDF)

Author Contributions

A.B. and J.B. designed and conceived the experiments. A.B. performed the experiments. A.B. and J.B. analyzed the data and wrote the paper. S.D. helped with the animal studies. All authors discussed the results and commented on the manuscript.

This work was supported by the Faculty Interdisciplinary Research Projects granted to J.B. by the Indian Institute of Technology Delhi (Grant Nos. MI02200G and MI02395G).

The authors declare no competing financial interest.

Notes

All animal experiments in this study have ethical approval from the NII Institutional Animal Ethics Committee.

Notes

All authors have agreed to publish this manuscript.

Supplementary Material

pt2c00214_si_001.pdf (338.2KB, pdf)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

pt2c00214_si_001.pdf (338.2KB, pdf)

Data Availability Statement

All materials used to obtain the data in this study are available from the corresponding authors upon reasonable request.

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