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. 2021 Dec 23;13(2):196–201. doi: 10.1039/d1md00324k

Designed hybrid anticancer nuclear-localized peptide inhibits aggressive cancer cell proliferation†

Prasenjit Mondal 1,2,‡,§, Saswat Mohapatra 1,2,‡, Debmalya Bhunia 1,‡, Prabir Kumar Gharai 1, Nabanita Mukherjee 3, Varsha Gupta 1, Satyajit Ghosh 3, Surajit Ghosh 1,2,3,✉
PMCID: PMC8864490  PMID: 35308028

Abstract

Cell proliferation is a crucial step that might promote cancer if deregulated. Therefore, this vital segment is critically controlled by a complicated cell-cycle process in normal cells that is regulated by some regulatory proteins. It has been observed that p16 protein, playing a crucial role in cell-cycle progression/regulation, remains inactivated in different cancer cells. This inactivity of p16 protein leads to the enhancement of cancer cell proliferation by allowing uncontrolled cancer cell division. Hence, the activity of p16 protein needs to be restored using new viral vectors, small molecules as well as peptides to control/suppress this type of abnormal cell proliferation. In this work, we have taken an interesting approach to increase the efficiency and bio-availability of p16 peptide (functional part of p16 protein) to be an aggressive anti-leukemia therapeutic agent by conjugating a nuclear-localized signal (NLS) sequence and a short peptide (AVPI) with it. Moreover, this newly designed NLS attached hybrid peptide greatly affects XIAP expressing but p16 lower expressing human chronic myelogenous leukemia (CML) cell proliferation by targeting both nuclear (CDK4/cyclin D) and cellular factors (XIAP) and promoting the caspase-3 dependent apoptosis pathway.

p16 and AVPI fused hybrid peptide containing NLS sequence promotes caspase-3 dependent apoptosis pathway by targeting both nuclear (CDK4/CyclinD) and cellular (XIAP) factors, and acts as a potent therapeutic agent against aggresive leukemia cells.graphic file with name d1md00324k-ga.jpg

Introduction

In tissue homeostasis, cell proliferation is a crucial step that might promote cancer if dysregulated. This vital segment is critically controlled by a complicated cell-cycle process through various stages, which are known as G0, G1, S, G2 and M phases, respectively. Among these, the G1 phase is a critical phase since many oncogenic perturbations have been observed as targeted amplification or mutations through various G1-specific protein regulators.1–3 Interestingly, cyclin-dependent kinases (CDKs) also play an important role to control the cell cycle processes. There are several types of CDKs (e.g., CDK1, CDK2, CDK4, CDK6 etc.), but among them, alteration of CDK4 and its associated cyclins are common in human cancers. Therefore, designing inhibitors that specifically bind and inhibit CDK4 activity can act as a promising anticancer therapeutic agent. It has already been documented that p16, a major G1-specific cell-cycle regulatory protein, binds with CDK4 and CDK6. The prime role of p16 protein is to prevent the cell-cycle progression and restrict the action of cyclin D at the G1/S checkpoint by blocking the phosphorylation of retinoblastoma tumor (RB) suppressor.4–8 The major function of cyclin D is to drive the cell cycle forward by binding with CDKs and forming a catalytically active complex.6 Surprisingly, it has been observed that p16 protein remains inactivated in different cancer cells and as a result, the crucial role of p16 protein to halt cell-cycle progression at the G1/S boundary phase is hindered. This inactivity of p16 protein function leads to enhancement of cancer cell proliferation by allowing uncontrolled division of cancer cells. Hence, viral vectors, small molecules, or peptide-based drug molecules are needed to stop this type of aggressive cancerous cell division/proliferation by restoring the activity of the p16 protein. A popularly known Trojan p16 peptide has already been developed, which specifically can restore the activity of p16 protein.4,9 Thus, any small molecule or peptides possessing inhibitory activity such as p16 against the CDK4:cyclin D complex might have an aggressive antitumor activity and can act as a potent therapeutic agent in tumors formed due to the altered p16 protein. However, the delivery of these small molecules or peptides towards the active site of action is a challenging task due to their poor penetration ability and the possibility of enzymatic degradation. Recently, transduction systems having protein transduction domain have gained immense considerations due to their therapeutic delivery, however, experimentally the efficiency of these systems is not up to the mark. Several other approaches to these types of malignancies have also been reported, such as the use of RI-TAT p53C′ peptide against Burkitt's lymphoma10 that needs a 48 h of treatment with a high concentration of peptide to target the p53 protein. Immunotherapeutic and genomic approaches are also reported.11 Therefore, designing new molecules is necessary to effectively tackle this type of challenging problem in aggressive cancer cell proliferation.

Previously, Kondo et al. has efficiently delivered the p16 peptide attached with novel transporter delivery systems (Wr-T, WR-T and r9) towards the hematopoietic neoplasm K562 cells.12 Although this Wr-T system itself is a long 30-mer peptide sequence with an attachment of r9 peptide to increase its cell-penetrating ability. It has often been observed that this type of large peptide system suffers from enzymatic degradation13 and aggregation inside the cell.14 In this work, we have attached a nuclear localizing signal (NLS) sequence, a class of highly cationic peptides widely used for cellular uptake of linked cargo, with the functional part of the p16 protein (p16 peptide).15 This was intended to have a better inhibitory activity of cyclin-dependent p16 by enhancing its uptake and availability in cells and targeting the CDK4 protein effectively. Further, a short tetrapeptide AVPI, derived from Smac/Diablo antagonises XIAP, was attached with this designed p16-NLS peptide to increase the efficacy of this peptide to be an aggressive anti-leukemia therapeutic agent as AVPI is known to promote caspase 3 dependent apoptotic cell death.16 Schematic representation of the inhibitory activity of p16 peptide and sequences of all the peptides used in this study are shown in Scheme 1a and b, respectively.

Scheme 1. (a) Schematic representation of the inhibition of CDK4 by p16 peptide. Left panel: CDK4 binds with cyclin D, forms CDK4–cyclin D complex that enhances cell regulation from G1 to S phase. Right panel: p16 binds with CDK4, forms CDK4–p16 complex, and inhibits the formation of CDK4–cyclin D complex that results in inhibition of cell differentiation process. (b) Sequences of different peptides (arranged in N-terminal to C-terminal) used in this work.

Scheme 1

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Results and discussion

Synthesis, characterization, and fluorescein labelling of designed peptides

We have synthesized p16, fluorescein-p16 (F-p16), p16-NLS, fluorescein-p16-NLS (F-p16-NLS), AVPI-FK-p16-NLS (AFPN), and fluorescein-AVPI-FK-p16-NLS (F-AFPN) peptides using a CEM microwave peptide synthesizer equipped with liberty-1 followed by purification and characterization using C18 reverse-phase HPLC and MALDI-TOF spectrometry (Fig. S1–S6, ESI†).

Various in vitro studies with fluorescein-p16 and fluorescein-p16-NLS peptide in U937 and K562 cells

After successful synthesis of all peptides, we performed various in vitro assays in two different cell lines (U937, lymphocyte p16 intrinsic expressing cells and K562, bone marrow lymphoblast p16 lower expressing cells). Meta-analysis of the RNAseq data for p16 expression in these two cell lines (U937 and K562) shows clear overexpression of p16/CDKN2A in U937 cells compared to K562 (Fig. S7, ESI†).17 Now, to compare the uptake efficacy, at first, we conducted the flow cytometric uptake study with fluorescein-attached p16 and p16-NLS peptide in U937 cells. Interestingly, flow cytometric analysis indicates that fluorescein-attached p16-NLS peptide exhibits higher uptake compared to fluorescein-p16 at 5 μM concentration (following previous work, Fig. 1a–d).12,18

Fig. 1. Flow cytometric uptake study of fluorescein-p16 and fluorescein-p16-NLS peptide performed in U937 cells. Untreated control (a), fluorescein-p16 (b), fluorescein-p16-NLS (c) and bar diagrams indicate the higher uptake of fluorescein-p16-NLS compared to fluorescein-p16 (d), scale bars correspond to standard deviation of value from mean ***p < 0.001 (n = 3), performing two-tailed student's t-test.

Fig. 1

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As nuclear localizing sequence (NLS) invigorates the uptake efficacy of the p16 peptides, we performed and compared the apoptosis study of p16-NLS and p16 peptide in U937 cells using Annexin V/PI staining after 24 h of treatment with both the peptides in a range of concentrations (5–25 μM). It has been observed that both the peptides exhibited similar apoptosis results (early and late apoptosis).19,20 Hence, p16-NLS peptide failed to exhibit significant apoptotic death upon treatment in U937 cells (Fig. S8, ESI†) as U937 already has intrinsic expression of p16. Therefore, we were interested in checking the effect of NLS attached p16 peptide in p16 lower expressing K562 cells. Henceforth, we checked the uptake of fluorescein-p16-NLS peptide in K562 cells, in suspension culture, after 4 h of treatment. Fluorescence microscopic images revealed a significant uptake (F-p16-NLS) of the peptide into K562 cells (Fig. 2a–d and S9, ESI†).

Fig. 2. (a–d) Microscopic images captured in different channels (DIC, DAPI, and FITC) and their merged image show good uptake of fluorescein-p16-NLS peptide in K562 cells, observed after 4 h of treatment. Scale bar corresponds to 100 μm. (e) Flow cytometric analysis indicates higher uptake of F-p16-NLS compared to F-p16 and (f) F-p16-NLS uptake undergoes through endocytosis pathway.

Fig. 2

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Then, we performed flow cytometric analysis in K562 cells upon 1 h treatment of both peptides (F-p16-NLS and F-p16 peptide) at 5 μM concentration, which signifies the higher uptake of NLS attached p16 peptide (Fig. 2e).

Flow cytometric analysis to elucidate the mechanistic pathway of peptide's cellular internalization

As the F-p16-NLS peptide exhibited higher uptake, we tried to unravel the mechanistic pathways (as in several ways peptides can enter the cell)21 during cellular uptake using flow cytometric analysis. For that purpose, two sets of cultured cells were treated with F-p16-NLS (5 μM) and incubated at 37 °C and 4 °C, separately, for 1 h. Intriguingly, FACS data revealed that the uptake of F-p16-NLS into K562 cells incubated at 37 °C was higher compared to that at 4 °C. This result suggests possible cellular uptake of F-p16-NLS following the endocytosis mechanism (Fig. 2f). This is because low temperature (4 °C) interrupts the enzymatic action, reducing the cellular ATP storage, and affecting endocytosis. This assay supports the involvement of endocytosis as the major mechanism of cellular uptake inside the K562 cells.21

FACS apoptosis study of p16 and p16-NLS peptide in K562 cells

After that, we performed and compared the apoptosis study between p16 and p16-NLS peptides from 1.5625 to 25 μM concentrations in K562 cells after 24 h of treatment. The FACS result indicates the higher amount of apoptosis death (early and late) after treatment with the p16-NLS peptide in K562 cells. From the quantification data of cell death, we have observed a higher cell population in both early and late phases of apoptosis, indicating the induction of apoptotic cell death upon treatment with p16-NLS compared to the p16 peptide (Fig. S10 and S11, ESI†). We also calculated the IC50 from the % apoptotic cells readouts against a gradient of peptide concentration, and by linear fitting of the curve. We observed that p16-NLS (5.583 μM) has a significantly lower IC50 value in K562 cells than in p16 (30.119 μM), whereas in U937 cells, both peptides show higher IC50 (373.636 μM for p16 peptide and 1391.7 μM for p16-NLS).

As such, we performed all our experiments using peptide concentrations well below the IC50 value to ensure maximal cell viability. But, for lower concentrations (<5 μM), NLS-p16 treated in K562 cells does not show much apoptotic value because for uptake via endocytosis or direct translocation of cell-penetrating peptides (CPPs), peptide concentration should be at least 10 μM or above, which allow CPPs to escape from endosomes into the cytosol or directly penetrate the plasma membrane.22

Caspase-3 dependent apoptosis pathway and designing of hybrid p16 peptide

Caspases exist as inactive pro-enzyme that undergo proteolytic processing to produce two subunits, large and small, that further dimerize to form the active enzyme. This sequential activation of caspases plays a central role in the execution phase during cell apoptosis process.23,24 Further it has been noticed that the activation of p16 was found to activate cell cycle arrest, senescence, and trigger apoptosis. Again, apoptosis in cancer cells can be inhibited through various mechanisms. Among these XIAP, a ubiquitous IAP (inhibitor of apoptosis protein) inhibits various caspases and importantly caspase 3 when present, hence inhibiting apoptosis. As K562 expresses XIAP,25 compromising the apoptotic pathway due to p16-NLS certainly appears feasible. To achieve this, AVPI, a short tetrapeptide derived from Smac/Diablo, known as the antagonist of XIAP and promotes caspase 3 dependent apoptotic cell death,16 was attached with p16-NLS peptide to develop an efficient peptide specific to leukemia target cells. In addition, we have intentionally inserted the Cathepsin B substrate FK sequence in-between AVPI and p16-NLS to increase the rate of enzymatic cleavage of the peptide on this position after reaching the cytoplasm. After cleavage of the peptide, the functional p16 peptide will be released and binds at its specific CDK target site. Therefore, AVPI-FK-p16-NLS peptide was synthesized using SPPS and further apoptosis study was performed up to 20 μM concentrations. Interestingly, the flow cytometric study indicates that our designed peptide (AVPI-NLS-p16) exhibited a significant enhancement of apoptotic cell death compared to that with the NLS-p16 peptide (Fig. 3a–e). We also observed that the NLS-P16-AVPI peptide exhibited a higher amount of caspase 3 activation (44 ± 1.6%) than NLS-P16 (36 ± 2%) and the control (31 ± 0.4%), which further confirmed the activation of the caspase 3 pathway (Fig. 3f–i).

Fig. 3. (Left panel) Apoptosis study using FACS. K562 cells treated with (a and c) p16-NLS, (b and d) AVPI-p16-NLS (AFPN) at 10 μM and 20 μM concentrations respectively and (e) bar diagram shows quantitatively that NLS-P16-AVPI causes higher apoptotic death of K562 cells. (Right panel) Caspase activation study using FACS. K562 cells treated with different peptides i.e. (f) control (g) p16-NLS, (h) AFPN and (i) bar diagram indicates the higher amount of caspase 3 activation upon treatment with AFPN compared to p16-NLS and untreated. Scale bars correspond to the standard deviation of the value from the mean (n = 3), *p < 0.05 and ***p < 0.001, after performing a two-tailed student's t-test.

Fig. 3

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Molecular docking of p16 peptide with CDK4 protein

We also performed the molecular docking experiment to further investigate the binding region of p16 peptide in CDK4 caspase (PDB ID-3G33).26 For this study, we did an unbiased blind docking, considering the whole CDK4 protein in the grid box. Interestingly, we observed that the p16 peptide binds at the activation segment of CDK4 with a high binding affinity (B.E −6.2 kcal mol−1) and inhibits the complex formation between CDK4 and cyclin D (Fig. 4).

Fig. 4. (a) Molecular docking image of only CDK4–cyclin D complex (PDB ID: 3G33) and (b) p16 peptide binding at the activation segment of CDK4 (B.E −6.2 kcal mol−1) and making complex with it (interacting partners are Y22, D145, A167, S171, Q173, Y196 and E224).

Fig. 4

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Conclusions

In summary, we have designed and developed a CDK-targeted hybrid peptide conjugated with NLS sequence to target the CDK4 protein mostly located at the cellular cytoplasm (when non-complexed), and AVPI to target XIAP.16 Initial results suggest that after the attachment of the NLS sequence with the p16 peptide, a significant enhancement of cellular uptake occurs. Further, we have checked the activity of p16 and p16-NLS peptides in both p16 higher expressing (U937) and lower expressing (K562) cells. FACS study revealed that there was no significant difference between these two peptides in p16 expressing cells though the uptake of p16-NLS was higher. On the other hand, in K562 cells, our designed peptide showed significant apoptotic death. This signifies the specific activity of our peptide towards p16 lowering expressing tumor cells. Further p16-NLS was fused with AVPI interlinked with FK, which was ultimately cleaved by lysosomal Cathepsin B to free up AVPI and inhibit cellular XIAP. The modified AVPI-p16-NLS peptide exhibited a higher amount of caspase 3 activation (44 ± 1.6%) compared to p16-NLS (36 ± 2%) and control (31 ± 0.4%). Finally, we can conclude that this designed sequence can enter the cell rapidly and specifically and act as a potent therapeutic agent against aggressive leukemia cells.

Experimental section

Synthesis of p16, nuclear-localized sequence (NLS) and p16-NLS peptides along with carboxy-fluorescein attachment

300 mg of wang resin was swelled overnight in DMF–DCM (1 : 1) solvent. Five equivalents of excess fmoc-protected amino acids were coupled successively followed by deprotection using 20% piperidine in a CEM microwave peptide synthesizer (liberty 1). Coupling and deprotection steps were maintained for eight and five min, respectively. N,N′-Diisopropylethylamine (DIPEA) and HBTU were used as an activator base and activator, respectively. DMF was used as a solvent. After that, the peptide-attached resin was washed with DMF and DCM solvents. Synthesized peptides were cleaved using standard resin cleavage cocktail solution containing 92.5% trifluoroacetic acid (TFA), 2.5% Milli Q water, 2.5% EDT and 2.5% phenol. The cleavage solution containing peptide-containing resin was kept on an automated shaker (Labnet International) for 3 h. Then, TFA was removed from the filtrate using nitrogen flow. Cold diethyl ether was added gradually to the remaining filtrate to ensure complete precipitation, which was then separated by centrifugation. Peptides were purified using the C-18 reverse phase HPLC column and expected masses were confirmed using MALDI-TOF spectrometry. Similarly, covalently attached fluorescein tagged peptides were synthesized and purified by the above-mentioned procedure.

Fluorescence microscopy imaging

Fluorescein attached p16 and p16-NLS (5 μM) were treated on K562 cells for 4 h. After that, treated cells were observed under an inverted fluorescence microscope with 40× objective (Olympus IX83 equipped with ANDOR iXON3 camera).

FACS apoptosis study

U937 and K562 cells (∼5 × 105 cells per mL) were harvested overnight in a 6-well plate and treated with either 45 μM of p16-NLS or p16 separately for 24 h. After that, cells were suspended in 100 μL solution of binding buffer contained with propidium iodide (PI) and annexin V and incubated at 37 °C for 15 min. The emission of annexin V and PI was analysed using FITC and PI channels of BD LSRFortessa™ flow cytometer using emission filters at 530 and 610 nm, respectively. Cells in Q1, Q2 and Q4 are considered necrotic, early and late apoptosis, respectively. Q3 quadrant cells are considered a healthy cell population.

Cell cycle study

A cell cycle study was conducted by treatment with either p16 or p16-NLS of 45 μM for 24 h. Next, cells were incubated with PI (100 μg mL−1) and RNase (10 μg mL−1) for 45 min at 37 °C temperature. After treatment, U937 and K562 cells were fixed with 70% ethanol at 20 °C overnight. Finally, cell cycle analysis was performed using PI channels of BD LSRFortessa™ flow cytometer with emission filters at 610 nm.

Cellular uptake study by microscopic imaging

K562 cells (∼2 × 103) were seeded in a DMEM medium containing 10% fetal bovine serum (FBS) on a confocal disc overnight prior to treatment. K562 cells were treated with fluorescein attached p16 and p16-NLS (5 μM) in DMEM containing media and incubated for 4 h (DMSO concentration maintained 0.4%). Next, 4% formaldehyde in PBS buffer was added for 30 min to fix the cells in each cover glass. Next, the formaldehyde solution was removed and washed with PBS buffer. The nucleus was stained with Hoechst 33258 (1 μg mL−1) for 1 h. Finally, Hoechst 33258 solution was removed and washed with PBS buffer three times. Thus, each confocal disc was ready for microscopic imaging. Cell imaging was performed using an Andor spinning disc confocal microscope with 40× objective (Olympus) equipped with Andor iXon 3897 EMCCD camera in bright field, 488 and 405 nm wavelength light.

Flow cytometry for cellular uptake

U937 and K562 cells were cultured in a 6-well plate at a density of ∼5 × 105 cells each well prior to 24 h of treatment. Cells were treated with fluorescein attached p16 and p16-NLS (5 μM) for 4 h. After that, cells were washed with phosphate buffer and trypsinized. Comparative cellular uptake of p16 and p16-NLS was performed and analyzed using FACS (Ex – 488 and Em – 500 to 600 nm).

Cellular internalization study using FACS

The cellular internalization mechanism of fluorescein-p16 and fluorescein-p16-NLS-treated cells was studied using the previously described method.21 In short, seeded K562 cells (1 × 106) were detached and collected in a suspension containing a serum-free DMEM (colourless) culture medium. After that, cells were incubated at 37 °C and 4 °C for 60 min, separately. Next, these cell suspensions were treated with both fluorescein-attached peptides separately of final concentration 5 μM and incubated at 37 °C or 4 °C for 4 h, separately. After that, the cell suspension was centrifuged to remove excess fluorescein-p16 and fluorescein-p16-NLS from the solution and resuspended in a DMEM culture medium containing trypsin (1 mg mL−1) and incubated for 15 min. Cells were washed with serum-free DMEM (colorless) culture medium and fluorescent signal was analysed using 488 nm channels of BD LSRFortessa™ flow cytometer and emission filters at 530 nm.

Molecular docking

Autodock-Vina software version 1.1.2 was used for blind docking.27 A 50 × 62 × 48 affinity grid box was centred on the receptor cyclin-dependent kinase (CDK4) (PDB ID: 3G33)28 for docking with p16 peptide.

Author contributions

P. M. performed the synthesis, purification, fluorescein labelling and characterization of all peptides. S. M. and D. B. performed the FACS uptake and apoptosis assay, microscopic imaging of peptide's cellular uptake, and analysed results with P. M. P. K. G. helped P. M. in peptide characterization, N. M., V. G., and S. G., helped in some in vitro assays. S. G. conceived the idea, supervised the project, and wrote the manuscript.

Conflicts of interest

The authors declare no competing financial interest.

Supplementary Material

MD-013-D1MD00324K-s001

Acknowledgments

The authors wish to thank NCCS-Pune for cell lines, CSIR-IICB Kolkata and IIT Jodhpur for infrastructure. P. M. and P. K. G. thank CSIR, S. M. thank UGC, D. B. and V. G. thanks to DST-Inspire, N. M. and S. G. thank IITJ for fellowship. SG kindly acknowledges SERB, DST-Grant (CRG/2019/000670 and SERB STAR) for financial assistance.

†

Electronic supplementary information (ESI) available. See DOI: 10.1039/d1md00324k

Notes and references

  1. Liggett W. H. Sidransky D. J. Clin. Oncol. 1998;16:1197–1206. doi: 10.1200/JCO.1998.16.3.1197. [DOI] [PubMed] [Google Scholar]
  2. Hunter T. Pines J. Cell. 1994;79:573–576. doi: 10.1016/0092-8674(94)90543-6. [DOI] [PubMed] [Google Scholar]
  3. Hall M. Peters G. Adv. Cancer Res. 1996;68:67–108. doi: 10.1016/S0065-230X(08)60352-8. [DOI] [PubMed] [Google Scholar]
  4. Hosotani R. Miyamoto Y. Fujimoto K. Doi R. Otaka A. Fujii N. Imamura M. Clin. Cancer Res. 2002;8:1271. [PubMed] [Google Scholar]
  5. Wang S. Wuu J. Savas L. Patwardhan N. Khan A. Hum. Pathol. 1998;29:1304–1309. doi: 10.1016/S0046-8177(98)90262-3. [DOI] [PubMed] [Google Scholar]
  6. Malumbres M. Barbacid M. Nat. Rev. Cancer. 2009;9:153. doi: 10.1038/nrc2602. [DOI] [PubMed] [Google Scholar]
  7. Ball K. L. Lain S. Fahraeus R. Smythe C. Lane D. P. Curr. Biol. 1996;1:71. doi: 10.1016/s0960-9822(06)00029-7. [DOI] [PubMed] [Google Scholar]
  8. Bertoli C. Skotheim J. M. de Bruin R. A. M. Nat. Rev. Mol. Cell Biol. 2013;14:518. doi: 10.1038/nrm3629. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Massaoka M. H. Matsuo A. L. Figueiredo C. R. Girola N. Faria C. F. Azevedo R. A. Travassos L. R. FEBS Open Bio. 2014;4:153. doi: 10.1016/j.fob.2014.01.007. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Snyder E. L. Meade B. R. Saenz C. C. Dowdy S. F. PLoS Biol. 2004;2:186. doi: 10.1371/journal.pbio.0020036. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Barrett A. J. Blanc K. Le. Clin. Exp. Immunol. 2010;161:223. doi: 10.1111/j.1365-2249.2010.04197.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kondo E. Seto M. Yoshikawa K. Yoshino T. Mol. Cancer Ther. 2004:1623. [PubMed] [Google Scholar]
  13. Dehsorski A. Hamley L. W. Seitsonen J. Ruokolainen J. Langmuir. 2013;29:6665. doi: 10.1021/la401025r. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Tyedmers J. Mogk A. Bukau B. Nat. Rev. Mol. Cell Biol. 2010;11:777. doi: 10.1038/nrm2993. [DOI] [PubMed] [Google Scholar]
  15. Ragin A. D. Morgan R. A. Chmielewski J. Chem. Biol. 2002;8:943. doi: 10.1016/S1074-5521(02)00189-8. [DOI] [PubMed] [Google Scholar]
  16. Lim H. Jin X. Kim J. Hwang S. Shin K. B. Choi J. Nam K. No K. T. Comput. Struct. Biotechnol. J. 2019;17:1217. doi: 10.1016/j.csbj.2019.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. CDKN2A (cyclin dependent kinase inhibitor 2A) expression 21Q4from Public database of DepMap (https://depmap.org/portal/gene/CDKN2A?tab=characterization)
  18. Cardozo A. K. Buchillier V. Mathieu M. Chen J. Ortis F. Ladrière L. Allaman-Pillet N. Poirot O. Kellenberger S. Beckmann J. S. Eizirik D. L. Bonny C. Maurer F. Biochim. Biophys. Acta, Biomembr. 2007;1768:2222. doi: 10.1016/j.bbamem.2007.06.003. [DOI] [PubMed] [Google Scholar]
  19. Mohapatra S. Saha A. Mondal P. Jana B. Ghosh S. Biswas A. Ghosh S. Adv. Healthcare Mater. 2017;6:1600718. doi: 10.1002/adhm.201600718. [DOI] [PubMed] [Google Scholar]
  20. Bhunia D. Pradhan K. Das G. Ghosh S. Mondal P. Ghosh S. Chem. Commun. 2018;54:9309. doi: 10.1039/C8CC05687K. [DOI] [PubMed] [Google Scholar]
  21. Wang Z. Xie Y. Zhang L. Zhang H. An X. Wang T. Meng A. Cell Struct. Funct. 2008;33:171–183. doi: 10.1247/csf.08019. [DOI] [PubMed] [Google Scholar]
  22. Skotland T. Geir Iversen T. Torgersen M. L. Sandvig K. Molecules. 2015;20:13313–13323. doi: 10.3390/molecules200713313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Greene B. T. Thorburn J. Wllingham M. C. Thorburn A. Planalp R. P. Brechbiel M. W. Jennings-Gee J. Wilkinson J. Torti F. M. Torti S. V. J. Biol. Chem. 2002;277:25568. doi: 10.1074/jbc.M110345200. [DOI] [PubMed] [Google Scholar]
  24. Hashimoto T. Kikkawa U. Kamada S. PLoS One. 2011;6:e18449. doi: 10.1371/journal.pone.0018449. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Uhlen M. Zhang C. Lee S. Sjöstedt E. Fagerberg L. Bidkhori G. Benfeitas R. Arif M. Liu Z. Edfors F. Sanli K. von Feilitzen K. Oksvold P. Lundberg E. Hober S. Nilsson P. Mattsson J. Schwenk J. M. Brunnström H. Glimelius B. Sjöblom T. Edqvist P. H. Djureinovic D. Micke P. Lindskog C. Mardinoglu A. Ponten F. Science. 2017;357:eaan2507. doi: 10.1126/science.aan2507. [DOI] [PubMed] [Google Scholar]
  26. Takakia T. Echalierb A. Brownb N. R. Hunta T. Endicottb J. A. Noble M. E. M. Proc. Natl. Acad. Sci. U. S. A. 2009:4171. doi: 10.1073/pnas.0809674106. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Trott O. Olson A. J. J. Comput. Chem. 2010;31:455. doi: 10.1002/jcc.21334. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Takaki T. Echalier A. Brown N. R. Hunt T. Endicott J. A. Noble M. E. Proc. Natl. Acad. Sci. U. S. A. 2009;106:4171–4176. doi: 10.1073/pnas.0809674106. [DOI] [PMC free article] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

MD-013-D1MD00324K-s001
MD-013-D1MD00324K-s001.pdf (1.8MB, pdf)

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