Polypharmacology-Driven Discovery of ZAK-I-57: A Potent Multi-Targeted Benzoxazinone Small Molecule for Hepatocellular Carcinoma Therapy

Abstract

Hepatocellular carcinoma (HCC) is a deadly disease characterized by a high mortality rate and resistance to conventional therapies, highlighting the need for novel therapeutic interventions. Given the multifaceted nature of HCC pathogenesis, a multitargeted and polypharmacological approach is crucial for effective treatment. This study reports the potent multitargeted and polypharmacological properties of ZAK-I-57, a benzoxazinone derivative, as a potential therapeutic option for HCC. In cell-based model, ZAK-I-57 demonstrated significant in vitro inhibition of proliferation in HCC cells. Utilizing PLC/PRF/5 tumor-bearing and HCC patient-derived tumor xenograft (PDTX) mouse models, we compared the efficacy of ZAK-I-57 with that of sorafenib, the current standard treatment. ZAK-I-57 demonstrated superior tumor suppressive effects at doses of 15 and 30 mg/kg, outperforming sorafenib. Western blot analysis revealed that ZAK-I-57 downregulated the oncogenic proteins EGFR and c-Myc, while promoting apoptosis by increasing Bax and decreasing Bcl-2 expression. Strikingly, ZAK-I-57 exhibited excellent ADMET properties, including high gastrointestinal absorption and good lipophilicity, along with an excellent safety profile, with no significant off-target toxicity in vital organs. In summary, our findings highlight ZAK-I-57 as a new and promising multitarget therapeutic agent for HCC, warranting further clinical investigation to improve patient outcomes.

Keywords

benzoxazinone derivatives / hepatocellular carcinoma / multi-targeted therapy / polypharmacology / tumor suppression

References

[1] W. Guo, J. Huang, N. Wang, et al., “Integrating Network Pharmacology and Pharmacological Evaluation for Deciphering the Action Mechanism of Herbal Formula Zuojin Pill in Suppressing Hepatocellular Carcinoma,” Frontiers in Pharmacology 10 (2019): 1185.

[2] J. Ferlay, I. Soerjomataram, R. Dikshit, et al., “Cancer Incidence and Mortality Worldwide: Sources, Methods and Major Patterns in GLOBOCAN 2012,” International Journal of Cancer 136, no. 5 (2015): E359-E386.

[3] J. M. Llovet, A. Villanueva, A. Lachenmayer, R. S. Finn, “Advances in Targeted Therapies for Hepatocellular Carcinoma in the Genomic Era,” Nature Reviews Clinical Oncology 12, no. 7 (2015): 408-424.

[4] M. Sherman, “Recurrence of Hepatocellular Carcinoma,” New England Journal of Medicine 359, no. 19 (2009): 2045-2047.

[5] Y. S. Ma, L. J. Bin, T. M. Wu, D. Fu, “New Therapeutic Options for Advanced Hepatocellular Carcinoma,” Cancer Control 27, no. 3 (2020): 1073274820945975.

[6] Z. Liu, Y. Lin, J. Zhang, et al., “Molecular Targeted and Immune Checkpoint Therapy for Advanced Hepatocellular Carcinoma,” Journal of Experimental and Clinical Cancer Research 38, no. 1 (2019): 1-13.

[7] K. S. Bhullar, N. O. Lagarón, E. M. McGowan, et al., “Kinase-Targeted Cancer Therapies: Progress, Challenges and Future Directions,” Molecular Cancer 17, no. 1 (2018): 1-20.

[8] L. Guo, C. Hu, M. Yao, G. Han, “Mechanism of Sorafenib Resistance Associated With Ferroptosis in HCC,” Frontiers in Pharmacology 14 (2023): 1207496.

[9] J. Guo, J. Zhao, Q. Xu, D. Huang, “Resistance of Lenvatinib in Hepatocellular Carcinoma,” Current Cancer Drug Targets 22, no. 11 (2022): 865-878.

[10] E. H. K. Mok, C. O. N. Leung, L. Zhou, et al., “Caspase-3-Induced Activation of SREBP2 Drives Drug Resistance via Promotion of Cholesterol Biosynthesis in Hepatocellular Carcinoma,” Cancer Research 82, no. 17 (2022): 3102-3115.

[11] A. Antolin, P. Workman, J. Mestres, B. Al-Lazikani, “Polypharmacology in Precision Oncology: Current Applications and Future Prospects,” Current Pharmaceutical Design 22, no. 46 (2016): 6935-6945.

[12] Z. A. Knight, H. Lin, K M. Shokat, “Targeting the Cancer Kinome Through Polypharmacology,” Nature Reviews Cancer 10, no. 2 (2010): 130-137.

[13] B. M. Kuenzi, L. L. Remsing Rix, P. A. Stewart, “Polypharmacology-Based Ceritinib Repurposing Using Integrated Functional Proteomics,” Nature Chemical Biology 13, no. 12 (2017): 1222-1231.

[14] B. P. Munson, M. Chen, A. Bogosian, et al., “De Novo Generation of Multi-Target Compounds Using Deep Generative Chemistry,” Nature Communications 15, no. 1 (2024): 1-12.

[15] A. Anighoro, J. Bajorath, G. Rastelli, “Polypharmacology: Challenges and Opportunities in Drug Discovery,” Journal of Medicinal Chemistry 57, no. 19 (2014): 7874-7887.

[16] Z. Xiong, M. Jeon, R. J. Allaway, et al., “Crowdsourced Identification of Multi-Target Kinase Inhibitors for RET- and TAU-Based Disease: The Multi-Targeting Drug DREAM Challenge,” PLoS Computational Biology 17, no. 9 (2021): e1009302.

[17] A. Bari, Z. A. Khan, S. A. Shahzad, et al., “Design and Syntheses of 7-Nitro-2-aryl-4H-benzo[d][1,3]Oxazin-4-ones as Potent Anticancer and Antioxidant Agents,” Journal of Molecular Structure 1214 (2020): 128252.

[18] B. P. Marasini, F. Rahim, S. Perveen, et al., “Synthesis, Structure-Activity Relationships Studies of Benzoxazinone Derivatives as α-Chymotrypsin Inhibitors,” Bioorganic Chemistry 70 (2017): 210-221.

[19] M. Gütschow, U. Neumann, “Inhibition of Cathepsin G by 4H-3,1-Benzoxazin-4-ones,” Bioorganic & Medicinal Chemistry 5, no. 10 (1997): 1935-1942.

[20] A. Krantz, T. F. Tam, T. J. Liak, et al., “Design and Synthesis of 4H-3,1-Benzoxazin-4-ones as Potent Alternate Substrate Inhibitors of Human Leukocyte Elastase,” Journal of Medicinal Chemistry 33, no. 2 (2002): 464-479.

[21] P. W. Hsieh, F. R. Chang, C. H. Chang, et al., “2-Substituted Benzoxazinone Analogues as Anti-human Coronavirus (anti-HCoV) and ICAM-1 Expression Inhibition Agents,” Bioorganic & Medicinal Chemistry Letters 14, no. 18 (2004): 4751-4754.

[22] M. Shariat, S. Abdollahi, “Synthesis of Benzoxazinone Derivatives: A New Route to 2 (N Phthaloylmethyl)-4H-3,1-benzoxazin-4-one,” Molecules (Basel, Switzerland) 9, no. 8 (2004): 705-712.

[23] S. Jiang, A. Awadasseid, S. Narva, et al., “Anti-Cancer Activity of Benzoxazinone Derivatives via Targeting c-Myc G-quadruplex Structure,” Life Sciences 258 (2020): 118252.

[24] T. F. Zhao, X. L. Xu, W. Y. Sun, Y. Lu, “Construction of Benzoxazinones From Anilines and Their Derivatives,” Organic Letters 25, no. 27 (2023): 4968-4973.

[25] A. Merecz-Sadowska, V. M. S. Isca, P. Sitarek, et al., “Exploring the Anticancer Potential of Semisynthetic Derivatives of 7α-Acetoxy-6β-hydroxyroyleanone From Plectranthus Sp.: An In Silico Approach,” International Journal of Molecular Sciences 25, no. 8 (2024): 4529.

[26] A. Daina, O. Michielin, V. Zoete, “SwissADME: A Free Web Tool to Evaluate Pharmacokinetics, Drug-Likeness and Medicinal Chemistry Friendliness of Small Molecules,” Scientific Reports 7, no. 1 (2017): 1-13.

[27] F. Zafar, A. Gupta, K. Thangavel, et al., “Physicochemical and Pharmacokinetic Analysis of Anacardic Acid Derivatives,” ACS Omega 5, no. 11 (2020): 6021-6030.

[28] D. F. Veber, S. R. Johnson, H. Y. Cheng, B. R. Smith, K. W. Ward, K D. Kopple, “Molecular Properties That Influence the Oral Bioavailability of Drug Candidates,” Journal of Medicinal Chemistry 45, no. 12 (2002): 2615-2623.

[29] C. A. Lipinski, F. Lombardo, B. W. Dominy, P J. Feeney, “Experimental and Computational Approaches to Estimate Solubility and Permeability in Drug Discovery and Development Settings,” Advanced Drug Delivery Reviews 64, (2012): 4-17.

[30] J. B. Baell, G A. Holloway, “New Substructure Filters for Removal of Pan Assay Interference Compounds (PAINS) From Screening Libraries and for Their Exclusion in Bioassays,” Journal of Medicinal Chemistry 53, no. 7 (2010): 2719-2740.

[31] R. Brenk, A. Schipani, D. James, et al., “Lessons Learnt From Assembling Screening Libraries for Drug Discovery for Neglected Diseases,” Chemmedchem 3, no. 3 (2008): 435-444.

[32] Y. Isyaku, A. Uzairu, S. Uba, “Computational Studies of a Series of 2-Substituted Phenyl-2-oxo-, 2-Hydroxyl- and 2-Acylloxyethylsulfonamides as Potent Anti-fungal Agents,” Heliyon 6, no. 4 (2020): e03724.

[33] A. Daina, O. Michielin, V. Zoete, “SwissTargetPrediction: Updated Data and New Features for Efficient Prediction of Protein Targets of Small Molecules,” Nucleic Acids Research 47, no. W1 (2019): W357-W364.

[34] W. H. Su, C. C. Chao, S. H. Yeh, D. S. Chen, P. J. Chen, Y. S. Jou, “OncoDB.HCC: An Integrated Oncogenomic Database of Hepatocellular Carcinoma Revealed Aberrant Cancer Target Genes and Loci,” Nucleic Acids Research 35, supplement, no. S1 (2007): D727-D731.

[35] L. Lee, K. Wang, G. Li, et al., “Liverome: A Curated Database of Liver Cancer-Related Gene Signatures With Self-Contained Context Information,” 10th International Conference on Bioinformatics - 1st ISCB Asia Joint Conference 2011, InCoB 2011/ISCB-Asia 2011: Computational Biology—Proceedings from Asia Pacific Bioinformatics Network (APBioNet), supplement, 12, no. S3 (2011): 1-13.

[36] Q. Wang, Y. Liang, C. Peng, P. Jiang, “Network Pharmacology-Based Study on the Mechanism of Scutellariae Radix for Hepatocellular Carcinoma Treatment,” Evidence-Based Complementary and Alternative Medicine 2020 (2020): 8897918, https://doi.org/10.1155/2020/8897918

[37] J. C. Oliveros, " Venny 2.1.0," 2007, https://bioinfogp.cnb.csic.es/tools/venny/

[38] C. von Mering, M. Huynen, D. Jaeggi, S. Schmidt, P. Bork, B. Snel, “STRING: A Database of Predicted Functional Associations Between Proteins,” Nucleic Acids Research 31, no. 1 (2003): 258-261.

[39] C. T. Lopes, M. Franz, F. Kazi, et al., “Cytoscape Web: An Interactive Web-Based Network Browser,” Bioinformatics 26, no. 18 (2010): 2347-2348.

[40] DAVID, "Functional Annotation Bioinformatics Microarray Analysis," accessed April 19, 2022, https://david.ncifcrf.gov/

[41] P. Boudou-Rouquette, S. Ropert, O. Mir, et al., “Variability of Sorafenib Toxicity and Exposure Over Time: A Pharmacokinetic/Pharmacodynamic Analysis,” Oncologist 17, no. 9 (2012): 1204.

[42] Y. Qin, S. Han, Y. Yu, et al., “Lenvatinib in Hepatocellular Carcinoma: Resistance Mechanisms and Strategies for Improved Efficacy,” Liver International 44, no. 8 (2024): 1808-1831.

[43] R. J. Motzer, M. H. Taylor, T. R. J. Evans, et al., “Lenvatinib Dose, Efficacy, and Safety in the Treatment of Multiple Malignancies,” Expert Review of Anticancer Therapy 22, no. 4 (2022): 383.

[44] J. M. Llovet, S. Ricci, V. Mazzaferro, et al., “Sorafenib in Advanced Hepatocellular Carcinoma,” New England Journal of Medicine 359, no. 4 (2008): 378-390.

[45] P. Ryszkiewicz, B. Malinowska, E. Schlicker, “Polypharmacology: Promises and New Drugs in 2022,” Pharmacological Reports 75, no. 4 (2023): 755.

[46] S. He, S. Tian, X. He, et al., “Multiple Targeted Self-emulsifying Compound RGO Reveals Obvious Anti-Tumor Potential in Hepatocellular Carcinoma,” Molecular Therapy Oncolytics 22 (2021): 604-616.

[47] C. P. Lin, C. R. Liu, C. N. Lee, T. S. Chan, H E. Liu, “Targeting c-Myc as a Novel Approach for Hepatocellular Carcinoma,” World Journal of Hepatology 2, no. 1 (2010): 16.

[48] S. Song, Z. Yu, Y. You, et al., “EGFR/MET Promotes Hepatocellular Carcinoma Metastasis by Stabilizing Tumor Cells and Resisting to RTKs Inhibitors in Circulating Tumor Microemboli,” Cell Death & Disease 13, no. 4 (2022): 1-12.

[49] H. Jin, Y. Shi, Y. Lv, et al., “EGFR Activation Limits the Response of Liver Cancer to Lenvatinib,” Nature 595, no. 7869 (2021): 730-734.

[50] Y. Yarden, “The EGFR Family and Its Ligands in Human Cancer: Signalling Mechanisms and Therapeutic Opportunities,” European Journal of Cancer 37, supplement, no. S4 (2001): 3-8.

[51] M. Bhat, E. Pasini, C. Pastrello, et al., “Estrogen Receptor 1 Inhibition of Wnt/β-Catenin Signaling Contributes to Sex Differences in Hepatocarcinogenesis,” Frontiers in Oncology 11 (2021): 4878.

[52] H. Ding, Y. Wang, H. Zhang, “CCND1 Silencing Suppresses Liver Cancer Stem Cell Differentiation and Overcomes 5-Fluorouracil Resistance in Hepatocellular Carcinoma,” Journal of Pharmacological Sciences 143, no. 3 (2020): 219-225.

[53] E. A. Toraih, H. G. Alrefai, M. H. Hussein, G. M. Helal, M. S. Khashana, M S. Fawzy, “Overexpression of Heat Shock Protein HSP90AA1 and Translocase of the Outer Mitochondrial Membrane TOM34 in HCV-induced Hepatocellular Carcinoma: A Pilot Study,” Clinical Biochemistry 63 (2019): 10-17.

[54] F. Rahmani, A. Ziaeemehr, S. Shahidsales, et al., “Role of Regulatory miRNAs of the PI3K/AKT/mTOR Signaling in the Pathogenesis of Hepatocellular Carcinoma,” Journal of Cellular Physiology 235, no. 5 (2020): 4146-4152.

[55] R. Yamaguchi, H. Yano, A. Iemura, S. Ogasawara, M. Haramaki, M. Kojiro, “Expression of Vascular Endothelial Growth Factor in human Hepatocellular Carcinoma,” Hepatology 28, no. 1 (1998): 68-77.

[56] Z. Qiang, J. Wan, X. Chen, H. Wang, “Mechanisms and Therapeutic Targets of ErbB Family Receptors in Hepatocellular Carcinoma: A Narrative Review,” Translational Cancer Research 13, no. 6 (2024): 3156-3178.

[57] N. Sueangoen, A. Tantiwetrueangdet, R. Panvichian, “HCC-Derived EGFR Mutants Are Functioning, EGF-Dependent, and Erlotinib-Resistant,” Cell & Bioscience 10, no. 1 (2020): 1-15.

[58] L. Jiang, L. Li, Y. Liu, et al., “Drug Resistance Mechanism of Kinase Inhibitors in the Treatment of Hepatocellular Carcinoma,” Frontiers in Pharmacology 14 (2023): 1097277.

[59] S. N. Steinway, H. Dang, H. You, C. B. Rountree, W. Ding, “The EGFR/ErbB3 Pathway Acts as a Compensatory Survival Mechanism Upon c-Met Inhibition in Human c-Met+ Hepatocellular Carcinoma,” PLoS ONE 10, no. 5 (2015): e0128159.

[60] N. Sueangoen, A. Tantiwetrueangdet, R. Panvichian, “HCC-Derived EGFR Mutants Are Functioning, EGF-Dependent, and Erlotinib-Resistant,” Cell & Bioscience 10, no. 1 (2020): 41.

[61] J. R. Whitfield, L. Soucek, “The Long Journey to Bring a Myc Inhibitor to the Clinic,” Journal of Cell Biology 220, no. 8 (2021): e202103090.

[62] J. Chauhan, H. Wang, J. L. Yap, et al., “Discovery of Methyl 4'-methyl-5-(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)-[1,1'-biphenyl]-3-carboxylate, an Improved Small-Molecule Inhibitor of c-Myc-max Dimerization,” Chemmedchem 9, no. 10 (2014): 2274-2285.

[63] R. L. Schroeder, C. L. Stevens, J. Sridhar, “Small Molecule Tyrosine Kinase Inhibitors of ErbB2/HER2/Neu in the Treatment of Aggressive Breast Cancer,” Molecules (Basel, Switzerland) 19, no. 9 (2014): 15196.

[64] M. García-Aranda, E. Pérez-Ruiz, M. Redondo, “Bcl-2 Inhibition to Overcome Resistance to Chemo- and Immunotherapy,” International Journal of Molecular Sciences 19, no. 12 (2018): 3950.

[65] J. Y. Zheng, G. S. Yang, W. Z. Wang, et al., “Overexpression of Bax Induces Apoptosis and Enhances Drug Sensitivity of Hepatocellular Cancer-9204 Cells,” World Journal of Gastroenterology : WJG 11, no. 23 (2005): 3498.

[66] A. K. M. Chow, L. Ng, C. S. C. Lam, et al., “The Enhanced Metastatic Potential of Hepatocellular Carcinoma (HCC) Cells With Sorafenib Resistance,” PLoS ONE 8, no. 11 (2013): e78675.

[67] H. Li, K. Xu, G. Pian, S. Sun, “Artesunate and Sorafenib: Combinatorial Inhibition of Liver Cancer Cell Growth,” Oncology Letters 18, no. 5 (2019): 4735-4743.

[68] C. D. Griffiths, B. Zhang, K. Tywonek, B. M. Meyers, P E. Serrano, “Toxicity Profiles of Systemic Therapies for Advanced Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis,” JAMA Network Open 5, no. 7 (2022): e2222721.

[69] J. J. Brown, B. Parashar, H. Moshage, et al., “A Long-Term Hepatitis B Viremia Model Generated by Transplanting Nontumorigenic Immortalized Human Hepatocytes in Rag-2-Deficient Mice,” Hepatology 31, no. 1 (2000): 173-181.

[70] S. Yuan, B. Zuo, S. C. Zhou, et al., “Integrating Network Pharmacology and Experimental Validation to Explore the Pharmacological Mechanism of Astragaloside IV in Treating Bleomycin-Induced Pulmonary Fibrosis,” Drug Design, Development and Therapy 17 (2023): 1289-1302.

[71] H. Deng, J. Jiang, S. Zhang, L. Wu, Q. Zhang, W. Sun, “Network Pharmacology and Experimental Validation to Identify the Potential Mechanism of Hedyotis diffusa Willd Against Rheumatoid Arthritis,” Scientific Reports 13, no. 1 (2023): 1-12.

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