Identification of Novel HPK1 Hit Inhibitors: From In Silico Design to In Vitro Validation
Abstract
1. Introduction
2. Results and Discussion
2.1. Structure-Based Virtual Screening
2.1.1. Rationale of Structure and Database Selection
2.1.2. Filtration by Lipinski’s Rule
2.1.3. Validation of Docking Methods
2.1.4. Glide Docking
2.2. Induced Fit Docking and Final Selection of Hits
2.3. Substructure Search
2.4. In Vitro Kinase Inhibition Assay Results
2.5. Analysis of Screening Results
2.6. Molecular Dynamics Simulation and Analysis
2.7. Analyzing Pharmaceutically Relevant Predicted Properties
2.8. A Posteriori Validation of Ligand Binding Mode
2.9. Limitations and Future Work
3. Materials and Methods
3.1. Preparation of Ligands
3.2. Preparation of Protein
3.3. Grid Generation
3.4. Structure-Based Virtual Screening Workflow
3.5. Induced Fit Docking Study
3.6. In Vitro Kinase Inhibition Assay
3.7. Molecular Dynamics Simulations
3.8. Physicochemical Properties Analysis
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
Abbreviations
Abbreviation | Definition |
Asp | Aspartic acid |
ATP | Adenosine Triphosphate |
BLNK | B-cell Linker Protein |
CTLA4 | Cytotoxic T-Lymphocyte-Associated protein 4 |
Cys | Cystine |
DMSO | Dimethylsulfoxide |
DS | Discovery Studio |
DTT | dithiothreitol |
EGTA | Ethylene Glycol-bis(β-aminoethyl ether)-N,N,N’,N’-Tetraacetic Acid |
Glu | Glutamic acid |
Gly | Glycine |
HPK1 | Hematopoietic Progenitor Kinase 1 |
HTS | High Throughput Screening |
HTVS | High Throughput Virtual Screening |
IC50 | Half-maximal inhibitory concentration |
IFD | Induced Fit Docking |
IL-2 | Interleukin-2 |
Leu | Leucine |
MAP4K1 | Mitogen-Activated Protein Kinase Kinase Kinase Kinase 1 |
MD | Molecular Dynamics |
Met | Methionine |
PD1 | Programmed Cell Death Protein 1 |
PDB | Protein Data Base |
PGE2 | Prostaglandin E2 |
Phe | Phenylalanine |
RMSD | Root Mean Square Deviation |
SAR | Structure-Activity Relationship |
SBVS | Structure-based Virtual Screening |
SLP76 | SH2 domain containing Leukocyte Protein of 76kDa |
SP | Standard Precision |
TIP3P | Transferable Intermolecular Potential With 3 Points |
Tyr | Tyrosine |
Val | Valine |
VMD | Visual Molecular Dynamic |
XP | Extra Precision |
Appendix A
References
- Farkona, S.; Diamandis, E.P.; Blasutig, I.M. Cancer Immunotherapy: The Beginning of the End of Cancer? BMC Med. 2016, 14, 73. [Google Scholar] [CrossRef] [PubMed]
- Kciuk, M.; Yahya, E.B.; Mohamed, M.M.I.; Rashid, S.; Iqbal, M.O.; Kontek, R.; Abdulsamad, M.A.; Allaq, A.A. Recent Advances in Molecular Mechanisms of Cancer Immunotherapy. Cancers 2023, 15, 2721. [Google Scholar] [CrossRef]
- Yang, L.; Ning, Q.; Tang, S.S. Recent Advances and Next Breakthrough in Immunotherapy for Cancer Treatment. J. Immunol. Res. 2022, 2022, 8052212. [Google Scholar] [CrossRef] [PubMed]
- Ribas, A.; Wolchok, J.D. Cancer Immunotherapy Using Checkpoint Blockade. Science 2018, 359, 1350–1355. [Google Scholar] [CrossRef]
- Colucci, M.; D’Alonzo, V.; Santangelo, F.; Miracco, C.; Valente, M.; Maio, M.; Di Giacomo, A.M. Successful Targeting of CTLA-4 in a Melanoma Clinical Case: A Long-Term “One Stop Therapeutic Shop”. OncoTargets Ther. 2022, 15, 1409–1415. [Google Scholar] [CrossRef] [PubMed]
- Davies, M.J. PD-1/PD-L1 Inhibitors for Non-Small Cell Lung Cancer: Incorporating Care Step Pathways for Effective Side-Effect Management. J. Adv. Pract. Oncol. 2019, 10, 21–35. [Google Scholar] [CrossRef]
- Hou, K.; Xu, X.; Ge, X.; Jiang, J.; Ouyang, F. Blockade of PD-1 and CTLA-4: A Potent Immunotherapeutic Approach for Hepatocellular Carcinoma. Biofactors 2024, 50, 250–265. [Google Scholar] [CrossRef]
- Huo, G.; Liu, W.; Chen, P. Efficacy of PD-1/PD-L1 Inhibitors in Gastric or Gastro-Oesophageal Junction Cancer Based on Clinical Characteristics: A Meta-Analysis. BMC Cancer 2023, 23, 143. [Google Scholar] [CrossRef]
- Li, Q.; Han, J.; Yang, Y.; Chen, Y. PD-1/PD-L1 Checkpoint Inhibitors in Advanced Hepatocellular Carcinoma Immunotherapy. Front. Immunol. 2022, 13, 1070961. [Google Scholar] [CrossRef]
- Fujiwara, Y.; Mittra, A.; Naqash, A.R.; Takebe, N. A Review of Mechanisms of Resistance to Immune Checkpoint Inhibitors and Potential Strategies for Therapy. Cancer Drug Resist. 2020, 3, 252–275. [Google Scholar] [CrossRef]
- Kawashima, S.; Togashi, Y. Resistance to Immune Checkpoint Inhibitors and the Tumor Microenvironment. Exp. Dermatol. 2023, 32, 240–249. [Google Scholar] [CrossRef] [PubMed]
- O’Donnell, J.S.; Long, G.V.; Scolyer, R.A.; Teng, M.W.L.; Smyth, M.J. Resistance to PD1/PDL1 Checkpoint Inhibition. Cancer Treat. Rev. 2017, 52, 71–81. [Google Scholar] [CrossRef]
- Shergold, A.L.; Millar, R.; Nibbs, R.J.B. Understanding and Overcoming the Resistance of Cancer to PD-1/PD-L1 Blockade. Pharmacol. Res. 2019, 145, 104258. [Google Scholar] [CrossRef]
- Sun, Y. Tumor Microenvironment and Cancer Therapy Resistance. Cancer Lett. 2016, 380, 205–215. [Google Scholar] [CrossRef] [PubMed]
- Wang, Q.; Wu, X. Primary and Acquired Resistance to PD-1/PDL1 Blockade in Cancer Treatment. Int. Immunopharmacol. 2017, 46, 210–219. [Google Scholar] [CrossRef] [PubMed]
- Runde, A.P.; Mack, R.; SJ, P.B.; Zhang, J. The Role of TBK1 in Cancer Pathogenesis and Anticancer Immunity. J. Exp. Clin. Cancer Res. 2022, 41, 135. [Google Scholar] [CrossRef] [PubMed]
- Kwantwi, L.B.; Tandoh, T. Focal Adhesion Kinase-Mediated Interaction between Tumor and Immune Cells in the Tumor Microenvironment: Implications for Cancer-Associated Therapies and Tumor Progression. Clin. Transl. Oncol. 2024, 26, 879–892. [Google Scholar] [CrossRef]
- Zhang, Z.; Bu, L.; Luo, J.; Guo, J. Targeting Protein Kinases Benefits Cancer Immunotherapy. Biochim. Biophys. Acta Rev. Cancer 2022, 1877, 188738. [Google Scholar] [CrossRef]
- Zhu, Q.; Chen, N.; Tian, X.; Zhou, Y.; You, Q.; Xu, X. Hematopoietic Progenitor Kinase 1 in Tumor Immunology: A Medicinal Chemistry Perspective. J. Med. Chem. 2022, 65, 8065–8090. [Google Scholar] [CrossRef]
- Sawasdikosol, S.; Burakoff, S. The Structure of HPK1 Kinase Domain: To Boldly Go Where No Immuno-Oncology Drugs Have Gone Before. Structure 2019, 27, 1–3. [Google Scholar] [CrossRef]
- Sawasdikosol, S.; Burakoff, S. A Perspective on HPK1 as a Novel Immuno-Oncology Drug Target. Elife 2020, 9, e55122. [Google Scholar] [CrossRef]
- Wang, Y.; Zhang, K.; Georgiev, P.; Wells, S.; Xu, H.; Lacey, B.M.; Xu, Z.; Laskey, J.; McLeod, R.; Methot, J.L.; et al. Pharmacological Inhibition of Hematopoietic Progenitor Kinase 1 Positively Regulates T-Cell Function. PLoS ONE 2020, 15, e0243145. [Google Scholar] [CrossRef] [PubMed]
- You, D.; Hillerman, S.; Locke, G.; Chaudhry, C.; Stromko, C.; Murtaza, A.; Fan, Y.; Koenitzer, J.; Chen, Y.; Briceno, S.; et al. Enhanced Antitumor Immunity by a Novel Small Molecule HPK1 Inhibitor. J. ImmunoTher. Cancer 2021, 9, e001402. [Google Scholar] [CrossRef] [PubMed]
- Hernandez, S.; Qing, J.; Thibodeau, R.H.; Du, X.; Park, S.; Lee, H.M.; Xu, M.; Oh, S.; Navarro, A.; Roose-Girma, M.; et al. The Kinase Activity of Hematopoietic Progenitor Kinase 1 Is Essential for the Regulation of T Cell Function. Cell Rep. 2018, 25, 80–94. [Google Scholar] [CrossRef] [PubMed]
- Sawasdikosol, S.; Zha, R.; Fisher, T.S.; Alzabin, S.; Burakoff, S.J. HPK1 Influences Regulatory T Cell Functions. Immunohorizons 2020, 4, 382–391. [Google Scholar] [CrossRef]
- Shui, J.W.; Boomer, J.S.; Han, J.; Xu, J.; Dement, G.A.; Zhou, G.; Tan, T.H. Hematopoietic Progenitor Kinase 1 Negatively Regulates T Cell Receptor Signaling and T Cell-Mediated Immune Responses. Nat. Immunol. 2007, 8, 84–91. [Google Scholar] [CrossRef]
- Chen, H.; Guan, X.; He, C.; Lu, T.; Lin, X.; Liao, X. Current Strategies for Targeting HPK1 in Cancer and the Barriers to Preclinical Progress. Expert Opin. Ther. Targets 2024, 28, 237–250. [Google Scholar] [CrossRef]
- Xu, J.; Li, Y.; Chen, X.; Yang, J.; Xia, H.; Huang, W.; Zeng, S. Opportunities and Challenges for Targeting HPK1 in Cancer Immunotherapy. Bioorg. Chem. 2024, 153, 107866. [Google Scholar] [CrossRef]
- Zhou, L.; Wang, T.; Zhang, K.; Zhang, X.; Jiang, S. The Development of Small-Molecule Inhibitors Targeting HPK1. Eur. J. Med. Chem. 2022, 244, 114819. [Google Scholar] [CrossRef]
- Linney, I.D.; Kaila, N. Inhibitors of Immuno-Oncology Target HPK1—A Patent Review (2016 to 2020). Expert Opin. Ther. Pat. 2021, 31, 893–910. [Google Scholar] [CrossRef]
- Chan, B.K.; Seward, E.; Lainchbury, M.; Brewer, T.F.; An, L.; Blench, T.; Cartwright, M.W.; Chan, G.K.Y.; Choo, E.F.; Drummond, J.; et al. Discovery of Spiro-Azaindoline Inhibitors of Hematopoietic Progenitor Kinase 1 (HPK1). ACS Med. Chem. Lett. 2022, 13, 84–91. [Google Scholar] [CrossRef]
- Guimarães, C.R.W.; Rai, B.K.; Munchhof, M.J.; Liu, S.; Wang, J.; Bhattacharya, S.K.; Buckbinder, L. Understanding the Impact of the P-Loop Conformation on Kinase Selectivity. J. Chem. Inf. Model. 2011, 51, 1199–1204. [Google Scholar] [CrossRef] [PubMed]
- Bhullar, K.S.; Lagarón, N.O.; McGowan, E.M.; Parmar, I.; Jha, A.; Hubbard, B.P.; Rupasinghe, H.P.V. Kinase-Targeted Cancer Therapies: Progress, Challenges and Future Directions. Mol. Cancer 2018, 17, 48. [Google Scholar] [CrossRef] [PubMed]
- Fabbro, D.; Cowan-Jacob, S.W.; Moebitz, H. Ten Things You Should Know About Protein Kinases: IUPHAR Review 14. Br. J. Pharmacol. 2015, 172, 2675–2700. [Google Scholar] [CrossRef]
- Zhang, M.; Liu, Y.; Jang, H.; Nussinov, R. Strategy Toward Kinase-Selective Drug Discovery. J. Chem. Theory Comput. 2023, 19, 1615–1628. [Google Scholar] [CrossRef] [PubMed]
- Zhu, H.; Zhang, Y.; Li, W.; Huang, N. A Comprehensive Survey of Prospective Structure-Based Virtual Screening for Early Drug Discovery in the Past Fifteen Years. Int. J. Mol. Sci. 2022, 23, 15961. [Google Scholar] [CrossRef]
- Nunes da Rocha, M.; de Sousa, D.S.; da Silva Mendes, F.R.; dos Santos, H.S.; Marinho, G.S.; Marinho, M.M.; Marinho, E.S. Ligand and Structure-Based Virtual Screening Approaches in Drug Discovery: Minireview. Mol. Divers. 2024, 28, 1172–1185. [Google Scholar] [CrossRef]
- Ge, H.; Peng, L.; Sun, Z.; Liu, H.; Shen, Y.; Yao, X. Discovery of Novel HPK1 Inhibitors Through Structure-Based Virtual Screening. Front. Pharmacol. 2022, 13, 850855. [Google Scholar] [CrossRef]
- Lipinski, C.A. Lead- and Drug-Like Compounds: The Rule-of-Five Revolution. Drug Discov. Today Technol. 2004, 1, 337–341. [Google Scholar] [CrossRef]
- Friesner, R.A.; Murphy, R.B.; Repasky, M.P.; Frye, L.L.; Greenwood, J.R.; Halgren, T.A.; Sanschagrin, P.C.; Mainz, D.T. Extra Precision Glide: Docking and Scoring Incorporating a Model of Hydrophobic Enclosure for Protein-Ligand Complexes. J. Med. Chem. 2006, 49, 6177–6196. [Google Scholar] [CrossRef]
- Sindikara, D.; Miller, E.B.; Murphy, R.B.; Borrelli, K.W.; Grisewood, M.J.; Ranalli, F.; Friesner, R.A. A Reliable and Accurate Solution to the Induced Fit Docking Problem for Protein-Ligand Binding. J. Chem. Theory Comput. 2021, 17, 2630–2639. [Google Scholar] [CrossRef]
- Loch, C.M.; Liang, S.; Wu, J.; Eason, M.; Goupil, A.; Acinapura, A.; Purcell, J.; Dominguez, M. Abstract 938: Screening the Entire Kinase-Directed, FDA-Approved Pharmacopeia Against the Largest Collection of Wild-Type and Mutant Kinases Reveals Many Opportunities for Drug Repurposing and Targeted Therapy. Cancer Res. 2024, 84 (Suppl. S6), 938. [Google Scholar] [CrossRef]
- Peng, J.; Ding, X.; Chen, C.X.; Shih, P.Y.; Meng, Q.; Ding, X.; Zhang, M.; Aliper, A.; Ren, F.; Lu, H.; et al. Design, Synthesis, and Biological Evaluation of a Series of Spiro Analogues as Novel HPK1 Inhibitors. ACS Med. Chem. Lett. 2024, 15, 1234–1245. [Google Scholar] [CrossRef]
- Peng, J.; Ding, X.; Chen, C.X.; Zhao, P.; Ding, X.; Zhang, M.; Aliper, A.; Ren, F.; Lu, H.; Zhavoronkov, A. Discovery of Pyridine-2-Carboxamides Derivatives as Potent and Selective HPK1 Inhibitors for the Treatment of Cancer. J. Med. Chem. 2024, 67, 2760–2775. [Google Scholar] [CrossRef]
- Vara, B.A.; Levi, S.M.; Achab, A.; Candito, D.A.; Fradera, X.; Lesburg, C.A.; Kawamura, S.; Lacey, B.M.; Lim, J.; Methot, J.L.; et al. Discovery of Diaminopyrimidine Carboxamide HPK1 Inhibitors as Preclinical Immunotherapy Tool Compounds. ACS Med. Chem. Lett. 2021, 12, 653–661. [Google Scholar] [CrossRef]
- Asinex Corp. Asinex Database: All Screening Compounds. 2024. Available online: https://www.asinex.com/screening-libraries-(all-libraries) (accessed on 26 January 2024).
- Otava Chemicals. Compound Libraries for HTS: Lead-Like Library. 2024. Available online: https://www.otavachemicals.com/products/compound-libraries-for-hts/lead-like-library (accessed on 26 January 2024).
- Schrödinger, LLC. LigPrep, Version 2024-3; Schrödinger, LLC: New York, NY, USA, 2024; Available online: https://www.schrodinger.com (accessed on 31 August 2024).
- Schrödinger, LLC. Epik, Version 2024-3; Schrödinger, LLC: New York, NY, USA, 2024; Available online: https://www.schrodinger.com (accessed on 31 August 2024).
- Madhavi Sastry, G.; Adzhigirey, M.; Day, T.; Annabhimoju, R.; Sherman, W. Protein and Ligand Preparation: Parameters, Protocols, and Influence on Virtual Screening Enrichments. J. Comput.-Aided Mol. Des. 2013, 27, 221–234. [Google Scholar] [CrossRef] [PubMed]
- Olsson, M.H.M.; Søndergaard, C.R.; Rostkowski, M.; Jensen, J.H. PROPKA3: Consistent Treatment of Internal and Surface Residues in Empirical pKa Predictions. J. Chem. Theory Comput. 2011, 7, 525–537. [Google Scholar] [CrossRef]
- Friesner, R.A.; Banks, J.L.; Murphy, R.B.; Halgren, T.A.; Klicic, J.J.; Mainz, D.T.; Repasky, M.P.; Knoll, E.H.; Shelley, M.; Perry, J.K.; et al. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 1. Method and Assessment of Docking Accuracy. J. Med. Chem. 2004, 47, 1739–1749. [Google Scholar] [CrossRef]
- Halgren, T.A.; Murphy, R.B.; Friesner, R.A.; Beard, H.S.; Frye, L.L.; Pollard, W.T.; Banks, J.L. Glide: A New Approach for Rapid, Accurate Docking and Scoring. 2. Enrichment Factors in Database Screening. J. Med. Chem. 2004, 47, 1750–1759. [Google Scholar] [CrossRef]
- Sherman, W.; Day, T.; Jacobson, M.P.; Friesner, R.A.; Farid, R. Novel Procedure for Modeling Ligand/Receptor Induced Fit Effects. J. Med. Chem. 2006, 49, 534–553. [Google Scholar] [CrossRef]
- Schrödinger, LLC. Prime, Version 2024-3; Schrödinger, LLC: New York, NY, USA, 2024; Available online: https://www.schrodinger.com (accessed on 31 August 2024).
- Jo, S.; Kim, T.; Iyer, V.G.; Im, W. CHARMM-GUI: A Web-Based Graphical User Interface for CHARMM. J. Comput. Chem. 2008, 29, 1859–1865. [Google Scholar] [CrossRef] [PubMed]
- Case, D.A.; Cheatham, T.E., III; Darden, T.; Gohlke, H.; Luo, R.; Merz, K.M., Jr.; Onufriev, A.; Simmerling, C.; Wang, B.; Woods, R. The Amber Biomolecular Simulation Programs. J. Comput. Chem. 2005, 26, 1668–1688. [Google Scholar] [CrossRef] [PubMed]
- Humphrey, W.; Dalke, A.; Schulten, K. VMD—Visual Molecular Dynamics. J. Mol. Graph. 1996, 14, 33–38. [Google Scholar] [CrossRef] [PubMed]
- Schrödinger, LLC. QikProp, Version 2024-3; Schrödinger, LLC: New York, NY, USA, 2024; Available online: https://www.schrodinger.com (accessed on 31 August 2024).
ID | Chemical Structure | Glide XP Docking Score (kcal/mol) | Induced Fit Docking Score (kcal/mol) | % Inhibition at 25 µM | % Inhibition at 50 µM | IC50 (µM) | Tanimoto Similarity Score b Relative to GEN-8 |
---|---|---|---|---|---|---|---|
ISR-03 | −9.70 | −11.26 | 26.64 | 52.39 | 43.9 | 0.016 | |
ISR-04 | −8.44 | −10.87 | 2.44 | 10.06 | >100 | 0.018 | |
ISR-05 a | ND | −10.47 | 0 | 13.92 | 24.2 | 0.019 |
ID | QP log S a | QP log BB b | QP P Caco c | QPP MDCK d | Percent Human Oral Absorption e | QP log Khsa f |
---|---|---|---|---|---|---|
ISR-03 | −4.685 | −0.975 | 656.244 | 313.776 | 95.024 | 0.201 |
ISR-05 | −4.567 | −1.451 | 150.376 | 91.940 | 77.169 | −0.051 |
GEN-8 | −4.955 | −1.235 | 210.370 | 164.248 | 84.130 | 0.290 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Isawi, I.H.; Obeidat, R.M.; Alnabulsi, S.; Al Zoubi, R. Identification of Novel HPK1 Hit Inhibitors: From In Silico Design to In Vitro Validation. Int. J. Mol. Sci. 2025, 26, 4366. https://doi.org/10.3390/ijms26094366
Isawi IH, Obeidat RM, Alnabulsi S, Al Zoubi R. Identification of Novel HPK1 Hit Inhibitors: From In Silico Design to In Vitro Validation. International Journal of Molecular Sciences. 2025; 26(9):4366. https://doi.org/10.3390/ijms26094366
Chicago/Turabian StyleIsawi, Israa H., Rayan M. Obeidat, Soraya Alnabulsi, and Rufaida Al Zoubi. 2025. "Identification of Novel HPK1 Hit Inhibitors: From In Silico Design to In Vitro Validation" International Journal of Molecular Sciences 26, no. 9: 4366. https://doi.org/10.3390/ijms26094366
APA StyleIsawi, I. H., Obeidat, R. M., Alnabulsi, S., & Al Zoubi, R. (2025). Identification of Novel HPK1 Hit Inhibitors: From In Silico Design to In Vitro Validation. International Journal of Molecular Sciences, 26(9), 4366. https://doi.org/10.3390/ijms26094366