Discovery of 4-Quinazolinone-Containing Phenylalanine Derivatives as Potent, Resistant-Tolerant HIV Capsid Inhibitors

Abstract

The HIV-1 capsid (CA) is a validated antiviral target that plays critical roles in both the early and late stages of the viral life cycle. Using structure-based strategy, we designed and synthesized a series of phenylalanine derivatives containing a 4-quinazolinone scaffold as novel HIV-1 CA inhibitors. Among them, IC-2i exhibited potent antiviral activity in MT-4 cells against HIV-1 NL4-3 (EC₅₀ = 0.65 ± 0.27 nM) and effectively protected cells from HIV-1 IIIB infection. SPR revealed that IC-2i interacts strongly with CA hexamers (K_D = 2.7 ± 0.5 nM) with an extended residence time and competes with host factors CPSF6 and NUP153, disrupting CA assembly and disassembly. IC-2i retained activity against lenacapavir (LEN)-resistant strains, such as N74D (11-fold shift vs. 20-fold for LEN). Crystallographic analysis revealed that IC-2i binds at the CA NTD–CTD interface and forms hydrogen bonds with Thr107 and Ser41 (NTD–NTD interface). Pharmacological evaluation demonstrated favorable properties, including good plasma stability, low toxicity (SI > 1571), and suitable pharmacokinetics with a prolonged half-life following subcutaneous administration (T_1/2 = 19.9 h). Overall, this study identifies 4-quinazolinone-based phenylalanine derivatives as promising HIV-1 CA inhibitors and highlights IC-2i as a potential long-acting therapeutic candidate for HIV-1 treatment.

Keywords

4-quinazolinone / drug design / HIV-1 capsid / long-acting drugs / phenylalanine derivatives

References

[1] P. J. McLaren and J. Fellay, “HIV-1 and human Genetic Variation,” Nature Reviews Genetics 22, no. 10 (2021): 645–657.

[2] V. Rojas-Celis, F. Valiente-Echeverría, R. Soto-Rifo, and D. Toro-Ascuy, “New Challenges of HIV-1 Infection: How HIV-1 Attacks and Resides in the central Nervous System,” Cells 8, no. 10 (2019): 1245.

[3] K. M. De Cock, H. W. Jaffe, and J. W. Curran, “Reflections on 40 Years of AIDS,” Emerging Infectious Diseases 27, no. 6 (2021): 1553–1560.

[4] WHO Data and statistics, https://www.unaids.org/en/resources/fact-sheet.

[5] N. L. Board, M. Moskovljevic, F. Wu, R. F. Siliciano, and J. D. Siliciano, “Engaging Innate Immunity in HIV-1 Cure Strategies,” Nature Reviews Immunology 22, no. 8 (2022): 499–512.

[6] L. Menéndez-Arias, S. Martín-Alonso, and E. Frutos-Beltrán, “An Update on Antiretroviral Therapy,” Advances in Experimental Medicine and Biology 1322 (2021): 31–61.

[7] L. Menéndez-Arias, “Molecular Basis of human Immunodeficiency Virus Type 1 Drug Resistance: Overview and Recent Developments,” Antiviral Research 98, no. 1 (2013): 93–120.

[8] J. Borrás-Blasco, A. Navarro-Ruiz, C. Borrás, and E. Casterá, “Adverse Cutaneous Reactions Associated With the Newest Antiretroviral Drugs in Patients With human Immunodeficiency Virus Infection,” Journal of Antimicrobial Chemotherapy 62, no. 5 (2008): 879–888.

[9] X. Tao, Y. Lu, Y. Zhou, L. Zhang, and Y. Chen, “Efficacy and Safety of the Regimens Containing Tenofovir Alafenamide versus Tenofovir Disoproxil Fumarate in Fixed-dose Single-tablet Regimens for Initial Treatment of HIV-1 Infection: A Meta-analysis of Randomized Controlled Trials,” International Journal of Infectious Diseases 93 (2020): 108–117.

[10] N. Apostolova, H. A. Funes, A. Blas-Garcia, M. J. Galindo, A. Alvarez, and J. V. Esplugues, “Efavirenz and the CNS: What We Already Know and Questions That Need to be Answered,” Journal of Antimicrobial Chemotherapy 70, no. 10 (2015): 2693–2708.

[11] X. Zhang, S. Xu, L. Sun, et al., “HIV-1 Capsid Inhibitors: A Sword to Destroy the Virus,” Future Medicinal Chemistry 14, no. 9 (2022): 605–607.

[12] W. M. McFadden, A. A. Snyder, K. A. Kirby, et al., “Rotten to the Core: Antivirals Targeting the HIV-1 Capsid Core,” Retrovirology 18, no. 1 (2021): 41.

[13] A. C. Francis and G. B. Melikyan, “Single HIV-1 Imaging Reveals Progression of Infection Through CA-dependent Steps of Docking at the Nuclear Pore, Uncoating, and Nuclear Transport,” Cell Host & Microbe 23, no. 4 (2018): 536–548. e6.

[14] T. G. Müller, V. Zila, K. Peters, et al., “HIV-1 Uncoating by Release of Viral cDNA From Capsid-Like Structures in the Nucleus of Infected Cells,” Elife 10 (2021): e64776.

[15] A. Selyutina, M. Persaud, K. Lee, V. KewalRamani, and F. Diaz-Griffero, “Nuclear Import of the HIV-1 Core Precedes Reverse Transcription and Uncoating,” Cell Reports 32, no. 13 (2020): 108201.

[16] A. Dharan, S. Opp, O. Abdel-Rahim, et al., “Bicaudal D2 Facilitates the Cytoplasmic Trafficking and Nuclear Import of HIV-1 Genomes During Infection,” Proceedings of the National Academy of Sciences of the United States of America 114, no. 50 (2017): E10707–E10716.

[17] S. K. Carnes, J. Zhou, and C. Aiken, “Hiv-1 Engages a Dynein-dynactin-BICD2 Complex for Infection and Transport to the Nucleus,” Journal of Virology 92, no. 20 (2018): e00358–18.

[18] J. Fernandez, D. M. Portilho, A. Danckaert, et al., “Microtubule-associated Proteins 1 (MAP1) Promote human Immunodeficiency Virus Type I (HIV-1) Intracytoplasmic Routing to the Nucleus,” Journal of Biological Chemistry 290, no. 8 (2015): 4631–4646.

[19] R. C. Burdick, C. Li, M. Munshi, et al., “HIV-1 Uncoats in the Nucleus near Sites of Integration,” Proceedings of the National Academy of Sciences of the United States of America 117, no. 10 (2020): 5486–5493.

[20] A. N. Engelman, “HIV Capsid and Integration Targeting,” Viruses 13, no. 1 (2021): 125.

[21] E. R. Wright, J. B. Schooler, H. J. Ding, et al., “Electron Cryotomography of Immature HIV-1 Virions Reveals the Structure of the CA and SP1 Gag Shells,” Embo Journal 26, no. 8 (2007): 2218–2226.

[22] P. Spearman, “HIV-1 Gag as an Antiviral Target: Development of Assembly and Maturation Inhibitors,” Current Topics in Medicinal Chemistry 16, no. 10 (2016): 1154–1166.

[23] F. K. Schur, W. J. Hagen, M. Rumlová, et al., “Structure of the Immature HIV-1 Capsid in Intact Virus Particles at 8.8 Å Resolution,” Nature 517, no. 7535 (2015): 505–508.

[24] P. W. Keller, C. S. Adamson, J. B. Heymann, E. O. Freed, and A. C. Steven, “HIV-1 Maturation Inhibitor Bevirimat Stabilizes the Immature Gag Lattice,” Journal of Virology 85, no. 4 (2011): 1420–1428.

[25] T. R. Gamble, F. F. Vajdos, S. Yoo, et al., “Crystal Structure of human Cyclophilin A Bound to the Amino-terminal Domain of HIV-1 Capsid,” Cell 87, no. 7 (1996): 1285–1294.

[26] T. Ni, S. Gerard, G. Zhao, et al., “Intrinsic Curvature of the HIV-1 CA Hexamer Underlies Capsid Topology and Interaction With Cyclophilin A,” Nature Structural & Molecular Biology 27, no. 9 (2020): 855–862.

[27] K. A. Matreyek, S. S. Yücel, X. Li, and A. Engelman, “Nucleoporin NUP153 Phenylalanine-glycine Motifs Engage a Common Binding Pocket Within the HIV-1 Capsid Protein to Mediate Lentiviral Infectivity,” Plos Pathogens 9, no. 10 (2013): e1003693.

[28] K. Bichel, A. J. Price, T. Schaller, G. J. Towers, S. M. Freund, and L. C. James, “HIV-1 Capsid Undergoes Coupled Binding and Isomerization by the Nuclear Pore Protein NUP358,” Retrovirology 10 (2013): 81.

[29] A. J. Price, A. J. Fletcher, T. Schaller, et al., “CPSF6 defines a Conserved Capsid Interface That Modulates HIV-1 Replication,” Plos Pathogens 8, no. 8 (2012): e1002896.

[30] L. Sun, X. Zhang, S. Xu, et al., “An Insight on Medicinal Aspects of Novel HIV-1 Capsid Protein Inhibitors,” European Journal of Medicinal Chemistry 217 (2021): 113380.

[31] A. T. Gres, K. A. Kirby, V. N. KewalRamani, J. J. Tanner, O. Pornillos, and S. G. Sarafianos, “Structural Virology. X-ray Crystal Structures of Native HIV-1 Capsid Protein Reveal Conformational Variability,” Science 349, no. 6243 (2015): 99–103.

[32] S. Mattei, B. Glass, W. J. Hagen, H. G. Kräusslich, and J. A. Briggs, “The Structure and Flexibility of Conical HIV-1 Capsids Determined Within Intact Virions,” Science 354, no. 6318 (2016): 1434–1437.

[33] A. S. Krebs, L. M. Mendonça, and P. Zhang, “Structural Analysis of Retrovirus Assembly and Maturation,” Viruses 14, no. 1 (2021): 54.

[34] W. S. Blair, C. Pickford, S. L. Irving, et al., “HIV Capsid Is a Tractable Target for Small Molecule Therapeutic Intervention,” Plos Pathogens 6, no. 12 (2010): e1001220.

[35] A. Saito, D. Ferhadian, G. A. Sowd, et al., “Roles of Capsid-interacting Host Factors in Multimodal Inhibition of HIV-1 by PF74,” Journal of Virology 90, no. 12 (2016): 5808–5823.

[36] S. V. Rebensburg, G. Wei, R. C. Larue, et al., “Sec24C is an HIV-1 Host Dependency Factor Crucial for Virus Replication,” Nature Microbiology 6, no. 4 (2021): 435–444.

[37] A. J. Price, D. A. Jacques, W. A. McEwan, et al., “Host Cofactors and Pharmacologic Ligands Share an Essential Interface in HIV-1 Capsid That Is Lost Upon Disassembly,” Plos Pathogens 10, no. 10 (2014): e1004459.

[38] S. R. Yant, A. Mulato, D. Hansen, et al., “A Highly Potent Long-acting Small-molecule HIV-1 Capsid Inhibitor With Efficacy in a Humanized Mouse Model,” Nature Medicine 25, no. 9 (2019): 1377–1384.

[39] J. O. Link, M. S. Rhee, W. C. Tse, et al., “Clinical Targeting of HIV Capsid Protein With a Long-acting Small Molecule,” Nature 584, no. 7822 (2020): 614–618.

[40] R. Begley, J. Lutz, and M. S. Rhee, “GS-6207 Sustained Delivery Formulation Supports 6-month Dosing Interval,” in presented at: 2020 International AIDS Conference, July 6–10 2020.

[41] H. Dvory-Sobol, N. Shaik, C. Callebaut, and M. S. Rhee, “Lenacapavir: A First-in-class HIV-1 Capsid Inhibitor,” Current Opinion in HIV and AIDS 17, no. 1 (2022): 15–21.

[42] S. Segal-Maurer, E. DeJesus, H. J. Stellbrink, et al., “Capsid Inhibition With lenacapavir in Multidrug-resistant HIV-1 Infection,” New England Journal of Medicine 386, no. 19 (2022): 1793–1803.

[43] T. L. Houston and B. Shi, inventors, “Gilead Sciences, Inc, USA. assignee. Preparation of Choline Salt Forms of a Cyclopropacyclopentapyrazolylacetamide Compound HIV Capsid Inhibitor,” “Patent WO2019035973A1. Patent Application Copyright (C) 2023 American Chemical Society (ACS). All Rights Reserved,” (2019).

[44] G. Alvarez, L. van Pul, X. Robert, et al., “Identification of 2-(4-N,N-Dimethylaminophenyl)-5-methyl-1-phenethyl-1H-benzimidazole Targeting HIV-1 CA Capsid Protein and Inhibiting HIV-1 Replication in Cellulo,” BMC Pharmacology and Toxicology 23, no. 1 (2022): 43.

[45] E. P. Gillis, K. Parcella, M. Bowsher, et al., “Potent Long-acting Inhibitors Targeting the HIV-1 Capsid Based on a Versatile Quinazolin-4-one Scaffold,” Journal of Medicinal Chemistry 66, no. 3 (2023): 1941–1954.

[46] M. S. Bowsher, E. P. Gillis, C. Iwuagwu, K. E. Parcella, M. Patel, and A. X. Wang, investors, “ViiV Healthcare UK (No.5) Limited, Assignee. Indazolylquinazolinones as Inhibitors of human Immunodeficiency Virus Replication and Their Preparation. Patent WO2020084491,” “Patent Application Copyright © 2023 American Chemical Society (ACS). All Rights Reserved,” (2020).

[47] L. Sun, A. Dick, M. E. Meuser, et al., “Design, Synthesis, and Mechanism Study of Benzenesulfonamide-containing Phenylalanine Derivatives as Novel HIV-1 Capsid Inhibitors With Improved Antiviral Activities,” Journal of Medicinal Chemistry 63, no. 9 (2020): 4790–4810.

[48] X. Zhang, L. Sun, and S. Xu, “Design, Synthesis, and Mechanistic Study of 2-piperazineone-bearing Peptidomimetics as Novel HIV Capsid modulators,” RSC Medicinal Chemistry 14 (2023), 1272–1295, https://doi.org/10.1039/D3MD00134B.

[49] S. M. Bester, D. Adu-Ampratwum, A. S. Annamalai, et al., “Structural and Mechanistic Bases of Viral Resistance to HIV-1 Capsid Inhibitor Lenacapavir,” MBio 13, no. 5 (2022): e0180422.

[50] C. Callebaut, L. VanderVeen, N. Margot, V. Naik, and M. Rhee, “Activity and Resistance Characterization of the HIV Capsid Inhibitor lenacapavir,” in presented at: 2021 Conference on Retroviruses and Opportunistic Infections, Boston, March 6–10 2021.

[51] HIV Drug Resistance Database. https://hivdb.stanford.edu/.

[52] T. Hisano, “Recent Studies on the Modified Niementowski 4-Quinazolone Synthesis. A Review,” Organic Preparations and Procedures International 5, no. 4 (1973): 145–193.

[53] F. Mustafa and H. L. Robinson, “Context-dependent Role of human Immunodeficiency Virus Type 1 Auxiliary Genes in the Establishment of Chronic Virus Producers,” Journal of Virology 67, no. 11 (1993): 6909–6915.

[54] G. Xiong, Z. Wu, J. Yi, et al., “ADMETlab 2.0: An Integrated Online Platform for Accurate and Comprehensive Predictions of ADMET Properties,” Nucleic Acids Research 49, no. W1 (2021): W5–W14.

[55] R. I. Connor, B. K. Chen, S. Choe, and N. R. Landau, “Vpr Is Required for Efficient Replication of human Immunodeficiency Virus Type-1 in Mononuclear Phagocytes,” Virology 206, no. 2 (1995): 935–944.

[56] S. Hoffenberg, R. Powell, A. Carpov, et al., “Identification of an HIV-1 Clade A Envelope That Exhibits Broad Antigenicity and Neutralization Sensitivity and Elicits Antibodies Targeting Three Distinct Epitopes,” Journal of Virology 87, no. 10 (2013): 5372–5383.

[57] R. Hadravová, M. Rumlová, and T. Ruml, “FAITH—fast Assembly Inhibitor Test for HIV,” Virology 486 (2015): 78–87.

[58] A. Dostalkova, R. Hadravova, F. Kaufman, et al., “A Simple, High-throughput Stabilization Assay to Test HIV-1 Uncoating Inhibitors,” Scientific Reports 9, no. 1 (2019): 17076.

[59] A. Dick, M. E. Meuser, and S. Cocklin, “Clade-Specific Alterations Within the HIV-1 Capsid Protein With Implications for Nuclear Translocation,” Biomolecules 12, no. 5 (2022): 695.

[60] S. Yoo, D. G. Myszka, C. Yeh, M. McMurray, C. P. Hill, and W. I. Sundquist, “Molecular Recognition in the HIV-1 Capsid/Cyclophilin A Complex,” Journal of Molecular Biology 269, no. 5 (1997): 780–795.

[61] D. Miranda and D. J. Sanchez, “Lenacapavir's Success: Revitalizing Antiviral Drug Discovery,” Virulence 16, no. 1 (2025): 2497902.

[62] J. K. Sodhi and L. Z. Benet, “Successful and Unsuccessful Prediction of Human Hepatic Clearance for Lead Optimization,” Journal of Medicinal Chemistry 64, no. 7 (2021): 3546–3559.

[63] Y. Song, C. Li, G. Liu, et al., “Drug-Metabolizing Cytochrome P450 Enzymes Have Multifarious Influences on Treatment Outcomes,” Clinical Pharmacokinetics 60, no. 5 (2021): 585–601.

[64] G. Wei, N. Iqbal, V. V. Courouble, et al., “Prion-Like Low Complexity Regions Enable Avid Virus-host Interactions During HIV-1 Infection,” Nature Communications 13, no. 1 (2022): 5879.

[65] C. Pannecouque, D. Daelemans, and E. De Clercq, “Tetrazolium-based Colorimetric Assay for the Detection of HIV Replication Inhibitors: Revisited 20 Years Later,” Nature Protocols 3, no. 3 (2008): 427–434.

[66] A. Björndal, H. Deng, M. Jansson, et al., “Coreceptor Usage of Primary human Immunodeficiency Virus Type 1 Isolates Varies According to Biological Phenotype,” Journal of Virology 71, no. 10 (1997): 7478–7487.

[67] I. Zentner, L. J. Sierra, A. K. Fraser, et al., “Identification of a Small-molecule Inhibitor of HIV-1 Assembly That Targets the Phosphatidylinositol (4,5)-bisphosphate Binding Site of the HIV-1 Matrix Protein,” Chemmedchem 8, no. 3 (2013): 426–432.

[68] S. Kortagere, N. Madani, M. K. Mankowski, et al., “Inhibiting Early-stage Events in HIV-1 Replication by Small-molecule Targeting of the HIV-1 Capsid,” Journal of Virology 86, no. 16 (2012): 8472–8481.

[69] F. W. Studier, “Protein Production by Auto-induction in High Density Shaking Cultures,” Protein Expression Purification 41, no. 1 (2005): 207–234.

[70] O. Pornillos, B. K. Ganser-Pornillos, S. Banumathi, Y. Hua, and M. Yeager, “Disulfide Bond Stabilization of the Hexameric Capsomer of human Immunodeficiency Virus,” Journal of Molecular Biology 401, no. 5 (2010): 985–995.

[71] S. Cocklin, H. Gopi, B. Querido, et al., “Broad-spectrum Anti-human Immunodeficiency Virus (HIV) Potential of a Peptide HIV Type 1 Entry Inhibitor,” Journal of Virology 81, no. 7 (2007): 3645–3648.

[72] J. He, S. Choe, R. Walker, P. Di Marzio, D. O. Morgan, and N. R. Landau, “Human Immunodeficiency Virus Type 1 Viral Protein R (Vpr) Arrests Cells in the G2 Phase of the Cell Cycle by Inhibiting p34cdc2 Activity,” Journal of Virology 69, no. 11 (1995): 6705–6711.

[73] I. Gross, H. Hohenberg, T. Wilk, et al., “A Conformational Switch Controlling HIV-1 Morphogenesis,” Embo Journal 19, no. 1 (2000): 103–113.

[74] I. Gross, H. Hohenberg, and H. G. Kräusslich, “In Vitro Assembly Properties of Purified Bacterially Expressed Capsid Proteins of human Immunodeficiency Virus,” European Journal of Biochemistry 249, no. 2 (1997): 592–600.

[75] L. S. Ehrlich, B. E. Agresta, and C. A. Carter, “Assembly of Recombinant human Immunodeficiency Virus Type 1 Capsid Protein in Vitro,” Journal of Virology 66, no. 8 (1992): 4874–4883.

[76] U. K. von Schwedler, T. L. Stemmler, V. Y. Klishko, et al., “Proteolytic Refolding of the HIV-1 Capsid Protein Amino-terminus Facilitates Viral Core Assembly,” Embo Journal 17, no. 6 (1998): 1555–1568.

[77] J. Lanman, J. Sexton, M. Sakalian, and P. E. Prevelige,, “Kinetic Analysis of the Role of Intersubunit Interactions in human Immunodeficiency Virus Type 1 Capsid Protein Assembly in Vitro,” Journal of Virology 76, no. 14 (2002): 6900–6908.

[78] S. Kortagere, J. P. Xu, M. K. Mankowski, R. G. Ptak, and S. Cocklin, “Structure-activity Relationships of a Novel Capsid Targeted Inhibitor of HIV-1 Replication,” Journal of Chemical Information and Modeling 54, no. 11 (2014): 3080–3090.

[79] S. Xu, L. Sun, M. Barnett, et al., “Discovery, Crystallographic Studies, and Mechanistic Investigations of Novel Phenylalanine Derivatives Bearing a Quinazolin-4-one Scaffold as Potent HIV Capsid Modulators,” Journal of Medicinal Chemistry 66, no. 23 (2023): 16303–16329.

[80] A. J. McCoy, “Solving Structures of Protein Complexes by Molecular Replacement With Phaser,” Acta Crystallographica Section D Biological Crystallography 63, no. Pt 1 (2007): 32–41.

[81] G. N. Murshudov, A. A. Vagin, and E. J. Dodson, “Refinement of Macromolecular Structures by the Maximum-likelihood Method,” Acta Crystallographica Section D Biological Crystallography 53, no. Pt 3 (1997): 240–255.

[82] P. Emsley and K. Cowtan, “Coot: Model-building Tools for Molecular Graphics,” Acta Crystallographica Section D Biological Crystallography 60, no. Pt 12 Pt 1 (2004): 2126–2132.

[83] N. W. Moriarty, R. W. Grosse-Kunstleve, and P. D. Adams, “electronic Ligand Builder and Optimization Workbench (eLBOW): A Tool for Ligand Coordinate and Restraint Generation,” Acta Crystallographica Section D Biological Crystallography 65, no. Pt 10 (2009): 1074–1080.

[84] S. J. Cottrell, T. S. Olsson, R. Taylor, J. C. Cole, and J. W. Liebeschuetz, “Validating and Understanding Ring Conformations Using Small Molecule Crystallographic Data,” Journal of Chemical Information and Modeling 52, no. 4 (2012): 956–962.

RIGHTS & PERMISSIONS

2026 The Author(s). MedComm published by Sichuan International Medical Exchange & Promotion Association (SCIMEA) and John Wiley & Sons Australia, Ltd.