CC chemokine receptor (CCR) 2 and 5 are G protein-coupled receptors that play a crucial role in immunohomeostasis. Accordingly, overactivation of their signaling pathways is involved in various immunopathologies and cancer. Extensive research focusing on discovering CCR2 and CCR5 orthosteric antagonists, ultimately resulted in some clinical success, but the area of intracellular allosteric modulators is still underexplored and the move from orthosteric to allosteric modulation could be an interesting paradigm shift. To this end, we document the development of novel CCR2 and CCR5 intracellular allosteric antagonists through a virtual screen on a small combinatorial library derived from existing CCR2, CCR5, and CCR4 ligands. Using a molecular docking approach, the created library was screened in its entirety utilizing a refined AlphaFold model of CCR5 based on the crystal structure of its close homologue, CCR2. The screening resulted in the identification of several virtual hits, out of which one was developed further by in-house synthesis. In total, 18 analogues were prepared and experimentally evaluated for their binding affinity for CCR2 and functional inhibition on CCR5. This expeditious and simple workflow beginning from docking to compound evaluation identified 3 hits for CCR2 (Ki = 1.3-6 μM) and 1 hit (IC50 = 10.8 μM) for CCR5. The obtained structure-activity relationships were also further rationalized using structural information available for both CCR5 and CCR2 providing valuable insights for future development of intracellular allosteric ligands.

CZ OPENSCREEN National Infrastructure for Chemical Biology Department of Informatics and Chemistry Faculty of Chemical Technology University of Chemistry and Technology Prague Technická 5 166 28 Prague Czech Republic

Department of Medicinal Chemistry Photopharmacology and Imaging Groningen Research Institute of Pharmacy Faculty of Science and Engineering Antonius Deusinglaan 1 9713 AV Groningen The Netherlands

Division of Medicinal Chemistry Leiden Academic Centre for Drug Research Leiden University 2333 CC Leiden The Netherlands

Oncode Institute 2333 CC Leiden The Netherlands

Zobrazit více v PubMed

Esche C., Stellato C., Beck L. A.. Chemokines: Key Players in Innate and Adaptive Immunity. Journal of Investigative Dermatology. 2005;125(4):615–628. doi: 10.1111/j.0022-202X.2005.23841.x. PubMed DOI

Hughes C. E., Nibbs R. J. B.. A Guide to Chemokines and Their Receptors. FEBS J. 2018;285(16):2944–2971. doi: 10.1111/febs.14466. PubMed DOI PMC

Junker A., Kokornaczyk A. K., Strunz A. K., Wunsch B.. Selective and dual targeting of CCR2 and CCR5 receptors: A current overview. Topics in Medicinal Chemistry. 2014;14:187–241. doi: 10.1007/7355_2014_40. DOI

Connor S. J., Paraskevopoulos N., Newman R., Cuan N., Hampartzoumian T., Lloyd A. R., Grimm M. C.. CCR2 Expressing CD4+ T Lymphocytes Are Preferentially Recruited to the Ileum in Crohn’s Disease. Gut. 2004;53(9):1287–1294. doi: 10.1136/gut.2003.028225. PubMed DOI PMC

Yerra V. G., Advani A.. Role of CCR2-Positive Macrophages in Pathological Ventricular Remodelling. Biomedicines 2022, Vol. 10, Page 661. 2022;10(3):661. doi: 10.3390/biomedicines10030661. PubMed DOI PMC

Bleul C. C., Wu L., Hoxie J. A., Springer T. A., Mackay C. R.. The HIV Coreceptors CXCR4 and CCR5 Are Differentially Expressed and Regulated on Human T Lymphocytes. Proc. Natl. Acad. Sci. U. S. A. 1997;94(5):1925–1930. doi: 10.1073/pnas.94.5.1925. PubMed DOI PMC

Barmania F., Pepper M. S.. C-C Chemokine Receptor Type Five (CCR5): An Emerging Target for the Control of HIV Infection. Appl. Transl Genom. 2013;2(1):3–16. doi: 10.1016/j.atg.2013.05.004. PubMed DOI PMC

Geissmann F., Jung S., Littman D. R.. Blood Monocytes Consist of Two Principal Subsets with Distinct Migratory Properties. Immunity. 2003;19(1):71–82. doi: 10.1016/S1074-7613(03)00174-2. PubMed DOI

Blanpain C., Migeotte I., Lee B., Vakili J., Doranz B. J., Govaerts C., Vassart G., Doms R. W., Parmentier M.. CCR5 Binds Multiple CC-Chemokines: MCP-3 Acts as a Natural Antagonist. Blood. 1999;94(6):1899–1905. doi: 10.1182/blood.V94.6.1899. PubMed DOI

Zhang H., Chen K., Tan Q., Shao Q., Han S., Zhang C., Yi C., Chu X., Zhu Y., Xu Y., Zhao Q., Wu B.. Structural Basis for Chemokine Recognition and Receptor Activation of Chemokine Receptor CCR5. Nature Communications 2021 12:1. 2021;12(1):1–12. doi: 10.1038/s41467-021-24438-5. PubMed DOI PMC

Shao Z., Tan Y., Shen Q., Hou L., Yao B., Qin J., Xu P., Mao C., Chen L. N., Zhang H., Shen D. D., Zhang C., Li W., Du X., Li F., Chen Z. H., Jiang Y., Xu H. E., Ying S., Ma H., Zhang Y., Shen H.. Molecular Insights into Ligand Recognition and Activation of Chemokine Receptors CCR2 and CCR3. Cell Discovery 2022 8:1. 2022;8(1):1–11. doi: 10.1038/s41421-022-00403-4. PubMed DOI PMC

Dawson J. R. D., Kufareva I.. Molecular Determinants of Antagonist Interactions with Chemokine Receptors CCR2 and CCR5. Biophys. J. 2023;122(3):185a. doi: 10.1016/j.bpj.2022.11.1140. DOI

Billen M., Schols D., Verwilst P.. Targeting Chemokine Receptors from the Inside-out: Discovery and Development of Small-Molecule Intracellular Antagonists. Chem. Commun. 2022;58(26):4132–4148. doi: 10.1039/D1CC07080K. PubMed DOI

Ortiz Zacarías N. V., Chahal K. K., Šimková T., Van Der Horst C., Zheng Y., Inoue A., Theunissen E., Mallee L., Van Der Es D., Louvel J., Ijzerman A. P., Handel T. M., Kufareva I., Heitman L. H.. Design and Characterization of an Intracellular Covalent Ligand for CC Chemokine Receptor 2. J. Med. Chem. 2021;64(5):2608–2621. doi: 10.1021/acs.jmedchem.0c01137. PubMed DOI PMC

Ortiz Zacarías N. V., Van Veldhoven J. P. D., Den Hollander L. S., Dogan B., Openy J., Hsiao Y. Y., Lenselink E. B., Heitman L. H., Ijzerman A. P.. Synthesis and Pharmacological Evaluation of Triazolopyrimidinone Derivatives as Noncompetitive, Intracellular Antagonists for CC Chemokine Receptors 2 and 5. J. Med. Chem. 2019;62(24):11035–11053. doi: 10.1021/acs.jmedchem.9b00742. PubMed DOI PMC

Singh P., Kumar V., Lee G., Jung T. S., Ha M. W., Hong J. C., Lee K. W.. Pharmacophore-Oriented Identification of Potential Leads as CCR5 Inhibitors to Block HIV Cellular Entry. Int. J. Mol. Sci. 2022;23(24):16122. doi: 10.3390/ijms232416122. PubMed DOI PMC

Mohamed Yusof N. I. S., Awaluddin N. A., Fauzi F. M.. Insight into the Structure and Physicochemical Properties of Potent Chemokine Receptor 5 Inhibitors for the Discovery of Novel Alzheimer’s Disease Drugs. Cent Nerv Syst. Agents Med. Chem. 2023;23(2):95–108. doi: 10.2174/1871524923666230726102846. PubMed DOI

Wu Q.-l., Cui L.-y., Ma W.-y., Wang S.-s., Zhang Z., Feng Z.-p., Sun H.-s., Chu S.-f., He W.-b., Chen N.-h.. A Novel Small-Molecular CCR5 Antagonist Promotes Neural Repair after Stroke. Acta Pharmacol Sin. 2023;44(10):1935–1947. doi: 10.1038/s41401-023-01100-y. PubMed DOI PMC

Appiah-Kubi P., Iwuchukwu E. A., Soliman M. E. S.. Structure-Based Identification of Novel Scaffolds as Potential HIV-1 Entry Inhibitors Involving CCR5. J. Biomol Struct Dyn. 2022;40(23):13115–13126. doi: 10.1080/07391102.2021.1982006. PubMed DOI

Karlshøj S., Amarandi R. M., Larsen O., Daugvilaite V., Steen A., Brvar M., Pui A., Frimurer T. M., Ulven T., Rosenkilde M. M.. Molecular Mechanism of Action for Allosteric Modulators and Agonists in CC-Chemokine Receptor 5 (CCR5) J. Biol. Chem. 2016;291(52):26860. doi: 10.1074/jbc.M116.740183. PubMed DOI PMC

Wang Y., Wang J., Shu M., Wang Y., Hu Y., Luo Y., Lin Z.. Identification of Potential CCR5 Inhibitors through Pharmacophore-Based Virtual Screening, Molecular Dynamics Simulation and Binding Free Energy Analysis. Mol. Biosyst. 2016;12(11):3396–3406. doi: 10.1039/C6MB00577B. PubMed DOI

Ji Y., Shu M., Lin Y., Wang Y., Wang R., Hu Y., Lin Z.. Combined 3D-QSAR Modeling and Molecular Docking Study on Azacycles CCR5 Antagonists. J. Mol. Struct. 2013;1045:35–41. doi: 10.1016/j.molstruc.2013.03.062. DOI

Ghasemi J. B., Nouri M.. Docking and CoMFA Study on Novel Human CCR5 Receptor Antagonists. Medicinal Chemistry Research. 2013;22(3):1356–1364. doi: 10.1007/s00044-012-0118-7. DOI

Gadhe C. G., Kothandan G., Cho S. J.. Binding Site Exploration of CCR5 Using in Silico Methodologies: A 3D-QSAR Approach. Arch Pharm. Res. 2013;36(1):6–31. doi: 10.1007/s12272-013-0001-1. PubMed DOI

Garcia-Perez J., Rueda P., Alcami J., Rognan D., Arenzana-Seisdedos F., Lagane B., Kellenberger E.. Allosteric Model of Maraviroc Binding to CC Chemokine Receptor 5 (CCR5) J. Biol. Chem. 2011;286(38):33409–33421. doi: 10.1074/jbc.M111.279596. PubMed DOI PMC

Ortiz Zacarías N. V., Van Veldhoven J. P. D., Portner L., Van Spronsen E., Ullo S., Veenhuizen M., Van Der Velden W. J. C., Zweemer A. J. M., Kreekel R. M., Oenema K., Lenselink E. B., Heitman L. H., Ijzerman A. P.. Pyrrolone Derivatives as Intracellular Allosteric Modulators for Chemokine Receptors: Selective and Dual-Targeting Inhibitors of CC Chemokine Receptors 1 and 2. J. Med. Chem. 2018;61(20):9146–9161. doi: 10.1021/acs.jmedchem.8b00605. PubMed DOI PMC

Zweemer A. J. M., Bunnik J., Veenhuizen M., Miraglia F., Lenselink E. B., Vilums M., De Vries H., Gibert A., Thiele S., Rosenkilde M. M., IJzerman A. P., Heitman L. H.. Discovery and Mapping of an Intracellular Antagonist Binding Site at the Chemokine Receptor CCR2. Mol. Pharmacol. 2014;86(4):358–368. doi: 10.1124/mol.114.093328. PubMed DOI

Korn M., Ehrt C., Ruggiu F., Gastreich M., Rarey M.. Navigating Large Chemical Spaces in Early-Phase Drug Discovery. Curr. Opin Struct Biol. 2023;80:102578. doi: 10.1016/j.sbi.2023.102578. PubMed DOI

Gorgulla C., Boeszoermenyi A., Wang Z. F., Fischer P. D., Coote P. W., Padmanabha Das K. M., Malets Y. S., Radchenko D. S., Moroz Y. S., Scott D. A., Fackeldey K., Hoffmann M., Iavniuk I., Wagner G., Arthanari H.. An Open-Source Drug Discovery Platform Enables Ultra-Large Virtual Screens. Nature 2020 580:7805. 2020;580(7805):663–668. doi: 10.1038/s41586-020-2117-z. PubMed DOI PMC

Sadybekov A. A., Sadybekov A. V., Liu Y., Iliopoulos-Tsoutsouvas C., Huang X. P., Pickett J., Houser B., Patel N., Tran N. K., Tong F., Zvonok N., Jain M. K., Savych O., Radchenko D. S., Nikas S. P., Petasis N. A., Moroz Y. S., Roth B. L., Makriyannis A., Katritch V.. Synthon-Based Ligand Discovery in Virtual Libraries of over 11 Billion Compounds. Nature 2021 601:7893. 2022;601(7893):452–459. doi: 10.1038/s41586-021-04220-9. PubMed DOI PMC

REAL Database SubsetsEnamine. https://enamine.net/compound-collections/real-compounds/real-database-subsets (accessed 2024. –07–26).

Hall B. W., Keiser M. J.. Retrieval Augmented Docking Using Hierarchical Navigable Small Worlds. J. Chem. Inf Model. 2024;64(19):7398–7408. doi: 10.1021/acs.jcim.4c00683. PubMed DOI PMC

Luttens A., Cabeza de Vaca I., Sparring L., Brea J., Martínez A. L., Kahlous N. A., Radchenko D. S., Moroz Y. S., Loza M. I., Norinder U., Carlsson J.. Rapid Traversal of Vast Chemical Space Using Machine Learning-Guided Docking Screens. Nature Computational Science 2025 5:4. 2025;5(4):301–312. doi: 10.1038/s43588-025-00777-x. PubMed DOI PMC

Marin E., Kovaleva M., Kadukova M., Mustafin K., Khorn P., Rogachev A., Mishin A., Guskov A., Borshchevskiy V.. Regression-Based Active Learning for Accessible Acceleration of Ultra-Large Library Docking. J. Chem. Inf Model. 2024;64(7):2612–2623. doi: 10.1021/acs.jcim.3c01661. PubMed DOI PMC

Gorgulla C., Cecchini D., Nigam A., Tang M., Reis J., Koop M., Gottinger A., Nicoll C. R., Jayaraj A., Çınaroğlu S. S., Törner R., Malets Y., Gehev M., Das K. M. P., Churion K., Kim J., Seo H.-S., Dhe-Paganon S., Secker C., Haddadnia M., Hasson A., Li M., Kumar A., Levin-Konigsberg R., Choi E.-B., Shapiro G. I., Cox H., Sebastian L., Braithwaite C., Bashyal P., Radchenko D. S., Kumar A., Aquilanti P.-Y., Gabb H., Alhossary A., Wagner G., Aspuru-Guzik A., Moroz Y. S., Kalodimos C. G., Fackeldey K., Mattevi A., Arthanari H.. AI-Enhanced Adaptive Virtual Screening Platform Enabling Exploration of 69 Billion Molecules Discovers Structurally Validated FSP1 Inhibitors. bioRxiv. 2025 doi: 10.1101/2023.04.25.537981. DOI

Yu Y., Cai C., Wang J., Bo Z., Zhu Z., Zheng H.. Uni-Dock: GPU-Accelerated Docking Enables Ultralarge Virtual Screening. J. Chem. Theory Comput. 2023;19(11):3336–3345. doi: 10.1021/acs.jctc.2c01145. PubMed DOI

Li C., Wang Y., Xing H., Wang Y., Wang Y., Ye J.. Vina-CUDA: An Efficient Program with in-Depth Utilization of GPU to Accelerate Molecular Docking. J. Chem. Inf Model. 2025;65(10):4751–4759. doi: 10.1021/acs.jcim.4c01933. PubMed DOI

Ding J., Tang S., Mei Z., Wang L., Huang Q., Hu H., Ling M., Wu J.. Vina-GPU 2.0: Further Accelerating AutoDock Vina and Its Derivatives with Graphics Processing Units. J. Chem. Inf Model. 2023;63(7):1982–1998. doi: 10.1021/acs.jcim.2c01504. PubMed DOI

Takács G., Havasi D., Sándor M., Dohánics Z., Balogh G. T., Kiss R.. DIY Virtual Chemical Libraries - Novel Starting Points for Drug Discovery. ACS Med. Chem. Lett. 2023;14(9):1188–1197. doi: 10.1021/ACSMEDCHEMLETT.3C00146. PubMed DOI PMC

Grotsch K., Sadybekov A. V., Hiller S., Zaidi S., Eremin D., Le A., Liu Y., Smith E. C., Illiopoulis-Tsoutsouvas C., Thomas J., Aggarwal S., Pickett J. E., Reyes C., Picazo E., Roth B. L., Makriyannis A., Katritch V., Fokin V. V.. Virtual Screening of a Chemically Diverse “Superscaffold” Library Enables Ligand Discovery for a Key GPCR Target. ACS Chem. Biol. 2024;19(4):866–874. doi: 10.1021/ACSCHEMBIO.3C00602. PubMed DOI PMC

Zhu T., Cao S., Su P. C., Patel R., Shah D., Chokshi H. B., Szukala R., Johnson M. E., Hevener K. E.. Hit Identification and Optimization in Virtual Screening: Practical Recommendations Based on a Critical Literature Analysis. J. Med. Chem. 2013;56:6560. doi: 10.1021/jm301916b. PubMed DOI PMC

Díaz-Rovira A. M., Martín H., Beuming T., Díaz L., Guallar V., Ray S. S.. Are Deep Learning Structural Models Sufficiently Accurate for Virtual Screening? Application of Docking Algorithms to AlphaFold2 Predicted Structures. J. Chem. Inf Model. 2023;63(6):1668–1674. doi: 10.1021/acs.jcim.2c01270. PubMed DOI

Coskun D., Lihan M., Rodrigues J. P. G. L. M., Vass M., Robinson D., Friesner R. A., Miller E. B.. Using AlphaFold and Experimental Structures for the Prediction of the Structure and Binding Affinities of GPCR Complexes via Induced Fit Docking and Free Energy Perturbation. J. Chem. Theory Comput. 2024;20(1):477–489. doi: 10.1021/acs.jctc.3c00839. PubMed DOI

Andrews G., Jones C., Wreggett K. A.. An Intracellular Allosteric Site for a Specific Class of Antagonists of the CC Chemokine G Protein-Coupled Receptors CCR4 and CCR5. Mol. Pharmacol. 2008;73(3):855–867. doi: 10.1124/mol.107.039321. PubMed DOI

Google DeepMind . C–C chemokine receptor type 5 - AlphaFold structure prediction. https://alphafold.ebi.ac.uk/entry/A0A089G6S6.

Zheng Y., Qin L., Zacarías N. V. O., De Vries H., Han G. W., Gustavsson M., Dabros M., Zhao C., Cherney R. J., Carter P., Stamos D., Abagyan R., Cherezov V., Stevens R. C., Ijzerman A. P., Heitman L. H., Tebben A., Kufareva I., Handel T. M.. Structure of CC Chemokine Receptor 2 with Orthosteric and Allosteric Antagonists. Nature 2016 540:7633. 2016;540(7633):458–461. doi: 10.1038/nature20605. PubMed DOI PMC

Sicho M.. Combining AlphaFold with Focused Virtual Library Design in the Development of Novel CCR2 and CCR5. Zenodo. 2024 doi: 10.5281/zenodo.13736590. PubMed DOI PMC

Eberhardt J., Santos-Martins D., Tillack A. F., Forli S.. AutoDock Vina 1.2.0: New Docking Methods, Expanded Force Field, and Python Bindings. J. Chem. Inf Model. 2021;61(8):3891–3898. doi: 10.1021/acs.jcim.1c00203. PubMed DOI PMC

Landrum G.. et al. RDKit: Open-Source Cheminformatics. 2023 doi: 10.5281/zenodo.591637. DOI

Amgen Inc. Patent WO2012/3264, 2012.

Smith P. K., Krohn R. I., Hermanson G. T., Mallia A. K., Gartner F. H., Provenzano M. D., Fujimoto E. K., Goeke N. M., Olson B. J., Klenk D. C.. Measurement of Protein Using Bicinchoninic Acid. Anal. Biochem. 1985;150(1):76–85. doi: 10.1016/0003-2697(85)90442-7. PubMed DOI

Invitrogen . Tango CCR5-bla U2OS Cell–Based Assay. https://assets.thermofisher.com/TFS-Assets/LSG/manuals/Tango_CCR5bla_U2OS_cells.pdf.

Adasme M. F., Linnemann K. L., Bolz S. N., Kaiser F., Salentin S., Haupt V. J., Schroeder M.. PLIP 2021: Expanding the Scope of the Protein-Ligand Interaction Profiler to DNA and RNA. Nucleic Acids Res. 2021;49(W1):W530–W534. doi: 10.1093/nar/gkab294. PubMed DOI PMC

ZINC. https://zinc15.docking.org/substances/home/ (accessed 2024. –08–02).

Bouz G., Juhás M., Otero L. P., de la Red C. P., Jand’Ourek O., Konečná K., Paterová P., Kubíček V., Janoušek J., Doležal M., Zitko J.. Substituted N-(Pyrazin-2-Yl)­Benzenesulfonamides; Synthesis, Anti-Infective Evaluation, Cytotoxicity, and In Silico Studies. Molecules. 2019;25(1):138. doi: 10.3390/MOLECULES25010138. PubMed DOI PMC

Wang G. Z., Haile P. A., Daniel T., Belot B., Viet A. Q., Goodman K. B., Sha D., Dowdell S. E., Varga N., Hong X., Chakravorty S., Webb C., Cornejo C., Olzinski A., Bernard R., Evans C., Emmons A., Briand J., Chung C. W., Quek R., Lee D., Gough P. J., Sehon C. A.. CCR2 Receptor Antagonists: Optimization of Biaryl Sulfonamides to Increase Activity in Whole Blood. Bioorg. Med. Chem. Lett. 2011;21:7291. doi: 10.1016/j.bmcl.2011.10.038. PubMed DOI

Šícho M.. Combining AlphaFold with Focused Virtual Library Design in the Development of Novel CCR2 and CCR5 Antagonists - LUF Compounds Poses. Zenodo. 2025 doi: 10.5281/zenodo.17206445. PubMed DOI PMC

Combining AlphaFold with Focused Virtual Library Design in the Development of Novel CCR2 and CCR5 Antagonists