Identification of Novel Rab27a/Melanophilin Blockers by Pharmacophore-Based Virtual Screening

Abstract

Melanocytes are unique cells that produce specific melanin-containing intracellular organelles called melanosomes. Melanosomes are transported from the perinuclear area of melanocytes toward the plasma membrane as they become more melanized in order to increase skin pigmentation. In this vesicular trafficking of melanosomes, Rab27a, melanophilin, and myosin Va play crucial roles in linking melanosomes to actin-based motors. To identify novel compounds to inhibit binding interface between Rab27a and melanophilin, a pharmacophore model was built based on a modeled 3D structure of the protein complex that describes the essential binding residues in the intermolecular interaction. A pharmacophore model was employed to screen a chemical library database. Finally, 25 virtual hits were selected for biological evaluations. The biological activities of 11 analogues were evaluated in a second assay. Two compounds were identified as having concentration-dependent inhibitory activity. By analyzing structure–activity relationships of derivatives of BMD-20, two hydroxyl functional groups were found to be critical for blocking the intermolecular binding between Rab27a and melanophilin.

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References

  1. 1.

    Marks, M. S., & Seabra, M. C. (2001). The melanosome: membrane dynamics in black and white. Nature Reviews Molecular Cell Biology, 2, 738–748.

    CAS  Article  Google Scholar 

  2. 2.

    Langford, G. M. (1995). Actin- and microtubule-dependent organelle motors: interrelationships between the two motility systems. Current Opinion in Cell Biology, 7, 82–88.

    CAS  Article  Google Scholar 

  3. 3.

    Wu, X., Bowers, B., Rao, K., Wei, Q., & Hammer, J. A., III. (1998). Visualization of melanosome dynamics within wild-type and dilute melanocytes suggests a paradigm for myosin v function in vivo. Journal of Cell Biology, 143, 1899–1918.

    CAS  Article  Google Scholar 

  4. 4.

    Provance, D. W., Jr., Wei, M., Ipe, V., & Mercer, J. A. (1996). Cultured melanocytes from dilute mutant mice exhibit dendritic morphology and altered melanosome distribution. Proceedings of the National Academy of Sciences of the United States of America, 93, 14554–14558.

    CAS  Article  Google Scholar 

  5. 5.

    Jordens, I., Westbroek, W., Marsman, M., Rocha, N., Mommaas, M., Huizing, M., Lambert, J., Naeyaert, J. M., & Neefjes, J. (2006). Rab7 and Rab27a control two motor protein activities involved in melanosomal transport. Pigment Cell Research, 19, 412–423.

    CAS  Article  Google Scholar 

  6. 6.

    Strom, M., Hume, A. N., Tarafder, A. K., Barkagianni, E., & Seabra, M. C. (2002). A family of Rab27-binding proteins. Melanophilin links Rab27a and myosin Va function in melanosome transport. Journal of Biological Chemistry, 277, 25423–25430.

    CAS  Article  Google Scholar 

  7. 7.

    Wu, X., Sakamoto, T., Zhang, F., Sellers, J. R., & Hammer, J. A., III. (2006). In vitro reconstitution of a transport complex containing Rab27a, melanophilin and myosin Va. FEBS Letters, 580, 5863–5868.

    CAS  Article  Google Scholar 

  8. 8.

    Chavas, L. M. G., Ihara, K., Kawasaki, M., Torii, S., Uejima, T., Kato, R., Izumi, T., & Wakatsuki, S. (2008). Elucidation of Rab27 recruitment by its effectors: structure of Rab27a bound to exophilin4/Slp2-a. Structure, 16, 1468–1477.

    CAS  Article  Google Scholar 

  9. 9.

    Kukimoto-Niino, M., Sakamoto, A., Kanno, E., Hanawa-Suetsugu, K., Terada, T., Shirouzu, M., Fukuda, M., & Yokoyama, S. (2008). Structural basis for the exclusive specificity of Slac2-a/melanophilin for the Rab27 GTPases. Structure, 16, 1478–1490.

    CAS  Article  Google Scholar 

  10. 10.

    Wilson, S. M., Yip, R., Swing, D. A., O'Sullivan, T. N., Zhang, Y., Novak, E. K., Swank, R. T., Russell, L. B., Copeland, N. G., & Jenkins, N. A. (2000). A mutation in Rab27a causes the vesicle transport defects observed in ashen mice. Proceedings of the National Academy of Sciences of the United States of America, 97, 7933–7938.

    CAS  Article  Google Scholar 

  11. 11.

    Singh, R. K., Mizuno, K., Wasmeier, C., Wavre-Shapton, S. T., Recchi, C., Catz, S. D., Futter, C., Tolmachova, T., Hume, A. N., & Seabra, M. C. (2013). Distinct and opposing roles for Rab27a/Mlph/MyoVa and Rab27b/Munc13-4 in mast cell secretion. FEBS Journal, 280, 892–903.

    CAS  Article  Google Scholar 

  12. 12.

    Menasche, G., Feldmann, J., Houdusse, A., Desaymard, C., Fischer, A., Goud, B., & de Saint Basile, G. (2003). Biochemical and functional characterization of Rab27a mutations occurring in Griscelli syndrome patients. Blood, 101, 2736–2742.

    CAS  Article  Google Scholar 

  13. 13.

    Fukuda, M. (2002). Synaptotagmin-like protein (Slp) homology domain 1 of Slac2-a/melanophilin is a critical determinant of GTP-dependent specific binding to Rab27A. Journal of Biological Chemistry, 277, 40118–40124.

    CAS  Article  Google Scholar 

  14. 14.

    Pereira-Leal, J. B., & Seabra, M. C. (2000). The mammalian Rab family of small GTPases: definition of family and subfamily sequence motifs suggests a mechanism for functional specificity in the Ras superfamily. Journal of Molecular Biology, 301, 1077–1087.

    CAS  Article  Google Scholar 

  15. 15.

    Agrafiotis, D. K., & Bandyopadhyay, D. (2008). A self-organizing algorithm for molecular alignment and pharmacophore development. Journal of Computational Chemistry, 29, 965–982.

    Article  Google Scholar 

  16. 16.

    Barillari, C., Marcou, G., & Rognan, D. (2008). Hot-spots-guided receptor-based pharmacophores (HS-pharm): a knowledge-based approach to identify ligand-anchoring atoms in protein cavities and prioritize structure-based pharmacophores. Journal of Chemical Information and Modeling, 48, 1396–1410.

    CAS  Article  Google Scholar 

  17. 17.

    (2010) Discovery Studio 3.0., Accelrys Inc., San Diego, CA. U.S.A.

  18. 18.

    (2005) Catalyst 4.10. Accelrys Inc, San Diego, CA, USA.

  19. 19.

    Venkatachalam, C. M., Jiang, X., Oldfield, T., & Waldman, M. (2003). LigandFit: a novel method for the shape-directed rapid docking of ligands to protein active sites. Journal of Molecular Graphics and Modelling, 21, 289–307.

    CAS  Article  Google Scholar 

  20. 20.

    Bohm, H. J. (1998). Prediction of binding constants of protein ligands: a fast method for the polarization of hits obtained from the de novo design on 3D database search programs. Journal of Computer Aided Molecular Design, 12, 309–323.

    CAS  Article  Google Scholar 

  21. 21.

    Gehlhaar, D. K., Verkhivker, G. M., Rejto, P. A., Sherman, C. J., Fogel, D. B., Fogel, L. J., & Freer, S. T. (1995). Molecular recognition of the inhibitor AG-1343 by HIV-1 protease: conformationally flexible docking by evolutionary programming. Chemical Biology, 2, 317–324.

    CAS  Article  Google Scholar 

  22. 22.

    Krammer, A., Kirchhoff, P. D., Jiang, X., Venkatachalam, C. M., & Waldman, M. (2005). LigScore: a novel scoring function for predicting binding affinities. Journal of Molecular Graphics and Modelling, 23, 395–407.

    CAS  Article  Google Scholar 

  23. 23.

    Gohlke, H., Hendlich, M., & Klebe, G. (2000). Knowledge-based scoring function to predict protein–ligand interactions. Journal of Molecular Biology, 295, 337–356.

    CAS  Article  Google Scholar 

  24. 24.

    Charifson, P. S., Corkery, J. J., Murcko, M. A., & Walters, W. P. (1999). Consensus scoring: a method for obtaining improved hit rates from docking databases of three-dimensional structures into proteins. Journal of Medicinal Chemistry, 42, 5100–5109.

    CAS  Article  Google Scholar 

  25. 25.

    Hume, A. N., Ushakov, D. S., Tarafder, A. K., Ferenczi, M. A., & Seabra, M. C. (2007). Rab27a and MyoVa are the primary Mlph interactors regulating melanosome transport in melanocytes. Journal of Cell Science, 120, 3111–3122.

    CAS  Article  Google Scholar 

  26. 26.

    Sukumar, N., & Das, S. (2011). Current trends in virtual high throughput screening using ligand-based and structure-based methods. Combinatorial Chemistry & High Throughput Screening, 14, 872–888.

    CAS  Article  Google Scholar 

  27. 27.

    Allen, J. G., Bourbeau, M. P., Wohlhieter, G. E., Bartberger, M. D., Michelsen, K., Hungate, R., Gadwood, R. C., Gaston, R. D., Evans, B., Mann, L. W., Matison, M. E., Schneider, S., Huang, X., Yu, D., Andrews, P. S., Reichelt, A., Long, A. M., Yakowec, P., Yang, E. Y., Lee, T. A., & Oliner, J. D. (2009). Discovery and optimization of chromenotriazolopyrimidines as potent inhibitors of the mouse double minute 2-tumor protein 53 protein–protein interaction. Journal of Medicinal Chemistry, 52, 7044–7053.

    CAS  Article  Google Scholar 

  28. 28.

    Eymin, B., Gazzeri, S., Brambilla, C., & Brambilla, E. (2002). Mdm2 overexpression and p14ARF inactivation are two mutually exclusive events in primary human lung tumors. Oncogene, 21, 2750–2761.

    CAS  Article  Google Scholar 

  29. 29.

    Michael, D., & Oren, M. (2003). The p53-Mdm2 module and the ubiquitin system. Seminars in Cancer Biology, 13, 49–58.

    CAS  Article  Google Scholar 

  30. 30.

    Momand, J., Jung, D., Wilczynski, S., & Niland, J. (1998). The MDM2 gene amplification database. Nucleic Acids Research, 26, 3453–3459.

    CAS  Article  Google Scholar 

  31. 31.

    Soussi, T., Dehouche, K., & Béroud, C. (2000). p53 website and analysis of p53 gene mutations in human cancer: forging a link between epidemiology and carcinogenesis. Human Mutation, 15, 105–113.

    CAS  Article  Google Scholar 

  32. 32.

    Hardcastle, I. R., Liu, J., Valeur, E., Watson, A., Ahmed, S. U., Blackburn, T. J., Bennaceur, K., Clegg, W., Drummond, C., Endicott, J. A., Golding, B. T., Griffin, R. J., Gruber, J., Haggerty, K., Harrington, R. W., Hutton, C., Kemp, S., Lu, X., McDonnell, J. M., Newell, D. R., Noble, M. E. M., Payne, S. L., Revill, C. H., Riedinger, C., Xu, Q., & Lunec, J. (2011). Isoindolinone inhibitors of the murine double minute 2 (MDM2)-p53 protein–protein interaction: structure–activity studies leading to improved potency. Journal of Medicinal Chemistry, 54, 1233–1243.

    CAS  Article  Google Scholar 

  33. 33.

    Merck. Study of MK-8242 alone and in combination with cytarabine in participants with acute myelogenous leukemia (http://clinicaltrials.gov/show/NCT01451437)

  34. 34.

    Rew, Y., Sun, D., Gonzalez-Lopez De Turiso, F., Bartberger, M. D., Beck, H. P., Canon, J., Chen, A., Chow, D., Deignan, J., Fox, B. M., Gustin, D., Huang, X., Jiang, M., Jiao, X., Jin, L., Kayser, F., Kopecky, D. J., Li, Y., Lo, M. C., Long, A. M., Michelsen, K., Oliner, J. D., Osgood, T., Ragains, M., Saiki, A. Y., Schneider, S., Toteva, M., Yakowec, P., Yan, X., Ye, Q., Yu, D., Zhao, X., Zhou, J., Medina, J. C., & Olson, S. H. (2012). Structure-based design of novel inhibitors of the MDM2–p53 interaction. Journal of Medicinal Chemistry, 55, 4936–4954.

    CAS  Article  Google Scholar 

  35. 35.

    A study of RO5045337 [RG7112] in patients with advanced solid tumors. Available from: http://www.clinicaltrials.gov/ct2/show/NCT00559533?term=RG7112&rank=1.

  36. 36.

    In, Y., Chai, H. H., & No, K. T. (2005). A partition coefficient calculation method with the SFED model. Journal of Chemical Information and Modeling, 45, 254–263.

    CAS  Article  Google Scholar 

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Acknowledgments

This study was supported by a grant of the Korea Healthcare technology R&D project, Ministry of Health & Welfare, Republic of Korea (grant no. A103017).

Author contribution

Jong Young Joung performed virtual screening, analyzed data, and wrote the paper; Ha Yeon Lee performed biological assays for hit compounds and wrote the paper; Jongil Park performed biological assays; Jee-Young Lee analyzed assay results; Byung Ha Chang analyzed SAR results; Kyoung Tai No designed experiments; Ky-Youb Nam designed experiments and wrote the paper; and Jae Sung Hwang designed the biological assays.

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Correspondence to Ky-Youb Nam or Jae Sung Hwang.

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J. Y. Joung and H. Y. Lee contributed equally to this work.

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Joung, J.Y., Lee, H.Y., Park, J. et al. Identification of Novel Rab27a/Melanophilin Blockers by Pharmacophore-Based Virtual Screening. Appl Biochem Biotechnol 172, 1882–1897 (2014). https://doi.org/10.1007/s12010-013-0615-2

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Keywords

  • Rab27a/melanophilin
  • Pharmacophore-based virtual screening
  • Skin pigmentation
  • Mekanosome tansport
  • Molecular docking