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Combinatorial chemistry identifies high-affinity peptidomimetics against α4β1 integrin for in vivo tumor imaging

Abstract

Small peptide–based agents have attracted wide interest as cancer-targeting agents for diagnostic imaging and targeted therapy. There is a need to develop new high-affinity and high-specificity peptidomimetic or small-molecule ligands against cancer cell surface receptors. Here we report on the identification of a high-affinity peptidomimetic ligand (LLP2A; IC50 = 2 pM) against α4β1 integrin using both diverse and highly focused one-bead-one-compound combinatorial peptidomimetic libraries in conjunction with high-stringency screening. We further demonstrate that LLP2A can be used to image α4β1-expressing lymphomas with high sensitivity and specificity when conjugated to a near infrared fluorescent dye in a mouse xenograft model. Thus, LLP2A provides an important tool for noninvasive monitoring of α4β1 expression and activity during tumor progression, and it shows great potential as an imaging and therapeutic agent for α4β1-positive tumors.

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Figure 1: Chemical structures and screening of the OBOC libraries for α4β1-targeting ligands.
Figure 2: The stability and specificity of LLP2A.
Figure 3: In vitro cell binding assays with LLP2A-biotin.
Figure 4: Effect of single amino acid mutation of α4 on its binding to LLP2A.
Figure 5: In vivo NIRF imaging of Molt-4 tumor–bearing mice.
Figure 6: Ex vivo NIRF images and microscopic analysis of tumors and organs that were excised from mice 24 h after receiving (a,ce) LLP2A-SA-Alexa680 or (b,f,g) SA-Alexa680 alone.
Figure 7: Specific accumulation of LLP2A probes in α4β1-expressing tumors.

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References

  1. Aina, O.H., Sroka, T.C., Chen, M.L. & Lam, K.S. Therapeutic cancer targeting peptides. Biopolymers 66, 184–199 (2002).

    Article  CAS  Google Scholar 

  2. Coiffier, B. et al. Rituximab (anti-CD20 monoclonal antibody) for the treatment of patients with relapsing or refractory aggressive lymphoma: a multicenter phase II study. Blood 92, 1927–1932 (1998).

    CAS  PubMed  Google Scholar 

  3. Wiseman, G.A. et al. Ibritumomab tiuxetan radioimmunotherapy for patients with relapsed or refractory non-Hodgkin lymphoma and mild thrombocytopenia: a phase II multicenter trial. Blood 99, 4336–4342 (2002).

    Article  CAS  Google Scholar 

  4. Okarvi, S.M. Peptide-based radiopharmaceuticals: future tools for diagnostic imaging of cancers and other diseases. Med. Res. Rev. 24, 357–397 (2004).

    Article  CAS  Google Scholar 

  5. Holzmann, B., Gosslar, U. & Bittner, M. α4 integrins and tumor metastasis. Curr. Top. Microbiol. Immunol. 231, 125–141 (1998).

    CAS  PubMed  Google Scholar 

  6. Vincent, A.M., Cawley, J.C. & Burthem, J. Integrin function in chronic lymphocytic leukemia. Blood 87, 4780–4788 (1996).

    CAS  PubMed  Google Scholar 

  7. Marco, R.A., Diaz-Montero, C.M., Wygant, J.N., Kleinerman, E.S. & McIntyre, B.W. Alpha 4 integrin increases anoikis of human osteosarcoma cells. J. Cell. Biochem. 88, 1038–1047 (2003).

    Article  CAS  Google Scholar 

  8. de la Fuente, M.T., Casanova, B., Garcia-Gila, M., Silva, A. & Garcia-Pardo, A. Fibronectin interaction with alpha4beta1 integrin prevents apoptosis in B cell chronic lymphocytic leukemia: correlation with Bcl-2 and Bax. Leukemia 13, 266–274 (1999).

    Article  CAS  Google Scholar 

  9. Damiano, J.S. & Dalton, W.S. Integrin-mediated drug resistance in multiple myeloma. Leuk. Lymphoma 38, 71–81 (2000).

    Article  CAS  Google Scholar 

  10. Matsunaga, T. et al. Interaction between leukemic-cell VLA-4 and stromal fibronectin is a decisive factor for minimal residual disease of acute myelogenous leukemia. Nat. Med. 9, 1158–1165 (2003).

    Article  CAS  Google Scholar 

  11. Olson, D.L., Burkly, L.C., Leone, D.R., Dolinski, B.M. & Lobb, R.R. Anti-alpha4 integrin monoclonal antibody inhibits multiple myeloma growth in a murine model. Mol. Cancer Ther. 4, 91–99 (2005).

    CAS  PubMed  Google Scholar 

  12. Garmy-Susini, B. et al. Integrin alpha4beta1-VCAM-1-mediated adhesion between endothelial and mural cells is required for blood vessel maturation. J. Clin. Invest. 115, 1542–1551 (2005).

    Article  CAS  Google Scholar 

  13. Osborn, L. et al. Arrangement of domains, and amino acid residues required for binding of vascular cell adhesion molecule-1 to its counter-receptor VLA-4 (alpha 4 beta 1). J. Cell Biol. 124, 601–608 (1994).

    Article  CAS  Google Scholar 

  14. Komoriya, A. et al. The minimal essential sequence for a major cell type-specific adhesion site (CS1) within the alternatively spliced type III connecting segment domain of fibronectin is leucine-aspartic acid-valine. J. Biol. Chem. 266, 15075–15079 (1991).

    CAS  PubMed  Google Scholar 

  15. Lin, K.C. & Castro, A.C. Very late antigen 4 (VLA4) antagonists as anti-inflammatory agents. Curr. Opin. Chem. Biol. 2, 453–457 (1998).

    Article  CAS  Google Scholar 

  16. Yusuf-Makagiansar, H., Anderson, M.E., Yakovleva, T.V., Murray, J.S. & Siahaan, T.J. Inhibition of LFA-1/ICAM-1 and VLA-4/VCAM-1 as a therapeutic approach to inflammation and autoimmune diseases. Med. Res. Rev. 22, 146–167 (2002).

    Article  CAS  Google Scholar 

  17. Lowman, H.B. Bacteriophage display and discovery of peptide leads for drug development. Annu. Rev. Biophys. Biomol. Struct. 26, 401–424 (1997).

    Article  CAS  Google Scholar 

  18. Weiner, R.E. & Thakur, M.L. Radiolabeled peptides in the diagnosis and therapy of oncological diseases. Appl. Radiat. Isot. 57, 749–763 (2002).

    Article  CAS  Google Scholar 

  19. Lam, K.S. et al. A new type of synthetic peptide library for identifying ligand-binding activity. Nature 354, 82–84 (1991).

    Article  CAS  Google Scholar 

  20. Lam, K.S., Lebl, M. & Krchnak, V. The “one-bead-one-compound” combinatorial library method. Chem. Rev. 97, 411–448 (1997).

    Article  CAS  Google Scholar 

  21. Meldal, M., Svendsen, I., Breddam, K. & Auzanneau, F.I. Portion-mixing peptide libraries of quenched fluorogenic substrates for complete subsite mapping of endoprotease specificity. Proc. Natl. Acad. Sci. USA 91, 3314–3318 (1994).

    Article  CAS  Google Scholar 

  22. Spring, D.R., Krishnan, S., Blackwell, H.E. & Schreiber, S.L. Diversity-oriented synthesis of biaryl-containing medium rings using a one bead/one stock solution platform. J. Am. Chem. Soc. 124, 1354–1363 (2002).

    Article  CAS  Google Scholar 

  23. Copeland, G.T. & Miller, S.J. Selection of enantioselective acyl transfer catalysts from a pooled peptide library through a fluorescence-based activity assay: an approach to kinetic resolution of secondary alcohols of broad structural scope. J. Am. Chem. Soc. 123, 6496–6502 (2001).

    Article  CAS  Google Scholar 

  24. Liu, R., Marik, J. & Lam, K.S. A novel peptide-based encoding system for “one-bead one-compound” peptidomimetic and small molecule combinatorial libraries. J. Am. Chem. Soc. 124, 7678–7680 (2002).

    Article  CAS  Google Scholar 

  25. Song, A.M., Zhang, J.H., Lebrilla, C.B. & Lam, K.S. A novel and rapid encoding method based on mass spectrometry for “One-Bead One-Compound” small molecule combinatorial libraries. J. Am. Chem. Soc. 125, 6180–6188 (2003).

    Article  CAS  Google Scholar 

  26. Affleck, R.L. Solutions for library encoding to create collections of discrete compounds. Curr. Opin. Chem. Biol. 5, 257–263 (2001).

    Article  CAS  Google Scholar 

  27. Park, S.I. et al. The use of one-bead one-compound combinatorial library method to identify peptide ligands for alpha-4-beta-1 integrin receptor in non-Hodgkin's lymphoma. Lett. Pept. Sci. 8, 171–178 (2002).

    Google Scholar 

  28. Lin, K. et al. Selective, tight-binding inhibitors of integrin alpha4beta1 that inhibit allergic airway responses. J. Med. Chem. 42, 920–934 (1999).

    Article  CAS  Google Scholar 

  29. Falcioni, R. et al. Expression of beta 1, beta 3, beta 4, and beta 5 integrins by human lung carcinoma cells of different histotypes. Exp. Cell Res. 210, 113–122 (1994).

    Article  CAS  Google Scholar 

  30. Irie, A., Kamata, T., Puzon-McLaughlin, W. & Takada, Y. Critical amino acid residues for ligand binding are clustered in a predicted beta-turn of the third N-terminal repeat in the integrin alpha 4 and alpha 5 subunits. EMBO J. 14, 5550–5556 (1995).

    Article  CAS  Google Scholar 

  31. Weissleder, R. & Ntziachristos, V. Shedding light onto live molecular targets. Nat. Med. 9, 123–128 (2003).

    Article  CAS  Google Scholar 

  32. Becker, A. et al. Receptor-targeted optical imaging of tumors with near-infrared fluorescent ligands. Nat. Biotechnol. 19, 327–331 (2001).

    Article  CAS  Google Scholar 

  33. Wilbur, D.S., Hamlin, D.K., Sanderson, J. & Lin, Y. Streptavidin in antibody pretargeting. 4. Site-directed mutation provides evidence that both arginine and lysine residues are involved in kidney localization. Bioconjug. Chem. 15, 1454–1463 (2004).

    Article  CAS  Google Scholar 

  34. Singh, J. et al. Identification of potent and novel alpha4beta1 antagonists using in silico screening. J. Med. Chem. 45, 2988–2993 (2002).

    Article  CAS  Google Scholar 

  35. Hynes, R.O. Integrins: bidirectional, allosteric signaling machines. Cell 110, 673–687 (2002).

    Article  CAS  Google Scholar 

  36. Shimaoka, M., Takagi, J. & Springer, T.A. Conformational regulation of integrin structure and function. Annu. Rev. Biophys. Biomol. Struct. 31, 485–516 (2002).

    Article  CAS  Google Scholar 

  37. Rivera-Nieves, J. et al. L-selectin, alpha 4 beta 1, and alpha 4 beta 7 integrins participate in CD4+ T cell recruitment to chronically inflamed small intestine. J. Immunol. 174, 2343–2352 (2005).

    Article  CAS  Google Scholar 

  38. Chen, X. et al. Micro-PET imaging of alphavbeta3-integrin expression with 18F-labeled dimeric RGD peptide. Mol. Imaging 3, 96–104 (2004).

    Article  CAS  Google Scholar 

  39. Venditto, V.J., Regino, C.A. & Brechbiel, M.W. PAMAM dendrimer based macromolecules as improved contrast agents. Mol. Pharm. 2, 302–311 (2005).

    Article  CAS  Google Scholar 

  40. McIntyre, J.O. et al. Development of a novel fluorogenic proteolytic beacon for in vivo detection and imaging of tumour-associated matrix metalloproteinase-7 activity. Biochem. J. 377, 617–628 (2004).

    Article  CAS  Google Scholar 

  41. Boerman, O.C., van Schaijk, F.G., Oyen, W.J. & Corstens, F.H. Pretargeted radioimmunotherapy of cancer: progress step by step. J. Nucl. Med. 44, 400–411 (2003).

    PubMed  Google Scholar 

  42. Rosebrough, S.F. Two-step immunological approaches for imaging and therapy. Q. J. Nucl. Med. 40, 234–251 (1996).

    CAS  PubMed  Google Scholar 

  43. Yang, J.T., Rayburn, H. & Hynes, R.O. Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development. Development 121, 549–560 (1995).

    CAS  PubMed  Google Scholar 

  44. Spies, S.M. Imaging and dosing in radioimmunotherapy with Yttrium 90 ibritumomab tiuxetan (Zevalin). Semin. Nucl. Med. 34, 10–13 (2004).

    Article  Google Scholar 

  45. Fields, G.B. & Noble, R.L. Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids. Int. J. Pept. Protein Res. 35, 161–214 (1990).

    Article  CAS  Google Scholar 

  46. Kaiser, E., Colescott, R.L., Bossinger, C.D. & Cook, P.I. Color test for detection of free terminal amino groups in the solid-phase synthesis of peptides. Anal. Biochem. 34, 595–598 (1970).

    Article  CAS  Google Scholar 

  47. Kamata, T., Puzon, W. & Takada, Y. Identification of putative ligand binding sites within I domain of integrin alpha 2 beta 1 (VLA-2, CD49b/CD29). J. Biol. Chem. 269, 9659–9663 (1994).

    CAS  PubMed  Google Scholar 

  48. Kamata, T., Wright, R. & Takada, Y. Critical threonine and aspartic acid residues within the I domains of beta 2 integrins for interactions with intercellular adhesion molecule 1 (ICAM-1) and C3bi. J. Biol. Chem. 270, 12531–12535 (1995).

    Article  CAS  Google Scholar 

  49. Eto, K. et al. Functional classification of ADAMs based on a conserved motif for binding to integrin alpha 9beta 1: implications for sperm-egg binding and other cell interactions. J. Biol. Chem. 277, 17804–17810 (2002).

    Article  CAS  Google Scholar 

  50. Tarui, T., Miles, L.A. & Takada, Y. Specific interaction of angiostatin with integrin alpha(v)beta(3) in endothelial cells. J. Biol. Chem. 276, 39562–39568 (2001).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by the National Institutes of Health (R33CA-86364, R33CA-99136 and U19CA113298) and the National Science Foundation (CHE-0302122). The 500-MHz NMR spectrometer was purchased in part with the grant NSF 9724412. We thank R. Wisdom, A. Lehman, S.M. Dixon and A. Enstrom for editorial assistance.

Author information

Authors and Affiliations

Authors

Contributions

L.P., study concept and design, acquisition and analysis of the majority of the data of the manuscript and drafting of the manuscript; R.L., design and synthesis of OBOC libraries and compounds, sequencing and decoding of positive beads and drafting of the manuscript; J.M., obtaining the NMR data of LLP2A and synthesis of CS-1 peptides; X.W., analysis of the NMR data of LLP1A, LLP1A-biotin and scrambled LLP2A-biotin; Y.T., providing all the CHO cell clones transfected with α4 mutants and critical revision of the manuscript; K.L., principal investigator of the project, study concept and design, critical revision of the manuscript, obtained funding and study supervision.

Note: Supplementary information is available on the Nature Chemical Biology website.

Corresponding author

Correspondence to Kit S Lam.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Characterization of LLP2A (PDF 94 kb)

Supplementary Table 1

The 30 isocyanates in the X1 position of the initial library (PDF 23 kb)

Supplementary Table 2

The 14 nitro-containing compounds in the X2 position of the initial library (PDF 21 kb)

Supplementary Table 3

The 20 Lys derivatives derived from carboxylic acids in the X3 position of the initial library (PDF 25 kb)

Supplementary Table 4

Building blocks at positions X3, X4, and X5 of the initial library (PDF 34 kb)

Supplementary Table 5

The 45 amino acids at positions X6, X7, and X8 of the initial library (PDF 25 kb)

Supplementary Table 6

Building blocks at positions of X3, X4, and X5 of the focused library (PDF 46 kb)

Supplementary Methods (PDF 430 kb)

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Peng, L., Liu, R., Marik, J. et al. Combinatorial chemistry identifies high-affinity peptidomimetics against α4β1 integrin for in vivo tumor imaging. Nat Chem Biol 2, 381–389 (2006). https://doi.org/10.1038/nchembio798

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