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


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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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




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).

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