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Molecular Imaging and Contrast Agent Database (MICAD) [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2004-2013.

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Molecular Imaging and Contrast Agent Database (MICAD) [Internet].

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Cy5.5-Knottin 2.5F

Cy5.5-Knottin 2.5F
, PhD
National Center for Biotechnology Information, NLM, NIH, Bethesda, MD 20894

Created: ; Last Update: May 28, 2009.

Chemical name:Cy5.5-Knottin 2.5F
Abbreviated name:Cy5.5-Knottin 2.5F
Agent Category:Ligand
Target:αvβ3, αvβ5, and α5β1 integrin receptors
Target Category:Receptors
Method of detection:Near-infrared fluorescence
Source of signal / contrast:Cy5.5
  • Checkbox In vitro
  • Checkbox Rodents
Structure not available in PubChem.



Integrin receptors that mediate tumor angiogenesis, growth, and metastasis through a complex network of signaling pathways are known to be expressed on the surface of cancerous tumor cells and neovasculature (1-3). Because of their role in tumor development and progression, inhibition of integrin receptor activity is being actively investigated in clinical trials for the treatment and imaging of various cancers (4, 5). Non-invasive imaging probes can be used to determine not only the efficacy of integrin-targeted anti-cancer drugs, but also to monitor disease progression and metastasis (6, 7). Although integrin receptors usually bind through the arginine-glycine-aspartic acid (RGD) motif of the extracellular matrix protein ligands, it is the amino acid residues surrounding the RGD motif that determine the receptor specificity and affinity for the ligand (4, 8, 9). As a result of the small size of the drugs, mainly peptides, that target the integrin receptor, it has been challenging to generate peptides containing the RGD motif that have an improved pharmacokinetic behavior, receptor affinity, and tumor uptake for in vivo imaging purposes. Any modification of the peptide structure has yielded integrin-targeted imaging agents that have only a limited advancement and application in the clinics (10, 11). Only one imaging compound with an RGD motif, a radioactive fluorine-labeled cyclic pentapeptide ([18F]-galacto-RGD), was determined to be suitable to identify integrin-positive tumors and to investigate αvβ3 integrin expression in humans. However, [18F]-galacto-RGD has been shown to have a low tumor uptake, and it generated a high background signal due to accumulation in the liver (12, 13).

In an effort to develop imaging agents for the detection of integrin receptors expressed on tumor cell surface and neovasculature, Kimura et al. (14) used the directed evolution technique (15, 16) to place the integrin-binding RGD motif into a cystine knot peptide (also known as knottins) trypsin inhibitor of the squash plant (Ecballium elaterium) (17). The peptide was reported to have a high affinity for the αvβ3 and αvβ5 or the αvβ3, αvβ5, and α5β1 integrin receptors. In general, knottins have a core structure containing a disulfide bond, are resistant to proteolysis, have a high thermodynamic stability, and are nonimmunogenic (14). The knottin peptide containing an RGD motif (designated as knottin 2.5F) was labeled with a near-infrared (NIR) fluorescent cyanine dye, Cy5.5, to obtain Cy5.5-knottin 2.5F, and the Cy5.5 conjugate was used to image integrin-expressing xenograft tumors in mice with NIR fluorescence imaging. It is pertinent to mention that the knottin 2.5F discussed in this chapter and the knottin 2.5D peptide discussed separately in MICAD ( were developed and investigated by Kimura et al. (14). The structural and amino acid sequence similarities and differences of the two peptides are described elsewhere (14).



The synthesis of knottin 2.5F was performed with the use of the 9-fluorenylmethylcarbonyl-based solid-phase technique as described by Kimura et al. (14). In addition, two other knottin peptides, FN-RGD2 (for use as positive a control) and FN-RDG2 (negative control) were synthesized using the same technique mentioned above. A third peptide, c(RGDyK), was obtained from commercial sources for use as a second control. The amino acid sequence, structure, and properties of the various integrin-binding peptides used for studies described in this chapter are discussed by Kimura et al. (14).

To conjugate knottin 2.5F to Cy5.5, the knottin was mixed with a solution of Cy5.5-N-hydroxysuccinimide ester in dimethylsulfoxide and triethylamine. The reaction was allowed to proceed at room temperature in the dark (duration not reported) with constant monitoring with reverse-phase high-performance liquid chromatography (RP-HPLC) on a C18 column (14). Upon completion of the reaction, the conjugated peptide was purified with semi-preparative RP-HPLC and freeze-dried for storage (conditions not reported). The peptide purity and molecular mass were respectively confirmed with analytical RP-HPLC and electrospray or matrix-assisted laser desorption/ionization time-of-flight mass spectrometery. Yield of the final product, the number of Cy5.5 molecules conjugated to each molecule of knottin 2.5F, and the stability of the fluorescent dye–labeled knottin were not reported.

Echistatin labeled with radioactive iodine (125I-echistatin), used as a control in some studies, was obtained from a commercial source (14).

In Vitro Studies: Testing in Cells and Tissues


To determine the relative integrin-binding affinities (reported as 50% inhibitory concentration (IC50)) of the modified and unmodified peptides, including knottin 2.5F, a competition binding assay was performed with 125I-echistatin (a strong antagonist of αvβ3, αvβ5, α5β1 and αiibβ3 integrins (14)) using U-87MG cells (a human glioblastoma cell line that expresses several types of integrins on the cell surface) as described by Kimura et al. (14). The IC50 for Cy5.5-knottin 2.5F was determined to be 3.5 ± 0.8 nmol/L compared with IC50 values of 26.5 ± 5.0 nmol/L, 370 ± 150 nmol/L, and 860 ± 400 nmol/L for the unconjugated knottin 2.5F, control FN-RGD2 and c(RGDyK) peptides, respectively. The negative control FN-RDG2 peptide, with or without Cy5.5 conjugation, did not compete with 125I-echistatin for binding to the U-87MG cells (14).

The integrin-binding specificity of Cy5.5-knottin 2.5F was investigated with a competition displacement assay using immobilized integrin receptors and 125I-echistatin (14). Different concentrations (5 and 50 nmol/L) of the conjugated knottin 2.5F and the control peptides were respectively incubated with 125I-echistatin in microtiter plates coated with detergent-solubalized αvβ3, αvβ5, α5β1, and αiibβ3 integrin receptors for 3 h at room temperature. The plates were washed three times to remove unbound radioactivity, and the contents were solubalized in 2 N sodium hydroxide to determine the amount of receptor-bound radioactivity. Compared with the conjugated FN-RGD2 and c(RGDyK) peptides, the Cy5.5-knottin 2.5F peptide exhibited a high competition for125I-echistatin binding to the different integrin receptors (varying from ~35 to 95% reduction in binding of labeled echistatin using a low (5nmol/L) and a high (50 nmol/L) concentration of the unlabeled peptides) except the αiibβ3 receptor, which is expressed mainly on blood platelet cells (14).

Animal Studies



The use of Cy5.5-knottin 2.5F as an NIR imaging probe was investigated in mice bearing human U-87MG cell xenograft tumors and compared with imaging results obtained with the Cy5.5-conjugated control peptides (14). The animals (n = 3 mice for each probe) were injected with the respective Cy5.5-conjugated peptides through the tail vein and imaged at various time points up to 24 h. Whole-body imaging was performed on the animals, and the tumor/normal tissue fluorescence ratios (T/N) were calculated. The T/N ratios generated (for details see Figure 3B in reference 14) with Cy5.5-knottin 2.5F had a low standard deviation (~3.0 at 1 h and ~3.75 at 24 h) and were significantly (P < 0.05) higher than those obtained from the control Cy5.5-conjugated peptides (~2.2 at 1 h and ~3.0 at 24 h) at all time points from 0 to 24 h after administration of the probes. Also, the signal obtained with the positive control Cy5.5-conjugated peptides was only slightly higher than that of the negative control, FN-RDG2 peptide (~2 at 1 h and 2.5 at 24 h). No in vivo biodistribution of the Cy5.5-conjugated knottin 2.5F peptide was reported.

From these studies the investigators concluded that the direct evolution–engineered integrin-binding peptides are probably suitable for the detection and diagnosis of various cancers (14).

Other Non-Primate Mammals


No references are currently available.

Non-Human Primates


No references are currently available.

Human Studies


No references are currently available.

Supplemental Information


NIH Support

  1. Some of the work presented in this chapter was supported by National Cancer Institute grants 5K01 CA104706, 5R25T CA118681, and R25T CA118681 and a National Institutes of Health grant P50 CA114747.


Legate K.R., Wickstrom S.A., Fassler R. Genetic and cell biological analysis of integrin outside-in signaling. Genes Dev. 2009;23(4):397–418. [PubMed: 19240129]
Pontier S.M., Muller W.J. Integrins in mammary-stem-cell biology and breast-cancer progression--a role in cancer stem cells? J Cell Sci. 2009;122(Pt 2):207–14. [PMC free article: PMC2714417] [PubMed: 19118213]
Streuli C.H. Integrins and cell-fate determination. J Cell Sci. 2009;122(Pt 2):171–7. [PMC free article: PMC2714415] [PubMed: 19118209]
Moschos S.J., Drogowski L.M., Reppert S.L., Kirkwood J.M. Integrins and cancer. Oncology. 2007;21(9) Suppl 3:13–20. [PubMed: 17927026]
Fujita Y., Abe R., Shimizu H. Clinical approaches toward tumor angiogenesis: past, present and future. Curr Pharm Des. 2008;14(36):3820–34. [PubMed: 19128235]
Zaccaro L., Del Gatto A., Pedone C., Saviano M. Peptides for tumour therapy and diagnosis: current status and future directions. Curr Med Chem. 2009;16(7):780–95. [PubMed: 19275595]
Haubner R., Decristoforo C. Radiolabelled RGD peptides and peptidomimetics for tumour targeting. Front Biosci. 2009;14:872–86. [PubMed: 19273105]
Chen, C.Y., J.H. Shiu, Y.H. Hsieh, Y.C. Liu, Y.C. Chen, Y.C. Chen, W.Y. Jeng, M.J. Tang, S.J. Lo, and W.J. Chuang, Effect of D to E mutation of the RGD motif in rhodostomin on its activity, structure, and dynamics: Importance of the interactions between the D residue and integrin. Proteins, 2009. [PubMed: 19280603]
Manzoni, L., L. Belvisi, D. Arosio, M. Civera, M. Pilkington-Miksa, D. Potenza, A. Caprini, E.M. Araldi, E. Monferini, M. Mancino, F. Podesta, and C. Scolastico, Cyclic RGD-Containing Functionalized Azabicycloalkane Peptides as Potent Integrin Antagonists for Tumor Targeting. ChemMedChem, 2009. [PubMed: 19212960]
Cai W., Rao J., Gambhir S.S., Chen X. How molecular imaging is speeding up antiangiogenic drug development. Mol Cancer Ther. 2006;5(11):2624–33. [PubMed: 17121909]
Haubner R. Alphavbeta3-integrin imaging: a new approach to characterise angiogenesis? Eur J Nucl Med Mol Imaging. 2006;33 Suppl 1:54–63. [PubMed: 16791598]
Haubner R., Weber W.A., Beer A.J., Vabuliene E., Reim D., Sarbia M., Becker K.F., Goebel M., Hein R., Wester H.J., Kessler H., Schwaiger M. Noninvasive visualization of the activated alphavbeta3 integrin in cancer patients by positron emission tomography and [18F]Galacto-RGD. PLoS Med. 2005;2(3):e70. [PMC free article: PMC1069665] [PubMed: 15783258]
Beer A.J., Haubner R., Sarbia M., Goebel M., Luderschmidt S., Grosu A.L., Schnell O., Niemeyer M., Kessler H., Wester H.J., Weber W.A., Schwaiger M. Positron emission tomography using [18F]Galacto-RGD identifies the level of integrin alpha(v)beta3 expression in man. Clin Cancer Res. 2006;12(13):3942–9. [PubMed: 16818691]
Kimura R.H., Cheng Z., Gambhir S.S., Cochran J.R. Engineered knottin peptides: a new class of agents for imaging integrin expression in living subjects. Cancer Res. 2009;69(6):2435–42. [PMC free article: PMC2833353] [PubMed: 19276378]
Otten L.G., Quax W.J. Directed evolution: selecting today's biocatalysts. Biomol Eng. 2005;22(1-3):1–9. [PubMed: 15857778]
Leemhuis H., Kelly R.M., Dijkhuizen L. Directed evolution of enzymes: Library screening strategies. IUBMB Life. 2009;61(3):222–8. [PubMed: 19180668]
Favel A., Mattras H., Coletti-Previero M.A., Zwilling R., Robinson E.A., Castro B. Protease inhibitors from Ecballium elaterium seeds. Int J Pept Protein Res. 1989;33(3):202–8. [PubMed: 2654042]


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