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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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GKVLAK–(IRIS Blue-(1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid t-butyl ester)-10-acetic acid monoamide))–GGGGTVQQEL

DCCP16-IRIS Blue
, PhD
National Center for Biotechnology Information, NLM, NIH

Created: ; Last Update: April 26, 2011.

Chemical name:GKVLAK–(IRIS Blue-(1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid t-butyl ester)-10-acetic acid monoamide))–GGGGTVQQELImage DCCP16IRIS.jpg
Abbreviated name:DCCP16-IRIS Blue
Synonym:
Agent Category:Peptides
Target:Transglutaminases
Target Category:Enzymes
Method of detection:Optical imaging
Source of signal / contrast:IRIS Blue
Activation:Yes
Studies:
  • Checkbox In vitro
  • Checkbox Rodents
Structure of DCCP16-IRIS Blue (1).

Background

[PubMed]

The fluorescently labeled 16-peptide agent GKVLAK-(IRIS Blue-(1,4,7,10-tetraazacyclododecane-1,4,7-tris(acetic acid t-butyl ester)-10-acetic acid monoamide (DOTAMA)))-GGGGTVQQEL, abbreviated as DCCP16-IRIS Blue, was developed by Tei et al. for optical imaging of the pathological processes associated with high transglutaminase activity (1).

Transglutaminases are a family of Ca2+-dependent enzymes that catalyze extracellular covalent cross-linking of proteins (2, 3). These enzymes participate in many important biological processes, such as blood coagulation, skin-barrier formation, hardening of the fertilization envelope, and extracellular matrix assembly (2, 4). They are also involved in multiple pathological processes, including wound healing, cancer, myocardial infarctions, and atherothrombosis (5-7). The fibrin-stabilizing factor XIII (FXIII, also known as plasma transglutaminase) and the tissue transglutaminase (TG2) are the two enzymes that have attracted the greatest interest in transglutaminase-targeted imaging and therapy (1, 8, 9). FXIII cross-links fibrin during blood clotting and subsequently produces a mechanically stronger clot with high fibrinolytic resistance, whereas TG2 catalyzes covalent cross-linking of the extracellular matrix. The enzyme-mediated cross-linking is achieved by forming an isopeptide bond between the γ-carbonyl group of a glutamine (Gln) in one protein and the ε-amino group of a lysine (Lys) residue in a nearby protein (3, 9). This cross-link can be blocked by covalent incorporation of an acyl-acceptor amine substrate into the fibrin units or an acyl-donor Gln-containing peptide complementary to the FXIII-reactive Lys donor cross-linking sites of the protein (1). FXIII and TG2 are much more sensitive toward the Gln-bearing substrates than to amine donor Lys residues. These transglutaminase features form the fundamental basis for the development of imaging agents for detection of transglutaminase activity. In recent years, peptides based either on β-casein and α2-antiplasmin for the Gln-donor substrate requirements or on the bovine αA-crystallin for the amine donor substrate requirements have been synthesized for in vitro transglutaminase assays, and imaging probes consisting of peptides from α2-antiplasmin have been investigated for mapping the activity of endogenous FXIII and TG2 (10-12).

Tei et al. designed a new model peptide, DCCP16, which was labeled with Gd for MRI and the fluorescent dye IRIS Blue for optical imaging (1). IRIS Blue is a cyanine dye that has an absorption maximum of 660 nm and an emission maximum of 680 nm in phosphate buffer at pH 7.4. The DCCP16 peptide consists of two moieties. The first moiety is the hexapeptide TVQQEL, which bears two Gln residues and one valine residue. The second moiety is the pentapeptide GKVLA, which is known to be a good substrate for transglutaminases. A four-glycine spacer and a Lys residue were inserted between the Lys and the Gln moieties for conjugation of the Gd-DOTAMA or the IRIS Blue-DOTAMA. This spacer-DOTAMA was chosen to keep the Gd- or IRIS Blue-complex far enough from interfering with the active site. The MRI and optical probes were therefore set as GKVLAK-(Gd-DOTAMA)-GGGGTVQQEL (Gd-DCCP16) and GKVLAK-(IRIS Blue-DOTAMA)-GGGGTVQQEL (DCCP16-IRIS Blue), respectively. In vivo effectiveness of the two agents was validated with MRI and optical mapping of transglutaminase-induced agent retention in mouse models of tumor xenografts and blood clotting (1). Noninvasive imaging of transglutaminase activity with Gd-DCCP16 or DCCP16-IRIS Blue provides an important tool for detecting and monitoring transglutaminase-targeted therapy in diverse pathologies including cancer, wound healing, myocardial infarction, and pregnancy failure associated with congenital FXIII deficiency (1). This chapter describes the results generated with DCCP16-IRIS Blue. The results obtained with Gd-DCCP16 were described in the chapter on Gd-DCCP16 in MICAD.

Synthesis

[PubMed]

The DCCP16 peptide GKVLAKGGGGTVQQEL and the DCCP16 control GAVLAKGGGGTVAAEL were synthesized with solid-phase peptide synthesis with a rink amide resin as solid support and the standard Fmoc strategy (1). As the negative control peptide, the amino acids responsible for the transglutaminase activity of DCCP16 (Gln and Lys) were replaced with alanine. DOTAMA was coupled to the ε-NH2 group of the Lys residue. The purity values of both GKVLAK-(DOTAMA)-GGGGTVQQEL (DCCP16) and GAVLAK-(DOTAMA)-GGGGTVAAEL (DCCP16 control) were >95%. The yields of DCCP16 and DCCP16 control were 25% (48 mg) and 20% (35 mg), respectively. The fluorescent dye IRIS Blue was then conjugated to DCCP16 and DCCP16 control through DOTAMA. The final product DCCP16-IRIS Blue was purified and characterized. The yield of DCCP16-IRIS Blue was 16%.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

Two experiments were performed to verify that DCCP16 is covalently linked by transglutaminases (1). TG2-Mediated covalent linking between DCCP16-IRIS Blue and casein was first demonstrated by incubation of both agents in the presence of TG2 and by analysis of the product with denaturing SDS gel electrophoresis. A strong fluorescent band with a molecular weight of 25–50 kDa was observed, which corresponds to the product generated by covalent binding of more than one moiety of DCCP16-IRIS Blue (~2 kDa) to N’,N’-dimethylcasein (180 amino acids, 25–30 kDa). The results indicated that DCCP16-IRIS Blue could serve as a substrate that allows covalent cross-linking to dimethylcasein, catalyzed by TG2 (1).

TG2-Mediated covalent linking was further verified with incubation of large MCF7 (human breast cancer cell line) spheroids (1 mm in diameter) with DCCP16-IRIS Blue for 48 h (1). Strong fluorescence was observed only from spheroids treated with DCCP16-IRIS Blue.

Animal Studies

Rodents

[PubMed]

Optical detection of transglutaminase activity with DCCP16-IRIS Blue was tested in animal models of thrombus (n = 6 mice) (1). Thrombi were induced on the ears of CD1 female nude mice with laser (n = 2 mice) and with thermal injury (n = 4 mice), and DCCP16-IRIS Blue was subsequently administered intravenously. During the first 20 min after administration, DCCP16-IRIS Blue distributed evenly throughout the entire tissue surrounding the wounds. After 24 h, clearance of nonbound DCCP16-IRIS Blue was complete, and bound DCCP16-IRIS Blue was seen in both wounds. After 72 h, the wounds had partly healed and the clots were partially degraded, and the fluorescence was less intense. Residual fluorescence could be detected in one of the wounds 13 days after administration. These results indicate that DCCP16-IRIS Blue could serve as an effective in vivo substrate for the detection of transglutaminase activity (1). No results were reported for DCCP16-IRIS Blue control and no blocking studies were presented.

Other Non-Primate Mammals

[PubMed]

No references are currently available.

Non-Human Primates

[PubMed]

No references are currently available.

Human Studies

[PubMed]

No references are currently available.

References

1.
Tei L., Mazooz G., Shellef Y., Avni R., Vandoorne K., Barge A., Kalchenko V., Dewhirst M.W., Chaabane L., Miragoli L., Longo D., Neeman M., Aime S. Novel MRI and fluorescent probes responsive to the Factor XIII transglutaminase activity. Contrast Media Mol Imaging. 2010;5(4):213–22. [PubMed: 20812289]
2.
Bergamini C.M., Griffin M., Pansini F.S. Transglutaminase and vascular biology: physiopathologic implications and perspectives for therapeutic interventions. Curr Med Chem. 2005;12(20):2357–72. [PubMed: 16181137]
3.
Park D., Choi S.S., Ha K.S. Transglutaminase 2: a multi-functional protein in multiple subcellular compartments. Amino Acids. 2010;39(3):619–31. [PubMed: 20148342]
4.
Aeschlimann D., Thomazy V. Protein crosslinking in assembly and remodelling of extracellular matrices: the role of transglutaminases. Connect Tissue Res. 2000;41(1):1–27. [PubMed: 10826705]
5.
McCarthy J.R., Patel P., Botnaru I., Haghayeghi P., Weissleder R., Jaffer F.A. Multimodal nanoagents for the detection of intravascular thrombi. Bioconjug Chem. 2009;20(6):1251–5. [PMC free article: PMC2733224] [PubMed: 19456115]
6.
Jaffer F.A., Tung C.H., Wykrzykowska J.J., Ho N.H., Houng A.K., Reed G.L., Weissleder R. Molecular imaging of factor XIIIa activity in thrombosis using a novel, near-infrared fluorescent contrast agent that covalently links to thrombi. Circulation. 2004;110(2):170–6. [PubMed: 15210587]
7.
Mehta K., Kumar A., Kim H.I. Transglutaminase 2: a multi-tasking protein in the complex circuitry of inflammation and cancer. Biochem Pharmacol. 2010;80(12):1921–9. [PubMed: 20599779]
8.
Wilhelmus M.M., van Dam A.M., Drukarch B. Tissue transglutaminase: a novel pharmacological target in preventing toxic protein aggregation in neurodegenerative diseases. Eur J Pharmacol. 2008;585(2-3):464–72. [PubMed: 18417122]
9.
Collighan R.J., Griffin M. Transglutaminase 2 cross-linking of matrix proteins: biological significance and medical applications. Amino Acids. 2009;36(4):659–70. [PubMed: 18982407]
10.
Miserus R.J., Herias M.V., Prinzen L., Lobbes M.B., Van Suylen R.J., Dirksen A., Hackeng T.M., Heemskerk J.W., van Engelshoven J.M., Daemen M.J., van Zandvoort M.A., Heeneman S., Kooi M.E. Molecular MRI of early thrombus formation using a bimodal alpha2-antiplasmin-based contrast agent. JACC Cardiovasc Imaging. 2009;2(8):987–96. [PubMed: 19679287]
11.
Mazooz G., Mehlman T., Lai T.S., Greenberg C.S., Dewhirst M.W., Neeman M. Development of magnetic resonance imaging contrast material for in vivo mapping of tissue transglutaminase activity. Cancer Res. 2005;65(4):1369–75. [PubMed: 15735023]
12.
Caccamo D., Curro M., Ientile R. Potential of transglutaminase 2 as a therapeutic target. Expert Opin Ther Targets. 2010;14(9):989–1003. [PubMed: 20670177]
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