The tri-amino acid sequence, Arg-Gly-Asp (RGD), is the cell adhesion motif displayed on extracellular matrix (ECM), blood and cell surface proteins such as laminin, vitronectin, fibrinogen, von Willebrand factor and osteopontin. As the specific recognition site of interaction between integrins and their ligands, RGD plays an important role in the interactions between cells and the ECM. RGD is involved in regulating many biological activities such as cell attachment, cell spreading, actin-skeleton formation and focal-adhesion formation with integrins. The RGD sequence has garnered much attention over the past two decades and the potential applications for RGD peptides are diverse such as the treatment and diagnosis of tumors, development of antithrombotic drugs and tissue engineering (1).
Integrin-binding can be reproduced by short RGD-containing peptides. Synthetic RGD peptides can be linear or cyclic; however, linear RGD peptides have some drawbacks such as low binding affinity and rapid degradation by proteases in serum. On the other hand, cyclic RGD peptides such as those with disulfide, thioether and rigid aromatic rings have shown improved integrin receptor binding affinity and selectivity compared to linear RGD peptides (1). Peptides that bind to specific integrins have been created by incorporating specific amino acids flanking the RGD sequence and cyclizing them. In addition, dimeric forms of cyclic RGD peptides have been produced to improve their tumor targeting properties (Figure 1).
Figure 1 E(c(RGDfK))₂, a dimeric form of c(RGDfK), Bachem H-7952
RGD has a high affinity for αvβ3 integrins overexpressed on tumor neovasculature and on the surface of tumor cells making RGD peptides interesting for the treatment and diagnosis of tumors. When conjugated to imaging agents, RGD peptides can provide high specificity and sensitivity in tumor imaging applications (2). For example, researchers have shown that RGD peptides conjugated to the near-infrared fluorescent dye Cy7 could be useful for noninvasive detection and semi quantification of tumor integrin expression (3). In addition, investigators have designed and tested RGD peptides to serve as targeting molecules that deliver radionuclides to integrins on tumor cells. RGD radiotracers are usually αvβ3 integrin targeted but many cyclic RGD peptide tracers are also able to bind to αvβ5, α5β1, α6β4, α4β1, and αvβ6 integrins and this ability can lead to enhanced tumor uptake. Preclinical and clinical studies have shown that radiolabeled cyclic RGD peptides such as 99mTc-3P-RGD2, 18F-Alfatide-I and 18F-Alfatide-II are useful probes for use in single photon emission computed tomography (SPECT) or positron emission tomography (PET) imaging for early cancer detection and for monitoring tumor response to anti-angiogenic treatments in a noninvasive manner (1).
The therapeutic efficacy of anti-cancer drugs can be limited by poor penetration into tumor tissue and by adverse effects on healthy cells. Consequently, interest has turned to tumor-penetrating peptides such as iRGD (cyclic CRGDKRGPDC) that are under development to deliver drugs into extravascular tumor tissue. When injected intravenously, iRGD targets αv integrins expressed on tumor vessels. iRGD peptide is proteolytically cleaved in the tumor exposing what is known as the CendR (C-end Rule) motif at the C-terminus of the peptide. CendR binds to neuropilins, molecules that regulate vascular permeability, to activate a bulk transport pathway through tumor tissue. This transport system allows drugs conjugated to iRGD and free drugs co-administered with iRGD to pass through vessel walls and spread within the tumor tissue (4). In research studies in mice, the co-administration of Gemcitabine, an approved chemotherapy drug for non-small cell lung cancer, with iRGD peptide resulted in enhanced tumor-penetrating ability and therapeutic efficacy of Gemcitabine. Reports from animal studies have shown that iRGD can enhance the intratumoral dissemination and efficacy of Doxorubicin, Paclitaxel and Endostatin when conjugated with these drugs or drug-loaded vehicles. Additional studies have shown that co-administration with iRGD enhances efficacy and accumulation of Paclitaxel, Doxorubicin, Trastuzumab and Cisplatin (5).
RGD peptides also hold promise as antithrombotic drugs. The glycoprotein (GP) IIb/IIIa receptor is a key integrin involved in platelet aggregation and thrombus formation. Fibrinogen is the main ligand that binds to the GP IIb/IIIa receptor and the binding of fibrinogen to this receptor occurs via RGD sequences located in fibrinogen. The binding of fibrinogen to GP IIb/IIIa results in cross-linking between adjacent platelets, which leads to thrombosis (6). Several types of GP IIb/IIIa receptor antagonists exist or are under development to inhibit platelet aggregation including monoclonal antibodies against GP IIb/IIIa and peptide and non-peptide antagonists of GP IIb/IIIa. The peptide antagonists include RGD-containing snake venoms, linear RGD peptides and cyclic RGD or KGD peptides (7). For example, the approved drug Integrilin® (Figure 2), a disulfide-linked cyclic KGD heptapeptide, acts as a competitive antagonist to fibrinogen and reversibly binds to the GP IIb/IIIa receptor. Several regulatory authorities have approved Integrilin® for the treatment of angina, acute coronary syndrome and myocardial infarction.
Figure 2 Eptifibatide, a GP IIb/IIIa receptor antagonist, Bachem H-6654
RGD peptides are widely studied for tissue engineering applications. Synthetic materials used in orthopedic and dental treatments often have useful properties such as 3-D architecture and mechanical strength but these materials do not support strong cell adhesion without the addition of an adhesive factor (8). Immobilization of RGD on the material’s surface helps increase cell adhesion and promotes new tissue formation. Researchers have studied attaching RGD to oxide-coated metals and titanium surfaces in order to enhance implant bone healing (2). In addition, RGD may lead to a biomimetic approach for cornea repair and other tissue needs. For example, researchers are developing an RGD-coupled silk protein-biomaterial to replicate native corneal stromal tissue architecture. Gil et al. reported that tissue-engineered cornea silk biomaterials replicated native corneal stromal lamellar architecture with the appropriate corneal stromal phenotypes and aligned collagen fibril lamellae in culture. The RGD coupling in this application enhanced cell attachment, proliferation, alignment and expression of corneal stroma markers (9).
Further study and development of RGD peptides in the fields of oncology, thrombosis and tissue engineering may lead to improved cancer treatments and imaging, antithrombotic drugs and novel tissue-engineering applications. Bachem offers over 50 different RGD peptides and analogs including many cyclic RGD peptides and a Tide FluorTM labelled RGD peptide to support researchers exploring these areas.
(1) S. Liu, Radiolabeled cyclic RGD peptide bioconjugates as radiotracers targeting multiple integrins, Bioconjug. Chem., 26(18), 1413-1438 (2016)
(2) F. Wang et al., The functions and applications of RGD in tumor therapy and tissue engineering, Int. J. Mol. Sci., 14(7), 13447–13462 (2013)
(3) Y. Wu et al., Near-infrared fluorescence imaging of tumor integrin alpha v beta 3 expression with Cy7-labeled RGD multimers, Mol. Imaging Biol., 8(4), 226-236 (2006)
(4) K. Sugahara et al., Tumor-penetrating iRGD peptide inhibits metastasis, Mol. Cancer Ther., 14(1), 120-128 (2015)
(5) Q. Zhang et al., A novel strategy to improve the therapeutic efficacy of gemcitabine for non-small cell lung cancer by the tumor-penetrating peptide iRGD, PLoS One, 10(6), (2015)
(6) T.A. Meadows and D.L. Bhatt, Clinical aspects of platelet inhibitors and thrombus formation, Circ. Res., 100(9), 1261-1275 (2007)
(7) A.I. Schafer, Antiplatelet therapy with glycoprotein IIb/IIIa receptor inhibitors and other novel agents, Tex. Heart Inst. J., 24(2), 90-96 (1997)
(8) S. Bellis, Advantages of RGD peptides for directing cell association with biomaterials, Biomaterials, 32(18), 4205-4210 (2011)
(9) E. Gil et al., Helicoidal multi-lamellar features of RGD-functionalized silk biomaterials for corneal tissue engineering, Biomaterials, 31(34), 8953-8963 (2010)