RGD Negative Control

An integrin αVβ3-targeting linear RGD mimetic containing a small-molecule drug conjugate (SMDC) was synthesized by combining the antimitotic agent monomethyl auristatin E (MMAE), an enzymatically cleavable Val-Ala-PABC linker with a linear conjugable RGD mimetic. The structure proposal for the conjugable RGD mimetic was suggested upon the DAD mapping analysis of a previously synthesized small-molecule RGD mimetic array based on a tyrosine scaffold. Therefore, a diversifying strategy was developed as well as a novel method for the partial hydrogenation of pyrimidines in the presence of the hydrogenolytically cleavable Cbz group. The small-molecule RGD mimetics were evaluated in an ELISA-like assay, and the structural relationships were analyzed by DAD mapping revealing activity differences induced by structural changes as visualized in dependence on special structural motifs. This provided a lead structure for generation of an SMDC containing the antimitotic drug MMAE. The resulting SMDC containing a linear RGD mimetic was tested in a cell adhesion and an in vitro cell viability assay in comparison to reference SMDCs containing cRGDfK or cRADfK as the homing device. The linear RGD SMDC and the cRGDfK SMDC inhibited adhesion of αVβ3-positive WM115 cells to vitronectin with IC50 values in the low µM range, while no effect was observed for the αVβ3-negative M21-L cell line. The cRADfK SMDC used as a negative control was about 30-fold less active in the cell adhesion assay than the cRGDfK SMDC. Conversely, both the linear RGD SMDC and the cRGDfK SMDC are about 55-fold less cytotoxic than MMAE against the αVβ3-positive WM115 cell line with IC50 values in the nM range, while the cRADfK SMDC is 150-fold less cytotoxic than MMAE. Hence, integrin binding also influences the antiproliferative activity giving a targeting index of 2.8.

Long noncoding RNA (lncRNA) differentiation antagonizing noncoding RNA (DANCR) is a proven oncogenic lncRNA across multiple cancer types. Its effects on cancer cell migration and invasion position it as a potential target for therapy on multiple levels of gene regulation. DANCR is overexpressed in non-small cell lung cancer (NSCLC), the most common lung cancer subtype with poor patient survival. To effectively deliver small interfering RNA (siRNA) against DANCR for NSCLC therapy, we used arginine-glycine-aspartic acid (RGD)-poly(ethylene glycol) (PEG)-(1-aminoethyl)-iminobis[N-oleicylcysteinyl-1-aminoethyl)propionamide] (ECO)/small interfering RNA against DANCR (siDANCR) nanoparticles to transfect A549 and NCI-H1299 cells. Over 90% DANCR silencing was observed along with inhibition of cell migration, invasion, and spheroid formation relative to transfection with negative control siRNA in RGD-PEG-ECO nanoparticles. DANCR knockdown further showed efficacy in reducing migration and invasion of epidermal growth factor receptor (EGFR)-inhibitor resistant NSCLC along with resensitization to the inhibitor. RGD-PEG-ECO/siDANCR demonstrated silencing for up to 7 d following a single transfection. The results suggest nanoparticle-mediated RNA interference against DANCR as a potential approach for NSCLC treatment by regulating cell migration and invasion in addition to improving EGFR inhibitor response.

Interferon (IFN) regulates immune responses and antitumor activity. Arginine-glycine-aspartic acid (RGD) peptides can specifically bind to integrin αvβ3, a transmembrane receptor that is highly expressed on the surface of various cancer cells. In this study, we expressed recombinant RGD-IFN-α2a-core fusion proteins and assessed their antitumor activity in vitro. Two RGD-IFN-α2a-core fusion proteins and a negative control protein were expressed in vitro. These two RGD-IFN-α2a-core fusion proteins could bind the tumor cell surface specifically and did not bind to normal cells. RGD-IFN-α2a-core fusion protein treatment of tumor cells significantly reduced cell viability and induced apoptosis in a dose-dependent manner. At the 'mRNA' level, both proteins could upregulate CASP3 expression. These data indicate that both laboratory-engineered RGD-IFN-α2a-core fusion proteins could bind the surface of tumor cells and induce apoptosis in vitro. Further studies will investigate the in-vivo antitumor activities of the RGD-IFN-α2a-core fusion proteins.