Abstract
Development of therapies that selectively inhibit cancer cells but leave normal cells untouched is an important impediment to progress in cancer therapy. This paper reports the novel strategy employing the R-Tf-D-LP4 peptide, an unconventional cell penetrating peptide selectively targeting liver cancer cells, combined with the potent antitumor cytokine interleukin 15 (IL-15), with selective activity towards liver cancer cells. The fusion protein was constructed as a chimera between the IL-15 and the targeting peptide R-Tf-D-LP4 to improve the targeting specificity of this protein to the desired cells. With the help of modern computer modeling technologies, a fusion protein was designed, and its interactions with the IL-15 receptor complex (IL-15Rα/IL-15Rβ/γc) were evaluated using a molecular docking technique. The structure and physicochemical properties of the chimeric protein were determined using programs such as trROSETTA, I-TASSER, and ProtParam, after which they were further verified using SAVES 6.1 server with an excellent ERRAT quality score of 99.35%. Molecular docking using ClusPro 2.0, along with interaction evaluation using PDBsum program, demonstrated stability of the complex and strong hydrogen bonds and salt bridge interactions. The results were verified using the Desmond module in Schrödinger software to simulate molecular dynamics, which revealed a stable structure and proper functioning of the complex in physiological conditions. The expression analysis forecasted good yields from E. coli fermentation. The construct of the IL15/R-Tf-D-LP4 protein gene was created and inserted into the pET-22b ( +) vector, which was then transformed in BL21 (DE3) cells. The recombinant fusion protein was obtained by affinity chromatography and tested for purity by SDS-PAGE and western blotting. Cytotoxicity activity of the fusion protein was evaluated against the HepG2 cell line with the MTT test. As a result, the cytotoxic effect with an IC50 value of 23 μg/ml was established, compared to the non-chimera protein's IC50 value of 62 μg/ml. The achieved results allow us to speak about the prospects of using IL-15/R-Tf-D-LP4 fusion protein for clinical purposes in the future.