Antitumor Effects of a Peptide Sequences Based on Gramicidin Molecule as Possible Inhibitor of PD-1 and PD-L-1

The new generation of anticancer compounds are peptide sequences that can lead to possible inhibition of PD-1 and PD-L-1 molecules. The objective of this study was to evaluate the antitumor effects of peptide sequences based on gramicidin molecule as potential inhibitors of PD-1 and PD-L-1 molecules in computerized and in vitro conditions. In this research, gramicidin sequence was used as the base molecule and its simpler derivatives were designed by adding or subtracting polar amino acids. For this, by using Askalaf software, first the sequences were created and then the structure was optimized. Then, using the molecular docking method (with the HDOCK online site), the interaction of all derivatives with PD1 and PDL1 molecules was evaluated individually, and their docking energy and RMSD were noted and compared. For the final selection of the best derivative, parameters of toxicity (with Toxinpred database), allergenicity (with Allertop database), antigenicity (with ABCpred database) were also considered using online databases. In the experimental part to evaluate the selected peptide in vitro, the best peptide identified in the computational part was selected and synthesized and purified by the solid phase technique by the French Proteogenics company. An ELISA kit was used to prove the involvement of the synthesized peptide in the connection between PD1 and PDL1 molecules. For this, PD1 molecules were first coated in the ELISA wells, and then PDL1 molecules were exposed to them, and the amount of color produced at 630 wavelength was evaluated by enzymatic method. Then, different concentrations of the selected peptide were added to the coated wells, and after incubation, PDL1 molecules were added, and as in the previous step, the amount of color produced was evaluated at 630 wavelength. Finally, the percentage of the effect of the selected peptide compared to the control was calculated. To understand the anticancer effect of the selected peptide on cancer cells, the toxicity test was used with the MTT method. For this, serial concentrations (100, 50, and 25 mg/ml) were exposed to MFC-7 cancer cells, and after 12, 24, and 48 hours, the rate of cell death was calculated compared to the control. In the bioinformatics section, only 8 peptides had antigenic strength less than one and were selected for the next step. Also, by examining the power of antigenicity, allergenicity, toxicity and homology, only 3 peptides had all the necessary characteristics. In the experimental part, it was clearly determined that by increasing the concentration of the selected peptide up to 42%, the interaction between PD1 and PDL1 is inhibited. Also, the examination of the percentage of death of breast cancer cells (MCF-7) by different concentrations of the selected peptide at 12, 24 and 48 hours showed that the rate of cell death increases with the increase in concentration and time. This study showed that by integrating bioinformatics tests and experimental tests, a suitable peptide can be designed for possible inhibition of PD-1 and PD-L-1 molecules. To check the effectiveness and find out the possible side effects of the selected peptide, additional tests should be done and then it should be tested in an animal model and in a clinical trial.