Abstract: Objective　The method of "molecular docking - molecular dynamics - in vitro pharmacology" was used to screen and optimize small molecule polypeptide compounds that can block PD-1/PD-L1 binding and have high anti-tumor activity. Methods Using the 5-peptide compound database constructed by our team and the three-dimensional structure data of the PD-L1 protein downloaded from the protein database (PDB), the peptide compounds obtained by flexible molecular docking using Molecular Operating Environment software. The molecular dynamics calculation and analysis of the compounds with the highest GWVI/WSA (generalized Born volume integral/weighted surface area) free energy change value (ΔG) were carried out, including the root mean square deviation (RMSD) of the position change of the weight atom and the interaction energy (the sum of Lennard-Jones potential and Coulombic energy). The blocking effect of ligand compounds on PD-1/PD-L1 binding was analyzed by the homogeneous time-resolved fluorescence (HTRF) technique. The co-culture system of Jurkat T lymphocytes and melanoma B16-F10 cells was established to explore the effect of compound ligands on the killing effect of T cells on tumors and the effect on the secretion level of IL-2 in the co-culture supernatant. Results The selected polypeptide compounds were analyzed by molecular dynamics, and the binding of RGGHA to RGGHH and PD-L1 was stable. There are many kinds of interactions between RGGHA and PD-L1，which can compete with PD-1 ligands to bind Asp122, Try123 and Lys124 sites of the PD-L1 protein and block PD-1/PD-L1 signal transduction. HTRF experiments showed that the binding inhibition rate of RGGHA to PD-1 and PD-L1 was 58.38%, and that of RGGHH was 42.73%. In addition, we established a co-culture system of Jurkat T lymphocytes and melanoma B16-F10 cells to explore the immunostimulatory effect and mechanism of peptides. The results show that RGGHA and RGGHH can significantly increase the secretion level of IL-2, improve the killing ability of T cells against tumor cells and activate the tumor immune microenvironment. Conclusions The study found that the polypeptide compounds RGGHA and RGGHH can effectively block the PD-1/PD-L1 interaction and reactivate the anti-cancer immune response, which can be used as lead compounds for new drug development.

Bitter taste is one of the important contents in the theory of"Five tastes"in traditional Chinese medicine(TCM).It has the functions of"releasing,dryness and firmness"and plays an important role in the treatment of respiratory diseases.Bitter taste receptors(T2Rs)is a kind of G-protein-coupled receptors,studies have shown that it can act on human airway smooth muscle,relax smooth muscle and dilate bronchus,and play a vital role in the treatment of bronchial asthma and chronic obstructive pulmonary disease.It can also inhibit the inflammatory reaction and reduce the damage of lung tissue by reducing the release of inflammatory factors and promoting the apoptosis of inflammatory cells.The therapeutic mechanism of bitter Chinese herbs action may depend on the effect of T2Rs,the effects of bitter Chinese herbs such as"lowering qi","releasing heat","eliminating dampness"are quite consistent with the cognitions of western medicine that bitter taste receptors could"relaxe bronchus","reduce airway resistance","inhibits inflammatory factors"and"reduces mucus secretion".Based on the understanding of bitter taste in TCM theory and modern research,this study aims to discuss the effect of bitter Chinese herbs depend on T2Rs as the starting point,so as to provide a new research direction for clinical treatment of pulmonary diseases such as bronchial asthma and chronic obstructive pulmonary disease.