Discovery On Small Molecule Inhibitors Of PD-1/PD-L1

Recently, many studies are focused on the application of immune system against cancer during the tumor therapy. The activation of immune system for enhancing antitumor immune responses and for restraining and killing tumor cells is called as tumor immunotherapy, which became the most promising method in the field of cancer research. The tumor immunotherapy is divided into several categories involving cellular immune therapy, tumor vaccine, and immune checkpoint inhibitors. Many gratifying results have achieved in the aforementioned fields.The immune checkpoint aiming at regulating pathways in T cells plays an important role in the maintenance of immune tolerance by adjusting autoimmune reaction. However, the activation of immune checkpoint is able to suppress autoimmune in the process of tumor invasion, the growth and the escape of tumor cells. Two immune check point drugs including CTLA-4 and PD-1 have been approved clinically. The programmed death receptor-1(programmed death-1, PD-1) can be expressed on the sensitized T cell, B cell, and NK cell. Two natural ligands, PD-L1 and PD-L2, are highly expressed in tumor cells and tumor infiltrating lymphocytes. The binding between PD-1 and PD-L exhibits negative regulation effect on the sensitized T cell, which can reduce the activity of T cell and can even induce its apoptosis. Therefore, blockading the PD-1/PD-L pathway is a promising protocol for cancer immunotherapy by enhancing the function of T cell. The signaling pathways have not only affected cancer development, but also possessed importantly biological significance towards autoimmune diseases as well as chronic viral infection.Three monoclonal antibodies including Opdivo, Keytruda, and Tecentriq based on the PD-1/PD-L1 pathway were recently approved by the FDA. Clinical effect on m Abs indicated impressive response rates in patients by prolonging overall survival, particularly, for melanoma, non-small-cell lung cancer(NSCLC), renal cell carcinoma(RCC), non-hodgkin's lymphoma and bladder cancer etc. Although m Abs of PD-1/PD-L1 pathway is very attractive, few small molecule inhibitors have been reported. Thus, small molecule inhibitors became an attractive topic nowadays. Under the certain treatment effect, small molecule inhibitors can reduce immunogenicity side effects, avoid deficiency as drugs, and decrease cost. Therefore, the work of this paper is based on the structure of PD-1/PD-L1 and computer-aided drug design to select promising small molecule inhibitors. The major work is carried out as the following aspects:1. Discovery on small molecule inhibitors based on the crystal structure of m PD-1/PD-L1Because of no report on PD-1/PD-L1 crystal structure at the beginning of the present study, we selected m PD-1/PD-L1 crystal(PDB code:3BIK) as the basis of computer-aided drug design-molecular docking and virtual selection. After screening results from our own database and Maybridge database, we chose the small molecule compounds with satisfying biological activity and selected tr149 as the lead compound. We designed and synthesized 13 compounds involving dimethyl amine benzoate derivatives, and two of them exhibited better biological activities than that of str149. The preliminary structure-activity relationships of these compounds were obtained by exploring the mechanism of small molecule bound to the target protein, and were worthy to be investigated in the further research.At this stage, no published reports were found at the molecular level testing system for compounds.Therefore, ELISA biological activity evaluation model was established for quick detection of small molecule inhibitory activity. Two kinds of ELISA(coat with PD-1 or PD-L1) verified the reliability each other. The results from the self-built ELISA detection model were in agreement with that from HTRF model. ELISA inhibitory activity was just slightly lower, due to ELISA operation caused by the limitation of the system itself. In general, self-built ELISA detection model was reliable because ELISA and HTRF can authenticate mutually with eliminating the detection error of a single method. This work established a ?computer-aided design-molecule synthesis-biological evaluation? system to be the foundation for rapid screening of bioactive molecules.2. Discovery on small molecule inhibitors based on the crystal structure of h PD-1/PD-L1In December 2015, the h PD-1/PD-L1 crystal structure(PDB code: 4 ZQK) was reported. In contrast to 4ZQK binding with 3BIK, the analysis on amino acids located at the interaction surface among proteins, docking with Maybridge database and FDA drug database. We selected 106 compounds to be evaluated by means of HTRF and found compound RH02220 and RJC04089 inhibited >50% combination of PD-1/PD-L1 at 100 mM. Similar research was performed on RH02220 and RJC04089, 36 compounds were found and evaluated. We chose the reasonable molecules RJC04089 and XBX00144 as the lead compounds. In addition, we designed 13 pentpeptides according to the functional importance residues 121-125 in PD-L1 and explored the mechanism of 5 amino acid residues in the combination of PD-1 with PD-L1.3. Discovery on small molecule inhibitors based on the crystal structure of h PD-L1/BMS-202In April 2016, a small molecule inhibitor(BMS-202) and PD-L1 compound crystal structure(PDB code:5J89) was reported. The structure reveals the mechanism of these inhibitors interfers with PD-1/PD-L1 combination by inducing PD-L1 dimer, the PD-L1 activity site was occupied. The mechanism provides us with a new perspective for designing small molecule inhibitors. Unlike 3BIK and 4ZQK with binding sites locating on the protein surface, the dimer of PD-L1 formed an active pocket as a pipeline and facilitated the interaction with small molecules. Therefore, docking and screening the same database, activities of small molecular compound increased significantly targeting 5J89. The inhibition rate(> 50% higher of the compounds) increased obviously at 100 mM. The compound str5730, indicated ~100% inhibition at 333 mM. The cellular level experiments also revealed that str5730 at 200 mM possessed obviously inhibitory effect against PD-1/PD-L1 combination. Compounds str5730 were selected as the lead compounds for structure transformation.During the period of this research, PD-1/PD-L1 protein crystal has two major breakthroughs. More accurate information of protein binding led to better results from the computer-aided drug design. Using DOLD, SYBYL, DS and Amber packages and different conditions of docking as well as screening from three databases(our own database 10000+, Maybridge55000 + compounds, and the FDA drug library), we systematically analyzed the results and chose some of compounds for evaluating biological activities in protein and cellular level.In conclusion, a computer-aided drug design strategy was effective in the discovery on small molecule inhibitors aiming at PD-1/PD-L1. With further understanding of protein crystal structure details and screening more molecular databases, small molecules will be useful for immune therapy in the future.