Study On Screening And Activity Evaluating Of BTK Or EGFR Inhibitors

Bruton tyrosine kinase?BTK?and epidermal growth factor receptor?EGFR?share the same conservative cysteine binding sites.Most of the inhibitors targeting BTK or EGFR T790M are covalent inhibitors,and have both BTK and EGFR T790M inhibitory activities.In this context,this study focus on the discovery and activity evaluation of potential BTK inhibitors and EGFR inhibitors.1.Screening and identification of novel BTK inhibitorsBruton's tyrosine kinase?BTK?is expressed at all stages of the development of B cell lymphocytes as a critical downstream effector of B cell antigen receptor?BCR?pathway.BTK regulates the proliferation,differentiation and apoptosis of B cells,which has been considered as an important target for the clinical treatment of B cell-related tumors or immune diseases.To date,two small molecule inhibitors targeting BTK have been listed.Ibrutinib as the first-generation BTK inhibitor,is a covelent BTK inhibitor,and has been approved in November 2013 for the treatment of chronic lymphocytic leukemia?CLL?,mantle cell lymphoma?MCL?and Waldenstrom's macroglobulinemia?WM?.However,its off-target effects caused large clinical side effects,therefore,the development of highly selective BTK inhibitors has attracted wide attention.Acalabrutinib?ACP-196?,also a covalent inhibitor,has been approved in November 2017 for the treatment of CLL and MCL.ACP-196 is considered to be a second-generation BTK inhibitor due to its high selectivity,but toxic side effects such as headaches,diarrhea and weight gain are found in some cases during its clinical treatment.Therefore,it is of great importance to develop selective BTK inhibitors with highly potency and low toxicity.In collaboration with chemists,we designed and synthesized a series of BTK inhibitors?compounds 1-8?with the core of pyrimido[4,5-d][1,3]oxazin-2-one.ELISA results showed that compound 1 and 2 had excellent inhibitory effects on BTK with an IC₅₀ value of 4.7 nM and 7.0 nM,respectively,which were weaker than that of the first-generation BTK inhibitor ibrutinib(IC₅₀=0.6 nM),but were better than that of the second-generation inhibitor ACP-196(IC₅₀=8.6 nM).Then,molecular docking showed that the electrophilic acrylamide groups of compound 1 or 2 could covalently bond to the Cys481 site of BTK protein.And this binding enhanced the affinity between compound and BTK,and thus caused the inhibition of BTK kinase activity.After comprehensive consideration of the structural novelty and physicochemical properties,compound 2 was chose for further research.In a preliminary kinase spectrum assay,compound 2 exhibted a inhibitory activity against BTK comparable to that of ibrutinib and better than that of ACP-196 at a concentration of 1000 nM.Meanwhile,compound2 showed weaker potency on most of the other kinases with a selectivity better than ibrutinib and comparable with that of ACP-196.In celluar level,compound 2 inhibited the phosphorylation of BTK of two B cell lymphoma cell lines?Ramos and TMD8?in a dose-dependent manner.And compound 2 also effectively inhibited the cell proliferation of Ramos and TMD8 with IC₅₀0 values of 15.07?M and 0.03?M,respectively,which was comparable with or better than the positive drugs.Furthermore,in TMD8 cells,compound 2 treatment caused a marked G1 phase arrest and decrease of p-Rb,Rb and cyclin D1 protein expression in a dose-dependent manner.Besides,cell apoptosis was induced in TMD8 cells after compound 2 treatment,accompanied by the increase of cleaved PARP and cleaved caspase 3.This work introduced compound 2 as a novel BTK inhibitor with high activity and selectivity,which is worthy of further study.The structural optimization of compound 2 and the screening of other BTK inhibitors is under way,which may help to develop potential BTK inhibitors with independent intellectual property rights for the treatment of B cell lymphoma.2.Screening and identification of the potential fourth-generation EGFR inhibitors targeting EGFR C797S mutationEpidermal growth factor receptor?EGFR?is an important target for the treatment of non-small cell lung cancer?NSCLC?.The first-generation EGFR inhibitors such as gefitinib and erlotinib have been approved for the treatment of NSCLC containing the EGFR sensitive mutations?exon 19Del,L858R?.However,acquired resistance severely limits their clinical use.Since EGFR T790M mutation has been identified as the most common cause of resistance,the third-generation EGFR inhibitors that effectively overcome EGFR T790M resistant mutation with selectivity over wild type EGFR?EGFR WT?has attracted much attention.AZD9291?osimertinib,Tagrisso?,is the only third-generation EGFR inhibitors approved by FDA,and it shows a dramatic clinical effects in NSCLC patients with EGFR T790M or sensitive mutations.However,drug resistance remains a major obstacle to the long-term clinical benefits of NSCLC targeted therapy.EGFR C797S mutation has been reported as one of the main causes of the clinical resistance to AZD9291?22-24%?,and then the discovery of new inhibitors targeting EGFR C797S,also known as the fourth-generation inhibitors,has become a new clinical requirement.EGFR allosteric inhibitor EAI045 and ALK/EGFR target inhibitor brigatinib were reported as representive fourth-generation EGFR inhibitors.However,they showed obvious in vivo anti-tumor activity only when combined with EGFR antibody.Although several fourth-generation EGFR inhibitors have been discovered,none of them has been in clinical research.Therefore,the development of the fourth-generation EGFR inhibitors with independent intellectual property rights that overcoming EGFR C797S mutation is of crucial and imperative significance.Collaborating with chemists,a total of 122 new fourth-generation EGFR inhibitors were designed and synthesized,and were screened for the kinase inhibitory activity against EGFR C797S using the ELISA method.Among them,79 compounds were found as active compounds(IC₅₀<100 nM),and 29 of them were identified as highly active compounds(IC₅₀<10 nM).In these highly active compounds,HCD210,HCD228,HCD229,HCD230,HCD233,HCD235,HCD239,HCD244,LS 2-145,LS3-16 and LS 3-19 showed considerable EGFR C797S selectivities with weaker kinase inhibitory activities on EGFR WT,and then subjected for futher evaluation in celluar level.In BaF3 cell lines highly expressing EGFR C797S?BaF3-EGFR L858R/T790M/C797S and BaF3-EGFR 19D/T790M/C797S?,HCD210,HCD228,HCD233 and HCD235 exhibted significant inhibition activities on the phosphorylation of EGFR and caused obvious inhibiton of cell prolifertion in vitro.This compound showed good EGFR C797S inhibition activity and anti-growth activity of EGFR C797S tumor cells,considered as potential fourth-generation EGFR inhibitors,were worthy of further structural modification and optimization and in vivo antitumor activity evaluation.At the same time,the activity screening of other new structure inhibitors is still in progress.This work is expected to develop candidate new four-generation EGFR inhibitors with independent intellectual property rights in China,so as to provide effective drugs for the treatment of NSCLC patients with EGFR C797S mutation,and also to give a reference for the research of similar inhibitors.