Discovery And Mechanistic Study Of Dual Inhibitors For ALK/EGFR Kinases

With the development of molecular biology,tumor biology and related disciplines,targeted drugs(molecular/genetic level)have gradually become research hotspots.More and more targeted drugs are used in clinical treatment.Compared with traditional cancer treatment methods(surgery,radiotherapy,chemotherapy,etc.),the adverse effects of targeted drugs are relatively small,and the anti-tumor effect is relatively high,which not only prolongs the survival of some patients,but also improves the quality of life of patients.Here,we get ALK/EGFR dual kinase inhibitors,CDK9 kinase inhibitors and BCR-ABL/V299L drug-resistant mutant kinase inhibitors through structure-guided drug design approach,and focused on the mechanism of drug action.Chapter 1:Discovery and Characterization of CHMFL-ALK/EGFR-050 as a potent ALK/EGFR dual kinase inhibitor capable of overcoming a variety of ALK/EGFR associated drug resistant mutants in NSCLCRecently,more and more concomitant EGFR mutations and ALK rearrangement are observed from the clinic,which still lacks effective single-agent therapy.Starting from ALK inhibitor 14(TAE684),we have developed a highly potent EGFR/ALK dual kinase inhibitor CHMFL-ALK/EGFR-050(compound 18),which potently inhibited EGFR L858R,del 19 and T790M mutants as well as EML4-ALK,R1275Q,L1196M,F1174L and C1156Y mutants biochemically.Compound 18 significantly inhibited the proliferation of EGFR mutant and EML4-ALK driven NSCLC cell lines.In the cellular context it strongly affected EGFR and ALK mediated signaling pathways,induced apoptosis and arrested cell cycle at G0/G1 phase.In the in vivo studies,18 significantly suppressed the tumor growth in H1975 cell inoculated xenograft model(40 mg/kg/d,TGI:99%)and H3122 cell inoculated xenograft model(40 mg/kg/d,TGI:78%).Compound 18 might be a potential drug candidate for EGFR-and or ALK-individual and as well as EGFR/ALK concomitant NSCLC.Chapter 2:Discovery and Characterization of CHMFL-ALK/EGFR-050 as a potent ALK/EGFR dual kinase inhibitor capable of overcoming a variety of ALK/EGFR associated drug resistant mutants in NSCLCEGFR targeted therapies yielded little to no efficacy in clinical trials.Co-targeting ALK and EGFR signaling may enhance the efficacy of EGFR targeted therapies.We have developed a highly potent ALK/EGFR dual kinase inhibitor CHMFL-ALK/EGFR-039,which significantly inhibited the proliferation of EGFR driven HNSCC cell lines.In the cellular context it strongly affected EGFR and ALK mediated signaling pathways,induced apoptosis and arrested cell cycle at G2/GMphase.In the in vivo studies,CHMFL-ALK/EGFR-039 significantly suppressed the tumor growth in HSC-3 cell inoculated xenograft model(100 mg/kg/d,TGI:87.6%)and CAL27 cell inoculated xenograft model(100mg/kg/d,TGI:65.8%).CHMFL-ALK/EGFR-039 might be a potential drug candidate for HNSCC.Chapter 3:Discovery and Characterization of JSH-150 as a Novel Highly Selective and Potent CDK9 Kinase InhibitorCDK9 is a key regulator of transcription elongation in eukaryotic cells and has been considered as a potential drug target for several diseases including cardiac hypertrophy and certain cancers such as a variety of blood cancers as well as solid tumors.Through a structure-guided rational drug design approach,we have discovered a highly selective inhibitor compound 39(JSH-150),which exhibited and IC50 of 1 nM against CDK9 kinase in the biochemical assay and achieved around 300-10000-fold selectivity over other CDK kinase family members.In addition,it also displayed high selectivity over other 468 kinases/mutants(Sscore(1)=0.01).Compound 39 displayed potent antiproliferative effect against melanoma,neuroblastoma,hepatoma,colon cancer,and lung cancer as well as leukemia cell lines.It could dose-dependently inhibit the phosphorylation of RNA Pol ?,suppress the expression of Mcl-1 and C-Myc,arrest the cell cycle and induce the apoptosis in the leukemia cells.In the MV4-11 cell-inoculated xenograft mouse model,10 mg/kg dosage of 39 could almost completely suppress the tumor progression.The high selectivity and good in vivo PK/PD profile suggested that 39 would be a good pharmacological tool to study CDK9-mediated physiology and pathology as well as a potential drug candidate for leukemia and other cancers.Chapter 4:Discovery and Characterization of a Novel Highly Potent and Selective Type ? Native and Drug Resistant V299L Mutant BCR-ABL Inhibitor(CHMFL-ABL-039)for Chronic Myeloid Leukemia(CML)BCR fused ABL kinase is the critical driving oncogene for chronic myeloid leukemia(CML)and has been extensively studied as the drug discovery target in the past decade.The successful introduction of tyrosine kinase inhibitors(TKI)such as Imatinib,Dasatinib and Bosutinib has greatly improved the CML patient survival rate.However,upon the chronic treatment,a variety of TKI resistant mutants,such as the V299L mutant which has been found in more and more patients with the high-throughput sequencing technology,are observed,although the incidence is still considered rare compared to the more prevalent gatekeeper T315I mutant.However,with the progress of the precision medicine concept,the rare mutation(or the orphan drug target)has attracted more and more attention.Here we report a novel type ?BCR-ABL kinase inhibitor,CHMFL-ABL-039,which not only displayed great potency(IC50:7.9 nM)and selectivity(S score(1)=0.02)against native ABL kinase among other kinases in the kinome,but also exhibited great potency(IC50:27.9 nM)and selectivity against Imatinib resistant V299L mutant among other frequently observed ABL kinase mutants.CHMFL-ABL-039 has demonstrated greater efficacies than Imatinib regarding to the anti-proliferation,inhibition of the signaling pathway,arrest of cell cycle progression,induction of apoptosis in vitro and suppression of the tumor progression in vivo in the native and V299L mutated BCR-ABL kinase driven cells/xenograft models.It would be a useful pharmacological tool to study the TKI resistant ABL V299L mutant mediated pathology and provide a potential precise treatment approach for this orphan CML subtype in the precision medicine era.