Re-discovery Of BTK Kinase Inhibitor QL47 As A Highly Potent And Novel HSP70 Inhibitor

Heat shock proteins are important components for maintaining the homeostasis of tumor cells.HSP stabilize the three-dimensional structure of various cancer-causing proto-oncogenes such as kinases and transcription factors and promote the development of cancer.Inhibition of heat shock proteins can cause structural instability of oncoproteins,which in turn can be degraded by the proteasome pathway.At present,most studies on heat shock protein inhibitors focus on HSP90 and HSP70.Most HSP90 inhibitors bind to the N-terminal ATP binding domain of HSP90,but such inhibitors can induce activation of HSF1 transcription factors which results in rapid expression of heat shock family protein such as HSP72.This negative feedback will reduce the effect of HSP90 inhbitor on tumor cells.HSP70 is a chaperone protein of HSP90.When a protein is expressed on ribosome,the naive protein is folded with the help of HSP70,then present to HSP90.HSP90 function is blocked by double inhibition of HSC70 and inducible HSP72,leading to degradation of proto-oncogenes.At the same time,HSP70 also has anti-apoptotic effect,which can help cancer cells inhibit the occurrence of apoptosis.Therefore,targeting HSP70 can exert anti-tumor effects by reducing the level of proto-oncoprotein,inhibiting the proliferation of tumor cells and causing apoptosis of tumor cells.Based on its important role in cancer progress,the development of new HSP70 inhibitors will add new tools and strategies for the diagnosis and treatment of cancers.Since heat shock proteins interact with a variety of proteins in the cell,especially the Driven-force oncoproteins.So we intended to establish a HSP90-based protein interaction detection system to detect interactions between heat shock proteins and client proteins.It can used to characterize the binding affinity of small molecules.The targets cover most of the clinical targets under development.The development of this system will contribute to the development of new drugs and the re-purposing of old drugs.Epidermal growth factor receptor(EGFR)is a transmembrane receptor tyrosine kinase.When EGF binds EGFR on the cell membrane,it will induce autophosphorylation of EGFR and activation of downstream signaling pathways,including RAS/RAF/ERK,PI3K/AKT and JAK/STAT signaling pathways.In China,lung cancer is the most malignant tumors with highest mortality rate.Non-small cell lung cancer accounts for about 80%of all lung cancers.10%-35%of patients with non-small cell lung cancer carry EGFR activation mutations such as exon 19 deletion and L858R point mutations,both of which cause excessive activation of the EGFR signaling pathway,thereby drivering tumor development.The first-generation EGFR inhibitor gefitinib has potent anti-tumor effect on EGFR driven NSCLC,but it often induces second T790M mutation in EGFR,which can cause resistance to gefitinib.Therefore,the development of selective inhibitors of EGFR T790M can overcome the resistance to first-generation EGFR inhibitor,develop new theurapy for EGFR driven NSCLC.Chapter 1:Re-discovery of BTK inhibitor QL47 as a highly potent and novel HSP70 inhibitorQL47 is an irreversible BTK kinase inhibitor.During high-throughput screening,we found that QL47 has a better anti-proliferative effect onFLT3-ITD mutant acute myeloid leukemia(AML)cells.Our previous studies showed that BTK knockout on FLT3-ITD mutant cells has no obvious effect on cell proliferation.It indicated that QL47 has other targets.By detecting FLT3 signaling pathway,we found that QL47 can induce the decreasing of FLT3-ITD protein level but without effect on FLT3-WT proteins,so we suspected that QL47 is working throught heat shock protein which can stable the mutant protein structure.Through proteomics and biochemistry analysis,we found that QL47 binds to the HSP70 protein irreversibly.The acrylamide of QL47 forms a covalent bond with HSP70 at Cys267.Although QL47 interact with HSP70 in the N-terminal ATP binding domain,but it does not inhibit the ATPase function of HSP70.It can affect the protein refolding function of HSP70,indicating that QL47 binds to HSP70 in a different way than the traditional HSP70 inhibitor VER155008,which means QL47 is a novel HSP70 inhibitor with distinct binding mode.QL47 provide theoretical and structural foundation for the development of future HSP70 inhibitors.Chapter 2 Mechanism Study Of QL47 against FLT3 mutant acute myeloid leukemiaThe highly potent anti-proliferation effect of QL47 in FLT3 mutant leukemia cells is mainly caused by the degradation of FLT3 protein,which blocks the STAT5-MYC signal pathway and finally inhibit the proliferation of FLT3-ITD AML cell lines.At the same time,HSP70 plays an important anti-apoptotic effect in tumor cells,inhibiting HSP70 can lead to the degradation of anti-apoptotic factor XIAP,inducing apoptosis and arresting cell cycle in G0-G1 phase.In addition,we tested the effect of QL47 on various mutant FLT3 on isogenic FLT3-BaF3 cell lines.The results showed that QL47 can not only degrade FLT3-ITD,but also has a strong degradation effect on various activating mutations and drug-resistant mutations such as N676D and F691L.The study on patient primary cells and mouse xenograft model showed that QL47 can inhibit the tumor growth effectively.Therefore,as a novel HSP70 inhibitor,QL47 provides new strategies for the treatment of FLT3-ITD AML patients,and have high potenial in overcome FLT3 mutation induced drug-resistance.Chapter 3:Establishment of a Protein-Protein Interaction System Based on HSP90 chaperonAssessment of the interactions between a drug and its protein target in a physiologically relevant cellular environment constitutes a major challenge in the pre-clinical drug discovery space.Given that HSP90 interacts with a multi of kinases and stabilizes their structures,and the interaction attenuates when a ligand binds to the kinase.Based on this principle,we intend to establish a HSP90-based protein interaction detection system based on the Split luciferase Complementation assay,which uses luciferase activity to characterize the interaction between HSP90 and kinase quantitatively.This system can cover most cancers related kinase targets and some transcription factor targets.These assay presented here enables target engagement studies for mediuM and high-throughput applications.Additionally,it provides a rapid assay development and screening platform for targets where phenotypic or other cell-based assays are not readily available.Meanwhile,it is also a supplement and expansion of our BaF3-kinase isogenic cell lines.Chapter 4:Discovery and Characterization of a Novel Irreversible EGFR Mutants Selective and Potent Kinase Inhibitor CHMFL-EGFR-26EGFR T790M mutation accounts for about 40-55%drug resistance for the first generation EGFR kinase inhibitors in the NSCLC.Starting from ibrutinib,a highly potent irreversible BTK kinase inhibitor,which was also found to be moderately active to EGFR T790M mutant,we discovered a highly potent irreversible EGFR inhibitor CHMFL-EGFR-26,which is selectively potent against EGFR mutants including L858R,del 19,and L858R/T790M.It displayed proper selectivity window between the EGFR mutants and the wide-type.CHMFL-EGFR-26 exhibited good selectivity profile among 468 kinases/mutants tested(S score(1)=0.02).In addition,X-ray crystallography revealed a distinct "DFG-in" and "cHelix-out" inactive binding mode between CHMFL-EGFR-26 and EGFR T790M protein.The compound showed highly potent anti-proliferative efficacy against EGFR mutant but not wide-type NSCLC cell lines through effective inhibition of the EGFR mediated signaling pathway,induction of apoptosis and arresting of cell cycle progression.CHMFL-EGFR-26 beard acceptable pharmacokinetic properties and demonstrated dose-dependent tumor growth suppression in the H1975(EGFR L858R/T790M)and PC-9(EGFR del 19)inoculated xenograft mouse models.Currently CHMFL-EGFR-26 was undergoing extensive pre-clinical evaluation for the clinical trial purpose.