Pharmacological Studies Of Kinase Inhibitors Against FLT3-ITD-positive Acute Myeloid Leukemia

Acute myeloid leukemia(AML)is a cancer of the blood and bone marrow,characterized by the rapid growth of abnormal white blood cells,which lead to interfere with the production of normal blood cells.FMS-like tyrosine kinase(FLT3)is a type ? receptor tyrosine kinase that plays important roles in the differentiation and survival of hematopoietic stem cells in bone marrow and has been observed overexpressed in the AML.A variety of gain-of-function mutations have been identified in?30%of the AML patients,such as FLT3-ITD has been actively pursued as a drug discovery target for AML.Recently PKC412 has received Food and Drug Administration(FDA)'s break through therapy designation for the FLT3-ITD+ AML.Here,we get a series of FLT3 kinase inhibitors through structure-guided drug design approach,and focused on the mechanism of drug action.Chapter 1:Research of a highly potent FLT3 kinase inhibitor CHMFL-FLT3-165 for FLT3-ITD-positive AML based on Ibrutinib structureThrough highthrought screening method,we discovered a BTK kinase inhibitor Ibrutinib,also displayed moderate against FLT3-ITD positive AML cell lines.To improve this drug activity against FLT3,we using a structure-guided drug design approach,discovered an type ? FLT3 kinase inhibitor CHMFL-FLT3-165.This Compound showed strongly activity against the human FLT3-ITD+ AML cell lines.Moreover,CHMFL-FLT3-165 suppressing the FLT3 auto-phosphorylation and mediated signaling in the FLT3-ITD-positive AML cancer cell lines,also it induced apoptosis and arrests cell cycle progression in the G0/G1 phase.In an MV4-11 cell-inoculated xenograft mouse model,CHMFL-FLT3-165 displayed dose-dependent tumor inhibition activity.However,the poor oral PK profile prevented it from further development,further medicinal chemistry effort is needed to improve the PK profile of this series of compounds.Chapter 2:Research of more potent and orally available FLT3 kinase inhibitor CHMFL-FLT3-122 for FLT3-ITD Positive Acute Myeloid LeukemiaAlthough CHMFL-FLT3-165 displayed strongly activity against FLT3-ITD mutant cells and xenograft mouse models,the poor oral PK still need to further improve for this new class of compounds.Starting from Ibrutinib,through a detailed SAR study,we discovered an orally available FLT3 kinase inhibitor CHMFL-FLT3-122.It significantly inhibited the proliferation of FLT3-ITD positive AML cancer cell lines MV4-11,MOLM13 and MOLM14.It also strongly affected FLT3-ITD mediated signaling pathways and induced apoptosis by arresting the cell cycle into the G0/G1 phase.Pharmacokinetic study revealed that CHMFL-FLT3-122 showed a good bioavailability F = 30%,T1/2(h)= 1.4h.In MV4-11 cell inoculated xenograft model,this compound showed significantly suppressed the tumor growth without exhibiting obvious toxicity.Those results showed CHMFL-FLT3-122 might be a potential drug candidate for FLT3-ITD positive AML.Chapter 3:Discovery of CHMFL-FLT3-213 as a highly Potent and overcome Multiple mutations type? FLT3 Kinase InhibitorDespite the clinical success of the first FLT3 kinase inhibitor PKC412,based on research and clinical trials showed this compound would develop resistance through acquisition of a secondary mutation in the FLT3 kinase domain.However,CHMFL-FLT3-122 showed resistance when a secondary mutation in the FLT3 kinase domain,eg,ITD-F691L,which located at the "gatekeeper" position in FLT3,analogous to residues in other kinases that are hotspots for drug-resistance to TKIs.Therefore,we contutine to explore the binding mode between Ibrutinib with FLT3,through a structure guided drug design approach,found a potent type II FLT3 kinase inhibitor CHMFL-FLT3-213,which exhibits highly potent against FLT3 wt/ITD mutant and other oncogenic mutations(such as FLT3-D835Y,FLT3-D835H,FLT3-D835V)of FLT3 kinases.Simultaneously,it also displays highly potent against drug resistant mutations FLT3-ITD-D835Y(GI50:2 nM)and FLT3-ITD-F691L(GI50:2 nM).In the cellular context it strongly affected FLT3-ITD mediated signaling pathways and induced apoptosis by arresting the cell cycle into the G0/G1 phase.In the in vivo studies CHMFL-FLT3-213 demonstrated a good bioavailability(19%)and significantly suppressed the tumor growth in MV4-11 cell inoculated xenograft model(3.75 mg/kg)without exhibiting obvious toxicity.CHMFL-FLT3-213 might be a potential drug candidate for FLT3 AML.Chapter 4:Dual Inhibition of AKT/FLT3-ITD by A674563 overcomes FLT3 ligand-induced drug resistance in FLT3-ITD positive AMLFor drug resistance,besides acquisition of a secondary mutation in the FLT3 kinase domain,one of the resistance mechanisms in the AML refractory patients involves FLT3-ligand induced reactivation of AKT and/or ERK signaling via FLT3 wt kinase.Thus,we were interested in fiding a dual inhibitor for FLT3-ITD and AKT.Through a screen of numerous AKT kinase inhibitors,we identifid a orally available AKT inhibitor,A674563,as a dual suppressor of AKT and FLT3-ITD.This compound exhibited strongly anti-proliferation activity in FLT3-ITD +AML,and able to overcome FLT3 ligand-induced(10ng/ml)drug resistance through simultaneous inhibition of AKT-mediated signaling.Our fidings suggest that A674563 might be a potential drug candidate for overcoming FLT3 ligand-mediated drug resistance in FLT3-ITD+ AML.