Discovery And Mechanistic Study Of Kinase Inhibitors For B Cell Related Malignancies

1. Characterization of Selective and Potent PI3Kδ Inhibitor and PI3Kδ/VPS34 dual inhibitor for B-Cell MalignanciesPI3Kδ is predominately expressed in leukocytes and has been found overexpressed in B-cell related malignancies such as CLL and AML. Hence it has been actively pursued as a drug discovery target. This portion of the work includes two chapters.Chapter 1:Discovery of a potent and selective PI3K8 inhibitor PI3KD-IN-015 for B-Cell Malignancies.Using computer aid-drug design and medicinal chemistry methods, we have discovered an ATP competitive PI3Kδ inhibitor PI3KD-IN-015, which demonstrates a high selectivity among other PI3K isoforms in both biochemical and cellular assays, meanwhile did not inhibit most of other protein kinases in the kinome. Through significantly affect the PI3K signaling. PI3KD-IN-015 exhibits moderately anti-proliferation efficacies against a variety of B-cell related cancer cell lines. It induced both autophagy and apoptosis in B-cell malignant cell lines. Moreover, combination of autophagy inhibitor Bafilomycin could enhance the anti-proliferation effect of PI3KD-IN-015. PI3KD-IN-015 moderately inhibited the proliferation of AML and CML patient primary cells. In short, these results indicate that PI3KD-IN-015 might be a useful drug candidate for further development of anti-B-cell related malignancies therapies.Chapter 2:Discovery of a potent PI3Kδ/Vps34 dual inhibitor PI3KD/V-IN-01 for B-Cell Malignancies.Despite the clinical success of the first selective PI3K8 inhibitor CAL-101, basic research and clinical trials have shown that inhibition of PI3K8 itself did not show too much cytotoxic efficacy against B-cell related malignances cells. In the characterization of our selective PI3K8 inhibitor PI3KD-IN-015 we also observed the same phenomenon. One possible reason is that PI3K8 inhibition induced autophagy that protects cancer cells from death. Since class III PI3K isoform PIK3C3/Vps34 plays critical roles in autophagy initiation and progression, we predicted that a PI3Kδ and Vps34 dual inhibitor might exhibite stronger anti-proliferative activity than PI3Kδ-targeted inhibitors. Through a structure-based drug design approach, we discovered a highly potent ATP-competitive PI3Kδ/Vps34 dual inhibitor, PI3KD/V-IN-01, which exhibits an IC50 of 6 nM against PI3Kδ and an IC50 of 19 nM against Vps34. PI3KD/V-IN-01 displayed 10-1500 fold selectivity over other PI3K isoforms and did not inhibit any other protein kinases in the kinome. In the cellular context, PI3KD/V-IN-01 showed 30-300 fold selectivity between PI3K5 and other class I PI3K isoforms. Compared with the selective PI3Kδ inhibitor CAL-101 and selective Vps34 inhibitor VPS34-IN-1, PI3KD/V-IN-01 exhibited better anti-proliferative activity against CLL, AML and Burkitt lymphoma cell lines. Significantly, FLT3-ITD AML cells are much more sensitive to PI3KD/V-IN-01 than the FLT3 wt expressing AML cells. PI3KD/V-IN-01 showed dose-dependent anti-tumor growth efficacies in the AML cell MV4-11 inoculated xenograft mouse model. These results suggest that dual inhibition of PI3Kδ and Vps34 might be a useful approach to improve the PI3Kδ inhibitor’s anti-tumor efficacy.2. Discovery of 2-((3-amino-4-methylphenyl)amino)-N-(2-methyl-5-(3-(trifluoromethyl)benzamido)phenyl)-4-(methylamino)pyrimidine-5-carboxamid e (CHMFL-ABL-053) as a potent, selective and orally available BCR-ABL inhibitor for Chronic Myeloid LeukemiaThe BCR-ABL fused tyrosine kinase onco-protein is a crucial driver for Philadelphia chromosome-positive chronic myeloid leukemia (Ph+CML), and has been extensively explored as a drug discovery target. Despite the FDA approved drugs such as Imatinib, Dasatinib, Nilotinib and Bosutinib have abtained great clinical success. Most of them are multi-target compounds, and all potently inhibit other targets which have similar structures as ABL1 such as c-KIT, DDR 1/2 and so on. Although the role of off-target inhibition is not very clear in clinical trials, the highly selective BCR-ABL inhibitor is still greatly demanded for both the clinical side effects and preclinical pathological mechanistic studies. Starting from a multiple-target dihydropyrimidopyrimidine core scaffold based on GNF-7 using medicinal chemistry approach we discovered CHMFL-ABL-053, a highly potent and selective BCR-ABL inhibitor which displayed an IC50 of 70 nM against ABL1 kinase. Compound CHMFL-ABL-053 exhibited high selectivity among other protein kinases with an S score (1)= 0.02 in the DiscoveRx’s KinomeScanTM selectivity profiling assay. Significantly, CHMFL-ABL-053 did not apparently inhibit c-KIT kinase, which is the common target of FDA approved BCR-ABL inhibitors like Nilotinib, Bosutinib, Imatinib and Dasatinib. CHMFL-ABL-053 exhibited anti-proliferation activity in CML cell lines such as KU812 (GI50:25nM), K562 (GI50:14nM) and MEG-01 (GI50:16nM) through suppressing the BCR-ABL auto-phosphorylation (EC50 about 100nM) and the phosphorylation of downstream signal mediators such as STAT5, Crkl and ERK. Also it induced apoptosis and arrested cell cycle progression in G0/G1 phase. Pharmacokinetic study revealed that CHMFL-ABL-053 had 24% bioavailability and over 4 hours of half-life in rats. In the K562 cells inoculated xenograft mouse model,50mg/kg/day dosage treatment could almost completely suppress the tumor progression. As a potential useful drug candidate for CML, CHMFL-ABL-053 is under extensive preclinical safety evaluation now.