The Pharmacological Study Of New Targeted Therapies For B Cell Related Malignancies

B cell related malignancies is a type of cancer that forms in B cells.There are many different types of B cell related malignancies, including B cell lymphoma.some leukaemias and myelomas.Bruton’s tyrosine kinase (BTK) kinase is a member of the TEC kinase family and is a key regulator of the B-cell receptor (BCR)-mediated signaling pathway. It is important for B-cell maturation, proliferation, survival and metastasis. Overexpression and gain of function mutations of BTK kinase has been observed in many types of B-cell malignancies and has been served as a validated drug discovery target. Ibrutinib (PCI-32765) is an irreversible BTK kinase inhibitor that has been extensively used as a tool compound to validate the role of BTK kinase in B cell related malignances. Ibrutinib was approved by the FDA for the clinical application on mantle cell lymphoma, chronic lymphocytic leukemia and Waldenstrom’s macroglobulinemia. So far, Ibrutinib is the most successful drug in the development BTK inhibitors. Our study focused on the drug research of new BTK inhibitors and new indication of the "old drug" Ibrutinib. This dissertation was divided into four parts.Part 1:Discovery of a Potent, Covalent BTK Inhibitor for B-Cell LymphomaBy employing structure-based drug design strategy, our team has successfully developed a small molecule inhibitor against BTK, named as QL47. QL47 works by covalently modifing BTK protein at Cysteine481 position to achieve a permanent inhibitory effect that finally results in target protein degradation, causing a more thorough inhibition on signaling transduction pathway. In a series of in vitro biochemical assays, this compound exhibits strong inhibition effects on BTK atuo-phosphorylation and downstream targets in B-cell lymphoma cells with overexpressed BTK. In addition, it shows high specificity against BTK kinase among over 520 kinases in the kinome. QL47 can effectively arrest cell cycle at the stage before DNA synthesis in cells, and showed potent growth inhibitory effects against a variety of B cell lymphoma cancer cell lines. Currently, the oncogenic roles of BTK kinase are still under further investigation, the development of selective inhibitor QL47 provides indispensible tools for revealing the biological and oncogenic functions of BTK kinase in human cells.Part2:Discovery of a BTK/MNK Dual Inhibitor for Lymphoma and LeukemiaMNK kinase plays a significant role in B cell related malignancies. However, inhibition of MNK activity has been observed to moderately inhibit the proliferation of AML cells. We hypothesized that simultaneously inhibiting BTK and MNKs kinases would exert combinatorial efficacies than targeting these kinases individually by analyzing the signalling pathway. Based on inspection of the available X-ray structures of BTK and MNK 1/2 kinases, we has successfully developed the first highly potent and relatively selective dual BTK/MNK inhibitor, QL-X-138, by employing structure-based drug design strategy. Simultaneous inhibition of BTK and MNK kinase activity shows greater efficacy in the cell lines with moderate sensitivity to selective BTK inhibitors. In addition, in the SKM-1 cell inoculated xenograft mouse model, QL-X-138 displayed a dose-dependent tumor inhibition activity.The rational design of multiple targeted drugs is usually challenging considering the difficulty of achieving a high level of selectivity, however it is an attractive approach to achieve improved efficacy against oncogene-driven diseases. This study again exemplifies that it is feasible to achieve a selective multiple targeted inhibitor through a rational design approach.Part3:Discovery of a Highly Potent and Selective Irreversible BTK/GST-O1 Dual Inhibitor for B-Cell MalignancesWith further exploration of BTK inhibitors, we replaced different kinds of chemical groups which could potentially form a covalent bond with the cysteine residue in BTK.With this method, we developed a novel covalent irreversible Btk inhibitor MM2-48. Compared with ibrutinib, MM2-48 enhanced the antiproliferative efficacies in vitro against a variety of B-cell cancer cell lines, AML and CLL primary patient cells and in vivo models through effective apoptosis induction and dual-action inhibitory mode of BTK activation. Proteomic study identified GST-O1 as another target of MM2-48, which might offer new opportunities beyond what ibrutinib has achieved. In addition, MM2-48 can induce the BTK protein degradation in the B-cell malignancy cell lines, causing a more thorough inhibition on BTK related signalling pathway. These primary results demonstrate that simultaneous inhibition of BTK and GSTO1 activities might be a new therapeutic strategy for B-cell malignances.Part4:Ibrutinib Selectively Targets FLT3-ITD in Mutant FLT3-positive AMLThe researchers observed that ibrutinb exhibited anti-proliferation activity against FLT3-ITD mutant AML cells but not WT-expressing AML cells by using a kinase target-based whole cell screening library. In a series of in vitro biochemical assays, this compound potently inhibited FLT3-ITD auto-phosphorylation and downstream targets in FLT3-ITD mutant AML cells, arrested cell cycle in G0/G1 phase and induced apoptotic cell death.The inhibitory effect of ibrutinib on those cells was independent of the original target BTK kinase. Currently, Ibrutinib is under extensive clinical investigation against a variety of different B-Cell malignancies. Given the fact that the safety profile of Ibrutinib is tolerated in the patients, our results might help to expand the application of this drug to AML patients harboring the FLT3-ITD mutant. This is another example of personalized targeted therpay in this precision medicine era.