Synthesis and pharmacology of novel inhibitors of histone deacetylases and of proteolysis targeting chimeras (PROTACs) for mutant FMS-like tyrosine kinase-3

点击翻译按钮获取中文摘要

Acute myeloid leukemia (AML) with mutations in the kinase FMS-like tyrosine kinase-3 (FLT3) is a clinically unsolved problem. The most common mutations of FLT3 occur in its juxtamembrane domain (FLT3-ITD). Several of the existing FLT3 inhibitors are very potent. However, they are not very effective against FLT3 with mutations in the tyrosine kinase domain (FLT3-TKD), which arise during the therapy with such inhibitors, or they are not specific for FLT3. A major goal in the development of new FLT3 inhibitors is to identify a molecule that potently inhibits mutant FLT3 in the active and inactive DFG-in and DFG-out conformations, while not acting against kinases that are necessary for normal hematopoiesis. Since inhibitors of the histone deacetylase (HDAC) family promote the degradation of mutant FLT3, we synthesized and tested novel inhibitors of this class of compounds. These agents specifically inhibit the tumor-relevant class I HDACs (HDAC1, HDAC2, HDAC3), and they are more selective and effective than clinically tested class I HDAC inhibitors against AML cells with FLT3-ITD and FLT3-TKD mutants. Other available, structurally related HDAC inhibitors will be analyzed for their effects against permanent and primary leukemia cells with mutated FLT3. In this context, we aim to molecularly explain our unexpected observation on a dose-dependent switch from a stabilization to a degradation of mutant FLT3 in cells that are treated with HDAC inhibitors. In addition, we have developed and tested protein-degrading inhibitors (so-called PROTACs, which cause proteasomal degradation of their target proteins) for FLT3. We have discovered for the first time a potent mutant-specific PROTAC for FLT3-ITD and FLT3-TKD, which we now aim to optimize with respect to its biological effects on leukemia cells. This will be done using structure-based optimization, innovative PROTAC synthesis concepts, in vitro inhibition/selectivity assays, and cellular characterization. We aim to link the best FLT3-ITD inhibitor scaffolds with different ubiqutin E3 ligase ligands to create even better PROTACs. As a further goal, we aim to test and molecularly understand the anti-leukemic effects of FLT3 PROTACs alone and in combination with novel HDAC inhibitors in permanent and primary AML cells. We will employ global transcriptome, proteome and phospho-proteome analyses, individual and kinome-wide selectivity studies, targeted protein analyses, flow cytometry and genetic knockout strategies. This will advance the preclinical establishment of HDAC inhibitors and FLT3 PROTACs and we can provide evidence for innovative, rationally designed combination therapies.