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

伴有FMS样酪氨酸激酶-3（FLT 3）突变的急性髓性白血病（AML）是临床上尚未解决的问题。FLT 3最常见的突变发生在其质膜结构域（FLT 3-ITD）。几种现有的FLT 3抑制剂非常有效。然而，它们对在酪氨酸激酶结构域（FLT 3-TKD）中具有突变的FLT 3不是非常有效，所述突变在用此类抑制剂治疗期间出现，或者它们对FLT 3不是特异性的。开发新的FLT 3抑制剂的主要目标是鉴定一种分子，该分子在活性和非活性DFG-in和DFG-out构象中有效抑制突变FLT 3，同时不对正常造血所必需的激酶起作用。由于组蛋白去乙酰化酶（HDAC）家族的抑制剂促进突变体FLT 3的降解，我们合成并测试了这类化合物的新型抑制剂。这些药物特异性抑制肿瘤相关的I类HDAC（HDAC 1、HDAC 2、HDAC 3），并且它们比临床测试的I类HDAC抑制剂对具有FLT 3-ITD和FLT 3-TKD突变体的AML细胞更具选择性和有效性。将分析其他可用的结构相关HDAC抑制剂对具有突变FLT 3的永久性和原发性白血病细胞的作用。在这种情况下，我们的目标是从分子上解释我们意外的观察到的剂量依赖性开关从一个稳定的突变FLT 3在细胞中的HDAC抑制剂处理的降解。此外，我们还开发并测试了FLT 3的蛋白质降解抑制剂（所谓的PROTAC，可导致其靶蛋白的蛋白酶体降解）。我们首次发现了一种针对FLT 3-ITD和FLT 3-TKD的有效的mu特异性PROTAC，我们现在的目标是优化其对白血病细胞的生物学效应。这将使用基于结构的优化、创新的PROTAC合成概念、体外抑制/选择性测定和细胞表征来完成。我们的目标是将最好的FLT 3-ITD抑制剂支架与不同的泛素E3连接酶配体连接起来，以创造更好的PROTAC。作为进一步的目标，我们的目标是测试和分子上理解FLT 3 PROTAC单独和与新型HDAC抑制剂组合在永久性和原发性AML细胞中的抗白血病作用。我们将采用全球转录组，蛋白质组和磷酸化蛋白质组分析，个体和激酶组范围的选择性研究，靶向蛋白质分析，流式细胞术和基因敲除策略。这将推进HDAC抑制剂和FLT 3 PROTAC的临床前建立，我们可以为创新的，合理设计的联合治疗提供证据。

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.