Inhibitors of Tyrosine Kinase-Dependent Signaling as Anti-Cancer Agents

被定义为抗癌治疗发展的分子靶点。Plk1在细胞分裂中起核心作用，Plk1活性的上调似乎与几种癌症的侵袭性和不良预后密切相关。这种蛋白在许多癌症中过度表达，其抑制作用可导致抗增殖作用。Plk1需要n端激酶结构域（KD）和c端polo-box结构域（PBD）的协同作用，前者执行其催化功能，后者与含磷丝氨酸（pS）和磷苏氨酸（pT）的序列进行蛋白-蛋白相互作用（PPIs）。虽然以Plk1 kd为导向的药物目前正处于治疗癌症的临床试验中，但与细胞毒性相关的问题已经出现，可能是脱靶效应的结果。靶向蛋白-蛋白相互作用（PPIs）已成为抗癌治疗发展的重要领域。对于磷酸化依赖性PPIs，如Plk1 PBD，磷酸化的蛋白残基可以提供高亲和力识别并结合靶蛋白热点。从5聚磷酸肽“PLHSpT”开始，与Kyung Lee博士的NCI实验室和Michael Yaffe博士的MIT实验室合作，我们最初确定了抑制肽，显示出从1000到10000倍的pbd结合亲和力。这些与Plk1 PBD结合的肽的x射线共晶结构显示了意想不到的结合模式，它利用了“隐式”结合通道，这种通道不存在于非配体PBD中，也不存在于母体五聚体磷酸肽中。通过连接到His咪唑环的N（pi）氮上的苯基烷基部分进入隐袋。多价性是实现高效和选择性配体-蛋白相互作用的有力手段。通过将atp结合间隙内结合的元件与间隙外结合的元件连接在一起，可以大大增加蛋白激酶（PK）抑制剂的选择性和亲和力。当二级组分进入辅助调控结构域时，所得到的配体可被描述为分子内“二价”配体。我们已经开展了开发二价配体的工作，旨在同时参与Plk1的KD和PBD区域。这导致二价结构体相对于单价pbd结合配体表现出超过100倍的Plk1亲和力增强，而单价pbd结合配体迄今为止表现出最高的pbd结合亲和力。令人惊讶的是，与广泛接受的KD- pbd相互作用的概念相反，我们发现即使KD和pbd结合组分之间的连接物最少，也可以保持极高的亲和力。除了显著推进pbd结合配体的发展外，我们的发现可能会引起对Plk1结构功能的重新思考以及该激酶所起生理作用的潜在影响。目的二：酪氨酸-DNA磷酸二酯酶1 （TDP1）通过修复停滞的TOP1与DNA的共价复合物来降低I型拓扑异构酶（TOP1）抑制剂的抗癌作用。它通过促进TOP1的Y723残基与其DNA底物的-磷酸之间的磷酸二酯键的水解来实现这一点。阻断TDP1功能将是提高TOP1抑制剂疗效和克服耐药性的一种有吸引力的手段。TDP1抑制剂将代表一种新的、有潜力的抗癌药物，可以与TOP1抑制剂一起用于抗癌治疗。虽然已经有关于TDP1抑制剂的报道，但迫切需要发现有效和特异性的TDP1抑制剂，这些抑制剂具有有效的结合和明确的作用机制。在与NCI实验室David Waugh博士和Yves Pommier博士的合作中，使用了超过600个片段的x射线晶体学屏幕来识别结合在TDP1催化口袋内的邻苯二甲酸和羟基喹啉基序的小分子变化。然而，这些化合物中的大多数显示有限的（毫摩尔）TDP1抑制能力。最近，我们与Jay Schneekloth博士的NCI实验室合作，对超过21,000种小分子微阵列（SMM）格式的药物样分子进行了TDP1小分子微阵列筛选，以检测它们与Alexa Fluor 647 （AF647）标记的TDP1的结合能力。该筛选从21,000个化合物中鉴定出109个（0.5%的命中率），并获得了首选的tdp1结合基序。其中有结构相似的N，2-二苯基咪唑[1,2-a]吡嗪-3胺，我们证明了它们的功能是TDP1的结合剂和催化抑制剂。然后，我们使用一锅Groebke-Blackburn-Bienayme多组分反应探索了核心杂环骨架，并获得了具有更高抑制能力的类似物。通过求解一组化合物的TDP1共晶结构，发现它们在TDP1催化位点结合，同时模拟了底物的相互作用。我们目前正在通过添加延伸到肽和DNA底物结合区域的功能来详细阐述母体smm衍生平台的结构。我们正在使用基于“点击”的肟多样化策略，我们已经在几个应用中成功地用于优化母体配体的结合相互作用。这种方法的关键是它能够采用单一的合成亲本结构，并使用易于获得的醛试剂库轻松地对其进行多样性。在这项工作中，我们通过添加aminooxy句柄来修改smm衍生的平台。这产生了两个含有氨基基的亲本结构。这些结构的苯甲酸部分旨在结合在催化位点磷酸化结合口袋内，而氨基基则位于此，以便产生的肟衍生物可以进入DNA或肽底物结合通道。通过这种方式，我们能够快速查询大约500个肟衍生物的结构。最有希望的化合物（低微摩尔IC50值）进一步衍生，以增加母体肟键的化学稳定性。通过这一过程，我们已经能够获得纳米级的TDP1抑制剂。我们最近收到了与TDP1催化位点结合的肟衍生抑制剂的晶体结构，它们的结合方式似乎与我们的分子对接研究预测的相似。展望未来，我们的目标是获得具有明确结合相互作用的有效抑制剂。这些抑制剂将为研究细胞环境中竞争性抑制TDP1功能的生化效应提供药理学工具。我们的工作对TDP1抑制剂的开发有一定的推动作用。最近提交了一份涵盖该技术各方面的PCT专利申请。

defined as a molecular target for anti-cancer therapy development. The Plk1 plays a central role in cell division and upregulation of Plk1 activity appears to be closely associated with aggressiveness and poor prognosis of several cancers. This protein is overexpressed in many cancers and its inhibition can result in antiproliferative effects. Plk1 requires the coordinated actions of both an N-terminal kinase domain (KD), which executes its catalytic function and a C-terminal polo-box domain (PBD), which engages in protein - protein interactions (PPIs) with phosphoserine (pS) and phosphothreonine (pT)-containing sequences. Although Plk1 KD-directed agents are currently in clinical trials for the treatment of cancers, issues related to cytotoxicity have arisen that may result from off-target effects. Targeting protein - protein interactions (PPIs) has emerged as an important area for anticancer therapeutic development. In the case of phospho-dependent PPIs, such as the Plk1 PBD, a phosphorylated protein residue can provide high-affinity recognition and bind to target protein hot spots. Starting from the 5-mer phosphopeptide "PLHSpT" and in collaboration with the NCI laboratory of Dr. Kyung Lee and the MIT laboratory of Dr. Michael Yaffe, we initially identified inhibitory peptides that showed from 1000- to more than 10,000-fold improved PBD-binding affinity. X-ray co-crystal structures of these peptides bound to Plk1 PBD indicated unanticipated modes of binding, which take advantage of a "cryptic" binding channel that is not present in the non-liganded PBD or engaged by the parent pentamer phosphopeptide. The cryptic pocket is accessed by means of a phenylalkyl moiety attached to the N(pi) nitrogen of the His imidazole ring. Multivalency can be a powerful means to achieve highly potent and selective ligand-protein interactions. The selectivity and affinity of protein kinase (PK) inhibitors can be greatly increased by linking an element that binds within the ATP-binding cleft together with a component that binds exterior to the cleft. When the secondary component accesses ancillary regulatory domains, the resulting ligand may be described as being intramolecular "bivalent." We have undertaken work to develop bivalent ligands, designed to simultaneously engage both KD and PBD regions of Plk1. This has resulted in bivalent constructs exhibiting more than 100-fold Plk1 affinity enhancement relative monovalent PBD-binding ligands, which had until this time, exhibited among the highest PBD-binding affinities yet reported. Startlingly, and in contradiction to widely accepted notions of KD-PBD interactions, we have found that extremely high affinities can be retained even with minimal linkers between KD and PBD-binding components. In addition to significantly advancing the development of PBD-binding ligands, our findings may cause a rethinking of the structure-function of Plk1 and potential implications for the physiological roles played by this kinase. Objective Two: Tyrosyl-DNA phosphodiesterase 1 (TDP1) it is capable of reducing the anticancer effects of type I topoisomerase (TOP1) inhibitors by repairing the stalled covalent complexes of TOP1 with DNA. It achieves this by promoting the hydrolysis of the phosphodiester bond between the Y723 residue of TOP1 and the -phosphate of its DNA substrate. Blocking TDP1 function would be an attractive means of enhancing the efficacy of TOP1 inhibitors and overcoming drug resistance. TDP1 inhibitors would represent a new and potentially promising class of anticancer agents that could be used with TOP1 inhibitors in anticancer therapy. Although there have been reports of TDP1 inhibitors, there is a pressing need for the discovery of effective and specific TDP1 inhibitors for which there is validated binding and a defined mechanism of actions. In collaboration with the NCI laboratories of Dr. David Waugh and Dr. Yves Pommier, used an X-ray crystallographic screen of more than 600 fragments to identify small molecule variations on phthalic acid and hydroxyquinoline motifs that bind within the TDP1 catalytic pocket. Yet, the majority of these compounds showed limited (millimolar) TDP1 inhibitory potencies. More recently, in collaboration with the NCI laboratory of Dr. Jay Schneekloth, we performed a TDP1 small molecule microarray screen of over 21,000 drug-like molecules in a small molecules microarray (SMM) format for their ability to bind Alexa Fluor 647 (AF647)-labeled TDP1. The screen identified 109 hits from 21,000 compounds (0.5% hit rate) and arrived at a preferred TDP1-binding motif. Among the hits were structurally similar N,2-diphenylimidazo[1,2-a]pyrazin-3-amines, which we demonstrated functioned as TDP1 binders and catalytic inhibitors. We then explored the core heterocycle skeleton using one-pot Groebke-Blackburn-Bienayme multicomponent reactions and arrived at analogs having higher inhibitory potencies. Solving TDP1 co-crystal structures of a subset of compounds showed their binding at the TDP1 catalytic site, while mimicking substrate interactions. We are currently elaborating the structure of the parent SMM-derived platform by adding functionality that extends into the peptide and DNA substrate binding regions. We are using a "click"-based oxime diversification strategy that we have used successfully in several applications to optimize the binding interactions of parent ligands. A key to this approach is its ability to take a single synthetic parent construct and easily diversity it using a library of readily obtainable aldehyde reagents. In this work, we are modifying our SMM-derived platforms by adding aminooxy handles. This yielded two parent aminooxy-containing constructs. The benzoic acid moieties of these constructs are intended to bind within the catalytic site phosphoryl-binding pocket while the aminooxy groups are situated so that the resulting oxime derivatives would access the DNA or peptide substrate-binding channels. In this way, we were able to rapidly interrogate the structures of approximately 500 oxime derivatives. The most promising compounds (low micromolar IC50 values) were further derivatized to increase the chemical stability of the parent oxime linkages. Through this process, we have been able to achieve TDP1 inhibitors with nanomolar potencies. We have recently received the crystal structure of oxime-derived inhibitors bound to the TDP1 catalytic site and it appears that they bind in a fashion that is similar to what was predicted by our molecular docking studies. Going forward our goal is to derive validated inhibitors with defined binding interactions. These inhibitors will provide pharmacological tools for studying the biochemical effects of competitively inhibiting TDP1 function in cellular settings. Our work should advance the general field of TDP1 inhibitor development. A PCT patent application has recently been filed covering aspects of this technology.