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

目的一：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。在Plk1成瘾的癌症中，靶向Plk1可能诱导癌细胞选择性有丝分裂阻断和凋亡细胞死亡。然而，针对Plk1激酶结构域（KD）的抑制剂的潜在限制可能源于激酶之间ATP结合间隙的高度相似性，特别是在Plk家族的其他成员（Plk1至5）中缺乏特异性。提高Plk1特异性是最迫切需要解决的问题之一，以实现更好的临床结果，减少毒理学问题。除了催化KD外，Plk1还含有一个非催化polo盒结构域（PBD），它与酶的生理底物结合，并将酶定位在着丝点内的离散位置。与ATP竞争性抑制剂不同，ATP竞争性抑制剂必须针对500多种其他细胞激酶获得特异性，PBD抑制剂靶向仅在四种蛋白质（Plk1至3和Plk5）中发现的结构独特结构域。单独抑制Plk1 PBD功能足以有效地在癌细胞中施加有丝分裂阻滞和凋亡细胞死亡，但在正常细胞中却没有，PBD结合相互作用的抑制剂可能作为开发抗Plk1治疗的靶标限制策略。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，我们最初确定了肽抑制剂，显示出从1000到10000倍以上的PBD结合亲和力改善。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，利用了“隐式”结合通道，该通道不存在于非配体PBD中或由母体五聚体磷酸肽参与。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。随后，我们使用基于肟连接的策略优化了这些PBD配体的相互作用。最近，我们利用树脂叠氮化炔烃环加成反应将1,2,3三唑官能性引入到有效的Plk1 PBD先导抑制剂中。三唑环的目的是诱导构象约束或作为他的模仿物。这些新配体中的某些保留了亲本肽的高Plk1 PBD结合亲和力，同时相对于Plk2和Plk3的PBD具有增强的Plk1 PBD选择性。值得注意的是，与分离PBD获得的值相比，某些肽在全长Plk1 ELISA检测中表现出明显高于预期的降低亲和力（一例为160倍，另一例为480倍）。较大的差异可能表明，这些含三唑的肽有效缓解KD和PBD之间结构域间相互作用引起的自抑制或在全长结构中参与PBD隐口袋的能力降低。这可能表明全长Plk1中KD和PBD的结构相互作用存在显著的纬度。目的二：蛋白酪氨酸磷酸酶（PTPases）使蛋白底物内的磷酸酪氨酸（pTyr）残基去磷酸化。PTPases协同蛋白酪氨酸激酶调节信号转导通路。由于信号转导通路在调节癌症和感染性疾病的病理过程中的重要作用，PTPases已成为开发治疗性抑制剂的重要靶点。我们之前使用EGFR衍生肽“VDADEpYL”作为非水解pTyr模拟残基的展示平台，从而鉴定出二氟膦甲乙基芳基片段，作为设计小分子抑制剂的起点。这部分可以用来将良好的底物转化为高亲和力的抑制剂。最近，在与Robert Ulrich博士的USAMRIID实验室和David Waugh博士的NCI实验室的合作中，我们使用这个肽序列作为支架来呈现药物样片段的微阵列文库，以确定抑制剂设计的基序。这项工作的目的是设计一种方法来开发蛋白质-蛋白质相互作用抑制剂（PPIs）是固有的PTPase催化特异性。通过在EGFR衍生肽的6个不同位置合并300个不同的药物样片段，创建了一个多样化的文库。利用天花大天花病毒H1 （VH1）、鼠疫芽孢杆菌耶尔森氏菌外蛋白H （YopH）和人双特异性蛋白磷酸酶DUSP14和DUSP22的PTPases检测与底物文库的详细相互作用。为了确定可能对抑制剂设计有用的片段，通过微阵列文库的催化分析获得了初步结果，并使用基于等离子体共振（SPR）的微阵列筛选从微阵列文库中获得了动力学结合数据。作为原理证明，利用两步催化和表面鉴定的高亲和力肟片段设计了低分子量、不含磷酸盐的肽，该肽能够在低微摩尔浓度下抑制PTP的催化作用。目的三：TDP1去除核和线粒体基因组中由链终止核苷和烷基化剂以及碱基氧化产生的DNA 3端阻断病变。与TDP1抑制剂联合治疗可能潜在地与拓扑异构酶抑制剂（TOP1）协同作用，以提高对癌细胞的选择性和效力。与David Waugh博士和Yves Pommier博士的NCI实验室合作，对TDP1的催化结构域进行了晶体学片段筛选，以确定构建TDP1抑制剂的新先导化合物。晶体结构鉴定出两个结合TDP1活性位点的片段，并对TDP1表现出可测量的抑制活性。这些片段的结合模式在TDP1活性位点的位置与先前的TDP1结合钒酸盐晶体结构相似，这是一种过渡态模拟物。利用对片段结合的结构见解，我们制备了几个片段衍生物，其中一些衍生物比亲本片段表现出明显更高的TDP1抑制能力。在与Jay Schneekloth博士的NCI实验室合作的另一项工作中，我们对超过20,000个药物类分子进行了TDP1小分子微阵列筛选，以确定新的TDP1结合基元。与博士的实验室合作。Pommier和Waugh，我们目前正在优化这些初始线索。

Objective One: 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. Targeting Plk1 may permit induction of cancer cell selective mitotic block and apoptotic cell death in Plk1 addicted cancers. However, a potential limitation of inhibitors directed at the Plk1 kinase domain (KD) may arise from a lack of specificity due to the high degree of similarity in the ATP binding clefts among kinases, particularly among other members of the Plk family (Plk1 to 5). Improving Plk1 specificity is one of the most pressing concerns to address to accomplish better clinical outcomes with less toxicological problems. In addition to its catalytic KD, Plk1 also contains a non catalytic polo box domain (PBD), which binds to the enzymes physiological substrates and localizes the enzyme to discrete locations within the kinetochore. Unlike ATP competitive inhibitors, whose specificities must be obtained against more than 500 other cellular kinases, PBD inhibitors target a structurally unique domain found in only four proteins (Plk1 to 3 and Plk5). Inhibition of Plk1 PBD function alone is sufficient for effectively imposing mitotic arrest and apoptotic cell death in cancer cells but not in normal cells and inhibitors of PBD binding interactions may serve as a target restricted strategy for developing anti Plk1 therapeutics. 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 peptidic inhibitors 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 that 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. Subsequently, we have optimized these PBD ligand interactions using an oxime ligation based strategy. Most recently, we have utilized on resin azide alkyne cycloaddition reactions to introduce 1,2,3 triazole functionality into potent lead Plk1 PBD inhibitors. The triazole rings were intended either to induce conformational constraint or to serve as His mimetics. Certain of these new ligands retain the high Plk1 PBD binding affinity of the parent peptide, while having enhanced selectivity for the PBD of Plk1 relative to the PBDs of Plk2 and Plk3. It is of note that certain peptides exhibit significantly greater than anticipated reduced affinities in full length Plk1 ELISA assays relative to values obtained with the isolated PBD (160 fold in one case and 480 fold in a second case). The larger differences may indicate a reduced ability of these triazole containing peptides to effectively relieve auto inhibition arising from interdomain interactions between the KD and PBD or to engage the PBD cryptic pocket in the full length construct. This may potentially indicate significant latitude in the structural interactions of the KD and PBD in full length Plk1. Objective Two: Protein tyrosine phosphatases (PTPases) dephosphorylate phosphotyrosine (pTyr) residues within protein substrates. PTPases work in concert with protein tyrosine kinases to regulate signal transduction pathways. Because of the critical involvement of signal transduction pathways in regulating pathological processes in cancer and infectious diseases, PTPases have emerged as important targets for the development of therapeutic inhibitors. We have previously used the EGFR derived peptide "VDADEpYL" as a display platform for nonhydrolyzable pTyr mimicking residues, which led to the identification of the difluorophosphonomethyl aryl moiety as a starting point for the design of small molecule inhibitors. This moiety could be used to convert a good substrate into a high affinity inhibitor. Most recently, in collaboration with the USAMRIID laboratory of Dr. Robert Ulrich and the NCI laboratory of Dr. David Waugh, we have used this peptide sequence as a scaffold for presenting microarrayed libraries of druglike fragments to identify motifs for inhibitor design. The goal of this work was to devise a method for developing inhibitors of protein protein interactions (PPIs) that are intrinsic to PTPase catalytic specificities. A diversified library was created by incorporating 300 different druglike fragments at six different positions on the EGFR derived peptide. Detailed interactions with the substrate library were examined using PTPases from smallpox virus Variola major H1 (VH1), the plague bacillus Yersinia outer protein H (YopH), and the human dual specificity protein phosphatases (DUSP14 and DUSP22). In order to identify fragments that could be useful in inhibitor design, primary results were obtained from catalytic assays with the microarrayed library and kinetic binding data were obtained from the microarrayed library using a plasmon resonance (SPR) based microarray screen. As proof of principle, a high affinity oxime fragment identified by the two step catalytic and surface was employed to design low molecular weight, non phosphate containing peptides, which were able to inhibit PTP catalysis at low micromolar concentrations. Object Three: TDP1 removes DNA 3 end blocking lesions generated by chain terminating nucleosides and alkylating agents, and by base oxidation both in the nuclear and mitochondrial genomes. Combination therapy with TDP1 inhibitors may potentially synergize with topoisomerase inhibitors (TOP1) to enhance selectivity and potency against cancer cells. In collaboration with the NCI laboratories of Dr. David Waugh and Dr. Yves Pommier, a crystallographic fragment screening campaign was performed against the catalytic domain of TDP1 to identify new lead compounds for the construction of TDP1 inhibitors. Crystal structures identified two fragments that bind to the TDP1 active site and exhibit measurable inhibitory activity against TDP1. The binding mode of these fragments is in a similar position in the TDP1 active site as seen in prior crystal structures of TDP1 with bound vanadate, a transition state mimic. Using structural insights into fragment binding, we prepared several fragment derivatives, some of which exhibited significantly higher TDP1 inhibitory potencies than the parent fragments. In a separate effort, in collaboration with the NCI laboratory of Dr. Jay Schneekloth, we performed a TDP1 small molecule microarray screen of over 20,000 drug like molecules to identify new TDP1 binding motifs. In collaboration with the laboratories of Drs. Pommier and Waugh, we are currently in the process of optimizing these initial leads.