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

目的一：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。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。我们通过使用超过600个片段的x射线晶体学屏幕开始这项工作，以确定在TDP1催化口袋内结合的邻苯二甲酸和羟基喹啉基序的小分子变化。最近，我们以小分子微阵列（SMM）格式对超过21,000个药物样分子进行了TDP1小分子微阵列筛选，以检测它们结合Alexa Fluor 647 （AF647）标记的TDP1的能力。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。这项工作的生物学方面是与NCI Yves Pommier博士的实验室合作完成的。x射线晶体学研究最初是在Dave Waugh博士的NCI实验室进行的，但最近这项工作转移到了George Lountos博士的FNLCR实验室。SMM的工作是与Jay Schneekloth博士的NCI实验室合作完成的。在报告期间，我们通过添加扩展到肽和DNA底物结合区域的功能，详细阐述了母体smm衍生平台的结构。我们采用了一种基于“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。这种方法的一个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多样化。这涉及到修改我们的smm衍生平台，通过添加氨基基句柄来产生两个包含氨基基的父结构。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，我们能够获得纳米级的TDP1抑制剂。我们获得了与TDP1催化位点结合的肟衍生抑制剂的晶体结构，并观察到它们的结合方式与我们的分子对接研究预测的相似。我们目前的工作是利用硫（VI）氟交换（SuFEx）生物相容性点击化学反应制备磺酰氟和含氟硫酸盐的共价配体，设计用于特异性靶向TDP1催化位点的Tyr204残基。我们在喹诺酮亲本平台的8位上制备并筛选了一个小文库，其中含有磺酰氟和含氟硫酸盐系链取代喹诺酮。重要的是，我们的TDP1与这些喹诺酮类药物结合的共晶结构表明，TDP1与催化位点Tyr204存在共价键。推进这些代理人的工作正在取得进展。目的二：LGR5增强典型Wnt/B-catenin信号通路。在结直肠癌的腺瘤-癌序列中发生的Wnt/B-catenin信号通路的解除，以及肿瘤内的特定亚群或分子可能被靶向治疗以防止复发和诱导长期缓解。这使得LGR5成为潜在的治疗靶点。蛋白质r - spontin -1 （RSPO1）作为配体结合到LGR5的细胞外结构域。RSPO1模拟物可能在结肠癌手术切除过程中作为靶向癌组织的亲和力标签。我的实验室正在与乔治城大学转化成像中心的Chris Albanese博士进行多方合作，开发一流的RSPO1肽模拟物，可以作为载体，在结肠癌手术切除过程中提供近红外（NIR）染料来照亮癌组织。我的实验室正在设计和合成荧光标记的lgr5结合肽模拟物。我们最初设计的RSPO-1模拟物是基于RSPO1结合LGR5与Ring Finger 43 （RNF43）的三元配合物的晶体结构。这表明RSPO1和LGR5之间的主要结合相互作用是通过位于其c端Furin 2 Domain （FU2）的含有双二硫的序列K96-K113中的残基F106和F110发生的。我们称之为“右侧”。二次结合是由其单二硫n端Furin 1结构域（FU1）的残基P77-I95内的R87残基介导的。我们称之为“左侧”。“Right Side”18-mer的结合相互作用尤为重要，重点将放在通过几种方法优化Right Side的合成上。“左侧”将被综合探索。在对右侧和左侧肽进行优化后，这些模拟物通过系聚部分连接形成二价结构。我们已经开发了固相肽合成（SPPS）化学方法来合成含有18聚体右侧的双二硫化物和含有18聚体左侧的单二硫化物。我们使用叠氮化物-炔点击化学将这些结构连接起来，形成完整的“37-mer”RSPO1模拟物，其中包含主要的RSPO1结合相互作用。37-mer的结合相互作用本质上可以看作是“二价”的，其左右两侧代表由桥接段连接的独立结合基团。二价配体的亲和力可以显著高于分离组分的亲和力。我的RSPO1模拟物与LGR5的特异性结合正在通过细胞染色、敲低和竞争实验得到证实。

Objective One: 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. 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. We began this work by using 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. More recently, 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. The biological aspects of this work are being done in collaboration with the NCI laboratory of Dr. Yves Pommier. X-ray crystallographic studies were initially conducted in the NCI laboratory of Dr. Dave Waugh, but more recently this work has shifted to the FNLCR laboratory of Dr. George Lountos. The SMM work was done in collaboration with the NCI laboratory of Dr. Jay Schneekloth. During the reporting period we have elaborated the structure of the parent SMM-derived platform by adding functionality that extends into the peptide and DNA substrate binding regions. We employed 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 using a library of readily obtainable aldehyde reagents. This involved modifying our SMM-derived platforms by adding aminooxy handles to yield 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 were able to achieve TDP1 inhibitors with nanomolar potencies. We obtained the crystal structure of oxime-derived inhibitors bound to the TDP1 catalytic site and observed that they bind in a fashion that is similar to what was predicted by our molecular docking studies. Our current work is using sulfur (VI) fluoride exchange (SuFEx) biocompatible click chemistry reactions to prepare sulfonyl fluoride and fluorosulfate-containing covalent ligands designed to site-specifically target the Tyr204 residue in the catalytic site of TDP1. We have prepared and screened a small library of substituted quinolines with sulfonyl fluorides and fluorosulfate-containing tethers at the 8-position of a parent quinolone platform. Importantly, our cocrystal structures of TDP1 bound to these quinolones suggest covalent bond to the Tyr204 at the catalytic site of TDP1. Work is progressing to advance these agents. Object Two: The LGR5 potentiates canonical Wnt/B-catenin signaling. The well characterized deregulation of Wnt/B-catenin signaling that occurs during the adenoma-carcinoma sequence in CRC and further that specific subpopulations or molecules within a tumor may be therapeutically targeted to prevent relapse and induce long-term remissions. This renders LGR5 as a potential therapeutic target. The protein, R-spondin-1 (RSPO1) serves as a ligand that binds to the extracellular domain of LGR5. RSPO1 mimetics could potentially serve as affinity tags for targeting cancerous tissues during surgical resection of colon cancers. My laboratory is participating in a multi-party collaboration with Dr. Chris Albanese (Center for Translational Imaging, Georgetown University) to develop first-in-class RSPO1 peptide mimetics that can serve as vehicles to deliver near IR (NIR) dyes to light up cancerous tissues during surgical resection of colon cancers. My laboratory is designing and syntgesizing fluorescently tagged LGR5-binding peptide mimetics. Our initial design of RSPO-1 mimetics is based on the crystal structure of RSPO1 bound to LGR5 in a ternary complex with Ring Finger 43 (RNF43). This shows that the major binding interactions between RSPO1 and LGR5 occur via residues F106 and F110 within the bis-disulfide containing sequence K96-K113 located in its C-terminal Furin 2 Domain (FU2). We term this the "Right Side." Secondary binding is mediated by the R87 residue contained within residues P77-I95 in its mono-disulfide N-terminal Furin 1 Domain (FU1). We term this the "Left Side." Binding interactions of the "Right Side" 18-mer are particularly important and emphasis will be placed on optimizing synthesis of the right side by several approaches. The "Left Side" will be synthetically explored. Following optimization of the Right and Left Side peptides, these mimetics were connected to form bivalent constructs using tethering moieties. We have already developed solid-phase peptide synthesis (SPPS) chemistries to synthesize both the bis-disulfide containing 18-mer Right Side and mono-disulfide containing Left Side. We are using azide-alkyne click chemistry to join the constructs to form the entire "37-mer" RSPO1 mimetic, which contains the major RSPO1 binding interactions. Binding interactions of the 37-mer may be viewed as being "bivalent" in nature with the Left and Right Sides representing independent binding moieties that are connected by a bridging segment. The affinity of bivalent ligands can be significantly higher than the affinities of the isolated components. Specific binding of my RSPO1 mimetics to LGR5 is being confirmed by cell staining, knockdown, and competition assays.