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

酪氨酸激酶依赖性信号传导抑制剂作为抗癌药物

• 批准号: 10262021
• 负责人: TERRENCE BURKE
• 金额: $ 113.36万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10262021
• 关键词: Affinity; Agreement; Alkylating Agents; Alkylation; Amides; Amino Acid Sequence; Amino Acids; Antibodies; Antibody-drug conjugates; Antineoplastic Agents; Apoptotic; Area; Binding; Binding Sites; Biological Assay; Bispecific Antibodies; C-terminal; Catalytic Antibodies; Catalytic Domain; Cations; Cell Death; Cell division; Cells; Charge; Chemicals; Chemistry; Chronic Lymphocytic Leukemia; Cleaved cell; Collaborations; Combined Modality Therapy; Complex; Crystallization; Cyclization; Cytotoxic Chemotherapy; Cytotoxic T-Lymphocytes; DNA; DNA Repair Enzymes; Data; Development; Drug Delivery Systems; Endothelial Cells; Enzymes; Exhibits; Fc Immunoglobulins; Florida; Fluorescence; Haptens; Human; Imidazole; Immunoglobulin M; Integrin alpha4; Kinetochores; Laboratories; Lead; Lesion; Ligands; Location; Malignant Neoplasms; Masks; Measures; Mediating; Membrane; Mind; Mitotic; Molecular Conformation; Monoclonal Antibodies; National Heart, Lung, and Blood Institute; Nitrogen; Normal Cell; Nuclear; Nucleosides; PLK1 gene; Parents; Penetration; Peptides; Periodicity; Pharmaceutical Preparations; Phosphopeptides; Phosphoserine; Phosphothreonine; Phosphotransferases; Phthalic Acids; Physiological; Plant Resins; Play; Polo-Box Domain; Positioning Attribute; Proteins; Reaction; Research; Roentgen Rays; Role; Selenocysteine; Series; Serine; Signal Transduction; Specificity; Speed; Structure; Structure-Activity Relationship; TOP1 gene; Therapeutic; Threonine; Topoisomerase; Topoisomerase Inhibitors; Tyrosine; Tyrosine Kinase Inhibitor; Up-Regulation; Work; analog; anti-cancer therapeutic; azetidinone; base; beta-Lactams; cancer cell; carbene; chimeric antigen receptor; cost; design; diketone; engineered T cells; folate-binding protein; functional group; improved; inhibitor/antagonist; inorganic phosphate; insight; kinase inhibitor; mitochondrial genome; neoplastic cell; novel strategies; outcome forecast; oxidation; peptide structure; peptidomimetics; pharmacophore; phosphodiester; polo-like kinase kinase 1; recruit; screening; small molecule; stable plasma protein solution; tumor; tyrosyl-DNA phosphodiesterase

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. In addition to its catalytic KD, Plk1 also contains a non-catalytic polo-box domain (PBD), which binds to the enzyme physiological substrates and localizes the enzyme to discrete locations within the kinetochore. PBD inhibitors target a structurally unique domain found in only four proteins (Plk1-3 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. In further work we discovered chemistry to install functionality at the His N(tau)-nitrogen using phospho-directed on-resin Mitsunobu alkylation conditions to produce peptidomimetics containing N(pi),N(tau)-bis-alkylated His residues. Importantly, the cationic bis-alkyl imidazolium species may increase membrane penetration via intramolecular "charge masking" of the anionic phosphate moiety. The X-ray co-crystal structures of bis-alkylated His-containing peptides bound to the PBD indicated that the His N(tau)-nitrogen is within 6 angstroms of the C-terminal carboxamide. We envisioned cyclic ligands could be prepared in which the N(pi),N(tau)-bis-alkylated His could serve as a bifurcated ring junction. We employed methylene linkers of various size between the N(tau)-nitrogen of imidazole and the C-terminus, utilizing an amide forming macrocyclization reaction. We eventually found that we were able to achieve a tripeptide macrocycle that retained high PBD-binding affinity. Inhibition data from the bis-alkyl His-containing cyclic ligands suggested that cyclization between the C-terminus and the pThr(-2) position is beneficial to activity. With this in mind, we investigated new functionality at the pThr(-2) position that could incorporate the -(CH2)8Ph required for high-affinity ligands and also an orthogonally-protected functional group for on-resin macrocyclization. We developed a new non-His-based amino acid that could serve as a macrocycle ring junction while accessing the critical cryptic pocket. Importantly, this new amino acid analog could be incorporated into SPPS on Rink resin to produce macrocyclic ligands that retained high PBD-binding affinities. In further work, we designed a series of probes based on the active pharmacophore of the Plk1 kinase inhibitor, BI2536 tethered to a fluorescent moiety. The probes provided a fluorescence-based measure of binding affinity, which could be used to determine the affinities of candidate Plk1 kinase inhibitors. We found that the assays were able to provide IC50 values of type 1 kinase inhibitors (inhibitors that compete with ATP-binding in the active conformation of the catalytic pocket) that are in accordance with values obtained from enzymatic assays. However, the probe was insensitive to other classes of kinase inhibitors. This rendered it potentially useful in conjunction with enzymatic kinase assays to distinguish type 1 inhibitors from these other classes of inhibitors. The assay may afford a facile means for initial screening of type 1 ATP-competitive Plk1 inhibitors that offers distinct advantages over kinase assays in terms of cost, speed and ease of handling. Objective Two: Tdp1 removes DNA 3-prime 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. Using structural insights into fragment binding, we prepared several fragment derivatives, some of which exhibited significantly higher Tdp1 inhibitory potencies than the parent molecules. 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. We identified 109 hits from 21,000 compounds (0.5% hit rate) and arrived at a preferred Tdp1-binding motif. Further structure activity relationship (SAR) work achieved a class of small molecules that showed low micromolar Tdp1-inhibitory potencies. X-ray co-crystal structures in the Waugh Laboratory showed that the promising leads bind at the catalytic site of Tdp1. In agreement with previous crystal structures of Tdp1-bound phthalic acid-containing fragments, these structures confirmed that the bis-carboxylic moieties recapitulate aspects of phosphate binding to the key catalytic residues. However, unlike simpler structures obtained in the earlier fragment screens, these more complex inhibitors orient distinct structural components into both the DNA and peptide substrate-binding regions. These are the first crystal structures of small molecules accessing the catalytic site as well as both the DNA substrate and peptide-binding regions. Objective Three: Antibody-drug conjugates (ADCs) constitute an important and emerging class of therapeutics. In a fourth area of research focus, we have a have a long-standing collaboration with the laboratory of Dr. Christoph Rader (Scripps Florida) to develop antibody-drug conjugates (ADCs). This capitalizes on our expertise in small molecule and peptide mimetic chemistry. Aspects of our approach employ monoclonal antibodies and antibody Fc fragments harboring a single C-terminal selenocysteine residue (Fc-Sec). In other work, we use the "catalytic antibody" h38C2 to effect selective covalent conjugation using azetidinone and beta-diketone-containing drug payloads. We are also contributing to the development of a platform of chemically programmed bispecific antibodies (biAbs). These endow target cell-binding small molecules with the ability to recruit and redirect cytotoxic T cells to eliminate cancer cells. In collaboration with Dr. Adrian Wiestner (NHLBI), we have developed a novel strategy for targeted cytotoxic therapy of chronic lymphocytic leukemia (CLL). This employs IgM-based Fc(mu)R-targeted antibody-drug delivery to effect potent and specific therapeutic activity against CLL. In parallel, we are using chimeric antigen receptor ("CAR")-engineered T cells that include the hapten-binding site of h38C2. These are being chemically programmed through covalent binding of the reactive h38C2 Lys residue with 1,3-diketone or beta-lactam moieties tethered by variable PEG spacers to cyclic RGDfK groups. This endows the CARs with the ability to bind to human integrin alpha4,beta3 with high affinity and specificity. These are anticipated to kill human integrin alpha4,beta3 - expressing tumor cells and tumor endothelial cells. We have also examined trifunctional folate receptor 1 (FOR1)-targeting moieties.

目标一：Plk1 在细胞分裂中发挥核心作用，Plk1 活性上调似乎与多种癌症的侵袭性和不良预后密切相关。除了其催化 KD 之外，Plk1 还包含一个非催化 polo-box 结构域 (PBD)，它与酶的生理底物结合并将酶定位到着丝粒内的离散位置。 PBD 抑制剂针对仅在四种蛋白质（Plk1-3 和 Plk5）中发现的结构独特的结构域。单独抑制 Plk1 PBD 功能足以有效地在癌细胞中产生有丝分裂停滞和细胞凋亡，但在正常细胞中则不然，PBD 结合相互作用的抑制剂可以作为开发抗 Plk1 疗法的靶点限制策略。从 5 聚体磷酸肽 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，我们初步鉴定出肽抑制剂，其 PBD 结合能力提高了 1000 至 10,000 倍以上亲和力。这些肽与 Plk1 PBD 结合的 X 射线共晶结构表明了意想不到的结合模式，该模式利用了非配体 PBD 中不存在的“神秘”结合通道或与亲本五聚体磷酸肽接合。通过连接至组氨酸咪唑环的 N(pi) 氮的苯基烷基部分可进入隐秘袋。在进一步的工作中，我们发现了使用磷定向树脂 Mitsunobu 烷基化条件在 His N(tau)-氮上安装功能性的化学方法，以产生含有 N(pi),N(tau)-双烷基化 His 残基的肽模拟物。重要的是，阳离子双烷基咪唑鎓类物质可以通过阴离子磷酸盐部分的分子内“电荷掩蔽”来增加膜渗透。与 PBD 结合的双烷基化含 His 肽的 X 射线共晶结构表明，His N(tau)-氮位于 C 端甲酰胺的 6 埃范围内。我们设想可以制备环状配体，其中 N(pi),N(tau)-双烷基化 His 可以作为分叉环连接。我们利用酰胺形成大环化反应，在咪唑的 N(tau)-氮和 C 末端之间采用了不同大小的亚甲基连接体。我们最终发现我们能够获得保留高 PBD 结合亲和力的三肽大环化合物。来自含双烷基组氨酸的环状配体的抑制数据表明，C 末端和 pThr(-2) 位之间的环化有利于活性。考虑到这一点，我们研究了 pThr(-2) 位置的新功能，该功能可以结合高亲和力配体所需的 -(CH2)8Ph 以及用于树脂大环化的正交保护官能团。我们开发了一种新的非组氨酸氨基酸，可以作为大环连接点，同时进入关键的隐秘口袋。重要的是，这种新的氨基酸类似物可以合并到 Rink 树脂上的 SPPS 中，以产生保留高 PBD 结合亲和力的大环配体。在进一步的工作中，我们基于 Plk1 激酶抑制剂 BI2536 的活性药效团设计了一系列探针，该抑制剂与荧光部分相连。这些探针提供了基于荧光的结合亲和力测量，可用于确定候选 Plk1 激酶抑制剂的亲和力。我们发现这些测定能够提供 1 型激酶抑制剂（在催化口袋的活性构象中与 ATP 结合竞争的抑制剂）的 IC50 值，该值与酶测定中获得的值一致。然而，该探针对其他类别的激酶抑制剂不敏感。这使得它可能与酶激酶测定结合使用，以区分 1 型抑制剂和其他类别的抑制剂。该测定可为 1 型 ATP 竞争性 Plk1 抑制剂的初步筛选提供一种简便的方法，与激酶测定相比，它在成本、速度和操作简便性方面具有明显的优势。目标二：Tdp1 消除由链终止核苷和烷化剂以及核和线粒体基因组中的碱基氧化产生的 DNA 3 引物末端封闭损伤。与 Tdp1 抑制剂的联合治疗可能与拓扑异构酶抑制剂 (Top1) 产生协同作用，以增强针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，针对 Tdp1 的催化结构域进行了晶体学片段筛选活动，以确定用于构建 Tdp1 抑制剂的新先导化合物。利用片段结合的结构见解，我们制备了几种片段衍生物，其中一些表现出比母体分子显着更高的 Tdp1 抑制效力。在另一项工作中，我们与 Jay Schneekloth 博士的 NCI 实验室合作，对 20,000 多个药物样分子进行了 Tdp1 小分子微阵列筛选，以鉴定新的 Tdp1 结合基序。我们从 21,000 种化合物中鉴定出 109 个命中（命中率 0.5%），并得出了首选的 Tdp1 结合基序。进一步的结构活性关系 (SAR) 工作获得了一类显示出低微摩尔 Tdp1 抑制效力的小分子。沃实验室的 X 射线共晶结构表明，有希望的先导化合物结合在 Tdp1 的催化位点上。与之前 Tdp1 结合的含邻苯二甲酸片段的晶体结构一致，这些结构证实双羧基部分概括了磷酸盐与关键催化残基结合的方面。然而，与早期片段筛选中获得的更简单的结构不同，这些更复杂的抑制剂将不同的结构成分定向到 DNA 和肽底物结合区域。这些是第一个进入催化位点以及 DNA 底物和肽结合区域的小分子晶体结构。目标三：抗体药物偶联物（ADC）构成一类重要的新兴疗法。在第四个研究重点领域，我们与 Christoph Rader 博士（佛罗里达州斯克里普斯）实验室长期合作开发抗体药物偶联物 (ADC)。这利用了我们在小分子和肽模拟化学方面的专业知识。我们的方法的各个方面采用单克隆抗体和含有单个 C 端硒代半胱氨酸残基 (Fc-Sec) 的抗体 Fc 片段。在其他工作中，我们使用“催化抗体”h38C2 使用氮杂环丁酮和含有 β-二酮的药物有效负载来实现选择性共价缀合。我们还致力于化学编程双特异性抗体 (biAb) 平台的开发。这些赋予靶细胞结合小分子招募和重定向细胞毒性 T 细胞以消灭癌细胞的能力。我们与 Adrian Wiestner 博士 (NHLBI) 合作，开发了一种针对慢性淋巴细胞白血病 (CLL) 的靶向细胞毒治疗的新策略。该技术采用基于 IgM 的 Fc(mu)R 靶向抗体药物递送来实现针对 CLL 的有效且特异的治疗活性。与此同时，我们正在使用嵌合抗原受体（“CAR”）工程化的 T 细胞，其中包含 h38C2 的半抗原结合位点。这些是通过反应性 h38C2 Lys 残基与 1,3-二酮或 β-内酰胺部分的共价结合进行化学编程的，1,3-二酮或 β-内酰胺部分通过可变 PEG 间隔基连接到环状 RGDfK 基团。这赋予了 CAR 以高亲和力和特异性结合人类整合素 α4、β3 的能力。这些预计可杀死表达人整合素α4、β3的肿瘤细胞和肿瘤内皮细胞。我们还检查了三功能叶酸受体 1 (FOR1) 靶向部分。