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。除了催化KD外，Plk1还含有一个非催化polo-box结构域（PBD），它与酶的生理底物结合，并将酶定位在着丝点内的离散位置。PBD抑制剂靶向仅在四种蛋白（Plk1-3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以作为开发抗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到超过10,000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，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。在进一步的工作中，我们发现了利用磷酸导向的树脂上Mitsunobu烷基化条件在His N(tau)-氮上安装功能的化学方法，以生产含有N(pi)，N(tau)-双烷基化His残基的肽模拟物。重要的是，阳离子双烷基咪唑可能通过阴离子磷酸盐部分的分子内“电荷掩蔽”来增加膜穿透。结合PBD的双烷基化含His肽的x射线共晶结构表明，His N(tau)-氮在c端羧基酰胺的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可以作为分岔环结。我们在咪唑的N(tau)-氮和c端之间使用了不同大小的亚甲基连接剂，利用酰胺形成的大环化反应。我们最终发现我们能够获得一个保留高pbd结合亲和力的三肽大环。含his的双烷基环配体的抑制数据表明，c端和pThr（-2）位置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，以及树脂上大环化所需的正交保护官能团。我们开发了一种新的非his基氨基酸，可以作为大环环结，同时进入关键的隐口袋。重要的是，这种新的氨基酸类似物可以结合到Rink树脂上的SPPS中，以产生保持高pbd结合亲和力的大环配体。在进一步的工作中，我们设计了一系列基于Plk1激酶抑制剂BI2536的活性药效团连接到荧光片段的探针。该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，这些分析能够提供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，与酶分析获得的值一致。然而，该探针对其他类型的激酶抑制剂不敏感。这使得它与酶激酶测定相结合，将1型抑制剂与其他类型的抑制剂区分开来。该检测方法可以为1型atp竞争性Plk1抑制剂的初始筛选提供一种简便的方法，在成本、速度和易于操作方面，它比激酶检测方法具有明显的优势。目的二：Tdp1去除由链终止核苷和烷基化剂以及核和线粒体基因组中的碱基氧化产生的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。利用片段结合的结构见解，我们制备了几种片段衍生物，其中一些衍生物比母体分子表现出更高的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个药物样分子进行了Tdp1小分子微阵列筛选，以确定新的Tdp1结合基序。我们从21,000个化合物中鉴定出109个（0.5%的命中率），并找到了首选的tdp1结合基序。进一步的构效关系（SAR）研究获得了一类具有低微摩尔tdp1抑制能力的小分子。Waugh实验室的x射线共晶结构表明，有希望的引线结合在Tdp1的催化位点。与先前tdp1结合的邻苯二甲酸片段的晶体结构一致，这些结构证实了双羧基部分再现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简单结构不同，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。这些是进入催化位点的小分子以及DNA底物和肽结合区域的第一个晶体结构。目的三：抗体-药物偶联物（adc）是一类重要的新兴治疗药物。在第四个研究重点领域，我们与Christoph Rader博士（Scripps Florida）的实验室长期合作开发抗体-药物偶联物（adc）。这充分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对含有氮杂二酮和-二酮的药物有效载荷进行选择性共价偶联。我们还致力于化学编程双特异性抗体（biAbs）平台的开发。这些赋予目标细胞结合小分子招募和重定向细胞毒性T细胞以消除癌细胞的能力。我们与Adrian Wiestner博士（NHLBI）合作，开发了一种靶向细胞毒治疗慢性淋巴细胞白血病（CLL）的新策略。该方法采用基于igm的Fc(mu) r靶向抗体-药物递送，对CLL产生有效和特异性的治疗活性。同时，我们正在使用嵌合抗原受体（CAR）工程T细胞，其中包括h38C2的半抗原结合位点。通过将活性h38C2赖氨酸残基与1,3-二酮或β -内酰胺部分共价结合，通过可变PEG间隔剂连接到环RGDfK基团上，对其进行化学编程。这使得car具有高亲和力和特异性结合人整合素α 4， β 3的能力。这些药物有望杀死表达人整合素α 4、β 3 -的肿瘤细胞和肿瘤内皮细胞。我们还研究了三功能叶酸受体1 （FOR1）靶向部分。