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在细胞分裂中起着核心作用，PLK1活性的上调似乎与几种癌症的侵略性和预后不良密切相关。除催化KD外，PLK1还包含一个非催化的polo盒结构域（PBD），该结构域（PBD）与酶生理底物结合，并将酶定位为动物学内部的离散位置。 PBD抑制剂靶向仅在四种蛋白质（PLK1-3和PLK5）中发现的结构独特结构域。仅抑制PLK1 PBD功能就足以有效地施加有丝分裂停滞和癌细胞中的凋亡细胞死亡，而在正常细胞和PBD结合相互作用的抑制剂中不足以作为开发抗PLK1疗法的目标限制策略。从5-MER磷酸肽PLHSPT并与Kyung Lee博士的NCI实验室和Michael Yaffe博士的MIT实验室合作开始，我们最初确定了肽抑制剂，这些肽抑制剂从1000-到10,000倍至10,000倍以上改善了PBD结合亲和力。与PLK1 PBD结合的这些肽的X射线共晶结构表明意外的结合模式，利用了非粘合PBD中不存在的“隐秘”结合通道或由亲本pentamer phophophoptide粘贴的。通过连接到咪唑环的N（PI）氮的苯基烷基部分来访问隐秘的口袋。在进一步的工作中，我们发现了化学性质在他的N（Tau） - 硝基激素上安装功能，并使用以磷酸化的方式定位了瑞糖类三菱烷基化条件，以产生含有N（pi），N（tau）的肽固定剂，将其残留物化为残留物。重要的是，阳离子双烷基咪唑种可能会通过阴离子磷酸盐部分的分子内“电荷掩模”增加膜渗透。与PBD结合的双烷基化肽的X射线共晶结构表明，他的N（Tau） - 氮基在C末端羧酰胺的6埃抗原范围内。我们设想可以准备循环配体，其中n（pi），n（tau）-bis烷基化他的烷基化可以用作分叉的环形连接。我们利用形成大环化反应的酰胺，利用N（Tau） - 咪唑和C末端的氮（Tau） - 硝基之间的各种大小的甲基接头。我们最终发现我们能够实现保留高PBD结合亲和力的三肽大环。来自双烷基His的循环配体的抑制数据表明，C末端与PTHR（-2）位置之间的环化对活动有益。考虑到这一点，我们研究了PTHR（-2）位置的新功能，该功能可能包含高亲和力配体所需的 - （CH2）8PH，以及用于牙龈上的宏观环化的正交函数组。我们开发了一种新的非HIS基氨基酸，该氨基酸可以作为大型循环连接处，同时访问关键的隐形袋。重要的是，这种新的氨基酸类似物可以掺入溜冰线树脂上的SPP中，以产生保留高PBD结合亲和力的大环体配体。在进一步的工作中，我们设计了一系列基于PLK1激酶抑制剂的活性药效团的探针，BI2536将其束缚在荧光部分中。这些探针提供了基于荧光的结合亲和力度量，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型激酶抑制剂的IC50值（与催化口袋的活性构象中的ATP结合竞争的抑制剂）与从酶试验中获得的值相符。但是，该探针对其他类别的激酶抑制剂不敏感。这使其与酶激酶测定法相结合，将其与其他类别的抑制剂区分开。该测定法可能具有轻松的手段，用于对1型ATP竞争PLK1抑制剂进行初步筛查，该抑制剂在成本，速度和易于处理方面具有与激酶分析相比具有不同优势的方法。目标两个：TDP1去除由链末端的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-PRIME末端阻断病变。与TDP1抑制剂结合疗法可能会与拓扑异构酶抑制剂（TOP1）协同，以提高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的催化域进行了晶体学片段筛选运动，以识别用于构建TDP1抑制剂的新铅化合物。使用对片段结合的结构见解，我们制备了几种片段衍生物，其中一些衍生物表现出比父分子明显更高的TDP1抑制效力。在另一项努力中，与Jay Schneekloth博士的NCI实验室合作，我们进行了TDP1小分子微阵列筛选，该筛选超过20,000个类似药物的分子，以识别新的TDP1结合图案。我们从21,000种化合物（0.5％的命中率）中确定了109次命中，并达到了首选的TDP1结合图案。进一步的结构活性关系（SAR）的工作获得了一类小分子，这些小分子显示出低微摩尔TDP1抑制作用。 Waugh实验室中的X射线共结晶结构表明，有希望的铅在TDP1的催化位点结合。这些结构与含TDP1结合的邻苯二甲酸片段的先前晶体结构一致，证实了双羧基部分的磷酸二羧基部分概括了磷酸盐与关键催化残基的结合。但是，与在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方向不同的结构成分均在DNA和肽底物结合区域中。这些是进入催化位点的小分子的第一个晶体结构，以及DNA底物和肽结合区域。目标三：抗体 - 药物缀合物（ADC）构成了一种重要且新兴的治疗疗法。在研究重点的第四个领域，我们与克里斯托夫·拉德（Christoph Rader）（Scripps Florida）的实验室进行了长期合作，以开发抗体 - 药物缀合物（ADC）。这利用了我们在小分子和肽模拟化学方面的专业知识。我们方法的各个方面采用带有单个C末端硒代半胱氨酸残基（FC-SEC）的单克隆抗体和抗体FC片段。在其他工作中，我们使用“催化抗体” H38C2使用偶氮二酮和含β-二酮的药物有效载荷来实现选择性共轭。我们还为化学编程双特异性抗体（BIABS）平台的发展做出了贡献。这些赋予靶细胞结合的小分子具有募集和重定向细胞毒性T细胞以消除癌细胞的能力。与Adrian Wiestner博士（NHLBI）合作，我们制定了一种新的策略，用于针对慢性淋巴细胞性白血病（CLL）的细胞毒性疗法。这采用了基于IgM的FC（MU）R靶向抗体 - 药物递送来对CLL有效和特定的治疗活性。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的触觉结合位点。这些是通过与1,3-二酮或β-内酰胺部分由可变的PEG垫片束缚到环状RGDFK基团的1,3-二酮或β-内酰胺部分的反应性H38C2 LYS残基对化学编程的。这赋予了汽车具有与人体整合素α4，具有高亲和力和特异性的beta3结合的能力。这些预计会杀死人内整合素α4，β3-表达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。