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A Leukemia Cell-Specific Coiled-Coil Protein for Treatment of Chronic Myeloid Leukemia

用于治疗慢性粒细胞白血病的白血病细胞特异性卷曲螺旋蛋白

基本信息

批准号：
10543539
负责人：
Carol S. Lim
金额：
$ 34.19万
依托单位：
UNIVERSITY OF UTAH
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-01-01 至 2025-12-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10543539
关键词：
Acute Lymphocytic Leukemia Affinity Amino Acids Apoptosis Apoptotic Area Bcr-Abl tyrosine kinase Binding Biology Blood Circulation Bypass Cell Line Cell physiology Cells Charge Chemicals Chromosomal translocation Chromosome abnormality Chronic Myeloid Leukemia Clinical Clinical Trials Collaborations Combination Drug Therapy Combined Modality Therapy Computer Models Dimerization Disease Drug resistance Event Experimental Leukemia Genes Genetic Diseases Goals Growth Heterodimerization Homo Homodimerization Hydrophobicity Individual Inflammatory Intravenous Lead Length Leukemic Cell Ligation Location Malignant Neoplasms Mediating Modeling Oncogenic Pain Patients Penetration Peptide Synthesis Peptides Permeability Phase Phosphotransferases Physiological Processes Production Protein Tyrosine Kinase Proteins Proteolysis Recurrence Research Personnel Resistance Sampling Serum Shapes Signal Transduction Solid Specificity Technology Tertiary Protein Structure Testing Therapeutic Therapeutic Intervention Translations Tyrosine Kinase Inhibitor Variant Viral cell killing chronic myeloid leukemia cell clinically relevant design dimer drug discovery improved in vitro testing in vivo inhibitor leukemia leukemia treatment mouse model mutant mutational status new technology novel novel therapeutic intervention pre-clinical protein oligomer resistance mutation solid state standard of care stapled peptide success targeted agent targeted treatment therapeutic protein tumorigenesis

项目摘要

Project Summary/Abstract The long-term goals of this proposal are to explore the protein dimerization interface as an area for therapeutic intervention. Protein dimerization/oligomerization is a recurring theme in biology representing the mechanism by which hundreds of proteins regulate key cellular processes such as enzymatic activity and signal transduction. This non-covalent protein homo- or heterodimerization is mediated by hydrophobicity and both shape and charge complementarity. Once thought to be undruggable, dimer interfaces are emerging as an area for powerful therapeutic intervention for inflammatory diseases, pain, genetic diseases, cancer, and other diseases. The goal of this proposal is to develop a clinically relevant small protein dimerization inhibitor. As a model, we will use our dimerization coiled-coil (cc) inhibitor of Bcr-Abl. Bcr-Abl is an example of a protein that must dimerize to enable its oncogenic activity. Bcr-Abl, results from an abnormal chromosomal translocation, manifests as a constitutively active tyrosine kinase and causes of 95% of chronic myeloid leukemias (CML). We build on our novel, computationally designed Bcr coiled-coil mutant (ccmut) that selectively dimerizes with Bcr-Abl and inhibits its activity. When virally delivered as a gene, ccmut is effective against wild-type and mutant forms of Bcr-Abl. Our ccmut specifically favors heterodimerization with Bcr-Abl to disrupt Bcr-Abl dimerization, a necessary step for oncogenesis, and thus represents a novel therapeutic strategy. We have also fused ccmut to a non-toxic cell- penetrating peptide with known leukemia cell specificity and showed that it disrupts Bcr-Abl dimerization and enters and kills leukemia cells. For this proposal, we will explore peptide stapling technologies (to increase proteolytic stability) and native chemical ligation to synthesize 2 shorter stapled peptides into a longer therapeutic protein domain. We will first computationally model possible staple locations that maintain target affinity. These stapled versions (CPP-St-ccmut) are predicted to enter cells, resist serum proteolysis, bind to Bcr-Abl and inhibit its activity. We will then test the activity of our constructs with and without TKI ponatinib to test “multidomain targeting” of Bcr-Abl in CML cell lines, CML patient samples, and a CML animal model. Aims are as follows: Aim 1: Computationally design and synthesize with solid state peptide synthesis and native chemical ligation, a leukemia-specific, stapled cc inhibitor (CPP-St-ccmut) against Bc-Abl. Aim 2: Determine cell internalization, binding, and apoptotic ability of CPP-St-ccmut candidates in leukemic cell lines including those with clinically relevant mutations that are resistant to TKIs, and cells derived from patient samples, alone and in combination with ponatinib. Aim 3: Demonstrate efficacy of CPP-St-ccmut with and without ponatinib in a simple, pre-clinical mouse model of CML (syngeneic mouse model using intravenously injected BaF/3 cells expressing drug- resistant Bcr-Abl variants, including compound mutants). Our goal is to develop a stapled protein domain targeting the protein-protein dimerization interface Bcr-Abl kinase.

项目概要/摘要该提案的长期目标是探索蛋白质二聚化界面作为治疗领域干涉。蛋白质二聚/寡聚化是生物学中反复出现的主题，代表了其机制数百种蛋白质通过其调节关键的细胞过程，例如酶活性和信号转导。这种非共价蛋白质同二聚或异二聚化是由疏水性以及形状和电荷介导的互补性。曾经被认为不可成药的二聚体界面正在成为一个强大的领域对炎症性疾病、疼痛、遗传性疾病、癌症和其他疾病的治疗干预。目标该提案的目的是开发一种临床相关的小蛋白二聚化抑制剂。作为模型，我们将使用我们的 Bcr-Abl 二聚化卷曲螺旋 (cc) 抑制剂。 Bcr-Abl 是必须二聚化的蛋白质的一个例子使其具有致癌活性。 Bcr-Abl 是异常染色体易位的结果，表现为组成型活性酪氨酸激酶，是 95% 慢性粒细胞白血病 (CML) 的病因。我们建立在我们的新颖的、计算设计的 Bcr 卷曲螺旋突变体 (ccmut)，选择性地与 Bcr-Abl 形成二聚体并抑制它的活动。当作为基因通过病毒传递时，ccmut 可有效对抗 Bcr-Abl 的野生型和突变型。我们的 ccmut 特别支持与 Bcr-Abl 的异二聚化，以破坏 Bcr-Abl 二聚化，这是肿瘤发生，因此代表了一种新的治疗策略。我们还将 ccmut 与无毒细胞融合- 具有已知白血病细胞特异性的穿透肽，并表明它会破坏 Bcr-Abl 二聚化，进入并杀死白血病细胞。对于本提案，我们将探索肽装订技术（以增加蛋白水解稳定性）和天然化学连接，将 2 个较短的钉合肽合成为较长的治疗肽蛋白质结构域。我们将首先对保持目标亲和力的可能的主食位置进行计算建模。这些预计钉合版本 (CPP-St-ccmut) 会进入细胞、抵抗血清蛋白水解、与 Bcr-Abl 结合并抑制它的活动。然后，我们将在使用和不使用 TKI ponatinib 的情况下测试我们的构建体的活性，以测试“多域” 在 CML 细胞系、CML 患者样本和 CML 动物模型中“靶向”Bcr-Abl。目标如下： 1：通过固态肽合成和天然化学连接进行计算设计和合成，针对 Bc-Abl 的白血病特异性钉合 cc 抑制剂 (CPP-St-ccmut)。目标 2：确定细胞内化， CPP-St-ccmut 候选物在白血病细胞系（包括临床上患有白血病的细胞系）中的结合和凋亡能力对 TKI 耐药的相关突变以及源自患者样本的细胞（单独或组合）与普纳替尼。目标 3：在简单的临床前试验中证明使用或不使用 ponatinib 的 CPP-St-ccmut 的功效 CML 小鼠模型（使用静脉注射表达药物的 BaF/3 细胞的同基因小鼠模型）抗性 Bcr-Abl 变体，包括复合突变体）。我们的目标是开发一个固定的蛋白质结构域靶向蛋白质-蛋白质二聚化界面 Bcr-Abl 激酶。