Development of a cell-penetrating beta-catenin antagonist peptide as a therapeutic candidate for Wnt-driven breast cancer

开发细胞穿透性 β-连环蛋白拮抗剂肽作为 Wnt 驱动乳腺癌的候选治疗药物

• 批准号: 10324076
• 负责人: Jim Rotolo
• 金额: $ 40.93万
• 依托单位: SAPIENCE THERAPEUTICS, INC.
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-02 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10324076
• 关键词: Adverse effects; Affinity; Amino Acid Sequence; Amino Acid Substitution; Amino Acids; BCL9 gene; Binding; Binding Sites; Biological Markers; Cancer Etiology; Cancer Patient; Cancerous; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Cells; Clinical; Development; Dose; Drug Targeting; Elements; Gene Expression; Genes; Human; In Vitro; Literature; Location; Malignant Neoplasms; Mediating; Metastatic Neoplasm to Lymph Nodes; Mus; Neoplasm Metastasis; Oncogenic; Pathologic; Pathway interactions; Peptides; Pharmaceutical Preparations; Phase; Phosphorylation; Predisposition; Prognosis; Proteolysis; Regimen; Reporter; Research; Research Design; Safety; Signal Transduction; Site; Small Business Innovation Research Grant; Specificity; Structure-Activity Relationship; Surrogate Markers; TCF7L2 gene; Testing; Therapeutic; Toxic effect; Transactivation; Transcription Coactivator; angiogenesis; antitumor effect; base; beta catenin; cancer cell; cancer initiation; cancer subtypes; cancer therapy; chemical stability; clinical efficacy; commercialization; cost; cytotoxicity; design; enantiomer; experimental study; improved; in vitro activity; in vivo; inhibitor/antagonist; innovation; lead candidate; malignant breast neoplasm; migration; mouse model; neoplastic cell; new therapeutic target; pharmacokinetics and pharmacodynamics; preclinical evaluation; programs; receptor; response; side effect; small molecule; small molecule inhibitor; targeted treatment; therapeutic candidate; tumor; Adverse effects; Affinity; Amino Acid Sequence; Amino Acid Substitution; Amino Acids; BCL9 gene; Binding; Binding Sites; Biological Markers; Cancer Etiology; Cancer Patient; Cancerous; Cell Death; Cell Line; Cell Proliferation; Cell Survival; Cells; Clinical; Development; Dose; Drug Targeting; Elements; Gene Expression; Genes; Human; In Vitro; Literature; Location; Malignant Neoplasms; Mediating; Metastatic Neoplasm to Lymph Nodes; Mus; Neoplasm Metastasis; Oncogenic; Pathologic; Pathway interactions; Peptides; Pharmaceutical Preparations; Phase; Phosphorylation; Predisposition; Prognosis; Proteolysis; Regimen; Reporter; Research; Research Design; Safety; Signal Transduction; Site; Small Business Innovation Research Grant; Specificity; Structure-Activity Relationship; Surrogate Markers; TCF7L2 gene; Testing; Therapeutic; Toxic effect; Transactivation; Transcription Coactivator; angiogenesis; antitumor effect; base; beta catenin; cancer cell; cancer initiation; cancer subtypes; cancer therapy; chemical stability; clinical efficacy; commercialization; cost; cytotoxicity; design; enantiomer; experimental study; improved; in vitro activity; in vivo; inhibitor/antagonist; innovation; lead candidate; malignant breast neoplasm; migration; mouse model; neoplastic cell; new therapeutic target; pharmacokinetics and pharmacodynamics; preclinical evaluation; programs; receptor; response; side effect; small molecule; small molecule inhibitor; targeted treatment; therapeutic candidate; tumor

ABSTRACT Dysregulation of Wnt/-catenin signaling is found in more than 20% of all cancers, including 50% of breast can- cers, and contributes to cancer initiation, angiogenesis, tumor migration and metastasis. However, attempts at developing a drug to inhibit -catenin have been thwarted by the inability of small molecule inhibitors to separate -catenin’s oncogenic from homeostatic functions, producing a toxic safety profile. BCL9 is a transcriptional co-activator that regulates oncogenic β-catenin-mediated gene transactivation. Disrup- tion of BCL9 interaction with β-catenin results in potent inhibition of -catenin-mediated oncogenic gene trans- activation without the GI toxicity associated with loss of signaling via APC or Axin. As BCL9 is highly expressed in tumors, but generally not normal cells, the β-catenin interaction with BCL9 represents an attractive, novel therapeutic target. Based on the above, we hypothesize that a peptide antagonist of β-catenin interaction with BCL9 will prove a safe and effective strategy for Wnt-driven cancers. Sapience has designed and synthesized a panel of β-catenin antagonist peptides (BCAPs) to disrupt the inter- action of β-catenin with BCL9, in which we introduced amino acid substitutions to the native BCL9 HD2 domain to enhance target affinity, included a non-toxic cell penetrating domain (CPD) for intracellular entry and D-amino acids to protect from proteolysis and confer chemical stability. Through structure-activity relationship (SAR)- based design, we developed a lead candidate, BCAP-58, with improved potency in vitro and in vivo. Through this SBIR program, we propose to develop an innovative inhibitor of -catenin/BCL9 interaction as a therapeutic for Wnt/-catenin-driven cancers, starting with breast cancer. We propose to characterize the in vitro (Specific Aim #1) and in vivo (Specific Aim #2) activity of BCAP-58. Initial experiments will evaluate the binding affinity of BCAP-58 with -catenin (Aim 1.1). Subsequently, the impact of BCAP-58 on Wnt/β-catenin activity will be determined by TOPFlash reporter, β-catenin phosphorylation and β-catenin subcellular localization (Aim 1.2). Finally, we will test the impact and target specificity of BCAP-58 on β-catenin-target gene expression by qPCR (Aim 1.3), and on cell proliferation and viability (Aim 1.4), against a panel of Wnt-dependent or -independent cell lines of various breast cancer subtypes and patient-derived tumoroids. To establish proof of concept for BCAP- 58 as a therapeutic for Wnt-dependent breast cancer, we will assess its repeat-dose toxicity and in vivo efficacy. First, we will establish a no adverse effect level (NOAEL) and PK profile in mice, to define the upper dosing limit (Aim 2.1). Next, we will investigate the dose-dependent activity of BCAP-58 in mouse models of the subtypes that demonstrate susceptibility to BCAP-58 in Aim 1 (Aim 2.2). Finally, we will characterize the impact of BCAP- 58 on β-catenin-mediated gene transactivation in vivo (Aim 2.3). Successful results will validate advancing BCAP-58 to IND-enabling preclinical evaluation, identify a maximally effective regimen and support a subsequent Phase II proposal, in support of an IND application.

抽象的 Wnt/-catenin信号的失调在所有癌症的20％以上都发现，包括50％的乳腺癌 CERS，并有助于癌症的启动，血管生成，肿瘤迁移和转移。但是，尝试 开发一种抑制-catenin的药物已被小分子抑制剂无法分离而阻碍了 -catenin从稳态功能发出的致癌性，产生有毒的安全性。 BCl9是一种转录共激活因子，可调节致癌β-catenin介导的基因反式激活。 dis Bcl9与β-catenin的相互作用导致潜在抑制-catenin介导的致癌基因反式 - 与通过APC或AXIN失去信号传导相关的GI毒性的激活。由于Bcl9高度表达 在肿瘤中，但通常不是正常细胞，β-catenin与Bcl9的相互作用代表了一个有吸引力的新型 治疗靶标。基于上述，我们假设β-catenin的肽拮抗剂与 BCL9将证明对WNT驱动的癌症是一种安全有效的策略。 Sapience设计并合成了一组β-catenin拮抗剂肽（BCAP），以破坏 β-catenin用BCl9的作用，在其中我们将氨基酸取代引入天然Bcl9 HD2结构域 为了增强目标亲和力，包括无毒细胞穿透域（CPD）用于细胞内进入和D-Amino 酸以防止蛋白水解和会议化学稳定性。通过结构活动关系（SAR） - 基于设计，我们开发了一个铅候选者BCAP-58，其体外和体内效力提高了。 通过这个SBIR计划，我们建议开发-catenin/bcl9相互作用的创新抑制剂 从乳腺癌开始的Wnt/-catenin驱动的癌症的治疗性。我们建议表征体外 （特定目标＃1）和体内（特定目标＃2）BCAP-58的活动。最初的实验将评估结合 BCAP-58与-catenin的亲和力（AIM 1.1）。随后，BCAP-58对Wnt/β-catenin活性的影响将 通过TopFlash报告基因，β-catenin磷酸化和β-catenin亚细胞定位确定（AIM 1.2）。 最后，我们将测试BCAP-58的影响和靶向特异性对qPCR的β-catenin-target基因表达 （AIM 1.3），在细胞增殖和生存力（AIM 1.4）上，依赖于Wnt依赖性或独立的细胞 各种乳腺癌亚型和患者衍生的肿瘤的线条。为BCAP建立概念证明 - 58作为Wnt依赖性乳腺癌的治疗性，我们将评估其重复剂量毒性和体内效率。 首先，我们将在小鼠中建立不良效应水平（noael）和PK剖面 （AIM 2.1）。接下来，我们将研究亚型小鼠模型中BCAP-58的剂量依赖性活性 这表明在AIM 1中对BCAP-58的敏感性（AIM 2.2）。最后，我们将表征BCAP的影响 58在体内β-catenin介导的基因反式激活（AIM 2.3）。成功的结果将验证前进 BCAP-58到达临床前评估，确定一种最大有效的方案，并支持随后的 第二阶段的建议，以支持IND应用。