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

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.

摘要 Wnt/β-catenin信号的失调在超过20%的所有癌症中被发现，包括50%的乳腺癌。 cers，并有助于癌症的发生、血管生成、肿瘤迁移和转移。然而， 开发抑制β-连环蛋白的药物一直受到阻碍，因为小分子抑制剂不能分离 β-连环蛋白的致癌性来自于体内平衡功能，产生毒性安全性。 BCL 9是一种转录共激活因子，调节致癌β-连环蛋白介导的基因反式激活。分散- BCL 9与β-catenin相互作用导致β-catenin介导的致癌基因trans-p53的有效抑制。 没有与通过APC或Axin的信号传导损失相关的GI毒性。由于BCL 9是高表达的， 在肿瘤中，但通常不是正常细胞中，β-连环蛋白与BCL 9的相互作用代表了一种有吸引力的，新的 治疗靶点基于上述，我们假设β-连环蛋白的肽拮抗剂与 BCL 9将被证明是Wnt驱动的癌症的安全有效的策略。 Sapience设计并合成了一组β-连环蛋白拮抗肽（BCAP），以破坏细胞间的相互作用。 β-连环蛋白与BCL 9的作用，其中我们将氨基酸取代引入天然BCL 9 HD2结构域 为了增强靶向亲和力，包括用于细胞内进入的无毒细胞穿透结构域（CPD）和D-氨基 酸，以防止蛋白水解并赋予化学稳定性。通过构效关系（SAR）- 基于设计，我们开发了一个领先的候选人，BCAP-58，在体外和体内具有改善的效力。 通过这个SBIR计划，我们建议开发一种创新的β-catenin/BCL 9相互作用抑制剂， 治疗Wnt/β-连环蛋白驱动的癌症，从乳腺癌开始。我们建议在体外表征 BCAP-58的体内（特异性目标#1）和体内（特异性目标#2）活性。最初的实验将评估结合 BCAP-58与β-连环蛋白的亲和力（Aim 1.1）。随后，BCAP-58对Wnt/β-连环蛋白活性的影响将 通过TOPFlash报告基因、β-catenin磷酸化和β-catenin亚细胞定位来确定（Aim 1.2）。 最后，我们将通过qPCR检测BCAP-58对β-连环蛋白靶基因表达的影响和靶特异性 (Aim 1.3），以及对细胞增殖和活力（目的1.4）的影响，针对一组Wnt依赖性或非依赖性细胞， 各种乳腺癌亚型和患者来源的类肿瘤的细胞系。为BCAP建立概念验证- 58作为Wnt依赖性乳腺癌的治疗剂，我们将评估其重复剂量毒性和体内疗效。 首先，我们将在小鼠中建立无不良作用水平（NOAEL）和PK特征，以定义剂量上限 (Aim 2.1）。接下来，我们将研究BCAP-58在亚型小鼠模型中的剂量依赖性活性 在目标1（目标2.2）中证明对BCAP-58敏感。最后，我们将描述BCAP的影响- 58对体内β-连环蛋白介导的基因反式激活的影响（目的2.3）。成功的结果将验证推进 BCAP-58到IND的临床前评价，确定最有效的方案，并支持后续的 II期提案，用于支持IND申请。