Developing novel LOX inhibitors to target chemotherapy resistant TNBC

项目摘要 三阴性乳腺癌（TNBC）是最具侵袭性的乳腺癌亚型。约占15%， 所有的乳腺癌导致了30%的乳腺癌死亡。TNBC主要通过常规治疗来治疗。 化疗;然而，对治疗的抵抗是常见的，导致高死亡率。重要的是 目前用于化学抗性TNBC的治疗策略的益处;即，免疫疗法和抗体- 药物缀合物仅限于一小部分患者，并且生存益处有限。因此有 迫切需要确定新的和有效的治疗策略，以克服耐药性化疗。 最近，我们确定了缺氧诱导的ECM重塑剂赖氨酰氧化酶（LOX）作为缺氧诱导ECM重塑的关键介质。 （Saatci等，Nature Communications，2020）。我们发现LOX 在化疗耐药肿瘤中过表达，其抑制使最具侵袭性的乳腺肿瘤重新敏感 使用几种临床相关的小鼠模型。然而，可用的LOX抑制剂是 非选择性或具有毒性。因此，我们在这个项目中的主要目标是开发有效的，具体的， 耐受性良好的LOX抑制剂，以克服具有高翻译潜力的TNBC中的化学抗性。 通过高通量化合物库筛选和命中到铅转换研究，我们确定了 具有LOX的有效靶向细胞接合的化合物，具有良好的口服药代动力学（PK）， 具有化学增敏作用而无主要毒性（US PTO 17/693，371和PCT/US 2022/20086，专利号1000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000000 待定）。从我们目前未优化的先导分子出发，我们的目标是开发具有以下特性的先导化合物： 增加效力、安全性和药物相似性。为了实现这一目标，在本快速通道STTR的第一阶段， 格兰特，我们将通过广泛的结构活性关系产生一个多样化的小分子库 (SAR)使用我们最初的药效团进行研究。我们将测试合成的抑制剂的程度 LOX酶活性抑制、LOX结合和对LOX的选择性。我们将执行脱靶 使用CEREP筛选以及激酶组分析评估抑制剂。入围的候选人将 进一步在ECM交联和3D化学敏化测定中使用细胞系和类器官进行测试。 有效性、选择性和稳定性更好的抑制剂将进入II期。在第二阶段，我们将执行几个 ADME测定，包括代谢稳定性/同一性、Caco-2渗透性和转运、心脏毒性和 遗传毒性、血浆蛋白结合、抑制/诱导/反应表型分析，以改善药物样 在TNBC细胞中保持靶向效力的同时，详细的PK/PD和毒性分析 最有前途的候选人将进行，然后通过测试他们的化疗增敏剂的效果， 最先进的免疫缺陷（细胞系和患者来源的异种移植物）和免疫活性 （同基因）小鼠模型。拟议项目的成功完成将导致有效和具体的， 铅优化LOX抑制剂，以克服TNBC的耐药性，最致命的乳腺癌形式。

PROJECT SUMMARY Triple negative breast cancer (TNBC) is the most aggressive breast cancer subtype. It accounts for ~15% of all breast cancer yet is responsible for 30% of breast cancer deaths. TNBC is treated primarily by conventional chemotherapy; however, resistance to therapy is common, leading to high mortality rates. Importantly, the benefit of current therapeutic strategies used in chemoresistant TNBC; i.e., immunotherapy and antibody- drug conjugates, is confined to only a fraction of patients, and survival benefit is limited. Therefore, there is an urgent need to identify novel and effective treatment strategies to overcome resistance to chemotherapy. Recently, we identified hypoxia-induced ECM re-modeler, lysyl oxidase (LOX) as a key mediator of chemoresistance in TNBC (Saatci et al, Nature Communications, 2020). We showed that LOX is overexpressed in chemoresistant tumors, and its inhibition re-sensitizes the most aggressive breast tumors to doxorubicin using several clinically-relevant mouse models. However, the available LOX inhibitors are either non-selective or has toxicity. Hence, our main objective in this project is to develop potent, specific and well-tolerated LOX inhibitors to overcome chemoresistance in TNBC that has a high translational potential. Through high-throughput compound library screening and hit-to-lead conversion studies, we identified compounds with potent on-target cellular engagement of LOX, with good oral pharmacokinetics (PK) and with chemosensitizer effect without major toxicity (US PTO 17/693,371 and PCT/US2022/20086, patent pending). Starting from our current non-optimized lead molecule, we aim to develop lead compounds with increased potency, safety and drug-likeness. To accomplish this goal, in Phase I of this Fast-Track STTR grant, we will generate a diverse library of small molecules via an extensive structure activity relationship (SAR) study using our initial pharmacophore. We will test the synthesized inhibitors with respect to the degree of LOX enzymatic activity inhibition, LOX binding and selectivity towards LOX. We will perform the off-target assessment of the inhibitors using CEREP screen as well as kinome profiling. The shortlisted candidates will further be tested in ECM crosslinking and 3D chemosensitization assays using both cell lines and organoids. Inhibitors with better efficacy, selectivity and stability will move to Phase II. In Phase II, we will perform several ADME assays, including metabolic stability/identity, Caco-2 permeability and transport, cardiotoxicity and genotoxicity, plasma protein binding, CYP inhibition/induction/reaction phenotyping to improve drug-like properties while maintaining on-target potency in TNBC cells. Detailed PK/PD and toxicity analyses of the most promising candidates will be carried out followed by testing their chemosensitizer effect using both state-of-the-art immunodeficient (cell line- and patient-derived xenografts) and immunocompetent (syngeneic) mice models. The successful completion of the proposed project will lead to potent and specific, lead-optimized LOX inhibitors to overcome chemoresistance in TNBC, the deadliest form of breast cancer.