Preclinical validation of a mitochondrial chaperonin as a target for the treatment of brain cancer

背景：胶质母细胞瘤(GBM)是最常见、最具侵袭性的原发性脑癌，患者预后极差。目前的治疗方法，包括手术去瘤和辅助放化疗，只能消除中心肿块。在脑浸润性、耐药和干细胞样脑基底膜肿瘤的新生过程中，目前还没有有效的治疗方法来阻止基底膜在治疗过程中/治疗后的进展和复发。虽然不同患者的脑肿瘤复发情况不同，但残留癌细胞的肿瘤再生有很高的代谢能量需求。因此，药物开发有希望的代谢靶点是非常可取的。通过表型药物发现/靶向去卷积的方法，我们证实了在体外和动物模型中，如果患者获得GBM细胞，化学抑制参与肿瘤增殖和凋亡的线粒体伴侣蛋白HSPD1会导致选择性的生物能量耗竭和死亡。随后的研究表明，我们的先导化合物KHS101通过HSPD1抑制剂特有的机制发挥作用：变构稳定构象状态，防止HSPD1的蛋白质-蛋白质相互作用。作为转译药物开发的下一步，我们的目标是开发患者来源的GBM细胞模型，用于基因救援实验，作为临床前验证的黄金标准，以及新生物临床靶点验证的成熟平台和具有临床影响的新生物发现的成熟平台。作为转译药物开发的下一步，我们的目标是开发患者来源的GBM细胞模型，用于基因救援实验，是临床前靶标验证的黄金标准，以及具有临床影响的新生物发现的成熟平台。我们的目标将是：1.鉴定功能性的、抗抑制剂的HSPD1变异体(GB细胞中的基因组修饰辅之以结构引导的蛋白质设计)2.体外结构表征抗抑制剂的HSPD1变异体3.该项目是跨学科的，涉及肿瘤生物学、化学生物学和结构生物学实验室的基础设施和专业知识。该项目建立在最近发表的一项工作的基础上，您将使用我们的方案来进行分子生物学(突变、蛋白质表达/纯化、慢病毒表达)、生化/细胞分析(如hSPD1酶/复性分析结合GBM细胞表型分析)和CryoEM，并辅之以CRISPR切片。通过这些方法，您将验证HSPD1作为未满足临床需求的合适靶点的药理抑制作用。除了针对GBM，新兴的工具和您的专业知识将使HSPD1抑制作为其他HSPD1依赖的恶性肿瘤的一流治疗策略成为可能，包括乳腺癌、肺癌、前列腺癌、胰腺癌、卵巢癌、肝癌、结肠癌和多发性骨髓瘤。

Background: Glioblastoma (GBM) is the most common and aggressive type of primary brain cancer, associated with extremely poor outcomes for patients. Current treatments including tumour removal by surgical 'debulking' and adjuvant chemoradiotherapy can only eliminate the central tumour mass. Dure to the brain-infiltrative, therapy-resistant and stem cell-like neology of GBM tumours, there are currently no effective treatments that stop GBM progression and recurrence during/ after therapy. While brain tumour recurrence varies from patient to patient, tumour repopulation from residual cancer cells has a high metabolic energy demand. Therefore, pharmaceutical exploitation of promising metabolic targets is highly desirable. Through a phenotypic drug discovery/ target deconvolution approach, we established that chemical inhibition of the mitochondrial chaperonin HSPD1 which has been implicated in cancer proliferation and apoptosis, causes selective bioenergetic exhaustion and death if patient derived GBM cells in vitro and in animal models. Subsequent studies suggest that our lead compound KHS101 works through a mechanism unique to HSPD1 inhibitors: allosteric stabilisation of a conformational state that prevents protein-protein interactions of HSPD1. As a next step towards translational drug development, we aim to develop patient-derived GBM cell models expressing inhibitor-resistant HSPD1 variants for genetic rescue experiments, the gold standard in pre-clinical validation and a proven platform for new biological clinical target validation and a proven platform for new biological discovery with clinical impact.As a next step towards translational drug development, we aim to develop patient derived GBM cell models expressing inhibitor resistant HSPD1 variants for genetic rescue experiments, the gold standard in pre-clinical target validation and a proven platform for new biological discovery with clinical impact. Our objectives will be:1. Identification of functional, inhibitor-resistant HSPD1 variants (genomic modification in GB cells complemented by structure-guided protein design)2. In vitro structural characterisation of inhibitor resistant HSPD1 variants3. Functional characterisation of inhibitor-resistant HSPD1 variants in GBM modelsThe project is interdisciplinary and involves infrastructure and expertise of tumour biology, chemical biology, and structural biology labs.The project builds on a recently published work, and you will use our protocols for molecular biology (mutagenesis, protein expression/ purification, lentiviral expression), biochemical/ cellular assays (such as hSPD1 enzymatic/ refolding assays combined with GBM cell phenotyping) and cryoEM, complemented by CRISPR tiling.Through these approaches you will validate pharmacological inhibition of HSPD1 as a suitable target for unmet clinical need. In addition to targeting GBM, the emerging tools and your expertise will enable the potential use of HSPD1 inhibition as a first in class treatment strategy for other HSPD1-dependant malignancies, including cancers of the breast, lung, prostate, pancreas, ovaries, liver, colon, and multiple myeloma.