Molecular characterisation of MC2R/MRAP antagonists for the treatment of ACTH excess in pituitary and neuroendocrine tumours.

已知垂体和神经内分泌肿瘤（NETs）分泌促肾上腺皮质激素（ACTH）， ACTH作用于肾上腺刺激糖皮质激素（GC）的产生。GC过量恶化预后(1)。识别和去除这种过量血浆ACTH的来源是优选的，但并不总是可能的（1,2）。因此，有效阻断ACTH作用的能力具有重要的临床应用价值。ACTH作用于其特异性受体，即黑素皮质素-2受体（MC2R）与MC2R辅助蛋白（MRAP）的复合物。该项目旨在开发和表征能够特异性抑制MC2R/MRAP复合物的小分子化合物，用于治疗垂体肿瘤和NETs。越来越多的人意识到需要开发ACTH拮抗剂来治疗ACTH过量(3)。MC2R/MRAP复合体对ACTH有独特的反应（4,5）。我们之前在共表达MC2R/MRAP的hek293细胞中发现了特异性抑制ACTH刺激cAMP生成的小分子(3)。总体目标是在分子和生理水平上表征MC2R/MRAP拮抗剂。从我们自己的先导化合物（788/0371/9018/6741）开始，再加上我们的工业合作伙伴鉴定的其他化合物。目的1。MC2R/MRAP拮抗剂的表征。该学生将实时描述拮抗剂对MC2R/MRAP生物相互作用、结合、信号传导和受体内化的影响。技术包括：纳米obret结合测定，使用荧光素酶生物传感器的cAMP积累测定，使用Gcamp6荧光生物传感器的p-Glo或Ca2+动员，使用纳米obit互补的β -阻滞蛋白募集和g蛋白募集。与我们的工业合作伙伴，在麻省理工学院，他们将体验药物建模和发现。目标2。MC2R/MRAP拮抗剂对促肾上腺皮质激素刺激的类固醇生成的影响。目前，还没有ACTH应答的、产生糖皮质激素的肾上腺细胞系可作为典型肾上腺皮质细胞的代表。因此，该学生将使用小鼠和人类原代肾上腺细胞培养来研究使用/不使用MC2R/MRAP拮抗剂的GC和类固醇生成。ELISA和质谱分析。目的3：确定MC2R/MRAP拮抗剂在ACTH过量动物模型中的有效性。在我们的ACTH过量小鼠模型中，学生将通过渗透泵输送ACTH，在给予MC2R/MRAP拮抗剂IP/IV/PO后测量GC/类固醇的产生。学生将接受最先进的药理学/受体信号技术的培训，并获得与原代细胞培养和最新小鼠模型相关的技能。指导团队由各自领域的专家组成，并有一个由技术人员、博士后和研究协调员组成的团队来支持学生。再加上在英国最令人兴奋的新生物技术公司之一的工业实习，他们将获得在学术界和工业界都追捧的技术和可转移技能。

Pituitary and neuroendocrine tumours (NETs) are known to secrete adrenocorticotropic hormone (ACTH), which acts on the adrenal gland to stimulate glucocorticoid (GC) production. GC excess worsens prognosis (1). Identifying and removal of the source of this excessive plasma ACTH is preferred but not always possible (1,2). Hence, the ability to effectively block the effects ACTH has important clinical utility. ACTH acts on its specific receptor, the melanocortin-2-receptor (MC2R) complexed to the MC2R accessory protein (MRAP). The project is to develop and characterise small molecule compounds that can specifically inhibit the MC2R/MRAP complex for the treatment of pituitary tumours and NETs. There is growing awareness of a need to develop ACTH antagonists to treat ACTH excess (3). The MC2R/MRAP complex is unique responding specifically to ACTH (4,5). We previously identified small molecules that specifically inhibited ACTH stimulated cAMP generation in HEK293-cells co-expressing MC2R/MRAP (3). The overarching aim is to characterize MC2R/MRAP antagonists at a molecular and physiological level. , starting with our own lead compounds (788/0371/9018/6741) with additional compounds as they are identified by our industrial partner. Aim 1. Characterization of MC2R/MRAP antagonists. The student will characterise the antagonist effect on MC2R/MRAP biologyinteraction, binding, signalling and receptor internalisation in real-time.. Techniques include: NanoBRET binding assays, cAMP accumulation assays using luciferase biosensors, p-Glo, or Ca2+ mobilization using the Gcamp6 fluorescent biosensor, beta-arrestin recruitment and G-protein recruitment using nanoBit complementation. With our industrial partners,At OMass they will experience drug modelling and discovery. Aim 2. Effects of MC2R/MRAP antagonists on ACTH stimulated steroid production. Currently, there are no ACTH responsive, glucocorticoid producing adrenal cell-lines available representative of typical adrenocortical cells. Therefore, the student will use murine and human primary adrenal cell cultures to study GC and steroid production with/without MC2R/MRAP antagonists. , by ELISA and mass-spectrometry. Aim 3: Determine effectiveness of MC2R/MRAP antagonists in animal models of ACTH excess. The student will measure GC/steroid production after administering MC2R/MRAP antagonists IP/IV/PO in our murine model of ACTH excess where ACTH is delivered via an osmotic pump. The student will be trained in state-of-the-art pharmacology/receptor signalling techniques and acquire skills working with primary cell cultures and the latest murine models. The supervising team are experts in their field and have a team of technicians, post-docs and research co-ordinators to support the student. Together with an industrial placement at one of the UKs most exciting new biotech companies, they will gain technical and transferable skills sought after in both academia and industry.