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

众所周知，垂体和神经内分泌肿瘤(Net)分泌促肾上腺皮质激素(ACTH)，作用于肾上腺刺激糖皮质激素(GC)的产生。糖皮质激素过量会恶化预后(1)。最好是确定和消除这种过量血浆ACTH的来源，但并不总是可能的(1，2)。因此，能够有效阻断ACTH的作用具有重要的临床应用价值。ACTH作用于其特定的受体，黑素皮质素-2受体(MC2R)与MC2R辅助蛋白(MRAP)形成络合物。该项目旨在开发和表征能够特异性抑制MC2R/MRAP复合体的小分子化合物，用于治疗垂体肿瘤和Net。人们越来越意识到有必要开发ACTH拮抗剂来治疗ACTH过剩(3)。MC2R/MRAP复合体是唯一对ACTH(4，5)特异反应的复合体。我们以前在共表达MC2R/MRAP(3)的HEK293细胞中发现了特异性抑制ACTH刺激的cAMP生成的小分子。总体目标是在分子和生理水平上表征MC2R/MRAP拮抗剂。首先是我们自己的先导化合物(788/0371/9018/6741)，以及我们的工业合作伙伴确定的其他化合物。目的1.MC2R/MRAP拮抗剂的性质。学生将实时描述拮抗剂对MC2R/MRAP生物相互作用、结合、信号和受体内化的影响。技术包括：NanoBRET结合分析，使用荧光素酶生物传感器的cAMP积累分析，使用Gcamp6荧光生物传感器的p-Glo或钙动员，使用Nanobit互补的β-arrestin募集和G蛋白募集。与我们的工业合作伙伴一起，他们将在OMassas体验药物建模和发现。目的2.MC2R/MRAP拮抗剂对ACTH刺激的类固醇生成的影响。目前，尚无ACTH反应性、分泌糖皮质激素的肾上腺细胞系作为典型肾上腺皮质细胞的代表。因此，学生将使用鼠和人的原代肾上腺细胞培养来研究在有/没有MC2R/MRAP拮抗剂的情况下GC和类固醇的产生。、酶联免疫吸附试验和质谱分析。目的3：确定MC2R/MRAP拮抗剂在ACTH过量动物模型中的作用。在我们的ACTH过量的小鼠模型中，学生将在应用MC2R/MRAP拮抗剂IP/IV/PO后测量GC/类固醇的产生，其中ACTH通过渗透泵输送。学生将接受最先进的药理学/受体信号技术方面的培训，并获得使用原代细胞培养和最新小鼠模型的技能。督导团队是各自领域的专家，并有一支由技术人员、博士后和研究协调员组成的团队为学生提供支持。再加上在英国最令人兴奋的新生物技术公司之一的工业安置，他们将获得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.