Synthetic Anionophores with Therapeutic Potential - a Coordinated Two-Centre Approach

常见的缩短寿命的遗传性疾病囊性纤维化（CF）的特点是有缺陷的阴离子运输穿过细胞边界（或膜）内衬管道和管道在整个身体。这项拟议中的研究旨在开发能够跨细胞膜运输阴离子的化学物质。这些合成转运蛋白可用于开发恢复阴离子转运至CF组织的疗法。他们也应该有价值的生物医学研究的工具。CF影响超过8,500个人在英国其中60%是小于20岁。在CF中，一种特定蛋白质的功能障碍，囊性纤维化跨膜传导调节因子（称为CFTR）导致整个身体的导管和管道被粘稠的粘液堵塞。在肺部，这会引发感染和炎症的恶性循环，破坏肺组织，导致呼吸困难，生活质量差和过早死亡。CFTR通常存在于导管和管道内衬细胞的表面，在那里它作为阴离子运动的被动门控途径，如氯离子和碳酸氢根。通过控制氯化物以及因此盐和水的运动，CFTR润滑导管和管道，而碳酸氢盐对于正常粘液形成和运动是至关重要的。CF治疗的一种新方法是用阴离子载体（对阴离子有选择性的离子转运蛋白）的“CFTR替代疗法”。阴离子载体是通过将阴离子转移穿过由生物膜呈现的屏障来模拟阴离子通道的作用的小分子。在通过吸入递送到肺部后，阴离子载体可以插入细胞膜，取代缺失的CFTR活性，并在一段时间内恢复正常的粘液运输。为了测试这种方法的可行性，必须开发合适的阴离子载体。特别是，活性必须高（以便可以使用少量），并且分子必须在活细胞中证明有效性（与合成模型膜相反）。独立工作的团队在布里斯托和南安普顿作出了重大贡献，这个问题，生产和研究阴离子载体是其中最活跃的可用。在拟议的工作中，他们将联手弥合“原理证明”和有意义的，潜在的治疗，生物活性之间的差距。为了开发用于CFTR旁路治疗的新型阴离子载体，我们寻求能够强烈结合阴离子，容易插入细胞膜并满足药物样分子规则的化学物质。我们将通过融合布里斯托和南安普顿阴离子载体的元素来制造这种小分子，以形成新型的阴离子载体。我们将确定阴离子载体结合氯离子和碳酸氢根的紧密程度，以及它们通过合成模型膜的难易程度。对于选定的化学品，我们将使用复杂的电学方法在模型膜中进行测试，以确定其确切的作用机制。为了确定在活细胞中有效的阴离子载体，我们将使用经过工程改造的表达阴离子敏感荧光蛋白的细胞来筛选大量具有转运活性的分子。将使用排列在CF肺的空气通道中的细胞对表现出最佳递送能力和阴离子转运活性的化合物进行进一步的生物学测试。首先，我们将研究阴离子载体是否恢复盐和水运输到这些细胞。然后，我们将确定阴离子载体是否促进粘液运输。最后，我们将对毒性和其他性质进行初步测试，以评估阴离子载体是否有可能作为药物获得成功。如果结果是有利的，我们的研究将为基于阴离子载体的CFTR替代疗法提供一个有说服力的案例，为与医疗和工业合作伙伴合作的药物开发计划铺平道路。

The common life-shortening inherited disease cystic fibrosis (CF) is characterised by defective anion transport across cell borders (or membranes) lining ducts and tubes throughout the body. The proposed research aims to develop chemicals capable of transporting anions across cell membranes. These synthetic transporters might be used to develop a therapy that restores anion transport to CF tissues. They should also have value as tools for biomedical research.CF affects over 8,500 individuals in the UK of whom 60% are less than 20 years old. In CF, malfunction of a particular protein, the cystic fibrosis transmembrane conductance regulator (termed CFTR) causes ducts and tubes throughout the body to become blocked by thick, sticky mucus. In the lungs, this triggers a vicious cycle of infection and inflammation that destroys lung tissue, leading to breathing difficulties, poor quality of life and premature death.CFTR is normally found on the surface of cells lining ducts and tubes, where it acts as a passive gated pathway for the movement of anions, such as chloride and bicarbonate. By controlling chloride and hence salt and water movements, CFTR lubricates ducts and tubes, while bicarbonate is critical for normal mucus formation and movement.A novel approach to CF treatment is "CFTR replacement therapy" with anionophores (ion transporters selective for anions). Anionophores are small molecules that mimic the actions of anion channels by transferring anions across the barrier presented by biological membranes. Following their delivery to the lungs by inhalation, anionophores could insert into the cell membrane, replace missing CFTR activity and restore, for a period, normal mucus transport. To test the feasibility of this approach suitable anionophores must be developed. In particular, activities must be high (so that small amounts can be used), and the molecules must have proven effectiveness in live cells (as opposed to synthetic model membranes).Working independently the teams at Bristol and Southampton have made significant contributions to this problem, producing and studying anionophores which are among the most active available. In the proposed work they will join forces to bridge the gap between "proof-of-principle" and meaningful, potentially therapeutic, biological activity.To develop novel anionophores for CFTR bypass therapy, we seek chemicals which bind anions strongly, insert easily into cell membranes and which satisfy rules for drug-like molecules. We will create such small molecules by fusing elements of the Bristol and Southampton anionophores to form novel anion carriers. We will determine how tightly the anionophores bind chloride and bicarbonate and how easily they pass these anions across synthetic model membranes. For selected chemicals, we will perform tests in model membranes using sophisticated electrical methods to establish their exact mechanism of action.To identify anionophores that are effective in live cells, we will use cells engineered to express an anion-sensitive fluorescent protein to screen large numbers of molecules for transport activity. Compounds that demonstrate optimal deliverability and anion transport activity will be subjected to further biological testing using cells that line the air passages of CF lungs. First, we will investigate whether anionophores restore salt and water transport to these cells. Then, we will determine whether anionophores promote mucus transport. Finally, we will perform preliminary tests for toxicity and other properties to assess whether the anionophores are likely to succeed as drugs. If results are favourable our studies will yield a persuasive case for anionophore-based CFTR replacement therapy, paving the way for a programme of drug development in collaboration with medical and industrial partners.