Enhancing the Pharmacology of Insulin through an expanded Genetic Code

描述（由申请人提供）：胰岛素是代谢稳态所需的关键激素。胰岛素的相对缺乏导致糖尿病（DM），这是一种以高血糖症和相关代谢异常为特征的病症。长期并发症包括视网膜病变和肾病。目前的胰岛素治疗被设计为通过模拟胰岛素的胰腺基础/餐时分泌来实现正常血糖。1型DM和新发2型DM的经典临床试验表明，血糖浓度的强化治疗导致微血管并发症显著减少，但低血糖事件增加。我们寻求设计新的胰岛素类似物，以提高胰岛素替代治疗的安全性和有效性。目前的胰岛素类似物制剂（尽管代表了20世纪90年代的显著进步）在药代动力学（PK）和物理稳定性方面是次优的。简而言之，短效类似物相对于餐后胰腺分泌太慢，而基础类似物不能提供真正的24小时PK。我的论文的目标是设计和表征一种新的超短效胰岛素类似物，用于（i）木里注射方案中的膳食控制和（ii）在闭环系统（“智能泵”）中的使用。我的研究计划的基本思想是利用扩展的遗传密码，使非标准胰岛素类似物的重组表达。对含有非天然氨基酸取代的胰岛素类似物（通过化学合成获得）的初步研究表明，在赖脯胰岛素（HumalogR的活性组分）的潜在二聚体界面处使用对-Cl-PheB 24（代替PheB 24）。我们假设，氯代芳香取代导致加速分解的锌胰岛素类似物六聚体由于电负性或大小的卤素取代（相对于苯丙氨酸）。在麻醉猪中进行的初步研究表明，相对于HumalogR，对氯苯丙氨酸24-赖脯胰岛素表现出加速的药效学。在我的论文中，我计划在酵母毕赤酵母中表达这种非标准的类似物，通过X射线晶体学确定其三维结构，并在潜在的制剂条件下表征其物理稳定性。据我所知，这将是第一个被确定的含有氯芳香取代的蛋白质结构，也是第一个这样的修饰如何增强蛋白质治疗特性的例子。

DESCRIPTION (provided by applicant): Insulin is a key hormone required for metabolic homeostasis. Relative lack of insulin results in diabetes mellitus (DM), a condition marked by hyperglycemia and associated metabolic abnormalities. Long-term complications include retinopathy and nephropathy. Current insulin therapies are designed to achieve normoglycemia by mimicking pancreatic basal/bolus secretion of insulin. Classical clinical trials in Type 1 DM and new-onset Type 2 DM have shown that intensive treatment of blood glucose concentration leads to a significant reduction in microvascular complications at the cost of increased hypoglycemic events. We seek to design novel insulin analogs to enhance the safety and efficacy of insulin replacement therapy. Current insulin analog formulations (although representing a significant advance in the 1990s) are sub- optimal with respect to pharmacokinetics (PK) and physical stability. In brief, short-acting analogs are too slow relative to post-prandial pancreatic secretion whereas basal analogs do not offer true 24-hour PK. The goal of my thesis is to design and characterize a novel ultra-short-acting insulin analog for (i) prandial control in muli- injection regiments and (ii) use in a closed-loop system ("smart pumps"). The essential idea underlying my research plan is exploitation of an expanded genetic code to enable recombinant expression of non-standard insulin analogs. Preliminary studies of insulin analogs containing unnatural amino-acid substitutions (obtained by chemical synthesis) suggest the utility of a para-Cl-PheB24 (in place of PheB24) at the potential dimer interface of insulin lispro (the active component of HumalogR). We hypothesize that the chloro-aromatic substitution leads to accelerated disassembly of the zinc insulin analog hexamer due to the electronegativity or size of the halogen substitution (relative to Phe). Pilot studies in anesthetized pigs indicate that para-Cl-PheB24-insulin lispro exhibits accelerated pharmacodynamics relative to HumalogR. In my thesis I plan to express this non-standard analog in the yeast Pichia pastoris, determine its three-dimensional structure by X-ray crystallography, and characterize its physical stability under conditions of potential formulation. To my knowledge, this will be the first structure to be determined of a protein containing a chloro-aromatic substitution and the first example of how such a modification can enhance the therapeutic properties of a protein.