The Structural and Functional Basis of HLA-associated Drug Hypersensitivity

描述(由申请人提供)：特殊的药物不良反应是不可预测的，与剂量无关，并可能危及生命；这使它们成为导致药物开发成本和不确定性的主要因素。临床数据表明，许多此类反应涉及免疫机制，遗传关联研究已经证实，几种药物的药物超敏反应与特定的HLA等位基因之间存在强烈的联系。抗病毒药物阿巴卡韦的遗传相关性最强，它只在表达人类白细胞抗原分子变异体B*57：01的患者中引起严重不良反应。阿巴卡韦诱导这种病理性T细胞反应的机制尚不清楚。我们有强有力的初步数据表明，在人类白细胞抗原相关药物超敏反应中，全身性药物不良反应可由一种改变的多肽库的形成而引起，该多肽库可触发T细胞免疫。通过体外结合试验、MHC配体洗脱和X射线结晶学分析，我们发现阿巴卡韦与人类白细胞抗原B*57：01的F-囊结合，从而改变了其特异性。这支持了一种新的假说，即全身性药物不良反应是由于在人类白细胞抗原相关药物超敏反应中触发T细胞免疫的改变的多肽库的形成所致。目的是利用这一方法来确定三种药物在改变与人类白细胞抗原结合的多肽中的作用，并指导降低引起人类白细胞抗原相关药物超敏反应的可能性的药物变体的开发。我们的具体目标是：1)以药物依赖的方式识别与人类白细胞抗原-B分子结合的多肽。我们将在药物存在或不存在的情况下对组合肽库进行与相关HLA-B分子的亲和力测量，并使用活细胞的MHC配体洗脱实验确认在MHC结合试验中检测到的药物效应。2)确定与超敏反应相关的与人类白细胞抗原B分子结合的药物的结构。我们将通过X射线结晶学来确定药物/多肽/人类白细胞抗原复合体的结构。如有可能，我们将利用目标1中确定的多肽进行结晶，在阿巴卡韦的情况下，我们发现这些多肽有助于药物/多肽/人类白细胞抗原复合体的形成。3)a.药物诱导的改变的多肽谱系的功能后果的表征。药物变异对人类白细胞抗原相关药物识别的功能影响的表征。我们将利用目标2的结构信息来设计不结合人类白细胞抗原分子的药物变体，因此预测不会引起T细胞免疫反应。我们将使用过敏症患者的样本测试药物变体的T识别能力。在分子结合试验中测试特定的药物组合对自身谱系的影响的能力可以极大地提高检测人类白细胞抗原连锁药物超敏反应的能力。此外，药物变化的方法可能对全球医疗保健努力具有吸引力，在这些努力中，经过验证的治疗方法中潜在的微小变化将简化治疗。

DESCRIPTION (provided by applicant): Idiosyncratic adverse drug reactions are unpredictable, dose independent and potentially life threatening; this makes them a major factor contributing to the cost and uncertainty of drug development. Clinical data suggests that many such reactions involve immune mechanisms, and genetic association studies have identified strong linkage between drug hypersensitivity reactions to several drugs and specific HLA alleles. One of the strongest such genetic associations has been found for the antiviral drug abacavir, which causes severe adverse reactions exclusively in patients expressing the HLA molecular variant B*57:01. The mechanism by which abacavir induces this pathologic T cell response remains unclear. We have strong preliminary data that systemic adverse drug reactions can be caused by the formation of an altered peptide repertoire that triggers T cell immunity in HLA associated drug hypersensitivity. Using in vitro binding assays, MHC ligand elution and X- ray crystallography, we have shown that abacavir binds inside the F-pocket of HLA B*57:01, thereby altering its specificity. This supports a novel hypothesis that systemic adverse drug reactions are due to the formation of an altered peptide repertoire that triggers T cell immunity in HLA associated drug hypersensitivity. The objective is to utilize this approach to define the roles of three drugs in altering peptide binding to HLA and to guide development of drug variants that have a reduced likelihood of causing HLA linked drug hypersensitivity. Our specific aims are: 1) Identify peptides that bind to HLA-B molecules in a drug dependent manner. We will perform affinity measurements of a combinatorial peptide library in the presence or absence of the drug to the relevant HLA-B molecule and confirm drug effects detected in the MHC binding assays using MHC ligand elution experiments with live cells. 2) Determine structures of drugs complexed to HLA-B molecules associated with hypersensitivity. We will determine the structure of drug/peptide/HLA complexes by X-ray crystallography. As available, we will utilize the peptides identified in Aim 1 for crystallization, which we have foun to facilitate drug/peptide/HLA complex formation in the case of abacavir. 3) a. Characterization of functional consequences of the drug-induced altered peptide repertoire. b. Characterization of the functional effects of drug variation on HLA associated drug recognition. We will utilize the structural information from Aim 2 to design drug variants that do not bind HLA molecules and are therefore predicted to not cause T cell immune responses. We will test the drug variants for T recognition using samples from hypersensitive patients. The ability to test specific drug HLA combinations in molecular binding assays for effects on the self repertoire could drastically improve the ability to detect HLA linked drug hypersensitivities. Moreover, the drug variation approach may be attractive to global healthcare endeavors where a potentially small change in a proven therapy would simplify treatment.