The Structural and Functional Basis of HLA-associated Drug Hypersensitivity

描述（由申请人提供）：特异性药物不良反应是不可预测的，剂量无关的，可能危及生命；这使它们成为造成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，遗传关联研究已经确定了几种药物超敏反应与特定HLA等位基因之间的强烈联系。抗病毒药物阿巴卡韦（abacavir）是这种遗传关联最强的药物之一，它只在表达HLA分子变异B*57:01的患者中引起严重的不良反应。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HLA相关的药物过敏中，系统药物不良反应可能是由改变的肽库的形成引起的，该肽库触发T细胞免疫。通过体外结合实验、MHC配体洗脱和X射线晶体学，我们发现阿巴卡韦结合在HLA B*57:01的f口袋内，从而改变了其特异性。这支持了一个新的假设，即全身性药物不良反应是由于在HLA相关的药物过敏中，肽库的改变引起T细胞免疫的形成。目的是利用这种方法来定义三种药物在改变肽与HLA结合中的作用，并指导药物变体的开发，这些药物变体降低了引起HLA相关药物超敏反应的可能性。我们的具体目标是：1)确定以药物依赖方式与HLA-B分子结合的肽。我们将在药物存在或不存在的情况下对组合肽库进行亲和力测量，并使用活细胞MHC配体洗脱实验确认MHC结合试验中检测到的药物效应。2)确定与过敏相关的HLA-B分子络合的药物结构。我们将通过x射线晶体学确定药物/肽/HLA复合物的结构。如有可能，我们将利用在Aim 1中鉴定的肽进行结晶，我们发现在阿巴卡韦的情况下，这有助于药物/肽/HLA复合物的形成。3) a.药物诱导的肽库改变的功能后果的表征。b.表征药物变异对HLA相关药物识别的功能影响。我们将利用来自Aim 2的结构信息来设计不结合HLA分子的药物变体，因此预计不会引起T细胞免疫反应。我们将使用来自过敏患者的样本来测试T识别的药物变体。在分子结合试验中检测特异性药物HLA组合对自身库的影响的能力可以大大提高检测HLA相关药物超敏反应的能力。此外，药物变化方法可能对全球医疗保健事业具有吸引力，因为在已证实的治疗方法中，潜在的小变化将简化治疗。

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.