Drug Resistance in HCV NS3/4A - Inhibitor binding versus substrate recognition

描述（由申请人提供）：全球估计有1.8亿人感染丙型肝炎病毒（HCV），每年新感染3-4百万，是美国肝脏疾病死亡的主要原因。对于那些意识到自己丙型肝炎病毒呈阳性的患者，治疗需要一年的门诊就诊，治疗方案往往难以耐受，即便如此，只有约50%的接受治疗的患者治愈。针对病毒蛋白NS3/4A（一种双功能蛋白酶/解旋酶）的新药正在临床试验中。然而，耐药性正在迅速出现，很可能使许多这些有前途的治疗方法提前过时。我们正在提出一种新的药物设计策略来避免耐药性，我们已经成功地将其应用于HIV-1蛋白酶，并开发出一种个位数的pM抑制剂，该抑制剂对一组耐药病毒保持亲和力。我们假设这种策略是有效的，并且适用于HCV NS3/4A的蛋白酶结构域，因为在分子水平上，耐药性是分子识别事件平衡的变化，选择性地削弱抑制剂结合，但保持底物识别和切割。为了了解和减少耐药的可能性，需要阐明底物识别的原子细节。因此，在本提案中，我们通过晶体结构、分子动力学模拟和酶动力学的结合来表征NS3/4A的四种不同的天然底物配合物。所得到的结构可能会定义一个底物包膜，它定义了底物识别所必需的区域，并将与抑制剂的结合和已知的耐药性模式进行比较。HCV NS3/4A底物包膜可以作为设计、合成和评估新型NS3/4A蛋白酶抑制剂的附加约束，这些抑制剂可能对HCV耐药不那么敏感。)

DESCRIPTION (provided by applicant): An estimated 180 million people are infected with hepatitis C virus (HCV) globally and 3-4 million are newly infected each year and is the leading cause of death from liver disease in the United States. For those aware of their positive HCV status, treatment involves a year of clinic visits with an often poorly tolerated treatment regime and even so only about 50% of treated patients are cured. New drugs targeting the viral protein, NS3/4A, which is a bifunctional protease/helicase are in clinical trials. However, drug resistance is arising quickly, likely rendering many of these promising therapeutics obsolete before their time. We are proposing a new drug design strategy to avoid drug resistance, which we have successfully applied to HIV-1 protease and developed single-digit pM inhibitors that retain affinity against a panel of drug resistant viruses. We hypothesize that this strategy works and is applicable to the protease domain of HCV NS3/4A, since at a molecular level drug resistance is a change in the balance of molecular recognition events that selectively weakens inhibitor binding but maintains substrate recognition and cleavage. To understand and reduce the likelihood of drug resistance, the atomic details of substrate recognition need to be elucidated. Thus in this proposal we are characterizing the four diverse natural substrate complexes of NS3/4A, through a combination of crystal structures, molecular dynamics simulations and enzyme kinetics. The resulting structures will likely define a substrate envelope, which defines the region necessary for substrate recognition and will be compared with the binding of inhibitors and the known patterns of drug resistance. The HCV NS3/4A substrate envelope can then be used as an added constraint in the design, synthesis and assessment of novel NS3/4A protease inhibitors that will likely be less susceptible to HCV drug resistance.)