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），每年有3-4百万人新感染，是美国肝病死亡的主要原因。对于那些意识到自己的HCV阳性状态的人，治疗需要一年的门诊，通常是耐受性差的治疗方案，即使如此，只有大约50%的治疗患者被治愈。靶向病毒蛋白NS 3/4A的新药正在临床试验中，NS 3/4A是一种双功能蛋白酶/解旋酶。然而，耐药性正在迅速出现，可能会使许多这些有前途的治疗方法在时间之前过时。我们正在提出一种新的药物设计策略，以避免耐药性，我们已经成功地应用于HIV-1蛋白酶，并开发了一位数的pM抑制剂，保留对一组耐药病毒的亲和力。我们假设，这种策略的工作，并适用于蛋白酶结构域的HCV NS 3/4A，因为在分子水平的耐药性是一个变化的分子识别事件，选择性地削弱抑制剂结合，但保持底物识别和切割的平衡。为了理解和减少耐药性的可能性，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组合来表征NS 3/4A的四种不同的天然底物复合物。所得到的结构将可能限定底物包膜，其限定底物识别所需的区域，并将与抑制剂的结合和已知的耐药性模式进行比较。然后，HCV NS 3/4A底物包膜可以用作设计、合成和评估可能对HCV耐药性不太敏感的新型NS 3/4A蛋白酶抑制剂的额外限制。

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.)