Directed evolution to diversify HIV protease function

描述(由申请人提供)： 治疗性抗体是治疗某些癌症、自身免疫和传染病的标准抗体，但当靶分子数量太大时，它们的疗效就会下降。对特定催化分解致病蛋白的蛋白酶进行工程改造，将使酶疗法能够治疗更广泛的疾病。HIV蛋白酶是这一目的的一个近乎理想的起点，因为有三条证据表明，这种酶具有异常的进化能力。首先，HIV蛋白水解酶在自然界中进化迅速，对合成的蛋白水解酶抑制剂有反应。第二，这种观察到的进化性与进化论是一致的。第三，研究人员已经采用了随机突变和筛选来创造一种针对肿瘤坏死因子-α(与类风湿性关节炎有关的靶蛋白)的变体。他现在提出的研究有以下具体目标： 1.通过模拟抗体亲和力成熟的定向进化过程，提高HIV蛋白酶变异体的活性和肿瘤坏死因子-α的特异性。 2.指导致癌蛋白HER2所特有的艾滋病毒蛋白水解酶变体的进化。 3.对野生型和变异型HIV蛋白进行纯化，并对其生物物理和动力学性质进行体外鉴定，以揭示适应性进化的结构机制。 这些实验是朝着酶疗法的新方法迈出的第一步。我们将展示HIV蛋白酶的不同寻常的进化能力，并更好地理解为什么某些酶比其他酶更适合蛋白质工程。为这些研究开发的高通量筛选也可以用于鉴定小分子蛋白酶抑制剂。

DESCRIPTION (provided by applicant): Therapeutic antibodies are the standard of care for certain cancers, auto-immune and infectious diseases, but their efficacy decreases when the number of target molecules is too large. The engineering of proteases that specifically catalyze the cleavage of disease-promoting proteins would enable enzyme therapies for a wider range of diseases. HIV protease is a nearly ideal starting point for this purpose because three lines of evidence suggest that this enzyme is unusually evolvable. First, HIV protease evolves rapidly in nature in response to synthetic protease inhibitors. Second, this observed evolvability is consistent with evolutionary theory. Third, the investigator has already employed random mutagenesis and screening to create a variant that is specific for TNF-alpha (target protein implicated in rheumatoid arthritis). He now proposes studies with the following specific aims: 1. to improve the activity and TNF-alpha specificity of the HIV protease variant through a directed evolution process that mimics antibody affinity maturation. 2. to direct the evolution of HIV protease variants specific for the oncogenic protein HER2. 3. to discover the structural mechanisms of adaptive evolution by purifying the wild-type and variant HIV proteases and characterizing their biophysical and kinetic properties in vitro. These experiments are a first step toward a new approach to enzyme therapy. We will demonstrate the unusual evolvability of HIV protease, and better understand why some enzymes are more amenable to protein engineering than others. The high throughput screens developed for these studies could also be used to identify small molecule protease inhibitors.