Procedures for producing and exploring Trypanosoma cruzi farnesyl pyrophosphate synthase (tcFPPS) for surface plasmon resonance (SPR) biosensor-driven fragment-based discovery have been established. The method requires functional sensor surfaces with high sensitivity for extended times and appropriate controls. Initial problems with protein stability and lack of useful reference compounds motivated optimization of experimental procedures and conditions. The improved methods enabled the production of pure, folded and dimeric protein, and identified procedures for storage and handling. A new coupled enzymatic assay, using luciferase for detection of pyrophosphate, was developed and used to confirm that the purified enzyme was active after purification and storage. It also confirmed that sensor surfaces prepared with structurally intact protein was active. An SPR-biosensor assay for fragment library screening and hit confirmation was developed. A thermal shift assay was used in parallel. A library of 90 fragments was efficiently screened by both assays at a single concentration in the presence and absence of the catalytic cofactor Mg2+ . Hits were selected on the basis of response levels or ΔT m  > 1°C and selectivity for tcFPPS in the presence of Mg2+ . Characterization of hits by SPR showed that all had low affinities and the relationships between steady-state responses and concentrations were not sufficiently hyperbolic for determination of KD -values. Instead, ranking could be performed from the slope of the linear relationship at low concentrations. This pilot screen confirms that the procedures developed herein enables SPR-biosensor driven fragment-based discovery of leads targeting tcFPPS, despite the lack of a reference compound. SIGNIFICANCE STATEMENT: To enable the discovery of drugs, it is essential to have access to relevant forms of the target protein and valid biochemical methods for studying the protein and effects of compounds that may be evolved into drugs. We have established methods for the discovery of drugs for treatment of American Trypanosomiasis (Chagas disease), using farnesyl pyrophosphate synthase from Trypanosoma cruzi as a target.

中文翻译：

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建立锥虫锥虫焦磷酸法呢基焦磷酸合酶作为生物传感器驱动的基于片段的铅发现的可行靶标。

已经建立了生产和探索用于表面等离振子共振（SPR）生物传感器驱动的基于片段的锥虫锥虫法尼基焦磷酸合酶（tcFPPS）的程序。该方法需要具有高灵敏度的功能性传感器表面，以延长时间并进行适当的控制。蛋白质稳定性的最初问题和缺乏有用的参考化合物促使优化实验程序和条件。改进的方法使得能够生产纯净，折叠和二聚体的蛋白质，并确定了存储和处理的程序。开发了一种使用荧光素酶检测焦磷酸盐的新型偶联酶检测方法，并用于确认纯化的酶在纯化和储存后具有活性。还证实了用结构完整的蛋白质制备的传感器表面是活性的。开发了用于片段文库筛选和命中确认的SPR生物传感器测定法。并行使用热位移测定。在存在和不存在催化辅因子Mg2 +的情况下，通过两种测定均以单一浓度有效筛选了90个片段的文库。根据反应水平或ΔTm> 1°C和在Mg2 +存在下对tcFPPS的选择性来选择命中。SPR命中的特征表明，它们均具有低亲和力，并且稳态响应与浓度之间的关系不足以确定KD值的双曲线。相反，可以在低浓度下根据线性关系的斜率进行排序。该初步筛选证实，尽管缺乏参考化合物，本文开发的程序仍能使靶向tcFPPS的潜在客户基于SPR-生物传感器驱动的基于片段的发现。重要声明：为了能够发现药物，必须获得相关形式的靶蛋白和有效的生化方法，以研究可能演变为药物的蛋白质和化合物的作用。我们已经建立了利用克鲁斯锥虫的法呢基焦磷酸合酶为靶标来发现治疗美国锥虫病（南美锥虫病）的药物的方法。获得相关形式的靶蛋白和有效的生化方法以研究蛋白和可能演变为药物的化合物的作用至关重要。我们已经建立了利用克鲁斯锥虫的法呢基焦磷酸合酶为靶标来发现治疗美国锥虫病（南美锥虫病）的药物的方法。获得相关形式的靶蛋白和有效的生化方法以研究蛋白和可能演变为药物的化合物的作用至关重要。我们已经建立了利用克鲁斯锥虫的法呢基焦磷酸合酶为靶标来发现治疗美国锥虫病（南美锥虫病）的药物的方法。