Bannister TD, Nair R, Spicer T, Fernandez Vega V, Eberhart C, Mercer BA, Cameron M, Schurer S, Amundsen SK, Karabulut A, Londoño LM, Smith GR, Hodder P
Scripps Research Institute Department of Chemistry, Translational Research Institute, Jupiter, Florida 33458
Scripps Research Institute Molecular Screening Center, Lead Identification Division, Translational Research Institute, Jupiter, Florida 33458
The AddAB and RecBCD helicase-nucleases are related bacterial enzyme complexes that are instrumental in the repair of DNA double-strand breaks and in genetic recombination. Although they have been extensively studied both genetically and biochemically, no inhibitors specific for this class of enzymes are known. We developed a high-throughput screen based on the ability of phage T4 mutants to grow in only if the host RecBCD enzyme, or a related helicase-nuclease, is inhibited or genetically inactivated. We optimized the assay for a 1,536-well plate format and screened the NIH molecular libraries sample collection (326,100 small molecules) for inhibitors of the AddAB enzyme expressed in an deletion strain. Two main inhibitor chemotypes, nitrofuran amides and pipemidic acid thioureas, emerged from this cell-based screening campaign, with half maximal effective concentration (EC) values ranging from 2.5–50 μM. The ultra-high throughput screening (uHTS) campaign was followed by medicinal chemistry structure activity relationship (SAR) optimization, biochemical secondary cell-based and cell-free screening efforts, and mechanism of action studies. These coordinated efforts resulted in the identification of ML328 as a molecular probe. ML328, a drug-like pipemidic acid thiourea, has dual AddAB/RecBCD activity in multiple cell-based and biochemical assays and should prove useful in further enzymatic, genetic, and physiological studies of these enzymes. Because ML328 is a first-in-class inhibitor—no significant prior art exists for potent and selective small molecule inhibition of bacterial AddAB and RecBCD helicase-nucleases—study of this molecular probe could lead to a novel class of drugs to combat infections, since this class of enzymes is widely distributed in bacteria but absent in eukaryotes and is necessary for successful bacterial infection of mammals.
AddAB和RecBCD解旋酶核酸酶是相关的细菌酶复合物，在DNA双链断裂修复和基因重组中发挥着重要作用。尽管它们已经在遗传学和生物化学方面得到了广泛研究，但目前还没有已知的针对这类酶的特异性抑制剂。我们基于噬菌体T4突变体只有在宿主RecBCD酶或相关解旋酶核酸酶被抑制或基因失活时才能生长的能力，开发了一种高通量筛选方法。我们针对1536孔板格式优化了该检测方法，并筛选了美国国立卫生研究院分子文库样本集（326,100种小分子），以寻找在缺失菌株中表达的AddAB酶的抑制剂。从这次基于细胞的筛选活动中出现了两种主要的抑制剂化学类型，即硝基呋喃酰胺和吡哌酸硫脲，其半数最大有效浓度（EC）值范围为2.5 - 50μM。在超高通量筛选（uHTS）活动之后，进行了药物化学结构活性关系（SAR）优化、基于细胞的生化二级筛选和无细胞筛选工作以及作用机制研究。这些协同努力导致鉴定出ML328作为一种分子探针。ML328是一种类药物吡哌酸硫脲，在多种基于细胞的和生化检测中具有双重AddAB/RecBCD活性，应该会在对这些酶的进一步酶学、遗传学和生理学研究中发挥作用。由于ML328是一类首创的抑制剂——此前不存在针对细菌AddAB和RecBCD解旋酶核酸酶的有效且选择性的小分子抑制的显著现有技术——对这种分子探针的研究可能会导致一类新型的抗感染药物，因为这类酶在细菌中广泛分布，但在真核生物中不存在，并且是细菌成功感染哺乳动物所必需的。
