ML328: A Novel Dual Inhibitor of Bacterial AddAB and RecBCD Helicase-nuclease DNA Repair Enzymes

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.