Identification of DNA primase inhibitors via a combined fragment-based and virtual screening.

Stefan Ilic,Gerhard Wagner,Charles C Richardson,Barak Akabayov,Sabine R Akabayov,Haribabu Arthanari

doi:10.1038/srep36322

Stefan Ilic, Gerhard Wagner + Show 4 more

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https://doi.org/10.1038/srep36322

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Abstract

The structural differences between bacterial and human primases render the former an excellent target for drug design. Here we describe a technique for selecting small molecule inhibitors of the activity of T7 DNA primase, an ideal model for bacterial primases due to their common structural and functional features. Using NMR screening, fragment molecules that bind T7 primase were identified and then exploited in virtual filtration to select larger molecules from the ZINC database. The molecules were docked to the primase active site using the available primase crystal structure and ranked based on their predicted binding energies to identify the best candidates for functional and structural investigations. Biochemical assays revealed that some of the molecules inhibit T7 primase-dependent DNA replication. The binding mechanism was delineated via NMR spectroscopy. Our approach, which combines fragment based and virtual screening, is rapid and cost effective and can be applied to other targets.

Highlights

Bacterial DnaG primase synthesizes RNA primers that are used by DNA polymerase in lagging strand synthesis during DNA replication
We describe the development of small molecule inhibitors of DnaG using the structurally similar primase domain of the bacteriophage T7 gene 4 protein (Fig. 1b) as a model for the bacterial primase
To find compounds that do not bind at high affinity but that have potential to become successful leads, we developed a platform for lead discovery comprising several complementary steps: [1] fragment based screening by saturation transfer difference (STD) spectroscopy of a small molecule library (Ro3), [2] hit optimization by virtual screening and the generation of a new set of drug-like compounds (Ro5), each of which contains a small molecule found in step 1, [3] docking of the drug-like compounds to the active site of T7 primase and selection of the compounds that will undergo functional and structural assays with the target T7 primase, and [4] further development of lead compounds

Summary

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We describe a technique for selecting small molecule inhibitors of the activity of T7 DNA primase, an ideal model for bacterial primases due to their common structural and functional features. Using NMR screening, fragment molecules that bind T7 primase were identified and exploited in virtual filtration to select larger molecules from the ZINC database. The profound differences between human and bacterial DNA primases (Fig. 1c) render the latter a selective target for drug design. Whether by fragment screening or HTS, can target key biochemical process or binding to an essential cellular component. We show that the use of fragment based virtual screening (FBVS, Fig. 2) can yield potent inhibitors, reduce costs, and provide more advanced information about lead binding properties prior to the medicinal chemistry phase of drug optimization

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