The process of DNA replication is central to the propagation of all bacteria. To date, no commercially available antibacterial targets any of the enzymes that make up the central replication system in bacteria. The DNA replication machinery of infectious organisms therefore stands as an unexplored target for drug development efforts and presents a significant opportunity. Replication has not previously been targeted in large part because its complexity poses a formidable technological barrier to setting up HTS assays. In addition, since activity of many individual subunits depends on proper association with other components of the replication apparatus, target-based assays using single subunits are generally not feasible. A substantial portion of the system must therefore be reconstituted for drug screening. In this proposal, we present a plan to develop and adapt assays where protein components of the bacterial replication apparatus work together in complex DNA replication reactions. The DNA Polymerase III Holoenzyme (Pol III HE) of bacteria contains approximately 10 subunits that undergo marked changes in protein-protein association during each of nearly 20 identified kinetic steps of the complex processive reaction they catalyze. Since all of these proteins must function together, the ca. 100 distinct interactions and catalytic events all provide targets for antibacterial action, and thus provide a multiple of the proteins present as targets. We have developed a simple fluorescence-based assay, and have adapted this format to a high throughput screen. Such assays are expected to be profoundly more efficient compared to traditional screening assays using single target enzymes, since activities of each of the proteins that comprise the holoenzyme are targeted simultaneously. We anticipate this will increase the number and the quality of hits obtained from screening the MLSCN collection, thereby providing a richer variety of inhibitors for further development and, in turn, appreciably increasing the odds of successfully identifying effective and novel antibacterials.

Early work in McHenry’s laboratory established the feasibility of assaying for double stranded DNA production of a reconstituted E. coli DNApol III holoenzyme system, consisting of DNA Polymerase III – (αεθ)2τ3δδ’χψ, β subunit – processivity factor, SSB – single-stranded DNA binding protein, and Primase. Inhibition of polymerase activity was screened by measuring the production of double-stranded DNA via PicoGreen fluorescence increase. A fully-automated qHTS experiment was performed against a collection of 71,028 compounds tested as 7- to 15-point concentration series at 4 uL reaction volume in 1536-well plate format. Actives identified from the screen were subjected to confirmatory experiments using the screening assay and subsequently against the individual targets in deconvolution assays.

1. qHTS Assay for Inhibitors of DNA Polymerase III Holoenzyme System
2. Marians KJ, Hiasa H, Kim DR, McHenry CS. Role of the core DNA polymerase III subunits at the replication fork. Alpha is the only subunit required for processive replication. J Biol Chem. 1998 Jan 23;273(4):2452-7. doi: 10.1074/jbc.273.4.2452. PMID: 9442096.
3. Kim DR, Pritchard AE, McHenry CS. Localization of the active site of the alpha subunit of the Escherichia coli DNA polymerase III holoenzyme. J Bacteriol. 1997 Nov;179(21):6721-8. doi: 10.1128/jb.179.21.6721-6728.1997. PMID: 9352922; PMCID: PMC179601.
4. Seville M, West AB, Cull MG, McHenry CS. Fluorometric assay for DNA polymerases and reverse transcriptase. Biotechniques. 1996 Oct;21(4):664, 666, 668, 670, 672. doi: 10.2144/96214st04. PMID: 8891218.