Fragment-Based Discovery of Inhibitors of the Bacterial DnaG-SSB Interaction

Abstract

In bacteria, the DnaG primase is responsible for synthesis of short RNA primers used to initiate chain extension by replicative DNA polymerase(s) during chromosomal replication. Among the proteins with which Escherichia coli DnaG interacts is the single-stranded DNA-binding protein, SSB. The C-terminal hexapeptide motif of SSB (DDDIPF; SSB-Ct) is highly conserved and is known to engage in essential interactions with many proteins in nucleic acid metabolism, including primase. Here, fragment-based screening by saturation-transfer difference nuclear magnetic resonance (STD-NMR) and surface plasmon resonance assays identified inhibitors of the primase/SSB-Ct interaction. Hits were shown to bind to the SSB-Ct-binding site using ¹⁵N-¹H HSQC spectra. STD-NMR was used to demonstrate binding of one hit to other SSB-Ct binding partners, confirming the possibility of simultaneous inhibition of multiple protein/SSB interactions. The fragment molecules represent promising scaffolds on which to build to discover new antibacterial compounds.

Keywords: SSB; antibacterial agents; fragment-based screening; primase; protein–protein interactions.

Figure 1

Superimposition of ¹⁵N–¹H HSQC spectra of DnaGC. The protein spectrum in the absence of fragment in black is compared with its spectrum after addition of fragment 4 (structure shown) in red. Representative assignments of resonances that showed the highest weighted chemical shift perturbation (CSP) (Figure S3d) are shown.

Figure 2

Modeled orientation of fragment 4. (a) The docked orientation of fragment 4 (green carbon atoms) in the single-stranded DNA-binding (SSB)-Ct binding pocket of DnaGC (gray carbon atoms). (b) A schematic representation of interactions between fragment 4 and its binding pocket. In all structural figures, the protein was visualized using visual molecular dynamics (VMD) [31].

Figure 3

(a) Structure of hits with binding affinities for further optimization. (b) ¹⁵N–¹H HSQC titration of fragment 4. Binding affinities (K_D values) were derived from the change in chemical shift, Δδ, with incremental additions of ligand.

Figure 4

Visualization of binding of compound 5. (a) The lowest energy binding poses of 5 (green carbon atoms) bound to DnaGC (gray carbon atoms). (b) Schematic representation of residues involved in interaction with compound 5.

Figure 5

1D ¹⁹F-NMR spectra of compound 5 at 1 mM in the presence (red trace) and absence (blue trace) of 50 μM DnaGC.

Figure 6

(a) Saturation transfer difference (STD) spectrum of compound 6 using DnaG-RCD. In red is a 1D ¹H-NMR reference spectrum, overlaid with a STD spectrum (blue). (b) Overlay of ¹⁵N–¹H HSQC spectra of ¹⁵N-DnaGC (black) with 5 (blue) and 6 (red), each at 1 mM. The apo-protein spectrum is shown in black. Representative assignments of resonances that showed the highest weighted CSP (Figure S3e,f) are shown.

Figure 7

(a) Docked binding pose of 6 (green carbon atoms) bound to DnaGC (gray carbon atoms). (b) Schematic representation of interactions.

Figure 8

Schematic representation of optimization of fragment 4. The red labeled groups were added during fragment-to-hit optimization. LE: Ligand efficiency (∆G/[number of heavy atoms]), n.d.: not determined.

Figure 9

1D ¹⁹F-NMR spectra. The blue spectrum is of fragment 4 alone and its spectrum in the presence of E. coli χ is shown in red.