Integrating Experimental and In Silico HTS in the Discovery of Inhibitors of Protein-Nucleic Acid Interactions

Abstract

Discovery of novel tool compounds and drug leads against a range of unorthodox protein targets has pushed both experimental screening methodologies as well as the field of structure-based design to the limit in recent years. Increasingly, it has been recognized that some of the most desirable targets for the development of small-molecule effectors are actually protein-protein and protein-nucleic acid interactions. There are numerous nontrivial challenges to pursuing small-molecule lead compounds directed toward PPIs and PNIs: relatively shallow cavities, large surface areas that are natively complexed to macromolecules, complex patterns of interstitial waters, a paucity of "hot spots," large conformational changes upon ligand binding, etc. Although there have been some notable successes targeting PPIs in the last decade, there has been distinctly less success in the realm of targeting PNIs. This chapter focuses on an approach, successfully applied by our group to address the challenge of gaining traction on the PPI target RAD52, which is a protein that binds both single-stranded and double-stranded DNA, and is an anticancer target for certain types of cancer. There are many approaches to tackling the difficult problems of finding effective small molecules that disrupt PPIs and PNIs, but the methods presented here offer a series of elegant solutions, which integrate experimental HTS, biophysical methods, docking, and molecular dynamics in a powerful way. Additionally, the structural knowledge gained from these studies provides a means for rationally understanding what features lead to ligand affinity in these fascinating and highly unorthodox pockets.

Keywords: In silico screening; Natural products; Protein–nucleic acid interactions; Structure-based drug discovery; Virtual screening.

Figure 1. FRET-based high throughput screening of the MicroSource Spectrum library

(A) Control lanes from a 384-well plate. The positive control (blue) containing stoichiometric RAD52 with Cy3-dT₃₀-Cy5 substrate challenged with an excess amount of unlabeled ssDNA (Poly dT100); while the negative control (red) consisted of unperturbed stoichiometric complex of RAD52 with Cy3-dT₃₀-Cy5. Red and blue solid lines represent the average and the standard deviation is shown with error bars respectively. (B) 12 identified hits (green) from cherry-picked rescreening of the initial screen of the MicroSource SPECTRUM collection, with several false positives shown in blue. Originally published as a part of Figure 1(DOI: 10.7554/eLife.14740) in Hengel et al, 2016 (Hengel et al., 2016); used under a CC-BY license.

Figure 2. WaterLOGSY experiments indicate direct interactions between HTS hit compounds and RAD52

A. Aromatic region of the 1D ¹H NMR spectrum of compound “1” (black) and the WaterLOGSY spectrum of 20 μM compound “1”in the presence of 3.3 μM RAD52 (red). The nonexchangeable proton peaks are labeled using atom names (blue) on the compound structure. B. Same as A, except representing the aromatic region of the 1D ¹H NMR spectrum of compound “6” (black) and the WaterLOGSY spectrum of 40 μM compound “6” in the presence of 3.3 μM RAD52 (red). The nonexchangeable proton peaks are labeled using atom names (blue) as indicated on the compound structure. Originally published as a part of Figure 2a and 3a (DOI: 10.7554/eLife.14740) in Hengel et al, 2016 (Hengel et al., 2016); used under a CC-BY license.

Figure 3. Docking workflow for screening small-molecule drugs against RAD52

Originally published as a part of Figure 8a (DOI: 10.7554/eLife.14740) in Hengel et al, 2016 (Hengel et al., 2016); used under a CC-BY license.

Figure 4. Implementation of the ROC analysis in our docking studies against the RAD52 target

Originally published as a part of Figure 8a (DOI: 10.7554/eLife.14740) in Hengel et al, 2016 (Hengel et al., 2016) ; used under a CC-BY license.

Figure 5. Spatial arrangement of inhibitors ‘1’ and ‘6’ with RAD52-ssDNA binding groove

A. Binding positions of inhibitors ‘1’ and ‘6’ along the sub-pockets of three individual monomers (yellow, green and blue) of the RAD52 ring (PDB 1KN0). Dotted lines indicate the approximate boundaries of ssDNA-binding groove. B. Ligand interactive maps of inhibitors ‘1’ and ‘6’ inside the sub-pockets of RAD52. Originally published as Figure 4 (DOI: 10.7554/eLife.14740) in Hengel et al, 2016 (Hengel et al., 2016) ; used under a CC-BY license.

Figure 6. Decision threshold selection is not absolute

Two simulated distribution (black and blue) of a quantity with one possible decision threshold (red) shown.

Figure 7

A typical conventional ROC curve with decreasing threshold strictness.

Figure 8. Application of ROC in combination with binding energy based docking scores for novel small molecule inhibitors for RAD52-ssDNA interaction

A. Calculated docking scores (kcal/mol) for the binding configurations of compound ‘1’ in comparison to decoys compounds. As the low docking scores indicate low binding energies, which correlate to favorable interactions, the histogram shows a clear distinction between true positives from compound ‘1’ and true negatives from ‘decoys’. B. Receiving-operating characteristics (Metz, 1978) curve used with the threshold shown yielding an AUC value of 0.9973. Originally published as a part of Figure 8b and 8c (DOI: 10.7554/eLife.14740) in Hengel et al, 2016 (Hengel et al., 2016); used under a CC-BY license.

Figure 9. Biochemical validation of NP-004255

A. Aromatic region of the 1D ¹H NMR spectrum of compound NP-004255 alone (black) and the WaterLOGSY spectrum of 40 μM compound NP-004255 in the presence of 3.3 μM RAD52 (red). The nonexchangeable proton peaks (blue) using atom names are shown on the structure of compound NP-004255. (B) IC50 values for inhibition of ssDNA binding and wrapping were determined using FRET-based assays that follow the change in geometry of a Cy3-dT30-Cy5 substrate (black circles). The computed IC50 value is shown above the curve. Titration of the RAD52–dsDNA with NP-004255 (gray boxes) shows that this inhibitor does not perturb the RAD52–dsDNA interaction. (C) Aromatic region of the 1D 1H NMR spectrum of compound NP-004255 alone (black) and the WaterLOGSY spectrum of 40 μM compound NP-004255 in the presence of 3.3 μM RPA (red). (D)Titration of the RAD52-RPA-Cy3-dT₃₀-Cy5 complex with NP-004255 (black circles). The computed IC₅₀ value is shown below the curve. Green squares show titration of the RPA-Cy3-dT₃₀-Cy5 complex with NP-004255 indicating NP-004255 does not perturb the RPA-ssDNA. Originally published as Figure 9 (DOI: 10.7554/eLife.14740) in Hengel et al, 2016 (Hengel et al., 2016); used under a CC-BY license.

Figure 10. Placement of RAD52 NP-004255 within the ssDNA binding groove

A. Electrostatic potential surface of three monomers of the RAD52-NTD (PDB 1KN0) positioning NP-004255 within the ssDNA binding groove. B. MOE ligand map of NP-004255 when binding RAD-52. Originally published as Figure 8d and 8e (DOI: 10.7554/eLife.14740) in Hengel et al, 2016 (Hengel et al., 2016); used under a CC-BY license.