Structure- and ligand-based virtual screening identifies new scaffolds for inhibitors of the oncoprotein MDM2

Abstract

A major challenge in the field of ligand discovery is to identify chemically useful fragments that can be developed into inhibitors of specific protein-protein interactions. Low molecular weight fragments (with molecular weight less than 250 Da) are likely to bind weakly to a protein's surface. Here we use a new virtual screening procedure which uses a combination of similarity searching and docking to identify chemically tractable scaffolds that bind to the p53-interaction site of MDM2. The binding has been verified using capillary electrophoresis which has proven to be an excellent screening method for such small, weakly binding ligands.

Fig 1. Crystal structures of MDM2 with bound small molecules.

Fig 2. Control docking results.

Crystallographic pose is coloured black, intermolecular hydrogen bonds are shown as orange dashes, Autodock result is red, Vina result is magenta. Pharmacaphore filter points are shown as black spheres. The protein is represented as a white transparent surface and cartoon secondary structure; residue side chains within 5 Å of the ligand are shown as black lines.

Fig 3. Structural formulae of the 5 hits identified through the initial virtual screen.

Fig 4. Nutlin-3 titration.

As the Nutlin-3 concentration increases the peak shifted back to p53-F control peak. Top trace is the p53-F alone followed by p53-F with MDM2-N. Nutlin-3 was then added into the sample with increasing concentration.

Fig 5. Graph showing inhibition (%) plotted against Nutlin-3 concentration.

From the data an IC50 of 37.3nM was determined.

Fig 6. Competition of Nutlin-3 with p53-F for MDM2-N measured by FP assay.

The graph shows anisotropy plotted against Nutlin-3 concentration. An IC₅₀ of 266 nM was determined.

Fig 7. Diphenylamine fragment based on the virtual screening hit 19.

Fig 8. Further exploration of analogues of virtual screening hit 19.

6 analogues selected on the basis of the hits showed inhibition in both the CE and FP assays.

Fig 9. Lowest energy Vina (magenta) and most populous cluster Autodock (green) docking poses of the active compounds.

The pharmacophore filter points are shown as black spheres. MDM2 is shown as a white transparent surface representation with the backbone visible as black secondary structure. Side chains of residues that line the active site are shown as black sticks. A) Compound 1; B) Compound 2; C) Compound 19 D) Compound 24

Fig 10. A) Vina docking poses of Compound 16 (magenta) and Compound 39 (orange); B) Vina docking poses of Compound 19 (magenta) and diphenylamine (orange).