Harnessing Fluorine-Sulfur Contacts and Multipolar Interactions for the Design of p53 Mutant Y220C Rescue Drugs

Abstract

Many oncogenic mutants of the tumor suppressor p53 are conformationally unstable, including the frequently occurring Y220C mutant. We have previously developed several small-molecule stabilizers of this mutant. One of these molecules, PhiKan083, 1-(9-ethyl-9H-carbazole-3-yl)-N-methylmethanamine, binds to a mutation-induced surface crevice with a KD = 150 μM, thereby increasing the melting temperature of the protein and slowing its rate of aggregation. Incorporation of fluorine atoms into small molecule ligands can substantially improve binding affinity to their protein targets. We have, therefore, harnessed fluorine-protein interactions to improve the affinity of this ligand. Step-wise introduction of fluorines at the carbazole ethyl anchor, which is deeply buried within the binding site in the Y220C-PhiKan083 complex, led to a 5-fold increase in affinity for a 2,2,2-trifluoroethyl anchor (ligand efficiency of 0.3 kcal mol(-1) atom(-1)). High-resolution crystal structures of the Y220C-ligand complexes combined with quantum chemical calculations revealed favorable interactions of the fluorines with protein backbone carbonyl groups (Leu145 and Trp146) and the sulfur of Cys220 at the mutation site. Affinity gains were, however, only achieved upon trifluorination, despite favorable interactions of the mono- and difluorinated anchors with the binding pocket, indicating a trade-off between energetically favorable protein-fluorine interactions and increased desolvation penalties. Taken together, the optimized carbazole scaffold provides a promising starting point for the development of high-affinity ligands to reactivate the tumor suppressor function of the p53 mutant Y220C in cancer cells.

Figure 1

Chemical structures of the known small-molecule stabilizers of p53-Y220C PhiKan083, PhiKan5196, and PhiKan7088.

Figure 2

Binding mode of the p53-Y220C stabilizer PhiKan083 and fluorinated model systems. (A) Experimentally determined binding mode of PhiKan083 (orange sticks) to the mutation-induced surface crevice of the p53 mutant Y220C (PDB code: 2VUK). (B) Snapshots of DFT-D optimized models of the PhiKan083 N-ethyl group and its fluorinated derivatives (orange sticks) bound to the Y220C surface crevice. For the DFT-D optimizations, truncated models of PhiKan083 (N-ethylpyrrole) and the p53-Y220C pocket (as depicted) were used (only non-hydrogen atoms and polar protons are shown). Interaction energies of each ligand model were compared to the N-ethyl reference interaction energy ΔE to calculate relative interaction energies (ΔΔE = ΔE_Ligand – ΔE_N-ethyl). The three distinct orientations of local minima of the 2-fluoroethyl anchor showed different interactions energies, indicating that orientation of the C–F vector toward the backbone carbonyl groups of Leu145 and Trp146 yields the most favorable interaction energy. Pictures were rendered using pymol (www.pymol.org).

Figure 3

Structures of compounds 1–6.

Scheme 1. Synthesis and Overall Yield of Compounds 3, 4, 5 and 6 Using the Two-Step Synthesis Described

Not applicable, precursor was bought from TCI UK.

Figure 4

Binding of compounds 4 (A) and 6 (B) to p53-Y220C as characterized by isothermal titration calorimetry (ITC).

Figure 5

X-ray structures of p53-Y220C with bound fluoro-derivatives of PhiKan083. Multipolar fluorine interactions are highlighted with magenta broken lines and fluorine–sulfur contacts with yellow broken lines. (A) Y220C in complex with monofluorinated compound 2. (B,C) Alternative conformations of the difluorinated compound 3 in chain B of the Y220C mutant. In chain A with only partial occupancy of 3, only one of the two side-chain conformations of Cys220 was observed, with a preferential orientation of 3 as highlighted in panel B. (D) Y220C in complex with 6 (chain B). Interactions with the main conformation of Cys220 are highlighted. The minor conformation of Cys220 is observed in chain B only.