Targeted rescue of a destabilized mutant of p53 by an in silico screened drug

Abstract

The tumor suppressor p53 is mutationally inactivated in approximately 50% of human cancers. Approximately one-third of the mutations lower the melting temperature of the protein, leading to its rapid denaturation. Small molecules that bind to those mutants and stabilize them could be effective anticancer drugs. The mutation Y220C, which occurs in approximately 75,000 new cancer cases per annum, creates a surface cavity that destabilizes the protein by 4 kcal/mol, at a site that is not functional. We have designed a series of binding molecules from an in silico analysis of the crystal structure using virtual screening and rational drug design. One of them, a carbazole derivative (PhiKan083), binds to the cavity with a dissociation constant of approximately 150 muM. It raises the melting temperature of the mutant and slows down its rate of denaturation. We have solved the crystal structure of the protein-PhiKan083 complex at 1.5-A resolution. The structure implicates key interactions between the protein and ligand and conformational changes that occur on binding, which will provide a basis for lead optimization. The Y220C mutant is an excellent "druggable" target for developing and testing novel anticancer drugs based on protein stabilization. We point out some general principles in relationships between binding constants, raising of melting temperatures, and increase of protein half-lives by stabilizing ligands.

Fig. 1.

In silico screening overview.

Fig. 2.

Compounds and numbering.

Fig. 3.

Effects of PhiKan083 on T-p53C-Y220C. (A) Changes in chemical shifts (normalized) vs. concentration for 15 resonances of T-p53C-Y220C in the presence of PhiKan083 at 20°C. The data are fitted to a single-site binding model. (B) Thermal denaturation of T-p53C-Y220C (10 μM) in the presence of PhiKan083. Denaturation is irreversible. However, at the very high heating rate of 270 K/h, the measured T_m is close to the reversible value. The data are fitted to the equation: T = T_m/(1 − (R/ΔS _D-N(Tm))ln(1 + [L]/K_d)), where T is the observed melting temperature, T_m that in the absence of ligand L, K_d its dissociation constant, and ΔS_D-N(Tm) the entropy of denaturation at T_m (the derivation is in the legend to Fig. S5). (C) Effect of PhiKan083 on kinetics of thermal denaturation at 37°C.

Fig. 4.

Isothermal titration calorimetry of PhiKan083 binding at 20°C showing raw data (Upper) and fit after integration (Lower).

Fig. 5.

Crystal structure of T-p53C-Y220C in complex with PhiKan083. (A) Ribbon representation of the overall structure of T-p53C-Y220C in complex with PhiKan083 (PDB ID code 2VUK, chain B). PhiKan083 is shown in green as a stick model with its molecular surface. It binds to the mutation-induced cleft on the protein surface that is distant from the known functional interfaces of the protein. The side chain of Cys-220 at the mutation site, which adopts two alternative conformations, is highlighted in orange. (B) Stereoview of the PhiKan083-binding site. p53 residues within a 5-Å distance of the ligand are shown as gray stick models. The protein surface is highlighted in semitransparent gray. (C) |F_o−F_c| simulated-annealing omit map of PhiKan083 bound to chain B of T-p53C-Y220C contoured at 3.0 σ. (D) Superposition of T-p53C-Y220C in its free (PDB ID code 2J1X chain B; green) (16) and PhiKan083-bound form (yellow), indicating small structural shifts upon ligand binding. PhiKan083 is depicted as a gray stick model. The small red spheres represent water molecules in the ligand-free structure that are displaced upon ligand binding. (E) In wild-type p53, Tyr-220 blocks part of the Phikan083-binding pocket, as shown for the structure of wild-type core domain (PDB code 2AC0, chain B; cyan) (42) superimposed onto Phikan083-bound T-p53C-Y220C (yellow protein chain and gray PhiKan083 molecule) and free T-p53C-Y220C (green). (F) Docking of Phikan083 to the structure of ligand-free T-p53C-Y220C (PDB ID code 2J1X, chain A, Thr-230 rotamer A; purple) and to the protein chain of the complex structure (yellow) compared with its actual binding mode in the crystal structure of the complex (green). All images were prepared with PYMOL (http://pymol.sourceforge.net).