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)
FoFc
simulated-annealing omit map of PhiKan083 bound to chain B of T-p53C-Y220C contoured at 3.0 . (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)

ost significant shift is observed for the side chain of Thr-150, which is displaced by up to 1.4 Å upon binding, thus widening the entrance of the pocket (Fig. 5D). The occupancy of PhiKan083 in the binding pocket upon soaking is significantly different in the two molecules in the asymmetric unit, even though the architecture of the crystal lattice would suggest a similar accessibility of both sites

bond with the main-chain carbonyl of Asp-228 (2.7-Å distance). Only very small structural shifts occur upon ligand binding to the mutant. The residues that are within 5 Å of PhiKan083 (residues 109, 145–147, 150, 151, 220–223, 228–230, and 257) superimpose with a rmsd of 0.3 Å (all atoms).

Binding would appear to have an important contri- bution from hydrophobic packing interactions. The ethyl group is in close contact to the sulfhydryl group of the mutated residue Cys-220, which adopts two alternative conformations, and a number of hydrophobic side chains (Phe-109, Leu-145, Val-147, and Leu-257), thus anchoring the ligand to the pocket. The planar carbazole ring system is sandwiched between the hydro- phobic side chains of Pro-222 and Pro-223 on one side, and Val-147 and Pro-151 on the other side of the binding cleft. The ring nitrogen sits close to the position of the hydroxyl group of the tyrosine residue in the wild-type structure (1.0-Å distance) (Fig. 5E).

We solved the crystal structure of the T-p53C-Y220C:PhiKan083 complex at 1.5-Å resolution by soak- ing the small molecule into crystals of the mutant (

The kinetics of denaturation of T-p53C-Y220C at 310 K (37°C) was fitted to a simple binding model for PhiKan083 (Fig. 3C). In the absence of ligand, the protein had a half-life of 3.8 min. This increased to 15.7 min at saturating concentrations of PhiKan083

We screened a secondary library of PhiKan059 ana- logues based on the carbazole scaffold, which yielded PhiKan083 (Fig. 2) among others with improved binding affinity to T-p53C- Y220C, with a Kd measured by NMR of 167  12
M for PhiKan083

PhiKan059 (Fig. 2) was discovered to bind to the mutation-induced cleft of T-p53C-Y220C but not to T-p53C

Y220C is the ninth most frequent p53 cancer mutant and accounts for an estimated 75,000 new cancer cases per annum worldwide based on cancer incidence statistics by the World Health Organization and reported mutation frequencies (www-p53.iarc.fr) (17). The mutation creates a surface crevice, and the protein is highly destabilized as a result (16)

we generated a total pool of 24.8 million conformers. We applied a series of increasingly more sophisti- cated filters including: