Small Molecule Inhibitors of Ca(2+)-S100B Reveal Two Protein Conformations.

The drug pentamidine inhibits calcium-dependent complex formation with p53 ((Ca)S100B·p53) in malignant melanoma (MM) and restores p53 tumor suppressor activity in vivo. However, off-target effects associated with this drug were problematic in MM patients. Structure-activity relationship (SAR) studies were therefore completed here with 23 pentamidine analogues, and X-ray structures of (Ca)S100B·inhibitor complexes revealed that the C-terminus of S100B adopts two different conformations, with location of Phe87 and Phe88 being the distinguishing feature and termed the "FF-gate". For symmetric pentamidine analogues ((Ca)S100B·5a, (Ca)S100B·6b) a channel between sites 1 and 2 on S100B was occluded by residue Phe88, but for an asymmetric pentamidine analogue ((Ca)S100B·17), this same channel was open. The (Ca)S100B·17 structure illustrates, for the first time, a pentamidine analog capable of binding the "open" form of the "FF-gate" and provides a means to block all three "hot spots" on (Ca)S100B, which will impact next generation (Ca)S100B·p53 inhibitor design.

Structure-Based Discovery of a Novel Pentamidine-Related Inhibitor of the Calcium-Binding Protein S100B.

Molecular Dynamics simulations of the pentamidine-S100B complex, where two molecules of pentamidine bind per monomer of S100B, were performed in an effort to determine what properties would be desirable in a pentamidine-derived compound as an inhibitor for S100B. These simulations predicted that increasing the linker length of the compound would allow a single molecule to span both pentamidine binding sites on the protein. The resulting compound, SBi4211 (also known as heptamidine), was synthesized and experiments to study its inhibition of S100B were performed. The 1.65 Å X-ray crystal structure was determined for Ca(2+)-S100B-heptamdine and gives high-resolution information about key contacts that facilitate the interaction between heptamidine and S100B. Additionally, NMR HSQC experiments with both compounds show that heptamidine interacts with the same region of S100B as pentamidine. Heptamidine is able to selectively kill melanoma cells with S100B over those without S100B, indicating that its binding to S100B has an inhibitory effect and that this compound may be useful in designing higher-affinity S100B inhibitors as a treatment for melanoma and other S100B-related cancers.