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Structure-based drug discovery is under way to identify and develop small-molecule S100B inhibitors (SBiXs). Such inhibitors have therapeutic potential for treating malignant melanoma, since high levels of S100B downregulate wild-type p53 tumor suppressor function in this cancer. Computational and X-ray crystallographic studies of two S100B-SBiX complexes are described, and both compounds (apomorphine hydrochloride and ethidium bromide) occupy an area of the S100B hydrophobic cleft which is termed site 3. These data also reveal novel protein-inhibitor interactions which can be used in future drug-design studies to improve SBiX affinity and specificity. Of particular interest, apomorphine hydrochloride showed S100B-dependent killing in melanoma cell assays, although the efficacy exceeds its affinity for S100B and implicates possible off-target contributions. Because there are no structural data available for compounds occupying site 3 alone, these studies contribute towards the structure-based approach to targeting S100B by including interactions with residues in site 3 of S100B. [ABSTRACT FROM AUTHOR]

The drug pentamidine inhibits calcium-dependent complex formation with p53 (CaS100B·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 CaS100B·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 (CaS100B·5a, CaS100B·6b) a channel between sites 1 and 2 on S100B was occluded by residue Phe88, but for an asymmetric pentamidine analogue (CaS100B·17), this same channel was open. The CaS100B·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 CaS100B, which will impact next generation CaS100B·p53 inhibitor design. [ABSTRACT FROM AUTHOR]

Elevated levels of the tumor marker S100B are observed in malignant melanoma, and this EF-hand-containing protein was shown to directly bind wild-type (wt) p53 in a Ca2+-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNAS100B) is sufficient to restore wild-type p53 levels and its downstream gene products and induce the arrest of cell growth and UV-dependent apoptosis in malignant melanoma. Therefore, it is a goal to develop S100B inhibitors (SBiXs) that inhibit the S100B-p53 complex and restore active p53 in this deadly cancer. Using a structure-activity relationship by nuclear magnetic resonance approach (SAR by NMR), three persistent binding pockets are found on S100B, termed sites 1-3. While inhibitors that simultaneously bind sites 2 and 3 are in place, no molecules that simultaneously bind all three persistent sites are available. For this purpose, Cys84 was used in this study as a potential means to bridge sites 1 and 2 because it is located in a small crevice between these two deeper pockets on the protein. Using a fluorescence polarization competition assay, several Cys84-modified S100B complexes were identified and examined further. For five such SBiX-S100B complexes, crystallographic structures confirmed their covalent binding to Cys84 near site 2 and thus present straightforward chemical biology strategies for bridging sites 1 and 3. Importantly, one such compound, SC1982, showed an Sl00B-dependent death response in assays with WM115 malignant melanoma cells, so it will be particularly useful for the design of SBiX molecules with improved affinity and specificity. [ABSTRACT FROM AUTHOR]

Vitamin A homeostasis is critical to normal cellular function. Retinol-binding protein (RBP) is the sole specific carrier in the bloodstream for hydrophobic retinol, the main form in which vitamin A is transported. The integral membrane receptor STRA6 mediates cellular uptake of vitamin A by recognizing RBP-retinol to trigger release and internalization of retinol. We present the structure of zebrafish STRA6 determined to 3.9-angstrom resolution by single-particle cryo-electron microscopy. STRA6 has one intramembrane and nine transmembrane helices in an intricate dimeric assembly. Unexpectedly, calmodulin is bound tightly to STRA6 in a noncanonical arrangement. Residues involved with RBP binding map to an archlike structure that covers a deep lipophilic cleft. This cleft is open to the membrane, suggesting a possible mode for internalization of retinol through direct diffusion into the lipid bilayer. [ABSTRACT FROM AUTHOR]