• Targeting Malignant Melanoma and Potential Off-target Effects in EC-coupling

      Melville, Zephan; Weber, David J., Ph.D. (2017)
      S100B belongs to the S100 family of Ca2+-binding proteins, a family known for calcium-dependent interactions that regulate biological processes. Upregulation of S100B in malignant melanoma (MM) downregulates p53 tumor suppressor function and is correlated with poor prognosis, making S100B a therapeutic target for MM. A fragment-based drug discovery program is underway to develop small-molecule S100B inhibitors. Compounds SC0025 and SC1990 occupy part of the S100B hydrophobic cleft, termed site 3, while compounds SBi5361 and 5363 occupy sites 1-3. Crystal structures show specific protein-inhibitor interactions to exploit in further studies for improving affinity and specificity. Heteronuclear RNA-binding protein (hnRNP) A18 is also involved in MM. A18 is upregulated in tumors and promotes tumor growth via coordination of pro-survival mRNA. The crystal structure for the RNA recognition motif (RRM) of A18 is reported here, with comparisons to the homologous RNA-binding protein, hnRNP A1. These comparisons show a conserved global fold and conservation of known RNA-binding residues. Given this, it would be impossible to design inhibitors specific for A18. Instead, it is the intrinsically disordered domain of A18 that must endow specificity, as this is not conserved. As such, this structure serves as a foundation for work with full-length A18 and drug-design efforts targeting A18 in MM. The sibling protein to S100B, S100A1 regulates several cellular processes, including Ca2+-signaling in striated muscle, through interaction with the ryanodine receptor. The crystal structure of S100A1, reported here, provides insights into S100A1-target binding specificity through key differences in the binding pockets of S100A1 and S100B. In cardiac cells, S100A1 increases Cav1 channel current amplitude, an effect blocked by inhibition of protein kinase A (PKA), implying a PKA-dependent process. As this did not require cAMP, its mechanism of activation remained unknown. Biochemical studies demonstrate that S100A1 directly activates PKA in a Ca2+-dependent manner. A functional role for this pathway is also established as PKA-dependent subcellular redistribution of HDAC4 was abolished in S100A1 knockout mice. Thus, the interaction between S100A1 and PKA provides a link between Ca2+- and PKA-signaling.