• Designing Specific Inhibitors to Target S100B in Melanoma

      Vera-Rodriguez, Darex J.; Young, Brianna; Spriggs, Shardell; Yu, Wenbo; Wilder, Paul T.; MacKerell, Alexander D., Jr.; Weber, David J., Ph.D. (2020)
      Malignant melanoma (MM) is defined as the most dangerous form of skin cancer, causing a large majority of skin cancer deaths. Previous studies demonstrate S100B as a tumor marker in MM, a protein that interacts with the tumor suppressor p53, inhibiting p53 function. With the goal of blocking this interaction, three binding sites on S100B for small molecules have been identified. However, developing drugs specific for S100B over other S100-family members remains a challenge. This project aims to identify S100B-specific small molecule inhibitors and understand the basis of their specificity over other S100 family members, specifically S100A1. A 2D-[1H,15N] NMR HSQC of S100B bound to a non-specific fragment showed multiple chemical shifts perturbations (CSPs) including residues A9, L10, F43, L40, F73, and C84. Interestingly, fewer and less pronounced CSPs were observed for a S100B-specific fragment. Site-Identified Ligand Competitive Saturation (SILCS) molecular dynamics (MD) simulations were performed to determine potential S100B binding sites that could explain the CSPs. Results show a strong hydrophobic pocket at low Grid Free Energy (GFE) levels comprised by the residues that showed CSPs for compounds that bind S100B. FDA-approved compounds were tested using SILCS-Monte Carlo (MC) to determine ligand binding poses at this pocket. Specific compounds were targeted with strong hydrophobic interactions and hydrogen bonds at low Ligand GFE (GFE). These data provide important information relevant to developing S100B-specific drugs to treat MM.
    • Discovery and Evaluation of Small Molecule Inhibitors of the S100B-p53 Interaction

      McKnight, Laura Emily; Weber, David J., Ph.D. (2012)
      S100B is a calcium-binding protein that is highly upregulated in malignant melanoma and is currently used as a prognostic indicator for the disease. S100B has been shown to bind to p53, decreasing p53 protein levels and inhibiting its function. Small molecule inhibitors are being investigated which use the S100B-p53 interaction as a therapeutic target, and the drug pentamidine was found to bind S100B; pentamidine-derived compounds were then designed, synthesized, and analyzed. Molecular Dynamics simulations of the pentamidine-S100B complex 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 or heptamidine, was synthesized and experiments to study its inhibition of S100B were performed. The 1.65 Å X-ray crystal structure was determined for Ca2+-loaded S100B bound to heptamidine 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 SBi4211 causes perturbations in the chemical shifts of the same residues of S100B as pentamidine. SBi4211 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.
    • The Role and Inhibition of S100B in Melanoma Cell Signaling

      Hartman, Kira Gianni; Weber, David J., Ph.D. (2012)
      The calcium–binding protein S100B is an effective and extensively used prognostic marker for melanoma, with increasing S100B being predictive of disease stage, increased recurrence, and low survival. Establishing the mechanism by which S100B alters cell signaling provides insight into how it may facilitate the progression of melanoma and aid in developing new pharmacological drugs to inhibit cancer advancement. To evaluate the significance of S100B in melanoma, knock–down and over–expression studies were conducted, finding a positive correlation between S100B expression and cell viability, as well as ERK phosphorylation. However, phosphorylation of RSK, a downstream ERK target, was determined to have an inverse relationship with S100B. Over–expression of a calcium–binding mutant S100B yields neither effect, indicating that each response is calcium–dependent. Pull–down experiments established the direct calcium–dependent binding of S100B to the C–terminus of RSK and kinase assays demonstrated that S100B prevents RSK phosphorylation at Thr573. Over–expression of S100B in melanoma cells reduces the phosphorylation of RSK, sequestering it in the cytosol. Conversely, cells with diminished S100B expression exhibited increased staining of phosphorylated RSK within the nucleus. Together these data are consistent with a mechanism in which elevated S100B binds RSK directly in a calcium–dependent manner, preventing ERK–mediated phosphorylation and subsequent nuclear translocation. Thus, S100B uniquely affects MAPK signaling by increasing levels of phosphorylated ERK while simultaneously preventing the phosphorylation of RSK. Capitalizing on this discovery, in addition to previously known S100B interactions such as with p53, we are searching for S100B inhibitors that will prevent cancer progression. To this end, in vitro FPCA was employed to rapidly screen 2,000 compounds, establishing whether they bind Ca<super>2+</super>–loaded S100B and inhibit S100B target complex formation. Building upon this, we developed a cell–based high throughput assay capable of screening an extensive library of 14,400 compounds, in addition to 26 putative S100B inhibitors identified through FPCA, by comparing their effects on cells expressing elevated S100B to cells where S100B has been significantly knocked–down. The desired endpoint of this research is the development of a drug with therapeutic activity for the treatment of malignant melanoma and/or other cancers with elevated S100B.
    • S100B regulation of p53 phosphorylation by PKC(alpha) and structural characterization of zinc(2+)-binding to calcium(2+)-S100B

      Wilder, Paul T.; Weber, David J., Ph.D. (2004)
      The S100B protein is a small acidic metal binding protein that has been found to be associated with neurological disorders and traumas, diabetes, and cancer. This work examines Zn2+ regulation of S100B Ca 2+-binding affinity and target protein interaction and also explores S100B interaction and regulation of an important target protein, tumor suppressor p53. It was found that S100B binds p53 preventing PKC phosphorylation of p53 (1), and this interaction connects the molecular and cellular functions of S100B with clinical data that associates S100B expression to several forms of cancer. In order to further characterize this S100B inhibition of PKC activities, peptides based on the PKC phosphorylation domains of p53 (residues 367--388), neuromodulin (residues 37--53), and the regulatory domain of PKC (residues 19--31) were synthesized and shown to be substrates for PKC and the catalytic domain of PKC, PKM, which lacks the Ca2+ and lipid regulatory region of PKC. It was necessary to use PKM in order to separate the influence of Ca2+ on the activation S100B from its effects on PKC allowing kinetic parameters of the PKC-dependent phosphorylation and its Ca2+-dependent inhibition by S100B to be determined for the peptides. While Ca2+-binding appears to be necessary for target protein binding by S100B, Zn2+-binding potentiates both S100B Ca2+-binding affinity and its interaction with certain target proteins. In order to determine how structural changes in S100B upon Zn2+ binding regulate its function, the high resolution structures of the Zn2+-Ca2+-S100B was completed using multi-dimensional heteronuclear NMR experiments. Studies were done to determine the amino acid ligands used for coordinating Zn2+ in S100B revealing what appears to be a conserved Zn2+/Cu2+-binding site at the dimer interface found in several S100 proteins. The structure shows that Zn2+-binding induces rearrangement of the Ca 2+-binding EF-hands in S100B that may contribute to the change in Ca2+-binding affinity found in S100B. Furthermore, there is an extension of the final helix IV and orienting of amino acids involved in target protein interaction upon Zn2+-binding by Ca2+ S100B that may influence target protein affinity.
    • Structure, Function, and Inhibition of S100B

      Charpentier, Thomas H.; Weber, David J., Ph.D. (2009)
      Aberrant levels of the small dimeric protein S100B have been found in malignant melanoma, renal cell cancer, and astrocytomas. S100B may aid in cancer progression via its interaction with and down regulation of the tumor suppressor p53, in a Ca²⁺ and possibly Zn²⁺ dependent manner. S100B bound to Ca²⁺ undergoes a conformational change exposing a hydrophobic cleft for the p53-S100B interaction. S100B binds to the C-terminus and tetramerization domains (319-393) of p53. Experiments reducing S100B expression via siRNA restores p53 levels in primary malignant melanoma cells. Thus, several small molecules have been identified that bind S100B and inhibit the Ca²⁺-S100B-p53 interaction. One of these small molecules is pentamidine, an FDA approved drug. We have characterized the interaction between Ca²⁺-S100B and pentamidine via nuclear magnetic resonance (NMR) and X-ray crystallography. We obtained crystal structures of pentamidine bound to Zn²⁺-Ca²⁺-S100B. The previously solved NMR structure of Zn²⁺-Ca²⁺-S100B was compared to the X-ray crystal structure solved here. We characterized the Zn²⁺ ligands in each structure to determine if Zn²⁺ binding changed the pentamidine interaction with S100B. A goal of the Weber lab has been to identify small molecules to inhibit the p53-S100B interaction and we have been moderately successful. We have identified three additional small molecules found through screens performed by our lab and the high throughput screening core (UMAB). SBi132, SBi279, and SBi523 (S100B inhibitor ###) were shown to interact with S100B through NMR and X-ray crystallography. Other small molecules derived from pentamidine or SBi132 interact with the "hinge" region of S100B, while other screening molecules were found to covalently bind to cysteine 84 on helix 4 of S100B. To characterize the S100B-p53 protein-protein interaction further, TRTK-12 a peptide derived from the CapZ protein, was used to study the effects of peptide bound to S100B. Surprisingly, the Ca²⁺ coordination for both EF-hands of S100B were not affected by TRTK-12. The X-ray structure of TRTK-12 peptide bound to S100B did show differences in temperature factor. These differences in peptide binding can aid us in identifying inhibitors of the S100B-p53 complex and restore p53 levels in malignant melanoma.
    • 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.
    • The three-dimensional structure and subunit association of S100B in the apo and Ca(2+)-bound states, and its Ca(2+)-dependent interaction with target proteins

      Drohat, Alexander Clark; Weber, David J., Ph.D. (1997)
      S100B(beta beta), a member of the S100 protein family, is a Ca2+-binding protein with noncovalent interactions at its dimer interface. The solution structure of S100B(beta beta) has been determined, in the apo- and Ca2+-loaded state, using 2D, 3D, and 4D NMR spectroscopy. In both states, the S100beta subunits (91 residue) contain four alpha-helices and a small antiparallel beta-sheet which brings together the two helix-loop-helix Ca2+-binding domains (EF-hands). Both structures are found to be globular and compact with an extensive hydrophobic core and a highly charged surface, consistent with the high solubility of S100B(beta beta). The antiparallel alignment of helix 1 with 1 prime (other subunit) and of helix 4 with 4 prime, and the perpendicular association of these pairs of antiparallel helices forms an X-type four helical bundle at the dimer interface in both structures. However, the orientation of helix 3 relative to helices 1, 2, and 4 in each subunit of apo-S100B(beta beta) differs significantly from that of Ca2+-loaded S100B(beta beta). This conformational change is well illustrated by the difference in interhelical angle (delta omega=112 degrees) of the C-terminal EF-hands (apo-S100beta, omega=140 degrees; Ca2+-loaded S100beta, omega=106 degrees). For comparison, omega ranges from 118 degrees to 145 degrees in the apo-state and from 84 degrees to 128 degrees in the Ca2+-bound states of the EF-hands in calbindin D subscript 9k, calcyclin, and calmodulin. The significant conformational change required of C-terminal EF-hand for it to adopt the Ca2+-bound structure readily explains the dramatic spectral effects previously observed for S100B({beta beta) upon the addition of Ca2+. The conformational change exposes a cleft defined by residues in the loop linking the EF-hands, helix 3, and the C-terminal loop of Ca2+-loaded S100B({beta beta). This cleft is absent in the apo-structure and is therefore likely to play a role in target protein binding. S100B(beta beta) has been shown to interact with the tumor suppressor protein, p53, and this interaction was examined with a peptide derived from the C-terminal regulatory domain of p53 (residues 367-388). Fluorescence and NMR spectroscopy experiments show that the p53 peptide binds to a location of S100B(beta beta) that probably involves residues in the hinge region (S41, L44, E45, E46) and the C-terminal loop (A83, C84, H85, E86, F87, F88) as previously predicted. However, residues in helix 3 (V52, V53, V56, T59) are also effected by p53 peptide binding, which supports the proposal that the conformational change observed in helix 3 plays a role in target protein binding. Finally, S100B(beta beta) was shown to exist (>99%) as a non-covalently associated dimer at concentrations as low as 1 nM (subunit concentration, 500 pM dimer) in apo and Ca2+-loaded state. Therefore, in reducing environments and at physiological concentrations, the noncovalent dimer is most likely the form of S100B presented to target proteins.