Covalent Small Molecule Inhibitors of Ca-Bound S100B

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 Ca-dependent manner, dissociate the p53 tetramer, and inhibit its tumor suppression functions. Likewise, inhibiting S100B with small interfering RNA (siRNA) 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 S100B-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. D improvements in chemotherapy, BRAF/MEK inhibitors, cytokine treatments, immunotherapies, vaccines, and combinatorial approaches for treating malignant melanoma (MM), long-term survival (>3 years) remains very poor for a majority of patients (>70%), and side effects from these treatments are sometimes quite severe. These issues are even more problematic after the onset of metastasis and/or when drug-resistant mechanisms arise. As with many cancers, survival from MM is most promising when it is detected early, so the development of useful biomarkers for detection and more recently for personalized medicine approaches is ongoing. One such marker, S100B, is especially important to monitor because its level is elevated in >90% of MM patients and its protein level correlates directly with poor survival (<1 year) and relapse, and it is especially predictive when used in combination with other diagnostic indicators. On the other hand, for the few MM patients (5−10%) who have low levels of S100B, the MM vaccine is most effective at providing longer survival times. The S100B protein is a marker for melanoma, and when its level is elevated, it contributes to disease progression. While the mechanism of elevated S100B levels toward MM progression is not fully understood, it contributes to lowering protein levels of the tumor suppressor p53 in a Ca-dependent manner. Specifically, p53 is sequestered by Ca-bound S100B (S100B), its phosphorylation in the C-terminal negative regulator domain blocked, its oligomerization disrupted, and its degradation promoted. Because p53 is typically wild-type in MM, efforts are underway to specifically inhibit formation of the S100B−p53 complex and restore p53 levels, particularly in cases in which the cancer is resistant to kinase inhibitors or other therapeutic options. As a proof of principle, blocking the S100B-dependent effect on p53 via RNA interference or by small molecule inhibitors (also known as SBiXs) restores p53 protein levels and its tumor suppression activities, including UV-activated apoptosis. One such inhibitor, pentamidine Received: May 9, 2014 Revised: September 30, 2014 Published: September 30, 2014 Article pubs.acs.org/biochemistry © 2014 American Chemical Society 6628 dx.doi.org/10.1021/bi5005552 | Biochemistry 2014, 53, 6628−6640 Terms of Use (also termed SBi1), entered stage II clinical trials for the treatment of relapsed or refractory malignant melanoma in patients with wild-type p53 and detectable S100B (www. clinicaltrials.gov, identifier NCT00729807). However, despite this promising line of inquiry, efficacy, specificity, and toxicity issues need to be improved significantly for SBiX lead molecules and warrant further investigation using drug design approaches. In previous structure−function studies of S100B, three persistent binding sites were identified in S100B−target and S100B−SBiX complexes (Figure 1). Site 1 interactions were first highlighted via the structure of S100B bound to the Cterminal regulatory domain of p53, while sites 2 and 3 were elucidated in the detailed characterization of the S100B−SBi1 complex. Here we describe a series of inhibitors, which occupy only the central binding site on S100B (site 2) through a covalent attachment to Cys84. To fully characterize this binding site, a series of “site 2” S100B−SBiX complexes were subjected to crystallization trials. Five new S100B−SBiX complexes were identified (i.e., for S100B−SC124, S100B− SBi4172, S100B−SC1982, and S100B−SC1475). As a group, these “site 2” inhibitors display a meaningful effect in cellular assays on their own, but as discussed here, they also provide promise for defining how to link SBiX molecules bound in sites 1 and 3, as part of a new chemical scaffold, which can occupy all three persistent binding pockets within S100B, simultaneously. These data also identify a common conformational change that occurs as a result of “site 2” occupation, which is necessary to consider in future therapeutic design efforts. ■ EXPERIMENTAL PROCEDURES Purification. N-labeled S100B (rat and bovine) was expressed and purified (>99%) with methods similar to those described previously. The concentrations of S100B stock solutions were determined using the Bio-Rad Protein Assay (Bio-Rad Inc., Hercules, CA). The S100B was stored at a concentration of ∼10 mM in 0.25 mM Tris (pH 7.2) and 0.25 mM DTT at −20 °C until use. Fluorescence Polarization Competition Assay (FPCA). The LOPAC1280 (Sigma-Aldrich) compound library was screened using an adaptation of a previously reported fluorescence polarization competition assay. Briefly, the compounds were screened for binding to Ca-loaded S100B by measuring changes in fluorescence polarization upon competition with the TAMRA-labeled version of peptide TRTK12, which is derived from CapZ protein residues 265− 276 (TRTKIDWNKILS). The FPCA was performed in 0.2 μM S100B (rat), 25 nM TAMRA-TRTK12, 50 mM HEPES (pH 7.2), 100 mM KCl, 15 mM NaCl, 10 mM CaCl2, 0.01% Triton X-100, and 0.3% DMSO in 1536-well plates with 8 μL per well. NMR Spectroscopy. Purified N-labeled S100B (rat) protein was dialyzed against 0.25 mM Tris (pH 7.5) and 0.25 mM DTT and concentrated to 10−15 mM using Amicon Ultra centrifugal filter units with a 10 kDa molecular weight cutoff; the concentration was determined using Bradford reagent (BioRad), and protein was then aliquoted and stored at −20 °C. The Ca-loaded S100B−SBiX heteronuclear single-quantum coherence (HSQC) samples contained 0.5 mM S100B subunit, 0.625 mM SBiX, 0.34 mM NaN3, 15 mM NaCl, 5% DMSO-d6, 10 mM CaCl2, 10% D2O, 0.2% TPEN, and 10 mM Hepes, adjusted to pH 7.2 with HCl. HSQC NMR data were collected at 37 °C with a Bruker Avance 800 US2 (800.27 MHz for protons) instrument equipped with pulsed-field gradients, four frequency channels, and triple-resonance, z-axis gradient cryogenic probes. Data were processed with NMRPipe, and proton chemical shifts were reported with respect to the H2O or HDO signal taken as 4.608 ppm relative to external TSP (0.0 ppm). The N chemical shifts were indirectly referenced as previously described using the following ratio of the zero-point frequency: 0.10132905 for N to H. A series of threedimensional HNCA, HNCOCA, N-edited NOESY-HSQC, and N-edited HMQC-NOESY-HSQC NMR experiments were conducted, and the data were sufficient to unambiguously assign all the observable backbone H, N, and C chemical shift values of S100B in the compound-bound state (Figures S1−S5 of the Supporting Information). Crystallographic Studies. Crystallization. All crystallization experiments were conducted using vapor diffusion methods and performed as follows using purified N-labeled S100B (bovine) protein. S100B−SBi4172 crystals were grown in sitting drops consisting of a 0.75:0.75 μL protein solution [20 mg/mL S100B, 10 mM cacodylate (pH 7.2), 7.5 mM CaCl2, and 2 mM SBi4172 prepared in DMSO-d6] and mother liquor [20% 2-methyl-2,4-pentanediol, 0.1 M Hepes (pH 7.0), and 7.5 mM CaCl2]. S100B−SBi4434 crystals were grown in sitting drops consisting of a 0.75:0.75 μL protein solution [40 mg/mL S100B, 10 mM cacodylate (pH 7.2), 7.5 mM CaCl2, and 4 mM SBi4434 prepared in DMSO-d6] and mother liquor [40% 2-methyl-2,4-pentanediol, 0.1 M cacodylate (pH 6.0), and 7.5 mM CaCl2]. S100B−SC1982 crystals were grown in sitting drops consisting of a 0.75:0.75 μL protein solution [40 mg/mL S100B, 10 mM cacodylate (pH 7.2), 7.5 mM CaCl2, and 4 mM SC1982 prepared in DMSO-d6] and mother liquor [40% 2-methyl-2,4-pentanediol, 0.1 M Tris (pH 8.0), and 7.5 mM CaCl2]. S100B−SC1475 crystals were grown in sitting drops consisting of a 0.75:0.75 μL protein solution [40 mg/mL S100B, 10 mM cacodylate (pH 7.2), 7.5 mM CaCl2, and 4 mM SC1475 prepared in DMSO-d6] and mother liquor [40% 2-methyl-2,4-pentanediol, 0.1 M Bis-Tris (pH 6.0), and 7.5 mM CaCl2]. S100B−SC124 crystals were grown in sitting drops consisting of a 0.75:0.75 μL protein solution [40 mg/mL S100B, 10 mM cacodylate (pH 7.2), 7.5 mM CaCl2, and 4 mM SC124 prepared in DMSO-d6] and mother liquor [40% 2-methyl-2,4-pentanediol, 0.1 M Hepes Figure 1. Binding sites 1−3. Shown is a ribbon diagram of the S100B dimer with the three persistent binding sites shaded. The sites were identified in S100B−target and S100B−SBiX complexes. Site 1 interactions were first highlighted via the structure of S100B bound to the C-terminal regulatory domain of p53, while sites 2 and 3 were elucidated in the detailed characterization of the S100B−SBi1 complex. Biochemistry Article dx.doi.org/10.1021/bi5005552 | Biochemistry 2014, 53, 6628−6640 6629 (pH 7.0), and 7.5 mM CaCl2]. The crystals were grown over a period of 1−14 days at 295 K. Crystals were flash-cooled directly from the crystallization drop. Data Collection and Processing. Diffraction data for S100B−SBi4172 crystals were collected at the Northeastern Collaborative Access Team (NE-CAT) 24ID-C beamline at the Advanced Photon Source (Argonne National Laboratory, Argonne, IL). Data were recorded at 100 K on a PILATUS detector and processed by NE-CAT’s RAPD automated processing (https://rapd.nec.aps.anl.gov/rapd), which uses XDS for integration and scaling. A 1.73 Å data set was collected at a wavelength of 0.97920 Å while the crystal was being oscillated by 1.0° each frame. The space group was determined to be P21. Diffraction data for S100B−SC124, S100B−SBi4434, and S100B−SC1982 crystals were collected remotely at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 7-1. Data were recorded at 100 K on an ADSC Q315 (315 mm × 315 mm) detector with collection strategies generated by BLU-ICE. S100B−SC124 and S100B−SC1982 data sets were processed and integrated by AUTOXDS, while S100B− SBi4434 data sets were processed and integrated by MOSFLM within the CCP4 program suite. A 1.58 Å S100B−SC124 data set was collected at a wavelength of 1.1271 Å while the crystal was being oscillated by 0.6° each frame. The space group was determined to be P21. A 1.08 Å S100B−SBi4434 data set was collected at a wavelength of 1.00 Å while the crystal was being oscillated by 0.35° each frame. The space group was determined to be P21. A 1.65 Å S100B−SC1982 data set was collected at a wavelength of 1.1271 Å while the crystal was being oscillated 0.45° each frame. The space group was determined to be C2221. Diffraction data for S100B−SC1475 crystals were collected remotely at Stanford Synchrotron Radiation Lightsource (SSRL) beamline 12-2. Data were recorded at 100 K on a PILATUS detector with collection strategies generated by BLU-ICE, and processing and integration were performed by AUTOXDS. A 2.18 Å data set was collected at a wavelength of 0.97950 Å while the crystal was being oscillated by 0.15° each frame. The space group was determined to be P21. Diffraction data statistics are summarized in Table 1. Structure Determination and Refinement. To determine the structure of S100B in complex with SBiXs, we performed molecular replacement (MR). A search model from a previous S100B structure [Protein Data Bank (PDB) entry 1MHO] was generated by removing coordinates for ligands and water. Molecular replacement was conducted within the AUTOMR function of the PHENIX software suite. The models were finished by manual building within COOT. The models were refined by the PHENIX.REFINE function of the PHENIX software suite. Ligands and waters were incorporated into the models by visual inspection of the |Fo| − |Fc| omit maps. The structure refinement statistics are summarized in Table 1. Cell-Based Assay with WM115 Malignant Melanoma Cells. The malignant melanoma cell line, WM115, was obtained from American Type Tissue Collection (ATCC) and cultured in minimum essential medium (Invitrogen) supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 100 units/mL penicillin-streptomycin (PS). The cells were infected with SMARTvector 2.0 lentiviral particles Table 1. Statistics of Reflection Data and Structure Refinements SC124 SC1475 SC1982 SBi4172 SBi4434 space group P21 P21 C2221 P21 P21 unit cell dimensions (Å) 34.7, 56.5, 48.3 35.0, 58.1, 47.9 34.6, 90.0, 60.8 35.0, 57.1, 48.0 35.2, 56.3, 48.1 unit cell angles (deg) 90.0, 110.1, 90.0 90.0, 111.2, 90.0 90.0, 90.0, 90.0 90.0, 111.0, 90.0 90.0, 108.8, 90.0 resolution range (Å) 35.35−1.58 35.40−2.18 36.17−1.65 44.82−1.73 21.69−1.08 no. of reflections observed 83472 (9120) 30768 (4167) 55035 (7875) 55396 (8136) 244228 (12126) no. of unique reflections 23617 (3115) 9152 (1259) 11752 (1673) 17118 (2517) 70592 (3504) no. of reflections in the Rfree set 1201 935 1173 869 2006 completeness (%) 97.8 (89.1) 96.7 (91.7) 99.9 (99.8) 92.6 (94.2) 92.2 (93.0) redundancy 3.5 (2.9) 3.4 (3.3) 4.7 (4.7) 3.2 (3.2) 3.5 (3.5) ⟨I/σ⟩ 22.6 (3.5) 11.6 (2.3) 22.7 (2.6) 13.8 (2.4) 9.0 (3.0) Rsym b 0.029 (0.325) 0.051 (0.461) 0.045 (0.613) 0.038 (0.452) 0.068 (0.351) Rcrys c 0.215 0.213 0.177 0.230 0.192 Rfree d 0.253 0.251 0.214 0.268 0.211 no. of amino acids 179 180 90 179 181 no. of protein atoms 1445 1454 751 1446 1464 no. of hetero atoms 20 46 29 30 36 no. of waters 173 19 137 76 321 rmsd for bond lengths (Å) 0.020 0.013 0.012 0.024 0.015 rmsd for bond angles (deg) 1.056 1.304 1.231 1.372 1.133 mean B factor (Å) 28.92 48.73 20.67 39.00 14.91 protein atoms (Å) 27.69 48.00 18.61 38.74 13.62 hetero atoms (Å) 44.54 71.95 28.82 38.00 22.01 water atoms (Å) 37.36 48.65 30.21 44.32 25.09 Ramachandran outliers (%) 0.0 0.0 0.0 0.0 0.0 Ramachandran favored (%) 100.0 98.9 100.0 98.9 99.4 The numbers in parentheses represent values from the highest-resolution shell (1.66−1.58 Å for SC124, 2.3−2.18 Å for SC1475, 1.74−1.65 Å for SC1982, 1.82−1.73 Å for SBi4172, and 1.10−1.08 Å for SBi4434). Atom counts do not include H. Rsym = ∑h(∑j|Ihj − ⟨Ih⟩|/∑Ih,j), where h is the set of Miller indices, j is the set of observations of reflection h, and ⟨Ih⟩ is the mean intensity. Rcrys = ∑h||Fo,h| − |Fc,h||/∑h|Fo,h|. Rfree was calculated using a percentage of the complete data set excluded from refinement. Deviations from ideal values. Biochemistry Article dx.doi.org/10.1021/bi5005552 | Biochemistry 2014, 53, 6628−6640 6630 containing either nontargeting scrambled or anti-S100B shRNA according to manufacturer’s recommendations (Thermo Scientific-Dharmacon). The following day, the medium containing lentivirus was removed, the cells were washed twice with PBS and trypsinized, and each well was expanded into a 24-well plate containing growth medium supplemented with puromycin (0.5 μg/mL). Upon confluence, the wells were trypsinized and single-cell diluted into 96-well plates. Positive clones with a reduction in the level of S100B protein expression were maintained in puromycin-containing medium. A cellular screen was then developed with these WM115 cell lines, and further test compounds were shown to bind S100B in vitro. In this cellular assay, the ability of SBiX molecules to inhibit the growth of WM115 melanoma cells infected with shRNA lentivirus (Dharmacon) targeting S100B (shRNA, i.e., low S100B) or a scrambled control (shRNA, i.e., high S100B) was examined quantitatively. The methods used were similar to those used previously and included the use of a Biomek FX Laboratory Automation Workstation (BeckmanCoulter) equipped with a 96-channel pipetting head. Specifically, 20 μL of MEM (Corning) supplemented with 10% fetal bovine serum, 0.5 μg/mL puromycin, and 1% Pen/ Strep was added to each well of a 384-well, clear bottom, tissue culture plate (Corning) containing 600 cells per well such that growing uninhibited they reach 80% confluence in 5 days. After the cells had grown for 24 h at 37 °C in a 5% CO2 humiditycontrolled incubator, 20 μL of the compound was added directly to the cell culture medium, while control cultures received an equivalent amount of DMSO. After being incubated for an additional 4 days, the cells were lysed by transferring 20 μL of lysis buffer consisting of 1.8% Igepal with a 1:10000 dilution of SYBR Green I (10000×, Invitrogen) to each well. The plates were then returned to the incubator for 24 h. The fluorescence intensity was then read through the bottom of the plate using a PolarStar fluorescent plate reader (BMG) using 485 nm excitation and 520 nm emission filters. The SYBR Green fluorescence is used to measure total DNA that in turn correlates with the cell number as previously described. The EC50 of each compound was determined using serial dilutions and performed in a minimum of three replicates. Hill curves of each replicate were generated using Origin Data Analysis Software. To analyze changes in total p53 protein levels upon treatment with SC1982, WM115 cells were seeded in triplicate at a density of 70 × 10 cells in 60 mm dishes in 1× MEM (Cellgro) supplemented with 10% FBS, 100 units/mL PS, and 0.5 μg/mL puromycin and allowed to adhere overnight. The following day, the old medium was removed and new medium containing 5 μM SC1982 or DMSO was added. The cells were harvested 4 h post-treatment using cold 1× RIPA lysis buffer [0.5 M Tris-HCl (pH 7.4), 1.5 M NaCl, 2.5% deoxycholic acid, 10% Igepal, and 10 mM EDTA] and subjected to Western blotting. Western blotting was performed using 30 μg of cell lysates loaded on a 12% sodium dodecyl sulfate−polyacrylamide gel electrophoresis gel (NuPage), which was subsequently transferred to PVDF membranes (Millipore) and reacted with p53 mouse monoclonal antibody (DO-1, Santa Cruz), mouse antiS100B antibody (BD Biosciences), and mouse anti-GAPDH antibody at dilutions recommended by the manufacturers. The blots were then reacted with goat anti-mouse secondary antibodies (Kirkegaard & Perry Laboratories) and treated with Immobilon Western Chemiluminescent HRP Substrate (Millipore) at dilutions recommended by the manufacturer. ■ RESULTS The binding of S100B to p53 downregulates tumor suppressor activity in cancer cells such as malignant melanoma, so a search for small molecule inhibitors that bind S100B and prevent formation of the S100B−p53 complex was undertaken. A fluorescence polarization competition assay (FPCA) was performed against the 1280-compound Library of Pharmacologica l ly Act ive Compounds (LOPAC1280). Because of the location of Cys84 within the S100B binding pocket (i.e., between sites 1 and 3), the focus of this investigation was a series of covalent complexes from this that were predicted to covalently bind at Cys84 of S100B. SBi4172 is a structural analogue of the covalent modifier SC0844 and was thought to bind similarly. SC124, or disulfiram, is a known covalent modifier. The chemical nature of SBi4434 suggests the possibility of adduct formation and is confirmed here. To gain additional opportunities for characterization of covalent adducts, compounds were also taken from a previously reported screen versus the 2000-compound Spectrum Collection. The compounds characterized in this study include SC1982 and SC1475, which were reported in the previous study as being dependent on Cys84 for binding, but the formed covalent adducts were not investigated. Cellular and biophysical characterization of these covalently bound inhibitors was explored, including their three-dimensional structures when bound to S100B (Tables 1−4). NMR Studies. Backbone resonance assignments were completed for each SBiX−S100B complex using standard multidimensional heteronuclear NMR data, and the chemical shift perturbations in S100B (with or without the compound) were evaluated as described for other S100B inhibitors. In general, the SBiX complexes studied here showed the largest perturbations within a well-defined binding pocket of S100B comprising helices 3 and 4 and loop 2 (termed the “hinge” Table 2. Cellular Assays high S100B EC50 (μM) low S100B EC50 (μM) compound mean SD mean SD T-test ratio (∓) n SC124 0.06 0.06 0.23 0.38 0.321 3.57 6 SC1475 13.87 1.70 13.81 5.00 0.980 1.00 6 SC1982 5.05 1.13 8.95 2.68 0.001 1.77 8 SBi4172 2.07 1.29 3.11 1.43 0.127 1.50 9