PMCC PMCC

Search tips
Search criteria

Advanced
Results 1-12 (12)
 

Clipboard (0)
None

Select a Filter Below

Journals
Year of Publication
Document Types
1.  Structure-Based Discovery of a Novel Pentamidine-Related Inhibitor of the Calcium-Binding Protein S100B. 
ACS medicinal chemistry letters  2012;3(12):975-979.
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 Ca2+-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.
doi:10.1021/ml300166s
PMCID: PMC3524579  PMID: 23264854
2.  Structure-Based Discovery of a Novel Pentamidine-Related Inhibitor of the Calcium-Binding Protein S100B 
ACS Medicinal Chemistry Letters  2012;3(12):975-979.
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 Ca2+–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.
doi:10.1021/ml300166s
PMCID: PMC3524579  PMID: 23264854
structure-based discovery; pentamidine-related inhibitor; calcium-binding protein S100B
3.  Structure of the S100A4/myosin-IIA complex 
Background
S100A4, a member of the S100 family of Ca2+-binding proteins, modulates the motility of both non-transformed and cancer cells by regulating the localization and stability of cellular protrusions. Biochemical studies have demonstrated that S100A4 binds to the C-terminal end of the myosin-IIA heavy chain coiled-coil and disassembles myosin-IIA filaments; however, the mechanism by which S100A4 mediates myosin-IIA depolymerization is not well understood.
Results
We determined the X-ray crystal structure of the S100A4Δ8C/MIIA1908-1923 peptide complex, which showed an asymmetric binding mode for the myosin-IIA peptide across the S100A4 dimer interface. This asymmetric binding mode was confirmed in NMR studies using a spin-labeled myosin-IIA peptide. In addition, our NMR data indicate that S100A4Δ8C binds the MIIA1908-1923 peptide in an orientation very similar to that observed for wild-type S100A4. Studies of complex formation using a longer, dimeric myosin-IIA construct demonstrated that S100A4 binding dissociates the two myosin-IIA polypeptide chains to form a complex composed of one S100A4 dimer and a single myosin-IIA polypeptide chain. This interaction is mediated, in part, by the instability of the region of the myosin-IIA coiled-coil encompassing the S100A4 binding site.
Conclusion
The structure of the S100A4/MIIA1908-1923 peptide complex has revealed the overall architecture of this assembly and the detailed atomic interactions that mediate S100A4 binding to the myosin-IIA heavy chain. These structural studies support the idea that residues 1908–1923 of the myosin-IIA heavy chain represent a core sequence for the S100A4/myosin-IIA complex. In addition, biophysical studies suggest that structural fluctuations within the myosin-IIA coiled-coil may facilitate S100A4 docking onto a single myosin-IIA polypeptide chain.
doi:10.1186/1472-6807-13-31
PMCID: PMC3924328  PMID: 24252706
X-ray crystallography; NMR; S100A4; Myosin-II; Cytoskeleton; Coiled-coil
4.  The novel BH3 α-helix mimetic JY-1-106 induces apoptosis in a subset of cancer cells (lung cancer, colon cancer and mesothelioma) by disrupting Bcl-xL and Mcl-1 protein–protein interactions with Bak 
Molecular Cancer  2013;12:42.
Background
It has been shown in many solid tumors that the overexpression of the pro-survival Bcl-2 family members Bcl-2/Bcl-xL and Mcl-1 confers resistance to a variety of chemotherapeutic agents. We designed the BH3 α-helix mimetic JY-1-106 to engage the hydrophobic BH3-binding grooves on the surfaces of both Bcl-xL and Mcl-1.
Methods
JY-1-106–protein complexes were studied using molecular dynamics (MD) simulations and the SILCS methodology. We have evaluated the in vitro effects of JY-1-106 by using a fluorescence polarization (FP) assay, an XTT assay, apoptosis assays, and immunoprecipitation and western-blot assays. A preclinical human cancer xenograft model was used to test the efficacy of JY-1-106 in vivo.
Results
MD and SILCS simulations of the JY-1-106–protein complexes indicated the importance of the aliphatic side chains of JY-1-106 to binding and successfully predicted the improved affinity of the ligand for Bcl-xL over Mcl-1. Ligand binding affinities were measured via an FP assay using a fluorescently labeled Bak-BH3 peptide in vitro. Apoptosis induction via JY-1-106 was evidenced by TUNEL assay and PARP cleavage as well as by Bax–Bax dimerization. Release of multi-domain Bak from its inhibitory binding to Bcl-2/Bcl-xL and Mcl-1 using JY-1-106 was detected via immunoprecipitation (IP) western blotting.
At the cellular level, we compared the growth proliferation IC50s of JY-1-106 and ABT-737 in multiple cancer cell lines with various Bcl-xL and Mcl-1 expression levels. JY-1-106 effectively induced cell death regardless of the Mcl-1 expression level in ABT-737 resistant solid tumor cells, whilst toxicity toward normal human endothelial cells was limited. Furthermore, synergistic effects were observed in A549 cells using a combination of JY-1-106 and multiple chemotherapeutic agents. We also observed that JY-1-106 was a very effective agent in inducing apoptosis in metabolically stressed tumors. Finally, JY-1-106 was evaluated in a tumor-bearing nude mouse model, and was found to effectively repress tumor growth. Strong TUNEL signals in the tumor cells demonstrated the effectiveness of JY-1-106 in this animal model. No significant side effects were observed in mouse organs after multiple injections.
Conclusions
Taken together, these observations demonstrate that JY-1-106 is an effective pan-Bcl-2 inhibitor with very promising clinical potential.
doi:10.1186/1476-4598-12-42
PMCID: PMC3663763  PMID: 23680104
Mcl-1; Bcl-xL; Small molecule inhibitor; Cancer; BH3 mimetic
5.  Targeting zymogen activation to control the matriptase-prostasin proteolytic cascade 
Journal of medicinal chemistry  2011;54(21):7567-7578.
Membrane-associated serine protease matriptase has been implicated in human diseases, and might be a drug target. In the present study, a novel class of matriptase inhibitors targeting zymogen activation is developed by a combination of the screening of compound library using a cell-based matriptase activation assay and a computer-aided search of commercially available analogs of a selected compound. Four structurally related compounds are identified that can inhibit matriptase activation with IC50 at low μM in both intact-cell and cell-free systems, suggesting that these inhibitors target the matriptase autoactivation machinery rather than the intracellular signaling pathways. These activation inhibitors can also inhibit prostasin activation, a downstream event that occurs in lockstep with matriptase activation. In contrast, the matriptase catalytic inhibitor CVS-3983 at a concentration 300-fold higher than its Ki fails to inhibit activation of either protease. Our results suggest that inhibiting matriptase activation is an efficient way to control matriptase function.
doi:10.1021/jm200920s
PMCID: PMC3214968  PMID: 21966950
6.  The effects of the CapZ peptide (TRTK-12) binding to S100B-Ca2+ as examined by NMR and X-ray crystallography 
Journal of molecular biology  2010;396(5):1227-1243.
Structure-based drug design is underway to inhibit the S100B-p53 interaction as a strategy for treating malignant melanoma. X-ray crystallography was used here to characterize an interaction between Ca2+-S100B and a target, TRTK-12, which binds to the p53 binding site on S100B. The structures of Ca2+-S100B (1.5 Å resolution) and S100B-Ca2+-TRTK12 (2.0 Å resolution) determined here indicate that the S100B-Ca2+-TRTK12 complex is dominated by an interaction between Trp-7 of TRTK-12 and a hydrophobic binding pocket exposed on Ca2+-S100B involving residues in helices 2 & 3 and loop 2. As with a S100B-Ca2+-p53 peptide complex, TRTK-12 binding to Ca2+-S100B was found to increase the proteins Ca2+ ion binding affinity. One explanation for this effect was that peptide binding introduced a structural change that increased the number of Ca2+ ligands and/or improved Ca2+ ion coordination geometry of S100B. This possibility was ruled out when the structures of S100B-Ca2+-TRTK12 and S100B-Ca2+ were compared and calcium ion coordination by the protein was found to be nearly identical in both EF-hand calcium-binding domains (RMSD=0.19). On the other hand, B-factors for residues in EF2 of Ca2+-S100B were found to be significantly lowered with TRTK-12 bound. This result is consistent with NMR 15N relaxation studies that showed that TRTK-12 binding eliminated dynamic properties observed in Ca2+-S100B. Such a loss of protein motion may also provide an explanation for how calcium ion binding affinity is increased upon binding a target. Lastly, it follows that any small molecule inhibitor bound to Ca2+-S100B would also have to cause an increase in calcium ion binding affinity to be effective therapeutically inside a cell, so these data need to be considered in future drug-design studies involving S100B.
doi:10.1016/j.jmb.2009.12.057
PMCID: PMC2843395  PMID: 20053360
S100B; TRTK-12; calcium; X-ray crystallography; NMR
7.  In vitro screening and structural characterization of inhibitors of the S100B-p53 interaction 
S100B is highly over-expressed in many cancers, including malignant melanoma. In such cancers, S100B binds wild-type p53 in a calcium-dependent manner, sequestering it, and promoting its degradation, resulting in the loss of p53-dependent tumor suppression activities. Therefore, S100B inhibitors may be able to restore wild-type p53 levels in certain cancers and provide a useful therapeutic strategy. In this regard, an automated and sensitive fluorescence polarization competition assay (FPCA) was developed and optimized to screen rapidly for lead compounds that bind Ca2+-loaded S100B and inhibit S100B target complex formation. A screen of 2000 compounds led to the identification of 26 putative S100B low molecular weight inhibitors. The binding of these small molecules to S100B was confirmed by nuclear magnetic resonance spectroscopy, and additional structural information was provided by x-ray crystal structures of several compounds in complexes with S100B. Notably, many of the identified inhibitors function by chemically modifying Cys84 in protein. These results validate the use of high-throughput FPCA to facilitate the identification of compounds that inhibit S100B. These lead compounds will be the subject of future optimization studies with the ultimate goal of developing a drug with therapeutic activity for the treatment of malignant melanoma and/or other cancers with elevated S100B.
doi:10.2147/IJHTS.S8210
PMCID: PMC2995924  PMID: 21132089
nuclear magnetic resonance; fluorescence polarization; melanoma; chlorpromazine; thimerosal; sanguinarine
8.  The Calcium-Dependent Interaction between S100B and the Mitochondrial AAA ATPase ATAD3A and the Role of This Complex in the Cytoplasmic Processing of ATAD3A▿  
Molecular and Cellular Biology  2010;30(11):2724-2736.
S100 proteins comprise a multigene family of EF-hand calcium binding proteins that engage in multiple functions in response to cellular stress. In one case, the S100B protein has been implicated in oligodendrocyte progenitor cell (OPC) regeneration in response to demyelinating insult. In this example, we report that the mitochondrial ATAD3A protein is a major, high-affinity, and calcium-dependent S100B target protein in OPC. In OPC, ATAD3A is required for cell growth and differentiation. Molecular characterization of the S100B binding domain on ATAD3A by nuclear magnetic resonance (NMR) spectroscopy techniques defined a consensus calcium-dependent S100B binding motif. This S100B binding motif is conserved in several other S100B target proteins, including the p53 protein. Cellular studies using a truncated ATAD3A mutant that is deficient for mitochondrial import revealed that S100B prevents cytoplasmic ATAD3A mutant aggregation and restored its mitochondrial localization. With these results in mind, we propose that S100B could assist the newly synthesized ATAD3A protein, which harbors the consensus S100B binding domain for proper folding and subcellular localization. Such a function for S100B might also help to explain the rescue of nuclear translocation and activation of the temperature-sensitive p53val135 mutant by S100B at nonpermissive temperatures.
doi:10.1128/MCB.01468-09
PMCID: PMC2876520  PMID: 20351179
9.  Solution structure of S100A1 bound to the CapZ peptide (TRTK12) 
Journal of molecular biology  2009;386(5):1265-1277.
Summary
As is typical for S100-target protein interactions, a Ca2+-dependant conformational change in S100A1 is required to bind to a 12-residue peptide (TRTK12) derived from the actin capping protein, CapZ. In addition, the Ca2+-binding affinity of S100A1 is found to be tightened (> 3-fold) when TRTK12 is bound. To examine the biophysical basis for these observations, the solution NMR structure of TRTK12 in a complex with Ca2+-loaded S100A1 was determined. When bound to S100A1, TRTK12 forms an amphipathic helix (residues N6 to S12) with several favorable hydrophobic interactions observed between W7, I10, and L11 of the peptide and a well-defined hydrophobic binding pocket in S100A1 that is only present in the Ca2+-bound state. Next, the structure of S100A1-TRTK12 was compared to that of another S100A1-target complex (i.e. S100A1-RyRP12), which illustrated how the binding pocket in Ca2+-S100A1 can accommodate peptide targets with varying amino acid sequences. Similarities and differences were observed when comparing the structures of S100A1-TRTK12 and S100B-TRTK12, providing insights regarding how more than one S100 protein can interact with the same peptide target. Such comparisons, including those to other S100-target and S100-drug complexes, provide the basis for designing novel small molecule inhibitors that could be specific for blocking one or more S100-target protein interaction(s).
doi:10.1016/j.jmb.2009.01.022
PMCID: PMC2768541  PMID: 19452629
S100A1; target binding; CapZ; S100 proteins; NMR
10.  Small Molecules Bound to Unique Sites in the Target Protein Binding Cleft of Calcium-Bound S100B As Characterized by Nuclear Magnetic Resonance and X-ray Crystallography† 
Biochemistry  2009;48(26):6202-6212.
Structural studies are part of a rational drug design program aimed at inhibiting the S100B–p53 interaction and restoring wild-type p53 function in malignant melanoma. To this end, structures of three compounds (SBi132, SBi1279, and SBi523) bound to Ca2+-S100B were determined by X-ray crystallography at 2.10 Å (Rfree = 0.257), 1.98 Å (Rfree = 0.281), and 1.90 Å (Rfree = 0.228) resolution, respectively. Upon comparison, SBi132, SBi279, and SBi523 were found to bind in distinct locations and orientations within the hydrophobic target binding pocket of Ca2+-S100B with minimal structural changes observed for the protein upon complex formation with each compound. Specifically, SBi132 binds nearby residues in loop 2 (His-42, Phe-43, and Leu-44) and helix 4 (Phe-76, Met-79, Ile-80, Ala-83, Cys-84, Phe-87, and Phe-88), whereas SBi523 interacts with a separate site defined by residues within loop 2 (Ser-41, His-42, Phe-43, Leu-44, Glu-45, and Glu-46) and one residue on helix 4 (Phe-87). The SBi279 binding site on Ca2+-S100B overlaps the SBi132 and SBi523 sites and contacts residues in both loop 2 (Ser-41, His-42, Phe-43, Leu-44, and Glu-45) and helix 4 (Ile-80, Ala-83, Cys-84, Phe-87, and Phe-88). NMR data, including saturation transfer difference (STD) and 15N backbone and 13C side chain chemical shift perturbations, were consistent with the X-ray crystal structures and demonstrated the relevance of all three small molecule–S100B complexes in solution. The discovery that SBi132, SBi279, and SBi523 bind to proximal sites on Ca2+-S100B could be useful for the development of a new class of molecule(s) that interacts with one or more of these binding sites simultaneously, thereby yielding novel tight binding inhibitors specific for blocking protein–protein interactions involving S100B.
doi:10.1021/bi9005754
PMCID: PMC2804263  PMID: 19469484
11.  Divalent Metal Ion Complexes of S100B in the Absence and Presence of Pentamidine 
Journal of molecular biology  2008;382(1):56-73.
As part of an effort to inhibit S100B, structures of pentamidine (Pnt) bound to Ca2+-loaded and Zn2+,Ca2+-loaded S100B were determined by X-ray crystallography at 2.15 Å (Rfree = 0.266) and 1.85 Å (Rfree = 0.243) resolution, respectively. These data were compared to X-ray structures solved in the absence of Pnt, including Ca2+-loaded S100B and Zn2+,Ca2+-loaded S100B determined here (1.88 Å; Rfree = 0.267). In the presence and absence of Zn2+, electron density corresponding to two Pnt molecules per S100B subunit was mapped for both drug-bound structures. One Pnt binding site (site 1) was adjacent to a p53 peptide binding site on S100B (±Zn2+), and the second Pnt molecule was mapped to the dimer interface (site 2; ±Zn2+) and in a pocket near residues that define the Zn2+ binding site on S100B. In addition, a conformational change in S100B was observed upon the addition of Zn2+ to Ca2+–S100B, which changed the conformation and orientation of Pnt bound to sites 1 and 2 of Pnt–Zn2+,Ca2+–S100B when compared to Pnt–Ca2+–S100B. That Pnt can adapt to this Zn2+-dependent conformational change was unexpected and provides a new mode for S100B inhibition by this drug. These data will be useful for developing novel inhibitors of both Ca2+-and Ca2+,Zn2+-bound S100B.
doi:10.1016/j.jmb.2008.06.047
PMCID: PMC2636698  PMID: 18602402
S100B; pentamidine; X-ray crystallography; zinc; calcium
12.  Structure of Ca2+-Bound S100A4 and Its Interaction with Peptides Derived from Nonmuscle Myosin-IIA† 
Biochemistry  2008;47(18):5111-5126.
S100A4, also known as mts1, is a member of the S100 family of Ca2+-binding proteins that is directly involved in tumor invasion and metastasis via interactions with specific protein targets, including nonmuscle myosin-IIA (MIIA). Human S100A4 binds two Ca2+ ions with the typical EF-hand exhibiting an affinity that is nearly 1 order of magnitude tighter than that of the pseudo-EF-hand. To examine how Ca2+ modifies the overall organization and structure of the protein, we determined the 1.7 Å crystal structure of the human Ca2+-S100A4. Ca2+ binding induces a large reorientation of helix 3 in the typical EF-hand. This reorganization exposes a hydrophobic cleft that is comprised of residues from the hinge region, helix 3, and helix 4, which afford specific target recognition and binding. The Ca2+-dependent conformational change is required for S100A4 to bind peptide sequences derived from the C-terminal portion of the MIIA rod with submicromolar affinity. In addition, the level of binding of Ca2+ to both EF-hands increases by 1 order of magnitude in the presence of MIIA. NMR spectroscopy studies demonstrate that following titration with a MIIA peptide, the largest chemical shift perturbations and exchange broadening effects occur for residues in the hydrophobic pocket of Ca2+-S100A4. Most of these residues are not exposed in apo-S100A4 and explain the Ca2+ dependence of formation of the S100A4–MIIA complex. These studies provide the foundation for understanding S100A4 target recognition and may support the development of reagents that interfere with S100A4 function.
doi:10.1021/bi702537s
PMCID: PMC2633413  PMID: 18410126

Results 1-12 (12)