[Show abstract][Hide abstract] ABSTRACT: The EphA4 receptor tyrosine kinase regulates a variety of physiological and pathological processes during neural development and the formation of tumor blood vessels; thus, it represents a new and promising therapeutic target. We used a combination of phage peptide display and computer modeling/docking approaches and discovered a novel cyclic nonapeptide, now designated TYY. This peptide selectively inhibits the binding of the ephrinA5 ligand with EphA4 and significantly blocks angiogenesis in a 3D matrigel culture system. Molecular docking reveals that TYY recognizes the same binding pocket on EphA4 that the natural ephrin ligand binds to and that the Tyr3 and Tyr4 side chains of TYY are both critical for the TYY/EphA4 interaction. The discovery of TYY introduces a valuable probe of EphA4 function and a new lead for EphA4-targeted therapeutic development.
PLoS ONE 11/2013; 8(11):e80183. DOI:10.1371/journal.pone.0080183 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: We have developed a novel class (2-amino-4-phenyl-4H-chromene-3-carboxylate) of inhibitors of tubulin assembly by modifying HA14-1, which is a Bcl-2 inhibitor discovered by our group. Three of these compounds, mHA1, mHA6, and mHA11, showed in vitro cytotoxicities against tumor cells that were more potent and more stable than authentic HA14-1, with IC50 values in the nM range. In contrast, cytotoxic effects of these mHAs on normal cells were slight. Computational docking, colchicine-tubulin competitive binding, and tubulin polymerization studies demonstrated that these compounds bind at the colchicine binding site on tubulin and inhibit the formation of microtubules. Treatment of HL-60/Bcl-2 leukemia and CRL5908 lung cancer cells with these mHAs led to pronounced microtubule density decreases, G2/M cell cycle arrest, and apoptosis, as determined by immunofluorescence microscopy, flow cytometry, and DNA fragmentation analysis. These results support the continued development of these compounds as potential anticancer agents.
Molecular Cancer Research 05/2013; 11(8). DOI:10.1158/1541-7786.MCR-12-0177 · 4.50 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Designing potent and selective peptides and small molecules that target Eph receptor tyrosine kinases remains a challenge and new strategies are needed for developing novel and potent ligands for these receptors. In this study, we performed a structure-activity relationship study of a previously identified 12 amino acid-long peptide, SWL, by alanine scanning to identify residues important for receptor binding. To further enhance and optimize the receptor binding affinity of the SWL peptide, we applied the concept of bivalent ligand design to synthesize several SWL-derived dimeric peptides as novel ligands capable of binding simultaneously to two EphA2 receptor molecules. The dimeric peptides possess higher receptor binding affinity than the original monomeric SWL peptide, consistent with bivalent binding. The most potent dimeric peptide, a SWL dimer with a 6 carbon linker, has about 13 fold increased potency compared to SWL. Furthermore, similar to SWL, the dimeric peptide is an agonist and can promote EphA2 tyrosine phosphorylation (activation) in cultured cells.
[Show abstract][Hide abstract] ABSTRACT: EphB4 is a member of the large Eph receptor tyrosine kinase family. By interacting with its preferred ligand ephrin-B2, which is also a transmembrane protein, EphB4 plays a role in a variety of physiological and pathological processes ranging from bone remodeling to cancer malignancy. EphB4-ephrin-B2 binding occurs at sites of contact between cells. Ephrin-B2 causes EphB4 clustering and increased kinase activity to generate downstream signals that affect cell behavior. Previous work identified a high-affinity antagonistic peptide that targets EphB4, named TNYL-RAW. This peptide is 15 amino acid long, has a molecular weight of ∼1,700Da and binds to the ephrin-binding pocket of EphB4. Here we report the structure-based design and chemical synthesis of two novel small molecules of ∼600-700Da, which were designed starting from the small and functionally critical C-terminal portion of the TNYL-RAW peptide. These compounds inhibit ephrin-B2 binding to EphB4 at low micromolar concentrations. Additionally, although the ephrin-B2 ligand can interact with multiple other Eph receptors besides EphB4, the two compounds retain the high selectivity of the TNYL-RAW peptide in targeting EphB4. TNYL-RAW peptide displacement experiments using the more potent of the two compounds, compound 5, suggest a competitive mode of inhibition. These EphB4 antagonistic compounds can serve as promising templates for the further development of small molecule drugs targeting EphB4.
[Show abstract][Hide abstract] ABSTRACT: Chemokine receptor CXCR4 is one of two principal coreceptors for the entry of HIV-1 into target cells. CXCR4 is known to form homodimers. We previously demonstrated that the amino terminus of viral macrophage protein II (vMIP-II) is the major determinant for CXCR4 recognition, and that V1 peptide derived from the N-terminus of vMIP-II (1-21 residues) showed significant CXCR4 binding. Interestingly, an all-d-amino acid analogue of V1 peptide, DV1 peptide, displayed an even higher binding affinity and strong antiviral activity in inhibiting the replication of CXCR4-dependent HIV-1 strains. In this study, we synthetically linked two DV1 peptides with the formation of a disulfide bond between the two cysteine residues present in the peptide sequence to generate a dimeric molecule potentially capable of interacting with two CXCR4 receptors. DV1 dimer exhibited enhanced binding affinity and antiviral activity compared with those of DV1 monomer. Ligand binding site mapping experiments showed that DV1 dimer overlaps with HIV-1 gp120 on CXCR4 binding sites, including several transmembrane (TM) residues located close to the extracellular side and the N-terminus of CXCR4. This finding was supported by the molecular modeling of CXCR4 dimer-DV1 dimer interaction based on the crystal structure of CXCR4, which showed that DV1 dimer is capable of interacting with the CXCR4 dimeric structure by allowing the N-terminus of each DV1 monomer to reach into the binding pocket of CXCR4 monomer. The development of this bivalent ligand provides a tool for further probing the functions of CXCR4 dimerization and studying CXCR4 heterodimerization with other receptors.
[Show abstract][Hide abstract] ABSTRACT: The replication of human immunodeficiency virus type 1 (HIV-1) can be profoundly inhibited by the natural ligands of two major HIV-1 coreceptors, CXCR4 and CCR5. Stromal cell-derived factor-1α (SDF-1α) is a natural ligand of CXCR4. We have recently developed a synthetic biology approach of using synthetically and modularly modified (SMM)-chemokines to dissect various aspects of the structure-function relationship of chemokines and their receptors. Here, we used this approach to design novel SMM-SDF-1α analogues containing unnatural N-methylated residues in the amino terminus to investigate whether the polypeptide main chain amide bonds in the N-terminus of SDF-1α play a role in SDF-1α signaling via CXCR4 and/or receptor internalization. The results show that SDF-1α analogues with a modified N-methylated main chain at position 2, 3, or 5 retain significant CXCR4 binding and yet completely lose signaling activities. Furthermore, a representative N-methylated analogue has been shown to be incapable of causing CXCR4 internalization. These results suggest that the ability of SDF-1α to activate CXCR4 signaling and internalization is dependent upon the main chain amide bonds in the N-terminus of SDF-1α. This study demonstrates the feasibility and value of applying a synthetic biology approach to chemically engineer natural proteins and peptide ligands as probes of important biological functions that are not addressed by other biological techniques.
[Show abstract][Hide abstract] ABSTRACT: More than 50 new diaminoquinazoline derivatives have been synthesized and evaluated in a colon carcinoma cell growth inhibition assay using HCT116 and SW480 cells. Twenty compounds with good cell growth inhibitory activities (<4 μM) were tested as inhibitors of the β-catenin/T cell transcription factor 4 (Tcf4) signaling pathway using a HCT116 cell-based luciferase reporter assay. Results from the biological activities as well as the comparative molecular field analysis (CoMFA) of the properties of the molecules yielded a preliminary structure-activity relationship (SAR). Three potent compounds, 74, 78, and 86, showed IC(50) values <1 μM for growth inhibition of HCT116 cells and ∼1 μM for SW480 cells, as well as IC(50) values of 1.5-2.5 μM for HCT116 cells with the luciferase reporter assay.
[Show abstract][Hide abstract] ABSTRACT: Overactivation or overexpression of β-catenin in the Wnt (wingless) signaling pathway plays an important role in tumorigenesis. Interaction of β-catenin with T-cell factor (Tcf) DNA binding proteins is a key step in the activation of the proliferative genes in response to upstream signals of this Wnt/β-catenin pathway. Recently, we identified a new small molecule inhibitor, named BC21 (C(32)H(36)Cl(2)Cu(2)N(2)O(2)), which effectively inhibits the binding of β-catenin with Tcf4-derived peptide and suppresses β-catenin/Tcf4 driven reporter gene activity. This inhibitor decreases the viability of β-catenin overexpressing HCT116 colon cancer cells that harbor the β-catenin mutation, and more significantly, it inhibits the clonogenic activity of these cells. Down-regulation of c-Myc and cyclin D1 expression, the two important effectors of the Wnt/β-catenin signaling, is confirmed by treating HCT116 cells with BC21. This compound represents a new and modifiable potential anticancer candidate that targets β-catenin/Tcf-4 interaction.
[Show abstract][Hide abstract] ABSTRACT: Aberrant activation of the Wnt/β-catenin signaling pathway is associated with a wide range of human cancers. The interaction of β-catenin with T cell factor (Tcf) is a key step in activation of proliferative genes in this pathway. Interruption of this interaction would be a valuable strategy as a tumor therapy. In this study, we developed a novel fluorescein isothiocyanate (FITC)-labeled Tcf4-derived probe for identification of inhibitors of the β-catenin/Tcf4 interaction using a fluorescence polarization assay. This assay shows high potential for use in high-throughput screening for the discovery of inhibitors of the β-catenin/Tcf4 interaction.
[Show abstract][Hide abstract] ABSTRACT: Over-activation or over-expression of β-catenin in the Wnt (wingless) signaling pathway plays an
important role in tumorigenesis. Interaction of β-catenin with T-cell factor (Tcf) DNA binding
proteins is a key step in the activation of the proliferative genes in response to upstream signals
of this Wnt/β-catenin pathway. Recently, we identified a new small molecule inhibitor, Named
BC21 (C32H36Cl2Cu2N2O2), which effectively inhibits the binding of β-catenin with Tcf4 derived
peptide and suppresses β-catenin/Tcf4 driven reporter gene activity. This inhibitor decreases the
viability of β-catenin over-expressing HCT116 colon cancer cells that harbor the β-catenin
mutation and, more significantly, it inhibits the clonogenic activity of these cells. Down
regulation of c-Myc and cyclin D1 expression, the two important effectors of the Wnt/β-catenin
signaling, is confirmed by treating HCT116 cells with BC21. This compound represents a new
and modifiable potential anticancer candidate that targets β-catenin/Tcf-4 interaction.