Expression and function of the human papillomavirus (HPV) early protein 6 (E6) is necessary for viral replication and oncogenesis in cervical cancers. HPV E6 targets the tumor suppressor protein p53 ...for degradation. To achieve this, "high-risk" HPV E6 proteins bind to and modify the target specificity of the ubiquitin ligase E6AP (E6 associated protein). This E6-dependent loss of p53 enables the virus to bypass host cell defenses and facilitates virally induced activation of the cell cycle progression during viral replication. Disruption of the interaction between E6 and E6AP and stabilization of p53 should decrease viability and proliferation of HPV positive cells. A new in vitro high-throughput binding assay was developed to assay binding between HPV-16 E6 and E6AP and to identify compounds that inhibit this interaction. The compound luteolin emerged from the screen and a library of novel flavones based on its structure was synthesized and characterized using this in vitro binding assay. The compounds identified in this study disrupt the E6/E6AP interaction, increase the levels of p53 and p21(Cip1/Waf1), and decrease proliferation of HPV positive cell lines. The new class of flavonoid E6 inhibitors displays a high degree of specificity for HPV positive cells. Docking analyses suggest that these compounds bind in a hydrophobic pocket at the interface between E6 and E6AP and mimic the leucines in the conserved α-helical motif of E6AP. The activity and specificity of these compounds represent a promising new lead for development as an antiviral therapy in the treatment of HPV infection and cervical cancer.
Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to ...identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid cycle and related metabolic processes as potential antidotes. In cyanide-sensitive 7 dpf larvae, we identified several such compounds that offer significant protection against cyanide toxicity. Modulators of the pyruvate dehydrogenase complex, as well as the small molecule sodium glyoxylate, consistently protected against cyanide toxicity in 7 dpf zebrafish larvae. Together, our results indicate that the resistance of zebrafish embryos to cyanide toxicity during early development is related to an altered regulation of cellular metabolism, which we propose may be exploited as a potential target for the development of novel antidotes against cyanide poisoning.
The human papillomavirus (HPV) HPV E6 protein has emerged as a central oncoprotein in HPV-associated cancers in which sustained expression is required for tumor progression. A majority of the E6 ...protein interactions within the human proteome use an alpha-helix groove interface for binding. The UBE3A/E6AP HECT domain ubiquitin ligase binds E6 at this helix-groove interface. This enables formation of a trimeric complex with p53, resulting in destruction of this tumor suppressor. While recent x-ray crystal structures are useful, examples of small molecule probes that can modulate protein interactions at this interface are limited. To develop insights useful for potential structure-based design of ligands for HPV E6, a series of 2,6-disubstituted benzopyranones were prepared and tested as competitive antagonists of E6-E6AP helix-groove interactions. These small molecule probes were used in both binding and functional assays to evaluate recognition features of the E6 protein. Evidence for an ionic functional group interaction within the helix groove was implicated by the structure-activity among the highest affinity ligands. The molecular topographies of these protein-ligand interactions were evaluated by comparing the binding and activities of single amino acid E6 mutants with the results of molecular dynamic simulations. A group of arginine residues that form a rim-cap over the E6 helix groove offer compensatory roles in binding and recognition of the small molecule probes. The flexibility and impact on the overall helix-groove shape dictated by these residues offer new insights for structure-based targeting of HPV E6.
Breast cancer is a collection of diseases in which molecular phenotypes can act as both indicators and mediators of therapeutic strategy. Therefore, candidate therapeutics must be assessed in the ...context of multiple cell lines with known molecular phenotypes. Docosahexaenoic acid (DHA) and curcumin (CCM) are dietary compounds known to antagonize breast cancer cell proliferation. We report that these compounds in combination exert a variable antiproliferative effect across multiple breast cell lines, which is synergistic in SK-BR-3 cells and triggers cell signaling events not predicted by the activity of either compound alone.
Dose response curves for CCM and DHA were generated for five breast cell lines. Effects of the DHA+ CCM combination on cell proliferation were evaluated using varying concentrations, at a fixed ratio, of CCM and DHA based on their individual ED₅₀. Detection of synergy was performed using nonlinear regression of a sigmoid dose response model and Combination Index approaches. Cell molecular network responses were investigated through whole genome microarray analysis of transcript level changes. Gene expression results were validated by RT-PCR, and western blot analysis was performed for potential signaling mediators. Cellular curcumin uptake, with and without DHA, was analyzed via flow cytometry and HPLC.
CCM+DHA had an antiproliferative effect in SK-BR-3, MDA-MB-231, MDA-MB-361, MCF7 and MCF10AT cells. The effect was synergistic for SK-BR-3 (ER⁻ PR⁻ Her2⁺) relative to the two compounds individually. A whole genome microarray approach was used to investigate changes in gene expression for the synergistic effects of CCM+DHA in SK-BR-3 cells lines. CCM+DHA triggered transcript-level responses, in disease-relevant functional categories, that were largely non-overlapping with changes caused by CCM or DHA individually. Genes involved in cell cycle arrest, apoptosis, inhibition of metastasis, and cell adhesion were upregulated, whereas genes involved in cancer development and progression, metastasis, and cell cycle progression were downregulated. Cellular pools of PPARγ and phospho-p53 were increased by CCM+DHA relative to either compound alone. DHA enhanced cellular uptake of CCM in SK-BR-3 cells without significantly enhancing CCM uptake in other cell lines.
The combination of DHA and CCM is potentially a dietary supplemental treatment for some breast cancers, likely dependent upon molecular phenotype. DHA enhancement of cellular curcumin uptake is one potential mechanism for observed synergy in SK-BR-3 cells; however, transcriptomic data show that the antiproliferation synergy accompanies many signaling events unique to the combined presence of the two compounds.
Intact, multiply protonated proteins of particular mass and charge were selected from ionized protein mixtures and gently landed at different positions on a surface to form a microarray. An array of ...cytochrome c, lysozyme, insulin, and apomyoglobin was generated, and the deposited proteins showed electrospray ionization mass spectra that matched those of the authentic compounds. Deposited lysozyme and trypsin retained their biological activity. Multiply charged ions of protein kinase A catalytic subunit and hexokinase were also soft-landed into glycerol-based liquid surfaces. These soft-landed kinases phosphorylated LRRASLG oligopeptide and D-fructose, respectively.
The expanding roles of PCNA in functional assembly of DNA replication and repair complexes motivated investigation of the structural and dynamic properties guiding specificity of PCNA-protein ...interactions. A series of biochemical and computational analyses were combined to evaluate the PIP Box recognition features impacting complex formation. The results indicate subtle differences in topological and molecular descriptors distinguishing both affinity and stoichiometry of binding among PCNA-peptide complexes through cooperative effects. These features were validated using peptide mimics of p85α and Akt, two previously unreported PCNA binding partners. This study characterizes for the first time a reverse PIP Box interaction with PCNA. Small molecule ligand binding at the PIP Box interaction site confirmed the adaptive nature of the protein in dictating overall shape and implicates allosterism in transmitting biological effects.
Early evaluation of new drug entities for their potential to cause mitochondrial dysfunction is becoming an important task for drug development. Multi-parametric high-content screening (mp-HCS) of ...mitochondrial toxicity holds promise as a lead in-vitro strategy for drug testing and safety evaluations. In this study, we have developed a mp-HCS and multi-parametric data analysis scheme for assessing cell responses to induced mitochondrial perturbation. The mp-HCS measurements are shown to be robust enough to allow for quantitative comparison of biological systems with different metabolic pathways simulated by alteration of growth media. Substitution of medium glucose for galactose sensitized cells to drug action and revealed novel response parameters. Each compound was quantitatively characterized according to induced phenotypic changes of cell morphology and functionality measured by fluorescent biomarkers for mitochondrial activity, plasma membrane permeability, and nuclear morphology. Descriptors of drug effects were established by generation of a SCRIT (Specialized-Cell-Response-to-Induced-Toxicity) vector, consisting of normalized statistical measures of each parameter at each dose and growth condition. The dimensionality of SCRIT vectors depends on the number of parameters chosen, which in turn depends on the hypothesis being tested. Specifically, incorporation of three parameters of response into SCRIT vectors enabled clustering of 84 training compounds with known pharmacological and toxicological activities according to the degree of toxicity and mitochondrial involvement. Inclusion of 6 parameters enabled the resolution of more subtle differences between compounds within a common therapeutic class; scoring enabled a ranking of statins in direct agreement with clinical outcomes. Comparison of drug-induced changes required variations in glucose for separation of mitochondrial dysfunction from other types of cytotoxicity. These results also demonstrate that the number of drugs in a training set, the choice of parameters used in analysis, and statistical measures are fundamental for specific hypothesis testing and assessment of quantitative phenotypic differences.
Total Synthesis of Iejimalide B Chen, Qingshou; Schweitzer, Dirk; Kane, John ...
Journal of organic chemistry,
07/2011, Letnik:
76, Številka:
13
Journal Article
Recenzirano
Odprti dostop
Iejimalide B, a structurally unique 24-membered polyene macrolide having a previously underutilized mode of anticancer activity, was synthesized according to a strategy employing Julia–Kocienski ...olefinations, a palladium-catalyzed Heck reaction, a palladium-catalyzed Marshall propargylation, a Keck-type esterification, and a palladium-catalyzed macrolide-forming, intramolecular Stille coupling of a highly complex cyclization substrate. The overall synthesis is efficient (19.5% overall yield for 15 linear steps) and allows for more practical scaled-up synthesis than previously reported strategies that differed in the order of assembly of key subunits and in the method of macrocyclization. The present synthesis paves the way for efficient preparation of analogues for drug development efforts.
While the fragment‐based drug design approach continues to gain importance, gaps in the tools and methods available in the identification and accurate utilization of protein subpockets have limited ...the scope. The importance of these features of small molecule–protein recognition is highlighted with several examples. A generalized solution for the identification of subpockets and corresponding chemical fragments remains elusive, but there are numerous advancements in methods that can be used in combination to address subpockets. Finally, additional examples of approaches that consider the relative importance of small‐molecule co‐dependence of protein conformations are highlighted to emphasize an increased significance of subpockets, especially at protein interfaces.
Fragment based drug design continues to gain importance in the field of drug discovery, but gaps in the tools and methods available in the identification and accurate utilization of protein subpockets have limited the scope. This review examines current computational methods for identifying ligand binding sites and predicting binding site similarity. The importance of considering binding site microenvironments is discussed as well as the significance of inducible subpockets as ways to improve future computational screening and fragment based design approaches..
Raman Detection of Proteomic Analytes Zhang, Dongmao; Xie, Yong; Mrozek, Melissa F ...
Analytical chemistry (Washington),
11/2003, Letnik:
75, Številka:
21
Journal Article
Recenzirano
The compatibility of nonenhanced Raman spectroscopy with chromatographic and mass spectroscopic proteomic sensing is demonstrated for the first time. High-quality normal Raman spectra are derived ...from protein solutions with concentrations down to 1 μM and 1 fmol of protein nondestructively probed within the excitation laser beam. These results are obtained using a drop coating deposition Raman (DCDR) method in which the solution of interest is microdeposited (or microprinted) on a compatible substrate, followed by solvent evaporation and backscattering detection. Representative applications include the DCDR detection of insulin derived from an HPLC fraction, nondestructive DCDR followed by MALDI-TOF of lysozyme, the DCDR detection of protein spots deposited using an ink-jet microprinter, and the identification of spectral differences between glycan isomers of equal mass (such as those derived from posttranslationally modified proteins).
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