Varatharasa Thiviyanathan

Curcumin–glutathione interactions and the role of human glutathione S-transferase P1-1

Curcumin (diferuloylmethane), a yellow pigment of turmeric with antioxidant properties has been shown to be a cancer preventative in animal studies. It contains two electrophilic alpha, beta-unsaturated carbonyl groups, which can react with nucleophilic compounds such as glutathione (GSH), but formation of the GSH-curcumin conjugates has not previously been demonstrated. In the present studies, we investigated the reactions of curcumin with GSH and the effect of recombinant human glutathione S-transferase(GST)P1-1 on reaction kinetics. Glutathionylated products of curcumin identified by FAB-MS and MALDI-MS included mono- and di-glutathionyl-adducts of curcumin as well as cyclic rearrangement products of GSH adducts of feruloylmethylketone (FMK) and feruloylaldehyde (FAL). The presence of GSTP1-1 significantly accelerated the initial rate of GSH-mediated consumption of curcumin in 10 mM potassium phosphate, pH 7.0, and 1 mM GSH. GSTP1-1 kinetics determined using HPLC indicated substrate inhibition (apparent K(m) for curcumin of 25+/-11 microM, and apparent K(i) for curcumin of 8+/-3 microM). GSTP1-1 was also shown to catalyze the reverse reaction leading to the formation of curcumin from GSH adducts of FMK and FAL.

Combinatorial Selection of DNA Thioaptamers Targeted to the HA Binding Domain of Human CD44

CD44, the primary receptor for hyaluronic acid, plays an important role in tumor growth and metastasis. CD44-hyaluronic acid interactions can be exploited for targeted delivery of anticancer agents specifically to cancer cells. Although various splicing variants of CD44 are expressed on the plasma membrane of cancer cells, the hyaluronic acid binding domain (HABD) is highly conserved among the CD44 splicing variants. Using a novel two-step process, we have identified monothiophosphate-modified aptamers (thioaptamers) that specifically bind to the CD44s HABD with high affinities. Binding affinities of the selected thioaptamers for the HABD were in the range of 180-295 nM, an affinity significantly higher than that of hyaluronic acid (K(d) above the micromolar range). The selected thioaptamers bound to CD44 positive human ovarian cancer cell lines (SKOV3, IGROV, and A2780) but failed to bind the CD44 negative NIH3T3 cell line. Our results indicated that thio substitution at specific positions of the DNA phosphate backbone results in specific and high-affinity binding of thioaptamers to CD44. The selected thioaptamers will be of great interest for further development as a targeting or imaging agent for the delivery of therapeutic payloads for cancer tissues.

X-aptamers: A bead-based selection method for random incorporation of druglike moieties onto next-generation aptamers for enhanced binding

By combining pseudorandom bead-based aptamer libraries with conjugation chemistry, we have created next-generation aptamers, X-aptamers (XAs). Several X-ligands can be added in a directed or random fashion to the aptamers to further enhance their binding affinities for the target proteins. Here we describe the addition of a drug (N-acetyl-2,3-dehydro-2-deoxyneuraminic acid), demonstrated to bind to CD44-HABD, to a complete monothioate backbone-substituted aptamer to increase its binding affinity for the target protein by up to 23-fold, while increasing the drugs level of binding 1-million fold.

Aptamers and the next generation of diagnostic reagents.

Antibodies have been extensively used as capture and detection reagents in diagnostic applications of proteomics‐based technologies. Proteomic assays need high sensitivity and specificity, a wide dynamic range for detection, and accurate, reproducible quantification with small confidence values. However, several inherent limitations of monoclonal antibodies in meeting the emerging challenges of proteomics led to the development of a new class of oligonucleotide‐based reagents. Natural and derivatized nucleic acid aptamers are emerging as promising alternatives to monoclonal antibodies. Aptamers can be effectively used to simultaneously detect thousands of proteins in multiplex discovery platforms, where antibodies often fail due to cross‐reactivity problems. Through chemical modification, vast range of additional functional groups can be added at any desired position in the oligonucleotide sequence, therefore the best features of small molecule drugs, proteins, and antibodies can be brought together into aptamers, making aptamers the most versatile reagent in proteomics. In this review, we discuss the recent developments in aptamer technology, including new selection methods and the aptamers’ application in proteomics.

Bone marrow endothelium-targeted therapeutics for metastatic breast cancer

Effective treatment of cancer metastasis to the bone relies on bone marrow drug accumulation. The surface proteins in the bone marrow vascular endothelium provide docking sites for targeted drug delivery. We have developed a thioaptamer that specifically binds to E-selectin that is overexpressed in the vasculature of tumor and inflammatory tissues. In this study, we tested targeted delivery of therapeutic siRNA loaded in the E-selectin thioaptamer-conjugated multistage vector (ESTA-MSV) drug carrier to bone marrow for the treatment of breast cancer bone metastasis. We evaluated tumor type- and tumor growth stage-dependent targeting in mice bearing metastatic breast cancer in the bone, and carried out studies to identify factors that determine targeting efficiency. In a subsequent study, we delivered siRNA to knock down expression of the human STAT3 gene in murine xenograft models of human MDA-MB-231 breast tumor, and assessed therapeutic efficacy. Our studies revealed that the CD31(+)E-selectin(+) population accounted for 20.8%, 26.4% and 29.9% of total endothelial cells respectively inside the femur of mice bearing early, middle and late stage metastatic MDA-MB-231 tumors. In comparison, the double positive cells remained at a basal level in mice with early stage MCF-7 tumors, and jumped to 23.9% and 28.2% when tumor growth progressed to middle and late stages. Accumulation of ESTA-MSV inside the bone marrow correlated with the E-selectin expression pattern. There was up to 5-fold enrichment of the targeted MSV in the bone marrow of mice bearing early or late stage MDA-MB-231 tumors and of mice with late stage, but not early stage, MCF-7 tumors. Targeted delivery of STAT3 siRNA in ESTA-MSV resulted in knockdown of STAT3 expression in 48.7% of cancer cells inside the bone marrow. Weekly systemic administration of ESTA-MSV/STAT3 siRNA significantly extended survival of mice with MDA-MB-231 bone metastasis. In conclusion, targeting the overexpressed E-selectin provides an effective approach for tissue-specific drug delivery to the bone marrow. Tumor growth in the bone can be effectively inhibited by blockage of the STAT3 signaling.

Hybrid-hybrid matrix structural refinement of a DNA three-way junction from 3D NOESY-NOESY

Homonuclear 3D NOESY-NOESY has shown great promise for the structural refinement of large biomolecules. A computationally efficient hybrid-hybrid relaxation matrix refinement methodology, using 3D NOESY-NOESY data, was used to refine the structure of a DNA three-way junction having two unpaired bases at the branch point of the junction. The NMR data and the relaxation matrix refinement confirm that the DNA three-way junction exists in a folded conformation with two of the helical stems stacked upon each other. The third unstacked stem extends away from the junction, forming an acute angle (∼60° ) with the stacked stems. The two unpaired bases are stacked upon each other and are exposed to the solvent. Helical parameters for the bases in all three strands show slight deviations from typical values expected for right-handed B-form DNA. Inter-nucleotide imino-imino NOEs between the bases at the branch point of the junction show that the junction region is well defined. The helical stems show mobility (± 20° ) indicating dynamic processes around the junction region. The unstacked helical stem adjacent to the unpaired bases shows greater mobility compared to the other two stems. The results from this study indicate that the 3D hybrid-hybrid matrix MORASS refinement methodology, by combining the spectral dispersion of 3D NOESY-NOESY and the computational efficiency of 2D refinement programs, provides an accurate and robust means for structure determination of large biomolecules. Our results also indicate that the 3D MORASS method gives higher quality structures compared to the 2D complete relaxation matrix refinement method.

RNA nanoparticles harboring annexin A2 aptamer can target ovarian cancer for tumor-specific doxorubicin delivery

A novel modified nucleic acid nanoparticle harboring an annexin A2 aptamer for ovarian cancer cell targeting and a GC rich sequence for doxorubicin loading is designed and constructed. The system utilizes a highly stable three-way junction (3WJ) motif from phi29 packaging RNA as a core structure. A phosphorothioate-modified DNA aptamer targeting annexin A2, Endo28, was conjugated to one arm of the 3WJ. The pRNA-3WJ motif retains correct folding of attached aptamer, keeping its functions intact. It is of significant utility for aptamer-mediated targeted delivery. The DNA/RNA hybrid nanoparticles remained intact after systemic injection in mice and strongly bound to tumors with little accumulation in healthy organs 6 h post-injection. The Endo28-3WJ-Sph1/Dox intercalates selectively enhanced toxicity to annexin A2 positive ovarian cancer cells in vitro. The constructed RNA/DNA hybrid nanoparticles can potentially enhance the therapeutic efficiency of doxorubicin at low doses for ovarian cancer treatment through annexin A2 targeted drug delivery.

Combinatorial selection and delivery of thioaptamers.

Oligonucleotide-based agents are emerging as potential therapeutic agents that can be attractive alternatives for the small-molecule chemical drugs. Monothiophosphate-backbone-modified DNA aptamers (thioaptamers) that specifically and tightly bind to the RNase H domain of the HIV RT (reverse transcriptase) have been isolated from nucleic acid libraries using combinatorial selection methods. The selected thioaptamer inhibited RNase H activity of the HIV RT in in vitro studies. In cell cultures, the transfected thioaptamer markedly reduced HIV production in a dose-dependent manner. Gel electrophoretic mobility-shift assays and NMR spectroscopy showed that the selected thioaptamer binds to the isolated RNase H domain, but did not bind to a structurally similar RNase H from Escherichia coli. In cell cultures, the transfected thioaptamer showed a dose-dependent inhibition of HIV replication, with a maximal inhibition of 83%. Using various liposome-delivery agents, the DNA thioaptamer was transfected into HIV-infected astrocytoma adherent cells with greater than 70% efficiency.

Potential double-flipping mechanism by E. coli MutY

To understand the structural basis of the recognition and removal of specific mismatched bases in double-stranded DNAs by the DNA repair glycosylase MutY, a series of structural and functional analyses have been conducted. MutY is a 39-kDa enzyme from Escherichia coli, which to date has been refractory to structural determination in its native, intact conformation. However, following limited proteolytic digestion, it was revealed that the MutY protein is composed of two modules, a 26-kDa domain that retains essential catalytic function (designated p26MutY) and a 13-kDa domain that is implicated in substrate specificity and catalytic efficiency. Several structures of the 26-kDa domain have been solved by X-ray crystallographic methods to a resolution of up to 1.2 A. The structure of a catalytically incompetent mutant of p26MutY complexed with an adenine in the substrate-binding pocket allowed us to propose a catalytic mechanism for MutY. Since reporting the structure of p26MutY, significant progress has been made in solving the solution structure of the noncatalytic C-terminal 13-kDa domain of MutY by NMR spectroscopy. The topology and secondary structure of this domain are very similar to that of MutT, a pyrophosphohydrolase. Molecular modeling techniques employed to integrate the two domains of MutY with DNA suggest that MutY can wrap around the DNA and initiate catalysis by potentially flipping adenine and 8-oxoguanine out of the DNA helix.

E-selectin Targeting PEGylated-thioaptamer Prevents Breast Cancer Metastases

E-selectin is an adhesion molecule expressed on the luminal surface of inflamed blood vessels that mediates hematogenous metastasis by assisting shear-resistant adhesion of circulating tumor cells to the vessel surface under dynamic blood flow. Previously, we developed an E-selectin antagonistic thioaptamer (ESTA) for the prevention of hematogenous metastasis through the blockade of CD44high breast cancer cells (BCa) adhesion to E-selectin-expressing premetastatic endothelial niche. The current study focuses on developing a PEGylated E-selectin targeting thioaptamer with improved pharmaceutical properties. A serial deletion of stem-loops reveled that loop-1 and -2 (ESTA7) are the minimally effective backbone structure necessary to obtain inhibition of the E-selectin/CD44 interaction and shear resistant adhesion of CD44high BCa to E-selectin-expressing human endothelial cells (HMVECs) at a level equal to ESTA. Chemical conjugation of methoxy-polyethylene-glycol (PEG) at the sizes of 5 and 10 kDa did not interfere with ESTA7-mediated shear-resistant adhesion. However, in vivo study demonstrated that only 10 kDa PEG-conjugated ESTA7 (ESTA7-p10) retains the activity to inhibit metastases at a level equal to parental ESTA. Additionally, a single intravenous injection of ESTA7-p10 inhibited the development of lung, brain, and bone metastases of MDA-MB-231, through the blockade of E-selectin. Moreover, PEGylation led to an extension of elimination half-life and increase of AUC, resulting in superior inhibition of metastasis development compared to parental ESTA with a longer interval between dosing in a spontaneous metastasis model. Lastly, repeated intravenous administration of ESTA7-p10 was tolerated in mice, highlighting the potential prophylactic application of ESTA7-p10 for metastasis prevention.E-selectin is an adhesion molecule expressed on the luminal surface of inflamed blood vessels that mediates hematogenous metastasis by assisting shear-resistant adhesion of circulating tumor cells to the vessel surface under dynamic blood flow. Previously, we developed an E-selectin antagonistic thioaptamer (ESTA) for the prevention of hematogenous metastasis through the blockade of CD44high breast cancer cells (BCa) adhesion to E-selectin-expressing premetastatic endothelial niche. The current study focuses on developing a PEGylated E-selectin targeting thioaptamer with improved pharmaceutical properties. A serial deletion of stem-loops reveled that loop-1 and -2 (ESTA7) are the minimally effective backbone structure necessary to obtain inhibition of the E-selectin/CD44 interaction and shear resistant adhesion of CD44high BCa to E-selectin-expressing human endothelial cells (HMVECs) at a level equal to ESTA. Chemical conjugation of methoxy-polyethylene-glycol (PEG) at the sizes of 5 and 10 kDa did not interfere with ESTA7-mediated shear-resistant adhesion. However, in vivo study demonstrated that only 10 kDa PEG-conjugated ESTA7 (ESTA7-p10) retains the activity to inhibit metastases at a level equal to parental ESTA. Additionally, a single intravenous injection of ESTA7-p10 inhibited the development of lung, brain, and bone metastases of MDA-MB-231, through the blockade of E-selectin. Moreover, PEGylation led to an extension of elimination half-life and increase of AUC, resulting in superior inhibition of metastasis development compared to parental ESTA with a longer interval between dosing in a spontaneous metastasis model. Lastly, repeated intravenous administration of ESTA7-p10 was tolerated in mice, highlighting the potential prophylactic application of ESTA7-p10 for metastasis prevention.