Arthur C. Watterson

Synthesis, Characterization and In Vitro Anti-invasive Activity Screening of Polyphenolic and Heterocyclic Compounds

Invasion is the hallmark of malignant tumors, and is responsible for the bad prognosis of the untreated cancer patients. The search for anti-invasive treatments led us to screen compounds of different classes for their effect in an assay for invasion. Thirty-nine new compounds synthesized in the present study along with 56 already reported compounds belonging mainly to the classes of lactones, pyrazoles, isoxazoles, coumarins, desoxybenzoins, aromatic ketones, chalcones, chromans, isoflavanones have been tested against organotypic confronting cultures of invasive human MCF-7/6 mammary carcinoma cells with embryonic chick heart fragments in vitro. Three of them (a pyrazole derivative, an isoxazolylcoumarin and a prenylated desoxybenzoin) inhibited invasion at concentrations as low as 1 microM; instead of occupying and replacing the heart tissue within 8 days, the MCF-7/6 cells grew around the heart fragments and left it intact, when treated with these compounds. At the anti-invasive concentration of 1 microM, the three compounds did not affect the growth of the MCF-7/6 cells, as shown in the sulforhodamine B assay. Aggregate formation on agar was not stimulated by any of the three anti-invasive compounds, making an effect on the E-cadherin/catenin complex improbable. This is an invasion suppressor that can be activated in MCF-7/6 cells by a number of other molecules. Our data indicate that some polyphenolic and heterocyclic compounds are anti-invasive without being cytotoxic for the cancer cells.

CHEMO-ENZYMATIC SYNTHESIS AND CHARACTERIZATION OF NOVEL FUNCTIONALIZED AMPHIPHILIC POLYMERS

ABSTRACT The condensation copolymerization of Dimethyl 5-hydroxyisophthalate (1) with Polyethylene glycols (PEGs) (2a–2d) of varying molecular weights, catalyzed by Novozyme-435 (immobilized Candida antarctica lipase B) in bulk is reported. The structures of the resulting polymers, Poly[(poly(oxyethylene)-oxy-5-hydroxyisophthaloyl] (3a–3c) were characterized by 1H (1D and 2D) and 13C-NMR spectroscopic experiments. Further, these polymers have been derivatized by attaching decanyl and 12-hydroxydodecanyl chains to the phenolic hydroxyl group. The resulting amphiphilic polymeric systems were characterized by detailed spectroscopic analysis. Light Scattering Photometry as well as Gel Per meation Chromatography were used to evaluate the particle size and molecular weights of the polymers. In principle, the method developed is flexible so that it can be used to generate a wide array of functionalized amphiphilic polymers. In the absence of biocatalytic transformation, such structural control would be extremely difficult or currently impossible to obtain.

Synthesis and solution properties of ampholytic acrylamide ionomers

Abstract The synthesis and semiconcentrated and dilute solution properties of two series of ampholytic acrylamide ionomers, poly(acrylamide MPTMA/AMPS) and poly(acrylamide METMA/MES), are reported as a function of the ionic content and added salt concentration. The viscosity dependence on shear rate was measured with a cone/plate Brookfield viscometer for sermiconcentrated solution and with a Cannon-Ubbelohde four-bulb shear dilution capillary viscometer for dilute solution at 25 ± 0.1°C. The two series of ampholytic acrylamide ionomers showed a characteristic pseudoplastic shear-thinning behavior under both conditions. The semiconcentrated solution viscosity parameters m and n in the power-law model, η = mγn-1, were determined and found to be functions of the ionic content. The viscosity at selected shear rates was found to be a complex function of the salt concentration. The intrinsic viscosity [η] was determined and compared with molecular parameters determined by light scattering under identical condi...

Design, synthesis and evaluation of novel PEGylated curcumin analogs as potent Nrf2 activators in human bronchial epithelial cells

Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a central transcription factor that regulates the anti-oxidant defense system and is considered as a modifier for several inflammatory diseases. Thus, activation of Nrf2 provides pivotal therapeutic target for developing therapy against these diseases. Herein, a chemo-enzymatic methodology is designed and developed to make PEGylated curcumins as water soluble drug candidates with enhanced aqueous solubility and bioavailability. For this, curcumin was judiciously converted to diester (1) using ethyl α-bromoacetate and potassium carbonate. The diester 1 in subsequent step was copolymerized with poly(ethylene glycol) using Candida antarctica lipase [CAL-B, Novozym 435] under solventless condition. C. antarctica selectively does trans-esterification and only catalyses reaction of the primary hydroxyls of poly(ethylene glycol). It does not affect the secondary enolic hydroxyls of curcumin, thus leaving behind the active group unaltered. A luciferase based reporter gene assay was used for primary screening for identifying a novel Nrf2 activator. Most of the PEGylated curcumin analogs strongly activate Nrf2 several folds higher than the free curcumin but copolymer 3a was identified as the most potent Nrf2 activator. Copolymer 3a induces Nrf2-driven NQO1 expression in a concentration dependent manner. Furthermore, a plausible mechanism for quantitative structure-activity relationship is also discussed.

Development of Poly(ethylene glycol) Based Amphiphilic Copolymers for Controlled Release Delivery of Carbofuran

The formation of micelles in a solvent that is selective for one of the blocks is one of the most important and useful properties of block copolymers. We had synthesized copolymers of polyethylene glycol and various dimethyl esters, which self assemble into nano micellar aggregates in aqueous media. In the present work, we have utilized these nano micelles for the encapsulation of carbofuran, [2,3–dihydro-2,2-dimethylbenzofuran-7-yl methylcarbamate], a systemic insecticide-nematicide, for the development of controlled release formulation.

Nanosphere-mediated delivery of vitamin E increases its efficacy against oxidative stress resulting from exposure to amyloid beta

Oxidative stress is an early hallmark of affected neurons in Alzheimers disease (AD). The antioxidant vitamin E provided limited neuroprotection in AD, which may have derived from its lipophilic nature and resultant inability to quench cytosolic reactive oxygen species (ROS), including those generated from antecedent membrane oxidative damage. We examined herein whether or not encapsulation into polyethylene glycol (PEG)-based nanospheres, which can enter the cytosol, improved the efficacy of vitamin E against amyloid-beta(Abeta)-induced ROS. Unexcapsulated vitamin E prevented Abeta-induced ROS in cultured SH-SY-5Y human neuroblastoma cells only if present prior to, or applied simultaneously with, Abeta treatment. By contrast, encapsulated vitamin E was equally effective if administered 1 hr after Abeta exposure. These findings suggest suggests that nanosphere-mediated delivery methods may be a useful adjunct for antioxidant therapy in AD.

Establishment of the enzymatic protein acetylation independent of acetyl CoA: recombinant glutathione S-transferase 3-3 is acetylated by a novel membrane-bound transacetylase using 7,8-diacetoxy-4-methyl coumarin as the acetyl donor

The current knowledge on biological protein acetylation is confined to acetyl CoA‐dependent acetylation of protein catalyzed by specific acetyl transferases and the non‐enzymatic acetylation of protein by acetylated xenobiotics such as aspirin. We have discovered a membrane‐bound enzyme catalyzing the transfer of acetyl groups from the acetyl donor 7,8‐diacetoxy‐4‐methyl coumarin (DAMC) to glutathione S‐transferase 3‐3 (GST3‐3), termed DAMC:protein transacetylase (TAase). The purified enzyme was incubated with recombinant GST3‐3 subunit and DAMC, the modified protein was isolated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) in gel digested with trypsin and the tryptic digest was analyzed by mass spectrometry. The N‐terminus and six lysines, Lys‐51, ‐82, ‐124, ‐181, ‐191 and ‐210, were found to be acetylated. The acetylation of GST3‐3 described above was not observed in the absence of either DAMC or TAase. These results clearly establish the phenomenon of protein acetylation independent of acetyl CoA catalyzed by a hitherto unknown enzyme (TAase) utilizing a certain xenobiotic acetate (DAMC) as the active acetyl donor.

Polymerization of Ion-Pair Comonomers of Related Structures

Abstract Two ion-pair comonomers of 2-methacryloyloxyethyltrimethyl-ammonium 2-methacryloyloxyethanesulfonate (METMAMES) and 3-acrylamido-3-methylbutyltrimethylammonium 2-acrylamido-2-methylpropanesulfonate have been synthesized. The spontaneous and radical homocopolymerizations of these monomer pairs, wherein no nonpolymerizable counterions are present, have also been studied. Moreover, the intrinsic viscosity of the homocopolymer derived from METMA MES was found to increase with increasing KCl concentration in aqueous solutions.

Synthesis of novel poly(ethylene glycol) based amphiphilic polymers

Abstract The synthesis of new amphiphilic polyesters based on poly(ethylene glycol) (PEGs) and studies on their solution properties are reported. Two novel monomers, dimethyl 5-n-butoxy isophthalate (2) and dimethyl 5-n-octoxy isophthalate (3) were synthesized. Three series of novel amphiphilic polyesters, i.e. poly(ethyleneoxy isophthalate)s (10–15), poly(ethyleneoxy n-butoxy isophthalate)s (16–21) and poly(ethyleneoxy n-octoxy isophthalate)s (22–27) have been synthesized from PEGs of different sizes and dimethyl isophthalates 1–3 via the transesterification–polycondensation using dibutyltin diacetate as a catalyst. The structures of the polyesters were established from a detailed analysis of their spectra, i.e. FTIR, 1H-NMR (one- and two-dimensional) and 13C-NMR. By adjusting the ratio of hydrophobic (diesters) and hydrophilic (PEGs) segments in polymers, their main chain structures and solution properties could be changed. The viscosity molecular weights (Mv) of polymers, obtained from Mark–Houwink–Sakurada relationship having poly(ethylene terephthalate) as a model, were in the range of 4500–32,000 g/mol. Intrinsic viscosities were studied based on polymer backbone length (PEGs effect) and pendant group (diesters effect) and these were found to be dependent on molecular weights of the PEGs used.

Novel function of calreticulin: Characterization of calreticulin as a transacetylase-mediating protein acetylator independent of acetyl CoA using polyphenolic acetates

Our earlier investigations culminated in the discovery of a unique membrane-bound enzyme in mammalian cells catalyzing the transfer of acetyl group from polyphenolic acetates (PAs) to certain functional proteins, resulting in the modulation of their activities. This enzyme was termed acetoxy drug:protein transacetylase (TAase) since it acted upon several classes of PAs. TAase was purified from rat liver microsomes to homogeneity and exhibited the molecular weight of 55 KDa. TAase-catalyzed protein acetylation by PAs was evidenced by the demonstration of immunoreactivity of the acetylated target protein such as nitric oxide synthase (NOS) with anti-acetyl lysine. The possible acetylation of human platelet NOS by PA as described above resulted in the enhancement of intracellular levels of nitric oxide (NO). PAs unlike the parent polyphenols were found to exhibit NO-related physiological effects. The N-terminal sequence was found to show 100 % homology with N-terminal sequence of mature calreticulin (CRT). The identity of TAase with CRT, an endoplasmic reticulum (ER) protein, was evidenced by the demonstration of the properties of CRT such as immunoreactivity with anti-calreticulin, binding to Ca2+ ions and being substrate for phosphorylation by protein kinase c (PKC), which are the hallmark characteristics of CRT. These observations for the first time convincingly attribute the transacetylase function to CRT, which possibly plays an important role in protein modification by way of carrying out acetylation of various enzymes through a biochemical mechanism independent of acetyl CoA.