Purification of recombinant catalase-peroxidase HPI from E. coli and its application in enzymatic polymerization reactions.

In this paper, a recombinant catalase-peroxidase HPI from Escherichia coli was prepared, purified, and used in enzymatic polymerization reactions for the production of several oligomeric products. We tested the enzyme on four different substrates, chosen as representative of phenols and anilines: phenol, 3-methoxyphenol, catechol, and aniline. The polymerization reactions were followed by SEC-HPLC analysis, and except for aniline, all the other substrates were completely converted into one or more polymerization products. Results showed that reactions performed with phenol and 3-methoxyphenol allowed the isolation of some oligomers of different weight: a 27-monomeric unit oligomer and a 23-U oligomer are the heaviest ones. Experiments performed with catechol showed the formation of oligomers of 7 U in the reaction with HPI. HPI polymerization reactions performed with aniline allowed the identification of two different oligomers, one of 4 U and one of 10 U. All the substrates have been also used in reactions catalyzed by HRP in the same reaction conditions. Several products were common to the two enzymes. This work suggests the use of HPI as an alternative enzyme in peroxidatic reactions for the production of different oligomers from phenols and other compounds.

Highly potent and selective 5-lipoxygenase inhibition by new, simple heteroaryl-substituted catechols for treatment of inflammation.

5-Lipoxygenase (LO) catalyzes the first steps in the formation of pro-inflammatory leukotrienes (LT) that are pivotal lipid mediators contributing to allergic reactions and inflammatory disorders. Based on its key role in LT biosynthesis, 5-LO is an attractive drug target, demanding for effective and selective inhibitors with efficacy in vivo, which however, are still rare. Encouraged by the recent identification of the catechol 4-(3,4-dihydroxyphenyl)dibenzofuran 1 as 5-LO inhibitor, simple structural modifications were made to yield even more effective and selective catechol derivatives. Within this new series, the two most potent compounds 3,4-dihydroxy-3'-phenoxybiphenyl (6b) and 2-(3,4-dihydroxyphenyl)benzo[b]thiophene (6d) potently inhibited human 5-LO in cell-free (IC506b and 6d = 20 nM) and cell-based assays (IC506b = 70 nM, 6d = 60 nM). Inhibition of 5-LO was reversible, unaffected by exogenously added substrate arachidonic acid, and not primarily mediated via radical scavenging and antioxidant activities. Functional 5-LO mutants expressed in HEK293 cells were still prone to inhibition by 6b and 6d, and docking simulations revealed distinct binding of the catechol moiety to 5-LO at an allosteric site. Analysis of 5-LO nuclear membrane translocation and intracellular Ca2+ mobilization revealed that these 5-LO-activating events are hardly affected by the catechols. Importantly, the high inhibitory potency of 6b and 6d was confirmed in human blood and in a murine zymosan-induced peritonitis model in vivo. Our results enclose these novel catechol derivatives as highly potent, novel type inhibitors of 5-LO with high selectivity and with marked effectiveness under pathophysiological conditions.

Micellar nanoreactors for hematin catalyzed synthesis of electrically conducting polypyrrole.

Enzymatic synthesis of doped polypyrrole (PPy) complexes using oxidoreductases (specifically peroxidases) is very well established "green" methods for producing conducting polypyrrole. The importance of this approach is realized by the numerous potential opportunities of using PPy in biological applications. However, due to very high costs and low acid stability of these enzymes, there is need for more robust alternate biomimetic catalysts. Hematin, a hydroxyferriprotoporphyrin, has a similar iron catalytic active center like the peroxidases and has previously shown to catalyze polymerization of phenol monomers at pH 12. The insolubility of hematin due to extensive self-aggregation at low pH conditions has prevented its use in the synthesis of conjugated polymers. In this study, we have demonstrated the use of a micellar environment with sodium dodecylbenzenesulfonate (DBSA) for biomimetic synthesis of PPy. The micellar environment helps solubilize hematin, generating nanometer size reactors for the polymerization of pyrrole. The resulting PPy is characterized using UV-visible, Fourier transform infrared, and X-ray photoelectron spectroscopy and reveals the formation of an ordered PPy/DBSA complex with conductivities approaching 0.1 S/cm.

Design and synthesis of functionalized 4-aryl-Catechol derivatives as new antiinflammtory agents with in vivo efficacy.

Oxidative stress and inflammation are two conditions that coexist in many multifactorial diseases and the discovery of antioxidants is an attractive approach that can simultaneously tackle two or more therapeutic targets of the arachidonic acid cascade. We report that the simple structural variations on the 4-aryl-benzene-1,2-diol side-arm of the scaffold significantly influence the selectivity against 5-LOX vs 12- and 15-LOX. Derivatives 4 a-l were evaluated for their antioxidant activity, using the DPPH, and ferric ion reducing antioxidant power (FRAP) methods. Docking simulations proposed concrete binding of the catechol series to 5-LO. Selected active compound 4-(3,4-dihydroxyphenyl)dibenzofuran (4l) was also tested in different in vivo mouse models of inflammation. 4l (0.1 mg/kg; i.p.) impaired (I) bronchoconstriction in ovalbumin-sensitized mice challenged with acetylcholine, (II) exudate formation in carrageenan-induced paw edema, and (III) zymosan-induced leukocyte infiltration in air pouches. These results pave the way for investigating the therapeutic potential of 4-aryl-benzene-1,2-diol, as novel multitarget therapeutic drugs, able to regulate the complex inflammatory cascade mechanisms.

Antioxidant Activity of Synthetic Polymers of Phenolic Compounds.

In recent years, developing potent antioxidants has been a very active area of research. In this context, phenolic compounds have been evaluated for their antioxidant activity. However, the use of phenolic compounds has also been limited by poor antioxidant activity in several in vivo studies. Polymeric phenols have received much attention owing to their potent antioxidant properties and increased stability in aqueous systems. To be truly effective in biological applications, it is important that these polymers be synthesized using benign methods. In this context, enzyme catalyzed synthesis of polymeric phenols has been explored as an environmentally friendly and safer approach. This review summarizes work in enzymatic syntheses of polymers of phenols. Several assays have been developed to determine the antioxidant potency of these polymeric phenols. These assays are discussed in detail along with structure-property relationships. A deeper understanding of factors affecting antioxidant activity would provide an opportunity for the design of versatile, high performing polymers with enhanced antioxidant activity.

Formulation and Characterization of Solid Lipid Nanoparticles Loading RF22-c, a Potent and Selective 5-LO Inhibitor, in a Monocrotaline-Induced Model of Pulmonary Hypertension.

Pulmonary arterial hypertension (PAH) is a rare but fatal disease characterized by persistent elevated blood pressure in the pulmonary circulation, due to increased resistance to blood flow, through the lungs. Advances in the understanding of the pathobiology of PAH clarify the role of leukotrienes (LTs) that appear to be an exciting new target for disease intervention. Over the years, our group has long investigated this field, detecting the 1,2-benzoquinone RF-22c as the most powerful and selective competitive inhibitor of the enzyme 5-lipoxygenase (5-LO). With the aim to improve the bioavailability of RF-22c and to confirm the role of 5-LO as therapeutic strategy for PAH treatment, we developed a solid lipid nanoparticle (SLN) loaded with drug. Therefore, in monocrotaline (MCT) rat model of PAH, the role of 5-LO has been investigated through the formulation of RF-22c-SLN. The rats were randomly grouped into control group, MCT group, and MCT + RF22-c group. After 21 days, all the animals were sacrificed to perform functional and histological evaluations. RF22-c-SLN treatment was able to significantly reduce the mean pulmonary arterial pressure (mPAP) and precapillary resistance (R-pre) compared to the MCT group. The MCT induced rise in medial wall thickness of pulmonary arterioles, and the cardiomyocytes width were significantly attenuated by RF22-c-SLN formulation upon treatment. The results showed that the selective inhibition of 5-LO improved hemodynamic parameters as well as vascular and cardiac remodeling by preventing induced pulmonary hypertension. The improved sustained release properties and targeting abilities achieved with the innovative nanotechnological approach may be therapeutically beneficial for PAH patients as a consequence of the increase of pharmacological effects and of the possible reduction and/or optimization of the drug frequency of administration.

Protective effect of piceatannol and bioactive stilbene derivatives against hypoxia-induced toxicity in H9c2 cardiomyocytes and structural elucidation as 5-LOX inhibitors.

Stilbenes with well-known antioxidant and antiradical properties are beneficial in different pathologies including cardiovascular diseases. The present research was performed to investigate the potential protective effect of resveratrol (1) and piceatannol (2), against hypoxia-induced oxidative stress in the H9c2 cardiomyoblast cell line, and the underlying mechanisms. Compounds 1 and 2 significantly inhibited the release of peroxynitrite and thiobarbituric acid levels at na no- or submicromolar concentrations, and this effect was more evident in piceatannol-treated cells, that significantly increased MnSOD protein level in a concentration dependent manner. Furthermore, since piceatannol, which is far less abundant in natural sources, displayed a higher bioactivity than the parent compound, we hereby report on a very fast synthesis and detailed structure-based design of a focused stilbene library. Finally, taking into account that hypoxia-induced ROS accumulation also increases expression and activity of 5-lipoxygenase (5-LOX) with production of leukotrienes, we have disclosed structural key factors crucial for 5-LOX activity. Among the synthesized analogues ( 3-7), compound 7 was the most effective in improving cardiomyocytes viability and in 5-LOX inhibition. In conclusion, modeling and experimental studies provided the basis for further optimization of stilbene analogues as multi-target inhibitors of the inflammatory and oxidative pathway.

Biocatalytically oligomerized epicatechin with potent and specific anti-proliferative activity for human breast cancer cells.

Catechins, naturally occurring flavonoids derived from wine and green tea, are known to exhibit multiple health benefits. Epigallocatechin gallate (EGCG) is one of the most widely investigated catechins, but its efficacy in cancer therapy is still inconsistent and limited. The poor stability of EGCG has contributed to the disparity in the reported anti-cancer activity and other beneficial properties. Here we report an innovative enzymatic strategy for the oligomerization of catechins (specifically epicatechin) that yields stable, water-soluble oligomerized epicatechins with enhanced and highly specific anti-proliferative activity for human breast cancer cells. This one-pot oxidative oligomerization is carried out in ambient conditions using Horseradish Peroxidase (HRP) as a catalyst yielding water-soluble oligo(epicatechins). The oligomerized epicatechins obtained exhibit excellent growth inhibitory effects against human breast cancer cells with greater specificity towards growth-inhibiting cancer cells as opposed to normal cells, achieving a high therapeutic differential. Our studies indicate that water-soluble oligomeric epicatechins surpass EGCG in stability, selectivity and efficacy at lower doses.

Recent advances in the search for novel 5-lipoxygenase inhibitors for the treatment of asthma.

The products of 5-lipoxygenase are synthesized and released in the airway when an asthmatic reaction occurs. 5-lipoxygenase via arachidonic acid metabolism produces leukotrienes that mediate bronchoconstriction and inflammatory modifications essential in the pathophysiology of asthma. Until to now, only one approved 5-LO inhibitor, zileuton, can be found as a potential therapy for asthma. With the increasing number of indications for anti-leukotriene (anti-LT) drugs, the development of 5-LO inhibitor agents becomes increasingly important. The present MiniReview reports an update on 5-LO inhibitors currently under clinical investigation. In addition, the latest advances focused on the development of new 5-lipoxygenase inhibitors as asthma anti-inflammatory agents are also discussed.

Structural insight into the optimization of ethyl 5-hydroxybenzo[g]indol-3-carboxylates and their bioisosteric analogues as 5-LO/m-PGES-1 dual inhibitors able to suppress inflammation.

The release of pro-inflammatory mediators, such as prostaglandines (PGs) and leukotrienes (LTs), arising from the arachidonic acid (AA) cascade, play a crucial role in initiating, maintaining, and regulating inflammatory processes. New dual inhibitors of 5-lipoxygenase (5-LO) and microsomal prostaglandin E2 synthase-1 (mPGES-1), that block, at the same time, the formation of PGE2 and LTs, are currently emerged as a highly interesting drug candidates for better pharmacotherapie of inflammation-related disorders. Following our previous studies, we here performed a detailed structure-based design of benzo[g]indol-3-carboxylate derivatives, disclosing several new key factors that affect both enzyme activity. Ethyl 2-(3,4-dichlorobenzyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (4b, RAF-01) and ethyl 2-(3,4-dichlorophenyl)-5-hydroxy-1H-benzo[g]indole-3-carboxylate (7h, RAF-02) emerged as the most active compounds of the series. Additionally, together with selected structure based analogues, both derivatives displayed significant in vivo anti-inflammatory properties. In conclusion, modeling and experimental studies lead to the discovery of new candidate compounds prone to further developments as multi-target inhibitors of the inflammatory pathway.

Novel series of benzoquinones with high potency against 5-lipoxygenase in human polymorphonuclear leukocytes.

5-Lipoxygenase (5-LO) is a potential target for pharmacological intervention with various inflammatory and allergic diseases. Starting from the natural dual 5-LO/microsomal prostaglandin E2 synthase (mPGES)-1 inhibitor embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone, 2) that suppresses 5-LO activity in human primary leukocytes with IC50 = 0.8-2 µM, we synthesized 48 systematically modified derivatives of 2. We modified the 1,4-quinone to 1,2-quinone, mono- or bimethylated the hydroxyl groups, and varied the C11-n-alkyl residue (C4- to C16-n-alkyl or prenyl) of 2. Biological evaluation yields potent analogues being superior over 2 and obvious structure-activity relationships (SAR) for inhibition of 5-LO. Interestingly, conversion to 1,2-benzoquinone and bimethylation of the hydroxyl moieties strongly improves 5-LO inhibition in polymorphonuclear leukocytes versus 2 up to 60-fold, exemplified by the C12-n-alkyl derivative 22c (4,5-dimethoxy-3-dodecyl-1,2-benzoquinone) with IC50 = 29 nM. Regarding inhibition of mPGES-1, none of the novel benzoquinones could outperform the parental compound 2 (IC50 = 0.21 µM), and only modest suppressive effects on 12- and 15-LOs were evident. Together, our detailed SAR study reveals 22c as highly potent 5-LO-selective lead compound in intact cells that warrants further preclinical evaluation as anti-inflammatory agent.

"Green"-enzymatic synthesis of pegylated phenolic macromer and polymer.

Environmentally benign synthesis of novel pegylated polyphenolics, by combining the extraordinary selectivities of a lipase and an oxidase to develop polymeric electrolytes for applications in dye sensitised solar cells.

Further studies on ethyl 5-hydroxy-indole-3-carboxylate scaffold: design, synthesis and evaluation of 2-phenylthiomethyl-indole derivatives as efficient inhibitors of human 5-lipoxygenase.

5-Lipoxygenase (5-LO), an enzyme that catalyzes the initial steps in the biosynthesis of pro-inflammatory leukotrienes, is an attractive drug target for the pharmacotherapy of inflammatory and allergic diseases. Here, we present the design, synthesis and biological evaluation of novel series of ethyl 5-hydroxyindole-3-carboxylate derivatives that efficiently inhibit human 5-LO. SAR analysis revealed that the potency of compounds is closely related to the positioning of the substituents at the phenylthiomethyl ring. The introduction of methyl or chlorine groups in ortho- and ortho/para-position of thiophenol represent the most favorable modifications. Among all tested compounds, ethyl 5-hydroxy-2-(mesitylthiomethyl)-1-methyl-1H-indole-3-carboxylate (19) is the most potent derivative which blocks 5-LO activity in cell-free assays with IC50 = 0.7 µM, and suppressed 5-LO product synthesis in polymorphonuclear leukocytes with IC50 = 0.23 µM.

Synthesis of covalently attached hexadecaanilines on carbon nanotubes: toward electronic nanocarbon preparation.

We describe the direct covalent-grafting synthesis of well-defined aniline oligomers, such as tetraaniline (A(4)) and hexadecaaniline (A(16), major)/eicosaaniline (A(20), minor), on the sidewalls of carbon nanotubes (CNTs), via dediazonization reaction, for achieving highly soluble nanomaterials suitable for printing purposes, with long-term physical stability. Chemically grafting a layer of electroactive hexadecaanilines on CNTs resembles semiconductive encapsulation of functionalized CNTs. The resulting covalent nanoconjugates SWNT-(A(4))(x), MWNT-(A(4))(x), SWNT-(A(16/20))(x), and MWNT-(A(16/20))(x) were characterized by various spectroscopic and microscopic mapping methods. The combination of transmission electron microscopy (TEM) and electron energy loss spectroscopy (EELS) analyses provided direct evidence for A(16/20) attachment to the CNTs, giving confirmation of the presence of heteroatoms surrounding the CNTs that was absent in the parent CNTs. Subsequent atom mapping in the vicinity of the tube structure allowed us to illustrate the 3D distribution of heteroatoms along the CNT surface.

Biomimetic synthesis of water-soluble conducting copolymers/homopolymers of pyrrole and 3,4-ethylenedioxythiophene.

A novel biomimetic route for the synthesis of electrically conducting homopolymers/copolymers of pyrrole and 3,4-ethylenedioxythiophene (EDOT) in the presence of a polyelectrolyte, such as polystyrene sulfonate (SPS), is presented. A poly(ethylene glycol)-modified hematin (PEG-hematin) was used to catalyze the homopolymerization of pyrrole and EDOT as well as copolymerization of EDOT and pyrrole in the presence of SPS to yield homopolymers of polypyrrole/SPS and PEDOT/SPS as well as a polypyrrole-co-poly(3,4-ethylenedioxythiophene)/SPS complex. Spectroscopic characterization [UV-visible, Fourier transform infrared (FTIR), and X-ray photoelectron spectroscopy (XPS)], thermal analysis, (TGA), and electrical conductivity studies for these complexes indicated the presence of a stable and electrically conductive form of these polymers. Furthermore, the presence of SPS that serves as a charge-compensating dopant in this complex provides a unique combination of properties such as processability and water solubility.

Photo-cross-linked immobilization of polyelectrolytes for enzymatic construction of conductive nanocomposites.

Horseradish peroxidase has been demonstrated to catalyze the oxidative polymerization of aniline to form conductive polyaniline. This process is facilitated by the presence of a macromolecular template with sulfonic acid groups that provide a unique environment for the formation of the conducting polymer. Here we report the use of a photo-cross-linkable thymine-based polymer with phenylsulfonate groups as a templating substrate. This allows for an aqueous-based environmentally benign photopatterning of a conductive material. A description and results of this process are presented.

Biomimetic synthesis of a water soluble conducting molecular complex of polyaniline and lignosulfonate.

A new biomimetic route for the synthesis of a conducting molecular complex of polyaniline (Pani) and a natural polyelectrolyte, lignosulfonate (LGS) is presented. A poly(ethylene glycol) modified hematin (PEG-hematin) was used to catalyze the polymerization of aniline in the presence of LGS to form a Pani/LGS complex. UV-vis, FTIR, conductivity and TGA studies for the LGS-polyaniline complex indicate the presence of a thermally stable and electrically conductive form of polyaniline. Also the presence of LGS in this complex, an inexpensive byproduct from pulp processing, provides a unique combination of properties such as electronic conductivity, processability and biodegradability. The use of this conductive complex for corrosion protection is also proposed.