Current Chemical Biology - Volume 7, Issue 2, 2013

Biofuels have been hailed as the fuels of the future with the potential to completely replace petroleum sourced fuels and also help in combating some of the current environmental issues associated with the intensive use of petroleum based fuels. Biofuels are of two types: primary and secondary. Primary biofuels are derived from sources like firewood, animal wastes, forest and crop residues etc. and are used in their unprocessed forms. Secondary biofuels are obtained by processing biomass and have been classified based on feedstock and processing technologies into first, second, third and fourth generation fuels, with the recently emerged fourth generation fuels having become the highlight of future research in the area. Among the different types of biofuels, biodiesel and bioethanol are now being used as transportation fuels. Biodiesel production is carried out via the transesterification reaction by using suitable catalysts. Lipases (triacyl glycerol hydrolases EC 3.1.1.3) have emerged as robust biocatalysts to accomplish the enzymatic synthesis of biodiesel. This review focuses on the need for biofuels and the most recent research developments in this field and also a brief overview of biodiesel fuel and lipase enzyme has been presented.

Biodiesel fuels (BDFs) have emerged as environmentally friendly substitutes for the fossil fuels being used extensively today. BDFs are essentially methyl esters of fatty acids and are produced by a reaction called as transesterification using catalysts. Alkali catalysis is widely applied for biodiesel production but suffers from certain drawbacks such as high energy consumption, difficulty in the transesterification of triglycerides with a high free fatty acid content and additional costs arising from downstream processes like glycerol recovery, treatment of highly alkaline waste water, etc. Enzymatic catalysis allows for the synthesis of specific alkyl esters, easy recovery of glycerol and the transesterification of triglycerides with high free fatty acid content but is impeded by the high cost of lipase enzyme. Enzyme immobilization, whole-cell biocatalysis, novel lipase expressing yeast cells and recombinant fungi are the approaches that are being used to reduce enzyme associated process costs for industrial scale production of biodiesel.

Lipases are versatile biocatalysts that catalyze a plethora of reactions such as hydrolysis, esterification, transesterification, aminolysis and acidolysis, making them one of the most sought biocatalysts for industrial applications. However, their widespread use in industry is hampered by problems arising mainly due to loss of activity, thermal denaturation, deactivation by organic solvents etc., which affect reaction yields and lead to impaired enzyme recyclability and reusability for successive runs. Methods such as enzyme immobilization, reaction engineering and molecular biology approaches are being used to alleviate these impediments. Lipase bio-imprinting and molecular biology techniques such as rational protein design and directed evolution have made it possible to engineer lipases having superior properties and selectivities, greatly enhancing their activities and operational stability thereby improving reaction yields. This review focuses on the applicability of the above-mentioned strategies to create tailor-designed lipase enzymes to suit specific reactions needs with an emphasis on the transesterification reaction to produce biodiesel.

Given the importance of butyl laurate in various industrial fields, its production by enzymatic catalysis has received greater interest. Butyl laurate was successfully synthesised by the esterification of lauric acid with butanol. This reaction was catalyzed by Rhizopus oryzae lipase immobilized onto silica aerogel in organic media. Response surface methodology was applied in order to approximate the effect of the butanol/ lauric acid molar ratio (1.2-5 mol/mol), the amount of lipase (100-700 IU) and the volume of hexane (3-9 mL) on the butyl laurate lipase-catalyzed esterification yield through an empirical model. Results clearly indicated that the lipase amount was the main factor influencing the synthesis yield. This yield increased with the lipase amount and decreased with the molar ratio of butanol/ lauric acid and the volume of hexane. The selected optimal conditions for synthesis were: a butanol/ lauric acid molar ratio of 1.2, a lipase amount of 550 IU and an hexane volume of 3 mL. The application of these optimized conditions led to a butyl laurate yield of 90.5%. The immobilized lipase was successfully reused for 26 cycles without a significant decrease of the conversion yield on butyl laurate. These results confirm the idea that employing Rhizopus oryzae lipase immobilized onto silica aerogel for a wide range of esterification reactions is feasible.

Nuclear sphingomyelin is mainly localized in specific lipid microdomains of the inner nuclear membrane in which the active chromatin is attached. Evidence of the presence of PKCξ in cell nucleus, where it acts on the chromatin remodeling, phosphorylation of histones, formation of the mitotic spindle is increasing. Although the pathway of the sphingomyelin in the cells was described as target of PKCξ or vice versa the PKCξ as target of sphingomyelin pathway, the relationship between the two molecules in cell nucleus has not been studied. Here, the possible nuclear PKCξ/ sphingomyelin metabolism enzymes interaction was investigated during liver regeneration. We found that the phospho PKCξ and sphingomyelin-synthase increase during the S phase of the cell cycle, while the sphingomyelinase is activated later. The incubation of H35 hepatoma cells with D45262, a compound with 87% PKCξ inhibitory activity at 10 microM concentration, increases specifically sphingomyelinase and inhibits sphingomyelin-synthase with the reduction of DNA and RNA synthesis as index of the delay of hepatoma cell growth. Current results indicate for the first time the presence of PKCξ isoform in the nucleus of hepatocytes and hepatoma cells and its relationship with the sphingomyelin metabolism during the S-phase of the cell cycle.

We present herein the results of a receptor independent 4D-QSAR analysis conducted on 4-aryl-2- trichloromethylquinazoline derivatives displaying in vitro antiplasmodial properties against the W2 multi-resistant Plasmodium falciparum strain. Conformational analysis was performed on a set of 14 molecules in order to obtain batches of lowest energy conformers. A linear regression model with Boltzmann-weighted descriptors was therefore applied. This QSAR approach is a two-step procedure which explores available experimental data to allow intelligent and accelerated screening of new chemical entities. In the first step, a multi-linear regression model including 14 molecules was built up involving molecular descriptors. In the second step, the model predicted the antiplasmodial activity (IC50) of 75 new molecules for a set of hypothesized structures whose molecular descriptors are available. Finally, among these 75 molecules, 5 new quinazolines which activity had been predicted by the model were synthesized and biologically evaluated. Their biological experimental activities were compared to the ones predicted by the QSAR model, so as to validate it. Out of these 5 quinazolines, there was a quite good correlation between the predicted and experimental IC50 values of 3 molecules while the results obtained for the 2 others pointed out two probable limitations for this QSAR model. Such 4DQSAR approach could open the way to a rational and faster preparation of more potent antiplasmodial quinazoline leads.

Flavonoids, a subclass of polyphenols, are abundant components of fruit and vegetables, prevalent in the diet and having an inverse association with the incidence of various degenerative diseases and cancer. Mechanisms underlying the beneficial effects of flavonoids on the human health are being investigated worldwide. Flavonoids have been found to reduce the risk of carcinogenesis by blocking the initiation and suppressing the promotion and progression of certain cancer cells. The accrued data suggests that the chemopreventive effects of flavonoids are exerted through induction of cytoprotective mechanisms to prevent the activation of pro-carcinogens and oxidants from damaging DNA (genome), and detoxify activated carcinogens by enhancing their conjugation and excretion. The balance of metabolic activation and detoxification of carcinogens is controlled through expression of drug-metabolizing Phase I and Phase II enzymes. If the detoxification pathway is saturated, the AhR-XRE-cytochrome P450s activation pathway produces arene oxides and the consequential additional damage promotes tumourigenesis. Fortunately, such oxidative damages can be prevented by CNC-bZIP transcription factors through differentially regulating antioxidant and detoxification genes, which contain ARE and its homologues in their promoters. Amongst the CNC-bZIP family, Nrf2 is a master regulator of expression of drugmetabolizing enzymes, and its activity is negatively regulated by Keap1 and β-TrCP. Expression of Nrf2 and downstream genes is tightly controlled by AhR and CNC-bZIP (e.g. Nrf1) family factors, whilst its negator Keap1 is also regulated by Nrf1 and Nrf2. Such crosstalks between AhR-XRE and Nrf2-ARE regulatory networks indicate that flavonoids trigger multiple signaling pathways to integrally activate cytoprotective genes against cytotoxic insults and oxidative stress. However, the unique chemopreventive role of Nrf1 in regulating antioxidant, detoxification and cytoprotective genes has yet to be fully elucidated and characterised.

Cadmium toxicity remains a major public health concern, despite a huge amount of work to explain its effects. The kidney is the most sensitive organ; and we recently provide the first evidence of a direct upregulation of autophagy by cadmium particularly in response to environmental relevant concentrations. Investigation of autophagy is greatly progressed and various strategies have been reported for studying this molecular process in different biological systems both in physiological and stress conditions. Furthermore, mechanisms of cadmium-induced autophagy in renal cells continue to be of interest given the unknown physiologic function of this metal. Cadmium is persistent within cytosol; it might damage proteins continuously and induces oxidative stress. The aim of this review is a critical analysis of knowledge about autophagic mechanisms induced by cadmium. We also report data obtained in different experimental studies, using cadmium and other xenobiotics, highlighting similarities in the induction of autophagic processes. A more detailed discussion will concern the role of autophagy in cadmium exposed renal proximal convoluted tubule since it is a suitable model system extremely sensitive to environmental stress and cadmium is one of the most nephrotoxic metals to which humans are exposed. We finally conclude that deficiency of autophagic process may be the origin of cadmium nephrotoxicity.

Phytochemical investigation of the methanolic extract of Eryngium campestre L. aerial parts led to isolation of eleven known flavonol glycosides. Structures were elucidated by spectroscopic and chemical methods. The methanol extract of E. campestre and the isolated flavonols exhibited moderate to strong antioxidant activity in DPPH radical scavenging and reducing power assays. Eryngium campestre extract showed significant inhibition of the A.A.β-amyloid Aβ 42 (IC50 = 155.75±7.43 ng/ml) without significant reduction in total Aβ (Aβ 40 + Aβ 42) levels in human H4 cell line, using sensitive sandwich enzyme linked immunosorbent assay (ELISA). The results showed no inhibition activity of the extract against COX-1 and COX-2 up to 400 ng/ml concentration.

The molecular mechanisms whereby small molecules that contaminate our environment cause physiological effects are largely unknown, in terms of both targets and mechanisms. The essential human enzyme porphobilinogen synthase (HsPBGS, a.k.a. 5-aminolevulinate dehydratase, ALAD) functions in heme biosynthesis. HsPBGS catalytic activity is regulated allosterically via an equilibrium of inactive hexamers and active octamers, and we have shown that certain drugs and drug-like small molecules can inhibit HsPBGS in vitro by stabilizing the hexamer. Here we address whether components of the National Toxicology Program library of environmental contaminants can stabilize the HsPBGS hexamer and inhibit activity in vitro. Native polyacrylamide gel electrophoresis was used to screen the library (1,408 compounds) for components that alter the oligomeric distribution of HsPBGS. Freshly purchased samples of 37 preliminary hits were used to confirm the electrophoretic results and to determine the dose-dependence of the perturbation of oligomeric distribution. Seventeen compounds were identified which alter the oligomeric distribution toward the hexamer and also inhibit HsPBGS catalytic activity, including the most potent HsPBGS inhibitor yet characterized (Mutagen X, IC50 = 1.4μM). PBGS dysfunction is associated with the inborn error of metabolism know as ALAD porphyria and with lead poisoning. The identified hexamer-stabilizing inhibitors could potentiate these diseases. Allosteric regulation of activity via an equilibrium of alternate oligomers has been proposed for many proteins. Based on the precedent set herein, perturbation of these oligomeric equilibria by small molecules (such as environmental contaminants) can be considered as a mechanism of toxicity.

Important roles for hydrogen sulfide (H2S) in physiological and pathological function are emerging. H2S is the fourth signaling gas molecule, following O2, NO and CO, that has been shown to be important for intra- and intermolecular signal transduction. These gas molecules should bind to the heme iron complex and regulate numerous important physiological functions such as transcription, guanylate cyclase, and phosphodiesterase. However, involvement of heme proteins in H2S-regulated functions has not been critically considered. This paper is to sort out the complicated interactions of H2S with heme proteins and to help advance our understanding of the molecular mechanism of the interaction between H2S and heme proteins. Importantly, H2S interacts with the heme iron complex of myoglobin and hemoglobin in the presence of H2O2, forming sulfheme (sulfur-incorporated porphyrin) iron complex with markedly different physicochemical characters (such as oxygen binding affinity) in contrast to those of the normal heme iron complex. It is suggested that involvement of the heme proteins in H2S-regulated physiological and pathological functions, in particular under oxidative stress conditions, is much more crucial than generally thought.