Inhibition of SARS-CoV 3CL protease by flavonoids

Abstract

There were severe panics caused by Severe Acute Respiratory Syndrome (SARS) and Middle-East Respiratory Syndrome-Coronavirus. Therefore, researches targeting these viruses have been required. Coronaviruses (CoVs) have been rising targets of some flavonoids. The antiviral activity of some flavonoids against CoVs is presumed directly caused by inhibiting 3C-like protease (3CLpro). Here, we applied a flavonoid library to systematically probe inhibitory compounds against SARS-CoV 3CLpro. Herbacetin, rhoifolin and pectolinarin were found to efficiently block the enzymatic activity of SARS-CoV 3CLpro. The interaction of the three flavonoids was confirmed using a tryptophan-based fluorescence method, too. An induced-fit docking analysis indicated that S1, S2 and S3′ sites are involved in binding with flavonoids. The comparison with previous studies showed that Triton X-100 played a critical role in objecting false positive or overestimated inhibitory activity of flavonoids. With the systematic analysis, the three flavonoids are suggested to be templates to design functionally improved inhibitors.     

The influence of cytochrome P450 enzyme activity on the composition and quantity of volatile organics in expired breath

Abstract

We have previously described a method to capture, identify and quantify volatile components in expired breath. The purpose of this research is to provide a non-invasive means to measure biomarkers of metabolism in vivo. In the present studies, the effect of 1-aminobenzotriazole (ABT), an inhibitor of diverse cytochrome P450 (P450) enzymes, on the composition of volatile organic chemicals (VOCs) expired in the breath of male F-344 rats was determined in parallel with the catalytic activities and total content of hepatic P450. lntraperitoneal administration of ABT (100 mg kg-¹) to rats resulted in markedly diminished hepatic microsomal P450 content and activities. The extent of inhibition was near maximal at 4 h, at which time approximately 50% of the total P450 content, about 65% of the CYPlA2 activity, 55% of the CYP2E1 activity, and about 80% of CYP2B activity were lost. Inhibition was maintained to 48 h post-dosing, but P450 content and activities had largely been restored by day 7. Concomitant with the inhibition of P450 were corresponding increases (up to several hundred-fold) in the molar amount of volatiles appearing in the breath of ABT-treated animals, and the rebound of P450 levels was attended by corresponding decreases in the appearance of breath volatiles. These studies indicate that P450 plays a major role in the metabolism of VOCs appearing in breath, and that these chemicals can serve as markers on P450 activity in vivo.     

Temperature stress causes different profiles of volatile compounds in two chemotypes of Salvia lavandulifolia Vahl.

In order to clarify volatile accumulation patterns along with their adaptability to temperature stress, replicas of two different accessions of Salvia lavandulifolia appointed as chemotypes (A and B) were subjected to increasing temperatures in environmental controlled cabins. Their respective volatile chemical profiles kept their integrity until reaching the growing temperature of 24 °C. Then, remarkable changes were observed. Stressed plants of both accessions showed an increase in the sesquiterpenic fraction and a decrease in the monoterpenic one, although changes in volatile percentages were highly related to the accession. Accession B overexpressed β-caryophyllene, caryophyllene oxide and manool when subjected to the higher temperature tested. On the other hand, accession A mainly showed a remarkable increase of β-bisabolene and viridiflorol. Furthermore, viridiflorol and manool have been confirmed as new chemotaxonomic markers for chemotypes A and B, respectively. The capacity to adjust the production of volatile compounds seems mostly attributed to the genetic background of the plant although more research is needed to understand the stress and its implications for the secondary metabolism. These results contribute remarkably to the chemical phenotyping of this species and may be useful for selection of genotypes.     

Complementation of a manganese-dependent superoxide dismutase-deficient yeast strain with Pneumocystis carinii sod2 gene

Manganese-dependent superoxide dismutase (MnSOD) is one of the key enzymes involved in the cellular defense against oxidative stress. Previously, the Pneumocystis carinii sod2 gene (Pcsod2) was isolated and characterized. Based on protein sequence comparison, Pcsod2 was suggested to encode a putative MnSOD protein likely to be targeted into the mitochondrion. In this work, the Pcsod2 was cloned and expressed as a recombinant protein in EG110 Saccharomyces cerevisiae strain lacking the MnSOD-coding gene (Scsod2) in order to investigate the function and subcellular localization of P. carinii MnSOD (PcMnSOD). The Pcsod2 gene was amplified by PCR and cloned into the pYES2.1/V5-His-TOPO^® expression vector. The recombinant construct was then transformed into EG110 strain. Once its expression had been induced, PcMnSOD was able to complement the growth defect of EG110 yeast cells that had been exposed to the redox-cycling compound menadione. N-term sequencing of the PcMnSOD protein allowed identifying the cleavage site of a mitochondrial targeting peptide. Immune-colocalization of PcMnSOD and yeast CoxIV further confirmed the mitochondrial localization of the PcMnSOD.Heterologous expression of PcMnSOD in yeast indicates that Pcsod2 encodes an active MnSOD, targeted to the yeast mitochondrion that allows the yeast cells to grow in the presence of reactive oxygen species (ROS).     

Expression of manganese superoxide dismutase is not altered in transgenic mice with elevated level of copper-zinc superoxide dismutase

In evaluating the relative expression of copper-zinc and manganese superoxide dismutase (CuZnSOD and MnSOD) in vivo in states like Down syndrome in which one dismutase is present at increased levels, we measured activities of both enzymes, in tissues of control and transgenic mice constitutively expressing increased levels of CuZnSOD, during exposure to normal and elevated oxygen tensions. Using SOD gel electrophoresis assay, CuZnSOD and MnSOD activities of brain, lung, heart, kidney, and liver from mice exposed to either normal (21%) or elevated (>99% oxygen, 630 torr) oxygen tensions for 120 h were compared. Whereas CuZnSOD activity was elevated in tissues of transgenic relative to control mice under both normoxic or hyperoxic conditions, MnSOD activities in organs of transgenic mice were remarkably similar to those of controls under both conditions. To confirm the accuracy of this method in quantitating MnSOD relative to CuZnSOD expression, two other methods were utilized. In lung, which is the organ exposed to the highest oxygen tension during ambient hyperoxia, a sensitive, specific ELISA for MnSOD was used. Again, MnSOD protein was not different in transgenic relative to control mice during exposure to air or hyperoxia. In addition, lung MnSOD protein was not changed significantly by exposure to hyperoxia in either group. In kidney, a mitochondrion-rich organ, SOD assay, before and after inactivation of CuZnSOD with diethyldithiocarbamate, was used. MnSOD activity was not different in organs from air-exposed transgenic relative to control mice. The data indicated that expression of MnSOD in vivo was not affected by overexpression of the CuZnSOD and, therefore, the two enzymes are probably regulated independently.     

Evaluation of new antimicrobial agents on Bacillus spp. strains: docking affinity and in vitro inhibition of glutamate-racemase

Three glutamic acid derivatives, two boron-containing and one imide-containing compound, were synthesized and tested for antimicrobial activity targeting glutamate-racemase. Antimicrobial effect was evaluated over Bacillus spp. Docking analysis shown that the test compounds bind near the active site of racemase isoforms, suggesting an allosteric effect. The boron derivatives had greater affinity than the imide derivative. In vitro assays shown good antimicrobial activity for the boron-containing compounds, and no effectiveness for the imide-containing compounds. The minimum inhibitory concentration of tetracycline, used as standard, was lower than that of the boron-containing derivatives. However, it seems that the boron-containing derivatives are more selective for bacteria. Experimental evidence suggests that the boron-containing derivatives act by inhibiting the racemase enzyme. Therefore, these test compounds probably impede the formation of the bacterial cell wall. Thus, the boron-containing glutamic acid derivatives should certainly be of interest for future studies as antimicrobial agents for Bacillus spp.     

Oncogenic potential of Nrf2 and its principal target protein heme oxygenase-1

Nuclear factor erythroid 2-related factor 2 (Nrf2) is an essential component of cellular defense against a vast variety of endogenous and exogenous insults, including oxidative stress. Nrf2 acts as a master switch in the circuits upregulating the expression of various stress-response proteins, especially heme oxygenase-1 (HO-1). Paradoxically, however, recent studies have demonstrated oncogenic functions of Nrf2 and its major target protein HO-1. Levels of Nrf2 and HO-1 are elevated in many different types of human malignancies, which may facilitate the remodeling of the tumor microenvironment making it advantageous for the autonomic growth of cancer cells, metastasis, angiogenesis, and tolerance to chemotherapeutic agents and radiation and photodynamic therapy. In this context, the cellular stress response or cytoprotective signaling mediated via the Nrf2–HO-1 axis is hijacked by cancer cells for their growth advantage and survival of anticancer treatment. Therefore, Nrf2 and HO-1 may represent potential therapeutic targets in the management of cancer. This review highlights the roles of Nrf2 and HO-1 in proliferation of cancer cells, their tolerance/resistance to anticancer treatments, and metastasis or angiogenesis in tumor progression.     

In-vitro evaluation of antioxidant,anti-elastase,anti-collagenase,anti-hyaluronidase activities of safranal and determination of its sun protection factor in skin photoaging

Safranal, a monoterpene aldehyde, is present as one of the main volatile constituents of Crocus sativus Linn. (saffron flowers). This volatile constituent not only contributes to the aroma of saffron but has been reported to possess antidiabetic, antiulcer, antiasthamatic, anticonvulsant, antidepressant, cardioprotective, anticancer and UV protective properties. Most of these therapeutic actions are contributed by its potential to quench reactive oxygen species (ROS). Antioxidant properties of phytoconstituents are now being explored for developing photoprotective skin formulations. These bioactives have the potential to protect the epidermal and dermal layers of the skin which mainly comprises of elastin and collagen. When UV rays penetrate the dermal layers, there is an increased production of elastase, collagenase and hyaluronidase leading to degradation of collagen, elastin and hyaluronic acid respectively. These dermal components are responsible to provide strength, elasticity and moisture to the skin. Due to frequent exposure to sunlight, these conditions tend to augment leading to wrinkle formation and sagging of skin.Although antioxidant properties of safranal have been established on various cell lines but till date no studies have been reported regarding the dermal enzyme inhibition activities. In the current research work, a comprehensive in vitro evaluation of antioxidant, anti-elastase, anti-collagenase, anti-hyaluronidase activities of safranal along with determination of sun protection factor (SPF) was carried out. The in vitro antioxidant activity was carried out by diphenylpicrylhydrazyl (DPPH) method and its IC₅₀ value was found to be 22.7 μg/ml. The enzyme inhibition IC₅₀ values of safranal for anti elastase activity were found to be 43.6 μg/ml, 70 μg/ml for antihyaluronidase activity and 9.4 μg/ml for anticollagenase activity. Photoprotective activity of safranal was determined by UV absorbance method and SPF calculated by Mansur equation which was found to be 6.6.The significant inhibitory activity of safranal on matrix metalloproteinases (MMPs) responsible for aging and a higher SPF established that this bioorganic molecule is a strong photoprotective agent. Its established free radical scavenging capability along with above characteristics make it a valuable component to be incorporated into herbal antiaging formulations.