Navigating the Unnatural Reaction Space: Directed Evolution of Heme Proteins for Selective Carbene and Nitrene Transfer.

Despite the astonishing diversity of naturally occurring biocatalytic processes, enzymes do not catalyze many of the transformations favored by synthetic chemists. Either nature does not care about the specific products, or if she does, she has adopted a different synthetic strategy. In many cases, the appropriate reagents used by synthetic chemists are not readily accessible to biological systems. Here, we discuss our efforts to expand the catalytic repertoire of enzymes to encompass powerful reactions previously known only in small-molecule catalysis: formation and transfer of reactive carbene and nitrene intermediates leading to a broad range of products, including products with bonds not known in biology. In light of the structural similarity of iron carbene (Fe═C(R1)(R2)) and iron nitrene (Fe═NR) to the iron oxo (Fe═O) intermediate involved in cytochrome P450-catalyzed oxidation, we have used synthetic carbene and nitrene precursors that biological systems have not encountered and repurposed P450s to catalyze reactions that are not known in the natural world. The resulting protein catalysts are fully genetically encoded and function in intact microbial cells or cell-free lysates, where their performance can be improved and optimized by directed evolution. By leveraging the catalytic promiscuity of P450 enzymes, we evolved a range of carbene and nitrene transferases exhibiting excellent activity toward these new-to-nature reactions. Since our initial report in 2012, a number of other heme proteins including myoglobins, protoglobins, and cytochromes c have also been found and engineered to promote unnatural carbene and nitrene transfer. Due to the altered active-site environments, these heme proteins often displayed complementary activities and selectivities to P450s.Using wild-type and engineered heme proteins, we and others have described a range of selective carbene transfer reactions, including cyclopropanation, cyclopropenation, Si-H insertion, B-H insertion, and C-H insertion. Similarly, a variety of asymmetric nitrene transfer processes including aziridination, sulfide imidation, C-H amidation, and, most recently, C-H amination have been demonstrated. The scopes of these biocatalytic carbene and nitrene transfer reactions are often complementary to the state-of-the-art processes based on small-molecule transition-metal catalysts, making engineered biocatalysts a valuable addition to the synthetic chemist's toolbox. Moreover, enabled by the exquisite regio- and stereocontrol imposed by the enzyme catalyst, this biocatalytic platform provides an exciting opportunity to address challenging problems in modern synthetic chemistry and selective catalysis, including ones that have eluded synthetic chemists for decades.

Heterobinuclear copper(II)­platinum(II) complexes with oxindolimine ligands: Interactions with DNA, and inhibition of kinase and alkaline phosphatase proteins.

Two mononuclear copper(II) compounds, [Cu(isad)(H2O)Cl]Cl 1 and [Cu(isah)(H2O)Cl]Cl 2, and its corresponding heterobinuclear species containing also platinum(II), [CuCl(isad)Pt(NH3)Cl2] 3 and [CuCl(isah)Pt(NH3)Cl2] 4 (where isad and isah are oxindolimine ligands, (E)-3-(2-(3-aminopropylamino)ethylimino)indolin-2-one, and (E)-3-(3-amino-2-hydroxypropylimino)indolin-2-one, respectively), have been previously synthesized and characterized by different spectroscopic techniques in our laboratory. Cytotoxicity assays performed with B16F10 murine cancer cells, and MES-SA human uterine sarcoma cells, showed IC50 values lower or in the same order of cisplatin. Herein, in order to better elucidate their probable modes of action, possible interaction and damage to DNA, as well as their effect on the activity of crucial proteins were verified. Both mononuclear complexes and the binuclear compound 4 displayed a significant cleavage activity toward plasmid DNA, while compound 3 tends to protect DNA from oxidative damage, avoiding degradation. Complementary experiments indicated a significant inhibition activity toward cyclin-dependent kinase (CDK1/cyclinB) activity in the phosphorylation of histone H1, and only moderate inhibition concerning alkaline phosphatase. Results also revealed that the reactivity is reliant on the ligand structure and on the nature of the metal present, in a synergistic effect. Simulation studies complemented and supported our results, indicating different bindings of the binuclear compounds to DNA. Therefore, the verified cytotoxicity of these complexes comprises multiple modes of action, including modification of DNA conformation, scission of DNA strands by reactive oxygen species, and inhibition of selected proteins that are crucial to the cellular cycle.

Quercetin reduced the formation of N-acetyl-p-benzoquinoneimine, a toxic metabolite of paracetamol in rats and isolated rat hepatocytes.

N-acetyl-p-benzoquinoneimine (NAPQI) is toxic metabolite of paracetamol formed primarily by cytochrome P4502E1 (CYP2E1) metabolic pathway when administered at therapeutic doses or overdose. The influence of quercetin (flavonoid) on the bioactivation of paracetamol to NAPQI was investigated using rat liver microsomes and rats in vivo. Paracetamol (80 mg/kg) was administered orally without or with silymarin (100 mg/kg), a known inhibitor of CYP2E1, CYP3A4 and quercetin (10 and 20 mg/kg) to rats for 15 consecutive days. Area under the plasma concentration-time curve (AUC0-∞ ) and the peakplasma concentration (Cmax ) of paracetamol were dose-dependently increased with quercetin (10 and 20 mg/kg) compared to paracetamol control group (p < 0.001). On the other hand, the AUC0-∞ and Cmax of NAPQI were decreased significantly with quercetin. The same results were observed with silymarin also. The elevated liver and kidney functional enzymes/compounds were significantly reduced by quercetin and silymarin compared to paracetamol control group. The formation of NAPQI was reduced in the incubation samples in presence of quercetin in experiment using isolated rat hepatocytes. The presentstudy results revealed that quercetin might be inhibited the CYP2E1-mediated metabolism of paracetamol; thereby decreased the formation of NAPQI and protected the liver and kidney.

Schisandrol B protects against acetaminophen-induced hepatotoxicity by inhibition of CYP-mediated bioactivation and regulation of liver regeneration.

Acetaminophen (APAP) overdose is the most frequent cause of drug-induced acute liver failure. Schisandra sphenanthera is a traditional hepato-protective Chinese medicine and Schisandrol B (SolB) is one of its major active constituents. In this study, the protective effect of SolB against APAP-induced acute hepatotoxicity in mice and the involved mechanisms were investigated. Morphological and biochemical assessments clearly demonstrated a protective effect of SolB against APAP-induced liver injury. SolB pretreatment significantly attenuated the increases in alanine aminotransferase and aspartate aminotransferase activity, and prevented elevated hepatic malondialdehyde formation and the depletion of mitochondrial glutathione (GSH) in a dose-dependent manner. SolB also dramatically altered APAP metabolic activation by inhibiting the activities of CYP2E1 and CYP3A11, which was evidenced by significant inhibition of the formation of the oxidized APAP metabolite NAPQI-GSH. A molecular docking model also predicted that SolB had potential to interact with the CYP2E1 and CYP3A4 active sites. In addition, SolB abrogated APAP-induced activation of p53 and p21, and increased expression of liver regeneration and antiapoptotic-related proteins such as cyclin D1 (CCND1), PCNA, and BCL-2. This study demonstrated that SolB exhibited a significant protective effect toward APAP-induced liver injury, potentially through inhibition of CYP-mediated APAP bioactivation and regulation of the p53, p21, CCND1, PCNA, and BCL-2 to promote liver regeneration.

Persistence and dissipation kinetics of trifloxystrobin and tebuconazole in onion and soil.

The persistence and dissipation kinetics of trifloxystrobin and tebuconazole on onion were studied after application of their combination formulation at a standard and double dose of 75 + 150 and 150 + 300 g a.i. ha(-1). The fungicides were extracted with acetone, cleaned-up using activated charcoal (trifloxystrobin) and neutral alumina (tebuconazole). Analysis was carried out by gas chromatograph (GC) and confirmed by gas chromatograph mass spectrometry (GC-MS). The recovery was above 80% and limit of quantification (LOQ) 0.05 mg kg(-1) for both fungicides. Initial residue deposits of trifloxystrobin were 0.68 and 1.01 mg kg(-1) and tebuconazole 0.673 and 1.95 mg kg(-1) from standard and double dose treatments, respectively. Dissipation of the fungicides followed first-order kinetics and the half life of degradation was 6-6.6 days. Matured onion bulb (and field soil) harvested after 30 days was free from fungicide residues. These findings suggest recommended safe pre-harvest interval (PHI) of 14 and 25 days for spring onion consumption after treatment of Nativo 75 WG at the standard and double doses, respectively. Matured onion bulbs at harvest were free from fungicide residues.

Fennel and raspberry leaf as possible inhibitors of acetaminophen oxidation.

In addition to CYP2E1, several CYP isoenzymes, notably CYP1A2, 2D6, and 3A4, are suggested to contribute in acetaminophen oxidation and formation of the hepatotoxic metabolite N-acetyl-p-benzoquinone imine (NAPQI). The in vitro CYP2E1 inhibitory potentials of fennel and raspberry leaf, herbs previously found to inhibit CYP1A2, 2D6, and 3A4 activities in vitro, were investigated. Extracts from commercially available herbal products were incubated with recombinant cDNA-expressed human CYP2E1. A validated LC/MS/MS methodology was applied for determination of 6-hydroxychlorzoxazone formation with disulfiram used as a positive inhibitory control. CYP2E1 IC50 inhibition constants were found to be 23 ± 4 and 27 ± 5 µg/ml for fennel and raspberry leaf, respectively, constants significantly lower than those presented in the literature for other herbal extracts. Together with previous findings, the presented in vitro data for CYP2E1 inhibition suggest that fennel and raspberry leaf have a significant potential of inhibiting all the major metabolic pathways for acetaminophen oxidation and NAPQI formation. Both herbs should be further investigated for their in vivo ability of inhibiting acetaminophen oxidation and NAPQI formation.

Abrogation by Trifolium alexandrinum root extract on hepatotoxicity induced by acetaminophen in rats.

OBJECTIVE: Acetaminophen (APAP) is a substance that harms human health by stimulating free radical production. This study investigated the ability of Trifolium alexandrinum root (TAR) extract to reduce the hepatotoxicity induced by APAP in rats. METHODS: Animals were classified into four groups and treated for 6 weeks. Group 1: normal control-treated (saline); Group 2: TAR extract-treated (100 mg/kg); Group 3: APAP-treated; Group 4: APAP plus TAR extract. RESULTS: APAP significantly elevated AST (aspartate amino transferase), ALT (amino alanine transferase), ALP (alkaline phosphatase), GGTP (gamma glutamyl transpeptidase), bilirubin, and malondialdehyde with a significant decrease in glutathione, superoxide dismutase, glutathione peroxidase, catalase, and glutathione S-transferase compared with the control group. Administration of TAR extract combined with APAP improved the liver damage induced by APAP. Histopathological evidence, together with observed DNA fragmentation, supported the detrimental effect of APAP and the ameliorating effect of TAR extract on liver toxicity. CONCLUSION: TAR extract has beneficial properties and can reduce the liver damage and toxicity induced by APAP. DISCUSSION: Free radical mediated processes have been implicated in the pathogenesis of many diseases. The protective effect of TAR root extract on APAP-induced hepatotoxicity in rats appears to be related to inhibition of lipid peroxidation and enhancement of antioxidant enzyme levels, in addition to a free radical scavenging action.

Combination of electrochemistry and nuclear magnetic resonance spectroscopy for metabolism studies.

During the development of new materials demonstrating biological activity, prediction and identification of reactive intermediates generated in the course of drug metabolism in the human liver is of great importance. We present a rapid and purely instrumental method for the structure elucidation of possible phase I metabolites. With electrochemical (EC) conversion adopting the oxidative function of liver-inherent enzymes and nuclear magnetic resonance (NMR) spectroscopy enabling structure elucidation, comprehensive knowledge on potential metabolites can be gained. Paracetamol (APAP) has been known to induce hepatotoxicity when exceeding therapeutic doses and was therefore selected as the test compound. The reactive metabolite N-acetyl-p-benzoquinone imine has long been proven to be responsible for the toxic side effects of APAP and can easily be generated by EC. EC coupled online to NMR is a straightforward technique for structure elucidation of reactive drug intermediates at an early stage in drug discovery.

An investigation of the bioactivation potential and metabolism profile of Zebrafish versus human.

The zebrafish model has been increasingly explored as an alternative model for toxicity screening of pharmaceutical drugs. However, little is understood about the bioactivation of drug to reactive metabolite and phase I and II metabolism of chemical in zebrafish as compared with human. The primary aim of our study was to establish the bioactivation potential of zebrafish using acetaminophen as a probe substrate. Our secondary aim was to perform metabolite profiling experiments on testosterone, a CYP3A probe substrate, in zebrafish and compare the metabolite profiles with that of human. The glutathione trapping assay of N-acetyl-p-benzoquinone imine demonstrated that zebrafish generates the same reactive metabolite as humans from the bioactivation of acetaminophen. Zebrafish possesses functional CYP3A4/5-like and UDP-glucuronosyltransferase metabolic activities on testosterone. Differential testosterone metabolism was observed among the two species. In silico docking studies suggested that the zebrafish CYP3A65 was responsible for the bioactivation of acetaminophen and phase I hydroxylation of testosterone. Our findings reinforce the need to further characterize the drug metabolism phenotype of zebrafish before the model can fully achieve its potential as an alternative toxicity screening model in drug research.

Fluorescence-based liver microsomal assay for screening of pharmaceutical reactive metabolites using a glutathione conjugated 96-well plate.

The purpose of our paper is to develop and validate a fluorescence-based mouse liver microsomal (MLM) assay in screening pharmaceutical reactive metabolites (RMs) using a glutathione (GSH)-conjugated 96-well plate. Poly(2-hydroxyethylmethacrylate) (pHEMA) polymeric membrane was coated on 96-well plates to provide a functional support for GSH conjugation. Oxidized GSH (GSSG) was conjugated on a cyanogen bromide (CNBr)-activated pHEMA surface. The conjugated GSH was regenerated after the reduction of GSSG using d,l-dithiothreitol (DTT). X-ray photoelectron spectroscopy, Ellman's, and fluorescence assays were applied to validate the chemistry and optimize the processes of GSH conjugation. The performance of the 96-well assay was further cross-validated using N-acetyl-p-benzo-quinone imine (NAPQI), a RM of acetaminophen (APAP), and the in vitro MLM assay of APAP. Finally, the developed method was applied to screen a batch of marketed drugs and chemicals on the formation of RMs. Our results indicated that optimum conditions were obtained for pHEMA loading, CNBr activation of pHEMA, and GSSG coupling and reduction. The detection limit of the assay for NAPQI was 500 nM with good specificity. In vitro MLM assay of APAP demonstrated a positive trapping index (TI) of 19.3%. The subsequent RM screening of a series of marketed drugs and chemical compounds resulted in a range of TI values (1.0-25.7%) that corroborated with their capacity in generating RMs. The differences of TI values are statistically significant between the compounds which are known to produce RMs and those that do not generate reactive intermediates. In conclusion, we successfully developed a fluorescence-based GSH-conjugated 96-well plate platform for the screening of RMs using MLM.

Prevention of acetaminophen-induced cataract by a combination of diallyl disulfide and N-acetylcysteine.

Injection of acetaminophen (APAP) (350 mg/kg body weight) into C57BL/6 mice in which cytochrome P450 (CYP) 1A1/1A2 had been induced produced acute cataract and other ocular tissue damage. Treatment of APAP-injected mice with one of the major organosulfides in garlic oil, diallyl disulfide (DADS) (200 mg/kg body weight), prevented cataract development and prolonged survival time. N-acetyl L-cysteine (NAC) (500 mg/kg body weight), a prodrug that stimulates glutathione synthesis, also prolonged survival time but was effective only weakly to prevent cataract formation. A combination of DADS and NAC completely prevented cataractogenesis, and all of the treated animals survived APAP toxicity. Neither DADS nor NAC inhibited CYP 1A1/1A2 induction as determined by their effect on the induction of hepatic microsomal ethoxyresorufin O-dealkylase (ERD) activity. However, in the in vitro enzyme assay, DADS, but not NAC, was a potent inhibitor of ERD activity (IC50 = 3.5 mM). Treatment with DADS or NAC slowed but did not stop the decrease of hepatic glutathione (GSH) content. At 4 hours after APAP injection, hepatic GSH began to increase only when DADS and NAC were administered together. These results suggest that the protective effect of DADS is due to its inhibition of biotransformation of APAP to the reactive metabolite N-acetyl-p-benzoquinone imine (NAPQI) by CYP 1A1/1A2 enzymes and that NAC provides protection by increasing cellular cysteine level and GSH synthesis, thus facilitating detoxification of NAPQI by glutathione conjugation. Assay of plasma glutamate-pyruvate transaminase activity, an indicator of liver necrosis, showed that treatment with DADS and NAC together effectively protected the liver. Therefore, the decrease of GSH as much as 30% of normal concentration, by itself, is not responsible for liver damage. The primary cause of hepatic necrosis is rapid accumulation of NAPQI.

Additive protection of cimetidine and N-acetylcysteine treatment against acetaminophen-induced hepatic necrosis in the rat.

Cimetidine protects against acetaminophen hepatotoxicity in the rat as evidenced by improved survival, lower serum aminotransferases, improved liver histology, decreased in vivo and in vitro covalent binding of acetaminophen to liver protein and decreased rate of glutathione depletion. This protection is best explained by inhibition of acetaminophen oxidation by cimetidine. N-acetylcysteine, the accepted antidote, protects against acetaminophen hepatotoxicity primarily by enhancing glutathione synthesis. Inhibition of acetaminophen oxidation by cimetidine has been demonstrated directly in vitro with both rat and human liver microsomes. The aim of the present study was to determine whether cimetidine and N-acetylcysteine might be additive in their protection against acetaminophen hepatotoxicity as cimetidine and N-acetylcysteine have different mechanisms of protective action. Treatment with either cimetidine or N-acetylcysteine improved survival and serum transaminases in a dose-related manner but protection by the combination was additive when compared to each agent alone. Cimetidine decreased the rate of hepatic glutathione depletion and acetaminophen covalent binding in vivo in a dose-dependent manner whereas only a high dose of N-acetylcysteine decreased covalent binding. However, the combination of cimetidine and N-acetylcysteine more effectively prevented glutathione depletion and covalent binding in vivo than either agent used alone. We conclude that protection against acetaminophen hepatotoxicity using a combination of cimetidine and N-acetylcysteine is better than that found with either agent alone. Inasmuch as cimetidine does not increase hepatic glutathione per se, or does N-acetylcysteine inhibit acetaminophen oxidation, the additive protection against acetaminophen hepatotoxicity is best explained by the above mentioned mechanisms of action for each agent.

Some pharmacological investigations of embelin and its semisynthetic derivatives.

Embelin, obtained from Embolin ribes was condensed with different primary amines. Depending on the conditions of reaction, disalts or diimines were formed. Ten such disalts and fourteen diimines were developed. Embelin and all its disalts showed analgesic activity whereas all the diimines derivatives were inactive. The disalt, 2:5 disobutyl amine embelin showed maximum action. Analgesic effect was noticed only after intraperitoneal administration but not after subcutaneous, intramuscular or oral administration. The compounds cause some local irritation. The possibility of peritoneal irritation rendering the animals unresponsive to experimental pain seems to deserve consideration. However, analgesic effect could be seen in dogs and cats after intravenous injection. Embelin and its disalt, 2:5 isobutyl amine embelin also exhibited antipyretic and antiinflammatory activities.

Oxidation of selenocystamine by diamineoxidase.

Selenocystamine is oxidatively deaminated by pig kidney diamineoxidase. The first product of the reaction is the corresponding cyclized aminoaldehyde, selenocystaldimine, which then undergoes further degradation. The oxidative deamination is thus the first step of a series of cyclic reactions which give rise to extensive cleavage of selenocystamine.