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Purpose: The purpose of this study was to evaluate the stability of angiotensin II in 0.9% sodium chloride for up to 5 days. Methods: We prepared angiotensin II dilutions, by aseptically diluting 2.5 mg (1 mL) in 249 mL 0.9% sodium chloride creating a solution of 10 000 ng/mL. Admixtures were stored under refrigeration (5 ± 3°C). Stability of the dilution was assessed by: preservation of clarity, consistency of pH, and retention of concentration. Solutions were sampled at times 0, 24, 48, 72, 96, 120 hours. Solutions were analyzed via High-Performance Liquid Chromatography (HPLC-UV) and Liquid Chromatography Mass Spectrometry (LC-MS/MS). Retention of concentration was set a priori at > 90% of initial concentration. Results: Clarity, color, and pH at all sample time points remained constant. Both methods of analysis confirmed similar results. When stored under refrigeration, the concentration of angiotensin II solution remained above 90% of initial concentration throughout the entire sampling period. Conclusions: Angiotensin II in 0.9% sodium chloride stored in infusion bags under refrigeration (5 ± 3°C) maintained at least 90% of their original concentrations for up to 5 days. Stability was also demonstrated based on turbidity, color, and pH assessment., Competing Interests: The author(s) declared the following potential conflicts of interest with respect to the research, authorship, and/or publication of this article: Dr. Benken serves on the speakers bureau for LaJolla Pharmaceutical Company, manufacturer of angiotensin II. LaJolla Pharmaceutical Company was not in consultation for the design, funding, or results of this study., (© The Author(s) 2023.)

Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms., (© 2023. The Author(s).)

We have previously reported a multipurpose silicone elastomer vaginal ring providing sustained release of dapivirine (an antiretroviral) and levonorgestrel (a progestin) for HIV prevention and hormonal contraception. During initial development, issues arose due to reaction between the ethynyl group in the levonorgestrel molecule and the hydride-functionalised polydimethylsiloxane components in the silicone elastomer formulation. This unwanted reaction occurred both during and to a lesser extent after ring manufacture, impacting the curing process, the mechanical properties of the ring, and the in vitro release of levonorgestrel. Recently, we reported custom silicone elastomer grades that minimise this reaction. In this follow-on study, we describe the manufacture, in vitro drug release, mechanical, and pharmaceutical stability testing of ring formulations prepared from a custom silicone elastomer and containing 200 mg dapivirine and 80, 160, 240 or 320 mg levonorgestrel. The rings showed mechanical properties similar to marketed ring products, sustained in vitro release of both drugs over 30 days in quantities deemed clinically relevant, offered acceptable assay values, and provided good product stability over 15 weeks at 40 °C and 75% relative humidity., Competing Interests: The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Prof. Karl Malcolm reports financial support was provided by 10.13039/501100000873Queen's University Belfast. Prof. Karl Malcolm has patent #US20190105192A1 pending to International Partnership for Microbicides. Prof. Karl Malcolm has patent #EP3209251A1 pending to International Partnership for Microbicides., (© 2022 The Author(s).)

Aiming to elucidate the system-wide effects of the alcohol-induced increase in the content of cytochrome P450 2E1 (CYP2E1) on drug metabolism, we explored the array of its protein-protein interactions (interactome) in human liver microsomes (HLM) with chemical crosslinking mass spectrometry (CXMS). Our strategy employs membrane incorporation of purified CYP2E1 modified with photoreactive crosslinkers benzophenone-4-maleimide and 4-( N -succinimidylcarboxy)benzophenone. Exposure of bait-incorporated HLM samples to light was followed by isolating the His-tagged bait protein and its crosslinked aggregates on Ni-NTA agarose. Analyzing the individual bands of SDS-PAGE slabs of thereby isolated protein with the toolset of untargeted proteomics, we detected the crosslinked dimeric and trimeric complexes of CYP2E1 with other drug-metabolizing enzymes. Among the most extensively crosslinked partners of CYP2E1 are the cytochromes P450 2A6, 2C8, 3A4, 4A11, and 4F2, UDP-glucuronosyltransferases (UGTs) 1A and 2B, fatty aldehyde dehydrogenase (ALDH3A2), epoxide hydrolase 1 (EPHX1), disulfide oxidase 1α (ERO1L), and ribophorin II (RPN2). These results demonstrate the exploratory power of the proposed CXMS strategy and corroborate the concept of tight functional integration in the human drug-metabolizing ensemble through protein-protein interactions of the constituting enzymes.

We investigated the correspondence between drug metabolism routes and the composition of the P450 ensemble in human liver microsomes (HLM). As a probe, we used Coumarin 152 (C152), a fluorogenic substrate metabolized by multiple P450 species. Studying the substrate-saturation profiles (SSP) in seven pooled HLM preparations, we sought to correlate them with the P450 pool's composition characterized by targeted proteomics. This analysis, complemented with the assays with specific inhibitors of CYP3A4 and CYP2C19, the primary C152 metabolizers, demonstrated a significant contrast between different HLM samples. To unveil the source of these differences, we implemented Principal Component Analysis (PCA) of the SSP series obtained with HLM samples with a known composition of the P450 pool. Our analysis revealed that the parameters of C152 metabolism are primarily determined by the content of CYP2A6, CYP2B6, CYP2C8, CYP2E1, and CYP3A5 of those only CYP2B6 and CYP3A5 can metabolize C152. To validate this finding, we studied the effect of enriching HLM with CYP2A6, CYP2E1, and CYP3A5. The incorporation of CYP3A5 into HLM decreases the rate of C152 metabolism while increasing the role of CYP2B6 in its turnover. In contrast, incorporation of CYP2A6 and CYP2E1 reroutes the C152 demethylation towards some P450 enzyme with a moderate affinity to the substrate, most likely CYP3A4. Our results reveal a sharp non-additivity of the individual P450 properties and suggest a pivotal role of P450-P450 interactions in determining drug metabolism routes. This study demonstrates the high potential of our new PCA-based approach in unveiling functional interrelationships between different P450 species., (Copyright © 2021 Elsevier Inc. All rights reserved.)

A dapivirine-releasing silicone elastomer vaginal ring for reducing women's risk of HIV acquisition has recently been approved. A next-generation multipurpose vaginal ring releasing dapivirine and levonorgestrel is currently in development, offering hormonal contraception and HIV prevention from a single device. Previously, we reported challenges with incorporating levonorgestrel into rings manufactured from addition-cure silicone elastomers due to an irreversible chemical reaction between the levonorgestrel molecule and the hydride-functionalised crosslinker component of the silicone elastomer formulation, leading to low drug content assay, cure inhibition, and reduced ring mechanical properties (which may account for the increased incidence of ring expulsion in vivo). Here, we report on the development and testing of various custom silicone elastomer materials specifically formulated to circumvent these issues. After extensive testing of the custom silicones and subsequent manufacture and testing (Shore M hardness, pot life, content assay, oscillatory rheology, mechanical testing) of rings containing both dapivirine and levonorgestrel, a lead candidate formulation was selected that was amenable to practical ring manufacture via injection molding, exhibited no substantial levonorgestrel binding, and offered suitable mechanical properties., Competing Interests: Leeanne Brown, Matthew Kihara, François Martin and Nicole McMullen are employees of Elkem Silicones. The other authors declare no competing financial or personal interest., (© 2021 The Author(s).)

Vaginal rings releasing antiretrovirals - either alone or in combination with contraceptive progestins - are being developed for prevention of human immunodeficiency virus (HIV) transmission via vaginal sex. Following Phase I trials, significant discolouration was observed on the surface of investigational silicone elastomer antiretroviral-contraceptive matrix-type vaginal rings containing either 25 mg dapivirine or 200 mg dapivirine plus levonorgestrel. In this study, potential causes of the discolouration have been assessed in vitro using simulated vaginal and menstrual fluids (SVF and SMF, respectively) to model in vivo exposure. The fluid compositions also included hydrogen peroxide (H 2 O 2 ), hydrogen peroxide plus a copper intrauterine device (IUD), or synthetic dyes (representing personal care and household cleaning products). No discolouration was observed for rings exposed to SVF + hydrogen peroxide (with or without an IUD). However, the SVF + dye compositions showed significant ring discolouration, with staining patterns similar to those observed with rings that had been exposed to highly-coloured personal care and household cleaning products during clinical trial use. Exposure of rings to SMF compositions invariably caused yellow surface discolouration, dark spotting and markings, similar to the staining patterns observed following clinical use. The darker marks on the ring surface were identified as blood debris derived from the SMF. The study indicates that surface discolouration of rings in vivo can be attributed to exposure to menstrual fluid or highly coloured personal care or household cleaning products. Discolouration of the rings was not associated with any specific safety risks for the user, though severe discolouration could potentially impact acceptability and adherence., Competing Interests: All authors declare no actual or potential conflicts of interest., (© 2021 The Author(s).)

We investigate the effect of the alcohol-induced increase in the content of CYP2E1 in human liver microsomes (HLM) on the function of CYP3A4. Membrane incorporation of the purified CYP2E1 into HLM considerably increases the rate of metabolism of 7-benzyloxyquinoline (BQ) and attenuates the homotropic cooperativity observed with this CYP3A4-specific substrate. It also eliminates the activating effect of α-naphthoflavone (ANF) seen in some HLM samples. To probe the physiological relevance of these effects, we compared three pooled preparations of HLM from normal donors (HLM-N) with a pooled preparation from ten heavy alcohol consumers (HLM-A). The composition of the P450 pool in all samples was characterized by the mass-spectrometric determination of 11 cytochrome P450 species. The fractional content of CYP2E1 in HLM-A was from 2.0 to 3.4 times higher than in HLM-N. In contrast, the content of CYP3A4 in HLM-A was the lowest among all samples. Despite that, HLM-A exhibited a much higher metabolism rate and a lower homotropic cooperativity with BQ, similar to CYP2E1-enriched HLM-N. To substantiate the involvement of interactions between CYP2E1 and CYP3A4 in these effects, we probed hetero-association of these proteins in CYP3A4-containing Supersomes™ with a technique employing CYP2E1 labeled with BODIPY-618 maleimide. These experiments evinced the interactions between the two enzymes and revealed an inhibitory effect of ANF on their association. Our results demonstrate that the functional properties of CYP3A4 are fundamentally dependent on the composition of the cytochrome P450 ensemble and suggest a possible impact of chronic alcohol exposure on the pharmacokinetics of drugs metabolized by CYP3A4., (Copyright © 2020 Elsevier Inc. All rights reserved.)

We closely characterized 7-Dimethylamino-4-trifluromethylcoumarin (Coumarin 152, C152), a substrate metabolized by multiple P450 species, to establish a new fluorogenic probe for the studies of functional integration in the cytochrome P450 ensemble. Scanning fluorescence spectroscopy and LC/MS-MS were used to characterize the products of N-demethylation of C152 and optimize their fluorometric detection. The metabolism of C152 by the individual P450 species was characterized using the microsomes containing cDNA-expressed enzymes. C152 metabolism in human liver microsomes (HLM) was studied in a preparation with quantified content of eleven P450 species. C152 is metabolized by CYP2B6, CYP3A4, CYP3A5, CYP2C19, CYP1A2, CYP2C9, and CYP2C8 listed in the order of decreasing turnover. The affinities exhibited by CYP3A5, CYP2C9, and CYP2C8 were lower than those characteristic to the other enzymes. The presumption of additivity suggests the participation of CYP3A4, CYP2B6, and CYP2C19 to be 84, 8, and 0.2%, respectively. Contrary to this prediction, inhibitory analysis identified CYP2C19 as the principal C152-metabolizing enzyme. We thoroughly characterize C152 for the studies of drug metabolism in HLM and demonstrate the limitations of the proportional projection approach by providing an example, where the involvement of individual P450 species cannot be predicted from their content.

In this study, we investigate the ability of ethanol-inducible CYP2E1 to interact with other cytochrome P450 species and affect the metabolism of their substrates. As a model system, we used CYP2E1-enriched human liver microsomes (HLM) obtained by the incorporation of purified CYP2E1. Using a technique based on homo-FRET in oligomers of CYP2E1 labeled with BODIPY 577/618 maleimide we demonstrated that the interactions of CYP2E1 with HLM result in the formation of its mixed oligomers with other P450 species present in the microsomal membrane. Incorporation of CYP2E1 results in a multifold increase in the rate of metabolism of CYP2E1-specific substrates p-Nitrophenol and Chlorzaxozone. The rate of their oxidation remains proportional to the amount of incorporated CYP2E1 up to the content of 0.3-0.4 nmol/mg protein (or ∼50% CYP2E1 in the P450 pool). The incorporated CYP2E1 becomes a fully functional member of the P450 ensemble and do not exhibit any detectable functional differences with the endogenous CYP2E1. Enrichment of HLM with CYP2E1 results in pronounced changes in the metabolism of 7-ethoxy-4-cyanocoumarin (CEC), the substrate of CYP2C19 and CYP1A2 suggesting an increase in the involvement of the latter in its metabolism. This effect goes together with an augmentation of the rate of dealkylation of CYP1A2-specific substrate 7-ethoxyresorufin. Furthermore, probing the interactions of CYP2E1 with model microsomes containing individual P450 enzymes we found that CYP2E1 efficiently interacts with CYP1A2, but lacks any ability to form complexes with CYP2C19. This finding goes inline with CYP2E1-induced redirection of the main route of CEC metabolism from CYP2C19 to CYP1A2., (© 2019 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Bacterial cytochrome P450 (CYP) enzymes are involved in the hydroxylation of various endogenous substrates while using a heme molecule as a cofactor. CYPs have gained biotechnological interest as useful biocatalysts capable of altering chemical structures by adding a hydroxyl group in a regiospecific manner. Here, we identified, purified, and characterized two CYP154C4 proteins from Streptomyces sp. W2061 (StCYP154C4-1) and Streptomyces sp. ATCC 11861 (StCYP154C4-2). Activity assays showed that both StCYP154C4-1 and StCYP154C4-2 can produce 2'-hydroxylated testosterone, which differs from the activity of a previously described NfCYP154C5 from Nocardia farcinica in terms of its 16α-hydroxylation of testosterone. To better understand the molecular basis of the regioselectivity of these two CYP154C4 proteins, crystal structures of the ligand-unbound form of StCYP154C4-1 and the testosterone-bound form of StCYP154C4-2 were determined. Comparison with the previously determined NfCYP154C5 structure revealed differences in the substrate-binding residues, suggesting a likely explanation for the different patterns of testosterone hydroxylation, despite the high sequence similarities between the enzymes (54% identity). These findings provide valuable insights that will enable protein engineering for the development of artificial steroid-related CYPs exhibiting different regiospecificity., (© 2018 Federation of European Biochemical Societies.)

Here, we report the first bacterial cytochrome P450, CYP154C8, that catalyzes the C-C bond cleavage reaction of steroids. A major change in product distribution is observed with CYP154C8, when the reactions are supported by NADPH and spinach redox partners ferredoxin and ferredoxin reductase, compared with previously reported reactions supported by NADH and redox partners containing putidaredoxin and putidaredoxin reductase. The NMR-based structural elucidation of reaction products reveals 21-hydroxyprednisone as the major product for prednisone, while the other product is identified as 1-dehydroadrenosterone obtained due to C-C bond cleavage. A similar pattern of product formation is observed with cortisone, hydrocortisone, and prednisone. The reaction catalyzed by CYP154C8 in the presence of oxygen surrogates also prominently shows the formation of C-C bond cleavage products., (© 2018 Federation of European Biochemical Societies.)

CYP154C8 catalyzes the hydroxylation of diverse steroids, as has previously been demonstrated, by using an NADH-dependent system including putidaredoxin and putidaredoxin reductase as redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8 reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase displayed catalytic activity different from that of the NADH-dependent system. The NADPH-dependent system showed multistep oxidation of progesterone and other substrates including androstenedione, testosterone, and nandrolone. (Diacetoxyiodo)benzene was employed to generate compound I (FeO 3+ ), actively supporting the redox reactions catalyzed by CYP154C8. In addition to 16α-hydroxylation, progesterone and 11-oxoprogesterone also underwent hydroxylation at the 6β-position in reactions supported by (diacetoxyiodo)benzene. CYP154C8 was active in the presence of high concentrations (>10 mm) of H 2 O 2 , with optimum conversion surprisingly being achieved at ≈75 mm H 2 O 2 . More importantly, H 2 O 2 tolerance by CYP154C8 was evident in the very low heme oxidation rate constant (K) even at high concentrations of H 2 O 2 . Our results demonstrate that alternative redox partners and oxidizing agents influence the catalytic efficiency and product distribution of a cytochrome P450 enzyme. More importantly, these choices affected the type and selectivity of reaction catalyzed by the P450 enzyme., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

CYP154C8 from Streptomyces sp. has been identified as a new cytochrome P450 with substrate flexibility towards different sets of steroids. In vitro treatment of these steroids with CYP154C8 revealed interesting product formation patterns with the same group of steroids. NMR study revealed the major product of corticosterone to be hydroxylated at the C21 position, whereas progesterone, androstenedione, testosterone, and 11-ketoprogesterone were exclusively hydroxylated at the 16α position. However, the 16α-hydroxylated product of progesterone was further hydroxylated to yield dihydroxylated products. 16-hydroxyprogesterone was hydroxylated at two positions to yield dihydroxylated products: 2α,16α-dihydroxyprogesterone and 6β,16α-dihydroxyprogesterone. To the best of our knowledge, this is the first report of generation of such products through enzymatic hydroxylation by a CYP450. In view of the importance of modified steroids as pharmaceutical components, CYP154C8 has immense potential for utilization in bioproduction of hydroxylated derivative compounds to be directly employed for pharmaceutical applications., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bacterial cytochrome P450 (CYP) steroid hydroxylases are effectively useful in the pharmaceutical industry for introducing hydroxyl groups to a wide range of steroids. We found a putative CYP steroid hydroxylase ( Ba CYP106A2) from the bacterium Bacillus sp. PAMC 23377 isolated from Kara Sea of the Arctic Ocean, showing 94% sequence similarity with Bm CYP106A2 ( Bacillus megaterium ATCC 13368). In this study, soluble Ba CYP106A2 was overexpressed to evaluate its substrate-binding activity. The substrate affinity ( K d value) to 4-androstenedione was 387 ± 37 µM. Moreover, the crystal structure of Ba CYP106A2 was determined at 2.7 Å resolution. Structural analysis suggested that the α8-α9 loop region of Ba CYP106A2 is intrinsically mobile and might be important for initial ligand binding. The hydroxyl activity of Ba CYP106A2 was identified using in vitro enzyme assays. Its activity was confirmed with two kinds of steroid substrates, 4-androstenedione and nandrolone, using chromatography and mass spectrometry methods. The main products were monohydroxylated compounds with high conversion yields. This is the second study on the structure of CYP106A steroid hydroxylases, and should contribute new insight into the interactions of bacterial CYP106A with steroid substrates, providing baseline data for studying the CYP106A steroid hydroxylase from the structural and enzymatic perspectives.

The present study reports an efficient in vitro micropropagation protocol for a medicinally important tree, Terminalia bellerica Roxb. from nodal segments of a 30 years old tree. Nodal segments taken from the mature tree in March-April and cultured on half strength MS medium gave the best shoot bud proliferation response. Combinations of serial transfer technique (ST) and incorporation of antioxidants (AO) [polyvinylpyrrolidone, PVP (50 mg l(-1)) + ascorbic acid (100 mg l(-1)) + citric acid (10 mg l(-1))] in the culture medium aided to minimize browning and improve explant survival during shoot bud induction. Highest multiplication of shoots was achieved on medium supplemented with 6-benzyladenine (BA, 8.8 μM) and α-naphthalene acetic acid (NAA, 2.6 μM) in addition to antioxidants. Shoot elongation was obtained on MS medium containing BA (4.4 μM) + phloroglucinol (PG, 3.9 μM). Elongated shoots were transferred to half strength MS medium containing indole-3-butyric acid (IBA, 2.5 μM) for root development. The acclimatization of plantlets was carried out under greenhouse conditions. The genetic fidelity of the regenerated plants was checked using inter simple sequence repeats (ISSR) and randomly amplified polymorphic DNA (RAPD) analysis. Comparison of the bands among the regenerants and mother plant confirmed true-to-type clonal plants.

Objective: This study was designed to determine the anti-inflammatory activity of docosahexaenoic acid (DHA), alone and in combination with eicosapentaenoic acid (EPA), in a murine model of rheumatoid arthritis, collagen induced arthritis (CIA)., Methods: The CIA was induced in DBA/1OlaHsd mice by the injection of bovine type II collagen in Freunds's complete adjuvant on days 0 and 21. Mice were fed modified diets containing DHA and/or EPA for 4 weeks prior to the initial collagen injection until study termination at day 45. The severity of CIA was assessed by measuring erythema, edema and mobility of the digits on the fore and hind paws, as well as histology. The level of serum anti-collagen antibodies was determined by ELISA. The ex vivo effects of DHA and/or EPA on splenocyte proliferation and cytokine production were evaluated by BrdU method and ELISA., Results: Prophylactic treatment with DHA, and not DHA/EPA, significantly reduced arthritis severity and joint damage. Treatment with DHA also decreased anti-collagen (CII) antibodies in vivo, downregulated interleukin-1β, interferonγ and upregulated protective interleukin-10 ex vivo., Conclusion: Prophylactic treatment with DHA was efficacious in a mouse model of rheumatoid arthritis and may be a useful intervention strategy against inflammatory arthritis.

Objective: Enzymatically oxygenated lipid products derived from omega-3 and omega-6 fatty acids play an important role in inflammation dampening. This study examined the anti-inflammatory effects of n-6 docosapentaenoic acid-derived (17S)-hydroxy-docosapentaenoic acid (17-HDPAn-6) and (10,17S)-dihydroxy-docosapentaenoic acid (10,17-HDPAn-6) as well as n-3 docosahexaenoic acid-derived 17(R/S)-hydroxy-docosahexaenoic acid (17-HDHA)., Materials and Methods: The effects of 17-HDPAn-6, 10,17-HDPAn-6 or 17-HDHA on activity and M1/M2 polarization of murine macrophage cell line RAW 264.7 were examined by phagocytosis assay and real-time PCR. To assess anti-inflammatory effects in vivo, dextran sodium sulfate (DSS) colitis was induced in mice treated with 17-HDPAn-6, 10,17-HDPAn-6, 17-HDHA or NaCl., Results: Our results show that 17-HDPAn-6, 10,17-HDPAn-6 and 17-HDHA increase phagocytosis in macrophages in vitro and promote polarization towards the anti-inflammatory M2 phenotype with decreased gene expression of TNF-α and inducible Nitric oxide synthase and increased expression of the chemokine IL-1 receptor antagonist and the Scavenger receptor Type A. Intraperitoneal treatment with 17-HDPAn-6, 10,17-HDPAn-6, or 17-HDHA alleviated DSS-colitis and significantly improved body weight loss, colon epithelial damage, and macrophage infiltration., Conclusion: These results suggest that DPAn-6-derived 17-HDPAn-6 and 10,17-HDPAn-6 as well as the DHA-derived 17-HDHA have inflammation-dampening and resolution-promoting effects that could be used to treat inflammatory conditions such as inflammatory bowel disease.

The active catalysts for the enantioselective ring opening (ARO) of meso-stilbene oxide, cis-butene oxide, cyclohexene oxide, cyclopentene oxide, and cyclooctene oxide with various substituted anilines were generated in situ by the reaction of Ti(O(i)Pr)(4) with poly-[(R,R)-N,N'-bis-{3-(1,1-dimethylethyl)-5-methylene salicylidene} cyclohexane-1,2-diamine]-1 and (1R,2R)-N,N'-bis[3,5-di(tert-butyl)salicylidene] cyclohexane-1,2-diamine-2. These catalysts in the presence of nonracemic imine as an additive provided β-amino alcohol in excellent yield (99%) and chiral purity (enantiomeric excess (ee) up to 99%) for the ARO of meso-stilbene oxide with aniline. The same protocol was less effective for the ARO of cyclic epoxides; however, when triphenylphosphine was used as an additive, there was a significant improvement in catalyst performance for the ARO of cyclohexene oxide (yield, 85-90%; ee, 63-67%). Both in situ generated polymeric and monomeric catalysts performed in a similar manner except that the polymeric catalyst Ti(IV)-1 was more active and recycled several times with retention of enantioselectivity when compared with the monomeric catalyst Ti(IV)-2, which was nonrecyclable., (Copyright © 2010 Wiley-Liss, Inc.)

17S-HDPAn-6 (17S-hydroxydocosa-4Z,7Z,10Z,13Z,15E-pentaenoic acid) and 10S,17S-HDPAn-6 (10S,17S-dihydroxydocosa-4Z,7Z,11E,13Z,15E-pentaenoic acid) are potent anti-inflammatory resolvins derived from DPAn-6 (docosapentaenoic acid n-6) and are analogous in structure and action to DHA (docosahexaenoic acid)-derived resolvins. These resolvins have proven to be potential drug candidates, albeit with therapeutic profiles that need optimization. The main objectives of this study were to evaluate key features of DPAn-6 derived resolvins that are important for therapeutic efficacy, demonstrate that these DPAn-6 resolvins could be produced naturally, and could therefore have physiological significance. Here we demonstrate biological production, examine pharmacokinetic profiles and identify key routes of metabolic inactivation of DPAn-6 derived resolvins. We compare their metabolic stability to a known resolvin, 17S-HDHA (17S-hydroxydocosa-4Z,7Z,10Z,13Z,15E,19Z-hexaenoic acid) and show that order of their stabilities is 10S,17S-HDPAn-6>17S-HDPAn-6>17S-HDHA. We show that both these compounds are not strong inhibitors of cytochrome-P450 enzymes. We evaluate activity of compounds in the delayed-type hypersensitivity model, results of which show that compounds need optimization for enhanced duration and magnitude of action. Analysis of the metabolic stability and identification of metabolites of these compounds could play an important role in the design of better analogs with longer durations of action and hence better efficacy.

Enzymatically oxygenated derivatives of the omega-3 fatty acids cis-4,7,10,13,16,19-docosahexaenoic acid (DHA) and cis-5,8,11,14,17-eicosapentaenoic acid, known as resolvins, have potent inflammation resolution activity (Serhan, C. N., Clish, C. B., Brannon, J., Colgan, S. P., Chiang, N., and Gronert, K. (2000) J. Exp. Med. 192, 1197-1204; Hong, S., Gronert, K., Devchand, P. R., Moussignac, R., and Serhan, C. N. (2003) J. Biol. Chem. 278, 14677-14687). Our objective was to determine whether similar derivatives are enzymatically synthesized from other C-22 fatty acids and whether these molecules possess inflammation resolution properties. The reaction of DHA, DPAn-3, and DPAn-6 with 5-, 12-, and 15-lipoxygenases produced oxylipins, which were identified and characterized by liquid chromatography coupled with tandem mass-spectrometry. DPAn-6 and DPAn-3 proved to be good substrates for 15-lipoxygenase. 15-Lipoxygenase proved to be the most efficient enzyme of the three tested for conversion of long chain polyunsaturated fatty acids to corresponding oxylipins. Since DPAn-6 is a major component of Martek DHA-S oil, we focused our attention on reaction products obtained from the DPAn-6 and 15-lipoxygenase reaction. (17S)-hydroxy-DPAn-6 and (10,17S)-dihydroxy-DPAn-6 were the main products of this reaction. These compounds were purified by preparatory high performance liquid chromatography techniques and further characterized by NMR, UV spectrophotometry, and tandem mass spectrometry. We tested both compounds in two animal models of acute inflammation and demonstrated that both compounds are potent anti-inflammatory agents that are active on local intravenous as well as oral administration. These oxygenated DPAn-6 compounds can thus be categorized as a new class of DPAn-6-derived resolvins.

Single amino acid substitutions rarely produce substantial changes in protein structure. Here we show that substitution of the C-cap residue in the alpha-helix of ubiquitin with proline (34P variant) leads to dramatic structural changes. The resulting conformational perturbation extends over the last two turns of the alpha-helix and leads to enhanced flexibility for residues 27-37. Thermodynamic analysis of this ubiquitin variant using differential scanning calorimetry reveals that the thermal unfolding transition remains highly cooperative, exhibiting two-state behavior. Similarities with the wild type in the thermodynamic parameters (heat capacity change upon unfolding and m-value) of unfolding monitored by DSC and chemical denaturation suggests that the 34P variant has comparable buried surface area. The hydrophobic core of 34P variant is not packed as well as that of the wild type protein as manifested by a lower enthalpy of unfolding. The increased mobility of the polypeptide chain of this ubiquitin variant allows the transient opening of the hydrophobic core as evidenced by ANS binding. Taken together, these results suggest exceptional robustness of cooperativity in protein structures.

The transcriptional activator, MarA, interacts with RNA polymerase (RNAP) to activate promoters of the mar regulon. Here, we identify the interacting surfaces of MarA and of the carboxy-terminal domain of the alpha subunit of RNAP (alpha-CTD) by NMR-based chemical shift mapping. Spectral changes were monitored for a MarA-DNA complex upon titration with alpha-CTD, and for alpha-CTD upon titration with MarA-DNA. The mapping results were confirmed by mutational studies and retention chromatography. A model of the ternary complex shows that alpha-CTD uses a '265-like determinant' to contact MarA at a surface distant from the DNA. This is unlike the interaction of alpha-CTD with the CRP or Fis activators where the '265 determinant' contacts DNA while another surface of the same alpha-CTD molecule contacts the activator. These results reveal a new versatility for alpha-CTD in transcriptional activation.

We have determined the high-resolution solution structure of the oxidized form of a chimeric human and Escherichia coli thioredoxin (TRX(HE)) by NMR. The overall structure is well-defined with a rms difference for the backbone atoms of 0.27 +/- 0.06 A. The topology of the protein is identical to those of the human and E. coli parent proteins, consisting of a central five-stranded beta-sheet surrounded by four alpha-helices. Analysis of the interfaces between the two domains derived from the human and E. coli sequences reveals that the general hydrophobic packing is unaltered and only subtle changes in the details of side chain interactions are observed. The packing of helix alpha(4) with helix alpha(2) across the hybrid interface is less optimal than in the parent molecules, and electrostatic interactions between polar side chains are missing. In particular, lysine-glutamate salt bridges between residues on helices alpha(2) and alpha(4), which were observed in both human and E. coli proteins, are not present in the chimeric protein. The origin of the known reduced thermodynamic stability of TRX(HE) was probed by mutagenesis on the basis of these structural findings. Two mutants of TRX(HE), S44D and S44E, were created, and their thermal and chemical stabilities were examined. Improved stability toward chaotropic agents was observed for both mutants, but no increase in the denaturation temperature was seen compared to that of TRX(HE). In addition to the structural analysis, the backbone dynamics of TRX(HE) were investigated by (15)N NMR relaxation measurements. Analysis using the model free approach reveals that the protein is fairly rigid with an average S(2) of 0.88. Increased mobility is primarily present in two external loop regions comprising residues 72-74 and 92-94 that contain glycine and proline residues.

The transcriptional activator Rob consists of an N-terminal domain (NTD) of 120 amino acids responsible for DNA binding and promoter activation and a C-terminal domain (CTD) of 169 amino acids of unknown function. Although several thousand molecules of Rob are normally present per Escherichia coli cell, they activate promoters of the rob regulon poorly. We report here that in cells treated with either 2,2"- or 4,4"-dipyridyl (the latter is not a metal chelator), Rob-mediated transcription of various rob regulon promoters was increased substantially. A small, growth-phase-dependent effect of dipyridyl on the rob promoter was observed. However, dipyridyl enhanced Rob's activity even when rob was regulated by a heterologous (lac) promoter showing that the action of dipyridyl is mainly posttranscriptional. Mutants lacking from 30 to 166 of the C-terminal amino acids of Rob had basal levels of activity similar to that of wild-type cells, but dipyridyl treatment did not enhance this activity. Thus, the CTD is not an inhibitor of Rob but is required for activation of Rob by dipyridyl. In contrast to its relatively low activity in vivo, Rob binding to cognate DNA and activation of transcription in vitro is similar to that of MarA, which has a homologous NTD but no CTD. In vitro nuclear magnetic resonance studies demonstrated that 2,2"-dipyridyl binds to Rob but not to the CTD-truncated Rob or to MarA, suggesting that the effect of dipyridyl on Rob is direct. Thus, it appears that Rob can be converted from a low activity state to a high-activity state by a CTD-mediated mechanism in vivo or by purification in vitro.

An unanswered question regarding gene regulation is how certain proteins are capable of binding to DNA with high affinity at specific but highly degenerate consensus sequences. We have investigated the interactions between the Escherichia coli transcription factor, MarA, and its diverse binding sites using NMR techniques. Complete resonance assignments for the backbone of the MarA protein complexed with DNA oligomers corresponding to its binding sites at the mar, fumC, micF and the fpr promoters were obtained. Secondary structure analysis based on chemical shifts reveals that regions identified as helical in the X-ray structure of the MarA-mar complex are present in the solution structure, although some of the helices are less well defined. The chemical shift differences between the four complexes confirm that helix 3 and helix 6 constitute the major DNA-binding elements. However, in striking contrast with the X-ray data: (i) the protein appears to be present in two or more conformations in each of the complexes; (ii) no slowly exchanging N(zeta)H(2) protons (indicative of hydrogen bonded groups) were observed by NMR for the two arginine residues proposed to form crucial hydrogen bonds in the X-ray structure; and (iii) regions at the N terminus, not observed in the X-ray structure, may be involved in DNA-binding. Taken together, the NMR results indicate that MarA in its complexes with DNA target sites is in a highly dynamic state, allowing for small but significant rearrangements of the side-chains and/or backbone to bind to the different DNA sequences.

Using surface-modified electrodes composed of omega-hydroxyalkanethiols, an experimentally based value for the inner-sphere reorganization energy of the bis(imidazole)iron porphyrin system has been obtained by examining the solvent dependence of the reorganization energy of bis(N-methylimidazole)meso-tetraphenyl iron porphyrin. The value obtained (0.41 +/- 0.06 eV) is remarkably similar to values we have recently reported for the reorganization energy of cytochrome b(5) (0.43 +/- 0.02 eV) and cytochrome c (0.58 +/- 0.06 eV). This strongly suggests that the protein matrix mimics the behavior of a low dielectric solvent and effectively shields the heme from the solvent. The effect of the orientation of the heme relative to the electrode was also explored by sytematically varying the steric bulk of the axial ligands. On the basis of a good linear correlation between the electronic coupling and the cosine of the angle between the heme plane and the surface of the electrode, it is suggested that a parallel orientation of the heme yields a maximum in the electronic coupling. Relevance to interheme protein electron transfer is discussed.

On the basis of a comparison of high-resolution solution structures calculated for both equilibrium forms of rat ferrocytochrome b5, differences in reduction potential and thermodyanmic stability have been characterized in terms of significant structural and dynamic differences between the two forms. The dominant difference between A and B conformations has long been known to be due to a 180 degrees rotation of the heme in the binding pocket about an axis defined by the alpha- and gamma-meso carbons, however, the B form has not been structurally characterized until now. The most significant differences observed between the two forms were the presence of a hydrogen bond between the 7-propionate and the S64 amide in the A form but not the B form and surprisingly a displacement of the heme out of the binding pocket by 0.9 A in the B form relative to the A form. The magnitude of other factors which could contribute to the known difference in reduction potentials in the bovine protein [Walker, F. A., Emrick, D., Rivera, J. E., Hanquet, B. J., and Buttlaire, D. H. (1988) J. Am. Chem. Soc. 110, 6234-6240], such as differences in the orientation of the axial imidazoles and differences in hydrogen bond strength to the imidazoles, have been evaluated. The dominant effector of the reduction potential would appear to be the lack of the hydrogen bond to the S64 amide in the B form which frees up the propionate to charge stabilize the iron in the oxidized state and thus lower the reduction potential of the B form. The structure we report for the A form, based on heteronuclear NMR restraints, involving a total of 1288 restraints strongly resembles both the X-ray crystal structure of the bovine protein and a recently reported structure for the A form of the rat protein based on homonuclear data alone [Banci, L., Bertini, I., Ferroni, F., and Rosato, A. (1997) Eur. J. Biochem. 249, 270-279]. The rmsd for the backbone atoms of the A form is 0.54 A (0.92 A for all non-hydrogens). The rmsd for the backbone of the B form is 0.51 A (0. 90 A for all non-hydrogen atoms). An analysis of backbone dynamics based on a model-free analysis of 15N relaxation data, which incorporated axially symmetric diffusion tensor modeling of the cytochrome, indicates that the protein is more rigid in the reduced state relative to the oxidized state, based on a comparison with order parameters reported for the bovine protein in the oxidized state [Kelly, G. P., Muskett, F. W., and Whitford, D. (1997) Eur. J. Biochem. 245, 349-354].

Mutants of cytochrome b5 were designed to achieve reorientation of individual axial imidazole ligands. The orientation of the axial ligand planes is thought to modulate the reduction potential of bis(imidazole) axially ligated heme proteins. The A67V mutation achieved this goal through the substitution of a bulkier, hydrophobic ligand for a residue, in the sterically hindered hydrophobic heme binding pocket. Solution structures of mutant and wild-type proteins in the region of the mutation were calculated using restraints obtained from 1H and 15N 2D homonuclear and heteronuclear NMR spectra and 1H-15N 3D heteronuclear NMR spectra. More than 10 restraints per residue were used in the refinement of both structures. Average local rmsd for 20 refined structures was 0.30 A for the wild-type structure and 0.38 A for the A67V mutant. The transfer of amide proton resonance assignments from wild-type to the mutant protein was achieved through overlays of 15N-1H heteronuclear correlation spectra of the reduced proteins. Side chain assignments and sequential assignments were established using conventional assignment strategies. Calculation of the orientation of the components of the anisotropic paramagnetic susceptibility tensor, using methods similar to procedures applied to the wild-type protein, shows that the orientation of the in-plane components are identical in the wild-type and mutant proteins. However, the orientation of the z-component of the susceptibility tensor calculated for the mutant protein differs by 17 degrees for the A-form and by 11 degrees for the B-form from the orientation calculated for the wild-type protein. The rotation of the z-component of the susceptibility tensor (toward the delta meso proton) is in the same direction and is of the same magnitude as the rotation of the H63 imidazole ring induced by mutation.