Nitric oxide synthases (NOS) are hemoproteins that catalyze the reaction of L-arginine to L-citrulline and nitric oxide. N-(3-(Aminomethyl)benzyl)acetamidine (1400W) was reported to be a slow, tight-binding, and highly selective inhibitor of iNOS in vitro and in vivo. Previous mechanistic studies reported that 1400W was recovered quantitatively after iNOS fully lost its activity and modification to iNOS was not detected. Here, it is shown that 1400W is a time-, concentration-, and NADPH-dependent irreversible inactivator of iNOS. HPLC-electrospray mass spectrometric analysis of the incubation mixture of iNOS with 1400W shows both loss of heme cofactor and formation of biliverdin, as was previously observed for iNOS inactivation by another amidine-containing compound, N5-(1-iminoethyl)-L-ornithine (L-NIO). The amount of biliverdin produced corresponds to the amount of heme lost by 1400W inactivation of iNOS. A convenient MS/MS-HPLC methodology was developed to identify the trace amount of biliverdin produced by inactivation of iNOS with either 1400W or L-NIO to be biliverdin IXalpha out of the four possible regioisomers. Two mechanisms were previously proposed for iNOS inactivation by L-NIO: (1) uncoupling of the heme peroxide intermediate, leading to destruction of the heme to biliverdin; (2) abstraction of a hydrogen atom from the amidine methyl group followed by attachment to the heme cofactor, which causes the enzyme to catalyze the heme oxygenase reaction. The second mechanistic proposal was ruled out by inactivation of iNOS with d3-1400W, which produced no d2-1400W. Detection of carbon monoxide as one of the heme-degradation products further excludes the covalent heme adduct mechanism. On the basis of these results, a third mechanism is proposed in which the amidine inactivators of iNOS bind as does substrate L-arginine, but because of the amidine methyl group, the heme peroxy intermediate cannot be protonated, thereby preventing its conversion to the heme oxo intermediate. This leads to a change in the enzyme mechanism to one that resembles that of heme oxygenase, an enzyme known to convert heme to biliverdin IXalpha. This appears to be the first example of a compound that causes irreversible inactivation of an enzyme without itself becoming modified in any way.

Introduction Major phenolics from licorice roots (Glycyrrhiza sp.) are glycosides of the flavanone liquiritigenin (F) and its 2′-hydroxychalcone isomer, isoliquiritigenin (C). As the F and C contents fluctuate between batches of licorice, both quality control and standardisation of its preparations become complex tasks. Objective To characterise the F and C metabolome in extracts from Glycyrrhiza glabra L. and Glycyrrhiza uralensis Fisch. ex DC. by addressing their composition in major F–C pairs and defining the total F:C proportion. Material and methods Three types of extracts from DNA-authenticated samples were analysed by a validated UHPLC/UV method to quantify major F and C glycosides. Each extract was characterised by the identity of major F–C pairs and the proportion of Fs among all quantified Fs:Cs. Results The F and C compositions and proportions were found to be constant for all extracts from a Glycyrrhiza species. All G. uralensis extracts contained up to 2.5 more Fs than G. glabra extracts. Major F–C pairs were B-ring glycosidated in G. uralensis, and A-/B-ring apiosyl-glucosidated in the G. glabra extracts. The F:C proportion was found to be linked to the glycosidation site: the more B-ring F-C glycosides were present, the higher was the final F:C proportion in the extract. These results enable the chemical differentiation of extracts from G. uralensis and G. glabra, which are characterised by total F:C proportions of 8.37:1.63 and 7.18:2.82, respectively. Conclusion Extracts from G. glabra and G. uralensis can be differentiated by their respective F and C compositions and proportions, which are both useful for further standardisation of licorice botanicals.

Nitric oxide synthase (NOS) catalyzes the conversion of l-arginine to l-citrulline and nitric oxide. N 5-(1-Iminoethyl)-l-ornithine (l-NIO, 5) is a natural product known to inactivate NOS, but the mechanism of inactivation is unknown. Upon incubation of iNOS with l-NIO a type I binding difference spectrum is observed, indicating that binding at the substrate binding site occurs. l-NIO is shown to be a time-dependent, concentration-dependent, and NADPH-dependent irreversible inhibitor of iNOS with K I and k inact values of 13.7 ± 1.6 μM and 0.073 ± 0.003 min-1, respectively. During inactivation the heme chromophore is partially lost (Figure ); HPLC shows that the loss corresponds to about 50% of the heme. Inclusion of catalase during incubation does not prevent heme loss. N 5-(1-Imino-2-[14C]ethyl)-l-ornithine (11) inactivates iNOS, but upon dialysis or gel filtration, no radioactivity remains bound to the protein or to a cofactor. The only radioactive product detected after enzyme inactivation is N ω-hydroxy-l-NIO (12); no C ω-hydroxy-l-NIO (13) or N δ-acetyl-l-ornithine (14) is observed (Figure ). The amount of 12 produced during the inactivation process is 7.7 ± 0.2 equiv per inactivation event. Incubations of 12 with iNOS show time-, concentration-, and NADPH-dependent inactivation that is not reversible upon dilution into the assay solution. Incubations that include an excess of l-arginine or with substitution of NADP+ for NADPH result in no significant loss of enzyme activity. The K I and k inact values for 12 are 830 ± 160 μM and 0.0073 ± 0.0007 min-1, respectively. The magnitude of these kinetic constants (compared with those of 5) suggest that 12 is not an intermediate of l-NIO inactivation of iNOS. Compound 12 also is a substrate for iNOS, exhibiting saturation kinetics with K m and k cat values of 800 ± 85 μM and 2.22 min-1, respectively; the product is shown to be N δ-acetyl-l-ornithine (14) (Figure ). The k cat and k inact values for 12 can be compared directly to give a partition ratio (k cat/k inact) for inactivation of 304; i.e., there are 304 turnovers to give NO per inactivation event. This high partition ratio further supports the notion that 12 is not involved in l-NIO inactivation of iNOS. C ω-Hydroxy-l-NIO (13) is not an inactivator of iNOS. These results suggest that l-NIO inactivation occurs after an oxidation step (NADPH is required for inactivation) but prior to a hydroxylation step (12 and 13 are not involved). Inactivation of iNOS by N 5-(1-imino-2-[2H3]ethyl)-l-ornithine (15) exhibits a kinetic isotope effect on H k inact/D k inact of 1.35 ± 0.08 and on H(k inact/K I)/D(k inact/K I) of 1.51 ± 0.3, suggesting that the methyl C−H bond is cleaved in a partially rate-determining step prior to hydroxylation, and that leads to inactivation. A new NADPH-dependent 400 nm peak in the HPLC of l-NIO-inactivated iNOS is produced (Figure ). LC−electrospray mass spectrometry (Figure ) demonstrates the m/z of the new metabolite to be 583, which is shown to correspond to biliverdin (23) (Figures and ...

This study introduces a flexible and compound targeted approach to Deplete and Enrich Select Ingredients to Generate Normalized Extract Resources, generating DESIGNER extracts, by means of chemical subtraction or augmentation of metabolites. Targeting metabolites based on their liquid–liquid partition coefficients (K values), K targeting uses countercurrent separation methodology to remove single or multiple compounds from a chemically complex mixture, according to the following equation: DESIGNER extract = total extract ± target compound(s). Expanding the scope of the recently reported depletion of extracts by immunoaffinity or solid phase liquid chromatography, the present approach allows a more flexible, single- or multi-targeted removal of constituents from complex extracts such as botanicals. Chemical subtraction enables both chemical and biological characterization, including detection of synergism/antagonism by both the subtracted targets and the remaining metabolite mixture, as well as definition of the residual complexity of all fractions. The feasibility of the DESIGNER concept is shown by K-targeted subtraction of four bioactive prenylated phenols, isoxanthohumol (1), 8-prenylnaringenin (2), 6-prenylnaringenin (3), and xanthohumol (4), from a standardized hops (Humulus lupulus L.) extract using specific solvent systems. Conversely, adding K-targeted isolates allows enrichment of the original extract and hence provides an augmented DESIGNER material. Multiple countercurrent separation steps were used to purify each of the four compounds, and four DESIGNER extracts with varying depletions were prepared. The DESIGNER approach innovates the characterization of chemically complex extracts through integration of enabling technologies such as countercurrent separation, K-by-bioactivity, the residual complexity concepts, as well as quantitative analysis by 1H NMR, LC-MS, and HiFSA-based NMR fingerprinting.