To what extent may aminochrome increase the vulnerability of dopaminergic neurons in the context of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disorder that progresses over time and is characterized by preferential reduction of dopaminergic neurons in the substantia nigra. Although the precise mechanisms leading to cell death in neurodegenerative disorders, such as PD, are not fully understood, it is widely accepted that increased oxidative stress may be a prevalent factor contributing to the deterioration of the nigrostriatal dopaminergic fibers in such conditions. Aminochrome, generated from dopamine (DA) metabolism, plays an important role in multiple pathogenic mechanisms associated with PD. Its capacity to induce a gradual reduction in dopaminergic neurons is due to its endogenous neurotoxicity. The formation of aminochrome results in the production of various reactive oxygen species (ROS), including pro-inflammatory factors, superoxide, nitric oxide, and hydroxyl radicals. This, in turn, causes loss of dopaminergic neurons, reducing DA uptake, and reduced numbers and shortened dendrites. Notably, o-quinones, which are more cytotoxic, arise from the oxidation of DA and possess a higher capacity to impede cellular defense mechanisms, thereby resulting in the death of neuronal cells. Aminochrome potentially contributes to the pathophysiology of PD by forming adducts with various proteins. All of the aforementioned effects suggest that aminochrome may play a crucial role in the pathophysiology of PD. Thus, aminochrome may serve as a more relevant preclinical model for PD, facilitating a better understanding of its pathophysiological processes and identification of novel therapeutic strategies aimed at preventing or slowing disease progression.

Moving Past Quinone-Methides: Recent Advances Toward Minimizing Electrophilic Byproducts from COS/H2S Donors.

Hydrogen sulfide (H2S) is an important biomolecule that plays key signaling and protective roles in different physiological processes. With goals of advancing both the available research tools and the associated therapeutic potential of H2S, researchers have developed different methods to deliver H2S on demand in different biological contexts. A recent approach to develop such donors has been to design compounds that release carbonyl sulfide (COS), which is quickly converted to H2S in biological systems by the ubiquitous enzyme carbonic anhydrase (CA). Although highly diversifiable, many approaches using this general platform release quinone methides or related electrophiles after donor activation. Many such electrophiles are likely scavenged by water, but recent efforts have also expanded alternative approaches that minimize the formation of electrophilic byproducts generated after COS release. This mini-review focuses specifically on recent examples of COS-based H2S donors that do no generate quinone methide byproducts after donor activation.

Functional and structural characterization of a flavoprotein monooxygenase essential for biogenesis of tryptophylquinone cofactor.

Bioconversion of peptidyl amino acids into enzyme cofactors is an important post-translational modification. Here, we report a flavoprotein, essential for biosynthesis of a protein-derived quinone cofactor, cysteine tryptophylquinone, contained in a widely distributed bacterial enzyme, quinohemoprotein amine dehydrogenase. The purified flavoprotein catalyzes the single-turnover dihydroxylation of the tryptophylquinone-precursor, tryptophan, in the protein substrate containing triple intra-peptidyl crosslinks that are pre-formed by a radical S-adenosylmethionine enzyme within the ternary complex of these proteins. Crystal structure of the peptidyl tryptophan dihydroxylase reveals a large pocket that may dock the protein substrate with the bound flavin adenine dinucleotide situated close to the precursor tryptophan. Based on the enzyme-protein substrate docking model, we propose a chemical reaction mechanism of peptidyl tryptophan dihydroxylation catalyzed by the flavoprotein monooxygenase. The diversity of the tryptophylquinone-generating systems suggests convergent evolution of the peptidyl tryptophan-derived cofactors in different proteins.

Unraveling Reversible DNA Cross-Links with a Biological Machine.

The reversible generation and capture of certain electrophilic quinone methide intermediates support dynamic reactions with DNA that allow for migration and transfer of alkylation and cross-linking. This reversibility also expands the possible consequences that can be envisioned when confronted by DNA repair processes and biological machines. To begin testing the response to such an encounter, quinone methide-based modification of DNA has now been challenged with a helicase (T7 bacteriophage gene protein four, T7gp4) that promotes 5' to 3' translocation and unwinding. This model protein was selected based on its widespread application, well characterized mechanism and detailed structural information. Little over one-half of the cross-linking generated by a bisfunctional quinone methide remained stable to T7gp4 and did not suppress its activity. The helicase likely avoids the topological block generated by this fraction of cross-linking by its ability to shift from single- to double-stranded translocation. The remaining fraction of cross-linking was destroyed during T7gp4 catalysis. Thus, this helicase is chemically competent to promote release of the quinone methide from DNA. The ability of T7gp4 to act as a Brownian ratchet for unwinding DNA may block recapture of the QM intermediate by DNA during its transient release from a donor strand. Most surprisingly, T7gp4 releases the quinone methide from both the translocating strand that passes through its central channel and the excluded strand that was typically unaffected by other lesions. The ability of T7gp4 to reverse the cross-link formed by the quinone methide does not extend to that formed irreversibly by the nitrogen mustard mechlorethamine.

The Uniqueness of Tryptophan in Biology: Properties, Metabolism, Interactions and Localization in Proteins.

Tryptophan (Trp) holds a unique place in biology for a multitude of reasons. It is the largest of all twenty amino acids in the translational toolbox. Its side chain is indole, which is aromatic with a binuclear ring structure, whereas those of Phe, Tyr, and His are single-ring aromatics. In part due to these elaborate structural features, the biosynthetic pathway of Trp is the most complex and the most energy-consuming among all amino acids. Essential in the animal diet, Trp is also the least abundant amino acid in the cell, and one of the rarest in the proteome. In most eukaryotes, Trp is the only amino acid besides Met, which is coded for by a single codon, namely UGG. Due to the large and hydrophobic π-electron surface area, its aromatic side chain interacts with multiple other side chains in the protein, befitting its strategic locations in the protein structure. Finally, several Trp derivatives, namely tryptophylquinone, oxitriptan, serotonin, melatonin, and tryptophol, have specialized functions. Overall, Trp is a scarce and precious amino acid in the cell, such that nature uses it parsimoniously, for multiple but selective functions. Here, the various aspects of the uniqueness of Trp are presented in molecular terms.

Pterocarpan synthase (PTS) structures suggest a common quinone methide-stabilizing function in dirigent proteins and proteins with dirigent-like domains.

The biochemical activities of dirigent proteins (DPs) give rise to distinct complex classes of plant phenolics. DPs apparently began to emerge during the aquatic-to-land transition, with phylogenetic analyses revealing the presence of numerous DP subfamilies in the plant kingdom. The vast majority (>95%) of DPs in these large multigene families still await discovery of their biochemical functions. Here, we elucidated the 3D structures of two pterocarpan-forming proteins with dirigent-like domains. Both proteins stereospecifically convert distinct diastereomeric chiral isoflavonoid precursors to the chiral pterocarpans, (-)- and (+)-medicarpin, respectively. Their 3D structures enabled comparisons with stereoselective lignan- and aromatic terpenoid-forming DP orthologs. Each protein provides entry into diverse plant natural products classes, and our experiments suggest a common biochemical mechanism in binding and stabilizing distinct plant phenol-derived mono- and bis-quinone methide intermediates during different C-C and C-O bond-forming processes. These observations provide key insights into both their appearance and functional diversification of DPs during land plant evolution/adaptation. The proposed biochemical mechanisms based on our findings provide important clues to how additional physiological roles for DPs and proteins harboring dirigent-like domains can now be rationally and systematically identified.

Trinuclear vanadium(iv) and vanadium(v) complexes derived from 2,4,6-triacetylphloroglucinol and study of their peroxidase mimicking activity.

Novel dibasic Schiff bases with three tridentate sites were obtained from the condensation of the triketone 2,4,6-triacetylphloroglucinol (H3ptk) with four different hydrazides, benzoyl hydrazide (bhz), furoyl hydrazide (fah), isonicotinoyl hydrazide (inh) and nicotinoyl hydrazide (nah): H6ptk(bhz)3I, H6ptk(fah)3II, H6ptk(inh)3III and H6ptk(nah)3IV. These ligand precursors I-IV, each being an ONO donor, are tricompartmental building blocks able to form trinuclear complexes having C3 symmetry. The reaction of I-IV with [VIVO(acac)2] leads to the formation of [{VIVO(H2O)}3(ptk(bhz)3)] 1, [{VIVO(H2O)}3(ptk(fah)3)] 2, [{VIVO(H2O)}3(ptk(inh)3)] 3, and [{VIVO(H2O)}3(ptk(nah)3)] 4. In methanol/aqueous solutions of M2CO3 (M+ = Na+, K+ and Cs+), these complexes are slowly converted into dioxidovanadium(v) compounds, namely, M3[(VVO2)3{ptk(bhz)3}]·6H2O [M+ = K+5, Na+9, Cs+13], M3[(VVO2)3{ptk(fah)3}]·6H2O [M+ = K+6, Na+10, Cs+14], M3[(VVO2)3{ptk(inh)3}]·6H2O [M+ = K+7, Na+11, Cs+15] and M3[(VVO2)3{ptk(nah)3}]·6H2O [M+ = K+8, Na+12, Cs+16]. All ligand precursors and complexes are characterized by various techniques such as FT-IR, UV/Visible, EPR, NMR (1H, 13C and 51V), elemental analysis, thermal studies, cyclic voltammetry (CV) and single-crystal X-ray analysis. X-ray diffraction studies of complexes K2.7[{(VVO2)3ptk(fah)3}]·11.5H2O·MeOH 6a, Cs3[{(VVO2)3ptk(bhz)3}]·7H2O 13a and Cs3[{(VVO2)3ptk(nah)3}]·7.3H2O 16a reveal their distorted square pyramidal geometry by coordinating through phenolate oxygen (of ptk), azomethine nitrogen and enolate oxygen (of hydrazide) atoms. The reactivity of complexes 5-16 and their catalytic potential were screened towards their peroxidase mimetic activity in the oxidation of dopamine to aminochrome driven by H2O2 as an oxidant. The conversion of dopamine to aminochrome with different catalysts was monitored by HPLC showing high activity under mild conditions with good conversions within 1 h. Kinetic studies using compounds 13-16 as catalyst precursors reveal that the reaction follows a Michaelis-Menten-like kinetics.

Late-stage difluoromethylation leading to a self-immobilizing fluorogenic probe for the visualization of enzyme activities in live cells.

Reported herein is a novel p-quinone methide-based self-immobilizing fluorogenic probe for the visualization of ß-galactosidase activities in live cells. This easily prepared imaging reagent massively increases the fluorescence intensity and covalently links to the activation site with high efficiency upon enzymatic trigger.

Highly Efficient Multiple-Labeling Probes for the Visualization of Enzyme Activities.

Quinone methide (QM) as a latent trapping unit has been widely explored in activity-based self-immobilizing reagents. However, further application of this strategy has been largely hampered by the limited labeling efficiency to proteins. In this study, a thorough investigation on the labeling efficiency and the structure of QM-based trapping unit is presented, from which a QM with multiple leaving groups was identified as an optimal trapping unit. An alkaline phosphatase (ALP) immobilizing reagent featured with this multiple-labeling trapping unit exhibited lower nonspecific binding and, remarkably, a significantly higher labeling efficiency over other immobilizing reagents upon enzymatic activation. The utility of this imaging reagent was further demonstrated with the in vitro and in vivo visualization of the ALP activities. Furthermore, the multiple functional trapping unit may find greater value in the other activity-based immobilizing probes.

MpTCP1 controls cell proliferation and redox processes in Marchantia polymorpha.

TCP transcription factors are key regulators of angiosperm cell proliferation processes. It is unknown whether their regulatory growth capacities are conserved across land plants, which we examined in liverworts, one of the earliest diverging land plant lineages. We generated knockout mutants for MpTCP1, the single TCP-P clade gene in Marchantia polymorpha, and characterized its function by conducting cell proliferation and morphological analyses as well as messenger RNA expression, transcriptome, chemical, and DNA binding studies. Mptcp1ge lines show a reduced vegetative thallus growth and extra tissue formation in female reproductive structures. Additionally, mutant plants reveal increased hydrogen peroxide (H2 O2 ) levels and an enhanced pigmentation in the thallus caused by formation of secondary metabolites, such as aminochromes. MpTCP1 proteins interact redox dependently with DNA and regulate the expression of a comprehensive redox network, comprising enzymes involved in H2 O2 metabolism. MpTCP1 regulates Marchantia growth in a context-dependent manner. Redox sensitivity of the DNA binding capacity of MpTCP1 proteins provides a mechanism to respond to altered redox conditions. Our data suggest that MpTCP1 activity could thereby have contributed to diversification of land plant morphologies and to adaptations to abiotic and biotic challenges, as experienced by liverworts during early land plant colonization.

CYP712K4 Catalyzes the C-29 Oxidation of Friedelin in the Maytenus ilicifolia Quinone Methide Triterpenoid Biosynthesis Pathway.

The native Brazilian plant Maytenus ilicifolia accumulates a set of quinone methide triterpenoids with important pharmacological properties, of which maytenin, pristimerin and celastrol accumulate exclusively in the root bark of this medicinal plant. The first committed step in the quinone methide triterpenoid biosynthesis is the cyclization of 2,3-oxidosqualene to friedelin, catalyzed by the oxidosqualene cyclase friedelin synthase (FRS). In this study, we produced heterologous friedelin by the expression of M. ilicifolia FRS in Nicotiana benthamiana leaves and in a Saccharomyces cerevisiae strain engineered using CRISPR/Cas9. Furthermore, friedelin-producing N. benthamiana leaves and S. cerevisiae cells were used for the characterization of CYP712K4, a cytochrome P450 from M. ilicifolia that catalyzes the oxidation of friedelin at the C-29 position, leading to maytenoic acid, an intermediate of the quinone methide triterpenoid biosynthesis pathway. Maytenoic acid produced in N. benthamiana leaves was purified and its structure was confirmed using high-resolution mass spectrometry and nuclear magnetic resonance analysis. The three-step oxidation of friedelin to maytenoic acid by CYP712K4 can be considered as the second step of the quinone methide triterpenoid biosynthesis pathway, and may form the basis for further discovery of the pathway and heterologous production of friedelanes and ultimately quinone methide triterpenoids.

The TYRP1-mediated protection of human tyrosinase activity does not involve stable interactions of tyrosinase domains.

Tyrosinases are melanocyte-specific enzymes involved in melanin biosynthesis. Mutations in their genes cause oculocutaneous albinism associated with reduced or altered pigmentation of skin, hair, and eyes. Here, the recombinant human intra-melanosomal domains of tyrosinase, TYRtr (19-469), and tyrosinase-related protein 1, TYRP1tr (25-472), were studied in vitro to define their functional relationship. Proteins were expressed or coexpressed in whole Trichoplusia ni larvae and purified. Their associations were studied using gel filtration and sedimentation equilibrium methods. Protection of TYRtr was studied by measuring the kinetics of tyrosinase diphenol oxidase activity in the presence (1:1 and 1:20 molar ratios) or the absence of TYRP1tr for 10 hr under conditions mimicking melanosomal and ER pH values. Our data indicate that TYRtr incubation with excess TYRP1tr protects TYR, increasing its stability over time. However, this mechanism does not appear to involve the formation of stable hetero-oligomeric complexes to maintain the protective function.

The unexplored potential of quinone methides in chemical biology.

Quinone methides (QMs) are transient reactive species that can be efficiently generated from stable precursors under a variety of biocompatible conditions. Due to their electrophilic nature, QMs have been widely explored as cross-linking agents of DNA and proteins under physiological conditions. However, QMs also have a diene character and can irreversibly react via Diels-Alder reaction with electron-rich dienophiles. This particular reactivity has been recently exploited to label biomolecules with fluorophores in living cells. QMs are characterised by two unique properties that make them ideal candidates for chemical biology applications: i) they can be efficiently generated in situ from very stable precursors by means of bio-orthogonal protocols ii) they are reversible cross-linking agents, making them suitable for "catch and release" target-enrichment experiments. Nevertheless, there are only few examples reported to date that truly take advantage of QMs unique chemistry in the context of chemical-biology assay development. In this review, we will examine the most relevant examples that illustrate the benefit of using QMs for chemical biology purposes and we will anticipate novel approaches to further their applications in biologically relevant contexts.

Enzymatic activation of indolequinone-substituted 5-fluorodeoxyuridine prodrugs in hypoxic cells.

Among the various enzymes, reductases that catalyze one-electron reduction are involved in the selective activation of functional compounds or materials in hypoxia, which is one of the well-known pathophysiological characteristics of solid tumors. Enzymatic one-electron reduction has been recognized as a useful reaction that can be applied in the design of tumor hypoxia-targeting drugs. In this report, we characterized the enzymatic reaction of 5-fluorodeoxyuridine (FdUrd) prodrug bearing an indolequinone unit (IQ-FdUrd), which is a substrate of reductases. IQ-FdUrd was activated to release FdUrd under hypoxic conditions after treatment with cytochrome NADPH P450 reductase. We also confirmed that IQ-FdUrd showed selective cytotoxicity in hypoxic tumor cells.

The Redox Properties of a Cysteine Tryptophylquinone-Dependent Glycine Oxidase Are Distinct from Those of Tryptophylquinone-Dependent Dehydrogenases.

GoxA is a cysteine tryptophylquinone (CTQ)-dependent glycine oxidase that is a member of a family of LodA-like proteins. The electrochemical midpoint potential ( Em) values for the quinone/semiquinone couple and the semiquinone/quinol couple were determined to be 111 and 21, respectively. The Em value for the overall two-electron quinone/quinol couple was similar to those of CTQ- and tryptophan tryptophylquinone (TTQ)-bearing dehydrogenases. However, for the well-studied TTQ-dependent methylamine dehydrogenase, the quinone/semiquinone couple is more negative than the semiquinone/quinol couple, the opposite of what was determined for GoxA. The change in Em value for the two-electron quinone/quinol couple of CTQ in GoxA with pH indicates that the overall two-electron transfer process is associated with the transfer of one proton. Thus, the quinol is anionic. The data reported herein further suggest that in GoxA the CTQ semiquinone is neutral, in contrast to the TTQ-dependent dehydrogenases, in which it is an anionic TTQ semiquinone. These results are discussed in the context of the structure and function of this glycine oxidase, compared to that of the tryptophylquinone-dependent dehydrogenases.

Identification of Quinone Methide Intermediate Resulting from Metabolic Activation of Icaritin in Vitro and in Vivo.

Many herbal medicines such as epimedium have been reported to cause adverse effects, and icaritin is the common aglycone of many glucosides in epimedium. Our present work aimed at the clarification of the metabolic activation of icaritin possibly responsible for the adverse effects of epimedium. A quinone methide metabolite (M1) was detected in icaritin-fortified microsomal incubations. A glutathione (GSH) conjugate (M2) and N-acetyl-l-cysteine (NAC) conjugate (M3) derived from icaritin were observed in GSH/NAC-supplemented rat/human liver microsomal incubations. CYP3A family was the predominant enzyme catalyzing the bioactivation of icaritin. In conclusion, sufficient evidence indicates the metabolic activation of icaritin to quinone methide metabolite.

Lignin-Biosynthetic Study: Reactivity of Quinone Methides in the Diastereopreferential Formation of p-Hydroxyphenyl- and Guaiacyl-Type ß- O-4 Structures.

p-Quinone methides are involved in lignin biosynthesis as transient intermediates, and the aromatization step has a great impact on the chemical structure of the resulting lignin. A series of quinone methides (QMs) were synthesized and allowed to react with water in pH 3-7 buffers at 25 °C to mimic the formation of p-hydroxyphenyl- and guaiacyl-type (H- and G-type, respectively) ß- O-4 structures in gymnosperm-plant cell walls. Water addition occurred in 3-methoxy-substituted QMs (G-type QMs) with half-lives of 1.4-15 min. In contrast, nonsubstituted QMs (H-type QMs) were very labile; they were aromatized to ß- O-4 products with half-lives of only 10-40 s. The rapid aromatization in H-type QMs may provide an advantage over G-type species for efficiently driving the lignin-polymerization cycle, which possibly contributes to the development of highly lignified compression wood. In the water-addition reaction, the threo isomers of the ß- O-4 products were stereopreferentially formed more than the erythro isomers from both G- and H-type QMs ( erythro/ threo ratios of 24:76 and 50:50, respectively). The proportion of erythro isomers was higher at lower-pH conditions. This pH-dependent trend agrees with findings from a previous study on 3,5-dimethoxy-substituted (syringyl-type, S-type) QMs; thus, this pH-dependent trend is common in H-, G-, and S-type lignin-related QMs. Higher threo-selectivity was obtained by changing the ß-etherified aromatic rings from G- to H-type. A similar but weaker effect was also observed by changing the QM moiety from G- to H-type.

Novel Alpha-Synuclein Oligomers Formed with the Aminochrome-Glutathione Conjugate Are Not Neurotoxic.

Aminochrome induces neurotoxic alpha-synuclein oligomer formation relevant to the etiology of Parkinson's disease. Oxidative stress produces aminochrome from dopamine, but conjugation with glutathione catalyzed by glutathione transferase M2-2 significantly decreases aminochrome-induced toxicity and alpha-synuclein oligomer formation. Notably, in the presence of the aminochrome-glutathione conjugate, previously unknown species of alpha-synuclein oligomers are formed. These aminochrome-glutathione oligomers of alpha-synuclein differ from formerly characterized oligomers and (i) have high molecular weight, and are stable and SDS-resistant, as determined by the Western blot method, (ii) show positive NBT-quinone-protein staining, which indicates the formation of alpha-synuclein adducts containing aminochrome. Furthermore, aminochrome-glutathione alpha-synuclein oligomers (iii) have distinctive shape and size, as determined by transmission electron microscopy, and (iv) are not toxic in U373MG cells. In conclusion, glutathione conjugated with aminochrome induces a new type of alpha-synuclein oligomers of a different size and shape, which have no demonstrable toxicity.

LC-MS/MS reveals the formation of iminium and quinone methide reactive intermediates in entrectinib metabolism: In vivo and in vitro metabolic investigation.

Entrectinib (RXDX-101) is orally available inhibitor of the tyrosine kinases including tropomyosin receptor kinases (Trk) A-C, C-ros oncogene 1 (ROS1) and anaplastic lymphoma kinase (ALK), with potential antineoplastic activity. Entrectinib (ENB) granted breakthrough designation by FDA for NTRK + Solid tumors. In vitro metabolism of ENB generates quinone methide and iminium reactive intermediates that were captured by potassium cyanide and GSH, respectively forming stable conjugates that were characterized by LC-MS/MS. Seven in vitro ENB metabolites were identified through four metabolic reactions including hydroxylation, N-dealkylation, N-oxidation and reduction. Furthermore, four reactive intermediates including two quinone methide and two iminium ions were detected and the bioactivation mechanisms were supposed. In vivo metabolism of ENB was done by giving single oral dose (35.2 mg/kg) to Sprague Dawley rats. In vivo metabolism generates five phase I metabolites similar to in vitro metabolism except no metabolic reactions were identified on indazole ring. One phase II metabolite was characterized in in vivo metabolism of ENB resulted from glucuronidation of hydroxyl metabolite of ENB. Reporting these data for ENB is very crucial in the development stage. Reviewing literatures revealed the absence of previous articles have been done for the ENB in vitro or in vivo metabolism study or structural characterization of the formed reactive intermediates.