Monoamine Oxidase Inhibitors Prevent Glucose-Dependent Energy Production, Proliferation and Migration of Bladder Carcinoma Cells.

Bladder cancer is the 10th most common cancer in the world and has a high risk of recurrence and metastasis. In order to sustain high energetic needs, cancer cells undergo complex metabolic adaptations, such as a switch toward aerobic glycolysis, that can be exploited therapeutically. Reactive oxygen species (ROS) act as key regulators of cancer metabolic reprogramming and tumorigenesis, but the sources of ROS remain unidentified. Monoamine oxidases (MAOs) are mitochondrial enzymes that generate H2O2 during the breakdown of catecholamines and serotonin. These enzymes are particularly important in neurological disorders, but recently, a new link between MAOs and cancer has been uncovered, involving their production of ROS. At present, the putative role of MAOs in bladder cancer has never been evaluated. We observed that human urothelial tumor explants and the bladder cancer cell line AY27 expressed both MAO-A and MAO-B isoforms. Selective inhibition of MAO-A or MAO-B limited mitochondrial ROS accumulation, cell cycle progression and proliferation of bladder cancer cells, while only MAO-A inhibition prevented cell motility. To test whether ROS contributed to MAO-induced tumorigenesis, we used a mutated form of MAO-A which was unable to produce H2O2. Adenoviral transduction of the WT MAO-A stimulated the proliferation and migration of AY27 cells while the Lys305Met MAO-A mutant was inactive. This was consistent with the fact that the antioxidant Trolox strongly impaired proliferation and cell cycle progression. Most interestingly, AY27 cells were highly dependent on glucose metabolism to sustain their growth, and MAO inhibitors potently reduced glycolysis and oxidative phosphorylation, due to pyruvate depletion. Accordingly, MAO inhibitors decreased the expression of proteins involved in glucose transport (GLUT1) and transformation (HK2). In conclusion, urothelial cancer cells are characterized by a metabolic shift toward glucose-dependent metabolism, which is important for cell growth and is under the regulation of MAO-dependent oxidative stress.

Crystallographic snapshots of UDP-glucuronic acid 4-epimerase ligand binding, rotation, and reduction.

UDP-glucuronic acid is converted to UDP-galacturonic acid en route to a variety of sugar-containing metabolites. This reaction is performed by a NAD+-dependent epimerase belonging to the short-chain dehydrogenase/reductase family. We present several high-resolution crystal structures of the UDP-glucuronic acid epimerase from Bacillus cereus The geometry of the substrate-NAD+ interactions is finely arranged to promote hydride transfer. The exquisite complementarity between glucuronic acid and its binding site is highlighted by the observation that the unligated cavity is occupied by a cluster of ordered waters whose positions overlap the polar groups of the sugar substrate. Co-crystallization experiments led to a structure where substrate- and product-bound enzymes coexist within the same crystal. This equilibrium structure reveals the basis for a "swing and flip" rotation of the pro-chiral 4-keto-hexose-uronic acid intermediate that results from glucuronic acid oxidation, placing the C4' atom in position for receiving a hydride ion on the opposite side of the sugar ring. The product-bound active site is almost identical to that of the substrate-bound structure and satisfies all hydrogen-bonding requirements of the ligand. The structure of the apoenzyme together with the kinetic isotope effect and mutagenesis experiments further outlines a few flexible loops that exist in discrete conformations, imparting structural malleability required for ligand rotation while avoiding leakage of the catalytic intermediate and/or side reactions. These data highlight the double nature of the enzymatic mechanism: the active site features a high degree of precision in substrate recognition combined with the flexibility required for intermediate rotation.

Assessment of Tractable Cysteines for Covalent Targeting by Screening Covalent Fragments.

Targeted covalent inhibition and the use of irreversible chemical probes are important strategies in chemical biology and drug discovery. To date, the availability and reactivity of cysteine residues amenable for covalent targeting have been evaluated by proteomic and computational tools. Herein, we present a toolbox of fragments containing a 3,5-bis(trifluoromethyl)phenyl core that was equipped with chemically diverse electrophilic warheads showing a range of reactivities. We characterized the library members for their reactivity, aqueous stability and specificity for nucleophilic amino acids. By screening this library against a set of enzymes amenable for covalent inhibition, we showed that this approach experimentally characterized the accessibility and reactivity of targeted cysteines. Interesting covalent fragment hits were obtained for all investigated cysteine-containing enzymes.

Monoamine Oxidases.

Monoamine oxidases A and B (MAO A and B) are mammalian flavoenzymes bound to the outer mitochondrial membrane. They were discovered almost a century ago and they have been the subject of many biochemical, structural and pharmacological investigations due to their central role in neurotransmitter metabolism. Currently, the treatment of Parkinson's disease involves the use of selective MAO B inhibitors such as rasagiline and safinamide. MAO inhibition was shown to exert a general neuroprotective effect as a result of the reduction of oxidative stress produced by these enzymes, which seems to be relevant also in non-neuronal contexts. MAOs were successfully expressed as recombinant proteins in Pichia pastoris, which allowed a thorough biochemical and structural characterization. These enzymes are characterized by a globular water-soluble main body that is anchored to the mitochondrial membrane through a C-terminal α-helix, similar to other bitopic membrane proteins. In both MAO A and MAO B the enzyme active site consists of a hydrophobic cavity lined by residues that are conserved in the two isozymes, except for few details that determine substrate and inhibitor specificity. In particular, human MAO B features a dual-cavity active site whose conformation depends on the size of the bound ligand. This article provides a comprehensive and historical review of MAOs and the state-of-the-art of these enzymes as membrane drug targets.

Structure-Based Engineering of Phanerochaete chrysosporium Alcohol Oxidase for Enhanced Oxidative Power toward Glycerol.

Glycerol is a major byproduct of biodiesel production, and enzymes that oxidize this compound have been long sought after. The recently described alcohol oxidase from the white-rot basidiomycete Phanerochaete chrysosporium (PcAOX) was reported to feature very mild activity on glycerol. Here, we describe the comprehensive structural and biochemical characterization of this enzyme. PcAOX was expressed in Escherichia coli in high yields and displayed high thermostability. Steady-state kinetics revealed that PcAOX is highly active toward methanol, ethanol, and 1-propanol ( kcat = 18, 19, and 11 s-1, respectively), but showed very limited activity toward glycerol ( kobs = 0.2 s-1 at 2 M substrate). The crystal structure of the homo-octameric PcAOX was determined at a resolution of 2.6 Å. The catalytic center is a remarkable solvent-inaccessible cavity located at the re side of the flavin cofactor. Its small size explains the observed preference for methanol and ethanol as best substrates. These findings led us to design several cavity-enlarging mutants with significantly improved activity toward glycerol. Among them, the F101S variant had a high kcat value of 3 s-1, retaining a high degree of thermostability. The crystal structure of F101S PcAOX was solved, confirming the site of mutation and the larger substrate-binding pocket. Our data demonstrate that PcAOX is a very promising enzyme for glycerol biotransformation.

Isolation and characterization of a thermostable F420:NADPH oxidoreductase from Thermobifida fusca.

F420H2-dependent enzymes reduce a wide range of substrates that are otherwise recalcitrant to enzyme-catalyzed reduction, and their potential for applications in biocatalysis has attracted increasing attention. Thermobifida fusca is a moderately thermophilic bacterium and holds high biocatalytic potential as a source for several highly thermostable enzymes. We report here on the isolation and characterization of a thermostable F420: NADPH oxidoreductase (Tfu-FNO) from T. fusca, the first F420-dependent enzyme described from this bacterium. Tfu-FNO was heterologously expressed in Escherichia coli, yielding up to 200 mg of recombinant enzyme per liter of culture. We found that Tfu-FNO is highly thermostable, reaching its highest activity at 65 °C and that Tfu-FNO is likely to act in vivo as an F420 reductase at the expense of NADPH, similar to its counterpart in Streptomyces griseus We obtained the crystal structure of FNO in complex with NADP+ at 1.8 Å resolution, providing the first bacterial FNO structure. The overall architecture and NADP+-binding site of Tfu-FNO were highly similar to those of the Archaeoglobus fulgidus FNO (Af-FNO). The active site is located in a hydrophobic pocket between an N-terminal dinucleotide binding domain and a smaller C-terminal domain. Residues interacting with the 2'-phosphate of NADP+ were probed by targeted mutagenesis, indicating that Thr-28, Ser-50, Arg-51, and Arg-55 are important for discriminating between NADP+ and NAD+ Interestingly, a T28A mutant increased the kinetic efficiency >3-fold as compared with the wild-type enzyme when NADH is the substrate. The biochemical and structural data presented here provide crucial insights into the molecular recognition of the two cofactors, F420 and NAD(P)H by FNO.

Two tyrosine residues, Tyr-108 and Tyr-503, are responsible for the deprotonation of phenolic substrates in vanillyl-alcohol oxidase.

A number of oxidoreductases from the VAO/para-cresol methylhydroxylase flavoprotein family catalyze the oxidation of para-substituted phenols. One of the best-studied is vanillyl-alcohol oxidase (VAO) from the fungus Penicillium simplicissimum For oxidation of phenols by VAO to occur, they must first be bound in the active site of the enzyme in their phenolate anion form. The crystal structure of VAO reveals that two tyrosine residues, Tyr-108 and Tyr-503, are positioned to facilitate this deprotonation. To investigate their role in catalysis, we created three VAO variants, Y108F, Y503F, and Y108F/Y503F, and studied their biochemical properties. Steady-state kinetics indicated that the presence of at least one of the tyrosine residues is essential for efficient catalysis by VAO. Stopped-flow kinetics revealed that the reduction of VAO by chavicol or vanillyl alcohol occurs at two different rates: kobs1, which corresponds to its reaction with the deprotonated form of the substrate, and kobs2, which corresponds to its reaction with the protonated form of the substrate. In Y108F, Y503F, and Y108F/Y503F, the relative contribution of kobs2 to the reduction is larger than in wild-type VAO, suggesting deprotonation is impaired in these variants. Binding studies disclosed that the competitive inhibitor isoeugenol is predominantly in its deprotonated form when bound to wild-type VAO, but predominantly in its protonated form when bound to the variants. These results indicate that Tyr-108 and Tyr-503 are responsible for the activation of substrates in VAO, providing new insights into the catalytic mechanism of VAO and related enzymes that oxidize para-substituted phenols.

The structure of monoamine oxidases: past, present, and future.

The first crystal structure of mammalian monoamine oxidases (MAOs) was solved in 2002; almost 65 years after, these FAD-dependent enzymes were discovered and classified as responsible for the oxidation of aromatic neurotransmitters. Both MAO A and MAO B feature a two-domain topology characterized by the Rossmann fold, interacting with dinucleotide cofactors, which is intimately associated to a substrate-binding domain. This globular body is endowed with a C-terminal α-helix that anchors the protein to the outer mitochondrial phospholipid bilayer. As monotopic membrane proteins, the structural elucidation of MAOs was a challenging task that required the screening of different detergent conditions for their purification and crystallization. MAO A and MAO B structures differ both in their oligomerization architecture and in details of their active sites. Purified human MAO B and rat MAO A are dimeric, whereas human MAO A was found to be monomeric, which is believed to result from the detergent treatments used to extract the protein from the membrane. The active site of MAOs consists of a hydrophobic cavity located in front of the flavin cofactor and extending to the protein surface. Some structural features are highly conserved in the two isozymes, such as a Tyr-Tyr aromatic sandwich in front of the flavin ring and a Lys residue hydrogen-bonded to the cofactor N5 atom, whereas a pair of gating residues (Phe208/Ile335 in MAO A; Ile199/Tyr326 in MAO B) specifically determines the different substrate and inhibitor properties of the two enzymes.

Kinetic Resolution of sec-Thiols by Enantioselective Oxidation with Rationally Engineered 5-(Hydroxymethyl)furfural Oxidase.

Various flavoprotein oxidases were recently shown to oxidize primary thiols. Herein, this reactivity is extended to sec-thiols by using structure-guided engineering of 5-(hydroxymethyl)furfural oxidase (HMFO). The variants obtained were employed for the oxidative kinetic resolution of racemic sec-thiols, thus yielding the corresponding thioketones and nonreacted R-configured thiols with excellent enantioselectivities (E≥200). The engineering strategy applied went beyond the classic approach of replacing bulky amino acid residues with smaller ones, as the active site was additionally enlarged by a newly introduced Thr residue. This residue established a hydrogen-bonding interaction with the substrates, as verified in the crystal structure of the variant. These strategies unlocked HMFO variants for the enantioselective oxidation of a range of sec-thiols.

Discovery and characterization of an F420-dependent glucose-6-phosphate dehydrogenase (Rh-FGD1) from Rhodococcus jostii RHA1.

Cofactor F420, a 5-deazaflavin involved in obligatory hydride transfer, is widely distributed among archaeal methanogens and actinomycetes. Owing to the low redox potential of the cofactor, F420-dependent enzymes play a pivotal role in central catabolic pathways and xenobiotic degradation processes in these organisms. A physiologically essential deazaflavoenzyme is the F420-dependent glucose-6-phosphate dehydrogenase (FGD), which catalyzes the reaction F420 + glucose-6-phosphate → F420H2 + 6-phospho-gluconolactone. Thereby, FGDs generate the reduced F420 cofactor required for numerous F420H2-dependent reductases, involved e.g., in the bioreductive activation of the antitubercular prodrugs pretomanid and delamanid. We report here the identification, production, and characterization of three FGDs from Rhodococcus jostii RHA1 (Rh-FGDs), being the first experimental evidence of F420-dependent enzymes in this bacterium. The crystal structure of Rh-FGD1 has also been determined at 1.5 Å resolution, showing a high similarity with FGD from Mycobacterium tuberculosis (Mtb) (Mtb-FGD1). The cofactor-binding pocket and active-site catalytic residues are largely conserved in Rh-FGD1 compared with Mtb-FGD1, except for an extremely flexible insertion region capping the active site at the C-terminal end of the TIM-barrel, which also markedly differs from other structurally related proteins. The role of the three positively charged residues (Lys197, Lys258, and Arg282) constituting the binding site of the substrate phosphate moiety was experimentally corroborated by means of mutagenesis study. The biochemical and structural data presented here provide the first step towards tailoring Rh-FGD1 into a more economical biocatalyst, e.g., an F420-dependent glucose dehydrogenase that requires a cheaper cosubstrate and can better match the demands for the growing applications of F420H2-dependent reductases in industry and bioremediation.

An unprecedented NADPH domain conformation in lysine monooxygenase NbtG provides insights into uncoupling of oxygen consumption from substrate hydroxylation.

N-Hydroxylating monooxygenases are involved in the biosynthesis of iron-chelating hydroxamate-containing siderophores that play a role in microbial virulence. These flavoenzymes catalyze the NADPH- and oxygen-dependent hydroxylation of amines such as those found on the side chains of lysine and ornithine. In this work we report the biochemical and structural characterization of Nocardia farcinica Lys monooxygenase (NbtG), which has similar biochemical properties to mycobacterial homologs. NbtG is also active on d-Lys, although it binds l-Lys with a higher affinity. Differently from the ornithine monooxygenases PvdA, SidA, and KtzI, NbtG can use both NADH and NADPH and is highly uncoupled, producing more superoxide and hydrogen peroxide than hydroxylated Lys. The crystal structure of NbtG solved at 2.4 Å resolution revealed an unexpected protein conformation with a 30° rotation of the NAD(P)H domain with respect to the flavin adenine dinucleotide (FAD) domain that precludes binding of the nicotinamide cofactor. This "occluded" structure may explain the biochemical properties of NbtG, specifically with regard to the substantial uncoupling and limited stabilization of the C4a-hydroperoxyflavin intermediate. Biological implications of these findings are discussed.

Biocatalytic Properties and Structural Analysis of Eugenol Oxidase from Rhodococcus jostii RHA1: A Versatile Oxidative Biocatalyst.

Eugenol oxidase (EUGO) from Rhodococcus jostii RHA1 had previously been shown to convert only a limited set of phenolic compounds. In this study, we have explored the biocatalytic potential of this flavoprotein oxidase, resulting in a broadened substrate scope and a deeper insight into its structural properties. In addition to the oxidation of vanillyl alcohol and the hydroxylation of eugenol, EUGO can efficiently catalyze the dehydrogenation of various phenolic ketones and the selective oxidation of a racemic secondary alcohol-4-(1-hydroxyethyl)-2-methoxyphenol. EUGO was also found to perform the kinetic resolution of a racemic secondary alcohol. Crystal structures of the enzyme in complexes with isoeugenol, coniferyl alcohol, vanillin, and benzoate have been determined. The catalytic center is a remarkable solvent-inaccessible cavity on the si side of the flavin cofactor. Structural comparison with vanillyl alcohol oxidase from Penicillium simplicissimum highlights a few localized changes that correlate with the selectivity of EUGO for phenolic substrates bearing relatively small p-substituents while tolerating o-methoxy substituents.

Kinetic and structural analysis of the irreversible inhibition of human monoamine oxidases by ASS234, a multi-target compound designed for use in Alzheimer's disease.

Monoamine oxidases (MAO) and cholinesterases are validated targets in the design of drugs for the treatment of Alzheimer's disease. The multi-target compound N-((5-(3-(1-benzylpiperidin-4-yl)propoxy)-1-methyl-1H-indol-2-yl)methyl)-N-methylprop-2-yn-1-amine (ASS234), bearing the MAO-inhibiting propargyl group attached to a donepezil moiety that inhibits cholinesterases, retained activity against human acetyl- and butyryl-cholinesterases. The inhibition of MAO A and MAO B by ASS234 was characterized and compared to other known MAO inhibitors. ASS234 was almost as effective as clorgyline (kinact/KI=3×10(6) min(-1)M(-1)) and was shown by structural studies to form the same N5 covalent adduct with the FAD cofactor.

Discovery and structural characterization of a thermostable bacterial monoamine oxidase.

Monoamine oxidases (MAOs) are pivotal regulators of neurotransmitters in mammals, while microbial MAOs have been shown to be valuable biocatalysts for enantioselective synthesis of pharmaceutical compounds or precursors thereof. To extend the knowledge of how MAOs function at the molecular level and in order to provide more biocatalytic tools, we set out to identify and study a robust bacterial variant: a MAO from the thermophile Thermoanaerobacterales bacterium (MAOTb ). MAOTb is highly thermostable with melting temperatures above 73 °C and is well expressed in Escherichia coli. Substrate screening revealed that the oxidase is most efficient with n-alkylamines with n-heptylamine being the best substrate. Presteady-state kinetic analysis shows that reduced MAOTb rapidly reacts with molecular oxygen, confirming that it is a bona fide oxidase. The crystal structure of MAOTb was resolved at 1.5 Å and showed an exceptionally high similarity with the two human MAOs, MAO A and MAO B. The active site of MAOTb resembles mostly the architecture of human MAO A, including the cysteinyl protein-FAD linkage. Yet, the bacterial MAO lacks a C-terminal extension found in human MAOs, which explains why it is expressed and purified as a soluble protein, while the mammalian counterparts are anchored to the membrane through an α-helix. MAOTb also displays a slightly different active site access tunnel, which may explain the specificity toward long aliphatic amines. Being an easy-to-express, thermostable enzyme, for which a high-resolution structure was elucidated, this bacterial MAO may develop into a valuable biocatalyst for synthetic chemistry or biosensing.

The DprE1 enzyme, one of the most vulnerable targets of Mycobacterium tuberculosis.

The re-emergence of tuberculosis in recent years led the World Health Organization (WHO) to launch the Stop TB Strategy program. Beside repurposing the existing drugs and exploring novel molecular combinations, an essential step to face the burden of tuberculosis will be to develop new drugs by identifying vulnerable bacterial targets. Recent studies have focused on decaprenylphosphoryl-D-ribose oxidase (DprE1) of Mycobacterium tuberculosis, an essential enzyme involved in cell wall metabolism, for which new promising molecules have proved efficacy as antitubercular agents. This review summarizes the state of the art concerning DprE1 in terms of structure, enzymatic activity and inhibitors. This enzyme is emerging as one of the most vulnerable target in M. tuberculosis.

LSD1: oxidative chemistry for multifaceted functions in chromatin regulation.

Three years after its discovery, lysine-specific demethylase 1 remains at the forefront of chromatin research. Its demethylase activity on Lys4 of histone H3 supports its role in gene repression. By contrast, the biochemical mechanisms underlying lysine-specific demethylase 1 involvement in transcriptional activation are not firmly established. Structural studies highlight a specific binding site for the histone H3 N-terminal tail and a catalytic machinery that is closely related to that of other flavin-dependent amine oxidases. These insights are crucial for the development of demethylation inhibitors. Furthermore, the exploration of putative non-histone substrates and potential signaling roles of hydrogen peroxide produced by the demethylation reaction could lead to new paradigms in chromatin biology.

The Peroxidase-Coupled Assay to Measure MAO Enzymatic Activity.

MAO activity measurement is generally performed following different spectroscopy methods, in most cases using peroxidase as a coupled reaction catalyst. In the presence of horseradish peroxidase (HRP), the assay follows the oxidation of the typical MAO substrate (aromatic amines) which generates hydrogen peroxide as a secondary product. There are several chromogens and fluorogens that, in the presence of hydrogen peroxide, are converted by HRP to detectable products. In the present chapter we describe the spectrophotometric 4-aminoantipyrine assay as well as the fluorogenic assay with the Amplex® Red chemical probe. These methods are applied on MAO activity and Michaelis-Menten curve determinations as well as inhibitory activity experiments.

Measurement of MAO Enzymatic Activity by Spectrophotometric Direct Assays.

MAO activity measurement can be monitored by direct peroxidase-free assays following different spectroscopy methods. Typically, these are assays that follow the conversion of different MAO substrates into its corresponding products monitored in either absorbance or fluorescence. Herein, we describe the assays for enzyme activity assessment with MAO B and particularly the MAO A substrate kynuramine, as well as the MAO B substrate benzylamine. Moreover, we also describe MAO activity determination using the tertiary amine substrate allyl amine 1-methyl-4-(1-methyl-1 H-pyrrol-2-yl)-1,2,3,6-tetrahydropyridine (MMTP). These are very useful methods for the investigation of MAO inhibitory activity by molecules known to be HRP-interfering. In the present chapter we demonstrate the application of these methods in MAO activity and Michaelis-Menten curve determinations as well as inhibitory activity experiments.

Crystallization of Human Monoamine Oxidase B.

The interest in monoamine oxidases A and B (MAO A and B) is due to their central role in regulating the balance of neurotransmitters, both in the central nervous system and in peripheral organs. As validated drug targets for depression and Parkinson's disease, the elucidation of their crystal structures was an essential step to guide drug design investigations. The development of the heterologous expression system of MAO B in Pichia pastoris and the identification of the detergent, buffer, and precipitant conditions allowed to determine the first crystal structure of human MAO B in 2002. A detailed protocol to obtain reproducible MAO B crystals is described.

Bioisosteric replacement based on 1,2,4-oxadiazoles in the discovery of 1H-indazole-bearing neuroprotective MAO B inhibitors.

Following a hybridization strategy, a series of 5-substituted-1H-indazoles were designed and evaluated in vitro as inhibitors of human monoamine oxidase (hMAO) A and B. Among structural modifications, the bioisostere-based introduction of 1,2,4-oxadiazole ring returned the most potent and selective human MAO B inhibitor (compound 20, IC50 = 52 nM, SI > 192). The most promising inhibitors were studied in cell-based neuroprotection models of SH-SY5Y and astrocytes line against H2O2. Moreover, preliminary drug-like features (aqueous solubility at pH 7.4; hydrolytic stability at acidic and neutral pH) were assessed for selected 1,2,4-oxadiazoles and compared to amide analogues through RP-HPLC methods. Molecular docking simulations highlighted the crucial role of molecular flexibility in providing a better shape complementarity for compound 20 within MAO B enzymatic cleft than rigid analogue 18. Enzymatic kinetics analysis along with thermal stability curves (Tm shift = +2.9 °C) provided clues of a tight-binding mechanism for hMAO B inhibition by 20.