[Metabolic Activities Catalyzed by Human Cytochrome P450 (CYP) 2D6 and CYP3A Subfamily Members and Effect of Various Compounds, Including Endogenous Steroid Hormones, on These Activities].

My research focused on the effects of various drugs on (1) dopamine formation from p-tyramine catalyzed by polymorphic cytochrome P450 (CYP or P450) 2D6 variants and (2) endogenous steroid hormone hydroxylation catalyzed by CYP3A subfamily members (CYP3A4, CYP3A5, CYP3A7). The activation (cooperativity) of metabolic reactions catalyzed by P450s was especially emphasized. The effects of various psychotropic agents on dopamine formation from p-tyramine, catalyzed by wild-type CYP2D6.1 and CYP2D6 variants, including CYP2D6.2 (Arg296Cys;Ser486Thr), CYP2D6.10 (Pro34Ser;Ser486Thr), and CYP2D6.39 (Ser486Thr) were compared. Michaelis (Km) and inhibition (Ki) constants of the psychotropic agents in the presence of CYP2D6.10 were higher than those observed in the presence of other CYP2D6 variants. Fluvoxamine, fluoxetine, milnacipran, and haloperidol activated CYP2D6-catalyzed dopamine formation [decreasing the Km and/or increasing the maximal velocity (kcat)], and this activation was CYP2D6 variant-dependent. Regarding the CYP3A subfamily, the effects of various compounds including endogenous steroid hormones on the 6ß-hydroxylation of steroid hormones, such as testosterone, progesterone, and cortisol, were determined; it was found that testosterone, dehydroepiandrosterone, and/or α-naphthoflavone activated 6ß-hydroxylation of cortisol and/or progesterone, but the effects varied in the presence of different CYP3A subfamily members. Further studies are required to confirm the mechanisms and therapeutic relevance of these activation phenomena.

Methylglyoxal-derived hydroimidazolone, MG-H1, increases food intake by altering tyramine signaling via the GATA transcription factor ELT-3 in Caenorhabditis elegans.

The Maillard reaction, a chemical reaction between amino acids and sugars, is exploited to produce flavorful food ubiquitously, from the baking industry to our everyday lives. However, the Maillard reaction also occurs in all cells, from prokaryotes to eukaryotes, forming advanced glycation end-products (AGEs). AGEs are a heterogeneous group of compounds resulting from the irreversible reaction between biomolecules and α-dicarbonyls (α-DCs), including methylglyoxal (MGO), an unavoidable byproduct of anaerobic glycolysis and lipid peroxidation. We previously demonstrated that Caenorhabditis elegans mutants lacking the glod-4 glyoxalase enzyme displayed enhanced accumulation of α-DCs, reduced lifespan, increased neuronal damage, and touch hypersensitivity. Here, we demonstrate that glod-4 mutation increased food intake and identify that MGO-derived hydroimidazolone, MG-H1, is a mediator of the observed increase in food intake. RNAseq analysis in glod-4 knockdown worms identified upregulation of several neurotransmitters and feeding genes. Suppressor screening of the overfeeding phenotype identified the tdc-1-tyramine-tyra-2/ser-2 signaling as an essential pathway mediating AGE (MG-H1)-induced feeding in glod-4 mutants. We also identified the elt-3 GATA transcription factor as an essential upstream regulator for increased feeding upon accumulation of AGEs by partially controlling the expression of tdc-1 gene. Furthermore, the lack of either tdc-1 or tyra-2/ser-2 receptors suppresses the reduced lifespan and rescues neuronal damage observed in glod-4 mutants. Thus, in C. elegans, we identified an elt-3 regulated tyramine-dependent pathway mediating the toxic effects of MG-H1 AGE. Understanding this signaling pathway may help understand hedonistic overfeeding behavior observed due to modern AGE-rich diets.

De novo biosynthesis of N-acetyltyramine in engineered Escherichia coli.

N-acetyltyramine as a tyramine alkaloid has drawn great attention because of its excellent anti-free radical, antithrombotic, and antitumour activity. Therefore, it is an attractive compound. In this study, we reported for the first time the construction a synthetic pathway of N-acetyltyramine in engineered Escherichia coli. First, the tyrosine decarboxylase tdc gene and arylalkylamine N-acyltransferase aanat gene were introduced into E. coli to generate a recombinant N-acetyltyramine producer with L-tyrosine as substrate. Subsequently, overexpressing aroGfbr and TyrAfbr enhance the availability of L-tyrosine to achieve de novo biosynthesis of N-acetyltyramine from glucose. Finally, overexpressing the transketolase I tktA and phosphoenolpyruvate synthase ppsA genes improved the N-acetyltyramine production to 854 mg/L.

Multiple Direct Effects of the Dietary Protoalkaloid N-Methyltyramine in Human Adipocytes.

Dietary amines have been the subject of a novel interest in nutrition since the discovery of trace amine-associated receptors (TAARs), especially TAAR-1, which recognizes tyramine, phenethylamine, tryptamine, octopamine, N-methyltyramine (NMT), synephrine, amphetamine and related derivatives. Alongside the psychostimulant properties of TAAR-1 ligands, it is their ephedrine-like action on weight loss that drives their current consumption via dietary supplements advertised for 'fat-burning' properties. Among these trace amines, tyramine has recently been described, at high doses, to exhibit an antilipolytic action and activation of glucose transport in human adipocytes, i.e., effects that are facilitating lipid storage rather than mobilization. Because of its close structural similarity to tyramine, NMT actions on human adipocytes therefore must to be reevaluated. To this aim, we studied the lipolytic and antilipolytic properties of NMT together with its interplay with insulin stimulation of glucose transport along with amine oxidase activities in adipose cells obtained from women undergoing abdominal surgery. NMT activated 2-deoxyglucose uptake when incubated with freshly isolated adipocytes at 0.01-1 mM, reaching one-third of the maximal stimulation by insulin. However, when combined with insulin, NMT limited by half the action of the lipogenic hormone on glucose transport. The NMT-induced stimulation of hexose uptake was sensitive to inhibitors of monoamine oxidases (MAO) and of semicarbazide-sensitive amine oxidase (SSAO), as was the case for tyramine and benzylamine. All three amines inhibited isoprenaline-induced lipolysis to a greater extent than insulin, while they were poorly lipolytic on their own. All three amines-but not isoprenaline-interacted with MAO or SSAO. Due to these multiple effects on human adipocytes, NMT cannot be considered as a direct lipolytic agent, potentially able to improve lipid mobilization and fat oxidation in consumers of NMT-containing dietary supplements.

Tyraminergic corollary discharge filters reafferent perception in a chemosensory neuron.

Interpreting sensory information requires its integration with the current behavior of the animal. However, how motor-related circuits influence sensory information processing is incompletely understood. Here, we report that current locomotor state directly modulates the activity of BAG CO2 sensory neurons in Caenorhabditis elegans. By recording neuronal activity in animals freely navigating CO2 landscapes, we found that during reverse crawling states, BAG activity is suppressed by tyraminergic corollary discharge signaling. We provide genetic evidence that tyramine released from the RIM reversal interneurons extrasynaptically activates the inhibitory chloride channel LGC-55 in BAG. Disrupting this pathway genetically leads to excessive behavioral responses to CO2 stimuli. Moreover, we find that LGC-55 signaling cancels out perception of self-produced CO2 and O2 stimuli when animals reverse into their own gas plume in ethologically relevant aqueous environments. Our results show that sensorimotor integration involves corollary discharge signals directly modulating chemosensory neurons.

Aromatic L-amino acid decarboxylases: mechanistic features and microbial applications.

Aromatic L-amino acid decarboxylases (AADCs) catalyze the conversion of aromatic L-amino acids into aromatic monoamines that play diverse physiological and biosynthetic roles in living organisms. For example, dopamine and serotonin serve as major neurotransmitters in animals, whereas tryptamine and tyramine are essential building blocks for synthesizing a myriad of secondary metabolites in plants. In contrast to the vital biological roles of AADCs in higher organisms, microbial AADCs are found in rather a limited range of microorganisms. For example, lactic acid bacteria are known to employ AADCs to achieve intracellular pH homeostasis and engender accumulation of tyramine, causing a toxic effect in fermented foods. Owing to the crucial pharmaceutical implications of aromatic monoamines and their derivatives, synthetic applications of AADCs have attracted growing attention. Besides, recent studies have uncovered that AADCs of human gut microbes influence host physiology and are involved in drug availability of Parkinson's disease medication. These findings bring the bacterial AADCs into a new arena of extensive research for biomedical applications. Here, we review catalytic features of AADCs and present microbial applications and challenges for biotechnological exploitation of AADCs. KEY POINTS: • Aromatic monoamines and their derivatives are increasingly important in the drug industry. • Aromatic L-amino acid decarboxylases are the only enzyme for synthesizing aromatic monoamines. • Microbial applications of aromatic L-amino acid decarboxylases have drawn growing attention.

The Prescriber's Guide to the MAOI Diet-Thinking Through Tyramine Troubles.

This review article features comprehensive discussions on the dietary restrictions issued to patients taking a classic monoamine oxidase inhibitor (phenelzine, tranylcypromine, isocarboxazid), or high-dose (oral or transdermal) selegiline. It equips doctors with the knowledge to explain to their patients which dietary precautions are necessary, and why that is so: MAOIs alter the capacity to metabolize certain monoamines, like tyramine, which causes dose-related blood pressure elevations. Modern food production and hygiene standards have resulted in large reductions of tyramine concentrations in most foodstuffs and beverages, including many cheeses. Thus, the risk of consequential blood pressure increases is considerably reduced-but some caution remains warranted. The effects of other relevant biogenic amines (histamine, dopamine), and of the amino acids L-dopa and L-tryptophan are also discussed. The tables of tyramine data usually presented in MAOI diet guides are by nature unhelpful and imprecise, because tyramine levels vary widely within foods of the same category. For this reason, it is vital that doctors understand the general principles outlined in this guide; that way, they can tailor their instructions and advice to the individual, to his/her lifestyle and situation. This is important because the pressor response is characterized by significant interpatient variability. When all factors are weighed and balanced, the conclusion is that the MAOI diet is not all that difficult. Minimizing the intake of the small number of risky foods is all that is required. Many patients may hardly need to change their diet at all.

Induced ligno-suberin vascular coating and tyramine-derived hydroxycinnamic acid amides restrict Ralstonia solanacearum colonization in resistant tomato.

Tomato varieties resistant to the bacterial wilt pathogen Ralstonia solanacearum have the ability to restrict bacterial movement in the plant. Inducible vascular cell wall reinforcements seem to play a key role in confining R. solanacearum into the xylem vasculature of resistant tomato. However, the type of compounds involved in such vascular physico-chemical barriers remain understudied, while being a key component of resistance. Here we use a combination of histological and live-imaging techniques, together with spectroscopy and gene expression analysis to understand the nature of R. solanacearum-induced formation of vascular coatings in resistant tomato. We describe that resistant tomato specifically responds to infection by assembling a vascular structural barrier formed by a ligno-suberin coating and tyramine-derived hydroxycinnamic acid amides. Further, we show that overexpressing genes of the ligno-suberin pathway in a commercial susceptible variety of tomato restricts R. solanacearum movement inside the plant and slows disease progression, enhancing resistance to the pathogen. We propose that the induced barrier in resistant plants does not only restrict the movement of the pathogen, but may also prevent cell wall degradation by the pathogen and confer anti-microbial properties, effectively contributing to resistance.

Male fire ant neurotransmitter precursors trigger reproductive development in females after mating.

Social insect queens have evolved mechanisms to prevent competition from their sexual daughters. For Solenopsis invicta, the fire ant, queens have evolved a primer pheromone that retards reproductive development in their winged reproductive daughters. If these daughters are removed from the influence of the queen, it takes about a week to start reproductive development; however, it starts almost immediately after mating. This dichotomy has been unsuccessfully investigated for several decades. Here we show that male fire ants produce tyramides, derivatives of the biogenic amine tyramine, in their reproductive system. Males transfer tyramides to winged females during mating, where the now newly mated queens enzymatically convert tyramides to tyramine. Tyramine floods the hemolymph, rapidly activating physiological processes associated with reproductive development. Tyramides have been found only in the large Myrmicinae ant sub-family (6,800 species), We suggest that the complex inhibition/disinhibition of reproductive development described here will be applicable to other members of this ant sub-family.

The impact of cell-free supernatants of Lactococcus lactis subsp. lactis strains on the tyramine formation of Lactobacillus and Lactiplantibacillus strains isolated from cheese and beer.

Tyramine is one of the most toxic biogenic amines and it is produced commonly by lactic acid bacteria in fermented food products. In present study, we investigated the influence of selected nisin-producing Lactococcus lactis subsp. lactis strains and their cell-free supernatants (CFSs) on tyramine production by four Lactobacillus and two Lactiplantibacillus strains isolated from cheese and beer. Firstly, we examined the antimicrobial effect of the CFSs from twelve Lactococcus strains against tested tyramine producers by agar-well diffusion assay. Six Lactococcus strains whose CFSs showed the highest antimicrobial effect on tyramine producers were further studied. Secondly, we investigated the influence of the selected six Lactococcus strains and their respective CFSs on tyramine production by tested Lactobacillus and Lactiplantibacillus strains in MRS broth supplemented with 2 g.L-1 of l-tyrosine. Tyramine production was monitored by HPLC-UV. The tyramine formation of all tested Lactobacillus and Lactiplantibacillus strains was not detected in the presence of Lc. lactis subsp. lactis CCDM 71 and CCDM 702, and their CFSs. Moreover, the remainder of the investigated Lactococcus strains (CCDM 670, CCDM 686, CCDM 689 and CCDM 731) and their CFSs decreased tyramine production significantly (P < 0.05) - even suppressing it completely in some cases - in four of the six tested tyramine producing strains.

Sensitivity to expression levels underlies differential dominance of a putative null allele of the Drosophila tßh gene in behavioral phenotypes.

The biogenic amine octopamine (OA) and its precursor tyramine (TA) are involved in controlling a plethora of different physiological and behavioral processes. The tyramine-ß-hydroxylase (tßh) gene encodes the enzyme catalyzing the last synthesis step from TA to OA. Here, we report differential dominance (from recessive to overdominant) of the putative null tßhnM18 allele in 2 behavioral measures in Buridan's paradigm (walking speed and stripe deviation) and in proboscis extension (sugar sensitivity) in the fruit fly Drosophila melanogaster. The behavioral analysis of transgenic tßh expression experiments in mutant and wild-type flies as well as of OA and TA receptor mutants revealed a complex interaction of both aminergic systems. Our analysis suggests that the different neuronal networks responsible for the 3 phenotypes show differential sensitivity to tßh gene expression levels. The evidence suggests that this sensitivity is brought about by a TA/OA opponent system modulating the involved neuronal circuits. This conclusion has important implications for standard transgenic techniques commonly used in functional genetics.

Tyramine induces dynamic RNP granule remodeling and translation activation in the Drosophila brain.

Ribonucleoprotein (RNP) granules are dynamic condensates enriched in regulatory RNA binding proteins (RBPs) and RNAs under tight spatiotemporal control. Extensive recent work has investigated the molecular principles underlying RNP granule assembly, unraveling that they form through the self-association of RNP components into dynamic networks of interactions. How endogenous RNP granules respond to external stimuli to regulate RNA fate is still largely unknown. Here, we demonstrate through high-resolution imaging of intact Drosophila brains that Tyramine induces a reversible remodeling of somatic RNP granules characterized by the decondensation of granule-enriched RBPs (e.g. Imp/ZBP1/IGF2BP) and helicases (e.g. Me31B/DDX-6/Rck). Furthermore, our functional analysis reveals that Tyramine signals both through its receptor TyrR and through the calcium-activated kinase CamkII to trigger RNP component decondensation. Finally, we uncover that RNP granule remodeling is accompanied by the rapid and specific translational activation of associated mRNAs. Thus, this work sheds new light on the mechanisms controlling cue-induced rearrangement of physiological RNP condensates.

Dopamine D2 Receptor Agonist Binding Kinetics-Role of a Conserved Serine Residue.

The forward (kon) and reverse (koff) rate constants of drug-target interactions have important implications for therapeutic efficacy. Hence, time-resolved assays capable of measuring these binding rate constants may be informative to drug discovery efforts. Here, we used an ion channel activation assay to estimate the kons and koffs of four dopamine D2 receptor (D2R) agonists; dopamine (DA), p-tyramine, (R)- and (S)-5-OH-dipropylaminotetralin (DPAT). We further probed the role of the conserved serine S1935.42 by mutagenesis, taking advantage of the preferential interaction of (S)-, but not (R)-5-OH-DPAT with this residue. Results suggested similar koffs for the two 5-OH-DPAT enantiomers at wild-type (WT) D2R, both being slower than the koffs of DA and p-tyramine. Conversely, the kon of (S)-5-OH-DPAT was estimated to be higher than that of (R)-5-OH-DPAT, in agreement with the higher potency of the (S)-enantiomer. Furthermore, S1935.42A mutation lowered the kon of (S)-5-OH-DPAT and reduced the potency difference between the two 5-OH-DPAT enantiomers. Kinetic Kds derived from the koff and kon estimates correlated well with EC50 values for all four compounds across four orders of magnitude, strengthening the notion that our assay captured meaningful information about binding kinetics. The approach presented here may thus prove valuable for characterizing D2R agonist candidate drugs.

Nonclinical Evaluation of Antibacterial Oxazolidinones Contezolid and Contezolid Acefosamil with Low Serotonergic Neurotoxicity.

Linezolid, the principal oxazolidinone antibiotic for therapy of Gram-positive infections, is limited by its myelosuppression and monoamine oxidase (MAO) inhibition, with the latter manifested as serotonergic neurotoxicity. The oral oxazolidinone contezolid and its injectable prodrug contezolid acefosamil are developed to overcome the above limitations. Serotonergic profiles for contezolid in vitro and for orally administered contezolid acefosamil in rodents are reported. Contezolid exhibited 2- and 148-fold reduction over linezolid reversible inhibition of MAO-A and MAO-B human enzyme isoforms. In the mouse head-twitch model, contezolid acefosamil was devoid of neurotoxicity at supratherapeutic oral doses of 40, 80, and 120 mg/kg. In the rat tyramine challenge model, no significant increase in arterial blood pressure was observed for contezolid acefosamil up to 120 mg/kg oral dosing. In these tests, the comparator linezolid has elicited serotonergic responses. Thus, contezolid and contezolid acefosamil exhibited an attenuated propensity to induce MAO-related serotonergic neurotoxicity. The data support a continued clinical evaluation of these agents, with potential to expand oxazolidinone therapies to patient populations on concurrent selective serotonin reuptake inhibitor medications or where MAO inhibitors are contraindicated.

Large-scale plasma-metabolome analysis identifies potential biomarkers of psoriasis and its clinical subtypes.

BACKGROUND: Psoriasis is an immune-mediated skin disease for which the crosstalk between genetic and environmental factors is responsible. To date, no definitive diagnostic criteria for psoriasis yet, and specific biomarkers are required. OBJECTIVE: We performed metabolome analysis to identify metabolite biomarkers of psoriasis and its subtypes such as psoriatic arthritis (PsA) and cutaneous psoriasis (PsC). METHODS: We constructed metabolomics profiling of 130 plasma samples (42 PsA patients, 50 PsC patients, and 38 healthy controls) using a nontargeted metabolomics approach. RESULTS: Psoriasis-control association tests showed that one metabolite (ethanolamine phosphate) was significantly increased in psoriasis samples than in the controls, whereas three metabolites decreased (false discovery rate [FDR] < 0.05; XA0019, nicotinic acid, and 20α-hydroxyprogesterone). In the association test between PsA and PsC, tyramine significantly increased in PsA than in PsC, whereas mucic acid decreased (FDR < 0.05). Molecular pathway analysis of the PsA-PsC association test identified enrichment of vitamin digestion and absorption pathway in PsC (P = 1.3 × 10-4). Correlation network analyses elucidated that a subnetwork centered on aspartate was constructed among the psoriasis-associated metabolites; meanwhile, the major subnetwork among metabolites with differences between PsA and PsC was primarily formed from saturated fatty acids. CONCLUSION: Our large-scale metabolome analysis highlights novel characteristics of plasma metabolites in psoriasis and the differences between PsA and PsC, which could be used as potential biomarkers of psoriasis and its clinical subtypes. These findings contribute to our understanding of psoriasis pathophysiology.

Metagenomic insights into the bacteria responsible for producing biogenic amines in sufu.

Harmful levels of biogenic amines (BAs) are frequently identified in sufu. The microorganisms and mechanisms responsible for BA production in sufu, however, are not well documented. In this study, sufu samples were randomly obtained from various regions of China. Putrescine, tyramine, and histamine were quantitated as the most abundant BAs. According to the metagenome sequencing, the abundances and diversities of genes encoding the critical enzymes in BA production were acquired. The results showed that genes encoding arginine-, ornithine-, tryptophan-, and histidine decarboxylases were the predominant amino acid decarboxylase genes. Furthermore, 34 metagenome-assembled genomes (MAGs) were generated, of which 23 encoded at least one gene involved in BA production. Genetic analysis of MAGs indicated genera affiliated with Enterococcus, Lactobacillus-related, and Lactococcus were the major histamine-synthesizing bacteria, and tyrosine may be utilized by Bacillus, Chryseobacterium, Kurthia, Lysinibacillus, Macrococcus, and Streptococcus to product tyramine. The critical species involved in two putrescine-producing pathways were also explored. In the ornithine decarboxylase pathway, Lactobacillus-related and Veillonella were predicted to be the main performers, whereas Sphingobacterium and unclassified Flavobacteriaceae were the dominant executors in the agmatine deiminase pathway. The present study not only explained the BAs formation mechanism in sufu but also identified specific bacteria used to control BAs in fermented soybean products.

Response of tyramine and glutamate related signals to nanoplastic exposure in Caenorhabditis elegans.

Neurotransmission related signals are involved in the control of response to toxicants. We here focused on the tyramine and the glutamate related signals to determine their roles in regulating nanoplastic toxicity in Caenorhabditis elegans. In the range of µg/L, exposure to nanopolystyrene (100 nm) increased the expression of tdc-1 encoding a tyrosine decarboxylase required for synthesis of tyramine, and decreased the expression of eat-4 encoding a glutamate transporter. Both TDC-1 and EAT-4 could act in the neurons to regulate the nanopolystyrene toxicity. Meanwhile, neuronal RNAi knockdown of tdc-1 induced a susceptibility to nanopolystyrene toxicity, and neuronal RNAi knockdown of eat-4 induced a resistance to nanopolystyrene toxicity. In the neurons, TYRA-2 functioned as the corresponding receptor of tyramine and acted upstream of MPK-1 signaling to regulate the nanopolystyrene toxicity. Moreover, during the control of nanopolystyrene toxicity, GLR-4 and GLR-8 were identified as the corresponding glutamate receptors, and acted upstream of JNK-1 signaling and DBL-1 signaling, respectively. Our results demonstrated the crucial roles of tyramine and glutamate related signals in regulating the toxicity of nanoplastics in organisms.

Determination of six underivatized biogenic amines by LC-MS/MS and study of biogenic amine production during trout (Salmo trutta) storage in ice.

Biogenic amines (BAs) are natural components of food produced mainly during metabolism in animals and plants. The determination of BAs is important because of their potential toxicity and their potential use as food spoilage indicators. In the present study, a method for the determination of six BAs (putrescine, cadaverine, histamine, ß-phenylethylamine, tyramine, and tryptamine) by Liquid Chromatography - Tandem Mass Spectrometry (LC-MS/MS) with Atmospheric Pressure Chemical Ionisation (APCI) source has been used on trout samples (Salmo trutta) stored in ice for 15 days. The results showed that on day 15 quite large amounts of putrescine (76.530 mg/kg), cadaverine (85.530 mg/kg), tryptamine (25.210 mg/kg), and histamine (15.975mg/kg) were detected, while the other BAs remained low (ß-phenylethylamine: 3.230 mg/kg, tyramine: 0.165mg/kg). Furthermore, microbiological data (Total Vial Count- TVC, Pseudomonas spp, and Shewanella putrefaciens) showed that trout samples became organoleptically unacceptable on day 12, while volatile compound analysis showed a significant increase in total amounts of alcohols, aldehydes, and ketones on days 12 and 15.

Search for Structural Basis of Interactions of Biogenic Amines with Human TAAR1 and TAAR6 Receptors.

The identification and characterization of ligand-receptor binding sites are important for drug development. Trace amine-associated receptors (TAARs, members of the class A GPCR family) can interact with different biogenic amines and their metabolites, but the structural basis for their recognition by the TAARs is not well understood. In this work, we have revealed for the first time a group of conserved motifs (fingerprints) characterizing TAARs and studied the docking of aromatic (ß-phenylethylamine, tyramine) and aliphatic (putrescine and cadaverine) ligands, including gamma-aminobutyric acid, with human TAAR1 and TAAR6 receptors. We have identified orthosteric binding sites for TAAR1 (Asp68, Asp102, Asp284) and TAAR6 (Asp78, Asp112, Asp202). By analyzing the binding results of 7500 structures, we determined that putrescine and cadaverine bind to TAAR1 at one site, Asp68 + Asp102, and to TAAR6 at two sites, Asp78 + Asp112 and Asp112 + Asp202. Tyramine binds to TAAR6 at the same two sites as putrescine and cadaverine and does not bind to TAAR1 at the selected Asp residues. ß-Phenylethylamine and gamma-aminobutyric acid do not bind to the TAAR1 and TAAR6 receptors at the selected Asp residues. The search for ligands targeting allosteric and orthosteric sites of TAARs has excellent pharmaceutical potential.

Phenoxy Radical Reactivity of Nucleic Acids: Practical Implications for Biotinylation.

Recent advances in peroxidase-mediated biotin tyramide (BT) signal amplification technology have resulted in high-resolution and subcellular compartment-specific mapping of protein and RNA localization. Horseradish peroxidase (HRP) in the presence of H2 O2 is known to activate phenolic compounds for phenoxy radical reaction with nucleic acids, where biotinylation by BT is a practical example. BT reactivity with RNA and DNA is not understood in detail. We report that BT phenoxy radicals react in a sequence-independent manner with guanosine bases in RNA. In contrast, DNA reactivity with BT cannot be detected by our methods under the same conditions. Remarkably, we show that fluorescein conjugates DNA rapidly and selectively reacts with BT phenoxy radicals, allowing convenient and practical biotinylation of DNA on fluorescein with retention of fluorescence.