Expanded Substrate Specificity in D-Amino Acid Transaminases: A Case Study of Transaminase from Blastococcus saxobsidens.

Enzymes with expanded substrate specificity are good starting points for the design of biocatalysts for target reactions. However, the structural basis of the expanded substrate specificity is still elusive, especially in the superfamily of pyridoxal-5'-phosphate-dependent transaminases, which are characterized by a conserved organization of both the active site and functional dimer. Here, we analyze the structure-function relationships in a non-canonical D-amino acid transaminase from Blastococcus saxobsidens, which is active towards D-amino acids and primary (R)-amines. A detailed study of the enzyme includes a kinetic analysis of its substrate scope and a structural analysis of the holoenzyme and its complex with phenylhydrazine-a reversible inhibitor and analogue of (R)-1-phenylethylamine-a benchmark substrate of (R)-selective amine transaminases. We suggest that the features of the active site of transaminase from B. saxobsidens, such as the flexibility of the R34 and R96 residues, the lack of bulky residues in the ß-turn at the entrance to the active site, and the short O-pocket loop, facilitate the binding of substrates with and without α-carboxylate groups. The proposed structural determinants of the expanded substrate specificity can be used for the design of transaminases for the stereoselective amination of keto compounds.

Recognition of methamphetamine and other amines by trace amine receptor TAAR1.

Trace amine-associated receptor 1 (TAAR1), the founding member of a nine-member family of trace amine receptors, is responsible for recognizing a range of biogenic amines in the brain, including the endogenous ß-phenylethylamine (ß-PEA)1 as well as methamphetamine2, an abused substance that has posed a severe threat to human health and society3. Given its unique physiological role in the brain, TAAR1 is also an emerging target for a range of neurological disorders including schizophrenia, depression and drug addiction2,4,5. Here we report structures of human TAAR1-G-protein complexes bound to methamphetamine and ß-PEA as well as complexes bound to RO5256390, a TAAR1-selective agonist, and SEP-363856, a clinical-stage dual agonist for TAAR1 and serotonin receptor 5-HT1AR (refs. 6,7). Together with systematic mutagenesis and functional studies, the structures reveal the molecular basis of methamphetamine recognition and underlying mechanisms of ligand selectivity and polypharmacology between TAAR1 and other monoamine receptors. We identify a lid-like extracellular loop 2 helix/loop structure and a hydrogen-bonding network in the ligand-binding pockets, which may contribute to the ligand recognition in TAAR1. These findings shed light on the ligand recognition mode and activation mechanism for TAAR1 and should guide the development of next-generation therapeutics for drug addiction and various neurological disorders.

25X-NBOMe compounds - chemistry, pharmacology and toxicology. A comprehensive review.

Recently, a growing number of reports have indicated a positive effect of hallucinogenic-based therapies in different neuropsychiatric disorders. However, hallucinogens belonging to the group of new psychoactive substances (NPS) may produce high toxicity. NPS, due to their multi-receptors affinity, are extremely dangerous for the human body and mental health. An example of hallucinogens that have been lately responsible for many severe intoxications and deaths are 25X-NBOMes - N-(2-methoxybenzyl)-2,5-dimethoxy-4-substituted phenethylamines, synthetic compounds with strong hallucinogenic properties. 25X-NBOMes exhibit a high binding affinity to serotonin receptors but also to dopamine, adrenergic and histamine receptors. Apart from their influence on perception, many case reports point out systemic and neurological poisoning with these compounds. In humans, the most frequent side effects are tachycardia, anxiety, hypertension and seizures. Moreover, preclinical studies confirm that 25X-NBOMes cause developmental impairments, cytotoxicity, cardiovascular toxicity and changes in behavior of animals. Metabolism of NBOMes seems to be very complex and involves many metabolic pathways. This fact may explain the observed high toxicity. In addition, many analytical methods have been applied in order to identify these compounds and their metabolites. The presented review summarized the current knowledge about 25X-NBOMes, especially in the context of toxicity.

Crosstalk between Pseudomonas aeruginosa antibiotic resistance and virulence mediated by phenylethylamine.

Multidrug efflux pumps are among the main Pseudomonas aeruginosa antibiotic-resistance determinants. Besides, efflux pumps are also involved in other relevant activities of bacterial physiology, including the quorum sensing-mediated regulation of bacterial virulence. Nevertheless, despite the relevance of efflux pumps in bacterial physiology, their interconnection with bacterial metabolism remains obscure. The effect of several metabolites on the expression of P. aeruginosa efflux pumps, and on the virulence and antibiotic resistance of this bacterium, was studied. Phenylethylamine was found to be both inducer and substrate of MexCD-OprJ, an efflux pump involved in P. aeruginosa antibiotic resistance and in extrusion of precursors of quorum-sensing signals. Phenylethylamine did not increase antibiotic resistance; however, the production of the toxin pyocyanin, the tissue-damaging protease LasB and swarming motility were reduced in the presence of this metabolite. This decrease in virulence potential was mediated by a reduction of lasI and pqsABCDE expression, which encode the proteins that synthesise the signalling molecules of two quorum-sensing regulatory pathways. This work sheds light on the interconnection between virulence and antibiotic-resistance determinants, mediated by bacterial metabolism, and points to phenylethylamine as an anti-virulence metabolite to be considered in the study of therapies against P. aeruginosa infections.

Ractopamine and age alter oxygen use and nitrogen metabolism in tissues of beef steers.

The objective was to quantify the effects of age and ractopamine (RAC) on whole body oxygen consumption and Leu flux, and oxygen flux and metabolism of nitrogenous compounds by the portal-drained viscera (PDV), liver, and hindquarters (HQ) of steers. Multicatheterized steers were fed a high energy diet every 2 h in 12 equal portions. Five younger steers (body weight, [BW] = 223 ±â€…10.1 kg) were 6 mo old and five older steers (BW = 464 ±â€…16.3 kg) were 14 mo old. Treatments were control (Cont) or 80 mg RAC per kg diet in a crossover design. Nitrogen (N) balance was measured on day 9 to 13. Whole body oxygen consumption and net flux were measured on day 11 and day 13, and net flux of N variables, Phe and Leu kinetics were measured on day 13. Whole body oxygen consumption increased (P < 0.05) in response to RAC in older but not younger steers. Retained N was greater (P = 0.009) for younger than older steers and increased (P = 0.010) with RAC in both ages of steers. Nitrogen retained as a percentage of N apparently absorbed increased (P < 0.05) in the older steers but not the younger steers in response to RAC. Oxygen uptake was greater (P < 0.05) in PDV, liver, and total splanchnic tissues in the younger steers and there was no response to RAC. In contrast, oxygen uptake in HQ increased (P < 0.05) with RAC in the older but not the younger steers. Concentration and net PDV release of α-amino N (AAN) were not affected by age or RAC. Uptake of AAN by liver decreased with RAC (P = 0.001). Splanchnic release of AAN was greater in younger steers (P = 0.020) and increased (P = 0.024) in response to RAC. For HQ tissues, uptake (P = 0.005) and extraction (P = 0.005) of AAN were lesser in older than younger steers and both increased (P = 0.001) in response to RAC. Based on Phe kinetics in HQ, RAC increased (P < 0.05) protein synthesis in older steers but not in younger steers. In contrast, protein breakdown decreased (P < 0.05) in response to RAC in younger steers. In response to RAC, protein degradation was less (P < 0.05) in younger than older steers. Based on Leu kinetics, whole body protein synthesis was greater in the younger steers (P = 0.022) but not altered in response to RAC. Ractopamine enhanced lean tissue growth by increasing supply of AAN to peripheral tissues and altering protein metabolism in HQ. These metabolic responses are consistent with established responses to RAC in production situations.

Ractopamine (RAC) is a feed additive that stimulates rate of gain, feed efficiency, and carcass leanness in finishing cattle. The objective of the present study was to quantify the effects of age and RAC on whole body metabolism and nutrient use by tissues of the portal-drained viscera, liver, and hindquarters of steers. Whole body oxygen consumption and oxygen uptake in hindquarters increased in response to RAC in older but not younger steers. Retained nitrogen was greater for younger than older steers and increased with RAC in both ages of steers. Uptake of α-amino nitrogen by liver decreased with RAC. For hindquarters tissues, uptake of α-amino nitrogen was lesser in older than younger steers and increased in both ages in response to RAC. Ractopamine increased protein synthesis in hindquarters of older steers but not in younger steers. In contrast, in response to RAC, protein breakdown decreased in younger steers and was less in younger than older steers. Ractopamine enhanced lean tissue growth by altering protein metabolism in hindquarters and increasing supply of α-amino nitrogen to peripheral tissues. The response to RAC was greater in older steers.

High-titre production of aromatic amines in metabolically engineered Escherichia coli.

AIMS: Aromatic amines with diverse physical characteristics are often employed as antioxidants and precursors to pharmaceutical products. As the traditional chemical methods pose serious environmental pollution, there is an arising interest in biomanufacturing aromatic amines from renewable feedstocks. MATERIALS AND RESULTS: We report the establishment of a bacterial platform for synthesizing three types of aromatic amines, namely, tyramine, dopamine and phenylethylamine. First, we expressed aromatic amino acid decarboxylase from Enterococcus faecium (pheDC) in an Escherichia coli strain with increasing shikimate (SHK) pathway flux towards L-tyrosine. We found that glycerol served as a better carbon source than glucose, resulting in 940 ± 46 mg/L tyramine from 4% glycerol. Next, the genes of lactate dehydrogenase (ldhA), pyruvate formate lyase (pflB), phosphate acetyltransferase (pta) and alcohol dehydrogenase (adhE) were deleted to mitigate the fermentation by-product formation. The tyramine level was further increased to 1.965 ± 0.205 g/L in the shake flask, which was improved by 2.1 times compared with that of the parental strain. By using a similar strategy, we also managed to produce 703 ± 21 mg/L dopamine and 555 ± 50 mg/L phenethylamine. CONCLUSIONS: We demonstrated that the knockout of ldhA-pflB-pta-adhE is an effective strategy for improving aromatic amine productions. SIGNIFICANCE AND IMPACT OF THE STUDY: This study achieved the highest aromatic amine titres in E. coli under shake flask reported to date.

Phenethylamine is a substrate of monoamine oxidase B in the paraventricular thalamic nucleus.

Monoamine oxidase (MAO) is a key enzyme responsible for the degradation of neurotransmitters and trace amines. MAO has two subtypes (MAO-A and MAO-B) that are encoded by different genes. In the brain, MAO-B is highly expressed in the paraventricular thalamic nucleus (PVT); however, its substrate in PVT remains unclear. To identify the MAO-B substrate in PVT, we generated Maob knockout (KO) mice and measured five candidate substrates (i.e., noradrenaline, dopamine, 3-methoxytyramine, serotonin, and phenethylamine [PEA]) by liquid chromatography tandem mass spectrometry. We showed that only PEA levels were markedly elevated in the PVT of Maob KO mice. To exclude the influence of peripheral MAO-B deficiency, we developed brain-specific Maob KO mice, finding that PEA in the PVT was increased in brain-specific Maob KO mice, whereas the extent of PEA increase was less than that in global Maob KO mice. Given that plasma PEA levels were elevated in global KO mice, but not in brain-specific KO mice, and that PEA passes across the blood-brain barrier, the substantial accumulation of PEA in the PVT of Maob KO mice was likely due to the increase in plasma PEA. These data suggest that PEA is a substrate of MAO-B in the PVT as well as other tissues.

Effects of ß-Phenylethylamine on Psychomotor, Rewarding, and Reinforcing Behaviors and Affective State: The Role of Dopamine D1 Receptors.

Beta-phenylethylamine (ß-PEA) is a well-known and widespread endogenous neuroactive trace amine found throughout the central nervous system in humans. In this study, we demonstrated the effects of ß-PEA on psychomotor, rewarding, and reinforcing behaviors and affective state using the open-field test, conditioned place preference (CPP), self-administration, and ultrasonic vocalizations (USVs) paradigms. We also investigated the role of the dopamine (DA) D1 receptor in the behavioral effects of ß-PEA in rodents. Using enzyme-linked immunosorbent assay (ELISA) and Western immunoblotting, we also determined the DA concentration and the DA-related protein levels in the dorsal striatum of mice administered with acute ß-PEA. The results showed that acute ß-PEA increased stereotypic behaviors such as circling and head-twitching responses in mice. In the CPP experiment, ß-PEA increased place preference in mice. In the self-administration test, ß-PEA significantly enhanced self-administration during a 2 h session under fixed ratio (FR) schedules (FR1 and FR3) and produced a higher breakpoint during a 6 h session under progressive ratio schedules of reinforcement in rats. In addition, acute ß-PEA increased 50-kHz USV calls in rats. Furthermore, acute ß-PEA administration increased DA concentration and p-DAT and TH expression in the dorsal striatum of mice. Finally, pretreatment with SCH23390, a DA D1 receptor antagonist, attenuated ß-PEA-induced circling behavior and ß-PEA-taking behavior in rodents. Taken together, these findings suggest that ß-PEA has rewarding and reinforcing effects and psychoactive properties, which induce psychomotor behaviors and a positive affective state by activating the DA D1 receptor in the dorsal striatum.

Ocean Acidification Amplifies the Olfactory Response to 2-Phenylethylamine: Altered Cue Reception as a Mechanistic Pathway?

With carbon dioxide (CO2) levels rising dramatically, climate change threatens marine environments. Due to increasing CO2 concentrations in the ocean, pH levels are expected to drop by 0.4 units by the end of the century. There is an urgent need to understand the impact of ocean acidification on chemical-ecological processes. To date, the extent and mechanisms by which the decreasing ocean pH influences chemical communication are unclear. Combining behaviour assays with computational chemistry, we explore the function of the predator related cue 2-phenylethylamine (PEA) for hermit crabs (Pagurus bernhardus) in current and end-of-the-century oceanic pH. Living in intertidal environments, hermit crabs face large pH fluctuations in their current habitat in addition to climate-change related ocean acidification. We demonstrate that the dietary predator cue PEA for mammals and sea lampreys is an attractant for hermit crabs, with the potency of the cue increasing with decreasing pH levels. In order to explain this increased potency, we assess changes to PEA's conformational and charge-related properties as one potential mechanistic pathway. Using quantum chemical calculations validated by NMR spectroscopy, we characterise the different protonation states of PEA in water. We show how protonation of PEA could affect receptor-ligand binding, using a possible model receptor for PEA (human TAAR1). Investigating potential mechanisms of pH-dependent effects on olfactory perception of PEA and the respective behavioural response, our study advances the understanding of how ocean acidification interferes with the sense of smell and thereby might impact essential ecological interactions in marine ecosystems.

The calcimimetic R-568 attenuates subarachnoid hemorrhage-induced vasospasm through PI3K/Akt/eNOS signaling pathway in the rat model.

Cerebral vasospasm (CVS) causes mortality and morbidity in patients after subarachnoid hemorrhage (SAH). The mechanism and adequate treatment of CVS are still elusive. R-568 is a calcimimetic agent known to exert a vasodilating effect. However, there is no report on its vasodilator effect against SAH-induced vasospasm. In the present study, we investigated the therapeutic effect of R-568 on the SAH-induced CVS model in rats. Seventy-two adult male Sprague-Dawley rats were divided into 8 groups: sham surgery; SAH only; SAH + Vehicle, SAH + R-568; SAH + R-568 + Wortmannin (the PI3K inhibitor); SAH + Wortmannin; SAH + R-568 + Calhex-231 (a calcilytic agent); SAH + Calhex-231. SAH was induced by blood (0.3 mL) given by intracisternal injection. R-568 (20 µM) was administered intracisternal immediately prior to experimental SAH. Basilar arteries (BAs) were obtained to evaluate PI3K/Akt/eNOS pathway (immunoblotting) and morphological changes 48 h after SAH. Perimeters of BAs were decreased by 24.1% in the SAH group compared to the control group and the wall thickness was increased by 75.3%. With R-568 treatment, those percentages were 9.6% and 29.6%, respectively, indicating that vasospasm was considerably improved when compared with the SAH group (P < 0.001 in both). While p-PI3K/PI3K and p-Akt/Akt ratio and eNOS protein expression were markedly decreased in the SAH rats, treatment with R-568 resulted in a significant increase in these levels. The beneficial effects of R-568 were partially blocked in the presence of Calhex-231 and completely blocked in the presence of Wortmannin. Herein, we found that treatment with R-568 would attenuate SAH-induced CVS through the PI3K/Akt/eNOS pathway and demonstrate therapeutic promise in CVS treatment following SAH.

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.

In-Source Fragmentation of Phenethylamines by Electrospray Ionization Mass Spectrometry: Toward Highly Sensitive Quantitative Analysis of Monoamine Neurotransmitters.

Electrospray ionization mass spectrometry (ESI-MS) is widely used to analyze biomolecules, which are usually detected as protonated and cation-adducted molecules in the positive-ion mode. However, phenethylamine derivatives, which are known as neurotransmitters and psychoactive drugs, undergo the protonation and subsequently lose NH3 during ESI. As a result, intense fragment-ion signals are observed in their ESI-MS spectra, which hamper the unambiguous identification of phenethylamine derivatives. To understand the mechanism of the loss of NH3 from these phenethylammoniums, the fragmentations of model 4-substituted phenethylamines were investigated and the fragment ions were identified as spiro[2.5]octadienyliums. Fragmentation was enhanced by the presence of electron-donating groups, and most substituted phenethylamines generated spiro[2.5]octadienyliums as fragment ions during ESI-MS, except those with strong electron-withdrawing groups. The quantitative analysis of phenethylamines by liquid chromatography tandem mass spectrometry is typically performed by multiple reaction monitoring using protonated molecules as the precursor. In contrast, the conversion of precursor ions from the protonated molecules into the spiro[2.5]octadienylium fragment improved the signal-to-noise ratio, allowing the quantitative analysis of phenethylamines with high sensitivity and accuracy.

Impaired neuronal and astroglial metabolic activity in chronic unpredictable mild stress model of depression: Reversal of behavioral and metabolic deficit with lanicemine.

Major depressive disorder is the leading cause of disability and suicidality worldwide. Here, we evaluated neural metabolic activity in prefrontal cortex (PFC) in C57BL6 mice undergoing a chronic unpredictable mild stress (CUMS) for three weeks to induce depression. Further, the efficacy of Lanicemine, a low trapping NMDA receptor antagonist, on behavioral and neurometabolic measures in CUMS mice was evaluated. The PFC neuronal and astroglial metabolic activity was evaluated by Proton Observed Carbon Edited (POCE) MR spectroscopy together with an infusion of [1,6-13C2]glucose and [2-13C]acetate, respectively. The rates of glutamatergic, GABAergic and astrocytic TCA cycles and neurotransmitter cycling were obtained by fitting a three-compartment metabolic model to 13C turnover of amino acids. Mice subjected to CUMS exhibited significantly reduced sucrose preference (CUMS 58.0 ± 12.5%, n = 29; Control 86.3 ± 6.4%, n = 30; p < 0.0001), and increased immobility (CUMS 146.1 ± 60.8s, n = 29; Control 29.9 ± 19.3s, n = 30; p < 0.0001) in the forced swim test. The concentrations of 13C labeled amino acids from [2-13C]acetate were decreased suggesting reduced astroglial metabolic activity in CUMS mice. The glutamatergic and GABAergic TCA cycle rates were decreased in CUMS mice when compared with controls. In addition, GABA-glutamine and glutamate-glutamine neurotransmitter cycling were reduced in mice subjected to CUMS regimen. Most interestingly, a short time intervention of lanicemine restored behavioral measures (sucrose preference and immobility), and rates of glucose oxidation in glutamatergic and GABAergic neurons in CUMS mice. In summary, our findings suggest that depression leads to a reduction in excitatory and inhibitory neurotransmission in PFC, and targeting glutamatergic pathway may have potential therapeutic role in chronic depression.

Pathway Engineering for Phenethylamine Production in Escherichia coli.

In this study, the metabolic pathway of phenethylamine synthesis was reconstructed by chromosomal integration and overexpression of the Enterococcus faecium pdc gene encoding phenylalanine decarboxylase in Escherichia coli. The genes encoding 3-deoxy-d-arabinoheptulosonate-7-phosphate synthase (aroG), shikimate kinase II (aroL), chorismate mutase/prephenate dehydratase (pheA), and tyrosine aminotransferase (tyrB) in the phenethylamine synthetic pathway were sequentially chromosomally overexpressed. The phosphotransferase system was replaced by deleting the ptsH-ptsI-crr genes and chromosomally overexpressing the genes encoding galactose permease (galP) and glucokinase (glk). In addition, the zwf gene encoding glucose-6-phosphate dehydrogenase in the pentose phosphate pathway was chromosomally overexpressed, generating the final engineered E. coli strain AUD9. The AUD9 strain produced 2.65 g L-1 phenethylamine with a yield of 0.27 g of phenethylamine g-1 glucose in batch fermentation; fed-batch fermentation of AUD9 produced 38.82 g L-1 phenethylamine with a productivity of 1.08 g L-1 h-1 phenethylamine, demonstrating its potential for industrial fermentative production of phenethylamine.

Nuclear quantum effects in enzymatic reactions: simulation of the kinetic isotope effect of phenylethylamine oxidation catalyzed by monoamine oxidase A.

The kinetic isotope effect (KIE) is arguably the most established experimental observable reflecting nuclear quantum effects in enzymatic reactions. The role of nuclear quantum effects in enzymes is rather intriguing and has long been a source of profound investigations. Herein, we present a computational study of monoamine oxidase A (MAO A) enzyme and its substrate phenylethylamine, focusing on the impact of nuclear quantum effects on the reaction free energy barrier. Two distinct schemes of quantization of nuclear motion were used, one being the established Quantum Classical Path (QCP) approach, and the other our own code for quantum treatment along the selected nuclear coordinate (hydrogen transfer coordinate) which reasonably mimics the reaction coordinate. In excellent agreement with the experimental value of 8.5 ± 0.3, H/D KIE was computed to 8.66, corresponding to the D-H barrier difference of 1.28 kcal mol-1. The magnitude of KIE implies that nuclear quantum effects probably have only a minor role in the reaction, which is in accordance with the features of potentials computed along the reaction coordinate and with the pertinent energy levels and wavefunctions. The computed H/D KIE for the same reaction in aqueous solution and in the gas phase was fairly similar to the one in the enzyme, suggesting that the role of tunneling in the catalytic function of MAO A is insignificant. The agreement between the computed and observed KIE supported by analysis of nuclear quantum effects implicitly validates the assumed hydride transfer reaction mechanism.

A Computational Pipeline to Predict Cardiotoxicity: From the Atom to the Rhythm.

RATIONALE: Drug-induced proarrhythmia is so tightly associated with prolongation of the QT interval that QT prolongation is an accepted surrogate marker for arrhythmia. But QT interval is too sensitive a marker and not selective, resulting in many useful drugs eliminated in drug discovery. OBJECTIVE: To predict the impact of a drug from the drug chemistry on the cardiac rhythm. METHODS AND RESULTS: In a new linkage, we connected atomistic scale information to protein, cell, and tissue scales by predicting drug-binding affinities and rates from simulation of ion channel and drug structure interactions and then used these values to model drug effects on the hERG channel. Model components were integrated into predictive models at the cell and tissue scales to expose fundamental arrhythmia vulnerability mechanisms and complex interactions underlying emergent behaviors. Human clinical data were used for model framework validation and showed excellent agreement, demonstrating feasibility of a new approach for cardiotoxicity prediction. CONCLUSIONS: We present a multiscale model framework to predict electrotoxicity in the heart from the atom to the rhythm. Novel mechanistic insights emerged at all scales of the system, from the specific nature of proarrhythmic drug interaction with the hERG channel, to the fundamental cellular and tissue-level arrhythmia mechanisms. Applications of machine learning indicate necessary and sufficient parameters that predict arrhythmia vulnerability. We expect that the model framework may be expanded to make an impact in drug discovery, drug safety screening for a variety of compounds and targets, and in a variety of regulatory processes.

Creation of (R)-Amine Transaminase Activity within an α-Amino Acid Transaminase Scaffold.

The enzymatic transamination of ketones into (R)-amines represents an important route for accessing a range of pharmaceuticals or building blocks. Although many publications have dealt with enzyme discovery, protein engineering, and the application of (R)-selective amine transaminases [(R)-ATA] in biocatalysis, little is known about the actual in vivo role and how these enzymes have evolved from the ubiquitous α-amino acid transaminases (α-AATs). Here, we show the successful introduction of an (R)-transaminase activity in an α-amino acid aminotransferase with one to six amino acid substitutions in the enzyme's active site. Bioinformatic analysis combined with computational redesign of the d-amino acid aminotransferase (DATA) led to the identification of a sextuple variant having a specific activity of 326 milliunits mg-1 in the conversion of (R)-phenylethylamine and pyruvate to acetophenone and d-alanine. This value is similar to those of natural (R)-ATAs, which typically are in the range of 250 milliunits mg-1. These results demonstrate that (R)-ATAs can evolve from α-AAT as shown here for the DATA scaffold.

Characterization of Arylalkylamine N-Acyltransferase from Tribolium castaneum: An Investigation into a Potential Next-Generation Insecticide Target.

The growing issue of insecticide resistance has meant the identification of novel insecticide targets has never been more important. Arylalkylamine N-acyltransferases (AANATs) have been suggested as a potential new target. These promiscuous enzymes are involved in the N-acylation of biogenic amines to form N-acylamides. In insects, this process is a key step in melanism, hardening of the cuticle, removal of biogenic amines, and in the biosynthesis of fatty acid amides. The unique nature of each AANAT isoform characterized indicates each organism accommodates an assembly of discrete AANATs relatively exclusive to that organism. This implies a high potential for selectivity in insecticide design, while also maintaining polypharmacology. Presented here is a thorough kinetic and structural analysis of AANAT found in one of the most common secondary pests of all plant commodities in the world, Tribolium castaneum. The enzyme, named TcAANAT0, catalyzes the formation of short-chain N-acylarylalkylamines, with short-chain acyl-CoAs (C2-C10), benzoyl-CoA, and succinyl-CoA functioning in the role of acyl donor. Recombinant TcAANAT0 was expressed and purified from E. coli and was used to investigate the kinetic and chemical mechanism of catalysis. The kinetic mechanism is an ordered sequential mechanism with the acyl-CoA binding first. pH-rate profiles and site-directed mutagenesis studies identified amino acids critical to catalysis, providing insights about the chemical mechanism of TcAANAT0. A crystal structure was obtained for TcAANAT0 bound to acetyl-CoA, revealing valuable information about its active site. This combination of kinetic analysis and crystallography alongside mutagenesis and sequence analysis shines light on some approaches possible for targeting TcAANAT0 and other AANATs for novel insecticide design.

Hexanol-Based Supramolecular Solvents Tool for the Determination of 11 Illicit Phenethylamines in Oral Fluid by LC-MS/MS.

Monitoring of new phenethylamine designer drugs in oral fluid (OF) is a crucial aim in workplace testing and driving under the influence of drug programs. In this study a simple and very quick method for the quantification of 11 illicit drugs in OF, which gave negative results to immunoassay tests, is proposed. Sample treatment and extraction of analytes were simultaneously achieved by applying supramolecular solvents (SUPRAS) tool. Chromatographic separation and compounds quantification were carried out by liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Efficacy of cleaning-up/extraction of this SUPRAS approach was fully confirmed by recovery and matrix effect results. The entire analytical procedure was validated following the international guidelines. The SUPRAS extraction coupled with LC-MS/MS resulted in powerful tool for the control of phenethylamines abuse, with rapid run time and minimal sample preparation. The use of this methodology could be easily extended to monitoring of other drugs of abuse.