Amine Oxidase Enzymes Research Articles

Identification and Evaluation of Olive Phenolics in the Context of Amine Oxidase Enzyme Inhibition and Depression: In Silico Modelling and In Vitro Validation.

The Mediterranean diet well known for its beneficial health effects, including mood enhancement, is characterised by the relatively high consumption of extra virgin olive oil (EVOO), which is rich in bioactive phenolic compounds. Over 200 phenolic compounds have been associated with Olea europaea, and of these, only a relatively small fraction have been characterised. Utilising the OliveNetTM library, phenolic compounds were investigated as potential inhibitors of the epigenetic modifier lysine-specific demethylase 1 (LSD1). Furthermore, the compounds were screened for inhibition of the structurally similar monoamine oxidases (MAOs) which are directly implicated in the pathophysiology of depression. Molecular docking highlighted that olive phenolics interact with the active site of LSD1 and MAOs. Protein-peptide docking was also performed to evaluate the interaction of the histone H3 peptide with LSD1, in the presence of ligands bound to the substrate-binding cavity. To validate the in silico studies, the inhibitory activity of phenolic compounds was compared to the clinically approved inhibitor tranylcypromine. Our findings indicate that olive phenolics inhibit LSD1 and the MAOs in vitro. Using a cell culture model system with corticosteroid-stimulated human BJ fibroblast cells, the results demonstrate the attenuation of dexamethasone- and hydrocortisone-induced MAO activity by phenolic compounds. The findings were further corroborated using human embryonic stem cell (hESC)-derived neurons stimulated with all-trans retinoic acid. Overall, the results indicate the inhibition of flavin adenine dinucleotide (FAD)-dependent amine oxidases by olive phenolics. More generally, our findings further support at least a partial mechanism accounting for the antidepressant effects associated with EVOO and the Mediterranean diet.

Amine oxidase copper-containing 3 aggravates cardiac remodelling by generating hydrogen peroxide after myocardial infarction.

Amine oxidase copper-containing 3 (AOC3) is a member of the semicarbazide-sensitive amine oxidase enzyme family. It acts as an ectoenzyme catalysing the oxidative deamination of primary amines and generating hydrogen peroxide (H2 O2 ). While AOC3 is implicated in cardiovascular diseases such as atherosclerosis, its role in cardiac remodelling after myocardial infarction (MI) is unclear. In this study, we first confirmed a long-term upregulation of AOC3 in both cardiac myofibroblasts after MI in vivo and angiotensin II (ANGII)-treated cardiac fibroblasts in vitro. AOC3 knockdown not only inhibited the activation of cardiac fibroblasts induced by ANGII but also alleviated cardiac fibrosis in mice after MI. Using sh-AOC3 lentiviruses, exogenous recombinant AOC3 (r-AOC3), semicarbazide (an AOC3 inhibitor), and catalase (a hydrogen peroxide scavenger) treatments, we also demonstrated that AOC3 promoted H2 O2 generation, increased oxidative stress, and enhanced ERK1/2 activation, which were responsible for the activation of cardiac fibroblasts. In particular, AOC3 knockdown also improved cardiac function and hypertrophy after MI. Through a coculture system, we confirmed that AOC3 expressed on cardiac myofibroblasts was able to enhance oxidative stress and induce hypertrophy of cardiomyocytes by promoting H2 O2 generation. Similarly, r-AOC3 promoted H2 O2 generation and resulted in oxidative stress and hypertrophy of cardiomyocytes, which were almost inhibited by both semicarbazide and catalase. In conclusion, AOC3 plays a critical role in cardiac fibrosis and hypertrophy after MI by promoting the generation of H2 O2 . AOC3 is a promising therapeutic target against cardiac remodelling. © 2023 The Pathological Society of Great Britain and Ireland.

Cytoprotective action of renalase in diabetic kidney disease: an update on its association with cardiovascular disease and COVID -19

Renalase is a mono amine oxidase enzyme that can metabolise the active catecholamines in the body. Renalase is typically expressed in the cardiovascular system, skeletal muscle, and renal system. Renalase gene is important in human diseases and is known to break down catecholamines in the blood. Gene for renalase as extreme frequency of congestive heart failure disease in people with long term renal disease and other disorders was shown to be explained by a protein that is released by the kidney. Recent advances have increased our understanding of its structure, enzymatic activity, mechanisms of action, relationships with human disease states, and potential therapeutic value. An enzyme description of renalase is provided in this review, along with COVID-19 infection.

Combining monoamine oxidase B and semicarbazide-sensitive amine oxidase enzyme inhibition to address inflammatory disease

The discovery of a dual MAO-B/SSAO inhibitor PXS-5131 is reported. The compound offers a compact and rigid three-dimensional structure with superior selectivity over MAO-A. Potency and selectivity are linked to both the double bond geometry and stereochemistry of the allylamine moiety, highlighting the importance of optimal set up of these features in the class of amine oxidase inhibitors. PXS-5131 possesses an attractive preclinical pharmacokinetic profile and has anti-inflammatory properties in models of acute inflammation and neuroinflammation.

Bovine Serum Amine Oxidase and Polyamine Analogues: Chemical Synthesis and Biological Evaluation Integrated with Molecular Docking and 3-D QSAR Studies

Natural polyamines (PAs) are key players in cellular homeostasis by regulating cell growth and proliferation. Several observations highlight that PAs are also implicated in pathways regulating cell death. Indeed, the PA accumulation cytotoxic effect, maximized with the use of bovine serum amine oxidase (BSAO) enzyme, represents a valuable strategy against tumor progression. In the present study, along with the design, synthesis, and biological evaluation of a series of new spermine (Spm) analogues (1-23), a mixed structure-based (SB) and ligand-based (LB) protocol was applied. Binding modes of BSAO-PA modeled complexes led to clarify electrostatic and steric features likely affecting the BSAO-PA biochemical kinetics. LB and SB three-dimensional quantitative structure-activity relationship (Py-CoMFA and Py-ComBinE) models were developed by means of the 3d-qsar.com portal, and their analysis represents a strong basis for future design and synthesis of PA BSAO substrates for potential application in oxidative stress-induced chemotherapy.

Inhibition of vascular adhesion protein-1 modifies hepatic steatosis in vitro and in vivo.

BACKGROUNDNon-alcoholic fatty liver disease (NAFLD) is associated with obesity, insulin resistance and dyslipidaemia and currently is estimated to affect up to a third of all individuals in developed countries. Current standard of care for patients varies according to disease stage, but includes lifestyle interventions common insulin sensitizers, antioxidants and lipid modifiers. However, to date specific therapies have shown little histological or fibrosis stage improvement in large clinical trials, and there is still no licensed therapy for NAFLD. Given the high prevalence, limited treatment options and significant screening costs for the general population, new treatments are urgently required.AIMTo assess the potential for inhibition of the amine oxidase enzyme vascular adhesion protein-1 (VAP-1) to modify hepatic lipid accumulation in NAFLD.METHODSWe have used immunochemical and qPCR analysis to document expression of VAP-1 and key functional proteins and transporters across the NAFLD spectrum. We then utilised hepatocytes in culture and human precision cut liver slices in concert with selective enzyme activity inhibitors to test the effects of activating the semicarbazide-sensitive amine oxidase activity of VAP-1 on hepatic lipid uptake and triglyceride export. A murine model of NAFLD was also used to determine the consequences of VAP-1 knockout and gene expression arrays were used to quantify the effects of VAP-1 activity on key lipid modifying and proinflammatory gene expression.RESULTSWe confirmed that increasing severity of NAFLD and progression to cirrhosis was associated with a significant increase in hepatocellular VAP-1 expression. Hepatocytes in vitro exposed to recombinant VAP-1 and its substrate methylamine showed increased lipid accumulation as determined by quantification of Oil Red O uptake. This was recapitulated using hydrogen peroxide, and lipid accumulation was accompanied by changes in expression of the lipid transporter molecules FABP3, FATP6, insulin receptor subunits and PPARα. Human liver tissue exposed to recombinant VAP-1 or substrates for endo/exogenous VAP-1 produced less triglyceride than untreated tissue and demonstrated an increase in steatosis. This response could be inhibited by using bromoethylamine to inhibit the SSAO activity of VAP-1, and mice deficient in VAP-1/AOC3 also demonstrated reduced steatosis on high fat diet. Exposure of human liver tissue to methylamine to activate VAP-1 resulted in increased expression of FABP2 and 4, FATP3-5, caveolin-1, VLDLR, PPARGC1 and genes associated with the inflammatory response.CONCLUSIONOur data confirm that the elevations in hepatic VAP-1 expression reported in nonalcoholic steatohepatitis can contribute to steatosis, metabolic disturbance and inflammation. This suggests that targeting the semicarbazide sensitive amine oxidase capacity of VAP-1 may represent a useful adjunct to other therapeutic strategies in NAFLD.

Evaluation of a Pan-Lysyl Oxidase Inhibitor, Pxs-5505, in Myelofibrosis: A Phase I, Randomized, Placebo Controlled Double Blind Study in Healthy Adults

BACKGROUND: Myeloproliferative neoplasms (MPNs) are clonal stem cell neoplasms characterized by terminal expansion of the myeloid cell lineage and include essential thrombocythemia (ET), polycythemia vera (PV), and myelofibrosis (MF). MF carries the worst prognosis and is characterized by reactive bone marrow fibrosis. Currently bone marrow transplant is the only known therapy that can reverse fibrosis and alter the disease course. Lysyl oxidases (LOX, LOXL1-4) are copper amine oxidase enzymes that facilitate the cross-linking of collagen and elastin through deamination and oxidization of lysine residues, yielding highly reactive aldehydes. This is essential for fibrotic tissue formation. An earlier study identified an important role for LOX in the development of MF in mice (Eliades et al, J. Biol. Chem. 2011; PMID: 21665949). Furthermore, small molecule pan-LOX inhibitors reduced spleen size and bone marrow fibrosis in mouse models of MF (Leiva et al, Int J. Hemat. 2019; PMID: 31637674). This provides clinical rationale for the use of pan-lysyl oxidase inhibition in MF. A Phase 1 study was conducted to establish the safety, tolerability, pharmacokinetics (PK), and pharmacodynamics (PD) of PXS-5505, a pan-lysyl oxidase inhibitor. Plasma levels of LOX and LOXL2 were investigated in a separate cohort of MPN patients, with the ultimate goal to develop PXS-5505-based therapy in MF patients. METHODS: PXS-5505 was dosed orally in a Phase I, randomized, placebo-controlled, single ascending (Part A) and multiple ascending dose (Part B) double-blind study in healthy male volunteers. Plasma LOX and LOXL2 levels were also measured in a separate cohort of MPN patients using ELISA-based Single Molecule Array technology (Simoa; Quanterix). RESULTS: Forty subjects were enrolled in Part A and 16 were enrolled in Part B. In Part A, there were 5 cohorts consisting of 8 subjects (6 active, 2 placebo) with doses of 10, 50, 100, 200 or 300 mg administered once. In Part B, there were 2 cohorts of 8 subjects each (6 active, 2 placebo) at doses of 100 or 200 mg daily for 14 days. There were no significant treatment-related adverse events. Mean age in Part A was 32.0 years (SD 11.14) and 32.1 years (SD 12.14) in Part B. Cmax and AUC increased linearly across the dose range of 10 to 300 mg for single dose administration. In multiple dosing at 200 mg daily, Cmax was 916 ng/mL and AUC0-24 was 7421 hr*ng/mL on Day 14, with median Tmax of 1 hour and t1/2 of 7 hours. When measured in plasma, LOX was dose-dependently inhibited and achieved very strong inhibition (median 80%) with a 200 mg dose around Tmax. The 300 mg dosing did not significantly increase inhibition. Multiple daily doses of 100-200 mg resulted in 60-70% and 50-60% inhibition of plasma LOX at 12 and 24 hours. Average LOX plasma levels were 3.61 ng/mL (range: 3.20-13.42 ng/mL; SD: 2.92 ng/mL). We measured plasma LOX and LOXL2 levels in a separate MPN cohort of 9 ET, 8 PV, and 13 MF patients (mean age 61.4, range:24-84, 65% males). LOXL2 levels were higher in MF (mean 415 pg/mL) compared to ET (mean 209 pg/mL) and PV (mean 322 pg/ml), although this was not significant. However, LOXL2 levels in all MPN patients (mean 333 pg/mL) were significantly higher than LOXL2 levels in normal controls (mean 152 pg/mL, p<0.01). There were no significant differences in LOX levels in the two groups. DISCUSSION: PXS-5505 demonstrated an excellent safety profile and was well tolerated in healthy human subjects. PK/PD properties are consistent with preclinical data and support once or twice daily >100 mg dosing over 14 days. PXS-5505 achieves long-lasting, strong inhibition of lysyl oxidases. Plasma LOXL2 levels are higher in MF patients compared to healthy controls, and we found no significant disease associations of LOX or LOXL2 among MPN subtypes in our small cohort. LOXL2 is likely a more sensitive MF biomarker as it is present at low concentrations in the blood, while LOX is constantly produced from major organs making detection due to disease more difficult. Based on previous mouse studies, it is possible that LOX levels would be higher in MF patients when compared to age-matched controls, which we will investigate further. We will open a Phase IB/II study of PXS-5505 in MF patients resistant to ruxolitinib. Disclosures Neuberg: Madrigak Pharmaceuticals: Current equity holder in publicly-traded company; Celgene: Research Funding; Pharmacyclics: Research Funding. Ravid:Pharmaxis: Research Funding. Jarolimek:Pharmaxis Ltd: Current Employment. Charlton:Pharmaxis Ltd: Current Employment. Hobbs:Novartis: Honoraria; Constellation: Honoraria, Research Funding; Jazz: Honoraria; Celgene/BMS: Honoraria; Merck: Research Funding; Incyte: Research Funding; Bayer: Research Funding.

UJI EFEKTIVITAS ANTIDEPRESAN EKSTRAK BATANG SERAI (Cymbopogon nardus (L.) Rendle) TERHADAP MENCIT PUTIH JANTAN (Mus musculus)

Depression is a mood disorder that is characterized by profound and sustained sadness and sadness until the loss of life passion. Lemongrass which contains eugenol is thought to have the potential as an antidepressant that works by inhibiting the Mono Amine Oxidase enzyme. This study aims to determine the effectiveness of antidepressant citronella extract (Cymbopogon nardus (L.) Rendle) on male white mice. This study used 5 treatment groups namely CMC Na 0.5%, amitriptilyn, lemongrass extract dose 1.5 mg/kgBB, 15 mg/kgBB and 150 mg/kgBB. Antidepressant testing was determined based on immobility time using the forsed swim test method. Data analysis using statistics was tested for normality and continued with the One Way Anova method with a 95% confidence level. The results showed that the most effective treatment group as an antidepressant was amitriptilyn followed by lemongrass extract dose 150 mg/kgBB. The conclusion of this study is that lemongrass extract dose 150 mg/kgBB has the effectiveness as an antidepressant but is no more effective than the comparison of amitrip

A novel thermostable alkaline histamine oxidase from Glutamicibacter sp. N1A3101, induced by histamine and its analogue betahistine

Biogenic amines (BAs) are low molecular weight organic bases formed by natural amino acids decarboxylation and trigger an array of toxicological effects in humans and animals. Bacterial amine oxidases enzymes are determined as practical tools to implement the rapid quantification of BAs in foods. Our study set out to obtain a new efficient, amine oxidase enzyme for developing new enzyme-based quantification of histamine. The soils from different sources were screened using histamine as sole carbon and nitrogen sources, and histamine oxidase producing bacteria were selected and identified using specific primers for histamine oxidase (HOD) gene. The HOD gene of six strains, out of 26 isolated histamine-utilizing bacteria, were amplified using our designed primers. The HOD enzyme from Glutamicibacter sp. N1A3101, isolated from nettle soil, was found to be thermostable and showed the highest substrate specificity toward the histamine and with no detected activity in the presence of putrescine, cadaverine, spermine, and spermidine. Its oxidation activity toward tyramine was lower than other HOD reported so far. The isolated enzyme was stable at 60 °C for 30 min and showed pH stability ranging from 6 to 9. Furthermore, we indicated the induction of identified HOD activity in the presence of betahistine as well, with nearly equal efficiency and without the consumption of the substrate.

Targeting the lysyl oxidases in tumour desmoplasia

The extracellular matrix (ECM) is a fundamental component of tissue microenvironments and its dysregulation has been implicated in a number of diseases, in particular cancer. Tumour desmoplasia (fibrosis) accompanies the progression of many solid cancers, and is also often induced as a result of many frontline chemotherapies. This has recently led to an increased interest in targeting the underlying processes. The major structural components of the ECM contributing to desmoplasia are the fibrillar collagens, whose key assembly mechanism is the enzymatic stabilisation of procollagen monomers by the lysyl oxidases. The lysyl oxidase family of copper-dependent amine oxidase enzymes are required for covalent cross-linking of collagen (as well as elastin) molecules into the mature ECM. This key step in the assembly of collagens is of particular interest in the cancer field since it is essential to the tumour desmoplastic response. LOX family members are dysregulated in many cancers and consequently the development of small molecule inhibitors targeting their enzymatic activity has been initiated by many groups. Development of specific small molecule inhibitors however has been hindered by the lack of crystal structures of the active sites, and therefore alternate indirect approaches to target LOX have also been explored. In this review, we introduce the importance of, and assembly steps of the ECM in the tumour desmoplastic response focussing on the role of the lysyl oxidases. We also discuss recent progress in targeting this family of enzymes as a potential therapeutic approach.

A Pilot Study on Biomarkers of Vascular Injury in Patients with Immune-Mediated Thrombotic Thrombocytopenic Purpura

Background: Immune-mediated thrombotic thrombocytopenic purpura (iTTP) is defined by thrombocytopenia and microangiopathic hemolytic anemia without an alternative explanation, confirmed by severely deficient ADAMTS13 to <10%. It is caused by autoantibodies against the ADAMTS13 protease. Following recovery from an acute iTTP episode, the patient is at risk for relapses and multiple long-term complications including hypertension, depression, headaches and neurocognitive impairment. The etiology of these complications is not well understood. The aim of this study was to identify biomarkers of vascular injury that could help to diagnose or predict the development of these long term complications of iTTP. Four plasma biomarkers of vascular injury were selected with the assistance of a multidisciplinary team of neurologists, pathologists and psychologists, based on their relevance in other chronic diseases and inflammation. Syndecan-1 (CD-138), is a cell surface heparan sulfate proteoglycan that interacts with extracellular matrix molecules and growth factors to maintain epithelial cell morphology. It has been reported to be a negative regulator of various inflammatory processes, with Syndecan-1 knockout (Sdc-1−/−) mice showing enhanced disease severity and impaired recovery.Thrombomodulin (CD141), is an endothelial surface transmembrane glycoprotein. It is involved in the activation of protein C in the inactivation of thrombin. Its expression has been associated with aging and cardiovascular disease.Vascular adhesion protein-1 (VAP) is a member of the copper-containing amine oxidase/semicarbazide-sensitive amine oxidase (AOC/SSAO) enzyme family. It is continuously expressed as a transmembrane glycoprotein in the vascular wall during development and facilitates the accumulation of inflammatory cells into the inflamed environment. It has been shown to be released in cerebral ischemia.E-selectin (CD62E), is a selectin cell adhesion molecule, expressed only in endothelial cells activated by cytokines, and is known to be increased in acute coronary syndrome and atherosclerosis. Methods: Patients with a diagnosis of iTTP functioning normally in their activities of daily living were recruited from existing IRB approved patient cohorts at both the Ohio State University (n = 10) (Columbus) and the University College London Hospitals (n = 15) (London, UK). These patients were studied and reported previously (Cataland, Scully M et al. AJH 2011, 86; 87-89). with a validated measure of neurocognitive function (Cogstate). Patients were characterized by this testing as having normal or abnormal cognitive function. Syndecan-1, thrombomodulin, vascular adhesion protein-1, and E-selectin were studied in plasma samples obtained at the time of the previous neurocognitive testing. All four biomarker assays were performed with commercially available Elisa-based assays. Results: There was a total of 25 patients that underwent neurocognitive testing previously; 11 patients had normal neurocognitive testing and 14 patients were characterized as abnormal. The median age was 41 (range 18-62). One patient was Asian, six were black and 18 were white. Nine patients were male. The demographic information for each group is listed in Table 1. The biomarker results for each group are listed on Table 2 and a comparison is seen on Figure 1. We did see a trend to lower levels of Syndecan-1, and higher levels of thrombomodulin and VAP-1 in patients with abnormal cognitive testing. The two groups had similar levels of CD62E. all biomarker testing was repeated twice and given consistent data. Conclusions: There is a need in the field for a better understanding of the pathophysiology of the long-term complications described in patients with iTTP. This pilot study is the first to look at biomarkers of vascular injury and inflammation that can correlate with neurocognitive impairment and potentially vascular injury in patients with iTTP, not when patients are having an acute episode but during follow up. In addition, we have shown that these biomarkers can be reliably tested in frozen samples from patients with iTPP, and important step for feasibility of future studies. These data are an important first step to develop specific biomarkers that correlate with the development of long-term complications in iTTP patients. Disclosures Masias: Rigel Pharmaceuticals: Consultancy. Scully:Alexion: Consultancy; Novartis: Consultancy; Ablynx/Sanofi: Consultancy; Shire/Takeda: Consultancy; Shire: Research Funding. Cataland:Ablynx/Sanofi: Consultancy, Research Funding; Alexion: Consultancy, Research Funding.

Aerobic catalytic systems inspired by copper amine oxidases

Abstract The goal of sustainable development has been accepted as a common policy in current society. In response to this challenge, the development of green processes which utilize environmentally benign oxidants, reduce chemical waste and handling costs, is highly desirable. Given the widespread importance of imines as pivotal synthetic intermediates and essential pharmacophores in numerous biologically active compounds, various catalytic methods allowing the aerobic oxidation of amines to imines have been developed. Recently, noticeable progress has arisen from the discovery of various quinone-based catalytic systems, inspired by copper amine oxidase enzymes (CuAOs), which are able to reproduce the selectivity of CuAOs for primary amines and even to expand the amine substrates scope. However, the need for synthesizing these catalysts prior use adversely affects the economics as well as the eco-friendly nature of the method. To surpass these drawbacks, the “second-order” biomimicry idea has been recently advanced to describe a system in which in situ modification of pre-catalyst components affords the active biomimetic catalyst. This minireview especially covers our recent contribution to the design of bioinspired quinone-based catalysts for the aerobic oxidation of amines to imines which has culminated in a dual bioinspired protocol as an example of “second-order” biomimicry.

Bifunctional Molecular Probes for Activity-Based Visualization of Quinone-Dependent Amine Oxidases.

The design, synthesis, and evaluation of two bifunctional molecular probes that can be used to visualize quinone-dependent amine oxidase enzymes in an activity-dependent manner are described. These probes use alkylhydrazines to irreversibly bind the target enzymes, which can then be visualized with either Western blotting or in-gel fluorescence. The results show that the Western blotting readout, which utilizes commercially available anti-nitrophenyl antibodies to detect a simple dinitrophenyl antigen, provides a stronger readout than the fluorescein-based fluorescence readout. This visualization strategy can be used to measure the potency of enzyme inhibitors by selectively visualizing the active enzyme that remains after treatment with an inhibitor. Looking forward, this probe molecule and visualization strategy will enable activity-based protein-profiling experiments, such as determining inhibitor selectivity values within full proteome mixtures, for this family of amine oxidase enzymes.

Imine Reductases, Reductive Aminases, and Amine Oxidases for the Synthesis of Chiral Amines: Discovery, Characterization, and Synthetic Applications.

Synthesis of the chiral amine moiety is a key challenge for synthetic organic chemistry due to its prevalence in many biologically active molecules. Imine reductase and amine oxidase enzymes have enabled the biocatalytic synthesis of a host of chiral amine compounds. In this chapter, procedures for the synthesis of chiral amines using imine reductases (IREDs), the recently discovered IRED homologues reductive aminases, and amine oxidases (AOs) are described. Amine oxidases have been the subject of mutagenesis approaches for improvement of substrate scope. The high-throughput screening method for determining active variants in amine oxidase libraries is illustrated. Finally, in an approach which takes inspiration from nature, many enzymes can be combined with each other in cascade reactions. The incorporation of imine reductase and monoamine oxidase biocatalysts into several cascade reactions, both in vitro and in vivo (where the approach moves toward synthetic biology), is reported.

Identification of Histidine 303 as the Catalytic Base of Lysyl Oxidase via Site-Directed Mutagenesis.

Lysyl oxidase (LOX) is a copper-dependent amine oxidase enzyme that catalyzes the formation of crosslinkages of collagen and elastin in connective tissues by oxidative deamination of lysine. Using site-directed mutagenesis, Histidine 303 has been shown to be a key residue that acts as the necessary catalytic base for this enzyme to function properly. Histidine 303 was mutated to isoleucine to remove catalytic activity and to aspartate and glutamate, respectively, in order to provide alternate residues that could act as a general base that could maintain catalytic activity. Overexpression of the H303I mutant yielded 3.9mg of enzyme per liter of media, the H303D mutant yielded 3.3mg of enzyme per liter of media, and the H303E mutant yielded 3.0mg/L of media. Overexpression of wildtype LOX yielded 4.5mg/L of media, which is a slight improvement from previous yields. Total copper incorporation for H303I was calculated to be 68% and no copper was detected for the H303D and H303E mutants. As LOX requires the self-processed cofactor lysyl tyrosyl quinone (LTQ) for activity, total LTQ content was obtained by reacting the enzyme with phenylhydrazine and using the previously reported extinction coefficient of 15.4mM/cm. LTQ content for the wildtype enzyme was determined to be 92%, for H303I the total LTQ content was determined to be 36%, and no LTQ was detected for the H303D and H303E mutants. No catalytic activity was detected for any mutants when compared to the wildtype which has a previously reported activity of 0.11 U/mg. Comparison of excitation-emission matrices (EEM) of each of the mutants as compared to the wildtype indicate that all the mutations cause a change in the internal environment of the enzyme, albeit to varying degrees, as evidenced by the observed shifts.

Abstract B98: Development and evaluation of selective, reversible LSD1 inhibitors from fragment startpoints.

Abstract There is currently considerable interest in lysine-specific histone demethylase 1 (LSD1) as a therapeutic target in human malignancies. Specifically LSD1 has been demonstrated to be an essential regulator of leukaemia stem cell potential, inhibiting differentiation and apoptosis in the MLL-AML setting. There are a variety of potent irreversible LSD1 inhibitors available but here we present two series of reversible aminothiazole inhibitors obtained through the expansion of hits derived from a high concentration biochemical screen of a fragment library. The potency of the initial fragment hits was increased 32-fold through synthesis, with one series of compounds showing clear structure activity relationships (SAR) and inhibitory activities in the range of 7 to 187 µM in a biochemical assay. This series also showed selectivity against the homologous amine oxidase enzyme monoamine oxidase A (MAO-A). This work represents one of the first reported examples of a reversible small molecule inhibitor of LSD1 with clear SAR and selectivity against MAO-A, and could provide a platform for the development of more potent reversible inhibitors. We also report the first Proof of Mechanism (POM) cell based assay utilizing CD86 expression as a surrogate marker of LSD1 activity in THP1 cells and its use to evaluate both our compounds and some recently reported reversible LSD1 inhibitors. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B98. Citation Format: Alex Stowell, Niall Hamilton, James Hitchin, Julian Blagg, Rosemary Burke, Samantha Burns, Mark J. Cockerill, Emma Fairweather, Colin Hutton, Allan Jordan, Daniel Mould, Graeme Thomson, Ian Waddell, Donald Ogilvie. Development and evaluation of selective, reversible LSD1 inhibitors from fragment startpoints. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B98.

Reactive oxygen species spermine metabolites generated from amine oxidases and radiation represent a therapeutic gain in cancer treatments

The most frequent interventions in cancer therapy are currently the destruction of cells by irradiation or administration of drugs both able to induce radical formation and toxic metabolites by enzyme-catalyzed reactions. The aim of this study was to determine the cell viability of cells undergoing a DNA damage threshold accomplished by ROS overproduction via both ectopic expression of murine spermine oxidase (mSMOX) and bovine serum amine oxidase (BSAO) enzymes. Low dose of X-irradiation delivers a challenging dose of damage as evaluated in proficient Chinese hamster AA8 cell line and both deficient transcription-coupled nucleotide excision repair (NER) UV61 cells and deficient base excision repair (BER) EM9 cells, at 6 and 24 h after exposure. The priming dose of ROS overexposure by mSMOX provokes an adaptive response in N18TG2, AA8 and EM9 cell lines at 24 h. Interestingly, in the UV61 cells, ROS overexposure by mSMOX delivers an earlier adaptive response to radiation. The enzymatic formation of toxic metabolites has mainly been investigated on wild-type (WT) and multidrug-resistant (MDR) cancer cell lines, using and spermine as substrate of the BSAO enzyme. MDR cells are more sensitive to the toxic polyamine metabolites than WT cells, thus indicating a new therapeutic strategy to overcome MDR tumors. Since SMOX in mammals is differentially activated in a tissue-specific manner and cancer cells can differ in terms of DNA repair and MDR capabilities, it could be of interest to simultaneously treat with very low dose of X-rays and/or to alter SMOX metabolism to generate a differential response in healthy and cancer tissues.

Potential of wine-associated lactic acid bacteria to degrade biogenic amines

Some lactic acid bacteria (LAB) isolated from fermented foods have been proven to degrade biogenic amines through the production of amine oxidase enzymes. Since little is known about this in relation to wine micro-organisms, this work examined the ability of LAB strains (n=85) isolated from wines and other related enological sources, as well as commercial malolactic starter cultures (n=3) and type strains (n=2), to degrade histamine, tyramine and putrescine. The biogenic amine-degrading ability of the strains was evaluated by RP-HPLC in culture media and wine malolactic fermentation laboratory experiments. Although at different extent, 25% of the LAB isolates were able to degrade histamine, 18% tyramine and 18% putrescine, whereas none of the commercial malolactic starter cultures or type strains were able to degrade any of the tested amines. The greatest biogenic amine-degrading ability was exhibited by 9 strains belonging to the Lactobacillus and Pediococcus groups, and most of them were able to simultaneously degrade at least two of the three studied biogenic amines. Further experiments with one of the strains that showed high biogenic amine-degrading ability (L. casei IFI-CA 52) revealed that cell-free extracts maintained this ability in comparison to the cell suspensions at pH 4.6, indicating that amine-degrading enzymes were effectively extracted from the cells and their action was optimal in the degradation of biogenic amines. In addition, it was confirmed that wine components such as ethanol (12%) and polyphenols (75 mg/L), and wine additives such as SO(2) (30 mg/L), reduced the histamine-degrading ability of L. casei IFI-CA 52 at pH 4.6 by 80%, 85% and 11%, respectively, in cell suspensions, whereas the reduction was 91%, 67% and 50%, respectively, in cell-free extracts. In spite of this adverse influence of the wine matrix, our results proved the potential of wine-associated LAB as a promising strategy to reduce biogenic amines in wine.

Monoamine oxidase activities in dissociated cell fractions from rat skeletal muscle.

Collagenase was used to dissociate rat skeletal muscle (gastrocnemius) into its constituent cells, from which a myocyte fraction enriched in striated muscle cells, and a non-myocyte fraction containing cells of connective tissue and vascular origin, were prepared. The activities of amine oxidase enzymes and alkaline phosphatase (AP) were then assayed in these fractions, and also in homogenates prepared from corresponding samples of non-dissociated tissue. The specific activities of the semicarbazide-sensitive amine oxidase (SSAO) and AP were considerably higher (30 to 35-fold) in non-myocyte than in myocyte fractions. AP is generally considered to be present predominantly in vascular cells of skeletal muscle, with little, if any, in skeletal muscle cells themselves. Thus, the results obtained may indicate a similar localization for SSAO activity. Support for this came from histochemical studies, which showed staining for SSAO primarily over the walls of larger blood vessels in the muscle. Unlike SSAO and AP, were marked differences in MAO-A activity between myocyte and non-myocyte fractions were not found, suggesting that MAO-A is more probably a constituent of cells within both fractions.

An Electrocatalytic System that Mimics the Catalytic Oxidation of Biogenic Mono- and Polyamines by Semicarbazide-sensitive Amine Oxidases (SSAOs)

Chemoselective oxidation of primary aliphatic biogenic mono- and polyamines into imines has been successfully achieved, under metal-free conditions, by using an electrocatalytic system that mimics the activity of semicarbazide-sensitive amine oxidase (SSAO) enzymes. Accordingly, high catalytic performance was observed with unbranched primary amines such as methylamine and aminoacetone, and with the primary amino groups of diamines and polyamines like putrescine and spermidine, all of them being reference substrates for SSAO enzymes. Furthermore, contrary to flavin-adenine dinucleotide (FAD)-dependent amine oxidase enzymes, no activity was found with secondary and tertiary amines. Finally, when compared with the direct electrochemical oxidation of primary aliphatic amines, which occurs at high anodic potential (Ean > + 1.5 V vs SCE) and led to unstable cation radicals that rapidly deprotonate and attach to the electrode surface (Pt, carbon), 1.0 V was gained using this electrocatalytic process (Ean = + 0.5 V vs SCE).