Synthesis and anti-cancer potential of potent peripheral MAOA inhibitors designed to limit blood:brain penetration.

Monoamine oxidases (MAOA/MAOB) are enzymes known for their role in neurotransmitter regulation in the central nervous system (CNS). Irreversible and non-selective MAO inhibitors (MAOi's) were the first class of antidepressants, thus subsequent work on drugs such as the selective MAOA inhibitor clorgyline has focussed on selectivity and increased CNS penetration. MAOA is highly expressed in high grade and metastatic prostate cancer with a proposed effect on prostate cancer growth, recurrence, and drug resistance. A Phase II Clinical Trial has demonstrated the therapeutic effects of the irreversible nonselective MAOi phenelzine for prostate cancer. However, neurologic adverse effects led to early withdrawal in 25% of the enrolled patient-population. In this work, we revised the clorgyline scaffold with the goal of decreasing CNS penetration to minimize CNS-related side effects while retaining or enhancing MAOA inhibition potency and selectivity. Using the known co-crystal structure of clorgyline bound with FAD co-factor in the hMAOA active site as a reference, we designed and synthesized a series of compounds predicted to have lower CNS penetration (logBB). All synthesized derivatives displayed favorable drug-like characteristics such as predicted Caco-2 permeability and human oral absorption, and exhibited highly selective hMAOA binding interactions. Introduction of an HBD group (NH2 or OH) at position 5 of the phenyl ring clorgyline resulted in 3x more potent hMAOA inhibition with equivalent or better hMAOB selectivity, and similar prostate cancer cell cytotoxicity. In contrast, introduction of larger substituents at this position or at the terminal amine significantly reduced the hMAOA inhibition potency, attributed in part to a steric clash within the binding pocket of the MAOA active site. Replacement of the N-methyl group by a more polar, but larger 2-hydroxyethyl group did not enhance potency. However, introduction of a polar 2-hydroxy in the propyl chain retained the highly selective MAOA inhibition and cancer cell cytotoxicity of clorgyline while reducing its CNS score from 2 to 0. We believe that these results identify a new class of peripherally directed MAOIs that may allow safer therapeutic targeting of MAOA for a variety of anti-cancer and anti-inflammatory indications.

Increased MAO-A Activity Promotes Progression of Pulmonary Arterial Hypertension.

Monoamine oxidases (MAOs), a class of enzymes bound to the outer mitochondrial membrane, are important sources of reactive oxygen species. Increased MAO-A activity in endothelial cells and cardiomyocytes contributes to vascular dysfunction and progression of left heart failure. We hypothesized that inhibition of MAO-A can be used to treat pulmonary arterial hypertension (PAH) and right ventricular (RV) failure. MAO-A levels in lung and RV samples from patients with PAH were compared with levels in samples from donors without PAH. Experimental PAH was induced in male Sprague-Dawley rats by using Sugen 5416 and hypoxia (SuHx), and RV failure was induced in male Wistar rats by using pulmonary trunk banding (PTB). Animals were randomized to receive either saline or the MAO-A inhibitor clorgyline at 10 mg/kg. Echocardiography and RV catheterization were performed, and heart and lung tissues were collected for further analysis. We found increased MAO-A expression in the pulmonary vasculature of patients with PAH and in experimental experimental PAH induced by SuHx. Cardiac MAO-A expression and activity was increased in SuHx- and PTB-induced RV failure. Clorgyline treatment reduced RV afterload and pulmonary vascular remodeling in SuHx rats through reduced pulmonary vascular proliferation and oxidative stress. Moreover, clorgyline improved RV stiffness and relaxation and reversed RV hypertrophy in SuHx rats. In PTB rats, clorgyline had no direct clorgyline had no direct effect on the right ventricle effect. Our study reveals the role of MAO-A in the progression of PAH. Collectively, these findings indicated that MAO-A may be involved in pulmonary vascular remodeling and consecutive RV failure.

Effect of Monoamine oxidase A (MAOA) inhibitors on androgen-sensitive and castration-resistant prostate cancer cells.

BACKGROUND: Monoamine oxidase A (MAOA) is best known for its role in neuro-transmitter regulation. Monoamine oxidase inhibitors are used to treat atypical depression. MAOA is highly expressed in high grade prostate cancer and modulates tumorigenesis and progression in prostate cancer. Here, we investigated the potential role of MAOA inhibitors (MAOAIs) in relation to the androgen receptor (AR) pathway and resistance to antiandrogen treatment in prostate cancer. METHODS: We examined MAOA expression and the effect of MAOI treatment in relation to AR-targeted treatments using the LNCaP, C4-2B, and 22Rv1 human prostate cancer cell lines. MAOA, AR-full length (AR-FL), AR splice variant 7 (AR-V7), and PSA expression was evaluated in the presence of MAOAIs (clorgyline, phenelzine), androgenic ligand (R1881), and antiandrogen (enzalutamide) treatments. An enzalutamide resistance cell line was generated to test the effect of MAOAI treatment in this model. RESULTS: We observed that MAOAIs, particularly clorgyline and phenelzine, were effective at decreasing MAOA activity in human prostate cancer cells. MAOAIs significantly decreased growth of LNCaP, C4-2B, and 22Rv1 cells and produced additive growth inhibitory effects when combined with enzalutamide. Clorgyline decreased expression of AR-FL and AR-V7 in 22Rv1 cells and was effective at decreasing growth of an enzalutamide-resistant C4-2B cell line with increased AR-V7 expression. CONCLUSIONS: MAOAIs decrease growth and proliferation of androgen-sensitive and castration-resistant prostate cancer cells. Clorgyline, in particular, decreases expression of AR-FL and AR-V7 expression and decreases growth of an enzalutamide-resistant cell line. These findings provide preclinical validation of MAOA inhibitors either alone or in combination with antiandrogens for therapeutic intent in patients with advanced forms of prostate cancer.

Discovery of novel 2,3-dihydro-1H-inden-1-amine derivatives as selective monoamine oxidase B inhibitors.

Inhibition of MAO-B has been an effective strategy for the treatment of Parkinson's disease. To find more potent and selective MAO-B inhibitors with novel chemical scaffold, we designed and synthesized a series of new 2,3-dihydro-1H-inden-1-amine derivatives on basis of our previous study. Furthermore, the corresponding structure-activity relationship (SAR) of these compounds is detailedly discussed. Compounds L4 (IC50 = 0.11 µM), L8 (IC50 = 0.18 µM), L16 (IC50 = 0.27 µM) and L17 (IC50 = 0.48 µM) showed similar MAO-B inhibitory activity as Selegiline. Moreover, L4, L16 and L17 also exhibited comparable selectivity with Selegiline, indicating that L4, L16 and L17 could be promising selective MAO-B inhibitors for further study.

Monoamine oxidase A is highly expressed in classical Hodgkin lymphoma.

Monoamine oxidase A (MAOA) is a mitochondrial enzyme that catalyzes oxidative deamination of neurotransmitters and dietary amines and produces H2 O2 . It facilitates the progression of gliomas and prostate cancer, but its expression and functional relevance have not been studied in lymphoma. Here, we evaluated MAOA in 427 cases of Hodgkin and non-Hodgkin lymphoma and in a spectrum of reactive lymphoid tissues by immunohistochemistry on formalin-fixed, paraffin-embedded specimens. MAOA was expressed by Hodgkin Reed-Sternberg (HRS) cells in the majority of classical Hodgkin lymphomas (cHLs) (181/241; 75%), with 34.8% showing strong expression. Weak MAOA was also noted in a minority of primary mediastinal large B-cell lymphomas (8/47; 17%) and in a mediastinal gray-zone lymphoma. In contrast, no MAOA was found in non-neoplastic lymphoid tissues, nodular lymphocyte-predominant Hodgkin lymphoma (NLPHL; 0/8) or any other non-Hodgkin lymphomas studied (0/123). MAOA was more common in Epstein-Barr virus (EBV)-negative compared to EBV-positive cHL (p < 0.0001) and was especially prevalent in the EBV-negative nodular sclerosing subtype. Similar to primary human lymphoma specimens, most cHL-derived cell lines displayed MAOA activity, whereas non-Hodgkin-lymphoma-derived cell lines did not. The MAOA inhibitor clorgyline reduced the growth of L1236 cells and U-HO1 cells, and shRNA knockdown of MAOA reduced the growth of L1236 cells. Conversely, ectopic overexpression of MAOA increased the growth of MAOA-negative HDLM2 cells. Combined treatment with clorgyline and ABVD (doxorubicin, bleomycin, vinblastine, dacarbazine) was more effective in reducing cell growth than either regimen alone. In summary, MAOA is highly expressed in cHL and may reflect the distinct biology of this lymphoma. Further studies on the potential utility of MAOA as a diagnostic marker and therapeutic target are warranted. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Design, synthesis, and biological evaluation of oxindole derivatives as antidepressive agents.

The 3-substituted oxindole derivatives were designed, synthesized, and evaluated for antidepressant activity by employing forced swimming test, tail suspension test, and MAO-A inhibition assay. Results of biological studies revealed that the majority of compounds exhibited potent to moderately potent activity and among them, 12 displayed potency comparable to that of the imipramine with %DID of 37.95 and 44.84 in the FST and TST, respectively. At the same time, imipramine showed %DID of 43.62 and 50.64 in the FST and TST, correspondingly. In the MAO-A inhibition assay, 12 showed an IC50 of 18.27 µmol, whereas the reference drug moclobemide displayed an IC50 of 13.1 µmol. The SAR study disclosed that the presence of bromo atom at the phenyl/furanyl or thienyl moiety in the oxindole derivatives was critical for the antidepressant activity.

Synthesis and evaluation of quinazoline amino acid derivatives as mono amine oxidase (MAO) inhibitors.

A series of quinazolinone amino acid ester and quinazolinone amino acid hydrazides were prepared under microwave irradiation as well as conventional condition. The microwave irradiation afforded the product in less reaction time, higher yield and purity. The structures of the synthesized compounds were confirmed by IR, NMR, and elemental analysis. The new synthesized compounds were studied for their monoamine oxidase inhibitory activity. They showed more selective inhibitory activity toward MAO-A than MAO-B. Compounds 7, 10, and 15 showed MAO-A inhibition activity (IC50=3.6×10(-9), 2.8×10(-9), 2.1×10(-9) M, respectively) comparable to that of the standard clorgyline (IC50=2.9×10(-9)M). 2-(2-(Benzo[d][1,3]dioxol-5-yl)-4-oxo-1,2-dihydroquinazolin-3(4H)-yl)acetohydrazide 15 showed selective MAO-A inhibition activity (SI=39524) superior to that of the standard clorgyline (SI=33793). The acute toxicity of the synthesized compounds was determined. In addition, computer-assisted simulated docking experiments were performed to rationalize the biological activity.

Acute inhibition of monoamine oxidase and ischemic preconditioning in isolated rat hearts: interference with postischemic functional recovery but no effect on infarct size reduction.

Monoamine oxidases (MAOs) have recently emerged as important mitochondrial sources of oxidative stress in the cardiovascular system. Generation of reactive oxygen species during the brief episodes of ischemic preconditioning (IPC) is responsible for the cardioprotection at reperfusion. The aim of this study was to assess the effects of two MAO inhibitors (clorgyline and pargyline) on the IPC-related protection in isolated rat hearts. Animals subjected to 30 min global ischemia and 120 min reperfusion were assigned to the following groups: (i) Control, no additional intervention; (ii) IPC, 3 cycles of 5 min ischemia and 5 min reperfusion before the index ischemia; (iii) IPC-clorgyline, IPC protocol bracketed for 5 min with clorgyline (50 µmol/L); (iv) IPC-pargyline, IPC protocol bracketed for 5 min with pargyline (0.5 mmol/L). The postischemic functional recovery was assessed by the left ventricular developed pressure (LVDP) and the indices of contractility (+dLVP/dt max) and relaxation (-dLVP/dt max). Infarct size (IS) was quantified by TTC staining. In both genders, IPC significantly improved functional recovery that was further enhanced in the presence of either clorgyline or pargyline. IS reduction was comparable among all the preconditioned groups, regardless of the presence of MAO inhibitors. In isolated rat hearts, acute inhibition of MAOs potentiates the IPC-induced postischemic functional recovery without interfering with the anti-necrotic protection.

Synthesis and Preliminary Biological Evaluation of 1,3,5-Triazine Amino Acid Derivatives to Study Their MAO Inhibitors.

Three series of 4,6-dimethoxy-, 4,6-dipiperidino- and 4,6-dimorpholino-1,3,5-triazin-2-yl) amino acid derivatives were synthesized and characterized. A preliminary study for their monoamine oxidase inhibitory activity showed that compounds 7, 18, and 25 had MAO-A inhibition activity comparable to that of the standard clorgyline, with apparently more selective inhibitory activity toward MAO-A than MAO-B and no significant acute toxicity.

Intrinsic cardiac adrenergic (ICA) cell density and MAO-A activity in failing rat hearts.

The efficiency (work/oxygen consumption) of isolated papillary muscles from failing hearts is reduced. We investigated whether this can be due to an increase of intrinsic cardiac adrenergic (ICA) cell density. The number of ICA cells in the septum and both ventricular walls was determined by tyrosine hydroxylase immunohistochemistry in rats with monocrotaline-induced pulmonary hypertension. We found that the number of ICA cells is about 200,000 per rat heart. ICA cell density was significantly lower in right ventricular myocardium of hypertrophied hearts (P < 0.01). MAO-A enzyme histochemistry and inhibition experiments with clorgyline in papillary muscles were performed to localize the enzyme and to determine its oxygen consumption. Upregulation of MAO-A was found in the right ventricular wall and papillary muscles of failing hearts (P = 0.018). A positive correlation between ICA cell density and MAO-A activity was absent. Clorgyline (2 µM) decreased the basal rate of oxygen consumption of right ventricular papillary muscles by 65 µM O(2)/s (P = 0.027). This rate can only be maintained for several seconds judging from the catecholamine content of the preparations reported previously. High ICA cell activity rather than density and/or recycling of oxidized catecholamines are discussed as alternative explanations for the low myocardial efficiency in experimental pulmonary hypertension.

Drug resistance is conferred on the model yeast Saccharomyces cerevisiae by expression of full-length melanoma-associated human ATP-binding cassette transporter ABCB5.

ABCB5, an ATP-binding cassette (ABC) transporter, is highly expressed in melanoma cells, and may contribute to the extreme resistance of melanomas to chemotherapy by efflux of anti-cancer drugs. Our goal was to determine whether we could functionally express human ABCB5 in the model yeast Saccharomyces cerevisiae, in order to demonstrate an efflux function for ABCB5 in the absence of background pump activity from other human transporters. Heterologous expression would also facilitate drug discovery for this important target. DNAs encoding ABCB5 sequences were cloned into the chromosomal PDR5 locus of a S. cerevisiae strain in which seven endogenous ABC transporters have been deleted. Protein expression in the yeast cells was monitored by immunodetection using both a specific anti-ABCB5 antibody and a cross-reactive anti-ABCB1 antibody. ABCB5 function in recombinant yeast cells was measured by determining whether the cells possessed increased resistance to known pump substrates, compared to the host yeast strain, in assays of yeast growth. Three ABCB5 constructs were made in yeast. One was derived from the ABCB5-ß mRNA, which is highly expressed in human tissues but is a truncation of a canonical full-size ABC transporter. Two constructs contained full-length ABCB5 sequences: either a native sequence from cDNA or a synthetic sequence codon-harmonized for S. cerevisiae. Expression of all three constructs in yeast was confirmed by immunodetection. Expression of the codon-harmonized full-length ABCB5 DNA conferred increased resistance, relative to the host yeast strain, to the putative substrates rhodamine 123, daunorubicin, tetramethylrhodamine, FK506, or clorgyline. We conclude that full-length ABCB5 can be functionally expressed in S. cerevisiae and confers drug resistance.

Mechanisms underlying increased serotonin-induced contraction in carotid arteries from chronic type 2 diabetic Goto-Kakizaki rats.

Serotonin (5-hydroxytryptamine; 5-HT) plays important roles in the cardiovascular system; however, the relationship between 5-HT-induced vasocontraction and the arterial 5-HT system including metabolism and signal transduction, in the presence of chronic type 2 diabetes (T2D) remains unclear. Therefore, we investigated 5-HT-induced contraction and associated mechanisms in carotid arteries from chronic T2D Goto-Kakizaki (GK) rats. Contractions in response to 5-HT were examined in carotid arteries from GK rats (42-46 weeks old). To investigate the response mechanisms of arterial smooth muscle, we constructed concentration-response curves for TCB2 (5-HT2A-receptor agonist), BW723C86 (5-HT2B-receptor agonist), and 5-HT in the presence of various inhibitors using endothelium-denuded preparations. Carotid arterial expressions of monoamine oxidase-A (MAO-A), serotonin transporter (SERT), and 5-HT2A were detected by immunoblotting. 5-HT-induced contraction was increased in carotid arteries from GK compared to control Wistar rats in both endothelium-intact and -denuded preparations. In denuded preparations, we found that: (1) TCB2-induced contraction was increased in GK rat arteries (vs. Wistar); (2) MAO-A inhibitor did not affect 5-HT-induced contraction, whereas SERT inhibitor augmented such contractions in both groups; and (3) differences in 5-HT-induced contractions were abolished by p38 MAPK, PI3K, and Rho kinase inhibitors. Carotid arterial expressions of MAO-A, SERT, and 5-HT2A remained unchanged in the groups. The results suggest that 5-HT-induced contraction is augmented in T2D GK rat carotid arteries. This augmentation is due to smooth muscle activation partly mediated by p38 MAPK, PI3K, and Rho kinases, and may also be partly due to arterial SERT activity.

Microdialysis perfusion of COA-Cl enhances dopamine metabolism in the dorsal striatum of freely moving mice.

We performed a microdialysis study to examine the effects of local perfusion of COA­Cl on the extracellular levels of dopamine (DA) and its metabolites in the dorsal striatum of mice in vivo. The mice were perfused with Ringer's solution (control) and COA­Cl (0.05, 0.1, or 0.5 mM) into the dorsal striatum. Dialysate samples were collected every 30 min and then analyzed using high­performance liquid chromatography coupled with an electrochemical detector. We found that local perfusion of COA­Cl (0.1 or 0.5 mM) into the dorsal striatum of living mice produced a significant and dose­dependent increase in extracellular levels of DA, 3­methoxytyramine (3­MT), and homovanillic acid (HVA), where only 0.5 mM COA­Cl increased dihydroxyphenylacetic acid (DOPAC) levels. However, 0.05 mM of COA­Cl did not significantly affect either DA levels or its metabolites. Then, we administered the monoamine oxidase (MAO) inhibitor clorgyline alone or in combination with COA­Cl (0.1 mM) to test whether COA­Cl­induced increases in DOPAC and HVA are mediated by increased MAO activity. Clorgyline alone increased 3­MT levels and decreased DOPAC and HVA levels but not DA levels. When combined with COA­Cl, clorgyline increased 3­MT levels and reversed the decrease in DOPAC and HVA levels caused by clorgyline. The increase in DA metabolism induced by COA­Cl suggests that some DA was further metabolized into DOPAC, 3­MT, and HVA. This indicates that COA­Cl plays a role in DA metabolism via increased DA release and/or activation of MAO, offering new insights into the effects of COA­Cl on DA metabolism in the brain.

Quantum-chemical approach to determining the high potency of clorgyline as an irreversible acetylenic monoamine oxidase inhibitor.

Density functional theory calculations were employed to investigate the nature of chemical bond formation between the flavin co-factor of the enzyme monoamine oxidase (MAO) and its irreversible acetylenic inhibitor clorgyline in its terminally deprotonated anionic form. Since MAOs regulate the level of neurotransmitters in living cells, this reaction is pharmacologically relevant for treating depression and other mood disorders. The results revealed that this pathway is associated with the activation free energy of ΔG act (#) = 17.4 kcal mol(-1), which, together with our previous results, suggests that clorgyline is intrinsically a more effective MAO inhibitor than antiparkinsonian drugs rasagiline and selegiline considering the preferred MAO isoforms in each case, thus displaying a trend in agreement with experimental data. The reaction is facilitated by the pronounced electrophilic character of the flavin moiety, due to its ability to efficiently accommodate excess negative charge from the approaching anionic inhibitor through resonance effect. The investigated mechanism was additionally validated by the inspection of the geometry of the flavin moiety in the formed adduct, which exhibit distortion from planarity consistent with experimental observations. These results offer valuable insight for mechanistic studies on other flavoenzymes and for the design of new antidepressants and antiparkinsonian drugs.

Coupling of a high-resolution monoamine oxidase-A inhibitor assay and HPLC-SPE-NMR for advanced bioactivity profiling of plant extracts.

INTRODUCTION: Depression is a mental disease causing large personal and socio-economic problems, and new improved drugs are therefore needed. Selective monoamine oxidase A (MAO-A) inhibitors are potential anti-depressants, but discovering new MAO-A inhibitors from natural sources by bioassay-guided approaches are a lengthy and time-consuming process. New analytical technologies that allow simultaneously chemical and biological screening of extracts are therefore urgently needed. METHOD: In the present study we describe coupling of a photometric microplate-based high-resolution MAO-A inhibitor assay with a hyphenated system consisting of high-performance liquid chromatography, solid-phase extraction and tube transfer nuclear magnetic resonance (HPLC-SPE-ttNMR). The standard compound clorgyline, and an extract of black pepper (Piper nigrum L.), representing a complex plant matrix, were used for proof-of-concept. RESULTS: The work with clorgyline showed that the microplate-based high-resolution assay produced MAO-A inhibition profiles that easily allowed detection of submicrogram amounts of this selective MAO-A inhibitor. Furthermore, the HPLC-SPE-ttNMR/high-resolution MAO-A inhibition assay platform allowed identification of piperine and two piperine analogues as the main MAO-A inhibitors in the black pepper petroleum ether extract. CONCLUSION: The HPLC-SPE-ttNMR/high-resolution MAO-A inhibition assay platform is a powerful tool for fast and efficient identification of new MAO-A inhibitors from complex extracts, and promise future advancement in the search for new anti-depressants from natural sources.

Inhibition of monoamine oxidase-a increases respiration in isolated mouse cortical mitochondria.

Monoamine oxidase (MAO) is an enzyme located on the outer mitochondrial membrane that metabolizes amine substrates like serotonin, norepinephrine and dopamine. MAO inhibitors (MAOIs) are frequently utilized to treat disorders such as major depression or Parkinson's disease (PD), though their effects on brain mitochondrial bioenergetics are unclear. These studies measured bioenergetic activity in mitochondria isolated from the mouse cortex in the presence of inhibitors of either MAO-A, MAO-B, or both isoforms. We found that only 10 µM clorgyline, the selective inhibitor of MAO-A and not MAO-B, increased mitochondrial oxygen consumption rate in State V(CI) respiration compared to vehicle treatment. We then assessed mitochondrial bioenergetics, reactive oxygen species (ROS) production, and Electron Transport Chain (ETC) complex function in the presence of 0, 5, 10, 20, 40, or 80 µM of clorgyline to determine if this change was dose-dependent. The results showed increased oxygen consumption rates across the majority of respiration states in mitochondria treated with 5, 10, or 20 µM with significant bioenergetic inhibition at 80 µM clorgyline. Next, we assessed mitochondrial ROS production in the presence of the same concentrations of clorgyline in two different states: high mitochondrial membrane potential (ΔΨm) induced by oligomycin and low ΔΨm induced by carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP). There were no changes in ROS production in the presence of 5, 10, 20, or 40 µM clorgyline compared to vehicle after the addition of oligomycin or FCCP. There was a significant increase in mitochondrial ROS in the presence of 80 µM clorgyline after FCCP addition, as well as reduced Complex I and Complex II activities, which are consistent with inhibition of bioenergetics seen at this dose. There were no changes in Complex I, II, or IV activities in mitochondria treated with low doses of clorgyline. These studies shed light on the direct effect of MAO-A inhibition on brain mitochondrial bioenergetic function, which may be a beneficial outcome for those taking these medications.

Design, synthesis, and biological activity of dual monoamine oxidase A and heat shock protein 90 inhibitors, N-Methylpropargylamine-conjugated 4-isopropylresorcinol for glioblastoma.

Monoamine oxidase A (MAO A) and heat shock protein 90 (HSP90) inhibitors have been shown to decrease the progression of glioblastoma (GBM) and other cancers. In this study, a series of MAO A/HSP90 dual inhibitors were designed and synthesized in the hope to develop more effective treatment of GBM. Compounds 4-b and 4-c are conjugates of isopropylresorcinol (pharmacophore of HSP90 inhibitor) with the phenyl group of clorgyline (MAO A inhibitor) by a tertiary amide bond substituted with methyl (4-b) or ethyl (4-c) group, respectively. They inhibited MAO A activity, HSP90 binding, and the growth of both TMZ-sensitive and -resistant GBM cells. Western blots showed that they increased HSP70 expression indicating reduced function of HSP90, reduced HER2 and phospho-Akt expression similar to MAO A or HSP90 inhibitor itself. Both compounds decreased IFN-γ induced PD-L1 expression in GL26 cells, suggesting they can act as immune checkpoint inhibitor. Further, they reduced tumor growth in GL26 mouse model. NCI-60 analysis showed they also inhibited the growth of colon cancer, leukemia, non-small cell lung and other cancers. Taken together, this study demonstrates MAO A/HSP90 dual inhibitors 4-b and 4-c reduced the growth of GBM and other cancers, and they have potential to inhibit tumor immune escape.

Myocardial ischemia-reperfusion injury is probably due to the excessive production of mitochondrial ROS caused by the activation of 5-HT degradation system mediated by PAF receptor.

AIM: Previously, we revealed a crucial role of 5-HT degradation system (5DS), consisting of 5-HT2A receptor (5-HT2AR), 5-HT synthases and monoamine oxidase A (MAO-A), in ischemia-reperfusion (IR)-caused organ injury. Whereas, platelet activating factor receptor (PAFR) also mediates myocardial ischemia-reperfusion injury (MIRI). Here, we try to clarify the relationship between 5DS and PAFR in mediating MIRI. METHODS: H9c2 cell injury and rat MIRI were caused by hypoxia/reoxygenation (H/R) or PAF, and by ligating the left anterior descending coronary artery then untying, respectively. 5-HT2AR and PAFR antagonists [sarpogrelate hydrochloride (SH) and BN52021], MAO-A, AKT, mTOR and 5-HT synthase inhibitors (clorgyline, perifosine, rapamycin and carbidopa), and gene-silencing PKCε were used in experiments RESULTS: The mitochondrial ROS production, respiratory chain damage, inflammation, apoptosis and myocardial infarction were significantly prevented by BN52021, SH and clorgyline in H/R and PAF-treated cells and in IR myocardium. BN52021 also significantly suppressed the upregulation of PAFR, 5-HT2AR, 5-HT synthases and MAO-A expression (mRNA and protein), and Gαq and PKCε (in plasmalemma) expression induced by H/R, PAF or IR; the effects of SH were similar to that of BN52021 except for no affecting the expression of PAFR and 5-HT2AR. Gene-silencing PKCε suppressed H/R and PAF-induced upregulation of 5-HT synthases and MAO-A expression in cells; perifosine and rapamycin had not such effects; however, clorgyline suppressed H/R and PAF-induced phosphorylation of AKT and mTOR. CONCLUSION: MIRI is probably due to PAFR-mediated 5-HT2AR activation, which further activates PKCε-mediated 5-HT synthesis and degradation, leading to mitochondrial ROS production.

Monoamine oxidase a expression is vital for embryonic brain development by modulating developmental apoptosis.

Monoamine oxidases (MAO-A, MAO-B) metabolize biogenic amines and have been implicated in neuronal apoptosis. Although apoptosis is an important process in embryo development, the role of MAO isoenzymes has not been investigated in detail. We found that expression of MAO-A and MAO-B can be detected early on during embryo development. Expression levels remained constant until around midgestation but then dropped to almost undetectable levels toward birth. Similar expression kinetics were observed in the brain. Isoform-specific expression silencing of MAO-A mediated by siRNA during in vitro embryogenesis induced developmental defects, as indicated by a reduction of the crown rump length and impaired cerebral development. These alterations were paralleled by elevated serotonin levels. Similar abnormalities were observed when embryos were cultured in the presence of the MAO-A inhibitor clorgyline or when the transcriptional inhibitor of MAO-A expression R1 was overexpressed. In contrast, no such alterations were detected when expression of MAO-B was knocked down. To explore the underlying mechanisms for the developmental abnormalities in MAO-A knockdown embryos, we quantified the degree of developmental apoptosis in the developing brain. MAO-A knockdown reduced the number of apoptotic cells in the neuroepithelium, which coincided with impaired activation of caspases 3 and 9. Moreover, we observed reduced cyclin D1 levels as an indicator of impaired cell proliferation in MAO-A knockdown embryos. This data highlights MAO-A as a vital regulator of embryonic brain development.

The monoamine oxidase A inhibitor clorgyline is a broad-spectrum inhibitor of fungal ABC and MFS transporter efflux pump activities which reverses the azole resistance of Candida albicans and Candida glabrata clinical isolates.

Resistance to the commonly used azole antifungal fluconazole (FLC) can develop due to overexpression of ATP-binding cassette (ABC) and major facilitator superfamily (MFS) plasma membrane transporters. An approach to overcoming this resistance is to identify inhibitors of these efflux pumps. We have developed a pump assay suitable for high-throughput screening (HTS) that uses recombinant Saccharomyces cerevisiae strains hyperexpressing individual transporters from the opportunistic fungal pathogen Candida albicans. The recombinant strains possess greater resistance to azoles and other pump substrates than the parental host strain. A flow cytometry-based HTS, which measured increased intracellular retention of the fluorescent pump substrate rhodamine 6G (R6G) within yeast cells, was used to screen the Prestwick Chemical Library (PCL) of 1,200 marketed drugs. Nine compounds were identified as hits, and the monoamine oxidase A inhibitor (MAOI) clorgyline was identified as an inhibitor of two C. albicans ABC efflux pumps, CaCdr1p and CaCdr2p. Secondary in vitro assays confirmed inhibition of pump-mediated efflux by clorgyline. Clorgyline also reversed the FLC resistance of S. cerevisiae strains expressing other individual fungal ABC transporters (Candida glabrata Cdr1p or Candida krusei Abc1p) or the C. albicans MFS transporter Mdr1p. Recombinant strains were also chemosensitized by clorgyline to other azoles (itraconazole and miconazole). Importantly, clorgyline showed synergy with FLC against FLC-resistant C. albicans clinical isolates and a C. glabrata strain and inhibited R6G efflux from a FLC-resistant C. albicans clinical isolate. Clorgyline is a novel broad-spectrum inhibitor of two classes of fungal efflux pumps that acts synergistically with azoles against azole-resistant C. albicans and C. glabrata strains.