Inhibition of DYRK1A attenuates vascular remodeling in pulmonary arterial hypertension via suppressing STAT3/Pim-1/NFAT pathway.

Pulmonary arterial hypertension (PAH) is characterized by progressive vascular remodeling caused by the excessive proliferation and survival of pulmonary artery smooth muscle cells (PASMCs). Dual-specificity tyrosine regulated kinase 1A (DYRK1A) is a pleiotropic kinase involved in the regulation of multiple biological functions, including cell proliferation and survival. However, the role and underlying mechanisms of DYRK1A in PAH pathogenesis remain unclear. We found that DYRK1A was upregulated in PASMCs in response to hypoxia, both in vivo and in vitro. Inhibition of DYRK1A by harmine significantly attenuated hypoxia-induced pulmonary hypertension and pulmonary artery remodeling. Mechanistically, we found that DYRK1A promoted pulmonary arterial remodeling by enhancing the proliferation and survival of PASMCs through activating the STAT3/Pim-1/NFAT pathway, because STAT3 gain-of-function via adeno-associated virus serotype 2 (AAV2) carrying the constitutively active form of STAT3 (STAT3C) nearly abolished the protective effect of harmine on PAH. Collectively, our results reveal a significant role for DYRK1A in pulmonary arterial remodeling and suggest it as a drug target with translational potential for the treatment of PAH.

ß-Carbolines norharman and harman change neurobehavior causing neurological damage in Caenorhabditis elegans.

ß-Carbolines norharman and harman, belonging to the class of heterocyclic aromatic amines (HAAs), are typical hazardous substances produced during the thermal processing of food. Compared to other HAAs, there have been limited reports on the toxicity of ß-carbolines. Nevertheless, the current studies are concerned with the neurotoxic effects of norharman and harman at high doses. It is still unknown whether the relatively low dose of ß-carbolines in foods induces neurotoxicity and the mechanism of the toxicity. In this study, C. elegans was exposed to a series of gradients of norharman and harman (0, 0.05, 5, and 10 mg L-1). The survival rate and indicators of ethology (locomotor behaviors, foraging behavior, and chemotaxis ability) were assessed. The antioxidant system and the contents of neurotransmitters, as well as the activity of acetylcholinesterase (AChE), were evaluated. Additionally, the RNA-seq screening of differentially expressed genes (DEGs) revealed the potential molecular mechanisms of norharman- and harman-induced toxic effects. Our results indicated that the risk of long-term exposure to norharman and harman at low doses (food-related doses) should be emphasized. Moreover, ß-carbolines might induce neurotoxicity by causing oxidative damage, regulating the content of neurotransmitters, and interfering with cytochrome P450 metabolism. This study would provide a toxicological basis for the neurotoxicity of ß-carbolines and lay the foundation for the risk assessment of endogenous pollutants in food.

Impact of genetic variants within serotonin turnover enzymes on human cerebral monoamine oxidase A in vivo.

Variants within the monoamine oxidase A (MAO-A, MAOA) and tryptophan hydroxylase 2 (TPH2) genes, the main enzymes in cerebral serotonin (5-HT) turnover, affect risk for depression. Depressed cohorts show increased cerebral MAO-A in positron emission tomography (PET) studies. TPH2 polymorphisms might also influence brain MAO-A because availability of substrates (i.e. monoamine concentrations) were shown to affect MAO-A levels. We assessed the effect of MAOA (rs1137070, rs2064070, rs6323) and TPH2 (rs1386494, rs4570625) variants associated with risk for depression and related clinical phenomena on global MAO-A distribution volume (VT) using [11C]harmine PET in 51 participants (21 individuals with seasonal affective disorder (SAD) and 30 healthy individuals (HI)). Statistical analyses comprised general linear models with global MAO-A VT as dependent variable, genotype as independent variable and age, sex, group (individuals with SAD, HI) and season as covariates. rs1386494 genotype significantly affected global MAO-A VT after correction for age, group and sex (p < 0.05, corr.), with CC homozygotes showing 26% higher MAO-A levels. The role of rs1386494 on TPH2 function or expression is poorly understood. Our results suggest rs1386494 might have an effect on either, assuming that TPH2 and MAO-A levels are linked by their common product/substrate, 5-HT. Alternatively, rs1386494 might influence MAO-A levels via another mechanism, such as co-inheritance of other genetic variants. Our results provide insight into how genetic variants within serotonin turnover translate to the cerebral serotonin system. Clinicaltrials.gov Identifier: NCT02582398. EUDAMED Number: CIV-AT-13-01-009583.

Discovery of YH677 as a cancer stemness inhibitor that suppresses triple-negative breast cancer growth and metastasis by regulating the TGFß signaling pathway.

Triple-negative breast cancer (TNBC) has a poor prognosis due to the lack of specific and highly effective therapeutic agents. Cancer stem cells (CSCs) are one of the main factors contributing to TNBC relapse and metastasis. Therefore, targeting CSCs selectively with small molecules is a novel strategy for drug development. In this study, the natural product harmine (HM) was identified as a hit compound from 2632 natural product monomers based on phenotypic screening of a 2D assay and patient-derived organoid (PDO) model that was established from a patient who had multiple drug resistance and various visceral and contralateral breast metastases. Next, harmine was further modified and optimized to obtain a lead compound (YH677) with a tetrahydro-ß-carboline scaffold. YH677 showed potent antiproliferative and antimigratory activities against several TNBC cell lines in vitro. In addition, YH677 inhibited epithelial mesenchymal transition (EMT) and stem cell marker expression in a dose-dependent manner. More importantly, YH677 suppressed breast cancer growth and metastasis in orthotopic, metastatic xenograft and patient-derived xenograft (PDX) models in vivo. Mechanistic studies showed that YH677 inhibits the expansion of CSCs by regulating the TGFß/Smad signaling pathway. These preclinical data provide a basis for the development of YH677 as a lead compound for TNBC treatment.

Harmine, an inhibitor of the type III secretion system of Salmonella enterica serovar Typhimurium.

New therapeutic strategies for clinical Salmonella enterica serovar Typhimurium (S. Typhimurium) infection are urgently needed due to the generation of antibiotic-resistant bacteria. Inhibition of bacterial virulence has been increasingly regarded as a potential and innovative strategy for the development of anti-infection drugs. Salmonella pathogenicity island (SPI)-encoded type III secretion system (T3SS) represents a key virulence factor in S. Typhimurium, and active invasion and replication in host cells is facilitated by the secretion of T3SS effector proteins. In this study, we found that harmine could inhibit T3SS secretion; thus, its potential anti-S. Typhimurium infection activity was elucidated. Harmine inhibits the secretion and expression of T3SS effector proteins and consequently attenuates the S. Typhimurium invasion function of HeLa cells. This inhibition may be implemented by reducing the transcription of pathogenesis-related SPI-1 transcriptional activator genes hilD, hilC, and rtsA. Harmine improves the survival rate and bacterial loads of mice infected with S. Typhimurium. In summary, harmine, an effective T3SS inhibitor, could be a leading compound for the development of treatments for Salmonella infection.

Evaluation of the Effects of Harmine on ß-cell Function and Proliferation in Standardized Human Islets Using 3D High-Content Confocal Imaging and Automated Analysis.

Restoration of ß-cell mass through the induction of proliferation represents an attractive therapeutic approach for the treatment of diabetes. However, intact and dispersed primary islets suffer from rapidly deteriorating viability and function ex vivo, posing a significant challenge for their experimental use in proliferation studies. Here, we describe a novel method for the assessment of compound effects on ß-cell proliferation and count using reaggregated primary human islets, or islet microtissues (MTs), which display homogeneous size and tissue architecture as well as robust and stable functionality and viability for 4 weeks in culture. We utilized this platform to evaluate the dose-dependent short- and long-term effects of harmine on ß-cell proliferation and function. Following compound treatment and EdU incorporation, islet MTs were stained and confocal-imaged for DAPI (nuclear marker), NKX6.1 (ß-cell marker), and EdU (proliferation marker), allowing automated 3D-analysis of number of total cells, ß-cells, and proliferating ß- and non-ß-cells per islet MT. In parallel, insulin secretion, intracellular insulin and ATP contents, and Caspase 3/7 activity were analyzed to obtain a comprehensive overview of islet MT function and viability. We observed that 4-day harmine treatment increased ß- and non-ß-cell proliferation, NKX6.1 expression, and basal and stimulated insulin secretion in a dose-dependent manner, while fold-stimulation of secretion peaked at intermediate harmine doses. Interestingly, 15-day harmine treatment led to a general reduction in harmine's proliferative effects as well as altered dose-dependent trends. The described methodology provides a unique tool for in vitro high-throughput evaluation of short- and long-term changes in human ß-cell proliferation, count and fraction along with a variety of functional parameters, in a representative 3D human islet model.

Inhibition of DYRK1a Enhances Cardiomyocyte Cycling After Myocardial Infarction.

BACKGROUND: DYRK1a (dual-specificity tyrosine phosphorylation-regulated kinase 1a) contributes to the control of cycling cells, including cardiomyocytes. However, the effects of inhibition of DYRK1a on cardiac function and cycling cardiomyocytes after myocardial infarction (MI) remain unknown. METHODS: We investigated the impacts of pharmacological inhibition and conditional genetic ablation of DYRK1a on endogenous cardiomyocyte cycling and left ventricular systolic function in ischemia-reperfusion (I/R) MI using αMHC-MerDreMer-Ki67p-RoxedCre::Rox-Lox-tdTomato-eGFP (RLTG) (denoted αDKRC::RLTG) and αMHC-Cre::Fucci2aR::DYRK1aflox/flox mice. RESULTS: We observed that harmine, an inhibitor of DYRK1a, improved left ventricular ejection fraction (39.5±1.6% and 29.1±1.6%, harmine versus placebo, respectively), 2 weeks after I/R MI. Harmine also increased cardiomyocyte cycling after I/R MI in αDKRC::RLTG mice, 10.8±1.5 versus 24.3±2.6 enhanced Green Fluorescent Protein (eGFP)+ cardiomyocytes, placebo versus harmine, respectively, P=1.0×10-3. The effects of harmine on left ventricular ejection fraction were attenuated in αDKRC::DTA mice that expressed an inducible diphtheria toxin in adult cycling cardiomyocytes. The conditional cardiomyocyte-specific genetic ablation of DYRK1a in αMHC-Cre::Fucci2aR::DYRK1aflox/flox (denoted DYRK1a k/o) mice caused cardiomyocyte hyperplasia at baseline (210±28 versus 126±5 cardiomyocytes per 40× field, DYRK1a k/o versus controls, respectively, P=1.7×10-2) without changes in cardiac function compared with controls, or compensatory changes in the expression of other DYRK isoforms. After I/R MI, DYRK1a k/o mice had improved left ventricular function (left ventricular ejection fraction 41.8±2.2% and 26.4±0.8%, DYRK1a k/o versus control, respectively, P=3.7×10-2). RNAseq of cardiomyocytes isolated from αMHC-Cre::Fucci2aR::DYRK1aflox/flox and αMHC-Cre::Fucci2aR mice after I/R MI or Sham surgeries identified enrichment in mitotic cell cycle genes in αMHC-Cre::Fucci2aR::DYRK1aflox/flox compared with αMHC-Cre::Fucci2aR. CONCLUSIONS: The pharmacological inhibition or cardiomyocyte-specific ablation of DYRK1a caused baseline hyperplasia and improved cardiac function after I/R MI, with an increase in cell cycle gene expression, suggesting the inhibition of DYRK1a may serve as a therapeutic target to treat MI.

Heme peroxidases are responsible for the dehydrogenation and oxidation metabolism of harmaline into harmine.

Harmaline and harmine are ß-carboline alkaloids with effective pharmacological effects. Harmaline can be transformed into harmine after oral administration. However, enzymes involved in the metabolic pathway remain unclear. In this study, harmaline was incubated with rat liver microsomes (RLM), rat brain microsomes (RBM), blood, plasma, broken blood cells, and heme peroxidases including horseradish peroxidase (HRP), lactoperoxidase (LPO), and myeloperoxidase (MPO). The production of harmine was determined by a validated UPLC-ESI-MS/MS method. Results showed that heme peroxidases catalyzed the oxidative dehydrogenation of harmaline. All the reactions were in accordance with the Hill equation. The reaction was inhibited by ascorbic acid and excess H2O2. The transformation of harmaline to harmine was confirmed after incubation with blood, plasma, and broken blood cells, rather than RLM and RBM. Harmaline was incubated with blood, plasma, and broken cells liquid for 3 h, and the formation of harmine became stable. Results indicated an integrated metabolic pathway of harmaline, which will lay foundation for the oxidation reaction of dihydro-ß-carboline. Moreover, the metabolic stability of harmaline in blood should not be ignored when the pharmacokinetics study of harmaline is carried out.

Structure-activity insights of harmine targeting DNA, ROS inducing cytotoxicity with PARP mediated apoptosis against cervical cancer, anti-biofilm formation and in vivo therapeutic study.

Harmine exhibits pH dependent structural equilibrium and possesses numerous biological and pharmacological activities. Mode and mechanism of DNA binding and its cytotoxicity were studied by multiple spectroscopic, calorimetric, molecular docking and in vitro apoptotic as well as in vivo biochemical and histological studies. It exists as cationic (structure I) and decationic form (structure II) in the pH range 3.0-7.8 and 8.5-12.4, respectively, with a pKa of 8.0. Structure I at pH 6.8 binds strongly to DNA with a cooperative mode of binding of Kiω 1.03 × 106 M-1and stoichiometry of 5.0 nucleotide phosphates. Structure I stabilized DNA by 10 °C, showed85%quenching of fluorescence intensity, perturbation in circular dichroism, partial intercalation and enthalpy driven exothermic binding. While, structure II at pH 8.5 has very weak interaction with CT DNA. Cytotoxic potencies of structure I was tested on four different cancer cell lines along with normal embryonic cell. It showed maximum cytotoxicity with GI50of 20 µM, against HeLa causing several apoptotic induction abilities. Harmine exhibited G2M arrest with ROS induced effective role in PARP mediated apoptosis as well as anti-inflammatory action on HeLa cells. Harmine further presented MIC and antibiofilm activity against Staphylococcus aureus in presence of <160 and 30 µg/ml, respectively. Mice with post harmine treatment (30 mg/kg b.w., I.P.) showed maximum recovery from damaged to near normal architecture of cervical epithelial cells. This study may be of prospective use in a framework to design novel beta carboline compounds for improved therapeutic applications in future against cervical cancer. HighlightsHarmine exists in structure I and structure II forms in the pH 6.8 and 8.5with a pKa of 8.0.Structure I at pH 6.8 binds strongly to DNA compared to structure II.Structure I showed maximum cytotoxicity with GI50 of 20 µM against HeLa.ROS mediated cytotoxicitywithG2M arrest with PARP mediated apoptosis was studied.Harmine (30µg/ml) exhibited antibiofilm activity against Staphylococcus aureus.Post harmine dose (30 mg/kg b.w., I.P.) in mice showed recovery of cervical epithelial cells.Communicated by Ramaswamy H. Sarma.

Coexpression of Gene Transcripts with Monoamine Oxidase A Quantified by Human In Vivo Positron Emission Tomography.

The monoamine oxidase A (MAO-A) is integral to monoamine metabolism and is thus relevant to the pathophysiology of various neuropsychiatric disorders; however, associated gene-enzyme relations are not well understood. This study aimed to unveil genes coexpressed with MAO-A. Therefore, 18 179 mRNA expression maps (based on the Allen Human Brain Atlas) were correlated with the cerebral distribution volume (VT) of MAO-A assessed in 36 healthy subjects (mean age ± standard deviation: 32.9 ± 8.8 years, 18 female) using [11C]harmine positron emission tomography scans. Coexpression analysis was based on Spearman's ρ, over-representation tests on Fisher's exact test with false discovery rate (FDR) correction. The analysis revealed 35 genes in cortex (including B-cell translocation gene family, member 3, implicated in neuroinflammation) and 247 genes in subcortex (including kallikrein-related peptidase 10, implicated in Alzheimer's disease). Significantly over-represented Gene Ontology terms included "neuron development", "neuron differentiation", and "cell-cell signaling" as well as "axon" and "neuron projection". In vivo MAO-A enzyme distribution and MAOA expression did not correlate in cortical areas (ρ = 0.08) while correlation was found in subcortical areas (ρ = 0.52), suggesting influences of region-specific post-transcriptional and -translational modifications. The herein reported information could contribute to guide future genetic studies, deepen the understanding of associated pathomechanisms and assist in the pursuit of novel therapeutic targets.

Evaluation of monoamine oxidase A and B type enzyme occupancy using non-radiolabelled tracers in rat brain.

Monoamine oxidase (MAO) enzymes, type A and B metabolise the amine neurotransmitters of the body. Selective inhibition of either enzyme is an approach for treating neurodegenerative and stress-induced disorders, and inhibition of an enzyme is proportional to the binding of the MAO inhibitor. Conventionally, the binding of test compounds to enzymes is assessed by radiolabelled ligands in ex vivo and in vivo occupancy assays. Regulatory restrictions and turnaround time are the limitations of the methods that use radiolabelled ligands. But the use of non-radiolabelled tracers and sensitive mass spectrometry (LC-MS/MS) based assays accelerated the determination of target occupancy in pre-clinical species. A report on use of non-radiolabelled ligand in in vivo MAO occupancy assay is not available. The objectives of the present study were to optimise non-radiolabelled harmine and deprenyl as selective tracers in MAO-A and MAO-B occupancy assays and evaluate MAO occupancy of test compounds in rat brain. Tracer optimisation resulted in a detectable, stable, and low ratio (<3.0) of tracer concentrations between any two brain tissues. In occupancy assay, tracer was intravenously administered (10 µg/kg, harmine or 60 µg/kg, L-deprenyl) after the treatment with test compound (clorgyline or tranylcypromine or pargyline or phenelzine or thioperamide). Specific brain tissues were isolated at a defined interval and tracer concentrations were quantified using LC-MS/MS method. Pre-treatment with MAO inhibitors resulted in a decrease (maximum, 80-85%) in harmine or an increase (maximum, 85-300%) in L-deprenyl concentrations. But we considered the change in tracer concentration, relative to the vehicle and positive control groups to calculate MAO occupancy. The observed selectivity and ratio of occupancies (ED50) of test compound towards MAO-A and MAO-B are comparable with the results from in vitro radiolabelled ligand-based inhibition assay. The results demonstrated the application of these non-radiolabelled tracers as suitable pre-clinical tools to determine MAO occupancy.

Harmine inhibits the proliferation and migration of glioblastoma cells via the FAK/AKT pathway.

AIMS: Glioblastoma is one of the most invasive tumors of the central nervous system, and has a high degree of malignancy and poor prognosis. Harmine, an active ingredient extracted from perennial herbs, has been reported to have obvious antitumor effects on various tumors. However, the effects of harmine on glioblastoma growth remain unknown. We here explored the effects of harmine on glioblastoma and its underlying molecular mechanisms related to tumorigenesis. MATERIALS AND METHODS: CCK-8 and immunofluorescent assay were performed to measure anti-proliferative effect of harmine on U251-MG and U373-MG cells. Wound healing assay was performed to measure the effects of harmine on cell migration. qRT-PCR and western blot were performed to detect the protein/gene expression. BALB/c nude mice bearing U251-MG xenografts was used to measure the effects of harmine on the growth of glioblastoma in vivo. KEY FINDINGS: Harmine treatment significantly suppressed the proliferation of U251-MG and U373-MG cells in a dose and time-dependent way. Mechanistically, harmine reduced the basal and EGF-enhanced the phosphorylation level of FAK and AKT. Moreover, harmine inhibited the cell viability of U251-MG and U373-MG cells by downregulating the phosphorylation of the FAK/AKT pathway. Besides, harmine significantly suppressed the migration of U251-MG cells by suppressing the expression of MMP2, MMP9 and VEGF. Subsequently, orthotopic xenograft models revealed that harmine treatment dramatically inhibited the growth of glioblastoma in vivo. SIGNIFICANCE: In conclusion, these results suggest that harmine suppresses the proliferation and migration of U251-MG and U373-MG cells by inhibiting the FAK/AKT signaling pathway. Our findings elucidate harmine could be a promising drug for glioblastoma therapy.

How to Separate Kinase Inhibition from Undesired Monoamine Oxidase A Inhibition-The Development of the DYRK1A Inhibitor AnnH75 from the Alkaloid Harmine.

The ß-carboline alkaloid harmine is a potent DYRK1A inhibitor, but suffers from undesired potent inhibition of MAO-A, which strongly limits its application. We synthesized more than 60 analogues of harmine, either by direct modification of the alkaloid or by de novo synthesis of ß-carboline and related scaffolds aimed at learning about structure-activity relationships for inhibition of both DYRK1A and MAO-A, with the ultimate goal of separating desired DYRK1A inhibition from undesired MAO-A inhibition. Based on evidence from published crystal structures of harmine bound to each of these enzymes, we performed systematic structure modifications of harmine yielding DYRK1A-selective inhibitors characterized by small polar substituents at N-9 (which preserve DYRK1A inhibition and eliminate MAO-A inhibition) and beneficial residues at C-1 (methyl or chlorine). The top compound AnnH75 remains a potent DYRK1A inhibitor, and it is devoid of MAO-A inhibition. Its binding mode to DYRK1A was elucidated by crystal structure analysis, and docking experiments provided additional insights for this attractive series of DYRK1A and MAO-A inhibitors.

Simultaneous generation of acrylamide, ß-carboline heterocyclic amines and advanced glycation ends products in an aqueous Maillard reaction model system.

The simultaneous formation of acrylamide; ß-carboline heterocyclic amines (HAs): harmane and norharmane; and advanced glycation end products (AGEs) (Nε-(carboxymethyl)lysine (CML) and Nε-(carboxyethyl)lysine (CEL)) was analyzed based on an aqueous model system. The model systems included lysine-glucose (Lys/Glu), asparagine-glucose (Asn/Glu), tryptophan-glucose (Trp/Glu), and a mixture of these amino acids (Mix/Glu). Only AGEs were generated when heated at 100 °C, Asn and Trp competed with Lys for glucose and methylglyoxal (MGO), and glyoxal (GO) decreased AGE content. The k value of CML, CEL, and acrylamide decreased when heated at 130 °C, whereas that of harmane increased in the Mix/Glu, owing to the competition between Lys and Asn for glucose, GO, and MGO. Harmane preferably formed via the Pictet-Spengler condensation between Trp and acetaldehyde, which further reduced acrylamide formation via the acrolein pathway.

Natural harmine negatively regulates the developmental signaling network of Drosophila melanogaster (Drosophilidae: Diptera) in vivo.

The widely distributed ß-carboline alkaloids exhibit promising psychopharmacological and biochemical effects. Harmine, a natural ß-carboline, can inhibit insect growth and development with unclear mechanisms. In this study, harmine (at 0-200 mg/L) showed a dose-dependent inhibitory effect on the pupal weight, length, height, pupation rate and eclosion rate of fruit flies Drosophila melanogaster, which was similar to the inhibition induced by the well-known botanical insect growth regulator azadirachtin. Moreover, the expression levels of major regulators from the developmental signaling network were down-regulated during the pupal stage except Numb, Fringe, Yorkie and Pten. The Notch, Wnt, Hedgehog and TGF-ß pathways mainly played vital roles in coping with harmine exposure in pupae stage, while the Hippo, Hedgehog and TGF-ß elements were involved in the sex differences. Notch, Hippo, Hedgehog, Dpp and Armadillo were proved to be suppressed in the developmental inhibition with fly mutants, while Numb and Punt were increased by harmine. In conclusion, harmine significantly inhibited the development of Drosophila by negatively affecting their developmental signaling network during different stages. Our results establish a preliminary understanding of the developmental signaling network subjected to botanical component-induced growth inhibition and lay the groundwork for further application.

Pharmacologic and genetic approaches define human pancreatic ß cell mitogenic targets of DYRK1A inhibitors.

Small molecule inhibitors of dual specificity, tyrosine phosphorylation-regulated kinase 1A (DYRK1A), including harmine and others, are able to drive human ß cell regeneration. While DYRK1A is certainly a target of this class, whether it is the only or the most important target is uncertain. Here, we employ a combined pharmacologic and genetic approach to refine the potential mitogenic targets of the DYRK1A inhibitor family in human islets. A combination of human ß cell RNA sequencing, DYRK1A inhibitor kinome screens, pharmacologic inhibitors, and targeted silencing of candidate genes confirms that DYRK1A is a central target. Surprisingly, however, DYRK1B also proves to be an important target: silencing DYRK1A results in an increase in DYRK1B. Simultaneous silencing of both DYRK1A and DYRK1B yields greater ß cell proliferation than silencing either individually. Importantly, other potential kinases, such as the CLK and the GSK3 families, are excluded as important harmine targets. Finally, we describe adenoviruses that are able to silence up to 7 targets simultaneously. Collectively, we report that inhibition of both DYRK1A and DYRK1B is required for induction of maximal rates of human ß cell proliferation, and we provide clarity for future efforts in structure-based drug design for human ß cell regenerative drugs.

The Human Fecal Microbiota Metabolizes Foodborne Heterocyclic Aromatic Amines by Reuterin Conjugation and Further Transformations.

SCOPE: Heterocyclic aromatic amines (HAAs) are process-induced food contaminants with high mutagenic and/or carcinogenic potential. Although the human gut microbiota is known to affect the metabolism of dietary constituents, its impact on HAA metabolism and toxicity has been little studied. Here, the glycerol-dependent metabolism of seven foodborne HAAs (AαC, Trp-P-1, harman, norharman, PhIP, MeIQx, and MeIQ) by the human fecal microbiota is investigated. METHODS AND RESULTS: As analyzed by HPLC-DAD/FLD, the extent of conversion is strongly dependent on glycerol supplementation and HAA structure. AαC (60-100%) and the 2-aminoimidazoazarenes (up to 58%) are especially prone to microbial conversion. Based on high-resolution MS and/or NMR spectroscopy data, 70 fecal metabolites are identified in total, mainly formed by chemical reactions with one or two molecules of microbially derived reuterin. Moreover, it has been demonstrated that the human fecal microbiota can further transform reuterin adducts by reduction and/or hydroxylation reactions. Upon isolation, some reuterin-induced HAA metabolites appear to be partially unstable, complicating structural identification. CONCLUSION: The formation of microbial metabolites needs to be incorporated into risk assessment considerations for HAAs in human health. In this study, several HAA metabolites, mainly reuterin-dependent, are identified in vitro, providing the basis for future human studies investigating microbial HAA metabolism.

Crystal Structure of Human Dual-Specificity Tyrosine-Regulated Kinase 3 Reveals New Structural Features and Insights into its Auto-phosphorylation.

Dual-specificity tyrosine-regulated kinases (DYRKs) auto-phosphorylate a critical tyrosine residue in their activation loop and phosphorylate their substrate on serine and threonine residues. The auto-phosphorylation occurs intramolecularly and is a one-off event. DYRK3 is selectively expressed at a high level in hematopoietic cells and attenuates erythroblast development, leading to anemia. In the present study, we determined the crystal structure of the mature form of human DYRK3 in complex with harmine, an ATP competitive inhibitor. The crystal structure revealed a phosphorylation site, residue S350, whose phosphorylation increases the stability of DYRK3 and enhances its kinase activity. In addition, our structural and biochemical assays suggest that the N-terminal auto-phosphorylation accessory domain stabilizes the DYRK3 protein, followed by auto-phosphorylation of the tyrosine of the activation loop, which is important for kinase activity. Finally, our docking analysis provides information for the design of novel and potent therapeutics to treat anemia.

A novel harmine derivative, N-(4-(hydroxycarbamoyl)benzyl)-1-(4- methoxyphenyl)-9H-pyrido[3,4-b]indole-3-carboxamide (HBC), as histone deacetylase inhibitor: in vitro antiproliferation, apoptosis induction, cell cycle arrest, and antimetastatic effects.

This study aims to design and synthesize a novel harmine derivative N-(4-(hydroxycarbamoyl) benzyl)-1-(4-methoxyphenyl)-9H-pyrido [3,4-b]indole-3-carboxamide (HBC) as histone deacetylase (HDAC) inhibitor, and evaluate its antitumor activities and anti-metastasis mechanism. HBC not only exerted significant ant-proliferation activity against five human cancer cell lines, especially for HepG2 cell with an IC50 value of 2.21 µM, which is nearly three-fold lower than SAHA (IC50 = 6.26 µM), but also showed selective HDAC1/6 inhibitory effects in vitro. However, HBC had little effect on normal hepatic cells LO2. Furthermore, HBC simultaneously increased the acetylation of histone H3, H4, and α-tubulin, induced hypochromism by electrostatical interaction with CT-DNA, triggered more significant cancer cell apoptosis and cell cycle arrest at G2/M than SAHA by inhibition of both CDK1 and cyclin B in a concentration dependent manner. In addition, scratch and invasion assay showed that HBC also dose-dependently suppressed migration and invasion capacities of highly metastatic HCC HepG2 cells through down-regulated the expression of tumor metastasis related proteins MMP-2 and MMP-9, significantly better than SAHA. Finally, HBC showed low acute toxicity to mice and significant growth inhibition of the hepatoma tumor in vivo. These results demonstrate that novel harmine-based HDAC inhibitor HBC not only exhibited selective HDAC1/6 inhibitory activity and significant in vitro and in vivo antitumor activity, but also possessed DNA binding effect, apoptosis induction, cell cycle arrest effects, and potent anti-metastasis mechanisms, which may hold great promise as therapeutic agent targeting HDAC1/6 for the intervention of human cancers.

A harmine-derived beta-carboline displays anti-cancer effects in vitro by targeting protein synthesis.

Growing evidence indicates that protein synthesis is deregulated in cancer onset and progression and targeting this process might be a selective way to combat cancers. While harmine is known to inhibit DYRK1A and intercalate into the DNA, tri-substitution was shown previously to modify its activity profile in favor of protein synthesis inhibition. In this study, we thus evaluated the optimized derivative CM16 in vitro anti-cancer effects unfolding its protein synthesis inhibition activity. Indeed, the growth inhibitory profile of CM16 in the NCI 60-cancer-cell-line-panel correlated with those of other compounds described as protein synthesis inhibitors. Accordingly, CM16 decreased in a time- and concentration-dependent manner the translation of neosynthesized proteins in vitro while it did not affect mRNA transcription. CM16 rapidly penetrated into the cell in the perinuclear region of the endoplasmic reticulum where it appears to target translation initiation as highlighted by ribosomal disorganization. More precisely, we found that the mRNA expression levels of the initiation factors EIF1AX, EIF3E and EIF3H differ when comparing resistant or sensitive cell models to CM16. Additionally, CM16 induced eIF2α phosphorylation. Those effects could explain, at least partly, the CM16 cytostatic anti-cancer effects observed in vitro while neither cell cycle arrest nor DNA intercalation could be demonstrated. Therefore, targeting protein synthesis initiation with CM16 could represent a new promising alternative to current cancer therapies due to the specific alterations of the translation machinery in cancer cells as recently evidenced with respect to EIF1AX and eIF3 complex, the potential targets identified in this present study.