7,8-Dihydroxyflavone is a direct inhibitor of human and murine pyridoxal phosphatase.

Vitamin B6 deficiency has been linked to cognitive impairment in human brain disorders for decades. Still, the molecular mechanisms linking vitamin B6 to these pathologies remain poorly understood, and whether vitamin B6 supplementation improves cognition is unclear as well. Pyridoxal 5'-phosphate phosphatase (PDXP), an enzyme that controls levels of pyridoxal 5'-phosphate (PLP), the co-enzymatically active form of vitamin B6, may represent an alternative therapeutic entry point into vitamin B6-associated pathologies. However, pharmacological PDXP inhibitors to test this concept are lacking. We now identify a PDXP and age-dependent decline of PLP levels in the murine hippocampus that provides a rationale for the development of PDXP inhibitors. Using a combination of small-molecule screening, protein crystallography, and biolayer interferometry, we discover, visualize, and analyze 7,8-dihydroxyflavone (7,8-DHF) as a direct and potent PDXP inhibitor. 7,8-DHF binds and reversibly inhibits PDXP with low micromolar affinity and sub-micromolar potency. In mouse hippocampal neurons, 7,8-DHF increases PLP in a PDXP-dependent manner. These findings validate PDXP as a druggable target. Of note, 7,8-DHF is a well-studied molecule in brain disorder models, although its mechanism of action is actively debated. Our discovery of 7,8-DHF as a PDXP inhibitor offers novel mechanistic insights into the controversy surrounding 7,8-DHF-mediated effects in the brain.

Vitamin B6 is an important nutrient for optimal brain function, with deficiencies linked to impaired memory, learning and mood in various mental disorders. In older people, vitamin B6 deficiency is also associated with declining memory and dementia. Although this has been known for years, the precise role of vitamin B6 in these disorders and whether supplements can be used to treat or prevent them remained unclear. This is partly because vitamin B6 is actually an umbrella term for a small number of very similar and interchangeable molecules. Only one of these is 'bioactive', meaning it has a biological role in cells. However, therapeutic strategies aimed at increasing only the bioactive form of vitamin B6 are lacking. Previous work showed that disrupting the gene for an enzyme called pyridoxal phosphatase, which breaks down vitamin B6, improves memory and learning in mice. To investigate whether these effects could be mimicked by drug-like compounds, Brenner, Zink, Witzinger et al. used several biochemical and structural biology approaches to search for molecules that bind to and inhibit pyridoxal phosphatase. The experiments showed that a molecule called 7,8-dihydroxyflavone ­ which was previously found to improve memory and learning in laboratory animals with brain disorders ­ binds to pyridoxal phosphatase and inhibits its activity. This led to increased bioactive vitamin B6 levels in mouse brain cells involved in memory and learning. The findings of Brenner et al. suggest that inhibiting pyridoxal phosphatase to increase vitamin B6 levels in the brain could be used together with supplements. The identification of 7,8-dihydroxyflavone as a promising candidate drug is a first step in the discovery of more efficient pyridoxal phosphatase inhibitors. These will be useful experimental tools to directly study whether increasing the levels of bioactive vitamin B6 in the brain may help those with mental health conditions associated with impaired memory, learning and mood.

Glycolytic flux control by drugging phosphoglycolate phosphatase.

Targeting the intrinsic metabolism of immune or tumor cells is a therapeutic strategy in autoimmunity, chronic inflammation or cancer. Metabolite repair enzymes may represent an alternative target class for selective metabolic inhibition, but pharmacological tools to test this concept are needed. Here, we demonstrate that phosphoglycolate phosphatase (PGP), a prototypical metabolite repair enzyme in glycolysis, is a pharmacologically actionable target. Using a combination of small molecule screening, protein crystallography, molecular dynamics simulations and NMR metabolomics, we discover and analyze a compound (CP1) that inhibits PGP with high selectivity and submicromolar potency. CP1 locks the phosphatase in a catalytically inactive conformation, dampens glycolytic flux, and phenocopies effects of cellular PGP-deficiency. This study provides key insights into effective and precise PGP targeting, at the same time validating an allosteric approach to control glycolysis that could advance discoveries of innovative therapeutic candidates.

Dissection and Explant Culture of Murine Allantois for the In Vitro Analysis of Allantoic Attachment.

The placenta is essential for the growth and development of mammalian embryos. For this reason, numerous genetic alterations and likely also environmental insults that disturb placenta development or function can cause early pregnancy loss in mice and humans. Nevertheless, simple in vitro assays to screen for potential effects on placenta formation are lacking. Here, we focus on modeling the first and critical step in placenta formation, which consists of the attachment of the allantois to the chorion. We describe a method to rapidly assess the attachment of allantoic explants on immobilized α4ß1 integrin, which serves as a chorio-mimetic substrate.This in vitro approach enables a qualitative evaluation of the attachment and spreading behavior of multiple allantois explants at different consecutive time points. The protocol may be used to investigate the effect of targeted mouse mutations, drugs, or various environmental factors that have been linked to pregnancy complications or fetal loss on allantois attachment ex vivo.

An essential developmental function for murine phosphoglycolate phosphatase in safeguarding cell proliferation.

Mammalian phosphoglycolate phosphatase (PGP) is thought to target phosphoglycolate, a 2-deoxyribose fragment derived from the repair of oxidative DNA lesions. However, the physiological role of this activity and the biological function of the DNA damage product phosphoglycolate is unknown. We now show that knockin replacement of murine Pgp with its phosphatase-inactive PgpD34N mutant is embryonically lethal due to intrauterine growth arrest and developmental delay in midgestation. PGP inactivation attenuated triosephosphate isomerase activity, increased triglyceride levels at the expense of the cellular phosphatidylcholine content, and inhibited cell proliferation. These effects were prevented under hypoxic conditions or by blocking phosphoglycolate release from damaged DNA. Thus, PGP is essential to sustain cell proliferation in the presence of oxygen. Collectively, our findings reveal a previously unknown mechanism coupling a DNA damage repair product to the control of intermediary metabolism and cell proliferation.

Genetic dissection of alpha2-adrenoceptor functions in adrenergic versus nonadrenergic cells.

Alpha(2)-adrenoceptors mediate diverse functions of the sympathetic system and are targets for the treatment of cardiovascular disease, depression, pain, glaucoma, and sympathetic activation during opioid withdrawal. To determine whether alpha(2)-adrenoceptors on adrenergic neurons or alpha(2)-adrenoceptors on nonadrenergic neurons mediate the physiological and pharmacological responses of alpha(2)-agonists, we used the dopamine beta-hydroxylase (Dbh) promoter to drive expression of alpha(2A)-adrenoceptors exclusively in noradrenergic and adrenergic cells of transgenic mice. Dbh-alpha(2A) transgenic mice were crossed with double knockout mice lacking both alpha(2A)- and alpha(2C)-receptors to generate lines with selective expression of alpha(2A)-autoreceptors in adrenergic cells. These mice were subjected to a comprehensive phenotype analysis and compared with wild-type mice, which express alpha(2A)- and alpha(2C)-receptors in both adrenergic and nonadrenergic cells, and alpha(2A)/alpha(2C) double-knockout mice, which do not express these receptors in any cell type. We were surprised to find that only a few functions previously ascribed to alpha(2)-adrenoceptors were mediated by receptors on adrenergic neurons, including feedback inhibition of norepinephrine release from sympathetic nerves and spontaneous locomotor activity. Other agonist effects, including analgesia, hypothermia, sedation, and anesthetic-sparing, were mediated by alpha(2)-receptors in nonadrenergic cells. In dopamine beta-hydroxylase knockout mice lacking norepinephrine, the alpha(2)-agonist medetomidine still induced a loss of the righting reflex, confirming that the sedative effect of alpha(2)-adrenoceptor stimulation is not mediated via autoreceptor-mediated inhibition of norepinephrine release. The present study paves the way for a revision of the current view of the alpha(2)-adrenergic receptors, and it provides important new considerations for future drug development.

Alpha2C-adrenoceptor polymorphism is associated with improved event-free survival in patients with dilated cardiomyopathy.

AIMS: The sympathetic nervous system plays a central role in cardiac growth but its overstimulation is associated with increased mortality in patients with chronic heart failure. Pre-synaptic alpha2-adrenoceptors are essential feedback regulators to control the release of norepinephrine from sympathetic nerves. In this study we tested whether a deletion polymorphism in the human alpha2C-adrenoceptor gene (alpha2CDel322-325) affects progression of heart failure in patients with dilated cardiomyopathy (DCM). METHODS AND RESULTS: We genotyped and phenotyped 345 patients presenting with DCM in the heart transplant unit of the German Heart Institute, starting in 1994. Patients were treated according to guidelines (99% ACEI, 76% beta-blockers) and were followed until December 2002 or until a first event [death, heart transplantation, or implantation of a left ventricular assist device (LVAD) for a life-threatening condition] occurred. Mean follow-up time was 249 weeks (4.9 years) in event-free patients and 104 weeks (2 years) in patients with events. During follow-up, 51% of the patients exhibited an event: death (18%), implantation of LVAD as bridging for transplantation (7%), or heart transplantation (25%). By Kaplan-Meier analysis, DCM patients with the deletion variant Del322-325 in the alpha2C-adrenoceptor showed significantly decreased event rates (P=0.0043). Cox regression analysis revealed that the presence of the deletion was associated with reduced death rate (relative risk: 0.129, 95% CI: 0.18-0.9441, P=0.044) and event rates (relative risk: 0.167, 95% CI: 0.041-0.685, P=0.012). CONCLUSION: Alpha2C-adrenoceptor deletion may be a novel, strong, and independent predictor of reduced event rates in DCM patients treated according to guidelines.

Feedback inhibition of catecholamine release by two different alpha2-adrenoceptor subtypes prevents progression of heart failure.

BACKGROUND: Elevated plasma norepinephrine levels are associated with increased mortality in patients and in animal models with chronic heart failure. To test which alpha2-adrenoceptor subtypes operate as presynaptic inhibitory receptors to control norepinephrine release in heart failure, we investigated the response of gene-targeted mice lacking alpha2-adrenoceptor subtypes (alpha2-KO) to chronic left ventricular pressure overload. In addition, we determined the functional consequences of genetic variants of alpha2-adrenoceptors in human patients with chronic heart failure. METHODS AND RESULTS: Cardiac pressure overload was induced by transverse aortic constriction. Three months after aortic banding, survival was dramatically reduced in alpha2A-KO (52%) and alpha2C-KO (47%) mice compared with wild-type and alpha2B-deficient (86%) animals. Excess mortality in alpha2A- and alpha2C-KO strains was attributable to heart failure with enhanced left ventricular hypertrophy and fibrosis and elevated circulating catecholamines. The clinical importance of this finding is emphasized by the fact that heart failure patients with a dysfunctional variant of the alpha2C-adrenoceptor had a worse clinical status and decreased cardiac function as determined by invasive catheterization and by echocardiography. CONCLUSIONS: Our results indicate an essential function of alpha2A- and alpha2C-adrenoceptors in the prevention of heart failure progression in mice and human patients. Identification of heart failure patients with genetic alpha2-adrenoceptor variants as well as new alpha2-receptor subtype-selective drugs may represent novel therapeutic strategies in chronic heart failure and other diseases with enhanced sympathetic activation.

Placental alpha(2)-adrenoceptors control vascular development at the interface between mother and embryo.

A substantial percentage of human pregnancies are lost as spontaneous abortions after implantation. This is often caused by an inadequately developed placenta. Proper development of the placental vascular system is essential to nutrient and gas exchange between mother and developing embryo. Here we show that alpha(2)-adrenoceptors, which are activated by adrenaline and noradrenaline, are important regulators of placental structure and function. Mice with deletions in the genes encoding alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors died between embryonic days 9.5 and 11.5 from a severe defect in yolk-sac and placenta development. In wildtype placentae, alpha(2)-adrenoceptors are abundantly expressed in giant cells, which secrete angiogenic factors to initiate development of the placental vascular labyrinth. In placentae deficient in alpha(2A)-, alpha(2B)- and alpha(2C)-adrenoceptors, the density of fetal blood vessels in the labyrinth was markedly lower than normal, leading to death of the embryos as a result of reduced oxygen and nutrient supply. Basal phosphorylation of the extracellular signal regulated kinases ERK1 and ERK2 was also lower than normal, suggesting that activation of the mitogen-activated protein kinase (MAP kinase) pathway by alpha(2)-adrenoceptors is required for placenta and yolk-sac vascular development. Thus, alpha(2)-adrenoceptors are essential at the placental interface between mother and embryo to establish the circulatory system of the placenta and thus maintain pregnancy.