Isel Pascual

Tanycyte pyroglutamyl peptidase II contributes to regulation of the hypothalamic-pituitary-thyroid axis through glial-axonal associations in the median eminence.

Pyroglutamyl peptidase II (PPII), a highly specific membrane-bound metallopeptidase that inactivates TRH in the extracellular space, is tightly regulated by thyroid hormone in cells of the anterior pituitary. Whether PPII has any role in the region where axons containing hypophysiotropic TRH terminate, the median eminence, is unknown. For this purpose, we analyzed the cellular localization and regulation of PPII mRNA in the mediobasal hypothalamus in adult, male rats. PPII mRNA was localized in cells lining the floor and infralateral walls of the third ventricle and coexpressed with vimentin, establishing these cells as tanycytes. PPII mRNA extended in a linear fashion from the tanycyte cell bodies in the base of the third ventricle to its cytoplasmic and end-feet processes in the external zone of the median eminence in close apposition to pro-TRH-containing axon terminals. Compared with vehicle-treated, euthyroid controls, animals made thyrotoxic by the i.p. administration of 10 microg L-T(4) daily for 1-3 d, showed dramatically increased accumulation of silver grains in the mediobasal hypothalamus and an approximately 80% increase in enzymatic activity. PPII inhibition in mediobasal hypothalamic explants increased TRH secretion, whereas i.p. injection of a specific PPII inhibitor increased cold stress- and TRH-induced TSH levels in plasma. We propose that an increase in circulating thyroid hormone up-regulates PPII activity in tanycytes and enhances degradation of extracellular TRH in the median eminence through glial-axonal associations, contributing to the feedback regulation of thyroid hormone on anterior pituitary TSH secretion.

Purification of a specific inhibitor of pyroglutamyl aminopeptidase II from the marine annelide Hermodice carunculata In vivo effects in rodent brain

An inhibitor of the metallo-ectoenzyme, pyroglutamyl aminopeptidase II (PPII), a thyrotropin releasing hormone-specific peptidase, was identified by screening extracts from marine species of the Cuban coast-line belonging to the phylla Chordata, Echinodermata, Annelida, Mollusca, Cnidaria, Porifera, Chlorophyta and Magnoliophyta. Isolation of the inhibitor (HcPI), from the marine annelide Hermodice carunculata, was achieved by trichloroacetic acid treatment of the aqueous extract, followed by ion-exchange chromatography on DEAE Sephacel, gel filtration on Sephadex G-25 and reverse phase-HPLC. HcPI had a small apparent molecular weight (below 1000 Da) and was not a peptide. It inhibited rat PPII (a membrane preparation with 8.5mg protein/ml) with an apparent K(i) of 51 nM. HcPI did not inhibit serine (trypsin, chymotrypsin, elastase and dipeptidyl aminopeptidase IV), cysteine (papain, bromelain and pyroglutamyl aminopeptidase I), aspartic (pepsin and recombinant human immunodeficiency virus 1 protease (HIV1-PR)) nor other metallo proteinases (collagenase, gelatinase, angiotensin converting enzyme, aminopeptidase N and carboxypeptidase A). HcPI was non-toxic and active in vivo. Intraperitoneal injection of HcPI reduced mouse pituitary and brain PPII activity. Potency of the effect was higher in hypophysis and hypothalamus than in other brain regions. Intrathecal administration to male rats reduced PPII activity in the spinal cord. In conclusion we have identified a specific inhibitor of PPII that is the first M1 family zinc metallo-peptidase inhibitor isolated from marine invertebrates. It may be useful for elucidating the in vivo role of PPII in the pituitary and central nervous system.

Anterior pituitary pyroglutamyl peptidase II activity controls TRH-induced prolactin release

Ecto-peptidases modulate the action of peptides in the extracellular space. The relationship between peptide receptor and ecto-peptidase localization, and the physiological role of peptidases is poorly understood. Current evidence suggests that pyroglutamyl peptidase II (PPII) inactivates neuronally released thyrotropin-releasing hormone (TRH). The impact of PPII localization in the anterior pituitary on the endocrine activities of TRH is unknown. We have studied whether PPII influences TRH signaling in anterior pituitary cells in primary culture. In situ hybridization (ISH) experiments showed that PPII mRNA was expressed only in 5-6% of cells. ISH for PPII mRNA combined with immunocytochemistry for prolactin, beta-thyrotropin, or growth hormone, showed that 66% of PPII mRNA expressing cells are lactotrophs, 34% somatotrophs while none are thyrotrophs. PPII activity was reduced using a specific phosphorothioate antisense oligodeoxynucleotide or inhibitors. Compared with mock or scrambled oligodeoxynucleotide-treated controls, knock-down of PPII expression by antisense targeting increased TRH-induced release of prolactin, but not of thyrotropin. Similar data were obtained with either a transition-state or a tight binding inhibitor. These results demonstrate that PPII expression in lactotrophs coincides with its ability to control prolactin release. It may play a specialized role in TRH signaling in the anterior pituitary. Anterior pituitary ecto-peptidases may fulfill unique functions associated with their restricted cell-specific expression.

Combinatorial Multicomponent Access to Natural-Products-Inspired Peptidomimetics: Discovery of Selective Inhibitors of Microbial Metallo-aminopeptidases

The development of selective inhibitors of microbial metallo‐aminopeptidases is an important goal in the pursuit of antimicrobials for therapeutic applications. Herein, we disclose a combinatorial approach relying on two Ugi reactions for the generation of peptidomimetics inspired by natural metallo‐aminopeptidase inhibitors. The library was screened for inhibitory activity against the neutral metallo‐aminopeptidase of Escherichia coli (ePepN) and the porcine kidney cortex metallo‐aminopeptidase (pAPN), which was used as a model of the M1‐aminopeptidases of mammals. Six compounds showed typical dose–response inhibition profiles toward recombinant ePepN, with two of them being very potent and highly selective for ePepN over pAPN. Another compound showed moderate ePepN inhibition but total selectivity for this bacterial enzyme over its mammalian orthologue at concentrations of physiological relevance. This strategy proved to be useful for the identification of lead compounds for further optimization and development.

Effect of divalent cations on the porcine kidney cortex membrane-bound form of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV is an ectopeptidase with multiple physiological roles including the degradation of incretins, and a target of therapies for type 2 diabetes mellitus. Divalent cations can inhibit its activity, but there has been little effort to understand how they act. The intact membrane-bound form of porcine kidney dipeptidyl peptidase IV was purified by a simple and fast procedure. The purified enzyme hydrolyzed Gly-Pro-p-nitroanilide with an average V(max) of 1.397±0.003 μmol min(-1) mL(-1), k(cat) of 145.0±1.2 s(-1), K(M) of 0.138±0.005 mM and k(cat)/K(M) of 1050 mM(-1) s(-1). The enzyme was inhibited by bacitracin, tosyl-L-lysine chloromethyl ketone, and by the dipeptidyl peptidase IV family inhibitor L-threo-Ile-thiazolidide (K(i) 70 nM). The enzyme was inhibited by the divalent ions Ca(2+), Co(2+), Cd(2+), Hg(2+) and Zn(2+), following kinetic mechanisms of mixed inhibition, with K(i) values of 2.04×10(-1), 2.28×10(-2), 4.21×10(-4), 8.00×10(-5) and 2.95×10(-5) M, respectively. According to bioinformatic tools, Ca(2+) ions preferentially bound to the β-propeller domain of the porcine enzyme, while Zn(2+) ions to the α-β hydrolase domain; the binding sites were strikingly conserved in the human enzyme and other homologues. The functional characterization indicates that porcine and human homologues have very similar functional properties. Knowledge about the mechanisms of action of divalent cations may facilitate the design of new inhibitors.

Effect of zinc and calcium ions on the rat kidney membrane-bound form of dipeptidyl peptidase IV.

Dipeptidyl peptidase IV (DPP-IV) is an ectopeptidase with many roles, and a target of therapies for different pathologies. Zinc and calcium produce mixed inhibition of porcine DPP-IV activity. To investigate whether these results may be generalized to mammalian DPP-IV orthologues, we purified the intact membrane-bound form from rat kidney. Rat DPP-IV hydrolysed Gly-Pro-p-nitroanilide with an average Vmax of 0.86±0.01 μmol min–1mL–1 and KM of 76±6 μM. The enzyme was inhibited by the DPP-IV family inhibitor l-threo-Ile-thiazolidide (Ki=64.0±0.53 nM), competitively inhibited by bacitracin (Ki=0.16±0.01 mM) and bestatin (Ki=0.23±0.02 mM), and irreversibly inhibited by TLCK (IC50 value of 1.20±0.11 mM). The enzyme was also inhibited by divalent ions like Zn2+ and Ca2+, for which a mixed inhibition mechanism was observed (Ki values of the competitive component: 0.15±0.01 mM and 50.0±1.05 mM, respectively). According to bioinformatic tools, Ca2+ ions preferentially bound to the β-propeller domain of the rat and human enzymes, while Zn2+ ions to the α-β hydrolase domain; the binding sites were essentially the same that were previously reported for the porcine DPP-IV. These data suggest that the cationic susceptibility of mammalian DPP-IV orthologues involves conserved mechanisms.

Rational design and synthesis of affinity matrices based on proteases immobilized onto cellulose membranes

ABSTRACT Discovery of new protease inhibitors may result in potential therapeutic agents or useful biotechnological tools. Obtainment of these molecules from natural sources requires simple, economic, and highly efficient purification protocols. The aim of this work was the obtainment of affinity matrices by the covalent immobilization of dipeptidyl peptidase IV (DPP-IV) and papain onto cellulose membranes, previously activated with formyl (FCM) or glyoxyl groups (GCM). GCM showed the highest activation grade (10.2 µmol aldehyde/cm2). We implemented our strategy for the rational design of immobilized derivatives (RDID) to optimize the immobilization. pH 9.0 was the optimum for the immobilization through the terminal α-NH2, configuration predicted as catalytically competent. However, our data suggest that protein immobilization may occur via clusters of few reactive groups. DPP-IV−GCM showed the highest maximal immobilized protein load (2.1 µg/cm2), immobilization percentage (91%), and probability of multipoint covalent attachment. The four enzyme-support systems were able to bind at least 80% of the reversible competitive inhibitors bacitracin/cystatin, compared with the available active sites in the immobilized derivatives. Our results show the potentialities of the synthesized matrices for affinity purification of protease inhibitors and confirm the robustness of the RDID strategy to optimize protein immobilization processes with further practical applications.

KBE009: An antimalarial bestatin-like inhibitor of the Plasmodium falciparum M1 aminopeptidase discovered in an Ugi multicomponent reaction-derived peptidomimetic library

Malaria is a global human parasitic disease mainly caused by the protozoon Plasmodium falciparum. Increased parasite resistance to current drugs determines the relevance of finding new treatments against new targets. A novel target is the M1 alanyl-aminopeptidase from P. falciparum (PfA-M1), which is essential for parasite development in human erythrocytes and is inhibited by the pseudo-peptide bestatin. In this work, we used a combinatorial multicomponent approach to produce a library of peptidomimetics and screened it for the inhibition of recombinant PfA-M1 (rPfA-M1) and the in vitro growth of P. falciparum erythrocytic stages (3D7 and FcB1 strains). Dose-response studies with selected compounds allowed identifying the bestatin-based peptidomimetic KBE009 as a submicromolar rPfA-M1 inhibitor (Ki=0.4μM) and an in vitro antimalarial compound as potent as bestatin (IC50=18μM; without promoting erythrocyte lysis). At therapeutic-relevant concentrations, KBE009 is selective for rPfA-M1 over porcine APN (a model of these enzymes from mammals), and is not cytotoxic against HUVEC cells. Docking simulations indicate that this compound binds PfA-M1 without Zn2+ coordination, establishing mainly hydrophobic interactions and showing a remarkable shape complementarity with the active site of the enzyme. Moreover, KBE009 inhibits the M1-type aminopeptidase activity (Ala-7-amido-4-methylcoumarin substrate) in isolated live parasites with a potency similar to that of the antimalarial activity (IC50=82μM), strongly suggesting that the antimalarial effect is directly related to the inhibition of the endogenous PfA-M1. These results support the value of this multicomponent strategy to identify PfA-M1 inhibitors, and make KBE009 a promising hit for drug development against malaria.

Use of principal component analysis and molecular docking to identify novel selective Plasmepsin II non-competitive inhibitors with antimalarial activity.

Reference EPFL-CONF-214088doi:10.1002/pro.2823View record in Web of Science Record created on 2015-12-02, modified on 2017-05-12

Use of principal component analysis and molecular docking to identify novel selective plasmepsin II inhibitors.

We have selected the mediterranean shrub Euphorbia characias as an experimental model to study the complexity of plant latex chemistry. Latex is a mixture with diversified composition, that includes alkaloids, terpenoid compounds, polymeric substances such as resins and gums, starch, oil and a large number of proteins and enzymatic activities. The aim of the present study is to contribute to the knowledge of this plant product evaluating the antioxidant properties of extracts of E. characias and searching for polymeric substances as natural rubber. We analyzed different extracts from the latex of E. characias and performed a new extraction method (involving the use of trichloacetic acid, TCA) that turned out to be easier, faster and higher reproducible if compared to common extraction methods involving organic solvents like methanol, ethanol, and petroleum ether/methanol. TCA extract of E. characias latex exhibits antioxidant activities determined as total content of free-radical scavenging, polyphenols and total flavonoids. GC-MS analysis confirms the presence of several compound identified as antioxidant molecules. E. characias latex contains a natural rubber. The optimum rubber extraction is achieved with acetic acid followed by cyclohexane/ethanol treatment. The rubber content was shown to be 14% (w/v) of the E. characias latex and the gel content is 2.5% of the rubber weight. E. characias natural rubber showed a molecular weight of 93000 and Mw/Mn of 2.9. On the basis of H NMR, C NMR and FT-IR spectroscopy, the structure of this rubber can be identified as cis-1,4-polyisoprene. This study was partially supported by a grant from Regione Autonoma della Sardegna, Progetti di ricerca di base CRP2-22.Session 1—Maternal Medicine 1. 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ALVEOLAR O2 AND CO2 TENSIONS DURING PREGNANCY, MEASURED WITH A NOVEL NON-INVASIVE TECHNIQUE Litos M, Hadjistavrou C, Antsaklis A, Xygakis A, Jordanoglou J; Athens, Greece 14. VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) LEVELS IN DIABETIC PREGNANCY: RELATIONS TO NORMAL PREGNANCY AND GLYCAEMIC CONTROL Manderson JG, McClure N, Patterson CC, Hadden DR, Traub AI, McCance DR; Belfast, UK 15. HYPOTHYROIDISM IN PREGNANCY: INCREASE IN THYROXINE DOSE MAY BE TOO LATE USING OUR CURRENT PROTOCOL Masheshwari S, Singh A, Trinder J; Bristol, UK 16. PREGNANCY CARE IN OBESE WOMEN. AN OBSERVATIONAL STUDY IN A DISTRICT GENERAL HOSPITAL Nawar-Youssef MN, Iqbal F, Odukoya OA; Scunthorpe, UK 17. MANAGEMENT OF POST-PARTUM HYPERTENSION (PHT) Ogunnoiki O, Hirsi-Farah S, Gray G, Nelson-Piercy C; London, UK 18. STUDY ON SCREENING FOR GESTATIONAL DIABETES ParveenAS,PeploeD,Bell-ThomasS;Abergavenny,UK 19. 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