In vitro and in vivo studies on UFP-112, a novel potent and long lasting agonist selective for the nociceptin/orphanin FQ receptor

[(pF)Phe(4)Aib(7)Arg(14)Lys(15)]N/OFQ-NH(2) (UFP-112) has been designed as a novel ligand for the nociceptin/orphanin FQ (N/OFQ) peptide receptor (NOP) by combining into the same peptide different chemical modifications reported to increase N/OFQ potency. In vitro data obtained in the electrically stimulated mouse vas deferens demonstrated that UFP-112 behaved as a high potency (pEC(50) 9.43) full agonist at the NOP receptor. UFP-112 effects were sensitive to the NOP antagonist UFP-101 but not to naloxone and no longer evident in tissues taken from NOP(-/-) mice. In vitro half life of UFP-112 in mouse plasma and brain homogenate was 2.6- and 3.5-fold higher than that of N/OFQ. In vivo, in the mouse tail withdrawal assay, UFP-112 (1-100pmol, i.c.v.) mimicked the actions of N/OFQ producing pronociceptive effects after i.c.v. administration and antinociceptive effects when given i.t.; in both cases, UFP-112 was approximately 100-fold more potent than the natural peptide and produced longer lasting effects. UFP-112 also mimicked the hyperphagic effect of N/OFQ producing a bell shaped dose response curve with the maximum reached at 10pmol. The hyperphagic effects of N/OFQ and UFP-112 were absent in NOP(-/-) mice. Equi-effective high doses of UFP-112 (0.1nmol) and N/OFQ (10nmol) were injected i.c.v. in mice and spontaneous locomotor activity recorded for 16h. N/OFQ produced a clear inhibitory effect which lasted for 60min while UFP-112 elicited longer lasting effects (>6h). In conscious rats, UFP-112 (0.1 and 10nmol/kg, i.v.) produced a marked and sustained decrease in heart rate, blood pressure, and urinary sodium excretion and a profound increase in urine flow. Collectively, these findings demonstrate that UFP-112 behaves in vitro and in vivo as a highly potent and selective ligand able to produce full and long lasting activation of NOP receptors.     

Adenosine Kinase of T. b. rhodesiense Identified as the Putative Target of 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine Using Chemical Proteomics

Background

Human African trypanosomiasis (HAT), a major parasitic disease spread in Africa, urgently needs novel targets and new efficacious chemotherapeutic agents. Recently, we discovered that 4-[5-(4-phenoxyphenyl)-2H-pyrazol-3-yl]morpholine (compound 1) exhibits specific antitrypanosomal activity with an IC₅₀ of 1.0 µM on Trypanosoma brucei rhodesiense (T. b. rhodesiense), the causative agent of the acute form of HAT.

Methodology/Principal Findings

In this work we show adenosine kinase of T. b. rhodesiense (TbrAK), a key enzyme of the parasite purine salvage pathway which is vital for parasite survival, to be the putative intracellular target of compound 1 using a chemical proteomics approach. This finding was confirmed by RNA interference experiments showing that down-regulation of adenosine kinase counteracts compound 1 activity. Further chemical validation demonstrated that compound 1 interacts specifically and tightly with TbrAK with nanomolar affinity, and in vitro activity measurements showed that compound 1 is an enhancer of TbrAK activity. The subsequent kinetic analysis provided strong evidence that the observed hyperactivation of TbrAK is due to the abolishment of the intrinsic substrate-inhibition.

Conclusions/Significance

The results suggest that TbrAK is the putative target of this compound, and that hyperactivation of TbrAK may represent a novel therapeutic strategy for the development of trypanocides.     

The crystal structure of PfFabZ, the unique beta-hydroxyacyl-ACP dehydratase involved in fatty acid biosynthesis of Plasmodium falciparum

The unique beta-hydroxyacyl-ACP dehydratase in Plasmodium falciparum, PfFabZ, is involved in fatty acid biosynthesis and catalyzes the dehydration of beta-hydroxy fatty acids linked to acyl carrier protein. The structure was solved by single anomalous dispersion (SAD) phasing using a quick-soaking experiment with potassium iodide and refined to a resolution of 2.1 A. The crystal structure represents the first structure of a Plasmodium beta-hydroxyacyl-ACP dehydratase with broad substrate specificity. The asymmetric unit contains a hexamer that appears as a trimer of dimers. Each dimer shows the known "hot dog" fold that has been observed in only a few other protein structures. Each of the two independent active sites in the dimer is formed by equal contributions from both subunits. The active site is mainly hydrophobic and looks like an L-shaped tunnel. The catalytically important amino acids His 133 and Glu 147' (from the other subunit), together with His98', form the only hydrophilic site in this tunnel. The inner end of the active site tunnel is closed by the phenyl ring of Phe 169, which is located in a flexible, partly visible loop. In order to explain the acceptance of substrates longer than ~C-7, the phenyl ring must move away to open the tunnel. The present structure supports an enzymatic mechanism consisting of an elimination reaction catalyzed by His 133 and Glu147'. 3-decynoyl-N-acetylcysteamine, an inhibitor known to interact with the E. coli dehydratase/isomerase, turned out to interact covalently with PfFabZ. A first model of PfFabZ with this potent inhibitor is presented.     

Pharmacological characterization of the nociceptin receptor which mediates reduction of alcohol drinking in rats

Chronic intracerebroventricular (ICV) treatment with nociceptin/orphanin FQ (NC), the endogenous ligand for the opioid receptor-like 1 (ORL1) receptor, reduces ethanol intake in alcohol-preferring rats and abolishes the rewarding properties of ethanol in the place conditioning paradigm. To pharmacologically characterize the receptor involved, the present study evaluated the effect on ethanol drinking in genetically selected Marchigian Sardinian alcohol-preferring (msP) rats of ICV injections for 8 days of NC or of the NC analogs NC(1-17)NH(2), NC(1-13)NH(2), NC(1-12)NH(2) and [Nphe(1)]NC(1-13)NH(2). In vitro studies indicate that NC, NC(1-17)NH(2), NC(1-13)NH(2) and NC(1-12)NH(2) are agonists, while [Nphe(1)]NC(1-13)NH(2) is a selective antagonist at the ORL1 receptor. Freely feeding and drinking rats were offered 10% ethanol 30 min/day at the beginning of the dark phase of the light cycle. NC significantly attenuated ethanol intake at 500 or 1000 ng/rat (210 or 420 pmol/rat). NC(1-17)NH(2), markedly reduced ethanol intake, but its effect was statistically significant at 1000 (420 pmol/rat), not at 500 ng/rat (210 pmol/rat). After the end of treatment ethanol drinking promptly came back to baseline level. Ethanol consumption was also reduced by NC(1-13)NH(2); however, its effect was less potent and pronounced. NC(1-12)NH(2) did not modify ethanol intake at doses up to 4000 ng/rat (2339 pmol/rat). Water and food consumption were not modified. Treatment with [Nphe(1)]NC(1-13)NH(2), 66 or 99 microg/rat, did not modify ethanol intake; however, [Nphe(1)]NC(1-13)NH(2), 66 microg/rat, given just before 1000 ng/rat of NC(1-17)NH(2), abolished the effect of the agonist. The present results show that the 13 amino acid N-terminal sequence of NC is essential for the effect on ethanol intake and indicate that [Nphe(1)]NC(1-13)NH(2) acts as an antagonist to block the effect of NC. These findings provide further evidence that selective agonists at the ORL-1 receptor attenuate ethanol intake in alcohol-preferring rats and suggest that the NC/ORL1 system may represent an interesting target for treatment of alcohol abuse.     

Impact of the isoelectric point of model parvoviruses on viral retention in anion‐exchange chromatography

Anion‐exchange chromatography (AEX) is used in the downstream purification of monoclonal antibodies to remove impurities and potential viral contamination based on electrostatic interactions. Although the isoelectric point (pI) of viruses is considered a key factor predicting the virus adsorption to the resin, the precise molecular mechanisms involved remain unclear. To address this question, we compared structurally homologous parvoviruses that only differ in their surface charge distribution. A single charged amino acid substitution on the capsid surface of minute virus of mice (MVM) provoked an increased apparent pI (pI_app) 6.2 compared to wild‐type MVM (pI_app = 4.5), as determined by chromatofocusing. Despite their radically different pI_app, both viruses displayed the same interaction profile in Mono Q AEX at different pH conditions. In contrast, the closely related canine parvovirus (pI_app = 5.3) displayed a significantly different interaction at pH 5. The detailed structural analysis of the intricate three‐dimensional structure of the capsids suggests that the charge distribution is critical, and more relevant than the pI, in controlling the interaction of a virus with the chromatographic resin. This study contributes to a better understanding of the molecular mechanisms governing virus clearance by AEX, which is crucial to enable robust process design and maximize safety.