Human P2Y 14 Receptor Agonists: Truncation of the Hexose Moiety of Uridine-5′-Diphosphoglucose and Its Replacement with Alkyl and Aryl Groups

Uridine-5′-diphosphoglucose (UDPG) activates the P2Y14 receptor, a neuroimmune system GPCR. P2Y14 receptor tolerates glucose substitution with small alkyl or aryl groups or its truncation to uridine-5′-diphosphate (UDP), a full agonist human P2Y14 receptor expressed in HEK-293 cells. 2-Thiouracil derivatives displayed selectivity for activation of the human P2Y14 vs. the P2Y6 receptor, such as 2-thio-UDP 4 (EC50 1.92 nM at P2Y14, 224-fold selectivity vs. P2Y6) and its β-propyloxy ester 18. EC50 of β-methyl ester of UDP and its 2-thio analogue were 2730 and 56 nM, respectively. β-t-Butyl ester of 4 was 11-fold more potent than UDPG, but β-aryloxy or larger, branched β-alkyl esters, such as cyclohexyl, were less potent. Ribose replacement of UDP with a rigid North or South methanocarba (bicyclo[3.1.0]hexane) group abolished P2Y14 receptor agonist activity. α,β-Methylene and difluoromethylene groups were well tolerated at the P2Y14 receptor and are expected to provide enhanced stability in biological systems. α,β-Methylene-2-thio-UDP 11 (EC50 0.92 nM) was 2160-fold selective versus P2Y6. Thus, these nucleotides and their congeners may serve as important pharmacological probes for the detection and characterization of the P2Y14 receptor

Structure−Activity Relationship of Uridine 5‘ - Diphosphoglucose Analogues as Agonists of the Human P2Y 14 Receptor

UDP-glucose (UDPG) and derivatives are naturally-occurring agonists of the Gi protein-coupled P2Y14 receptor, which occurs in the immune system. We synthesized and characterized pharmacologically novel analogues of UDPG modified on the nucleobase, ribose, and glucose moieties, as the basis for designing novel ligands in conjunction with modeling. The recombinant human P2Y14 receptor expressed in COS-7 cells was coupled to phospholipase C through an engineered Gα-q/i protein. Most modifications of the uracil or ribose moieties abolished activity; this is among the least permissive P2Y receptors. However, a 2-thiouracil modification in 15 (EC50 49 ± 2 nM) enhanced the potency of UDPG (but not UDP-glucuronic acid) by 7-fold. 4-Thio analogue 13 was equipotent to UDPG, but S-alkylation was detrimental. Compound 15 was docked in a rhodposin-based receptor homology model, which correctly predicted potent agonism of UDP-fructose, UDP-mannose, and UDP-inositol. The hexose moiety of UDPG interacts with multiple H-bonding and charged resides and provides a fertile region for agonist modification

Epidermal growth factor receptor mutations and anaplastic lymphoma kinase rearrangements in lung cancer with nodular ground-glass opacity

Vår uppsats handlar om formativ bedömning och vad det innebär att arbeta så i undervisningen. Vi tolkar att rådande läroplaner beskriver ett uppdrag som ligger i linje med ett formativt arbetssätt. Syftet var att ta reda på om lärare i samhällskunskap som arbetar på gymnasiet arbetar formativt och hur de gör det. Ett annat vanligt namn för formativ bedömning är bedömning för lärande (BFL). Genom kvalitativa intervjuer har vi frågat gymnasielärare i samhällskunskap hur de arbetar med bedömning, om de arbetar formativt och om det finns någon samsyn på deras respektive gymnasieskolor kring bedömning. Vi ville också veta vad formativt arbete innebär. Bedömning för lärande fokuserar på lärandet och hur eleven kan utvecklas och nå framgång i lärandet. Genom att konkretisera mål, syfte och kunskapskvaliteter så att eleven förstår dessa kan eleven få syn på sitt eget lärande. När eleven ges makt över sitt eget lärande leder det till ökad motivation. I formativ bedömning arbetar läraren bland annat med feedback och kamrat- och självvärdering. Vårt resultat visar att lärarna vi intervjuat i stor utsträckning arbetar summativt med formativa inslag i varierande grad, förutom en av lärarna som arbetar på en gymnasieskola i Borås som arbetar enligt bedömning för lärande. Studien visar att det råder brist på samsyn kring bedömning på gymnasie-skolorna, förutom på skolan i Borås där det finns tid avsatt för möten och samtal kring formativ bedömning. Litteraturen visar på de positiva effekterna av bedömning för lärande vilket också styrks av intervjun med läraren i Borås

Fabrication of uniform layer-by-layer assemblies with complementary protein cage nanobuilding blocks via simple His-tag/metal recognition

A capsid-forming enzyme, lumazine synthase isolated from hyperthermophile Aquifex aeolicus (AaLS), is prepared and utilized as a template for constructing nanobuilding blocks to fabricate uniform layer-by-layer (LbL) assemblies. Two functionally complementary AaLS protein cage nanoparticles (PCNs) are generated either by genetically introducing His-tags on the surface of wild-type AaLS PCNs or by chemically attaching metal chelates (Ni-NTA moiety) to the surface of cysteine-bearing AaLS PCNs individually. The multivalent displays of His-tags (AaLS-His6 PCN) and Ni-NTA ligands (AaLS-NTA-Ni PCN) on the surface of each complementary AaLS PCN are successfully demonstrated by mass spectrometric and surface plasmon resonance analyses. By using these two complementary AaLS PCNs, uniform LbL assemblies are constructed via simple recognition between His-tags and metal chelates without the aid of additional binding mediators. This approach illustrates the potential of fabricating uniform nanostructures using protein-based hybrid functional nanobuilding blocks.close3

Structure−Activity Relationships of Uridine 5‘-Diphosphate Analogues at the Human P2Y 6 Receptor

The structure activity relationships and molecular modeling of the uracil nucleotide-activated P2Y6 receptor have been studied. A series of UDP analogues bearing substitutions of the ribose moiety, the uracil ring, and the diphosphate group was synthesized and assayed for activity at the human P2Y6 receptor. The uracil ring was modified at the 4-position, with the synthesis of 4-substituted-thiouridine-5′-diphosphate analogues, as well as at positions 3 and 5. The effect of modifications at the level of the phosphate chain was studied by preparing a cyclic 3′,5′-diphosphate analogue, a 3′-diphosphate analogue and several dinucleotide diphosphates. 5-Iodo-UDP 32 (EC50 0.15 μM) was equipotent to UDP, while substitutions of the 2′-hydroxyl (amino, azido) greatly reduce potency. 2- and 4-Thio analogues, 20 and 21, respectively, were also relatively potent in comparison to UDP. However, most other modifications greatly reduced potency. Molecular modeling indicates that the β-phosphate of 5′-UDP and analogs is essential for the establishment of electrostatic interactions with two of the three conserved cationic residues of the receptor. Among 4-thioether derivatives, a 4-ethylthio analogue 23 displayed an EC50 of 0.28 μM, indicative of favorable interactions predicted for a small 4-alkylthio moiety with the aromatic ring of Y33 in TM1. The activity of analogue 19 in which the ribose was substituted with a 2-oxabicyclohexane ring in a rigid (S) conformation (P= 126°, 1′-exo) was consistent with molecular modeling. These results provide a better understanding of molecular recognition at the P2Y6 receptor and will be helpful in designing selective and potent P2Y6 receptor ligand

Molecular Modeling of the Human P2Y2 Receptor and Design of a Selective Agonist, 2′-Amino-2′-deoxy-2-thio-UTP

A rhodopsin-based homology model of the nucleotide-activated human P2Y2 receptor, including loops, termini, and phospholipids, was optimized with Monte Carlo Multiple Minimum. Docked UTP formed a nucleobase π–π complex with conserved Phe3.32. Selectivity-enhancing 2′-amino-2′-deoxy substitution interacted through π-hydrogen bonding with aromatic Phe6.51 and Tyr3.33. A “sequential ligand composition” approach for docking the flexible dinucleotide agonist Up4U demonstrated a shift of conserved cationic Arg3.29 from the UTP γ position to δ position of Up4U and Up4ribose. Sysnthesized nucleotides were tested as agonists at human P2Y receptors expressed in 1321N1 astrocytoma cells. 2′-Amino and 2-thio modifications synergized to enhance potency and selectivity; compound 8 (8 nM EC50) was 300-fold P2Y2-selective versus P2Y4. 2′-Amine acetylation reduced potency, and trifluoroacetylation produced intermediate potency. 5-Amino nucleobase substitution did not enhance potency through a predicted hydrophilic interaction, possibly because of destabilization of the receptor-favored (N)-ribose conformation. This detailed view of P2Y2 receptor recognition suggests mutations for model validation

Quantification of G i -Mediated Inhibition of Adenylyl Cyclase Activity Reveals That UDP Is a Potent Agonist of the Human P2Y 14 Receptor

ABSTRACT The P2Y 14 receptor was initially identified as a G protein-coupled receptor activated by UDP-glucose and other nucleotide sugars. We have developed several cell lines that stably express the human P2Y 14 receptor, allowing facile examination of its coupling to native G i family G proteins and their associated downstream signaling pathways (J Pharmacol Exp Ther 330: [162][163][164][165][166][167][168] 2009). In the current study, we examined P2Y 14 receptor-dependent inhibition of cyclic AMP accumulation in human embryonic kidney (HEK) 293, C6 glioma, and Chinese hamster ovary (CHO) cells stably expressing this receptor. Not only was the human P2Y 14 receptor activated by UDP-glucose, but it also was activated by UDP. The apparent efficacies of UDP and UDP-glucose were similar, and the EC 50 values (74, 33, and 29 nM) for UDP-dependent activation of the P2Y 14 receptor in HEK293, CHO, and C6 glioma cells, respectively, were similar to the EC 50 values (323, 132, and 72 nM) observed for UDP-glucose. UDP and UDP-glucose also stimulated extracellular signal-regulated kinase (ERK) 1/2 phosphorylation in P2Y 14 receptor-expressing HEK293 cells but not in wild-type HEK293 cells. A series of analogs of UDP were potent P2Y 14 receptor agonists, but the naturally occurring nucleoside diphosphates, CDP, GDP, and ADP exhibited agonist potencies over 100-fold less than that observed with UDP. Two UDP analogs were identified that selectively activate the P2Y 14 receptor over the UDP-activated P2Y 6 receptor, and these molecules stimulated phosphorylation of ERK1/2 in differentiated human HL-60 promyeloleukemia cells, which natively express the P2Y 14 receptor but had no effect in wild-type HL-60 cells, which do not express the receptor. We conclude that UDP is an important cognate agonist of the human P2Y 14 receptor. The metabotropic P2Y receptors include a subgroup of five receptors, the P2Y 1 , P2Y 2 , P2Y 4 , P2Y 6 , and P2Y 11 receptors, that primarily signal through G q -activated signaling pathways and a subgroup of three receptors, the P2Y 12 , P2Y 13 , and P2Y 14 receptors, that primarily signal by activating heterotrimeric G proteins of the G i famil

Pyrimidine Ribonucleotides with Enhanced Selectivity as P2Y 6 Receptor Agonists: Novel 4-Alkyloxyimino, (S)-Methanocarba, and 5′-Triphosphate γ-Ester Modifications †

The P2Y6 receptor is a cytoprotective G protein-coupled receptor (GPCR) activated by UDP (EC50, 0.30 μM). We compared and combined modifications to enhance P2Y6 receptor agonist selectivity, including ribose ring constraint, 5-iodo and 4-alkyloxyimino modifications, and phosphate modifications such as α,β-methylene and extension of the terminal phosphate group into γ-esters of UTP analogues. The conformationally constrained (S)-methanocarba UDP is a full agonist (EC50 0.042 μM). 4-Methoxyimino modification of pyrimidine enhanced P2Y6, preserved P2Y2 and P2Y4, and abolished P2Y14 receptor potency, in the appropriate nucleotide. N4-Benzyloxy-CDP (15, MRS2964) and N4-methoxy-Cp3U (23, MRS2957) were potent, selective P2Y6 receptor agonists (EC50 0.026 μM and 0.012 μM, respectively). A hydrophobic binding region near the nucleobase was explored with receptor modeling and docking. UTP-γ-aryl and cycloalkyl phosphoesters displayed only intermediate P2Y6 receptor potency, but had enhanced stability in acid and cell membranes. UTP-glucose was inactive, but its (S)-methanocarba analogue and N4-methoxy-cytidine 5′-triphospho-γ-[1]glucose were active (EC50 of 2.47 μM and 0.18 μM, respectively). Thus, the potency, selectivity, and stability of pyrimidine nucleotides as P2Y6 receptor agonists may be enhanced by modest structural changes

Synthesis and potency of novel uracil nucleotides and derivatives as P2Y2 and P2Y6 receptor agonists

The phosphate, uracil, and ribose moieties of uracil nucleotides were varied structurally for evaluation of agonist activity at the human P2Y2, P2Y4, and P2Y6 receptors. The 2-thio modification, found previously to enhance P2Y2 receptor potency, could be combined with other favorable modifications to produce novel molecules that exhibit high potencies and receptor selectivities. Phosphonomethylene bridges introduced for stability in analogues of UDP, UTP and uracil dinucleotides markedly reduced potency. Truncation of dinucleotide agonists of the P2Y2 receptor, in the form of Up4-sugars, indicated that a terminal uracil ring is not essential for moderate potency at this receptor and that specific SAR patterns are observed at this distal end of the molecule. Key compounds reported in this study include: 9, α,β-methylene-UDP, a P2Y6 receptor agonist; 30, Up4-phenyl ester and 34, Up4-[1]glucose, selective P2Y2 receptor agonists; 43, the 2-thio analogue of INS37217 (P1-(uridine 5′)-P4- (2′-deoxycytidine 5′) tetraphosphate), a potent and selective P2Y2 receptor agonist