Pyrimidine Nucleotides with 4-Alkyloxyimino and Terminal Tetraphosphate δ-Ester Modifications as Selective Agonists of the P2Y 4 Receptor

P2Y2 and P2Y4 receptors are G protein-coupled receptors, activated by UTP and dinucleoside tetraphosphates, which are difficult to distinguish pharmacologically for lack of potent and selective ligands. We varied structurally phosphate and uracil moieties in analogues of pyrimidine nucleoside 5′-triphosphates and 5′-tetraphosphate esters. P2Y4 receptor potency in phospholipase C stimulation in transfected 1321N1 human astrocytoma cells was enhanced in N4-alkyloxycytidine derivatives. OH groups on a terminal δ-glucose phosphoester of uridine 5′-tetraphosphate were inverted or substituted with H or F to probe H-bonding effects. N4-(Phenylpropoxy)-CTP 16 (MRS4062), Up4-[1]3′-deoxy-3′-fluoroglucose 34 (MRS2927) and N4-(phenylethoxy)-CTP 15 exhibit ≥10-fold selectivity for human P2Y4 over P2Y2 and P2Y6 receptors (EC50 values 23, 62 and 73 nM, respectively). δ-3-Chlorophenyl phosphoester 21 of Up4 activated P2Y2 but not P2Y4 receptor. Selected nucleotides tested for chemical and enzymatic stability were much more stable than UTP. Agonist docking at CXCR4-based P2Y2 and P2Y4 receptor models indicated greater steric tolerance of N4-phenylpropoxy group at P2Y4. Thus, distal structural changes modulate potency, selectivity, and stability of extended uridine tetraphosphate derivatives, and we report the first P2Y4 receptor-selective agonists

Functionalized Congeners of P2Y 1 Receptor Antagonists: 2-Alkynyl ( N )-Methanocarba 2′-Deoxyadenosine 3′,5′-Bisphosphate Analogues and Conjugation to a Polyamidoamine (PAMAM) Dendrimer Carrier

The P2Y1 receptor is a prothrombotic G protein-coupled receptor (GPCR) activated by ADP. Preference for the North (N) ring conformation of the ribose moiety of adenine nucleotide 3′,5′-bisphosphate antagonists of the P2Y1 receptor was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute. A series of covalently linkable N6-methyl-(N)-methanocarba-2′-deoxyadenosine-3′,5′-bisphosphates containing extended 2-alkynyl chains was designed and binding affinity at the human (h) P2Y1 receptor determined. The chain of these functionalized congeners contained hydrophilic moieties, a reactive substituent, or biotin, linked via an amide. Variation of the chain length and position of an intermediate amide group revealed high affinity of carboxylic congener 8 (Ki 23 nM) and extended amine congener 15 (Ki 132 nM), both having a 2-(1-pentynoyl) group. A biotin conjugate 18 containing an extended ε-aminocaproyl spacer chain exhibited higher affinity than a shorter biotinylated analogue. Alternatively, click coupling of terminal alkynes of homologous 2-dialkynyl nucleotide derivatives to alkyl azido groups produced triazole derivatives that bound to the P2Y1 receptor following deprotection of the bisphosphate groups. The preservation of receptor affinity of the functionalized congeners was consistent with new P2Y1 receptor modeling and ligand docking. Attempted P2Y1 antagonist conjugation to PAMAM dendrimer carriers by amide formation or palladium-catalyzed reaction between an alkyne on the dendrimer and a 2-iodopurine-derivatized nucleotide was unsuccessful. A dialkynyl intermediate containing the chain length favored in receptor binding was conjugated to an azide-derivatized dendrimer, and the conjugate inhibited ADP-promoted human platelet aggregation. This is the first example of attaching a strategically functionalized P2Y receptor antagonist to a PAMAM dendrimer to produce a multivalent conjugate exhibiting a desired biological effect, i.e. antithrombotic action

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

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities

Survey of Period Variations of Superhumps in SU UMa-Type Dwarf Novae

We systematically surveyed period variations of superhumps in SU UMa-type dwarf novae based on newly obtained data and past publications. In many systems, the evolution of superhump period are found to be composed of three distinct stages: early evolutionary stage with a longer superhump period, middle stage with systematically varying periods, final stage with a shorter, stable superhump period. During the middle stage, many systems with superhump periods less than 0.08 d show positive period derivatives. Contrary to the earlier claim, we found no clear evidence for variation of period derivatives between superoutburst of the same object. We present an interpretation that the lengthening of the superhump period is a result of outward propagation of the eccentricity wave and is limited by the radius near the tidal truncation. We interpret that late stage superhumps are rejuvenized excitation of 3:1 resonance when the superhumps in the outer disk is effectively quenched. Many of WZ Sge-type dwarf novae showed long-enduring superhumps during the post-superoutburst stage having periods longer than those during the main superoutburst. The period derivatives in WZ Sge-type dwarf novae are found to be strongly correlated with the fractional superhump excess, or consequently, mass ratio. WZ Sge-type dwarf novae with a long-lasting rebrightening or with multiple rebrightenings tend to have smaller period derivatives and are excellent candidate for the systems around or after the period minimum of evolution of cataclysmic variables (abridged).Comment: 239 pages, 225 figures, PASJ accepte