RESUMO
The UL24 family of proteins is widely conserved among herpesviruses. We demonstrated previously that UL24 of herpes simplex virus 1 (HSV-1) is important for the dispersal of nucleolin from nucleolar foci throughout the nuclei of infected cells. Furthermore, the N-terminal portion of UL24 localizes to nuclei and can disperse nucleolin in the absence of any other viral proteins. In this study, we tested the hypothesis that highly conserved residues in UL24 are important for the ability of the protein to modify the nuclear distribution of nucleolin. We constructed a panel of substitution mutations in UL24 and tested their effects on nucleolin staining patterns. We found that modified UL24 proteins exhibited a range of subcellular distributions. Mutations associated with a wild-type localization pattern for UL24 correlated with high levels of nucleolin dispersal. Interestingly, mutations targeting two regions, namely, within the first homology domain and overlapping or near the previously identified PD-(D/E)XK endonuclease motif, caused the most altered UL24 localization pattern and the most drastic reduction in its ability to disperse nucleolin. Viral mutants corresponding to the substitutions G121A and E99A/K101A both exhibited a syncytial plaque phenotype at 39 degrees C. vUL24-E99A/K101A replicated to lower titers than did vUL24-G121A or KOS. Furthermore, the E99A/K101A mutation caused the greatest impairment of HSV-1-induced dispersal of nucleolin. Our results identified residues in UL24 that are critical for the ability of UL24 to alter nucleoli and further support the notion that the endonuclease motif is important for the function of UL24 during infection.
Assuntos
Sequência Conservada , Herpesvirus Humano 1/química , Fosfoproteínas/metabolismo , Proteínas de Ligação a RNA/metabolismo , Proteínas Virais/fisiologia , Motivos de Aminoácidos , Substituição de Aminoácidos , Humanos , Mutagênese Sítio-Dirigida , Proteínas Nucleares , Fosfoproteínas/análise , Proteínas de Ligação a RNA/análise , Proteínas Virais/química , Proteínas Virais/genética , NucleolinaRESUMO
A weak, UDP-competitive antagonist of the pyrimidinergic receptor P2RY(14) with a naphthoic acid core was identified through high-throughput screening. Optimization provided compounds with improved potency but poor pharmacokinetics. Acylglucuronidation was determined to be the major route of metabolism. Increasing the electron-withdrawing nature of the substituents markedly reduced glucuronidation and improved the pharmacokinetic profile. Additional optimization led to the identification of compound 38 which is an 8 nM UDP-competitive antagonist of P2Y(14) with a good pharmacokinetic profile.
Assuntos
Ácidos Carboxílicos/síntese química , Naftalenos/síntese química , Antagonistas do Receptor Purinérgico P2/síntese química , Receptores Purinérgicos P2 , Difosfato de Uridina , Animais , Ligação Competitiva , Ácidos Carboxílicos/química , Ácidos Carboxílicos/farmacocinética , Ácidos Carboxílicos/farmacologia , Camundongos , Estrutura Molecular , Naftalenos/química , Naftalenos/farmacocinética , Naftalenos/farmacologia , Pan troglodytes , Ligação Proteica/efeitos dos fármacos , Antagonistas do Receptor Purinérgico P2/química , Antagonistas do Receptor Purinérgico P2/farmacocinética , Antagonistas do Receptor Purinérgico P2/farmacologia , Receptores Purinérgicos P2Y , Relação Estrutura-AtividadeRESUMO
A weak antagonist of the pyrimidinergic receptor P2Y(14) containing a dihydropyridopyrimidine core was identified through high-throughput screening. Subsequent optimization led to potent, non-UTP competitive antagonists and represent the first reported non-nucleotide antagonists of this receptor. Compound 18q was identified as a 10 nM P2Y(14) antagonist with good oral bioavailability and provided sufficient exposure in mice to be used as a tool for future in vivo studies.
Assuntos
Antagonistas do Receptor Purinérgico P2/síntese química , Pirimidinas/síntese química , Receptores Purinérgicos P2/química , Administração Oral , Animais , Disponibilidade Biológica , Camundongos , Estrutura Molecular , Pan troglodytes , Antagonistas do Receptor Purinérgico P2/química , Pirimidinas/administração & dosagem , Pirimidinas/química , Receptores Purinérgicos P2Y , Relação Estrutura-AtividadeRESUMO
P2Y14 is a member of the pyrimidinergic GPCR family. UDP-Glc has been previously shown to activate human P2Y14, whereas UDP was unable to activate the receptor. In this study, the authors used conventional and nonconventional methods to further characterize P2Y14 and its ligands. Conventional calcium mobilization and nonconventional cellular impedance functional assays revealed that UMP and UDP selectively activated HEK cells coexpressing P2Y14 and Gα(qi5). In the impedance assays, the presence of exogenous Gα(qi5) resulted in agonist-induced Gq signaling, whereas in the absence of exogenous Gα(qi5), the signal was indicative of Gi. The authors established the first P2Y14 membrane filtration binding assay using a novel optimized expression vector and [(3)H]UDP as radioligand. UDP-Glc, UMP, and UDP dose dependently inhibited [(3)H]UDP binding in the binding assay, and saturation analysis revealed that UDP bound P2Y14 with a K(D) = 10 nM and a B(max) = 110 pmol/mg. The authors screened a phosphonate library and identified compound A, which inhibited UDP-Glc-mediated calcium signaling in the fluorometric imaging plate reader assay (IC(50) = 2.3 µM) and competed for [(3)H]UDP binding in the novel binding assay with a K(i) = 1280 nM.