RESUMEN
Rotavirus infection remains a great health burden worldwide especially in some developing countries. It causes severe dehydrating diarrhea in infants, young children, as well as immunocompromised and organ transplanted patients. Viral replication heavily relies on the host to supply nucleosides. Thus, host enzymes involved in nucleotide biosynthesis represent potential targets for antiviral development. Dihydroorotate dehydrogenase (DHODH) is the rate-limiting enzyme in the de novo biosynthesis pathway of pyrimidines. In this study, we demonstrated that two specific DHODH enzyme inhibitors, brequinar (BQR) and leflunomide (LFM) robustly inhibited rotavirus replication in conventional human intestinal Caco2 cell line as well as in human primary intestinal organoids. The antiviral effect is conserved in both laboratory strain SA11 and rotavirus strain 2011K isolated from clinical sample. Mechanistic study indicated that BQR and LFM exerted their anti-rotavirus effect through targeting DHODH to deplete pyrimidine nucleotide pool. Therefore, targeting pyrimidine biosynthesis represents a potential approach for developing antiviral strategies against rotavirus.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Nucleósidos de Pirimidina , Rotavirus/efectos de los fármacos , Antivirales/metabolismo , Antivirales/farmacología , Compuestos de Bifenilo/metabolismo , Compuestos de Bifenilo/farmacología , Células CACO-2/enzimología , Células CACO-2/virología , Dihidroorotato Deshidrogenasa , Desarrollo de Medicamentos , Humanos , Leflunamida/metabolismo , Leflunamida/farmacología , Cultivo Primario de Células , Nucleósidos de Pirimidina/antagonistas & inhibidores , Nucleósidos de Pirimidina/biosíntesis , Rotavirus/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Recently we reported that the pyridinylimidazole class of p38 mitogen-activated protein (MAP) kinase inhibitors potently inhibited the facilitated transport of nucleosides and nucleoside analogs in K562 cells. These compounds competed with the binding of nitrobenzylthioinosine (NBMPR) to K562 cells, consistent with inhibition of the NBMPR-sensitive equilibrative transporter (ENT1). In this study we examined a large number of additional protein kinase inhibitors for their effects on nucleoside transport. We find that incubation of K562 cells with tyrosine kinase inhibitors (AG825, AG1517, AG1478, STI-571), protein kinase C (PKC) inhibitors (staurosporine, GF 109203X, R0 31-8220, arcyriarubin A), cyclin-dependent kinase inhibitors (roscovitine, olomoucine, indirubin-3'-monoxime), or rapamycin resulted in a dose-dependent reduction of intracellular uptake of [3H]uridine. In contrast, neither the MAP kinase kinase inhibitors (U0126, PD 98059) nor the phosphatidyl inositol-3 kinase inhibitors (wortmannin, LY 294002) affected this process. Furthermore, both transient uptake and prolonged [3H]thymidine incorporation in K562 cells were inhibited by protein kinase inhibitors, inactive analogs of kinase inhibitors (R0 31-6045, SB202474), and NBMPR, independently of effects on cell proliferation as determined by MTT assay. These studies demonstrate that a wide variety of protein kinase inhibitors affect nucleoside uptake through selective inhibition of nucleoside transporters, independently of kinase inhibition.
Asunto(s)
Proteínas Portadoras/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular , Nucleósidos de Pirimidina/antagonistas & inhibidores , Nucleósidos de Pirimidina/metabolismo , Transporte Biológico/efectos de los fármacos , Transporte Biológico/fisiología , Proteínas Portadoras/química , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Células K562 , Timidina/antagonistas & inhibidores , Timidina/metabolismo , Uridina/antagonistas & inhibidores , Uridina/metabolismoRESUMEN
We have recently reported the discovery of an entirely new category of potent antivaricella-zoster virus agents based on novel deoxynucleoside analogues bearing unusual fluorescent bicyclic furo base moieties. Initial studies revealed an absolute requirement of a long alkyl side-chain, with an optimal length of C8-C10, for antiviral activity. However, the impact of this requirement on the physical properties of these compounds is high: inherent lipophilicity and extremely poor aqueous solubility, which may limit the use of these nucleosides as drugs. In order to address this issue, we have now prepared a new series of analogues, bearing ether and glycol type side-chains, designed to improve the aqueous solubility of the compounds. Synthesis of target nucleosides involved Pd-catalysed coupling of terminal alkynes with 5-iodo-2'-deoxyuridine. The 5-alkynyl nucleosides thus obtained were then treated with copper (I) iodide to produce the desired bicyclic analogues. As anticipated, the new compounds exhibited a dramatic increase in aqueous solubility, although antiviral activity was significantly reduced. A possible correlation between antiviral activity and overall compound lipophilicity is discussed.
Asunto(s)
Antivirales/síntesis química , Antivirales/farmacología , Herpesvirus Humano 3/efectos de los fármacos , Nucleósidos de Pirimidina/antagonistas & inhibidores , Nucleósidos de Pirimidina/síntesis química , HumanosRESUMEN
Inhibition of DNA synthesis by ara-sangivamycin was antagonized by adenosine. The 50% inhibitory concentrations increased 1.6- to 32-fold in the presence of 1.0 to 50 microM adenosine, respectively. In contrast, the inhibition of human cytomegalovirus replication by ara-sangivamycin was not antagonized by as much as 50 microM adenosine. This suggests that different enzymes were responsible for the phosphorylation of ara-sangivamycin in uninfected and infected cells.
Asunto(s)
Adenosina/farmacología , Antivirales/antagonistas & inhibidores , Supervivencia Celular/efectos de los fármacos , Nucleósidos de Pirimidina/antagonistas & inhibidores , Adenosina Quinasa/antagonistas & inhibidores , Células Cultivadas , Citomegalovirus/efectos de los fármacos , ADN/biosíntesis , Humanos , Células KB/efectos de los fármacos , Cinética , Nucleósidos de Pirimidina/farmacología , Timidina/metabolismo , Ensayo de Placa ViralRESUMEN
The synthesis of 5-(azidomethyl)-2'-deoxyuridine (10) has been accomplished by two independent methods. The first involved tosylation of 5-(hydroxymethyl)-2'-deoxyuridine (1) to furnish a mixture of two mono- and a ditosyl nucleosides which were converted into the corresponding 5-(azidomethyl) (10), 5-(azidomethyl)-5'-azido (14), and 5-(hydroxymethyl)-5'-azido (15) derivatives of 2'-deoxyuridine. The second method was more selective and required the formation of the intermediate 5-(bromomethyl)-3',5'-di-O-acetyl-2'-deoxyuridine (8), followed by displacement of the bromo group by lithium azide and deacetylation. Catalytic hydrogenation of the azides 9, 10, 14, and 15 gave the corresponding amines 16, 2, 6, and 7, respectively. Compounds 1, 2, 10, and 16 inhibited the growth of murine Sarcoma 180 and L1210 in culture, and the activity of 2 was prevented by 2'-deoxypyrimidine nucleosides but not by purine nucleosides. The replication of herpes simplex virus type 1 (HSV-1) was strongly inhibited only by 1 and 10. Studies on the binding of the various thymidine analogues to HSV-1 encoded pyrimidine deoxyribonucleoside kinase indicate that 1 and 10 have good affinity for the enzyme.