engleski

9-(2-Hydroxypropyl)purine analogs as novel fraudulent substrates of HSV1-Thymidine Kinase

Thymidine kinase (TK, EC 2.7.1.21) is a key enzyme in the pyrimidine salvage pathway catalyzing the gama-phosphate transfer from ATP to thymidine, in order to synthesize thymidine monophosphate (TMP). Herpes viruses encode their own thymidine kinases which differ considerably from the human host cell isoenzyme. In particular, viral TKs are able to phosphorylate a broad spectrum of pyrimidine and purine substrate analogues including those carrying an acyclic sugar moiety such as the antiviral drugs acyclovir (ACV) and ganciclovir (GCV). Those molecules act as fraudulent substrates blocking virus proliferation by dead end complexes with the viral DNA after being activated by the HSV-specific TK. The difference in substrate specificity between the human cellular and the herpesviral TK isoenzymes is the molecular basis of selective antiviral therapy. Furthermore, HSV 1 TK was recently used in combination with ACV or GCV as suicide enzyme in gene therapy of cancer and AIDS. Intensive efforts have been directed towards the search of new compounds with general antiviral activity. With this aim the acyclic analogs of purine nucleosides containing 2-hydroxypropyl aliphatic side chains attached to the N-9 position of adenine, 6-chloropurine, 2-amino-6-chloropurine and guanine have been prepared. The structures of all compounds have been established by means of one- and two-dimensional 1H and 13C NMR spectroscopy. These compounds have been submitted to theoretical study aiming to understand the interactions with HSV1-TK. The results suggested that 9-(2-hydroxypropyl)adenine (9-HPA), 9-(2-hydroxypropyl)-6-chloropurine (9-HPCP), 9-(2-hydroxypropyl)-2-amino-6-chloropurine (9-HPaCP) and 9-(2-hydroxypropyl)guanine (9-HPG) could possibly be fraudulent substrates of TK. To verify this prediction, kinetics as well as analytical studies have been undertaken. The results of the kinetic experiments show that the four compounds compete with the natural substrate thymidine for the binding site (Ki (9-HPA) = 5.3 mM, Ki (9-HPCP) = 2.15 mM , Ki (9-HPaCP) = 4.8 mM, Ki (9-HPG) = 0.95 mM). High performance liquid chromatography has been used to verify if the four compounds were inhibitors or substrates by monitoring the phosphorylation reaction. The results of this experiment indicate that TK dependent phosphorylation of all four compounds occurs. The results of this interdisciplinary study indicate that 9-(2-hydroxypropyl)purine analogs represent a new class of compounds acting as fraudulent substrates of HSV1 TK and may be a useful lead compound for structure-based drug design of more potent therapeutic compounds.

9-(2-Hydroxypropyl)purine analogs; HSV1-thymidine kinase; fraudulent substrates

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