Rega Institute for Medical Research, KU Leuven, Leuven, Belgium (L.N., J.V.V., J.B.); School of Chemistry and Molecular Biosciences, University of Queensland, Brisbane, Queensland, Australia (L.W.G., D.T.K.); and Laboratory of Genetic Metabolic Diseases, Academic Medical Center, Amsterdam, The Netherlands (A.B.P.v.K., J.M.).
Mol Pharmacol. 2013 Oct;84(4):615-29. doi: 10.1124/mol.113.087247. Epub 2013 Aug 1.
6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and diverse RNA viruses. Its active metabolite, T-705-ribose-5'-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-5'-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide; the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution. Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having Km(app) values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.
6-氟-3-羟基-2-吡嗪甲酰胺（T-705）是一种新型的抗病毒化合物，对流感病毒和多种 RNA 病毒具有广泛的活性。其活性代谢物 T-705-核糖-5′-三磷酸（T-705-RTP）被流感病毒 RNA 聚合酶识别为与 GTP 竞争的底物，从而抑制病毒 RNA 的合成和致命的病毒诱变。哪些酶参与 T-705 的激活尚不清楚。我们在此证明，人次黄嘌呤鸟嘌呤磷酸核糖转移酶（HGPRT）将 T-705 转化为其核糖-5′-单磷酸（RMP），然后再形成 T-705-RTP。在 HGPRT 缺陷型 Madin-Darby 犬肾细胞中，T-705 和 T-1105（3-羟基-2-吡嗪甲酰胺；缺少 6-氟原子的类似物）的抗流感病毒活性丧失。在人胚肾 293T 细胞中进行 HGPRT 特异性基因敲低，然后再进行流感病毒核糖核蛋白重建，证实了这种 HGPRT 依赖性。腺嘌呤磷酸核糖转移酶（APRT）或烟酰胺磷酸核糖转移酶的敲低不会改变 T-705 和 T-1105 的抗病毒活性。酶促测定表明，T-705 和 T-1105 是人 HGPRT 的不良底物，其 Km(app) 值分别为 6.4 和 4.1 mM。APRT 形成 RMP 代谢物的量可以忽略不计，而人嘌呤核苷磷酸化酶形成核糖基代谢物的量也可以忽略不计。2-吡嗪甲酰胺衍生物的磷酸核糖化和抗病毒活性需要 3-羟基的存在，但不需要 6-氟取代基。T-705-RMP 与人 HGPRT 复合物的晶体结构显示了该化合物如何结合在活性部位。由于 HGPRT 似乎不能有效地转化 T-705，因此可以设计 T-705-RMP 前药来提高这种新型抗病毒药物的抗病毒效力。
