Gentry Brian G, Phan Quang, Hall Ellie D, Breitenbach Julie M, Borysko Katherine Z, Kamil Jeremy P, Townsend Leroy B, Drach John C
Department of Pharmaceutical, Biomedical and Administrative Sciences, College of Pharmacy and Health Sciences, Drake University, Des Moines, Iowa, USA
Department of Pharmaceutical, Biomedical and Administrative Sciences, College of Pharmacy and Health Sciences, Drake University, Des Moines, Iowa, USA.
Antimicrob Agents Chemother. 2015 Jan;59(1):226-32. doi: 10.1128/AAC.03686-14. Epub 2014 Oct 27.
Human cytomegalovirus (HCMV) infection can cause severe illnesses, including encephalopathy and mental retardation, in immunocompromised and immunologically immature patients. Current pharmacotherapies for treating systemic HCMV infections include ganciclovir, cidofovir, and foscarnet. However, long-term administration of these agents can result in serious adverse effects (myelosuppression and/or nephrotoxicity) and the development of viral strains with reduced susceptibility to drugs. The deoxyribosylindole (indole) nucleosides demonstrate a 20-fold greater activity in vitro (the drug concentration at which 50% of the number of plaques was reduced with the presence of drug compared to the number in the absence of drug [EC50] = 0.34 μM) than ganciclovir (EC50 = 7.4 μM) without any observed increase in cytotoxicity. Based on structural similarity to the benzimidazole nucleosides, we hypothesize that the indole nucleosides target the HCMV terminase, an enzyme responsible for packaging viral DNA into capsids and cleaving the DNA into genome-length units. To test this hypothesis, an indole nucleoside-resistant HCMV strain was isolated, the open reading frames of the genes that encode the viral terminase were sequenced, and a G766C mutation in exon 1 of UL89 was identified; this mutation resulted in an E256Q change in the amino acid sequence of the corresponding protein. An HCMV wild-type strain, engineered with this mutation to confirm resistance, demonstrated an 18-fold decrease in susceptibility to the indole nucleosides (EC50 = 3.1 ± 0.7 μM) compared to that of wild-type virus (EC50 = 0.17 ± 0.04 μM). Interestingly, this mutation did not confer resistance to the benzimidazole nucleosides (EC50 for wild-type HCMV = 0.25 ± 0.04 μM, EC50 for HCMV pUL89 E256Q = 0.23 ± 0.04 μM). We conclude, therefore, that the G766C mutation that results in the E256Q substitution is unique for indole nucleoside resistance and distinct from previously discovered substitutions that confer both indole and benzimidazole nucleoside resistance (D344E and A355T).
人巨细胞病毒（HCMV）感染可在免疫功能低下和免疫未成熟的患者中引发严重疾病，包括脑病和智力发育迟缓。目前用于治疗全身性HCMV感染的药物疗法包括更昔洛韦、西多福韦和膦甲酸钠。然而，长期使用这些药物会导致严重的不良反应（骨髓抑制和/或肾毒性）以及对药物敏感性降低的病毒株的出现。脱氧核糖吲哚（吲哚）核苷在体外表现出比更昔洛韦（半数有效浓度[EC50]=7.4μM）高20倍的活性（在有药物存在的情况下，与无药物时相比，空斑数量减少50%时的药物浓度[EC50]=0.34μM），且未观察到细胞毒性增加。基于与苯并咪唑核苷的结构相似性，我们推测吲哚核苷靶向HCMV末端酶，该酶负责将病毒DNA包装进衣壳并将DNA切割成基因组长度单位。为验证这一假设，分离出一株对吲哚核苷耐药的HCMV毒株，对编码病毒末端酶的基因的开放阅读框进行测序，鉴定出UL89外显子1中的G766C突变；该突变导致相应蛋白质的氨基酸序列发生E256Q变化。一株经过该突变改造以确认耐药性的HCMV野生型毒株，与野生型病毒（EC50=0.17±0.04μM）相比，对吲哚核苷的敏感性降低了18倍（EC50=3.1±0.7μM）。有趣的是，该突变并未赋予对苯并咪唑核苷的耐药性（野生型HCMV的EC50=0.25±0.04μM，HCMV pUL89 E256Q的EC50=0.23±0.04μM）。因此，我们得出结论，导致E256Q替代的G766C突变对于吲哚核苷耐药性而言是独特的，且不同于先前发现的赋予吲哚和苯并咪唑核苷耐药性的替代突变（D344E和A355T）。
