Short Communication
Identification and characterization of Zika virus NS5 RNA-dependent RNA polymerase inhibitors

https://doi.org/10.1016/j.ijantimicag.2019.07.010Get rights and content

ABSTRACT

The current outbreak of Zika virus (ZIKV) is the impetus for novel, safe and efficacious anti-ZIKV agents. ZIKV non-structural protein 5 RNA-dependent RNA polymerase (RdRp) is essential for viral replication and is logically regarded as an attractive drug target. This study used a fluorescence-based polymerase assay to find an anti-infective drug 10-undecenoic acid zinc salt (UA) which could inhibit RdRp activity with a half maximal inhibitory concentration (IC50) of 1.13–1.25 µM. Molecular docking and site-directed mutagenesis analyses identified D535 as the key amino acid in the interaction between RdRp and UA. Importantly, the surface plasmon resonance assay showed that UA had strong direct binding with ZIKV wild-type RdRp and a relatively weak interaction with D535A-RdRp. As a control, the nucleoside inhibitor sofosbuvir triphosphate (PSI-7409) conferred insensitivity to the fluorescence-based RdRp assay and cannot bind directly with RdRp. Moreover, UA showed anti-ZIKV activity comparable to sofosbuvir. All these results indicate that UA is likely to be a promising lead compound against ZIKV, exhibiting a different mechanism than sofosbuvir.

Introduction

Zika virus (ZIKV) causes neurological injury and congenital brain impacts when women are infected during pregnancy. The explosive spread of ZIKV through the Americas has caused worldwide attention and promoted the World Health Organization to declare ZIKV a global public health emergency. However, there are no vaccines or drugs for the prevention or treatment of ZIKV infection [1]. It may be a challenge to develop anti-ZIKV agents.

ZIKV is a single-stranded positive-sense RNA encoding a polyprotein of ∼3400 amino acids. This polyprotein includes three structural proteins (C, prM and E) and seven non-structural (NS) proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b and NS5) [2]. NS5 is the largest enzyme and the most conserved protein component. It contains a methyltransferase (MT) domain at its N-terminus and an RNA-dependent RNA polymerase (RdRp) domain at its C-terminus. The NS5 MT domain methylates the RNA cap to form N7-methyl-guanosine and 2’-O-methyl adenosine [3]. The NS5 RdRp domain carries out viral RNA synthesis through a de-novo initiation mechanism and is responsible for synthesizing RNA copies of both plus and minus polarity [4]. Since NS5 RdRp is absent in mammalian hosts and essential for viral replication, it is a significant drug target for anti-ZIKV agents.

For viral RdRp inhibitors, both nucleoside polymerase inhibitors (NIs) and non-nucleoside polymerase inhibitors (NNIs) have been approved in the case of viral infections [5]. NIs, when converted into a triphosphate form, can compete with natural NTP substrates and terminate the RNA chain elongation. Sofosbuvir was the first anti-HCV NI to achieve clinical success. The active chemical composition of sofosbuvir is phosphorylated intracellularly to 2’-C-methyl-2’-fluoro-uridine-5′-triphosphate (UTP) (PSI-7409), which binds at the active site of RdRp to compete with natural substrates for RNA synthesis [6]. NNIs usually bind with RdRp, either inactivating the enzyme or preventing the conformational changes required for RNA synthesis. For NNIs, both metal chelating agents and allosteric inhibitors have been shown to be successful for suppressing RdRp [7].

This study found that 10-undecenoic acid zinc salt (UA) could inhibit ZIKV RdRp by binding to its active centre D535 with potential anti-ZIKV activity. UA has a different mechanism of action than the approved clinical drug sofosbuvir, which may make it a potent anti-ZIKV agent.

Section snippets

ZIKV NS5 RdRp activity assay

The cDNA sequence of ZIKV NS5 RdRp (nucleotides 8419–10374; KU321639.1) was synthesized de novo by Invitrogen (Waltham, MA, USA). NS5 wild-type (WT)-RdRp, D535A-RdRp and D692A-RdRp proteins were purified as described [8]. The RNA template, 3’UTR-A30 (5’-A30-AACAGGUUCUAGAACCUGUU-3’) was resuspended to 200 µM in a buffer consisting of 50 mM Tris-HCl (pH 8.0) and 150 mM NaCl in 0.1% diethyl pyrocarbonate water. The solution was incubated at 55–60°C for 5 min and placed at room temperature to allow

Results

ZIKV NS5 RdRp protein was purified (Fig. 1A). A fluorescence-based alkaline phosphatase-coupled polymerase assay was used to characterize RdRp activity. RdRp catalyses nucleotidyl transfer of adenosine-5’ monophosphate from BBT-ATP to the RNA chain, generating BBT-ppi that is subsequently hydrolysed to the fluorescent BBT. Measurement of the fluorescent BBT indirectly represented RdRp activity. As shown in Fig. 1B, WT-RdRp showed strong activity with noticeable FLU 430/560 nm signals. The

Discussion

Currently, there are several effective ZIKV inhibitors, such as envelope glycoprotein inhibitor nanchangmycin, NS2B/NS3 protease inhibitor temoporfin, NS3 helicase inhibitor suramin and NS5 RdRp inhibitor sofosbuvir [8], [12]. Although considerable efforts have been made to discover anti-ZIKV drugs, no approved drugs are available to date in the clinic. Many of the compounds were unsuitable for clinical use because of their high inhibition concentration to ZIKV or their cytotoxicity.

NS5 RdRp

Funding

This work was supported by the Zika Special Project of MOST (273-2016), NSFC (81773784), Beijing Nova Program (Z181100006218075), Basic Scientific Research Program of CAMS (2018RC350005), CAMS Major Collaborative Innovation Project (2016-I2M-1-011) and Drug Innovation Major Project (2018ZX09711001-002-002).

Declaration of Competing Interest

None declared.

Ethical approval

Not required.

References (20)

There are more references available in the full text version of this article.

Cited by (26)

  • Sofosbuvir and its tri-phosphate metabolite inhibit the RNA-dependent RNA polymerase activity of non-structural protein 5 from the Kyasanur forest disease virus

    2023, Biochemical and Biophysical Research Communications
    Citation Excerpt :

    Hence, RdRp of NS5 plays a crucial role in viral genome replication and can serve as a potential antiviral drug target [17]. Nucleoside and non-nucleoside inhibitors against NS5 RdRp from different viruses belonging to the Flaviviridae family have been identified [18–25]. Sofosbuvir is an FDA-approved drug currently in use for the treatment of Hepatitis C virus (HCV) infection [26,27].

  • Chalcones from Angelica keiskei (ashitaba) inhibit key Zika virus replication proteins

    2022, Bioorganic Chemistry
    Citation Excerpt :

    The ZIKV NS5 RNA-dependent RNA-polymerase (RdRp), which generates double stranded RNA, has also been identified as a key target for therapeutic development. Several nucleotide-like inhibitors (NIs) of the ZIKV RdRp have been identified such as 2′-C-methylated nucleoside triphosphates [10], 2′-C-methyl- and 2′-C-ethynyl-substituted analog 5′-triphosphates [11], 10-undecenoic acid zinc salt [12] and sofosbuvir triphosphate [13,14]. Therapeutically, non-nucleotide inhibitors (NNIs), which block enzyme activity at the allosteric or “N-pocket”, are more desirable since they are more selective for viral over mammalian targets and display fewer side-effects [14,15].

  • Insights on Dengue and Zika NS5 RNA-dependent RNA polymerase (RdRp) inhibitors

    2021, European Journal of Medicinal Chemistry
    Citation Excerpt :

    In addition, this promising inhibitor showed an EC50 value of 7.43 μM on infected Huh7 cells with ZIKV. Moreover, the authors suggested that 10-undecenoic acid zin salt (56) is the first non-nucleoside polymerase inhibitor able to bind directly with Asp535 residue at the active site from ZIKV NS5/RdRp (PDB: 5U0C) [166]. In search of new NS5 RdRp inhibitors useful against Flaviviruses' replication, Tarantino et al. perform an in silico screening involving a library of 203 previously reported and new compounds HCV inhibitors [111].

View all citing articles on Scopus
1

These two authors contributed equally to this article.

View full text