Suramin inhibits chikungunya virus replication through multiple mechanisms

Highlights

• Suramin inhibits the activity of chikungunya virus (CHIKV) replication and transcription complexes in vitro.

• Suramin inhibits replication of CHIKV and other alphaviruses in cell culture.

• Suramin inhibits CHIKV RNA synthesis and -even stronger-an early step in the CHIKV replication cycle, likely entry.

• Testing of suramin-related compounds provided insight into the structure–activity relationship.

Abstract

Chikungunya virus (CHIKV) is a mosquito-borne alphavirus that causes severe and often persistent arthritis. In recent years, millions of people have been infected with this virus for which registered antivirals are still lacking. Using our recently established in vitro assay, we discovered that the approved anti-parasitic drug suramin inhibits CHIKV RNA synthesis (IC₅₀ of ∼5 μM). The compound inhibited replication of various CHIKV isolates in cell culture with an EC₅₀ of ∼80 μM (CC₅₀ > 5 mM) and was also active against Sindbis virus and Semliki Forest virus. In vitro studies hinted that suramin interferes with (re)initiation of RNA synthesis, whereas time-of-addition studies suggested it to also interfere with a post-attachment early step in infection, possibly entry. CHIKV (nsP4) mutants resistant against favipiravir or ribavirin, which target the viral RNA polymerase, did not exhibit cross-resistance to suramin, suggesting a different mode of action. The assessment of the activity of a variety of suramin-related compounds in cell culture and the in vitro assay for RNA synthesis provided more insight into the moieties required for antiviral activity. The antiviral effect of suramin-containing liposomes was also analyzed. Its approved status makes it worthwhile to explore the use of suramin to prevent and/or treat CHIKV infections.

Introduction

Chikungunya virus (CHIKV) is a mosquito-borne arthritogenic alphavirus that has infected millions of people since its re-emergence in 2005. In November 2013, CHIKV emerged in the Caribbean (Weaver, 2014, Weaver and Lecuit, 2015), starting an outbreak that has thus far resulted in over 1.2 million cases in the Americas (http://www.paho.org/hq/index.php?Itemid=40931).

CHIKV replication occurs in the cytoplasm on modified endosomal membranes and is driven by replication and transcription complexes (RTCs) that contain CHIKV nonstructural proteins (nsP) nsP1-4, of which nsP4 is the RNA-dependent RNA polymerase (RdRp). Early in infection negative-stranded RNA (−RNA) complementary to the viral genome is synthesized, which serves as template for the production of genomic and subgenomic RNA (sgRNA). The genome serves as mRNA for the production of nsPs and the sgRNA is translated into the structural proteins that are required for the biogenesis of new virions.

Despite intensified research efforts over the past years and the identification of a variety of compounds with anti-CHIKV activity in preclinical studies (Thiberville et al., 2013), there are still no registered drugs on the market for treating CHIKV infections. Suramin is a symmetrical sulfonated naphthylurea compound that was approved for the treatment of parasitic infections in 1921, but its anti-cancer and antiviral potential were discovered only 60 years later (reviewed in De Clercq, 2015, Liu and Zhuang, 2011, Voogd et al., 1993). It was shown that suramin had anti-reverse transcriptase activity against tumor-inducing viruses (De Clercq, 1979) and it was actually the first documented HIV reverse transcriptase inhibitor that was tested in human patients (Broder et al., 1985), but the compound’s side effects outweighed the clinical benefit due to the required long term treatment (Kaplan et al., 1987). A later study revealed that suramin’s anti-HIV-1 activity in vivo was actually due to its inhibitory effect on the interaction between the viral gp120 and the CD4 receptor (Schols et al., 1990). Suramin has also been shown to block the binding or early steps of infection of several DNA and RNA viruses, like herpes simplex virus type-1 (Aguilar et al., 1999), cytomegalovirus (Baba et al., 1993), human hepatitis B virus (Schulze et al., 2007), hepatitis delta virus (Petcu et al., 1988), hepatitis C virus (Garson et al., 1999), dengue virus (Chen et al., 1997), several bunyaviruses (Crance et al., 1997, Ellenbecker et al., 2014, Iqbal et al., 2000, Jiao et al., 2013), norovirus-like particles (Tamura et al., 2004) and enterovirus 71 (Wang et al., 2014), for which the antiviral activity of suramin was also confirmed in an animal model (Ren et al., 2014). In recent in vitro studies suramin was identified as a hepatis C virus and dengue virus helicase inhibitor (Basavannacharya and Vasudevan, 2014, Mukherjee et al., 2012) and also as a norovirus RdRp inhibitor by virtual screening and biochemical assays with purified enzymes (Mastrangelo et al., 2012, Tarantino et al., 2014). In the present study we assessed the effect of suramin on CHIKV RNA synthesis using our recently established in vitro assay that relies on RTCs isolated from infected cells (Albulescu et al., 2014). We found that suramin inhibits both CHIKV RNA synthesis in vitro as well as an early step in CHIKV infection of cultured cells. In addition to describing the inhibition of CHIKV replication through two independent mechanisms, we provide more insight into the moieties required for suramin’s antiviral activity.