Interference of small compounds and Mg2+ with dsRNA-binding fluorophores compromises the identification of SARS-CoV-2 RdRp inhibitors

Abstract

The COVID-19 pandemic highlighted the need for the rapid development of antiviral therapies. Viral RNA-dependent RNA polymerases (RdRp) are promising targets, and numerous virtual screenings for potential inhibitors were conducted without validation of the identified hits. Here we have tested a set of presumed RdRp inhibitors in biochemical assays based on fluorometric detection of RdRp activity or on the electrophoretic separation or RdRp products. We find that fluorometric detection of RdRp activity is unreliable as a screening method because many small compounds interfere with fluorophore binding to dsRNA, and this effect is enhanced by the Mg²⁺ metal ions used by nucleic acid polymerases. The fact that fluorimetric detection of RdRp activity leads to false-positive hits underscores the requirement for independent validation methods. We also show that suramin, one of the proposed RdRp inhibitors that could be validated biochemically, is a multi-polymerase inhibitor. While this does not hinder its potential as an antiviral agent, it cannot be considered an specific inhibitor of SARS-CoV-2 RdRp.

Keywords: Fluorophore; High throughput screening; In vitro RNA polymerization assay; RNA-dependent RNA polymerase; SARS-CoV-2; Small molecule inhibitors.

Fig. 1

Putative RdRp inhibitors display different activity in fluorophore-dependent and fluorophore-independent assays. A Left, schematic of the purification procedure of the nsp12-nsp8-nsp7 complex comprising the functional SARS-CoV-2 RdRp. Right, Coomassie Blue staining following electrophoretic separation of the purified RdRp complex. The positions of the three subunits are indicated. Different amounts of bovine serum albumin (BSA) are shown and were used to estimate nsp12 concentration. B Top, schematic of the fluorophore (SYBR Green)-dependent detection of RdRp activity. Bottom, dose-response assay with purified recombinant SARS-CoV-2 RdRp. C Histogram showing the inhibitory effect of the 17 test compounds as assessed in a SYBR Green-based assay. D Top, schematic of the electrophoretic-based detection of RdRp polymerization activity. Bottom, dose-response assay with purified SARS-CoV-2 RdRp. E Inhibitory activity of the 17 test compounds on RdRp activity using the electrophoretic method. For quantification purposes, the signal contained in areas A and B (see control lane) was measured with ImageJ, and polymerization efficiency (PE) was estimated by the formula PE = A/(A + B). The PE value in the absence of inhibitors (control lane) was approx. 68–70%. Histogram represents the percentage of polymerization in the presence of each inhibitor, relative to the control value (plotted as 100%). The quantification includes data from two independent electrophoretic experiments. Original gel images for parts A, D and E are presented in Supplementary Fig. 3.

Fig. 2

SYBR Green fluorescence displacement assays from dsRNA and dsDNA. A Schematic of the SYBR Green displacement assay from double-stranded nucleic acid molecules. B Histogram shows the SYBR Green displacement from a poly-(AU)₁₅ dsRNA by each of the 17 test compounds. C Same as (B), testing SYBR Green displacement from a poly-(AT)₁₅ DNA molecule. See text for details.

Fig. 3

Mg²⁺affects the association of fluorophores with nucleic acids. A Dose-response activity of suramin, corilagin, and simeprevir on the fluorophore displacement of SYBR Green from poly-(AU)₁₅ dsRNA (in the presence or absence of 6 mM MgCl₂. B Same as (A), using poly-(AT)₁₅ DNA. C Effect of MgCl₂ concentration on the fluorescent emission of SYBR Green, PicoGreen, and Quantifluor DNA bound to dsRNA (blue) or dsDNA (green).

Fig. 4

Suramin is a multi-polymerase inhibitor. A Dose-response assays showing the RdRp inhibitory activity of suramin, corilagin, and simeprevir in the electrophoretic-based polymerase assay. B Dose-response effect of suramin on the activity of Φ29pol, Polγ, and PrimPol. C Left, schematic of pCDNA3 BglII-XmaI fragment (2,078 bp) depicting the restriction enzymes whose target sequences are present in the polylinker region. Right, agarose gel electrophoretic analysis of the pCDNA3 BglII-XmaI fragment following incubation with the indicated restriction enzymes in the presence or absence of suramin (200 µM). Original gel images are presented in Supplementary Fig. 3.

Fig. 5

Model illustrating different modes of interference of small compounds and Mg²⁺ with fluorophore molecules associated to dsRNA. See text for details.