A Genetic Trap in Yeast for Inhibitors of SARS-CoV-2 Main Protease

Abstract

The ongoing COVID-19 pandemic urges searches for antiviral agents that can block infection or ameliorate its symptoms. Using dissimilar search strategies for new antivirals will improve our overall chances of finding effective treatments. Here, we have established an experimental platform for screening of small molecule inhibitors of the SARS-CoV-2 main protease in Saccharomyces cerevisiae cells, genetically engineered to enhance cellular uptake of small molecules in the environment. The system consists of a fusion of the Escherichia coli toxin MazF and its antitoxin MazE, with insertion of a protease cleavage site in the linker peptide connecting the MazE and MazF moieties. Expression of the viral protease confers cleavage of the MazEF fusion, releasing the MazF toxin from its antitoxin, resulting in growth inhibition. In the presence of a small molecule inhibiting the protease, cleavage is blocked and the MazF toxin remains inhibited, promoting growth. The system thus allows positive selection for inhibitors. The engineered yeast strain is tagged with a fluorescent marker protein, allowing precise monitoring of its growth in the presence or absence of inhibitor. We detect an established main protease inhibitor by a robust growth increase, discernible down to 1 μM. The system is suitable for robotized large-scale screens. It allows in vivo evaluation of drug candidates and is rapidly adaptable for new variants of the protease with deviant site specificities. IMPORTANCE The COVID-19 pandemic may continue for several years before vaccination campaigns can put an end to it globally. Thus, the need for discovery of new antiviral drug candidates will remain. We have engineered a system in yeast cells for the detection of small molecule inhibitors of one attractive drug target of SARS-CoV-2, its main protease, which is required for viral replication. The ability to detect inhibitors in live cells brings the advantage that only compounds capable of entering the cell and remain stable there will score in the system. Moreover, because of its design in yeast cells, the system is rapidly adaptable for tuning the detection level and eventual modification of the protease cleavage site in the case of future mutant variants of the SARS-CoV-2 main protease or even for other proteases.

Keywords: COVID-19; MazF toxin; SARS-CoV-2; Saccharomyces cerevisiae; antiviral agents; genetic engineering; genetic selection system; small molecules.

FIG 1

Principle of the genetic selection system. (A) Situation without Mpro inhibitor. SARS-CoV-2 Mpro is active and cuts the inactive MazEF fusion protein at the synthetic Mpro cleavage site in the linker region connecting the MazF (toxin) and MazE (toxin inhibitor) moieties, releasing active MazF. The RNase activity of MazF can then exert its toxic effect by degrading cellular RNAs, thus preventing growth of the cells carrying the construct. The output signal from the red fluorescent protein tag in these cells will be weak. (B) Situation with Mpro inhibitor. The activity of SARS-CoV-Mpro is reduced, and the inactive MazEF fusion stays mostly intact. The toxic RNase activity will be reduced, and these cells can grow faster, resulting in a stronger fluorescent signal output.

FIG 2

Titration of methionine concentration with a yeast strain expressing Mpro and MazEF chimera (strain HA_SC_Met17_Mpro carrying PSMv4) in the presence or absence of GC376. Growth measurements were obtained using the Bioscreen C reader, with the starting absorbance adjusted to 0 (mean ± SE, n = 3). (A) Cell cultures were grown in SD medium without uracil (SD–ura) and with various concentrations of methionine from 350 μM to 0 μM (see the legend). (B) Same conditions as for panel A but treated with the protease inhibitor GC376 (50 μM).

FIG 3

Titration of inhibitor GC376. Growth measurements were obtained as in Fig. 2 (mean ± SE, n = 3). Cell cultures were grown in SD–ura and 7.5 μM methionine and various concentrations of GC376 (see legend). (A) Strain HA_SC_Met17_Mpro carrying PSMv4 expressing Mpro and MazEF chimera. (B) Strain HA_SC_Met17_Mpro expressing Mpro (EV; empty vector). (C) Strain HA_SC_1352control carrying PSMv4 expressing the MazEF chimera only. (D) Control strain HA_SC_1352control with empty vector.

FIG 4

Growth of mCherry-tagged strain expressing Mpro and MazEF chimera in the presence of different candidate protease inhibitors. Strain HA_SC_Met17_Mpro_RED carrying PSMv4 was used. Growth measurements (fluorescence and absorbance) were obtained using the Eve robot (see Materials and Methods), with the starting measurement adjusted to 0 (mean ± SE, n = 16). RFU, relative fluorescence units. Cell cultures were grown in SD–ura and 15 μM methionine; 1.25% DMSO; and 0, 10, and 30 μM concentrations of the respective compound to be tested (see the legend).

FIG 5

Titration of GC376 and Boceprevir over wider concentration ranges. The same yeast strain, culture conditions, and growth measurements (fluorescence), and statistics were used described as for Fig. 4. GC376 was tested at 0, 1, 3, 10, 30, and 100 μM; boceprevir was tested at 0, 1, 3, 10, 30, 100, and 200 μM.