Effect of farnesyltransferase inhibitors on SARS-CoV-2

Abstract

Objectives

The emergence of SARS-CoV-2 in 2019 led to a severe pandemic situation. Treatment options are limited, and the efficacy of vaccines decreases due to mutations in SARS-CoV-2 strains. Therefore, new treatment options are urgently needed, and computational compound screenings are used to predict drugs quickly. One of these screenings revealed farnesyltransferase inhibitors (FTIs) as potential candidates.

Methods

SARS-CoV-2 infected Calu-3 cells were treated with lonafarnib and tipifarnib and fold change viral replication of SARS-CoV-2 was measured using RT-qPCR. Furthermore, morphological changes, like CPE formation, were evaluated. Effects on Calu-3 cells were analyzed using MTT assay.

Results

We demonstrated that the FTIs lonafarnib and tipifarnib have an effect on SARS-CoV-2 Wildtype and the Delta variant. Both FTIs dose-dependently reduced morphological changes and the formation of cytopathic effects in SARS-CoV-2 infected Calu-3 cells. The effect of the FTIs on Omicron needs to be further elucidated because of inefficient viral replication.

Conclusions

The FTI lonafarnib and tipifarnib might be effective drugs against different SARS-CoV-2 strains.

1. Introduction

The SARS-CoV-2 pandemic has necessitated the fast development of vaccines and new treatment options. Computational compound screenings are a common method to identify new drugs quickly. Such screenings revealed that the FTIs lonafarnib and tipifarnib might have antiviral potency against SARS-CoV-2 [1], [2]. Both are farnesyltransferase inhibitors (FTIs), with applications in different cancer therapies and hepatitis D virus infection [3].

In consideration of such predictions based on computational screenings, we investigated the putative effect of FTIs on different variants.

2. Methods

We infected Calu-3 cells with Wildtype, Delta, or Omicron variants, treated them with FTIs, and evaluated the change in viral replication, the morphological changes and cytopathic effects (CPEs) [4]. For a detailed description of the experimental procedure, please see supplementary material.

3. Results

The infection of Calu-3 cells with Wildtype and Delta variants led to massive morphological changes and CPEs, which were dose-dependently reduced after FTI treatment (Fig. 1 A). Both FTIs show stronger reductions in CPEs for the Delta variant compared to the Wildtype variant (Fig. 1A). The effect of remdesivir used as positive control was less pronounced (Fig. 1A).

Fig. 1.

(A) Representative morphological images of SARS-CoV-2 infected Calu-3 cells after treatment with lonafarnib and tipifarnib for 30 h. Cells infected with Wildtype, Delta or Omicron were treated with lonafarnib and tipifarnib (0.1 µM, 20 µM, and 30 µM), DMSO and Remdesivir (2 µM) as controls. Scale bar: 300 µM. (B)–(C) Cell viability test on Calu-3 cells after treatment with lonafarnib (B) and tipifarnib (C). Cells were incubated with lonafarnib and tipifarnib in different concentrations, ranging from 0.1 µM–50 µM for 48 h. (D)–(I) Effect of FTIs on fold change viral replication of SARS-CoV-2 on Calu-3 cells. Effect of lonafarnib (left) and tipifarnib (right) on Wildtype (D, E), Delta (F, G) and Omicron (H, I). A triangle for the fold-change in viral replication for remdesivir was added in (D)–(I).

DMSO, Dimethyl sulfoxide; FTI, Farnesyltransferase inhibitor; Wildtype, SARS-CoV-2 non-VOC/B.1.1; Delta, SARS-CoV-2 Delta/B.1.617.2; Omicron, SARS-CoV-2 Omicron/B.1.1.529.

After the optical evaluation, we quantified the effect of the FTIs on the virus replication in Calu-3 cells. We first elucidated the effect of the FTIs on the cells using MTT assays (Fig. 1B-C). Both FTIs evoked a dose-dependent reduction in cell viability, which was more prominent in higher concentrations. The CC₅₀ values were 31.28 for lonafarnib and 37.78 for tipifarnib.

After the evaluation of the virus-independent effects of the FTIs, we infected Calu-3 with Wildtype, Delta, or Omicron, treated them with FTIs, and calculated the fold change viral replication. To address this question, we measured extracellular viral RNA by reverse transcription-quantitative polymerase chain reaction. We observed a dose-dependent inhibitory effect of both FTIs on Wildtype and Delta variants. In Wildtype, lonafarnib and tipifarnib showed IC₅₀ values of 3.978 and 4.362, respectively. In Delta, lonafarnib and tipifarnib showed IC₅₀ values of 6.024 and 7.287, respectively (Fig. 1D–G). The effect of the FTIs on Omicron was not assessable because of insufficient viral replication (Fig. 1H-I). Remdesivir was used as a control drug to reduce viral replication similar to IC₅₀ values of previous studies at 2 µM [5]. As displayed in Figure 1D–I, remdesivir reduces but does not completely inhibit viral replication.

4. Discussion

Here, we aimed to characterize the effect of the FTIs lonafarnib and tipifarnib on SARS-CoV-2 Wildtype, Delta, and Omicron infected cells. We demonstrated a dose-dependent antiviral effect of both FTIs on Wildtype and Delta, whereas the effect on Omicron was not assessable because of the insufficient infection of Calu-3 cells. The reduction of CPEs on infected Calu-3 cells corresponds to the quantification of the fold change viral replication via reverse transcription-quantitative polymerase chain reaction.

Drug repurposing is commonly used to identify drugs against rapidly spreading diseases, and this approach hastens the approval of drugs and uses existing production capacities quickly [6]. In a high-throughput virtual screening approach [2], lonafarnib was identified as a potential drug candidate that might disrupt the binding of viral cofactors NSP7 and NSP8 to the highly active NSP12 polymerase complex [2]. In another computational screening of DrugBank compounds, lonafarnib was capable of simultaneously inhibiting the three viral targets 3CLpro, PLpro, and RdRp [1].

However, to the best of our knowledge, lonafarnib has never been tested in cell culture approaches on SARS-CoV-2. In this study, we demonstrated an antiviral effect on viral replication of both Wildtype and Delta variants, whereas the effect on Omicron was not measurable. The second FTI, tipifarnib, was found to be potentially effective against SARS-CoV-2 in a virtual screening [5]. Furthermore, its antiviral activity against the Wildtype strain was validated in Vero cells and Calu-3 cells, showing even synergistic effects when combined with omipalisib and remdesivir [5].

We confirmed these findings and, furthermore, demonstrated similar antiviral effects on Delta. Here, the IC₅₀ value for tipifarnib (7.29) is in accordance with the IC₅₀ value generated by Jang et al. in Wildtype (11.01) [5].

Funding

The authors would like to acknowledge the financial support of the Bavarian State Ministry of the Environment and Consumer Protection to LM and MH.

Competing interests

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Editor: Prof Carlo F Perno