Study identifies Etravirine and Dolutegravir as effective entry inhibitors of wild-type and predominant variants of SARS-CoV-2

By Tarun Sai LomteReviewed by Danielle Ellis, B.Sc.Apr 18 2022

In a recent study published in the International Journal of Molecular Sciences, researchers identified drugs inhibiting the entry of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

Background

SARS-CoV-2 enters host cells by attaching to the angiotensin-converting enzyme 2 (ACE2) receptor through its spike (S) protein. Blocking the interactions between ACE2 and S protein is critical to inhibiting infection. Coronavirus disease 2019 (COVID-19) vaccination has been (mostly) effective in curtailing the infection outbreak. Nevertheless, several variants of concern (VOCs) have emerged, exhibiting significant resistance to the vaccine- or infection-induced immunity. Hence, there is a pressing need to develop broad-spectrum drugs which inhibit the wild-type strain and mutant variants of SARS-CoV-2.

The study and findings

In the present study, researchers developed an assay for detecting the molecular interactions between the S protein’s receptor-binding domain (RBD) and the ACE2 receptor. A drug library was screened to identify potential (entry) inhibitors.

The authors employed the protein complementation technology, i.e., NanoLuc binary technology (NanoBiT), to develop the ACE2-RBD attachment assay. The human ACE2 was fused with the Small BiT (SmBiT) subunit of NanoLuc luciferase, transfected into HeLa cells, and a stable HeLa cell line expressing SmBiT-ACE2 was established. Subsequently, five large BiT (LgBiT)-RBD or S1 subunit fusion constructs were generated.

The recombinant fusion products (LgBiT-RBD, LgBiT-S1) were incubated with the cells expressing SmBiT-ACE2. No luminescence was detected with LgBiT-RBD, LgBiT-S1, and S1-LgBiT fusion constructs, while a robust luminescence signal was observed for the RBD-LgBiT construct. Another (fusion) construct with a linker between RBD and LgBiT (RBD-linker-LgBiT) had no noticeable improvements in the luciferase activity, and subsequent experiments were performed using the RBD-LgBiT construct.

Next, a Food and Drug Administration (FDA)-approved drug library was screened to identify potential drug candidates with inhibitory effects on the attachment of SARS-CoV-2 RBD. Of the 1068 drugs, 16 demonstrated RBD attachment inhibition by over 50%. Negative screening of the top 20 drugs with the highest inhibitory activity using the HiBiT-NanoLuc assay eliminated 11 candidates from further analysis. The nine candidates were Aripiprazole, ABT-199, Dolutegravir Sodium, Etravirine, Gramicidin, Ivermectin, Miconazole, Miconazole Nitrate, and Ospemifene.

A cytotoxic assay was performed on VeroE6 cells using these (nine) candidates, and only three: Dolutegravir, Etravirine, and Sspemifene, were non-toxic. Further, the candidate drugs were subject to the RBD-ACE2 attachment assay, which found Etravirine as the most effective, with a half-maximal effective concentration (EC₅₀) of 2.3 µM. Ivermectin which inhibits SARS-CoV-2 replication in cell cultures exhibited a weak EC₅₀ of 22.8 µM.

Pseudoviruses with SARS-CoV-2 S protein and NanoLuc reporter gene were produced. SmBiT-ACE2 cells were incubated with pseudovirus and candidate inhibitors for six hours, followed by the removal of pseudovirus and continued incubation for 16 hours. Dolutegravir and Etravirine neutralized S-typed pseudoviruses with EC₅₀ values of 40 nM and 5.8 nM, respectively. The neutralization efficiency of the candidates was determined using the plaque reduction neutralization test (PRNT).

The (authentic) SARS-CoV-2 was pre-incubated with the candidate drugs before infecting VeroE6 cells. Neutralization was estimated by quantitating the plaques five days-post infection (dpi). Dolutegravir neutralized SARS-CoV-2 infection with an EC₅₀ of 4.2 µM and Etravirine at 7.7 µM, whereas ivermectin inhibited 60% of infection; the authors presumed that the result might have been biased due to its cytotoxicity.

Molecular docking of Dolutegravir, Etravirine, and Ivermectin on S RBD and ACE2 was carried out. With ACE2-inhibitor(s) docking, the predictive binding sites were not at the RBD interaction interphase, indicating that it was not the primary target of the inhibitors. Unsurprisingly, high affinity was detected between S RBD and inhibitors at the ACE2-S protein interphase. The mean affinity score of Dolutegravir to RBD was -7.52 kilocalories per mole (kcal/mol), and it was -7.8 kcal/mol for Etravirine.

Moreover, when S RBD harboring N501Y substitution was tested, docking results were unaffected. The researchers investigated the neutralization potential of Dolutegravir and Etravirine against pseudoviruses carrying the S protein of Alpha, Beta, Delta, or Omicron VOCs. No significant changes were noted in the EC₅₀ values of the two drugs against SARS-CoV-2 VOCs. Of note was the highly potent neutralization of the Omicron VOC by both the drugs.

Conclusions

The research team identified two potent drugs for inhibiting the entry of SARS-CoV-2 into host cells. Overall, the RBD-ACE2 attachment developed by the researchers is robust and offers attachment results in just 10 minutes without the need for biosafety level (BSL)-2 facilities. The two drugs were effective and inhibited wild-type virus and mutant variants. Since the drugs can be administered orally, the authors posit their use as pre-or post-exposure prophylactic COVID-19 treatment.