TMPRSS2 inhibitor found to be a novel and effective therapeutic option against pan-variant SARS-CoV-2 infections

By Pooja Toshniwal PahariaReviewed by Aimee MolineuxApr 1 2022

In a recent study published in Nature, researchers assessed the efficacy of small molecule protease inhibitors of transmembrane serine protease 2 (TMPRSS2) against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants of concern (VOCs) Alpha, Beta, Gamma, and Delta in mice and human lung cells.

Background

Studies have reported that SARS-CoV-2 depends on host cellular pathways, such as the hijacking of TMPRSS2-related proteases for viral entry. This indicates that TMPRSS2 are potential therapeutic targets to prevent SARS-CoV-2 infection. The development of such host-directed antivirals (HDA) could protect against SARS-CoV-2 variant infections, for which vaccines developed during the initial coronavirus disease 2019 (COVID-19) period may have limited efficacy.

The authors of the present study previously designed peptidomimetic compounds that exhibited potent antiviral efficacy at impeding influenza A H1N1 virus infection of cultured human airway epithelial cells (Calu-3) by inhibition of serine proteases. The team hypothesized that these compounds would also be effective against SARS-CoV-2 VOCs since the TMPRSS2 cleavage sites of SARS-CoV-2 and the H1N1 virus are similar.

About the study

In the present study, researchers expanded on previous work they had conducted and developed a library of peptidomimetic compounds that inhibited TMPRSS2 activity and investigated their efficacy against SARS-CoV-2 VOCs in human lung cells and mice.

The peptidomimetics’ efficacy was evaluated using cellular assays that measured TMPRSS2-dependant inhibition of proteolytic activity. The compounds tested were Camostat mesylate (Cm), N-0130, N-0438, N-0678, N-0676, N-0386, N-1296, and N-0385 and their corresponding inhibition efficacies were 56%, 72%, 84%, 5%, 8%, 73%, 16% and 83%, respectively. N-0100 did not inhibit TMPRSS2 proteolytic activity.

The half-maximal inhibitory concentrations (IC50) of Cm, N-0130, N-0438, N-0386 and N-0385 compounds were17.5, 3.1 ,5.2, 3.9, 1.9 nM, respectively. As a result, N-0130, N-0385, N-0386, and N-0438 were the four most promising peptidomimetic compounds.

These four peptidomimetic compounds were subsequently tested at 100 nM concentrations for anti- SARS-CoV-2 activity using Calu-3 cells. The cells were subjected to immunohistochemistry (IHC) analysis for staining of double-stranded ribonucleic acid (dsRNA) and nucleocapsid, which are markers of viral replication and translation, respectively.

The compounds Cm, N-0100, N-0678, N-0676, N-0386, N-1296, N-0385 and N-0385(OH) inhibited SARS-CoV-2 by >83%, 93%, <23%, <53%,>99%, <44%, >99%, and <23%, respectively. Thus, TMPRSS2-inhibiting peptidomimetics were also inhibitors of SARS-CoV-2 replication and translation in Calu-3 cells. Additionally, consistent inhibitory profiles across dsRNA and nucleocapsid were observed.

The peptidomimetics that demonstrated >75% inhibition of SARS-CoV-2 (N-0385, N-0385(OH), and Cm) were further assessed for the extracellular release of SARS-CoV-2 virions from Calu-3 cells using plaque assays with 40 nM and 200 nM doses of the compounds. Both concentrations of N-0385 reduced viral titers by almost 97%.

The efficacy of the lead candidate, N-0385 (100nM) was further assessed in donor-derived human colonoids against SARS-CoV-2 Alpha, Beta, Gamma, and Delta VOCs. The team quantified messenger RNA (mRNA) expression of TMPRSS2 and angiotensin-converting enzyme 2 (ACE2) in the colonoids using quantitative polymerase chain reaction (qPCR) and subsequently investigated the colonoids’ susceptibility to SARS-CoV-2 infection. N-0385 demonstrated a selectivity index of >10⁶ and >99% inhibition of SARS-CoV-2 in human lung cells and patient-derived colonoids.

After establishing N-0385 efficacy in cellulo and in vitro, the team investigated whether administering N-0385 intranasally could improve survival and morbidity in vivo. K18-hACE2 was administered one intranasal dose of 7.2 mg/kg daily of N-0385(OH), N-0385, or control (0.9% saline) for eight days. The animals were challenged with SARS-CoV-2 (1 x 10³ plaque-forming units (PFU)/mouse) and monitored till 14 days post-infection (dpi).

Findings

The N-0385(OH)-treated mice and saline-control mice demonstrated 15% and 14% weight loss, respectively, whereas the N-0385 treated mice only lost 3% of their weight. This weight loss was lowest when N-0385 was administered at the time of infection. Moreover, most N-0385(OH)-treated mice and saline control mice were dead at 14 dpi whereas most of the N-0385-treated mice (70%) survived.

Additionally, histological examination revealed no brain lesions and minimal lung pathologies in N-0385-treated mice.

Based on these results, the team evaluated a shorter four-day N-0385 treatment regimen using K18-hACE2 mice. Furthermore, they also investigated single dose N-0385 pan-variant effectiveness of N-0385 against SARS-CoV-2 in mice. N-0385 demonstrated substantial cross-protection, in equivalent amounts when used as the short (four-day) or standard (eight-day) regimens.

Conclusions

Overall, the study findings showed that N-0385 had a low nanomolar pan-variant anti-SARS-CoV-2 activity and could be used as an effective prophylactic and therapeutic option when used in multiple or single-dose regimens.