The TMPRSS2 protein, a new target for drugs against SARS-CoV-2

As scientists continue to develop preventive and therapeutic compounds to treat severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, a new Signal transduction and targeted therapy A journal study reports the activity of a compound that inhibited host cell transmembrane serine protease serine protease 2 (TMPRSS2), ultimately preventing severe coronavirus disease 2019 (COVID-19).

Study: TMPRSS2, a new drug target directed against SARS-CoV-2. Image Credit: Kateryna Kon/Shutterstock.com

Introduction

The emergence of SARS-CoV-2 led to the devastating COVID-19 pandemic. Despite the rapid development and subsequent distribution of COVID-19 vaccines, they are only capable of eliciting short-term antibody-mediated immunity.

Additionally, some patient subgroups cannot be vaccinated safely or do not respond to current vaccines. Breakthrough or repeat infections are also common, especially due to the emergence of several new variants like the Beta and Omicron variants which can escape or evade immunity.

Repeated doses of booster vaccine have been recommended to keep antibody levels high enough. Monoclonal antibodies have also been approved to prevent serious illness in infected people at high risk; however, most have lost their effectiveness in Omicron infections.

Nonetheless, researchers have continued to explore new avenues or repurpose drugs for their potential utility in treating COVID-19. These include oral drugs like paxlovid, which inhibits the viral protease 3CLpro, as well as molnupiravir, which introduces errors when copying viral ribonucleic acid (RNA).

In the present study, compound N-0385 was described as a peptidomimetic directed against a host characteristic required by SARS-CoV-2 for infection.

SARS-CoV-2 enters host cells via the receptor binding domain (RBD) within its spike protein, which binds to the angiotensin-converting enzyme 2 (ACE2) receptor of the host cell. Importantly, this binding event must be accompanied by cleavage of the spike protein at two locations. Cleavage is performed by host cell furin at a polybasic site, which produces two distinct spike subunits S1 and S2, which are always covalently linked.

The second cleavage involves TMPRSS2 at the S2′ site and occurs after receptor binding, exposing the cleavage site upstream of the fusion peptide. This cleavage allows membrane fusion to occur, which then leads to entry of the virus into the host cell by endocytosis.

Inhibition of furin and TMPRSS2 have previously been shown to synergistically block viral replication in vitro. In the current paper, nanomolar concentrations of N-0385 effectively blocked SARS-CoV-2 leaving the virus in an incomplete cleavage state, in which it is unable to fuse and infect new host cells.

Study results

The researchers found that N-0385 had a highly selective action exceeding 106 without affecting other enzymes. Additionally, this compound suppressed infection with four SARS-CoV-2 (COV) variants of concern, including Alpha, Beta, P.1, and Delta variants.

Inhibition of SARS-CoV-2 spike cleavage by TMPRSS2 inhibits virus entry.  a Upper panel: Schematic representation of SARS-CoV-2 S cleavage by furin at the S1/S2 site and subsequent cleavage at the S2' site by TMPRSS2.  Cleavage at the S2' site exposes the fusion peptide (FP), priming S for membrane fusion (S colored cyan).  Inhibition of TMPRSS2 by N-0385 (blue hexagon) causes incomplete S cleavage (red colored S).  Bottom panel: Complete S cleavage supports membrane fusion and release of the viral genome into the target cell.  Inhibition of TMPRSS2 results in incomplete S cleavage and thus prevents virus fusion and entry.  b Alignment of amino acid sequences at the S1/S2 and S2' sites of different SARS-CoV-2, zoonotic SARS-CoV and MERS-CoV variants of concern.  Amino acid motifs highlighted in blue and orange are cleaved by furin and TMPRSS2, respectively.  The motifs highlighted in pink are most likely cleaved by TMPRSS2.  c Crystal structure of human TMPRSS2 (SRCR and serine protease domain) in complex with nafamostat (cyan, PDB: 7MEQ; upper left panel).  Catalytic domain (cartoon style) of TMPRSS2 (upper right panel).  The catalytic triad (green stick model) and nafamostat (cyan) are shown.  Structures of nafamostat (PubChem CID 4413) and N-0385 (PubChem CID 135169285, lower panels)Inhibition of SARS-CoV-2 spike cleavage by TMPRSS2 inhibits virus entry. a Upper panel: Schematic representation of SARS-CoV-2 S cleavage by furin at the S1/S2 site and subsequent cleavage at the S2′ site by TMPRSS2. Cleavage at the S2′ site exposes the fusion peptide (FP), priming S for membrane fusion (S colored cyan). Inhibition of TMPRSS2 by N-0385 (blue hexagon) causes incomplete S cleavage (red colored S). Bottom panel: Complete S cleavage supports membrane fusion and release of the viral genome into the target cell. Inhibition of TMPRSS2 results in incomplete S cleavage and thus prevents virus fusion and entry. b Alignment of amino acid sequences at the S1/S2 and S2′ sites of different variants of concern of SARS-CoV-2, zoonotic SARS-CoV and MERS-CoV. Amino acid motifs highlighted in blue and orange are cleaved by furin and TMPRSS2, respectively. The motifs highlighted in pink are most likely cleaved by TMPRSS2. vs Crystal structure of human TMPRSS2 (SRCR and serine protease domain) in complex with nafamostat (cyan, PDB: 7MEQ; upper left panel). Catalytic domain (cartoon style) of TMPRSS2 (upper right panel). The catalytic triad (green stick model) and nafamostat (cyan) are shown. Structures of nafamostat (PubChem CID 4413) and N-0385 (PubChem CID 135169285, lower panels)

In mouse models expressing the humanized K18-hACE2 receptor, pretreatment with N-0385 reduced disease severity and mortality rate from SARS-CoV-2 infection. Similar results were obtained when the treatment was given after the viral inoculation, with a single dose at 12 hours from the Delta inoculation being able to reduce weight loss and viral loads.

In fact, when four doses of the compound were administered prior to intranasal inoculation, lung tissue showed much lower viral titers, with no deaths reported among treated mice. Thus, treatment should be given before exposure or within a short time after exposure.

Female mice appeared to be more resistant to infection, or more susceptible to the compound, than male mice.

One of the advantages of treating SARS-CoV-2 infection via host targets rather than viral targets is that escape mutations are much less likely, as they will inhibit cleavage at the cleavage site. TMPRSS2 highly conserved from the tip. Thus, N-0385 will likely retain activity against future variants.

Second, N-0385 might be useful in treating infections with other viruses that also use the TMPRSS2 host, such as influenza virus or other coronaviruses. Recently, the structure of this enzyme has been unveiled, which could pave the way for more specifically designed inhibitors.

Importantly, TMPRSS2 does not have a crucial role in mice and its physiological role in humans is currently unknown. Thus, its inhibition in humans may not cause serious adverse effects.

Future directions

Further research will be needed to extend the therapeutic window, which at present seems short, like that of most antivirals approved for this use today. However, the use of N-0385 to prevent severe disease appears to be realistic, especially given its specific efficacy at nanomolar concentrations.

Future studies should also determine the timeliness and basis of sex differences in response to infection with SARS-CoV-2 and this compound. The extent to which N-0385 can be used to produce a synergistic effect with other antivirals acting by different mechanisms also needs to be investigated.

Broad-acting serine protease inhibitors have been evaluated in clinical trials for COVID-19 activity, including nafamostat and aprotinin. Nafamostat must be administered intravenously and is associated with venous inflammation at the catheter site, elevated sodium levels and respiratory failure. Moreover, the clinical effects of this treatment are weak, although favorable, in high-risk patients on supplemental oxygen.

Aprotinin reduced hospital stay and oxygen requirements without significant adverse effects being observed. This drug is delivered by inhalation, which might be a better route as it can inhibit TMPRSS2 on target airway cells exposed to SARS-CoV-2.

conclusion

[The current study] provided strong evidence that host-directed inhibition of TMPRSS2 by peptidomimetic compound N-0385, thereby preventing spike maturation, is effective against SARS-CoV-2 in vitro and in vivo, including variants of concern.”

N-0385 may be potentially useful for treating people who do not have vaccine immunity or who are at increased risk for severe COVID-19, with TMPRSS2 inhibitor therapy started soon after exposure.

Journal reference:

  • Keller, C., Bottcher-Friebertshauser, E., Lohoff, M. (2022). TMPRSS2, a novel drug target against SARS-CoV-2. Signal transduction and targeted therapy. doi: 10.1038/s41392-022-01084-x.

Irene B. Bowles