Study discovers inhibition of SARS-CoV-2 entry priming protein by approved drug

The COVID-19 pandemic caused by the severe acute respiratory syndrome -coronavirus 2 (SARS-CoV-2) has spread to most of the world, causing millions of cases and hundreds of thousands of deaths. A new study published on the preprint server bioRxiv* shows that a protease inhibitor drug already approved by the Food and Drug Administration (FDA) could inhibit viral entry.

What is TMPRSS2?

The SARS-CoV-2 enters the host cell via the angiotensin-converting enzyme 2 (ACE2) receptor, to which it attaches via the spike (S) protein on the virus envelope. Another host protein called transmembrane protein serine protease TMPRSS2 also plays a vital role in processing the S protein and receptor. This is necessary for further interaction of the S protein and ACE2 receptor leading to infection.

When the TMPRSS2 protein is blocked by a molecule called camostat mesylate, the virus cannot enter the host cell. This offers a promising avenue for the therapy of this infection.

TMPRSS2 plays a role in several coronavirus infections, such as the earlier SARS, MERS and the current SARS-CoV-2, and also some influenza infections. It is also important biologically as a prostate cancer oncogene.

It is a promising antiviral drug target because it is a host protein. Drugs that are designed to fit viral proteins are always subject to the risk of becoming obsolete due to the high rate of viral genomic mutations that can change the protein characteristics. However, agents that are directed against host proteins are less likely to counter rapid drug resistance.

This type of drug does carry one risk, however. This is the risk that alterations in the protein pathway involved will also cause undesirable changes in the host physiology.

Researchers have been concerned that TMPRSS2 is the starting point for a series of amplifying proteolytic activation events. These are important in regulating seminal fluid and lung proteins since TMPRSS2 is a sodium channel regulator.

However, mouse experiments in which the mice lack this protein do not show any apparent deficits. Scientists hope that this means other proteases can replace the protein with respect to its function.

This could mean it is safe to target TMPRSS2 during viral infections. Moreover, there are already approved drugs that act to inhibit the proteolytic activity of this enzyme, such as camostat mesylate, nafamostat, and BHH.

TMPS2_HUMAN (Transmembrane protease serine 2) Human ball and stick model isolated on white. Entry receptor for SARS-CoV-2. Covid-19, Coronavirus vaccine research & development. 3D rendering. Image Credit: OMMB / Shutterstock
TMPS2_HUMAN (Transmembrane protease serine 2) Human ball and stick model isolated on white. Entry receptor for SARS-CoV-2. Covid-19, Coronavirus vaccine research & development. 3D rendering. Image Credit: OMMB / Shutterstock

How was the study done?

The researchers did a functional screening to find TMPRSS2 inhibitors and compared their efficiency. The tested molecules include secretory leukocyte peptidase inhibitor (SLPI), 4-(2-aminomethyl) benzenesulfonyl fluoride (AEBSF), Boc-Gln-Ala-Arg-7-Amino-4-methylcoumarin (BOC-QAR-AMC), camostat mesylate, bromhexine hydrochloride (BHH) and alpha 1 antitrypsin (A1AT).

They used a human cell line in which TMPRSS2 was expressed at very high levels, at 2.5 times that of control cells. The rate of proteolysis was increased over 3.5 fold compared to that of controls and remained more than double after hours.

What did the study show?

The researchers used camostat mesylate, a known TMPRSS2 inhibitor, in control cells. At concentrations as low as 100 nM, it showed inhibitory action.

Next, they tested for the inhibitory action of SLPI, at various concentrations, without success. However, A1AT showed dose-dependent inhibition of the proteolysis induced by TMPRSS2 with the highest effects at a concentration of 1 μM.

With AEBSF, a protease inhibitor capable of inhibiting influenza infection in a murine experiment, dose-dependent inhibition of TMPRSS2 occurred with a peak at 1 μM.

BHH is an FDA-approved drug used as a mucolytic and cough suppressant, which inhibits TMPRSS2 in a dose-dependent manner, but is less efficient than A1AT or AEBSF.

What do the findings imply?

In the current study, the researchers shortlisted two novel inhibitor molecules, namely, AEBSF and A1AT. It is possible that these inhibitors could potentially block viral activity by preventing TMPRSS2’s action on S protein processing.

A1AT is a small protein manufactured in the liver and present in the blood at high levels. It can shoot up six times in acute inflammation or injury. It is delivered to many organs when administered as a drug.

In the lungs, it blocks the action of the protease enzymes neutrophil elastase, proteinase 3, and cathepsin G. it also enhances the clearance of apoptotic cells, and so protects the body against tissue damage and overactive inflammation.

Mutations in the A1AT gene could lead to subnormal levels of the protein, causing the lung to suffer protein breakdown and widespread emphysema of the lobes. The FDA approved the use of A1AT as a replacement in cases of deficiency.

While A1AT was previously known to block H3N2 infection and influenza B virus in animal models, this is the first time that it is being shown to inhibit TMPRSS2. The earlier viruses do not need to be primed by TMPRSS2, and their blocking was presumed to be via hepsin inhibition.

The use of A1AT may reduce the disease severity in COVID-19 by dampening inflammation within the alveoli as well as inhibiting TMPRSS2. Earlier experiments have shown that the levels of truncated A1AT found in peripheral blood in SARS patients are higher than in controls, and rise in proportion to disease severity. This could mean that this protein is a component of a natural immune response against CoV infection as well as acute lung disease.

A1AT is a serine protease inhibitor (SERPIN) and thus has a reactive center loop that is broken apart by interaction with proteases. This breakage causes A1AT to undergo a conformational change, which results in irreversible covalent bonding with the target protease, inhibiting its activity permanently.

It is thought that the truncated A1AT seen in SARS patients arise from this type of cleavage caused by TMPRSS2 and other proteases.

AEBSF also blocks the activity of TMPRSS2, being a small molecule that inhibits proteases nonspecifically. It has caused a decrease in the levels of both H1N1 and H7N7 nuclear proteins within the lung tissue of mice infected with influenza. Its mechanism of action is via a covalent bonding, which adds a sulfonyl group to the active site.

The use of protease inhibitors – camostat mesylate, A1AT, BHH, and AEBSF – could help develop antivirals against COVID-19. The researchers say, “A1AT may be particularly effective as it has the dual capacity, inhibiting TMPRSS2 (and hence viral uptake and subsequent replication) and possessing anti-inflammatory activity. The ready availability of A1AT calls attention to its potential clinical use for the COVID-19 pandemic.”

*Important Notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Journal reference:
Dr. Liji Thomas

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.

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