Pinning down Pin1: Potential COVID-19 therapy

By Namita MitraSep 22 2021

In their recent study published in Nature Scientific Reports, scientists from Japan have demonstrated that Prolyl isomerase Pin1 is one of the key cellular molecules necessary for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) propagation.

Study: Prolyl isomerase Pin1 plays an essential role in SARS-CoV-2 proliferation, indicating its possibility as a novel therapeutic target. Image Credit: Dana.S/ Shutterstock

Pin1 belongs to the family Parvulin of 'peptidyl-prolyl isomerases (PPIases)' that comprise two more families, the FKBP and Cyclophilin families. Pin1 binds to phosphoproteins motifs in its target molecule and catalyzes a cis-to-trans orientation of proline to affect either the protein stability, function, or structure. The roles of Pin1 have been revealed in cancers, Alzheimer's disease, and some metabolic diseases.

The team from Japan had previously observed markedly increased Pin1 expression levels in obese and diabetic mice in several tissues, including liver, muscle, adipose tissue, and kidney. Notably, Pin1 is also known to accelerate the proliferation of several viruses via molecular mechanisms that differ among virus types. Hence, the study's authors speculated that increased Pin1 levels might be involved in the severity of coronavirus disease 2019 (COVID-19) in patients with underlying disease conditions like obesity and diabetes.

What did the researchers do?

The team utilized two siRNA molecules to mediate suppression of Pin1 in SARS-CoV2 infected VeroE6/TMPRSS2 cells to elucidate the role of Pin1 in SARS-CoV-2 proliferation. Both siRNAs markedly reduced the expression of Pin1 protein and subsequently reduced the proliferation of SARS-CoV-2 in the cells, as assessed by SARS-CoV-2 nucleocapsid (N) protein levels detected in the cell lysates.

Subsequently, several Pin1 inhibitors, developed by the team in the past, were probed for their negative effect on SARS-CoV-2 proliferation. These compounds also exhibited strong suppressive effect on SARS-CoV-2 proliferation, precisely at a concentration of 10 μM, as demonstrated by complete prevention of syncytium formation, a cytopathic effect (CPE), in VeroE6/TMPRSS2 cells infected by SARS-CoV-2.

One such inhibitor, H-77, which seemed to be the most potent, showed an effective concentration of 50% (EC₅₀) of just 3.2 μM. The concentration-dependent inhibitory effect of H-77 was also shown by measuring viral protein levels in cell lysates or viral RNA isolated from the culture supernatants. Membrane fusion or syncytium formation, caused by a viral infection, became less apparent as the drug concentration was increased to 5 μM and was almost absent at concentrations above 7.5 μM.

By reflecting on the data obtained by disrupting Pin1 activity through siRNA and Pin1 inhibitory compounds, the team established that Pin1 is essential for SARS-CoV-2 proliferation.

Additionally, H-77 almost completely blocked SARS-CoV-2 proliferation when added two hours after infection and showed a weaker but still significant inhibitory effect when added six hours after infection. Intracellular viral nucleocapsid (N) mRNA levels were significantly reduced, providing evidence that H-77 inhibits viral proliferation at the viral RNA transcription step or earlier.

Similarly, applying H-77 for two hours, followed by washing out before virus infection, also strongly inhibited N-protein synthesis of SARS-CoV-2, indicating that the Pin inhibitor is also effective if the cells are pretreated immediately before virus infection.

The team also tested all-trans retinoic acid (ATRA), an agonist of the retinoic acid receptor (RAR) used medically to treat acute promyelocytic leukemia (APL) and was recently reported to inactivate Pin1 isomerase activity. ATRA similarly suppressed SARS-CoV-2 proliferation as shown by marked reductions in protein and viral RNA levels in a concentration-dependent manner and alleviated the CPEs. However, the EC₅₀ of ATRA (17.9 μM) was higher than that of H-77 (3.2 μM).

The inhibitory activities of Pin1 inhibitors, developed by the team, were confirmed through in vitro PPIase assay. However, their potential non-specific effects on other PPIase enzymes or kinases have not yet been ruled out. On the other hand, ATRA specifically inhibits the activity Pin1 and not FKBP or cyclophilin.

Implications of the study

The team proposed that Pin1 has a critical role in viral gene transcription or earlier steps after the invasion of SARS-CoV-2 into cells. This suggests the molecule is indispensable for SARS-CoV-2 proliferation. They further hypothesize that Pin1 inhibitors may be an effective therapy against COVID-19.

Limitations

Further studies are necessary to identify the target protein of Pin1 and its functions in the life cycle of SARS-CoV-2. The study also indicates the necessity for optimizing and developing novel compounds with potent Pin1 inhibitory activity and high specificity.