Prolyl isomerase Pin1 plays an essential role in SARS-CoV-2 proliferation, indicating its possibility as a novel therapeutic target

Novel coronavirus disease 2019 (COVID-19) has emerged as a global pandemic with far-reaching societal impact. Here we demonstrate that Pin1 is a key cellular molecule necessary for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) propagation. In this study, siRNA-mediated silencing of Pin1 expression markedly suppressed the proliferation of SARS-CoV-2 in VeroE6/TMPRSS2 cells. In addition, several recently generated Pin1 inhibitors showed strong inhibitory effects on SARS-CoV-2 proliferation, measured by both viral mRNA and protein synthesis, and alleviated the cytopathic effect (CPE) on VeroE6/TMPRSS2 cells. One compound, termed H-77, was found to block SARS-CoV-2 proliferation at an EC50 below 5 μM regardless of whether it was added to the culture medium prior to or after SARS-CoV-2 infection. The inhibition of viral N protein mRNA synthesis by H-77 implies that the molecular mechanism underlying SARS-CoV-2 inhibition is likely to be associated with viral gene transcription or earlier steps. Another Pin1 inhibitor, all-trans retinoic acid (ATRA)—a commercially available drug used to treat acute promyelocytic leukemia (APL) and which both activates the retinoic acid receptor and inhibits the activity of Pin1—similarly reduced the proliferation of SARS-CoV-2. Taken together, the results indicate that Pin1 inhibitors could serve as potential therapeutic agents for COVID-19.

www.nature.com/scientificreports/ of target proteins by catalyzing a cis-to-trans orientation of proline in its substrate's protein structure. Many studies have revealed roles of Pin1 in cancers, metabolism, and Alzheimer's disease 8,9 . Evidence suggests that Pin1 expression in cancer cells is closely related to the degree of their malignancy, as Pin1 enhances cell proliferation and inhibits apoptosis 10,11 . Nevertheless, Pin1 is not indispensable for the survival or growth of normal cells. Pin1 KO mice are born and become mature without any defects in size and appearance 12,13 . We have observed that Pin1 expression levels are markedly increased in several tissues including the liver, muscle, adipose tissue, and kidney in obese or diabetic mice 6 . Interestingly, Pin1 reportedly accelerates the proliferation of several viruses, although the molecular mechanism underlying Pin1-induced promotion of virus proliferation seems to differ among virus types [14][15][16][17][18] . Taken together, we speculate that the increased Pin1 expression in obese or diabetic patients may be involved in the rapid progress and/or severity of infection with SARS-CoV-2.

Results
Essential role of Pin1 in SARS-CoV-2 proliferation. We first examined the contribution of Pin1 to SARS-CoV-2 proliferation using VeroE6/TMPRSS2 cells, which are highly susceptible to SARS-CoV-2 infection due to their constitutive expression of transmembrane serine protease TMPRSS2 19 . Initially, we examined the effect of siRNA-mediated suppression of Pin1 expression on SARS-CoV-2 proliferation in VeroE6/TMPRSS2 cells. Treatment of the cells with either of two Pin1 siRNAs markedly reduced the expression of Pin1 protein and 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 (Fig. 1A). Notably, the degree of reduction was more pronounced in our study than in a previous study in which feline coronavirus replication was partially suppressed by treatment with Pin1 siRNA 14 . Subsequently, we investigated the effect of Pin1 inhibitors on SARS-CoV-2 proliferation.

Inhibition of SARS-CoV-2 proliferation by Pin1 inhibitors.
We have recently developed many novel compounds with Pin1 inhibitory activity, and they were experimentally characterized for their effects on SARS-CoV-2. Our experiments revealed that at least 20 of these compounds exhibit a strong suppressive effect on SARS-CoV-2 proliferation at a concentration of 10 μM. The chemical structures and the results for five representative compounds are shown in Table 1 and Fig. 1B, respectively. Studies on the viability of infected cells revealed that a cytopathic effect (CPE), syncytium formation, of VeroE6/TMPRSS2 cells by SARS-CoV-2 was also almost completely prevented by the addition of Pin1 inhibitors to the culture medium (The results of H-77 are shown in Fig. 2C,D.).
The 50% effective concentration (EC 50 ) was calculated using the virus production at different drug concentrations as an indicator ( Supplementary Fig. 1), and the values were entered in Table 1. Since H-77 showed the smallest value of 3.2 µM, more detailed studies were performed using H-77 as a potent suppressor of SARS-CoV-2 proliferation.
The concentration-dependent effect of H-77 against SARS-CoV-2 was shown by measuring viral protein levels in VeroE6/TMPRSS2 cells ( Fig. 2A) or viral RNA isolated from the culture medium (Fig. 2B). Membrane fusion, a CPE caused by viral infection, became less apparent as the drug concentration was increased to 5 μM and was almost absent at concentrations above 7.5 μM (Fig. 2D). This trend was evident in the fusion index, which quantifies the degree of membrane fusion (Fig. 2C). Considering the data obtained by disrupting Pin1 activity with siRNA and various Pin1 inhibitory compounds, it can be concluded that Pin1 is essential for SARS-CoV-2 proliferation.
We therefore next investigated whether H-77 can exert its inhibitory effect even when added at the same time as the SARS-CoV-2 infection or after in order to determine its applicability as a therapeutic agent (Fig. 3A). Our results showed that H-77 almost completely blocked SARS-CoV-2 proliferation when added 2 h after infection and showed a weaker but still significant inhibitory effect when added 6 h after infection (Fig. 3B,C). The amount of genomic RNA released from the cells was significantly reduced by H-77 treatment (Fig. 3D). In addition, intracellular viral N mRNA was significantly reduced, although some genome RNA was also mixed in (Fig. 3E), providing evidence that H-77 inhibits viral proliferation at the viral RNA transcription step or earlier.
Five potent Pin1 inhibitors, including H-77, were applied for 2 h before virus infection, followed by washing out the Pin1 inhibitors before virus infection. N-protein synthesis of SARS-CoV-2 was strongly inhibited even after washout (Fig. 4A,B). These results indicate that the Pin inhibitor is effective if the cells are pretreated immediately before virus infection.

Inhibition of viral replication by a medical agent.
At present, no highly specific Pin1 inhibitor is commercially available for either medical or experimental purposes. Although Juglone is the most commonly used Pin1 inhibitor compound for basic research, it reportedly binds to and inhibits the activity of many proteins, including tubulin, in addition to Pin1, and it was found that VeroE6/TMPRSS2 cells were unable to survive incubation with 2 μM Juglone for more than 12 h. As an alternative, we tested all-trans retinoic acid (ATRA), an agonist of the retinoic acid receptor (RAR) that is used medically to treat acute promyelocytic leukemia (APL) and was recently reported to inactivate Pin1 isomerase activity 20 . The activities of ATRA as an RAR agonist and a Pin1 inhibitor both contribute to the suppression of APL cell growth 20 . As a result, it was found that ATRA similarly suppressed SARS-CoV-2 proliferation as shown by marked reductions in protein and viral RNA levels in a concentration-dependent manner (Fig. 5A,B) and alleviated its CPE ( Supplementary Fig. 2), although the EC 50 of ATRA (17.9 µM, Supplementary Fig. 1) was higher than that of H-77 (3.2 µM).

Discussion
This study is the first study to demonstrate the essential role of Pin1 in SARS-CoV-2 proliferation and the possibility of Pin1 inhibition as a promising therapy against COVID-19. In the present study, the knockdown of Pin1 by siRNA inhibited the growth of SARS-CoV-2. On the other hand, we performed overexpression experiments using a Pin1 expression plasmid, but the results were unclear. Pin1 is upregulated in VeroE6/TMPRSS2 cells, which may have made it difficult to get results from the overexpression experiment. Pin1 tends to be upregulated in cancer cells and cells with active proliferation 10,11 . The inhibitory activities of our Pin1 inhibitors, H-77,  Table 1). However, potential non-specific effects on other proteins such as other PPIase enzymes or kinases have not yet been sufficiently ruled out. On the other hand, ATRA reportedly inhibits the activity Pin1 but does not affect the activity of FKBP or cyclophilin 20 . Thus, our results using Pin1 siRNAs and ATRA strongly support the involvement of Pin1 rather than that of other PPIases in the proliferation of SARS-CoV-2. We hypothesize that it is highly likely that the inhibitory effects of our five compounds on SARS-CoV-2 proliferation are mediated specifically through Pin1 inhibition, although the possibility of the existence of an additional mechanism(s) cannot be ruled out.
Interestingly, Pin1 has also been reported to enhance the proliferation of several other viruses including human immunodeficiency virus type 1 (HIV-1) 15 , hepatitis C virus (HCV) 16 , Epstein-Barr virus (EBV) 17 , human T-lymphotropic virus type 1 (HTLV-1) 18 , and feline coronavirus 14 . The molecular mechanisms underlying Pin1induced enhancement of viral proliferation can be largely divided into two mechanisms. One is mediation by enhanced production of oncogenic or inflammatory proteins in the host cells via association of Pin1 with cyclin D1, NF-kB, and Tax 18 . The other is direct involvement of Pin1 in various aspects of the life cycle of viruses such as core exuviation, genome integration, and RNA or DNA replication. For example, Pin1 has been shown to In conclusion, our study clearly showed an essential role of Pin1 in SARS-CoV-2 proliferation. Accordingly, the use of Pin1 inhibitors might be an effective therapy against COVID-19. Our study also indicated the necessity for optimizing and/or developing novel compounds with both potent Pin1 inhibitory activity and high specificity.  Table 1. These Pin1 inhibitors inhibit isomerase activity by more than 80% at a concentration of 20 μM, based on an in vitro assay using recombinant Pin1 protein. However, it should be noted that the results of such an in vitro assay usually differ significantly from the results obtained by in vivo experiments. The compounds were solubilized in DMSO. Before the infection experiments, the culture medium of VeroE6/TMPRSS2 cells was changed to DMEM without FBS and G-418, and virus and/or Pin1 inhibitors were added at the indicated titer or concentrations.

SARS-CoV-2 infection.
The SARS-CoV-2/JP/Hiroshima-46059T/2020 strain (accession number MZ853926), which was isolated from a cluster infection in Hiroshima 21 , was used. To prepare virus suspensions, VeroE6/TMPRSS2 cells were infected with the virus and incubated in DMEM. The virus titer was determined by the standard 50% tissue culture infectious dose (TCID 50 ) method and expressed as TCID 50 /ml as described previously 22 . SARS-CoV-2 infection was performed in the BSL3 facility of Hiroshima University. Unless oth-