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. 2024 Sep 26;19(9):e0310915.
doi: 10.1371/journal.pone.0310915. eCollection 2024.

The heat shock protein 90 inhibitor RGRN-305 attenuates SARS-CoV-2 spike protein-induced inflammation in vitro but lacks effectiveness as COVID-19 treatment in mice

Hakim Ben Abdallah  1 , Giorgia Marino  2 , Manja Idorn  2 , Line S Reinert  2 , Anne Bregnhøj  1 , Søren Riis Paludan  2 , Claus Johansen  1
Affiliations

The heat shock protein 90 inhibitor RGRN-305 attenuates SARS-CoV-2 spike protein-induced inflammation in vitro but lacks effectiveness as COVID-19 treatment in mice

Hakim Ben Abdallah et al. PLoS One. .

Abstract

The inhibition of heat shock protein 90 (HSP90), a molecular chaperone, has been proposed to be a potential novel treatment strategy for Coronavirus disease 2019 (COVID-19). In contrast to other studies, our data demonstrated that RGRN-305, a HSP90 inhibitor, exacerbated the cytopathic effect and did not reduce the viral shedding in VeroE6-hTMPRSS2 cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Likewise in a murine model of SARS-CoV-2, transgenic mice treated orally with RGRN-305 exhibited reduced survival by the end of the experiment (day 12) as 14% (1/7) survived compared to 63% (5/8) of those treated with drug-vehicle. Animal weight was not reduced by the RGRN-305 treatment. Interestingly, we demonstrated that inhibition of HSP90 by RGRN-305 significantly dampened the inflammatory response induced by SARS-CoV-2 spike protein in human macrophage-like cells (U937) and human lung epithelial cells (A549). Measured by quantitative real-time PCR, the mRNA expression of the proinflammatory cytokines TNF, IL1B and IL6 were significantly reduced. Together, these data suggest that HSP90 inhibition by RGRN-305 exacerbates the SARS-CoV-2 infection in vitro and reduces the survival of mice infected with SARS-CoV-2, but exhibits strong anti-inflammatory properties. This data shows that while RGRN-305 may be helpful in a 'cytokine storm', it has no beneficial impact on viral replication or survival in animals as a monotherapy. Further animal studies with HSP90 inhibitors in combination with an anti-viral drug may provide additional insights into its utility in viral infections and whether HSP90 inhibition may continue to be a potential treatment strategy for COVID-19 disease.

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Conflict of interest statement

I have read the journal’s policy and the authors of this manuscript have the following competing interests: H.B.A has received research grants from Aage Bangs Fund and participated in clinical trials sponsored by Galderma, Regranion and UCB. C.J. has served as a paid speaker for LEO Pharma, Eli Lilly and L’Oréal This does not alter our adherence to PLOS ONE policies on sharing data and materials.

Figures

Fig 1
Fig 1. ED50/LD50 for RGRN-305.
Toxicity of RGRN-305 and efficacy on inhibition of SARS-CoV2 infection were assessed in VeroE6-hTMPRSS2 cells in an ED50%/LD50% assay. The VeroE6-hTMPRSS2 cells were preincubated with RGRN-305 (0.1 μM, 1.0 μM and 10 μM) or vehicle 1 hour prior to infection with SARS-CoV2 B.1. (FR4286) isolate at a multiplicity of infection of 0.05 and 0.5. Cytopathic effect was assessed 72 hours post infection in cells stained with crystal violet (CV) solution. (A) Infection and toxicity were quantified by microscopy. Infection with the SARS-CoV2 B.1. strain caused cytopathological changes in the cells that were stained by CV solution as visible CV-dense spots/areas under the microscope. The SARS-CoV2 B.1. strain did not cause lytic replication or cell death in VeroE6-hTMPRSS2 under these conditions. Drug toxicity caused cell death resulting in the detachment of the VeroE6-hTMPRSS2 cells. Toxicity (cell death) was visualized as CV-negative (blank) areas under the microscope, corresponding to the detachment of dead cells. (B) % cytopathic effect caused by toxicity in uninfected wells (black line), % cytopathic effect caused by SARS-CoV2 infection (blue line), and % cytopathic toxicity scored in infected wells (Grey). % cytopathic effect (infection and toxicity) was calculated on biological triplicates of 8 wells. Abbreviations: CV, crystal violet UI, uninfected.
Fig 2
Fig 2. TCID 50% on release of viral progeny.
The release of new viral progeny from SARS-CoV-2 infected VeroE6-hTMPRSS2 cells treated with RGRN-305 or 17-AAG. The VeroE6-hTMPRSS2 cells were pretreated with vehicle, RGRN-305 or 17-AAG (0.01 μM, 0.1 μM and 1.0 μM) 1 hour prior to infection with B.1. (FR4286) isolate at a multiplicity of infection of 0.05. After 48 hours, the supernatant was collected and the release of virus was quantified by a Tissue Culture Infectious Dose (TCID50) assay and the Reed-Muench method. The release of SARS-CoV-2 was increased by RGRN-305 and 17-AAG treatment.
Fig 3
Fig 3. HSP90 inhibition by RGRN-305 decreased the gene expression of proinflammatory cytokines induced by SARS-CoV-2 S protein.
RGRN-305 (HSP90 inhibitor) inhibited the mRNA expression of TNF, IL1β and IL6 induced by SARS-CoV-2 S protein in human macrophage-like U937 and human lung epithelial A549 cells. (A) U937 macrophage-like cells were pretreated with RGRN-305 (5 μM) or water (drug-vehicle) for 8 hours prior to stimulation with SARS-CoV-2 S protein at a concentration of 500 ng/mL and 5000 ng/mL. After 8- and 24-hours post-stimulation, the mRNA expression of TNF, IL1β and IL6 was measured by RT-qPCR (n = 4) (B) A549 cells were pretreated with RGRN-305 or water (drug-vehicle) for 8 hours before stimulation with SARS-CoV-2 S protein at a concentration of 1000 ng/mL for 24 and 48 hours. The expression of the indicated cytokines was measured by RT-qPCR (n = 5). Data represent mean ± SEM. * p < 0.05 compared to vehicle. ** p < 0.05 compared to corresponding stimulation without preincubation with RGRN-305.
Fig 4
Fig 4. HSP90 inhibition by RGRN-305 and 17-AAG decreased proinflammatory protein secretion.
RGRN-305 and 17-AAG reduced the secretion of TNF, IL-1β and IL-6 in U937 and A549 cells challenged with SARS-CoV-2 S protein. (A) U937 cells and (B) A549 cells were pretreated with drug-vehicle (water), RGRN-305 (5 μM) or 17-AAG (1 μM) for 8 hours followed by SARS-CoV-2 S protein challenge (U937, 5000 ng/ml; A549, 1000 ng/ml) for 24 hours. The supernatant was collected and the secreted protein expression of TNF, IL-1β and IL-6 was measured with ELISA. Data are shown as mean ± SEM. * p < 0.05 compared to vehicle. ** p < 0.05 compared to corresponding SARS-CoV-2 S protein stimulation without preincubation with RGRN-305 or 17-AAG.
Fig 5
Fig 5. Effect of SARS-CoV-2 infection on mortality and body weight of K18-hACE C57BL/6J mice treated with RGRN-305 or vehicle.
(A-B) Mice were inoculated with 1.5*103 PFU of SARS-CoV-2 and treated once daily with 40 mg/kg body weight RGRN-305 (n = 7) or vehicle (n = 8) by oral gavage till the end of the experiment (day 12). (A) Kaplan-Meier survival curves of the mice treated with RGRN-305 (dashed line) and vehicle (black line) (B) The relative change of body weight compared to the weight before inoculation with SARS-CoV-2 of mice treated with RGRN-305 (black squares) compared to mice treated with vehicle (white squares). The dotted line corresponds to the 20% bodyweight reduction human endpoint criterion for euthanasia. Abbreviations: n.s., not significant. PFU, plaque-forming unit.
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Grants and funding

This study was supported by a research grant to HBA received from Aage Bangs Fundation. The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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