Proxalutamide reduces SARS-CoV-2 infection and associated inflammatory response

Early in the COVID-19 pandemic, data suggested that males had a higher risk of developing severe disease and that androgen deprivation therapy might be associated with protection. Combined with the fact that TMPRSS2, a host entry factor for the SARS-CoV-2 virus, was a well-known androgen-regulated gene, this led to an upsurge of research investigating androgen receptor (AR)-targeting drugs. Proxalutamide, a newer AR antagonist, was shown in initial clinical studies to benefit COVID-19 patients; however, further validation is needed as one study was retracted. Due to continued interest in proxalutamide, which is in several phase 3 trials, we examined its ability to impact SARS-CoV-2 infection and associated downstream inflammatory responses. Proxalutamide exerted similar effects as enzalutamide, an AR antagonist commonly prescribed for advanced prostate cancer treatment, in decreasing AR signaling and expression of TMPRSS2 and ACE2, the SARS-CoV-2 receptor. However, proxalutamide led to degradation of AR protein, an effect that was not observed with enzalutamide. Proxalutamide inhibited SARS-CoV-2 infection with an IC50 value of 97 nM, compared to 281 nM for enzalutamide. Importantly, proxalutamide inhibited infection by multiple SARS-CoV-2 variants and synergized with remdesivir. Proxalutamide also protected against cell death in response to TNF and IFN, and overall survival of mice was increased with proxalutamide treatment prior to cytokine exposure. Mechanistically, we found that proxalutamide increased levels of NRF2, an essential transcription factor that mediates antioxidant responses, and could decrease lung inflammation. These data provide compelling evidence that proxalutamide can prevent SARS-CoV-2 infection and cytokine-induced lung damage, suggesting that promising clinical data may emerge from ongoing phase 3 trials. To compare the transcriptomic changes associated with proxalutamide and enzalutamide, RNA-sequencing (RNA-Seq) analysis was carried out in androgen receptor (AR)-positive prostate cancer LNCaP cells using either 20 µM proxalutamide or enzalutamide for eight hours of treatment.