Structure-based phylogeny identifies avoralstat as a TMPRSS2 inhibitor that prevents SARS-CoV-2 infection in mice

Abstract

Drugs targeting host proteins can act prophylactically to reduce viral burden early in disease and limit morbidity, even with antivirals and vaccination. Transmembrane serine protease 2 (TMPRSS2) is a human protease required for SARS-CoV-2 viral entry and may represent such a target. We hypothesized that drugs selected from proteins related by their tertiary structure, rather than their primary structure, were likely to interact with TMPRSS2. We created a structure-based phylogenetic computational tool named 3DPhyloFold to systematically identify structurally similar serine proteases with known therapeutic inhibitors and demonstrated effective inhibition of SARS-CoV-2 infection in vitro and in vivo. Several candidate compounds, Avoralstat, PCI-27483, Antipain, and Soybean-Trypsin-Inhibitor, inhibited TMPRSS2 in biochemical and cell infection assays.Avoralstat, a clinically tested Kallikrein-related B1 inhibitor, inhibited SARS-CoV-2 entry and replication in human airway epithelial cells. In an in vivo proof of principle, Avoralstat significantly reduced lung tissue titers and mitigated weight-loss when administered prophylactically to SARS-CoV-2 susceptible mice indicating its potential to be repositioned for COVID-19 prophylaxis in humans.

The Transmembrane Protease TMPRSS2 as a Therapeutic Target for COVID-19 Treatment

“…In line with these findings, CM efficiently inhibits TMPRSS2-dependent entry of SARS-CoV-2 [74,83,97,117,[119][120][121][122][123][124]. Further in vitro studies on cell-associated or purified TMPRSS2 protein confirmed a reduction of TMPRSS2 protease activity by CM (Table 1) [83,120,[123][124][125][126][127]. Moreover, oral CM treatment protected mice from SARS-CoV-induced mortality [96].…”

“…Subsequently, CM has been reported to reduce the infectivity of various respiratory viruses including SARS-CoV, MERS-CoV, NL63, 229E, OC43, HKU-1, and influenza virus at micromolar concentrations [66,70,71,74,85,87,88,91,92,97,[116][117][118]. In line with these findings, CM efficiently inhibits TMPRSS2-dependent entry of SARS-CoV-2 [74,83,97,117,[119][120][121][122][123][124]. Further in vitro studies on cell-associated or purified TMPRSS2 protein confirmed a reduction of TMPRSS2 protease activity by CM (Table 1) [83,120,[123][124][125][126][127].…”

“…Other small molecule compounds were identified in two in silico screenings for TMPRSS2 inhibitors: the factor Xa inhibitor Otamixaban, the urokinase-type plasminogen activator UKI-1, the kallikrein inhibitor Avoralstat, and the factor VIIa inhibitor PCI-27483 all decreased TMPRSS2 activity (Table 1) [120,125]. However, only PCI-27483 and Avoralstat were shown to efficiently curb SARS-CoV-2-mediated entry and infection, with avoralstat being the more potent inhibitor [120]. Subsequent infection studies on hACE2-transduced BALB/c mice revealed that intraperitoneal administration of Avoralstat reduces SARS-CoV-2 titer in the lung and weight loss in a similar manner to CM [120].…”

“…However, only PCI-27483 and Avoralstat were shown to efficiently curb SARS-CoV-2-mediated entry and infection, with avoralstat being the more potent inhibitor [120]. Subsequent infection studies on hACE2-transduced BALB/c mice revealed that intraperitoneal administration of Avoralstat reduces SARS-CoV-2 titer in the lung and weight loss in a similar manner to CM [120].…”

A novel highly potent inhibitor of TMPRSS2-like proteases blocks SARS-CoV-2 variants of concern and is broadly protective against infection and mortality in mice

“…Plitidepsin is a promising HDA, which targets the ubiquitously expressed elongation factor 1-alpha and has demonstrated high potency (ED = 1.62 nM and SI = 40.4) against SARS-CoV-2 infection in pneumocyte-like cells 21 . Cm and nafamostat mesylate are also HDAs targeting serine proteases, including host TTSPs, that are undergoing human trials against SARS-CoV-2; however, no significant protection against infection was observed in the adenovirus hACE2 model (Cm) or hamster model (nafamostat mesylate) of SARS-CoV-2 infection 4,51,52 . Recently reported clinical trial data for Cm treatment of hospitalized COVID-19 patients demonstrated a lack of impact on time to recovery and incidence of death following SARS-CoV-2 infection 19 .…”