Protease inhibitors targeting coronavirus and filovirus entry

Yanchen Zhou, Punitha Vedantham, Kai Lu, Juliet Agudelo, Ricardo Carrion Jr, Jerritt W Nunneley, Dale Barnard, Stefan Pöhlmann, James H McKerrow, Adam R Renslo, Graham Simmons, Yanchen Zhou, Punitha Vedantham, Kai Lu, Juliet Agudelo, Ricardo Carrion Jr, Jerritt W Nunneley, Dale Barnard, Stefan Pöhlmann, James H McKerrow, Adam R Renslo, Graham Simmons

Abstract

In order to gain entry into cells, diverse viruses, including Ebola virus, SARS-coronavirus and the emerging MERS-coronavirus, depend on activation of their envelope glycoproteins by host cell proteases. The respective enzymes are thus excellent targets for antiviral intervention. In cell culture, activation of Ebola virus, as well as SARS- and MERS-coronavirus can be accomplished by the endosomal cysteine proteases, cathepsin L (CTSL) and cathepsin B (CTSB). In addition, SARS- and MERS-coronavirus can use serine proteases localized at the cell surface, for their activation. However, it is currently unclear which protease(s) facilitate viral spread in the infected host. We report here that the cysteine protease inhibitor K11777, ((2S)-N-[(1E,3S)-1-(benzenesulfonyl)-5-phenylpent-1-en-3-yl]-2-{[(E)-4-methylpiperazine-1-carbonyl]amino}-3-phenylpropanamide) and closely-related vinylsulfones act as broad-spectrum antivirals by targeting cathepsin-mediated cell entry. K11777 is already in advanced stages of development for a number of parasitic diseases, such as Chagas disease, and has proven to be safe and effective in a range of animal models. K11777 inhibition of SARS-CoV and Ebola virus entry was observed in the sub-nanomolar range. In order to assess whether cysteine or serine proteases promote viral spread in the host, we compared the antiviral activity of an optimized K11777-derivative with that of camostat, an inhibitor of TMPRSS2 and related serine proteases. Employing a pathogenic animal model of SARS-CoV infection, we demonstrated that viral spread and pathogenesis of SARS-CoV is driven by serine rather than cysteine proteases and can be effectively prevented by camostat. Camostat has been clinically used to treat chronic pancreatitis, and thus represents an exciting potential therapeutic for respiratory coronavirus infections. Our results indicate that camostat, or similar serine protease inhibitors, might be an effective option for treatment of SARS and potentially MERS, while vinyl sulfone-based inhibitors are excellent lead candidates for Ebola virus therapeutics.

Keywords: Cathepsin; Coronavirus; Filovirus; Vinylsulfones.

Copyright © 2015. Published by Elsevier B.V.

Figures

Scheme 1
Scheme 1
Synthesis of K11777 and P3-modified vinylsulfone analogs.
Fig. 1
Fig. 1
Identification of protease inhibitor K11777 as a broad-spectrum antiviral drug candidate. (a) Structure of K11777; N-methyl-piperazine-phenylalanyl-homophenylalanyl-vinylsulfone phenyl. (b) Dose response curves of compound K11777 against pseudoviruses with a variety of different viral envelopes. Data shown as mean ± SD of triplicate measurements. Representative experiments of at least three experiments are shown.
Fig. 2
Fig. 2
Inhibition of serine and/or cysteine proteases in cells transfected with, or endogenously expressing, TMPRSS2. 293T-CD13 cells transiently expressing TMPRSS2 (a) or Caco2 cells (b) were pretreated with serially diluted compound K11777, or a combination of serially diluted K11777 and camostat at two different concentrations (1 or 10 μM), followed by incubation with infectivity-normalized pseudoviruses in the presence of the inhibitors. The cells were then cultured at 37 °C/5% CO2 for two days and luciferase expression was measured. (a) Simultaneous treatment with both K11777 and camostat for 229E-S, EBOV or VSV-G pseudovirus infection. (b) Enhanced inhibition by a combination of K11777 and camostat for 229E-S mediated viral entry using Caco2 cells.
Fig. 3
Fig. 3
Effects of per os administered SMDC256160 and/or camostat on survival of BALB/c mice infected with a lethal SARS-CoV. Ten mice per group were dosed twice a day by oral gavage with SMDC256160 and/or camostat or diluent alone (sterile water) for 9 days beginning 10 h prior to infection with 10,000 pfu of mouse-adapted SARS-CoV.

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Source: PubMed

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