Learning from SARS: Preparing for the Next Disease Outbreak: Workshop Summary.
IN VITRO ANTIVIRAL ACTIVITY OF HUMAN RHINOVIRUS 3C PROTEASE INHIBITORS AGAINST THE SARS CORONAVIRUS
David A. Matthews, Amy K. Patick, Robert O. Baker, Mary A. Brothers, Peter S. Dragovich, Chris J. Hartmann, Theodore O. Johnson, Eric M. Mucker, Siegfried H. Reich, Paul A. Rejto, Peter W. Rose, Susan H. Zwiers, and John W. Huggins.
Rhinovirus Protease Inhibitor May Point Way To SARS Drugs
Published: Jan 14, 2004
NEW YORK (Reuters Health) – Although no longer considered a candidate for treating the common cold, an experimental nasal spray decreases viral shedding and reduces symptoms of experimental rhinoviral infection. And now, new research suggests the drug provides a base for developing drugs to fight severe acute respiratory syndrome (SARS), according to Pfizer scientists.
“This study was monumental, in that it provides the first proof of concept that you can take an inhibitor directed toward a novel virus target and in an experimental setting produce a reduction in symptomology and a reduction in levels of virus,” Dr. Amy K. Patick, at Pfizer in San Diego, told Reuters Health.
The compound, ruprintrivir, inhibits human rhinovirus replication by blocking the 3C protease, according to a study published in the December issue of Antimicrobial Agents and Chemotherapy.
Dr. Patick’s team analyzed the prophylactic potential of ruprintrivir in 103 healthy subjects. When administered 6 hours prior to rhinovirus challenge, ruprintrivir reduced the frequency of having at least one positive viral culture 35% to 37% compared with placebo, and viral titer and RNA levels were significantly reduced. Nasal discharge weights were reduced by 41% to 55% over the 5-day study period.
And in a phase II trial of treatment efficacy, ruprintrivir administered 12 hours after viral challenge reduced symptom scores by 33% compared with placebo (p = 0.01). But Dr. Patick said that in a natural setting, where the drug was administered within 24 to 36 hours of symptom onset, results were disappointing.
“At that point, Pfizer decided to put further research on ruprintrivir on hold,” she said. “We were asking too much of a disease that has such a short window” between rhinovirus infection and symptom onset.
However, that was shortly before the SARS epidemic broke out. In response to an appeal by the U.S. government, Pfizer submitted ruprintrivir along with hundreds of other compounds they had already tested against rhinovirus, to examine their effect against SARS.
It turns out that “quite a few had moderate activity” against the SARS coronavirus, including ruprintrivir, Dr. Patick said.
As a result, Pfizer is attempting to redesign a drug to more effectively inhibit the 3c protease of the SARS coronavirus. Their research team “is working day and night… designing, synthesizing and testing inhibitors in cell culture,” Dr. Patick said.
“If we develop an inhibitor of the SARS 3c protease, there is a good likelihood that we’ll be able to show efficacy in humans,” she added.
Source: Antimicrob Agents Chemother 2003;47:3907-3916.
Inhibitors of cathepsin L prevent severe acute respiratory syndrome coronavirus entry
Graham Simmons, Dhaval N. Gosalia, Andrew J. Rennekamp, Jacqueline D. Reeves, Scott L. Diamond, and Paul Bates
PNAS August 16, 2005 102 (33) 11876-11881; https://doi.org/10.1073/pnas.0505577102
Abstract
Severe acute respiratory syndrome (SARS) is caused by an emergent coronavirus (SARS-CoV), for which there is currently no effective treatment. SARS-CoV mediates receptor binding and entry by its spike (S) glycoprotein, and infection is sensitive to lysosomotropic agents that perturb endosomal pH. We demonstrate here that the lysosomotropic-agent-mediated block to SARS-CoV infection is overcome by protease treatment of target-cell-associated virus. In addition, SARS-CoV infection was blocked by specific inhibitors of the pH-sensitive endosomal protease cathepsin L. A cell-free membrane-fusion system demonstrates that engagement of receptor followed by proteolysis is required for SARS-CoV membrane fusion and indicates that cathepsin L is sufficient to activate membrane fusion by SARS-CoV S. These results suggest that SARS-CoV infection results from a unique, three-step process: receptor binding and induced conformational changes in S glycoprotein followed by cathepsin L proteolysis within endosomes. The requirement for cathepsin L proteolysis identifies a previously uncharacterized class of inhibitor for SARS-CoV infection.
Boceprevir, GC-376, and calpain inhibitors II, XII inhibit SARS-CoV-2 viral replication by targeting the viral main protease
- Chunlong Ma, Michael Dominic Sacco, Brett Hurst, Julia Alma Townsend, Yanmei Hu, Tommy Szeto, Xiujun Zhang, Bart Tarbet, Michael Thomas Marty, Yu Chen & Jun Wang
- Cell Research volume 30, pages678–692(2020)
Abstract
A new coronavirus SARS-CoV-2, also called novel coronavirus 2019 (2019-nCoV), started to circulate among humans around December 2019, and it is now widespread as a global pandemic. The disease caused by SARS-CoV-2 virus is called COVID-19, which is highly contagious and has an overall mortality rate of 6.35% as of May 26, 2020. There is no vaccine or antiviral available for SARS-CoV-2. In this study, we report our discovery of inhibitors targeting the SARS-CoV-2 main protease (Mpro). Using the FRET-based enzymatic assay, several inhibitors including boceprevir, GC-376, and calpain inhibitors II, and XII were identified to have potent activity with single-digit to submicromolar IC50 values in the enzymatic assay. The mechanism of action of the hits was further characterized using enzyme kinetic studies, thermal shift binding assays, and native mass spectrometry. Significantly, four compounds (boceprevir, GC-376, calpain inhibitors II and XII) inhibit SARS-CoV-2 viral replication in cell culture with EC50 values ranging from 0.49 to 3.37 µM. Notably, boceprevir, calpain inhibitors II and XII represent novel chemotypes that are distinct from known substrate-based peptidomimetic Mpro inhibitors. A complex crystal structure of SARS-CoV-2 Mpro with GC-376, determined at 2.15 Å resolution with three protomers per asymmetric unit, revealed two unique binding configurations, shedding light on the molecular interactions and protein conformational flexibility underlying substrate and inhibitor binding by Mpro. Overall, the compounds identified herein provide promising starting points for the further development of SARS-CoV-2 therapeutics.