Postinfection treatment with a protease inhibitor increases survival of mice with a fatal SARS-CoV-2 infection

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection continues to be a serious global public health threat. The 3C-like protease (3CLpro) is a virus protease encoded by SARS-CoV-2, which is essential for virus replication. We have previously reported a series of small-molecule 3CLpro inhibitors effective for inhibiting replication of human coronaviruses including SARS-CoV-2 in cell culture and in animal models. Here we generated a series of deuterated variants of a 3CLpro inhibitor, GC376, and evaluated the antiviral effect against SARS-CoV-2. The deuterated GC376 displayed potent inhibitory activity against SARS-CoV-2 in the enzyme- and the cell-based assays. The K18-hACE2 mice develop mild to lethal infection commensurate with SARS-CoV-2 challenge doses and were proposed as a model for efficacy testing of antiviral agents. We treated lethally infected mice with a deuterated derivative of GC376. Treatment of K18-hACE2 mice at 24 h postinfection with a derivative (compound 2) resulted in increased survival of mice compared to vehicle-treated mice. Lung virus titers were decreased, and histopathological changes were ameliorated in compound 2–treated mice compared to vehicle-treated mice. Structural investigation using high-resolution crystallography illuminated binding interactions of 3CLpro of SARS-CoV-2 and SARS-CoV with deuterated variants of GC376. Taken together, deuterated GC376 variants have excellent potential as antiviral agents against SARS-CoV-2.

Structure-Guided Design of Potent Inhibitors of SARS-CoV-2 3CL Protease: Structural, Biochemical, and Cell-Based Studies

“… 32 An alternative synthesis was used in the case of compounds 6 – 8 , 10 – 16 , 23 , and 24 , which involved the reaction of the precursor alcohol with (L) leucine methyl ester isocyanate, as described in detail previously. 33 The synthesis of precursor amino alcohol A was readily accomplished by coupling (L) Z-Leu with a glutamine surrogate, followed by sequential reduction with LiBH 4 and removal of the protective group (H 2 /Pd) ( Scheme 1 /panel C).…”

“…The expression and purification of the 3CLpro of MERS-CoV, as well as the FRET enzyme assays, were performed as described previously. 19 − 21 , 33 …”

“…The inhibition curve ( Figure 1 /panel C) for each compound was prepared, and the 50% effective concentration (EC 50 ) values were determined by GraphPad Prism software using a variable slope (GraphPad, La Jolla, CA). Compounds 4c and 15c were selected and examined for the antiviral effects with live SARS-CoV-2 in Vero E6 cells as described before, 33 and the EC 50 was calculated by the same method described above. A known SARS-CoV-2 3CLpro inhibitor, GC376, was used as the positive control in each experiment.…”

Drug Combinations as a First Line of Defense against Coronaviruses and Other Emerging Viruses

“…The reported synergies of the HCV drugs with RDV do demonstrate the potential utility of combinations targeting the SARS-CoV-2 polymerase and its proteases with DAAs (e.g., MPV plus Paxlovid [ 18 , 22 ]). Other new oral CoV protease inhibitors ( 22 , 112 , 113 , 125 – 127 ) and polymerase inhibitors ( 97 , 128 , 129 ) should also be considered if they have suitable C max /EC 50 ratios. Similarly, new TMPRSS2 inhibitors being explored ( 130 – 134 ) may have more suitable PK properties than camostat.…”

“…While approved drugs have the advantage that FDA guidance on the development of drug combinations allows for more rapid development ( https://www.fda.gov/media/80100 ), drugs that have passed human safety trials should also be considered as information about them becomes available. In this respect, it will be important to follow the development of AT-527 ( 128 ), other potential oral SARS-CoV-2 polymerase inhibitors ( 97 , 129 ) including oral forms of RDV ( 139 – 141 , 200 ) as well as the oral protease inhibitor GC-376 ( 102 , 125 ). As new oral/inhaled drugs with the same targets but better efficacy and PK become available, they should be analyzed as substitutes in previously characterized drug synergies.…”