The Mac1 ADP-ribosylhydrolase is a Therapeutic Target for SARS-CoV-2

Abstract

SARS-CoV-2 continues to pose a threat to public health. Current therapeutics remain limited to direct acting antivirals that lack distinct mechanisms of action and are already showing signs of viral resistance. The virus encodes an ADP-ribosylhydrolase macrodomain (Mac1) that plays an important role in the coronaviral lifecycle by suppressing host innate immune responses. Genetic inactivation of Mac1 abrogates viral replication in vivo by potentiating host innate immune responses. However, it is unknown whether this can be achieved by pharmacologic inhibition and can therefore be exploited therapeutically. Here we report a potent and selective lead small molecule, AVI-4206, that is effective in an in vivo model of SARS-CoV-2 infection. Cellular models indicate that AVI-4206 has high target engagement and can weakly inhibit viral replication in a gamma interferon- and Mac1 catalytic activity-dependent manner; a stronger antiviral effect for AVI-4206 is observed in human airway organoids. In an animal model of severe SARS-CoV-2 infection, AVI-4206 reduces viral replication, potentiates innate immune responses, and leads to a survival benefit. Our results provide pharmacological proof of concept that Mac1 is a valid therapeutic target via a novel immune-restoring mechanism that could potentially synergize with existing therapies targeting distinct, essential aspects of the coronaviral life cycle. This approach could be more widely used to target other viral macrodomains to develop antiviral therapeutics beyond COVID-19.

Figure 1:. Iterative structure-based design and optimization of AVI-4206 activity against Mac1.

(A) Evolution of the early lead AVI-219 to AVI-4206 by introducing and optimizing urea functionality as found in AVI-92 to contact Asp22 and introducing geminal dimethyl substitution of the pyrrolidinone ring. HTRF-based IC₅₀ values from (B) and (C), and PDB codes from (E) are indicated. (B and C) HTRF-based dose response curves showing peptide displacement of an ADPr-conjugated peptide from Mac1 by compounds from the urea (B) and the pyrrolidinone ring (C) optimization paths. Data is plotted as % competition mean ± SD of three technical replicates. Data were fitted with a sigmoidal dose-response equation using non-linear regression and the IC₅₀ values are quoted with 95% confidence intervals. (D) Mac1 catalytic activity dose response curve for indicated compounds. Data is plotted as % inhibition mean ± SD of four technical replicates. IC₅₀ values are quoted with 95% confidence intervals. (E) X-ray structures indicating conserved interactions during the optimization path from AVI-92 and AVI-219 (left) to AVI-4206 (right). Structures of compounds from the urea and the pyrrolidinone ring optimization paths are presented in the top and bottom middle panels, respectively. Multiple ligand conformations were observed for AVI-3367, AVI-3762 and AVI-4636 (labeled A and B). The F_O-F_C difference electron density map calculated prior to ligand modeling is shown for AVI-4206 (purple mesh contoured at 5 σ). Electron density maps used to model ligand other ligands are shown in Supplementary Figure 1.

Figure 2:. AVI-4206 engages Mac1 with high potency and selectivity in cells.

(A) CETSA-nLuc shows differential Mac1 stabilization after treatment of A549 cells with 10 μM of indicated compounds. Data are presented as mean ± SD of two technical replicates. Data were fitted with a sigmoidal dose-response equation using non-linear regression (gray line) and the T_agg values are quoted with 95% confidence intervals. (B) CETSA-nLuc shows a dose-dependent thermal stabilization of Mac1 after treatment of A549 cells with increasing concentrations of AVI-4206. Data are presented as mean ± SD of two technical replicates. (C and D) HTRF-based dose response curves showing displacement of an ADPr-conjugated peptide from indicated proteins by ADP-ribose (C) or AVI-4206 (D). ADP-ribose was used as a positive control. Data are presented as mean ± SD of three technical replicates. IC₅₀ values are quoted with 95% confidence intervals. (E) Structural modeling of MacroD2 (top, PDB code 4IQY) and Targ1 (bottom, PDB code 4J5S) showing design elements that prevent AVI-4206 cross reactivity. The atoms of clashing residues (Cys140 in MacroD2, Arg122 in Targ1) are shown with a dot representation. The ADP-ribose present in both human macrodomain structures has been omitted for clarity.

Figure 3:. AVI-4206 shows limited efficacy in cellular models of SARS-CoV-2 infection.

(A and B) Vero-TMPRSS2 (A) or A549-ACE2^h (B) cells were pretreated with compounds and infected with mNeonGreen reporter SARS-CoV-2. mNeonGreen expression was measured by the Incucyte system. Graphs represent mean +/− SD of % replication normalized to the DMSO control 24 post-infection of three independent experiments performed in triplicate. Data were fitted with a sigmoidal dose-response equation using non-linear regression (gray line) and the EC₅₀ values are quoted with 95% confidence intervals. (C) Schematic of the replicon assay to test the efficacy of AVI-4206 in A549 ACE2^h cells. (D) Luciferase readout of A549 ACE2^h cells infected with WA1 or WA1 Mac1 N40D replicons and treated with or without AVI-4206 and IFN-ɣ at indicated concentrations; *, P < 0.05 by two-tailed Student’s t-test relative to the no AVI-4206 and no IFN-ɣ control. Results are plotted as normalized mean ± standard deviation luciferase values of a representative biological experiment containing two technical replicates. (E) Schematic of the HAO experiment. (F) Viral particle production was measured by plaque assay at indicated time points and AVI-4206 concentrations. Error bars indicate s.e.m. **, P < 0.01; *, P < 0.05 by two-tailed Student’s t-test relative to the vehicle control.

Figure 4:. AVI-4206 has a favorable pharmacological profile.

(A) Pharmacokinetic properties of AVI-4206. (B) Unbound plasma exposure time course of AVI-4206, corrected for plasma protein binding, following administration by IV, PO, or IP routes in male CD-1 mice at the indicated doses. (C) Free plasma exposure of AVI-4206 and total exposure in lung homogenate following an IP dose of 10 mg/kg in female C57BL/6 mice. (D) Inhibition of CYP isoforms by AVI-4206 at a fixed concentration of 10 μM. Two experiments were performed with CYP3A4 using different positive controls. (E) Heatmap of AVI-4206 activity in an off-target safety panel including receptors, ion channels, and proteases, showing no antagonist response >15% at 10 μM.

Figure 5:. AVI-4206 reduces viral replication and increases survival and cytokine abundance in vivo.

(A) K18-hACE2 mice were intranasally infected and dosed as indicated with either AVI-4206 (n=15, intraperitoneally), nirmatrelvir (n=5, per os) or vehicle (n=10 for the AVI-4206 group or n=5 for the nirmatrelvir group). Mice infected with WA1 N40D mutant, which lacks Mac1 catalytic activity, served as a positive control (n=10). Lungs were harvested at indicated time points for virus titration by plaque assay. (B) The percent body weight loss for all animals treated with AVI-4206 (100 mg/kg IP) (C) or nirmatrelvir (300 mg/kg PO). The data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; ***, P < 0.001 by two-tailed Student’s t-test relative to the vehicle control at each timepoint. (D) Survival curve plotted based on the percent weight loss humane endpoint (20%) for AVI-4206 and (E) nirmatrelvir. (F) Viral load in the lungs and brain of infected mice at the indicated time points. The data are shown as mean ± s.e.m. *, P < 0.05; **, P < 0.01 by Mann Whitney’s test relative to the vehicle control. (G) Schematics and graphs demonstrating the abundance of indicated cytokines at 4 and 7 days post-infection in the lungs of infected mice. The data are presented as mean ± s.e.m. *, P < 0.05; **, P < 0.01 by two-tailed Student’s t-test relative to the vehicle control at each timepoint. None of the mice reached the humane endpoint at day 4 post-infection. For mice that reached the humane endpoint before day 7 post-infection, the tissues were collected and analyzed with mice at the 7 day time point.