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[Preprint]. 2025 Oct 14:2025.10.11.681833.
doi: 10.1101/2025.10.11.681833.

Discovery of AVI-6451, a Potent and Selective Inhibitor of the SARS-CoV-2 ADP-Ribosylhydrolase Mac1 with Oral Efficacy in vivo

Priyadarshini Jaishankar  1 , Galen J Correy  2 , Yusuke Matsui  3 , Takaya Togo  1 , Moira M Rachman  1 , Maisie G V Stevens  4 , Eric R Hantz  1 , Jeffrey Zheng  1 , Morgan E Diolaiti  4 , Mauricio Montano  3 , Taha Y Taha  3 , Julia Rosecrans  3 , Julius Pampel  2 , Nevan J Krogan  2 , Brian K Shoichet  1 , Alan Ashworth  4 , Melanie Ott  3 , James S Fraser  2 , Adam R Renslo  1   4
Affiliations

Discovery of AVI-6451, a Potent and Selective Inhibitor of the SARS-CoV-2 ADP-Ribosylhydrolase Mac1 with Oral Efficacy in vivo

Priyadarshini Jaishankar et al. bioRxiv. .

Update in

Abstract

The COVID-19 pandemic made plain the need for effective antivirals acting on novel antiviral targets, among which viral macrodomains have attracted considerable attention. We recently described AVI-4206 (1), a potent and selective inhibitor of the SARS-CoV-2 ADP-ribosylhydrolase Mac1 based on a 9H-pyrimido[4,5-b]indole core, the first Mac1 inhibitor to demonstrate antiviral efficacy in mouse models of SARS-CoV-2 infection, but requiring IP administration and frequent dosing. Herein we describe an extensive, structurally enabled medicinal chemistry effort to identify orally bioavailable Mac1 inhibitors by addressing permeability and efflux liabilities of 1 and many of its analogs. Multiple strategies were pursued to overcome these issues, including replacing a urea function to reduce hydrogen bond donor count. While heterocyclic urea mimetics could deliver analogs like AVI-6318 (3) with potencies and ADME profiles similar to 1, abrogation of the P-gp liability was finally achieved with entirely non-polar substituents in place of urea. Thus, AVI-6451 (4) is a potent Mac1 inhibitor lead with low intrinsic clearance, high oral bioavailability, and antiviral efficacy with once-daily oral administration in a mouse model of SARS-CoV-2 infection.

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Figures

Figure 1.
Figure 1.
Structures and properties (top) of Mac1 inhibitor AVI-4206 (1) and a ligand-efficient deazapurine fragment AVI-1504 (2), described by our groups previously. Structures and properties (bottom) of optimized leads 3 (AVI-6318) and 4 (AVI-6451), the discovery of which is detailed herein.
Figure 2.
Figure 2.
Superposition of the complex structures of 1 (9CY0) and ADP-ribose (7KQP) bound to Mac1 illustrating important polar contacts made with the backbone amides of F156/D157 in the ribose binding subsite and multiple hydrogen bonds with Asp22. Major hydrophobic contacts in the adenine site including F156, L126, L160, and V155 are indicated. Note the structured water that mediates hydrogen bonding between substrate and backbone residues in the ribose binding site.
Figure 3.
Figure 3.
Complex structures of analogs from Chart 1 bound to Mac1. Panel A) from left to right, AVI-4094, AVI-4100, AVI-4097, AVI-4268, AVI-4214. Panel B) left to right, AVI-4272, AVI-4678, AVI-4683, AVI-5707, AVI-4054. PanDDA event maps are contoured around ligands at 2 σ (blue mesh). Hydrogen bonds are shown with dashed black lines.
Figure 4.
Figure 4.
Complex structures of analogs from Chart 2 bound to Mac1. From left to right, AVI-4052, AVI-6318, AVI-6319, and AVI-6344. PanDDA event maps are contoured around ligands at 2 σ (blue mesh). Hydrogen bonds are shown with dashed black lines.
Figure 5.
Figure 5.
Unbound plasma exposure of 1 (AVI-4206) and AVI-4052 following a single IP dose of 10 mg/kg in mice. †Extrapolation of the free plasma exposure of AVI-4052 to a 10-fold higher dose, assuming linear pharmacokinetics. The Mac1 IC50 value of AVI-4052 is shown as an orange dotted line.
Figure 6.
Figure 6.
Complex structures of C8 urea and non-urea analogs bound to Mac1. From left to right, AVI-4206, AVI-6249, AVI-6354, AVI-6372, and AVI-6451 (4). PanDDA event maps are contoured around ligands at 2 σ (blue mesh) and FO-FC difference electron density maps calcucalculated prior to ligand placement are contoured at 4 σ (purple mesh). Hydrogen bonds are shown with dashed black lines.
Figure 7.
Figure 7.
Heatmaps showing the activity of 10 μM AVI-4206 and AVI-6451 in A) a Eurofins SafetySceen 44 panel of potential enzyme and receptor off targets and B) a panel of CYP isoforms.
Figure 8.
Figure 8.
AVI-6451 shows enhanced PK and cellular and in vivo antiviral efficacy compared to AVI-4206. Panel A. Single-dose mouse PK experiments demonstrate the reduced clearance and higher oral exposure of AVI-6451 (4, in red) compared to AVI-4206 (1, in blue). Compounds formulated in 10% DMSO|50% PEG400|40% (20% HP-β-CD in water). Panel B. Dose escalation study of 4 formulated as a suspension in 1% (hydroxypropyl)methyl cellulose|1% Tween 80 in water. Data from prior studies with 1 and 4 in DMSO|PEG400|HP-β-CD are shown for comparison. C. Dose-dependent antiviral activity of AVI-6451 (4, in orange) in monocyte derived macrophages infected with SARS-CoV-2 MA-WA1. panel D. Viral load in the lungs of wild-type mice (N=10/group) at days 2 and 4 post-infection with 1 x 104 PFU of SARS-CoV-2 MA-WA1. Mice were treated from day -1 to day 3 with vehicle, with 100 mg/kg AVI-4206 IP BID, or 100 mg/kg AVI-6451 PO QD. Viral load of mice infected with SARS CoV-2 MA-WA1 Mac1 N40D (mutant lacking Mac1 activity) are shown for comparison.
Scheme 1.
Scheme 1.
Synthesis of deazapurine analogs described below in Chart 1 (top scheme) and Chart 2 (bottom scheme). Conditions: (a) (R)-2-amino-3-methylbutan-1-ol, Et3N, DMSO, 110°C, 16 h, 45%. (b) R-B(OH)2, Pd(dppf)Cl2, Cs2CO3, dioxane-water (10:1), 110 °C; (c) 1-methyl-4-(pinacolborane)-1H-pyrazole-5-carbonitrile, dioxane-water (10:1), Pd(dppf)Cl2, K2CO3, 95 °C, 2 h, 72%; (d) R-NH2, iPrOH, aq. HCl, 100 °C.
Chart 1.
Chart 1.
Novel analogs bearing aryl and heteroaryl substitutions at C6 of the 5-deazapurine ring. Mac1 HTRF IC50, kinetic solubility and mouse liver microsome clearance values are indicated below the AVI-ID. See Figure S1 for dose-response curves.
Chart 2.
Chart 2.
Novel analogs bearing cyanopyrazole substitution at C6 and diverse substitutions at C4 of the 5-deazapurine ring. Mac1 HTRF IC50, kinetic solubility and mouse liver microsome clearance values are indicated below the AVI-ID. See Figure S1 for dose-response curves.
Chart 4
Chart 4
Structure of 9H-pyrimidoindole leads bearing optimized C4 heterocycles and non-urea substituents at C8. See Figure S1 for dose-response curves.
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