AVI-4773 and related MPro inhibitors outperform existing treatments like nirmatrelvir in preclinical studies, offering a promising path toward pan-coronavirus therapies.

Study: Structure-based discovery of highly bioavailable, covalent, broad-spectrum coronavirus M^Pro inhibitors with potent in vivo efficacy. Image Credit: H_Ko / Shutterstock.com

Since the start of the coronavirus disease 2019 (COVID-19) pandemic, numerous novel antiviral therapeutic agents have been developed to target key proteases involved in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) replication. Nirmatrelvir and ensitrelvir, for example, are main protease (M^pro) inhibitors that are currently approved for the clinical treatment of COVID-19.

The continuous emergence of highly transmissible, pathogenic, and immune-evading SARS-CoV-2 variants has weakened the efficacy of existing antiviral agents. Moreover, the looming threat of future pandemics due to coronaviral reservoirs in bats and other small mammals emphasizes the importance of identifying novel therapeutics with pan-coronavirus activity.

A recent study published in Science Advances reports the recent discovery of M^pro inhibitors that exhibit broad activity against SARS-CoV-2 and other coronaviruses.

Building the scaffold

Previously, the researchers of the current study screened 862 million custom molecules against the SARS-CoV-2 M^pro structure, which led to the identification of several scaffolds with micromolar inhibitory activity. AVI-1084, for example, exhibited a median inhibitory concentration (IC₅₀) of 29 μM.

This scaffold was subsequently used to contract a library of over 17,000 analogs that were evaluated for their binding activity to and docking into M^pro. Seven of these analog compounds exhibited in vitro M^pro activity, the most potent of which was AVI-3570 with an IC₅₀ of 1.5 μM. The improved potency of these compounds was attributed to fluoro- and chloro- substitutions in the thiophene ring of AVI-1084 that increased their interactions with the M^pro S2 pocket.

However, none of the seven identified compounds were capable of modifying the key pyridinone group occupying the S1 pocket. When an isoquinoline group was used to replace the pyridinone ring, AVI-3318 was created and found to be 50-fold more potent than AVI-1084.

X-ray crystallography of the AVI-3318-M^Pro complex provided similar results to the docking simulation assay and confirmed all hypothesized major interactions between the compounds and both the S1 and S2 pockets of M^pro. Additional structure-activity relationship (SAR) expansion involved adding various side chains at the remaining two positions of the dihydrouracil core in an effort to further improve compound potency.

AVI-4303, which is a C5 benzotriazole analog, exhibited ten-fold higher potency than AVI-3318. Comparatively, N1 propargyl analogs including AVI-4516 and AVI-4773 successfully bound to the S1 site with a nanomolar IC₅₀, which is 100-fold more potent than the des-propargyl molecule AVI-4375, which had an IC₅₀ of 7.4 μM.

Activity against SARS-CoV-2

Mouse in vivo studies demonstrated powerful antiviral activity of AVI-4516 against the SARS-CoV-2 Beta variant of concern (VOC). AVI-4692 and AVI-4694 also exhibited potent activity against human common cold coronaviruses like α-HCoV 229E and β-HCoV OC43, as well as the Middle East Respiratory Syndrome (MERS)-CoV, SARS-CoV, and the SARS-CoV-2 Beta, Delta, and BA.2 variants.

Most analogs lost activity against nirmatrelvir-resistant mutants; however, AVI-4516 remained highly potent against the SARS-CoV-2 E166Q variant. AVI-4516 and AVI-4773 retained similar potency to nirmaltrevir, whereas AVI-4692 and AVI-4694 were more potent than nirmatrelvir when tested against a triple mutant containing the most common mutations associated with drug resistance.

AVI-4773 reduced viral titers to undetectable levels  after three doses, which is a three-log reduction in viral load as compared to ensitrelvir treatment.

Mechanism of action

The combination of C6-aryl substitution with the propargyl warhead [AVI-4694] has great potential to produce an agent that effectively targets recently emergent variants of SARS-CoV-2.”

The inhibitory activity of AVI-4773 and other compounds in this series is likely due to the unactivated N-propargyl side chain. Despite being a weak electryophyl, the N-location of the terminal alkyne of this side chain helps to stabilize the compound to facilitate its reaction with the M^pro Cys145. The alkyne warheads cause powerful and permanent inhibition, which is comparable to the reversible inhibition produced by the noncovalent binding of the nitrile warheads in nirmatrelvir.

Conclusions

AVI-4516 and AVI-4773 exhibit potent M^Pro inhibition, which warrants additional research to optimize the chemical structure of these tool compounds for the development of future pan-coronavirus therapeutics.

Some of the key advantages of this distinct chemotype include easy modification by functional groups or substitutions at different binding sites, simple synthesis protocol, reduced manufacturing costs, potent pan-coronaviral activity, as well as superior pharmacokinetic properties and tissue distribution.