New antiviral compounds show broad protection against Covid-19 variants

New antiviral compounds show broad protection against Covid-19 variants

AVI-4773 and related MPRO inhibitors outperform existing treatments such as nirmatrelvir in preclinical studies, providing a promising path toward pan-coronavirus therapies.

Since the beginning of the coronavirus disease 2019 (COVID-19) pandemic, numerous new antiviral therapeutic agents have been developed to target key proteases involved in severe replication of acute respiratory syndrome coronavirus 2 (SARS-CoV-2). For example, nirmalatrelvir and ensitrelvir are major protease inhibitors (MPROs) currently approved for the clinical treatment of Covid-19.

The continued emergence of highly transmissible, pathogenic and immune-evading SARS-CoV-2 variants has weakened the effectiveness of existing antivirals. Furthermore, the looming threat of future pandemics due to coronaviral reservoirs in bats and other small mammals emphasizes the importance of identifying new therapeutics with pan-coronavirus activity.

A recently published study inScience advancesreports the recent discovery of MPRO inhibitors that exhibit broad activity against SARS-CoV-2 and other coronaviruses.

Building the scaffolding

Previously, researchers in the current study searched 862 million custom molecules against the SARS-CoV-2 MPRO structure, resulting in the identification of several scaffolds with micromolar inhibitory activity. For example, AVI-1084 showed a median inhibitory concentration (IC50) of 29 μm.

This scaffold was subsequently used to construct a library of over 17,000 analogs that were evaluated for their binding activity to MPRO. Seven of these analogous compounds appearedin vitroMPRO activity, the strongest of which was AVI-3570 with an IC50 of 1.5 μm. The improved potency of these compounds was attributed to fluorine and chlorine substitutions in the thiophene ring of AVI-1084, which increased their interactions with the Mpro S2 pocket.

However, none of the seven identified compounds were able to modify the key pyridinone group containing the S1 pocket. When an isoquinoline group was used to replace the pyridinone ring, AVI-3318 was created and was found to be 50 times stronger than AVI-1084.

X-ray crystallography of the AVI-3318-Mpro complex provided similar results to the docking simulation assay, confirming all hypothesized major interactions between the compounds and both the S1 and S2 pockets of MPRO. Additional expansion of the structure-activity relationship (SAR) included the addition of various side chains in the remaining two positions of the dihydrowour core to further improve the compound potency.

AVI-4303, a C5 benzotriazole analogue, demonstrated ten times greater potency than AVI-3318. In comparison, N1-propargyl analogs including AVI-4516 and AVI-4773 successfully bound to the S1 site with a nanomolar IC50 that is 100-fold stronger than the des-propargyl molecule AVI-4375, which had an IC50 of 7.4 μm.

Activity against SARS-CoV-2

MouseIn vivoStudies showed strong antiviral activity of AVI-4516 against the SARS-CoV-2 beta variant (VOC). AVI-4692 and AVI-4694 also demonstrated potent activity against human cold coronaviruses such as α-HCOV 229E and β-HCOV-OC43, as well as Middle East respiratory syndrome (MERS)-COV, SARS-CoV, and the SARS-COV-2 beta, delta, and BA.2 variants.

Most analogues lost activity against nirmatrelvir-resistant mutants; However, AVI-4516 remained very strong against the SARS-COV-2 E166Q variant. AVI-4516 and AVI-4773 retained similar potency to nirmatrelvir, while 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, representing a reduction in viral load 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 emerging variants of SARS-COV-2. “”

The inhibitory activity of AVI-4773 and other compounds in this series is likely due to the nonactivated N-propargyl side chain. Although a weak electryophyl, the N site of the terminal alkyne of this side chain helps to stabilize the compound to facilitate its reaction with the Mpro Cys145. The alkyne warheads cause strong and lasting inhibition comparable to the reversible inhibition produced by the noncovalent binding of the nitrile warheads in nirmatrelvir.

Conclusions

AVI-4516 and AVI-4773 exhibit potent MPRO inhibition, warranting 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 different chemotype include easy modification by functional groups or substitutions at different binding sites, simple synthesis protocol, reduced manufacturing cost, potent pan-coronaviral activity, and superior pharmacokinetic properties and tissue distribution.

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