PF-07321332 is an active ingredient in Paxlovid, which is a treatment for COVID-19. This molecule inhibits the activity of the main protease of the virus SARS-CoV-2. This main protease is responsible for producing 12 functional proteins which are highly conserved across several coronaviruses. This includes several of the well-known COVID-19 variants.
Finding a treatment that can remain effective against various mutant varieties as well as the wild-type is key to combating this virus. Recently, the D48N mutant was investigated in comparison to the wild-type for effectiveness of the treatment. The D48N mutant contains a single amino acid substitution near the main protease active site. This substitution has already been confirmed to exist in several COVID-19 variants.
To begin the study, first the D48N mutant was cultured and purified. The next step was to run enzymatic inhibition assays to test the inhibitory activities of both shikonin and PF-07321332. Shikonin was also included in the test because the research team’s previous work shows that it also appears to be promising for main protease inhibition.
Next, the purified D48N was mixed together with either of these molecules and allowed time to bond, resulting in the formation of a complex. The complex was then crystallized and the crystals collected for examination. By this method the structures of the complexes can be determined and the interaction of the molecule with D48N confirmed.
The results showed that both PF-07321332 and shikonin have potent inhibitory effects against both D48N mutant and wild-type main protease. Interestingly, shikonin demonstrated non-covalent binding while PF-07321332 demonstrated covalent binding. The data from this study may be useful in further work to develop additional treatments for COVID-19.
Zhao Z, Zhu Q, Zhou X, et al. Structural basis for the inhibition of SARS-CoV-2 Mpro D48N mutant by shikonin and PF-07321332. Viruses. 2023 Dec 30;16(1):65. PMID: 38257765