SARS-CoV-2 is the causative agent of COVID-19 and is responsible for the current global
pandemic. The viral genome contains 5 major open reading frames of which the largest
ORF1ab codes for two polyproteins, pp1ab and pp1a, which are subsequently cleaved into
16 nonstructural proteins (nsp) by two viral cysteine proteases encoded within the
polyproteins. The main protease (Mpro, nsp5) cleaves the majority of the nsp’s,
making it essential for viral replication and has been successfully targeted for the
development of antivirals. The first oral Mpro inhibitor, nirmatrelvir, was approved for
treatment of COVID-19 in late December 2021 in combination with ritonavir as Paxlovid.
Increasing the arsenal of antivirals and development of protease inhibitors and other
antivirals with a varied mode of action remains a priority to reduce the likelihood for
resistance emerging. Here, we report results from an artificial intelligence-driven
approach followed by
in vitro
validation, allowing the identification
of five fragment-like Mpro inhibitors with IC
50
values ranging from 1.5 to
241 μM. The three most potent molecules (compounds 818, 737, and 183) were tested
against SARS-CoV-2 by
in vitro
replication in Vero E6 and Calu-3 cells.
Compound 818 was active in both cell models with an EC
50
value comparable to
its measured IC
50
value. On the other hand, compounds 737 and 183 were only
active in Calu-3, a preclinical model of respiratory cells, showing selective indexes
twice as high as those for compound 818. We also show that our
in
silico
methodology was successful in identifying both reversible and covalent
inhibitors. For instance, compound 818 is a reversible chloromethylamide analogue of
8-methyl-γ-carboline, while compound 737 is an
N
-pyridyl-isatin
that covalently inhibits Mpro. Given the small molecular weights of these fragments,
their high binding efficiency
in vitro
and efficacy in blocking viral
replication, these compounds represent good starting points for the development of
potent lead molecules targeting the Mpro of SARS-CoV-2.