The continuous spread of SARS-CoV-2 calls for more direct-acting antiviral agents to
combat the highly infectious variants. The main protease (M
pro
) is an
promising target for anti-SARS-CoV-2 drug design. Here, we report the discovery of
potent non-covalent non-peptide M
pro
inhibitors featuring a
1,2,4-trisubstituted piperazine scaffold. We systematically modified the non-covalent
hit MCULE-5948770040 by structure-based rational design combined with multi-site binding
and privileged structure assembly strategies. The optimized compound
GC-14
inhibits M
pro
with high potency (IC
50
= 0.40 μM) and
displays excellent antiviral activity (EC
50
= 1.1 μM), being more
potent than Remdesivir. Notably,
GC-14
exhibits low cytotoxicity
(CC
50
> 100 μM) and excellent target selectivity for SARS-CoV-2
M
pro
(IC
50
> 50 μM for cathepsins B, F, K, L, and
caspase 3). X-ray co-crystal structures prove that the inhibitors occupy multiple
subpockets by critical non-covalent interactions. These studies may provide a basis for
developing a more efficient and safer therapy for COVID-19.
The
spread of SARS-CoV-2 keeps threatening human life and health,
and small-molecule antivirals are in demand. The main protease (Mpro) is an effective and highly conserved target for anti-SARS-CoV-2
drug design. Herein, we report the discovery of potent covalent non-peptide-derived
Mpro inhibitors. A series of covalent compounds with a
piperazine scaffold containing different warheads were designed and
synthesized. Among them, GD-9 was identified as the most
potent compound with a significant enzymatic inhibition of Mpro (IC50 = 0.18 μM) and good antiviral potency against
SARS-CoV-2 (EC50 = 2.64 μM), similar to that of remdesivir
(EC50 = 2.27 μM). Additionally, GD-9 presented favorable target selectivity for SARS-CoV-2 Mpro versus human cysteine proteases. The X-ray co-crystal structure
confirmed our original design concept showing that GD-9 covalently binds to the active site of Mpro. Our nonpeptidic
covalent inhibitors provide a basis for the future development of
more efficient COVID-19 therapeutics.
The Hepatitis B Virus (HBV) ribonuclease H (RNase H) is an attractive but unexploited drug target. Here, we addressed three limitations to the current state of RNase H inhibitor development: i) Efficacy has been assessed only in transfected cell lines; ii) Cytotoxicity data are from transformed cell lines rather than primary cells; and iii) It is unknown how the compounds work against nucleos(t)ide analog resistant HBV strains. Three RNase H inhibitors from different chemotypes,
110
(α-hydroxytropolone),
1133
(N-hydroxypyridinedione), and
1073
(N-hydroxynapthyridinone), were tested in HBV-infected HepG2-NTCP cells for inhibition of cccDNA accumulation and HBV product formation. 50% effective concentrations (EC
50
s) were 0.049-0.078 μM in the infection studies compared to 0.29-1.6 μM in transfected cells. All compounds suppressed cccDNA formation by >98% at 5 μM when added shortly after infection. HBV RNA, intracellular and extracellular DNA, and HBsAg secretion were all robustly suppressed. The greater efficacy of the inhibitors when added shortly after infection is presumably due to blocking amplification of the HBV cccDNA, which suppresses events downstream of cccDNA formation. The compounds had 50% cytotoxic concentrations (CC
50
s) of 16-100 μM in HepG2-derived cell lines but were non-toxic in primary human hepatocytes, possibly due to the quiescent state of the hepatocytes. The compounds had similar EC
50
s against replication of wild-type, Lamivudine-resistant and Adefovir/Lamivudine-resistant HBV, as expected because the RNase H inhibitors do not target the viral reverse transcriptase active site. These studies expand confidence in inhibiting the HBV RNase H as a drug strategy and support inclusion of RNase H inhibitors in novel curative drug combinations for HBV.
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