Discovery of oseltamivir-based novel PROTACs as degraders targeting neuraminidase to combat H1N1 influenza virus

Abstract

Annual and sporadic influenza outbreaks pose a great threat to human health and the economy worldwide. Moreover, the frequent mutation of influenza viruses caused by antigen drift complicates the application of antiviral therapeutics. As such, there is an urgent need for novel antiviral agents to tackle the problem of insufficient efficacy of licensed drugs. Inspired by the success of the newly emerged PROTACs (PROteolysis TArgeting Chimeras) strategy, we report herein the design and synthesis of novel PROTAC molecules based on an oseltamivir scaffold to combat severe annual influenza outbreaks. Among these, several compounds showed good anti-H1N1 activity and efficient influenza neuraminidase (NA) degradation activity. The best compound, 8e, effectively induced influenza NA degradation in a dose-dependent manner and relied on the ubiquitin-proteasome pathway. Moreover, Compound 8e exhibited potent antiviral activity toward both wild-type H1N1 virus and an oseltamivir-resistant strain (H1N1, H274Y). A molecular docking study demonstrated that Compound 8e had good hydrogen-bonding and hydrophobic interactions with both the active sites of NA and Von Hippel-Lindau (VHL) proteins, which could effectively drive the favorable interaction of these two proteins. Thus, as the first report of a successful anti-influenza PROTAC, this proof of concept will greatly widen the application range of the PROTAC technique to antiviral drug discovery.

Keywords: Antiviral drug; Influenza; Neuraminidase; Oseltamivir; PROTACs.

Graphical abstract

Fig. 1

Chemical structures of representative approvedanti-influenzadrugs.

Fig. 2

Design strategy ofoseltamivir-basedPROTACs with diverse linkers through use of different E3 ligase ligands.

Fig. 3

Degradation activity of NA protein in the presence of PROTACs 8e and 11 at different concentrations. (A, C) The NA degradation level was evaluated by western blot. (B, D) Western blot bands were quantified by ImageJ. The data were obtained from at least two independent assays.

Fig. 4

In vitroefficacy against H1N1 replication of Compound 8e; OSP was used as the positive control. (A) Western blot assay, (B) qRT–PCR assay and (C) plaque formation assay were used to evaluate the antiviral activity of Compound 8e. The concentration of OSP in the qRT–PCR assay was 5 μM. Mock, blank control; NC, negative control (treated with DMSO). (D) The cytotoxicity of Compound 8e was determined with the CCK-8 reagent.

Fig. 5

Compound 8e induced NA degradation via the ubiquitin−proteasome system. (A) NA degradation activity of Compound 8e was determined after treatment with 8e-neg (10 μM) or OSP (10 μM). (B) NA degradation activity of Compound 8e (10 μM) after treatment with MG132 (10 μM) or CH₃-VHL (10 μM).

Fig. 6

Antiviral activity of Compound 8e against anoseltamivir-resistantstrain (H1N1, H274Y). Western blot (A) and qRT–PCR (B) were used to evaluate the NP protein or mRNA level, respectively, after treatment with Compound 8e or OSP.

Fig. 7

Predicted binding mode of the NA-8e-VHL ternary complex (NA protein PDB: 2HU0, VHL protein PDB: 5NW2). (A) The backbone of the protein was rendered in a tube and colored green (NA protein) and blue (E3 protein). (B, C) The binding mode of 8e with E3 and NA. Hydrogen bond distance and π-stacking distance are represented in yellow.