Discovery of Potent and Broad-Spectrum Pyrazolopyridine-Containing Antivirals against Enteroviruses D68, A71, and Coxsackievirus B3 by Targeting the Viral 2C Protein

Abstract

The enterovirus genus of the picornavirus family contains many important human pathogens. EV-D68 primarily infects children, and the disease manifestations range from respiratory illnesses to neurological complications such as acute flaccid myelitis (AFM). EV-A71 is a major pathogen for the hand, foot, and mouth disease (HFMD) in children and can also lead to AFM and death in severe cases. CVB3 infection can cause cardiac arrhythmias, acute heart failure, as well as type 1 diabetes. There is currently no FDA-approved antiviral for any of these enteroviruses. In this study, we report our discovery and development of pyrazolopyridine-containing small molecules with potent and broad-spectrum antiviral activity against multiple strains of EV-D68, EV-A71, and CVB3. Serial viral passage experiments, coupled with reverse genetics and thermal shift binding assays, suggested that these molecules target the viral protein 2C. Overall, the pyrazolopyridine inhibitors represent a promising class of candidates for the urgently needed nonpolio enterovirus antivirals.

Figure 1.

Structurally diverse enterovirus 2C inhibitors. The hit compound 7a identified from our high-throughput screening served as a starting point for the lead optimization in this study.

Figure 2.

Synthesis of pyrazolopyridine analogs. (A) Synthesis of 1H-pyrazolo[3,4,-b]pyridine-4-carboxylic acid. (B) Representative synthesis of pyrazolopyridines with either tertiary amine (7g), secondary or primary amine (7k).

Figure 3.

Summary of SAR studies of pyrazolopyridine. Substitutions led to compounds with a low selectivity index (SI) are labeled in red. Potent leads have each of the shown substituents R₁, R₃, R₄, and R₆.

Figure 4.

Antiviral activity of compounds 7d, 7h, 10a, and 19 against EV-D68 US/MO/14-18947 in plaque assay. The plaque areas were quantified in Image J and EC₅₀ values were determined through curve fittings in Graphpad Prism 8 using log(concentration of inhibitors) vs percentage of plaque area with variable slopes. The results are the mean ± standard deviation of two repeats.

Figure 5.

Antiviral activity of compounds 7d, 7h, 10a, and 19 against EV-D68 US/MO/14-18947 in neuronal cell line SH-SY5Y using immunofluorescence assay. The immunofluorescence signals were quantified in Image J and EC₅₀ values were determined through curve fittings in Graphpad Prism 8 using log(concentration of inhibitors) vs percentage of positive control with variable slopes. The results are the mean ± standard deviation of two repeats.

Figure 6.

Mechanism of action of the broad-spectrum antiviral 7d. (A) Drug sensitivity of 7d against different strains of recombinant EV-D68 viruses in plaque assay. (B) Thermal shift binding assay of 7d against purified EV-D68 2C protein and the mutants selected from viral passage experiments. (C) Thermal shift binding assay of 7d against EV-D68 2C, EV-A71 2C, and CVB3 2C. (D) Melting temperature T_m and thermal shift ΔT_m of EV-D68 2C, EV-A71 2C and CVB3 2C in the presence of different concentrations of 7d.

Figure 7.

(A) Competition growth assay of rD183V/D323G with rWT EV-D68 virus. Electropherogram traces of 2C protein coding region were exported from Chromas Lite 2.6, and residues 183 and 323 from viruses at each passage were shown on the left. Codon GAT is for Asp, condon GTT is for Val, codon GGT is for Gly. The percentages of the mutant and WT virus populations at both positions were determined by measuring the height of the nucleotide peaks in the sequencing trace and shown on the right. (B) Stability of recombinant virus rD183V/D323G propagated in cell culture. The recombinant virus rD183V/D323G was propagated in RD cells sequentially for four passages, and 2C protein coding region from the viruses collected from Passage 2, 3, and 4 (P2, P3, P4) were sequenced. Electropherogram traces at residues 183 and 323 from viruses at each passage were shown.