Pyrazolopyrimidines: Potent Inhibitors Targeting the Capsid of Rhino‐ and Enteroviruses

Abstract

There are currently no drugs available for the treatment of enterovirus (EV)-induced acute and chronic diseases such as the common cold, meningitis, encephalitis, pneumonia, and myocarditis with or without consecutive dilated cardiomyopathy. Here, we report the discovery and characterization of pyrazolopyrimidines, a well-tolerated and potent class of novel EV inhibitors. The compounds inhibit the replication of a broad spectrum of EV in vitro with IC50 values between 0.04 and 0.64 μm for viruses resistant to pleconaril, a known capsid-binding inhibitor, without affecting cytochrome P450 enzyme activity. Using virological and genetics methods, the viral capsid was identified as the target of the most promising, orally bioavailable compound 3-(4-trifluoromethylphenyl)amino-6-phenylpyrazolo[3,4-d]pyrimidine-4-amine (OBR-5-340). Its prophylactic as well as therapeutic application was proved for coxsackievirus B3-induced chronic myocarditis in mice. The favorable pharmacokinetic, toxicological, and pharmacodynamics profile in mice renders OBR-5-340 a highly promising drug candidate, and the regulatory nonclinical program is ongoing.

Cryo-EM structure of pleconaril-resistant rhinovirus-B5 complexed to the antiviral OBR-5-340 reveals unexpected binding site

“…Most of the residues are highly conserved within the genus Enterovirus (23,24), enabling the inhibition of a broad spectrum of RVs and EVs with capsid binders (25). Capsid binders can exert virucidal activity against RVs and EVs (24,26), impede viral adsorption by modifying the conformation of the receptor binding site (24,27,28), and/or prevent structural changes required for RNA uncoating (29). As suggested by the example of pleconaril, occupancy of the hydrophobic pocket can be higher when the capsid binder is introduced during viral assembly (25).…”

“…Pyrazolopyrimidines are relatively new capsid-binding inhibitors. They show strong activity against pleconaril-resistant EVs and RVs (24). Like pleconaril and vapendavir, they are three-ring compounds.…”

“…The structural similarity and inhibition profile, together with results of computational modeling of CVB3 in complex with 3-(4-trifluoromethylphenyl)amino-6-phenylpyrazolo[3,4-d]pyrimidine-4-amine (OBR-5-340) (SI Appendix, Fig. S1), taken as an example, suggested that pyrazolopyrimidines would also bind to the hydrophobic pocket (24). However, the differences in the spectrum of pleconaril-and OBR-5-340-susceptible RVs suggest differences in the binding.…”

“…Because RV-B5 is highly susceptible to OBR-5-340 in cytopathic effect (CPE) inhibition assays (50% inhibitory concentration, IC 50 = 0.08 μM; compound 36 in the Supporting Information of ref. 24) and fully resistant to pleconaril (IC 50 > 12.5 μM; higher concentrations were cytotoxic) (24, 30), we selected it for the present study. Single-step growth curves with RV-B5 were recorded in the absence and presence of the highly effective OBR-5-340 concentration of 1 μM in HeLa cells.…”

“…Mechanism of Viral Inhibition. We investigated which stage of the infection cycle was most affected by OBR-5-340 by using a previously established plaque-reduction assay (24). OBR-5-340 (0.5 μM) was added at different time points before and after challenging the HeLa cells with RV-B5.…”