Abstract
Heteroaryldihydropyrimidine (HAP) and sulfamoylbenzamide (SBA) are promising non-nucleos(t)ide HBV replication inhibitors. HAPs are known to promote core protein mis-assembly, but the molecular mechanism of abnormal assembly is still elusive. Likewise, the assembly status of core protein induced by SBA remains unknown. Here we show that SBA, unlike HAP, does not promote core protein mis-assembly. Interestingly, two reference compounds HAP_R01 and SBA_R01 bind to the same pocket at the dimer-dimer interface in the crystal structures of core protein Y132A hexamer. The striking difference lies in a unique hydrophobic subpocket that is occupied by the thiazole group of HAP_R01, but is unperturbed by SBA_R01. Photoaffinity labeling confirms the HAP_R01 binding pose at the dimer-dimer interface on capsid and suggests a new mechanism of HAP-induced mis-assembly. Based on the common features in crystal structures we predict that T33 mutations generate similar susceptibility changes to both compounds. In contrast, mutations at positions in close contact with HAP-specific groups (P25A, P25S, or V124F) only reduce susceptibility to HAP_R01, but not to SBA_R01. Thus, HAP and SBA are likely to have distinctive resistance profiles. Notably, P25S and V124F substitutions exist in low-abundance quasispecies in treatment-naïve patients, suggesting potential clinical relevance.
Highlights
It is estimated that hepatitis B virus (HBV) causes 4 million acute infections and 686,000 deaths annually worldwide
We identified SBA_R01, a novel capsid assembly modulator belonging to sulfamoylbenzamide (SBA) series through a focused library screening (Fig. 1a)
It is technically challenging to obtain complex structures of HBV capsid with inhibitors at a reasonable resolution to unambiguously define the molecular interactions
Summary
It is estimated that hepatitis B virus (HBV) causes 4 million acute infections and 686,000 deaths annually worldwide. Different chemical classes of inhibitors targeting the HBV capsid are under development: heteroaryldihydropyrimidines (HAPs) and sulfamoylbenzamides (SBAs) (Fig. 1a). Published high-resolution complex structures[23,24], using a non-assembled core protein mutant Y132A as a surrogate, greatly facilitate structure-based drug design of next-generation HAPs24. To evaluate potential differences of these two classes of core protein modulators, we characterize the mechanisms of HAP and SBA and present the crystal structures of two reference compounds HAP_R01 and SBA_R01 in complex with the core protein mutant hexamer (Y132A).
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