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Heteroaryldihydropyrimidine (HAP) and Sulfamoylbenzamide (SBA) Inhibit Hepatitis B Virus Replication by Different Molecular Mechanisms

View Article: PubMed Central - PubMed

ABSTRACT

Hap_r01sba_r01hap_r01sba_r01hap_r01hap_r01,sba_r01: 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 and 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 , but is unperturbed by . Photoaffinity labeling confirms the 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 but not to . 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.

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Identification of photoaffinity labeling site Y118 by HAP_R01_PL1.(a) Intact mass measurement of the active photolabel HAP_R01_PL1-treated sample. Mw increase of 477 is highlighted in red box indicating the covalent labeling of HAP_R01_PL1 on core protein assembly. (b) Intact mass measurement of the control photolabel HAP_R02_PL1-treated sample. (c,d) MS2 data of pepsin-digested peptide 118–122: YLVSF from photoaffinity label-treated samples. Inlet tables show the calculated mass of fragmented ions. Grey shading denotes observed fragment ions. (c) HAP_R01_PL1-treated sample. (d) HAP_R02_PL1-treated sample.
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f4: Identification of photoaffinity labeling site Y118 by HAP_R01_PL1.(a) Intact mass measurement of the active photolabel HAP_R01_PL1-treated sample. Mw increase of 477 is highlighted in red box indicating the covalent labeling of HAP_R01_PL1 on core protein assembly. (b) Intact mass measurement of the control photolabel HAP_R02_PL1-treated sample. (c,d) MS2 data of pepsin-digested peptide 118–122: YLVSF from photoaffinity label-treated samples. Inlet tables show the calculated mass of fragmented ions. Grey shading denotes observed fragment ions. (c) HAP_R01_PL1-treated sample. (d) HAP_R02_PL1-treated sample.

Mentions: Y132A mutation generates two binding sites per dimer for HAP molecules in the crystal structure reported here, while there is only one HAP1 site for two dimers from the low resolution capsid crystal structure22. Furthermore, it remains to be evaluated whether the HAP-bound Y132A structure is consistent with the interactions between HAP and misassembled capsid in solution. We defined the orientation of HAP upon binding to capsid assembly using a photoaffinity study coupled with liquid chromatography/tandem mass spectrometry (LC/MSMS). Three active photoaffinity probes containing an R-configuration at the 4th position of the dihydropyrimidine ring were generated to map out the HAP_R01-interacting residues on core protein. Each of these probes possesses a photoreactive function group at different positions (6th, 5th, and 4th) of the dihydropyrimidine core of HAP_R01, named by HAP_R01_PL1, HAP_R01_PL2, and HAP_R01_PL3, respectively. It is well known that the corresponding 4thS-diastereomer of HAP is less active than the 4thR-diastereometer1323. Thus the S-diastereomers were also synthesized as the negative controls, named by HAP_R02_PL1, HAP_R02_PL2, and HAP_R02_PL3, respectively (Fig. 4, Supplementary Figs S4 and S6). The R-configuration compounds retain reasonable activity in promoting capsid assembly and reducing viral DNA replication (Supplementary Table S2). A peak with molecular weight (Mw) increase of 477 Daltons was detected by LC/MS in samples treated with HAP_R01_PL1, but not in samples treated with HAP_R02_PL1 (Fig. 4a,b). The intact mass increase exactly corresponds to the Mw of the carbene species derived from photo-activation (subtraction of 28 due to loss of two nitrogen atoms). HAP_R01_PL2 and HAP_R01_PL3 can also covalently label core protein with additional mass of 556 and 529 respectively. The labeling signals in the control HAP_R02_PL2 and HAP_R02_PL3-treated samples are significantly lower than the active R-configured photolabels (Supplementary Figs S4 and S6).


Heteroaryldihydropyrimidine (HAP) and Sulfamoylbenzamide (SBA) Inhibit Hepatitis B Virus Replication by Different Molecular Mechanisms
Identification of photoaffinity labeling site Y118 by HAP_R01_PL1.(a) Intact mass measurement of the active photolabel HAP_R01_PL1-treated sample. Mw increase of 477 is highlighted in red box indicating the covalent labeling of HAP_R01_PL1 on core protein assembly. (b) Intact mass measurement of the control photolabel HAP_R02_PL1-treated sample. (c,d) MS2 data of pepsin-digested peptide 118–122: YLVSF from photoaffinity label-treated samples. Inlet tables show the calculated mass of fragmented ions. Grey shading denotes observed fragment ions. (c) HAP_R01_PL1-treated sample. (d) HAP_R02_PL1-treated sample.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5304331&req=5

f4: Identification of photoaffinity labeling site Y118 by HAP_R01_PL1.(a) Intact mass measurement of the active photolabel HAP_R01_PL1-treated sample. Mw increase of 477 is highlighted in red box indicating the covalent labeling of HAP_R01_PL1 on core protein assembly. (b) Intact mass measurement of the control photolabel HAP_R02_PL1-treated sample. (c,d) MS2 data of pepsin-digested peptide 118–122: YLVSF from photoaffinity label-treated samples. Inlet tables show the calculated mass of fragmented ions. Grey shading denotes observed fragment ions. (c) HAP_R01_PL1-treated sample. (d) HAP_R02_PL1-treated sample.
Mentions: Y132A mutation generates two binding sites per dimer for HAP molecules in the crystal structure reported here, while there is only one HAP1 site for two dimers from the low resolution capsid crystal structure22. Furthermore, it remains to be evaluated whether the HAP-bound Y132A structure is consistent with the interactions between HAP and misassembled capsid in solution. We defined the orientation of HAP upon binding to capsid assembly using a photoaffinity study coupled with liquid chromatography/tandem mass spectrometry (LC/MSMS). Three active photoaffinity probes containing an R-configuration at the 4th position of the dihydropyrimidine ring were generated to map out the HAP_R01-interacting residues on core protein. Each of these probes possesses a photoreactive function group at different positions (6th, 5th, and 4th) of the dihydropyrimidine core of HAP_R01, named by HAP_R01_PL1, HAP_R01_PL2, and HAP_R01_PL3, respectively. It is well known that the corresponding 4thS-diastereomer of HAP is less active than the 4thR-diastereometer1323. Thus the S-diastereomers were also synthesized as the negative controls, named by HAP_R02_PL1, HAP_R02_PL2, and HAP_R02_PL3, respectively (Fig. 4, Supplementary Figs S4 and S6). The R-configuration compounds retain reasonable activity in promoting capsid assembly and reducing viral DNA replication (Supplementary Table S2). A peak with molecular weight (Mw) increase of 477 Daltons was detected by LC/MS in samples treated with HAP_R01_PL1, but not in samples treated with HAP_R02_PL1 (Fig. 4a,b). The intact mass increase exactly corresponds to the Mw of the carbene species derived from photo-activation (subtraction of 28 due to loss of two nitrogen atoms). HAP_R01_PL2 and HAP_R01_PL3 can also covalently label core protein with additional mass of 556 and 529 respectively. The labeling signals in the control HAP_R02_PL2 and HAP_R02_PL3-treated samples are significantly lower than the active R-configured photolabels (Supplementary Figs S4 and S6).

View Article: PubMed Central - PubMed

ABSTRACT

Hap_r01sba_r01hap_r01sba_r01hap_r01hap_r01,sba_r01: 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 and 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 , but is unperturbed by . Photoaffinity labeling confirms the 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 but not to . 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.

No MeSH data available.


Related in: MedlinePlus