Metal binding 6-arylthio-3-hydroxypyrimidine-2,4-diones inhibited human cytomegalovirus by targeting the pUL89 endonuclease of the terminase complex

Lei Wang; Tiffany C. Edwards; Rajkumar Lalji Sahani; Jiashu Xie; Hideki Aihara; Robert J. Geraghty; Zhengqiang Wang

doi:10.1016/j.ejmech.2021.113640

Metal binding 6-arylthio-3-hydroxypyrimidine-2,4-diones inhibited human cytomegalovirus by targeting the pUL89 endonuclease of the terminase complex

Lei Wang, Tiffany C. Edwards, Rajkumar Lalji Sahani, Jiashu Xie, Hideki Aihara, Robert J. Geraghty, Zhengqiang Wang

Research output: Contribution to journal › Article › peer-review

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Abstract

The genome packaging of human cytomegalovirus (HCMV) requires a divalent metal-dependent endonuclease activity localized to the C-terminus of pUL89 (pUL89-C), which is reminiscent of RNase H-like enzymes in active site structure and catalytic mechanism. Our previous work has shown that metal-binding small molecules can effectively inhibit pUL89-C while conferring significant antiviral activities. In this report we generated a collection of 43 metal-binding small molecules by repurposing analogs of the 6-arylthio-3-hydroxypyrimidine-2,4-dione chemotype previously synthesized for targeting HIV-1 RNase H, and by chemically synthesizing new N-1 analogs. The analogs were subjected to two parallel screening assays: the pUL89-C biochemical assay and the HCMV antiviral assay. Compounds with significant inhibition from each assay were further tested in a dose-response fashion. Single dose cell viability and PAMPA cell permeability were also conducted and considered in selecting compounds for the dose-response antiviral testing. These assays identified a few analogs displaying low μM inhibition against pUL89-C in the biochemical assay and HCMV replication in the antiviral assay. The target engagement was further evaluated via a thermal shift assay using recombinant pUL89-C and molecular docking. Overall, our current work identified novel inhibitors of pUL89-C with significant antiviral activities and further supports targeting pUL89-C with metal-binding small molecules as an antiviral approach against HCMV.

Original language	English (US)
Article number	113640
Journal	European Journal of Medicinal Chemistry
Volume	222
DOIs	https://doi.org/10.1016/j.ejmech.2021.113640
State	Published - Oct 15 2021

Bibliographical note

Funding Information:
Based on the screening results, 23 analogs were selected for further target-specific biochemical and biophysical studies against pUL89-C including the dose-response testing in the ELISA assay, and a thermal shift assay (TSA) that has been used to characterize target binding in drug discovery [58–60]. For the dose-response biochemical assay, previously reported pUL89-C inhibitors (7r, 8i, and 9b, structures shown in Table 2 header) were used as controls. For the TSA, two non-pUL89-C targeting HCMV inhibitors (GCV and BDCRB, Fig. 3) were included as additional controls. Results from these studies are summarized in Table 2. Dose-response curves, TSA traces and ΔTm bar graphs for representative compounds are also shown in Fig. 4 A–C. In the dose-response ELISA assay, all three positive controls potently inhibited pUL89-C (IC50 = 3.3–3.5 μM). Of the 23 new analogs, 8 (10b, 10e, 11f, 11g, 11m, 11n, 12a and 12e) inhibited pUL89-C with single-digit μM IC50s (IC50 = 1.9–8.1 μM), and 4 additional analogs of subtype 11 (11a, 11c, 11i and 11j) exhibited significant inhibition (IC50 = 11.8–14.1 μM). Modest inhibition (IC50 = 22–48 μM) was observed with all other compounds tested (Table 2). Consistent with the biochemical results, 7 of the 8 most potent biochemical inhibitors produced significant right shift (positive ΔTm) of pUL89-C melting point (ΔTm = 0.74–2.8 °C) in the TSA. The sole exception is 11n, which did not produce a significant shift (ΔTm = 0.16 °C). Interestingly, all three selected analogs with a biphenyl Ar caused a left shift: 10n (ΔTm = −0.55 °C), 10p (ΔTm = −1.7 °C), and 10r (ΔTm = −1.4 °C). This association of the biphenyl Ar and the left shift in TSA was congruent with previously reported pUL89-C inhibitors, where large left shift was observed with the two biphenyl containing HPD-NH analogs [55] (8i: ΔTm = −2.2 °C; 9b: ΔTm = −2.8 °C), whereas the phenyl bearing 7r (ΔTm = 2.4 °C) produced a strong right shift. These results collectively suggest that a bulky Ar may negatively impact the enzymatic activity by destabilizing pUL89-C. Importantly, the two non pUL89-C-targeting HCMV inhibitors, GCV and BDCRB, did not confer significant shift in the TSA, supporting the pUL89-C target engagement of our new inhibitors. Finally, we also measured the permeability of 13 selected analogs in a PAMPA permeability assay (Table 2). Of these, 10 analogs showed good permeability (Pe = 2.4–6.6 × 10−6 cm/s), two (10e: Pe = 0.21 × 10−6 cm/s; and 10p: Pe = 1.1 × 10−6 cm/s) exhibited moderately low permeability, and one (10b) displayed prohibitively low PAMPA permeability (Pe = 0.017 × 10−6 cm/s).This research was supported by the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, grant number R01AI136982 (to RJG and ZW), and the National Institute of General Medical Sciences, the National Institutes of Health, grant number R35 GM118047 (to HA). We thank the Minnesota Supercomputing Institute for molecular modeling resources. We thank Christine Dreis from the Center for Drug Design at the University of Minnesota for technical assistance.

Funding Information:
This research was supported by the National Institute of Allergy and Infectious Diseases , the National Institutes of Health , grant number R01AI136982 (to RJG and ZW), and the National Institute of General Medical Sciences , the National Institutes of Health , grant number R35 GM118047 (to HA). We thank the Minnesota Supercomputing Institute for molecular modeling resources. We thank Christine Dreis from the Center for Drug Design at the University of Minnesota for technical assistance.

Publisher Copyright:
© 2021 Elsevier Masson SAS

Keywords

Human cytomegalovirus
Metal-binding compounds
Terminase complex
pUL89-C
thermal shift assay

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@article{59362e3232f04a618ea805192c0971d1,

title = "Metal binding 6-arylthio-3-hydroxypyrimidine-2,4-diones inhibited human cytomegalovirus by targeting the pUL89 endonuclease of the terminase complex",

abstract = "The genome packaging of human cytomegalovirus (HCMV) requires a divalent metal-dependent endonuclease activity localized to the C-terminus of pUL89 (pUL89-C), which is reminiscent of RNase H-like enzymes in active site structure and catalytic mechanism. Our previous work has shown that metal-binding small molecules can effectively inhibit pUL89-C while conferring significant antiviral activities. In this report we generated a collection of 43 metal-binding small molecules by repurposing analogs of the 6-arylthio-3-hydroxypyrimidine-2,4-dione chemotype previously synthesized for targeting HIV-1 RNase H, and by chemically synthesizing new N-1 analogs. The analogs were subjected to two parallel screening assays: the pUL89-C biochemical assay and the HCMV antiviral assay. Compounds with significant inhibition from each assay were further tested in a dose-response fashion. Single dose cell viability and PAMPA cell permeability were also conducted and considered in selecting compounds for the dose-response antiviral testing. These assays identified a few analogs displaying low μM inhibition against pUL89-C in the biochemical assay and HCMV replication in the antiviral assay. The target engagement was further evaluated via a thermal shift assay using recombinant pUL89-C and molecular docking. Overall, our current work identified novel inhibitors of pUL89-C with significant antiviral activities and further supports targeting pUL89-C with metal-binding small molecules as an antiviral approach against HCMV.",

keywords = "Human cytomegalovirus, Metal-binding compounds, Terminase complex, pUL89-C, thermal shift assay",

author = "Lei Wang and Edwards, {Tiffany C.} and Sahani, {Rajkumar Lalji} and Jiashu Xie and Hideki Aihara and Geraghty, {Robert J.} and Zhengqiang Wang",

note = "Funding Information: Based on the screening results, 23 analogs were selected for further target-specific biochemical and biophysical studies against pUL89-C including the dose-response testing in the ELISA assay, and a thermal shift assay (TSA) that has been used to characterize target binding in drug discovery [58–60]. For the dose-response biochemical assay, previously reported pUL89-C inhibitors (7r, 8i, and 9b, structures shown in Table 2 header) were used as controls. For the TSA, two non-pUL89-C targeting HCMV inhibitors (GCV and BDCRB, Fig. 3) were included as additional controls. Results from these studies are summarized in Table 2. Dose-response curves, TSA traces and ΔTm bar graphs for representative compounds are also shown in Fig. 4 A–C. In the dose-response ELISA assay, all three positive controls potently inhibited pUL89-C (IC50 = 3.3–3.5 μM). Of the 23 new analogs, 8 (10b, 10e, 11f, 11g, 11m, 11n, 12a and 12e) inhibited pUL89-C with single-digit μM IC50s (IC50 = 1.9–8.1 μM), and 4 additional analogs of subtype 11 (11a, 11c, 11i and 11j) exhibited significant inhibition (IC50 = 11.8–14.1 μM). Modest inhibition (IC50 = 22–48 μM) was observed with all other compounds tested (Table 2). Consistent with the biochemical results, 7 of the 8 most potent biochemical inhibitors produced significant right shift (positive ΔTm) of pUL89-C melting point (ΔTm = 0.74–2.8 °C) in the TSA. The sole exception is 11n, which did not produce a significant shift (ΔTm = 0.16 °C). Interestingly, all three selected analogs with a biphenyl Ar caused a left shift: 10n (ΔTm = −0.55 °C), 10p (ΔTm = −1.7 °C), and 10r (ΔTm = −1.4 °C). This association of the biphenyl Ar and the left shift in TSA was congruent with previously reported pUL89-C inhibitors, where large left shift was observed with the two biphenyl containing HPD-NH analogs [55] (8i: ΔTm = −2.2 °C; 9b: ΔTm = −2.8 °C), whereas the phenyl bearing 7r (ΔTm = 2.4 °C) produced a strong right shift. These results collectively suggest that a bulky Ar may negatively impact the enzymatic activity by destabilizing pUL89-C. Importantly, the two non pUL89-C-targeting HCMV inhibitors, GCV and BDCRB, did not confer significant shift in the TSA, supporting the pUL89-C target engagement of our new inhibitors. Finally, we also measured the permeability of 13 selected analogs in a PAMPA permeability assay (Table 2). Of these, 10 analogs showed good permeability (Pe = 2.4–6.6 × 10−6 cm/s), two (10e: Pe = 0.21 × 10−6 cm/s; and 10p: Pe = 1.1 × 10−6 cm/s) exhibited moderately low permeability, and one (10b) displayed prohibitively low PAMPA permeability (Pe = 0.017 × 10−6 cm/s).This research was supported by the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, grant number R01AI136982 (to RJG and ZW), and the National Institute of General Medical Sciences, the National Institutes of Health, grant number R35 GM118047 (to HA). We thank the Minnesota Supercomputing Institute for molecular modeling resources. We thank Christine Dreis from the Center for Drug Design at the University of Minnesota for technical assistance. Funding Information: This research was supported by the National Institute of Allergy and Infectious Diseases , the National Institutes of Health , grant number R01AI136982 (to RJG and ZW), and the National Institute of General Medical Sciences , the National Institutes of Health , grant number R35 GM118047 (to HA). We thank the Minnesota Supercomputing Institute for molecular modeling resources. We thank Christine Dreis from the Center for Drug Design at the University of Minnesota for technical assistance. Publisher Copyright: {\textcopyright} 2021 Elsevier Masson SAS",

year = "2021",

month = oct,

day = "15",

doi = "10.1016/j.ejmech.2021.113640",

language = "English (US)",

volume = "222",

journal = "European Journal of Medicinal Chemistry",

issn = "0223-5234",

publisher = "Elsevier Masson SAS",

}

TY - JOUR

T1 - Metal binding 6-arylthio-3-hydroxypyrimidine-2,4-diones inhibited human cytomegalovirus by targeting the pUL89 endonuclease of the terminase complex

AU - Wang, Lei

AU - Edwards, Tiffany C.

AU - Sahani, Rajkumar Lalji

AU - Xie, Jiashu

AU - Aihara, Hideki

AU - Geraghty, Robert J.

AU - Wang, Zhengqiang

N1 - Funding Information: Based on the screening results, 23 analogs were selected for further target-specific biochemical and biophysical studies against pUL89-C including the dose-response testing in the ELISA assay, and a thermal shift assay (TSA) that has been used to characterize target binding in drug discovery [58–60]. For the dose-response biochemical assay, previously reported pUL89-C inhibitors (7r, 8i, and 9b, structures shown in Table 2 header) were used as controls. For the TSA, two non-pUL89-C targeting HCMV inhibitors (GCV and BDCRB, Fig. 3) were included as additional controls. Results from these studies are summarized in Table 2. Dose-response curves, TSA traces and ΔTm bar graphs for representative compounds are also shown in Fig. 4 A–C. In the dose-response ELISA assay, all three positive controls potently inhibited pUL89-C (IC50 = 3.3–3.5 μM). Of the 23 new analogs, 8 (10b, 10e, 11f, 11g, 11m, 11n, 12a and 12e) inhibited pUL89-C with single-digit μM IC50s (IC50 = 1.9–8.1 μM), and 4 additional analogs of subtype 11 (11a, 11c, 11i and 11j) exhibited significant inhibition (IC50 = 11.8–14.1 μM). Modest inhibition (IC50 = 22–48 μM) was observed with all other compounds tested (Table 2). Consistent with the biochemical results, 7 of the 8 most potent biochemical inhibitors produced significant right shift (positive ΔTm) of pUL89-C melting point (ΔTm = 0.74–2.8 °C) in the TSA. The sole exception is 11n, which did not produce a significant shift (ΔTm = 0.16 °C). Interestingly, all three selected analogs with a biphenyl Ar caused a left shift: 10n (ΔTm = −0.55 °C), 10p (ΔTm = −1.7 °C), and 10r (ΔTm = −1.4 °C). This association of the biphenyl Ar and the left shift in TSA was congruent with previously reported pUL89-C inhibitors, where large left shift was observed with the two biphenyl containing HPD-NH analogs [55] (8i: ΔTm = −2.2 °C; 9b: ΔTm = −2.8 °C), whereas the phenyl bearing 7r (ΔTm = 2.4 °C) produced a strong right shift. These results collectively suggest that a bulky Ar may negatively impact the enzymatic activity by destabilizing pUL89-C. Importantly, the two non pUL89-C-targeting HCMV inhibitors, GCV and BDCRB, did not confer significant shift in the TSA, supporting the pUL89-C target engagement of our new inhibitors. Finally, we also measured the permeability of 13 selected analogs in a PAMPA permeability assay (Table 2). Of these, 10 analogs showed good permeability (Pe = 2.4–6.6 × 10−6 cm/s), two (10e: Pe = 0.21 × 10−6 cm/s; and 10p: Pe = 1.1 × 10−6 cm/s) exhibited moderately low permeability, and one (10b) displayed prohibitively low PAMPA permeability (Pe = 0.017 × 10−6 cm/s).This research was supported by the National Institute of Allergy and Infectious Diseases, the National Institutes of Health, grant number R01AI136982 (to RJG and ZW), and the National Institute of General Medical Sciences, the National Institutes of Health, grant number R35 GM118047 (to HA). We thank the Minnesota Supercomputing Institute for molecular modeling resources. We thank Christine Dreis from the Center for Drug Design at the University of Minnesota for technical assistance. Funding Information: This research was supported by the National Institute of Allergy and Infectious Diseases , the National Institutes of Health , grant number R01AI136982 (to RJG and ZW), and the National Institute of General Medical Sciences , the National Institutes of Health , grant number R35 GM118047 (to HA). We thank the Minnesota Supercomputing Institute for molecular modeling resources. We thank Christine Dreis from the Center for Drug Design at the University of Minnesota for technical assistance. Publisher Copyright: © 2021 Elsevier Masson SAS

PY - 2021/10/15

Y1 - 2021/10/15

N2 - The genome packaging of human cytomegalovirus (HCMV) requires a divalent metal-dependent endonuclease activity localized to the C-terminus of pUL89 (pUL89-C), which is reminiscent of RNase H-like enzymes in active site structure and catalytic mechanism. Our previous work has shown that metal-binding small molecules can effectively inhibit pUL89-C while conferring significant antiviral activities. In this report we generated a collection of 43 metal-binding small molecules by repurposing analogs of the 6-arylthio-3-hydroxypyrimidine-2,4-dione chemotype previously synthesized for targeting HIV-1 RNase H, and by chemically synthesizing new N-1 analogs. The analogs were subjected to two parallel screening assays: the pUL89-C biochemical assay and the HCMV antiviral assay. Compounds with significant inhibition from each assay were further tested in a dose-response fashion. Single dose cell viability and PAMPA cell permeability were also conducted and considered in selecting compounds for the dose-response antiviral testing. These assays identified a few analogs displaying low μM inhibition against pUL89-C in the biochemical assay and HCMV replication in the antiviral assay. The target engagement was further evaluated via a thermal shift assay using recombinant pUL89-C and molecular docking. Overall, our current work identified novel inhibitors of pUL89-C with significant antiviral activities and further supports targeting pUL89-C with metal-binding small molecules as an antiviral approach against HCMV.

AB - The genome packaging of human cytomegalovirus (HCMV) requires a divalent metal-dependent endonuclease activity localized to the C-terminus of pUL89 (pUL89-C), which is reminiscent of RNase H-like enzymes in active site structure and catalytic mechanism. Our previous work has shown that metal-binding small molecules can effectively inhibit pUL89-C while conferring significant antiviral activities. In this report we generated a collection of 43 metal-binding small molecules by repurposing analogs of the 6-arylthio-3-hydroxypyrimidine-2,4-dione chemotype previously synthesized for targeting HIV-1 RNase H, and by chemically synthesizing new N-1 analogs. The analogs were subjected to two parallel screening assays: the pUL89-C biochemical assay and the HCMV antiviral assay. Compounds with significant inhibition from each assay were further tested in a dose-response fashion. Single dose cell viability and PAMPA cell permeability were also conducted and considered in selecting compounds for the dose-response antiviral testing. These assays identified a few analogs displaying low μM inhibition against pUL89-C in the biochemical assay and HCMV replication in the antiviral assay. The target engagement was further evaluated via a thermal shift assay using recombinant pUL89-C and molecular docking. Overall, our current work identified novel inhibitors of pUL89-C with significant antiviral activities and further supports targeting pUL89-C with metal-binding small molecules as an antiviral approach against HCMV.

KW - Human cytomegalovirus

KW - Metal-binding compounds

KW - Terminase complex

KW - pUL89-C

KW - thermal shift assay

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JO - European Journal of Medicinal Chemistry

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ER -

Metal binding 6-arylthio-3-hydroxypyrimidine-2,4-diones inhibited human cytomegalovirus by targeting the pUL89 endonuclease of the terminase complex

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