TY - JOUR
T1 - Activation of 6-alkoxy-substituted methylenecyclopropane nucleoside analogs requires enzymatic modification by adenosine deaminase-like protein 1
AU - Vollmer, Kathryn J.
AU - Burns, Anna C.
AU - Sauer, Hannah E.
AU - Williams, John D.
AU - Komazin-Meredith, Gloria
AU - Cardinale, Steve
AU - Butler, Michelle
AU - Aron, Zachary
AU - Hussein, Islam
AU - Busch, Marc G.
AU - Bowlin, Terry L.
AU - Gentry, Brian G.
N1 - Funding Information:
This work was funded by NIH grant R44 AI082799, the Drake University College of Pharmacy and Health Sciences Harris Endowment, the Drake Undergraduate Science Collaborative Institute, and the Iowa Space Grant Consortium (award NNX16AL88H).
Publisher Copyright:
Copyright © 2019 American Society for Microbiology. All Rights Reserved.
PY - 2019
Y1 - 2019
N2 - To determine the mechanism of action of third-generation methylenecyclopropane nucleoside analogs (MCPNAs), DNA sequencing of herpes simplex virus 1 (HSV-1) isolates resistant to third-generation MCPNAs resulted in the discovery of G841S and N815S mutations in HSV-1 UL30. Purified HSV-1 UL30 or human cytomegalovirus (HCMV) UL54 was then subjected to increasing concentrations of MBX-2168-triphosphate (TP), with results demonstrating a 50% inhibitory concentration (IC50) of ~200 μM, indicating that MBX-2168-TP does not inhibit the viral DNA polymerase. Further metabolic studies showed the removal of a moiety on the guanine ring of MBX-2168. Therefore, we hypothesized that enzymatic removal of a moiety at the 6-position of the guanine ring of third-generation MCPNAs is an essential step in activation. To test this hypothesis, pentostatin (deoxycoformycin [dCF]), an adenosine deaminase-like protein 1 (ADAL-1) inhibitor, was coincubated with MBX-2168. The results showed that dCF antagonized the effect of MBX-2168, with a >40-fold increase in the 50% effective concentration (EC50) at 50 μM dCF (EC50 of 63.1 ± 8.7 μM), compared with MBX-2168 alone (EC50 of 0.2 ± 0.1 μM). Purified ADAL-1 demonstrated time-dependent removal of the moiety on the guanine ring of MBX-2168-monophosphate (MP), with a Km of 17.5 ± 2.4 μM and a Vmax of 0.12 ± 0.04 nmol min−1. Finally, synguanol-TP demonstrated concentration-dependent inhibition of HSV-1 UL30 and HCMV UL54, with IC50s of 0.33 ± 0.16 and 0.38 ± 0.11 μM, respectively. We conclude that ADAL-1 is the enzyme responsible for removing the moiety from the guanine ring of MBX-2168-MP prior to conversion to a TP, the active compound that inhibits the viral DNA polymerase.
AB - To determine the mechanism of action of third-generation methylenecyclopropane nucleoside analogs (MCPNAs), DNA sequencing of herpes simplex virus 1 (HSV-1) isolates resistant to third-generation MCPNAs resulted in the discovery of G841S and N815S mutations in HSV-1 UL30. Purified HSV-1 UL30 or human cytomegalovirus (HCMV) UL54 was then subjected to increasing concentrations of MBX-2168-triphosphate (TP), with results demonstrating a 50% inhibitory concentration (IC50) of ~200 μM, indicating that MBX-2168-TP does not inhibit the viral DNA polymerase. Further metabolic studies showed the removal of a moiety on the guanine ring of MBX-2168. Therefore, we hypothesized that enzymatic removal of a moiety at the 6-position of the guanine ring of third-generation MCPNAs is an essential step in activation. To test this hypothesis, pentostatin (deoxycoformycin [dCF]), an adenosine deaminase-like protein 1 (ADAL-1) inhibitor, was coincubated with MBX-2168. The results showed that dCF antagonized the effect of MBX-2168, with a >40-fold increase in the 50% effective concentration (EC50) at 50 μM dCF (EC50 of 63.1 ± 8.7 μM), compared with MBX-2168 alone (EC50 of 0.2 ± 0.1 μM). Purified ADAL-1 demonstrated time-dependent removal of the moiety on the guanine ring of MBX-2168-monophosphate (MP), with a Km of 17.5 ± 2.4 μM and a Vmax of 0.12 ± 0.04 nmol min−1. Finally, synguanol-TP demonstrated concentration-dependent inhibition of HSV-1 UL30 and HCMV UL54, with IC50s of 0.33 ± 0.16 and 0.38 ± 0.11 μM, respectively. We conclude that ADAL-1 is the enzyme responsible for removing the moiety from the guanine ring of MBX-2168-MP prior to conversion to a TP, the active compound that inhibits the viral DNA polymerase.
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U2 - 10.1128/AAC.01301-19
DO - 10.1128/AAC.01301-19
M3 - Article
C2 - 31332074
AN - SCOPUS:85072573515
SN - 0066-4804
VL - 63
JO - Antimicrobial agents and chemotherapy
JF - Antimicrobial agents and chemotherapy
IS - 10
M1 - e01301-19
ER -