Activation of 6-alkoxy-substituted methylenecyclopropane nucleoside analogs requires enzymatic modification by adenosine deaminase-like protein 1

Kathryn J. Vollmer, Anna C. Burns, Hannah E. Sauer, John D. Williams, Gloria Komazin-Meredith, Steve Cardinale, Michelle Butler, Zachary Aron, Islam Hussein, Marc G. Busch, Terry L. Bowlin, Brian G. Gentry

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
Article numbere01301-19
JournalAntimicrobial agents and chemotherapy
Volume63
Issue number10
DOIs
StatePublished - Jan 1 2019

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Adenosine Deaminase
Nucleosides
Pentostatin
Guanine
Human Herpesvirus 1
Proteins
Viral DNA
DNA-Directed DNA Polymerase
Cytomegalovirus
Inhibitory Concentration 50
methylenecyclopropane
alkoxyl radical
MBX 2168
DNA Sequence Analysis
Mutation
triphosphoric acid
Enzymes

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

Cite this

Vollmer, Kathryn J. ; Burns, Anna C. ; Sauer, Hannah E. ; Williams, John D. ; Komazin-Meredith, Gloria ; Cardinale, Steve ; Butler, Michelle ; Aron, Zachary ; Hussein, Islam ; Busch, Marc G. ; Bowlin, Terry L. ; Gentry, Brian G. / Activation of 6-alkoxy-substituted methylenecyclopropane nucleoside analogs requires enzymatic modification by adenosine deaminase-like protein 1. In: Antimicrobial agents and chemotherapy. 2019 ; Vol. 63, No. 10.
@article{cb0aad71ba274a75b4f68bc2cd1c1a05,
title = "Activation of 6-alkoxy-substituted methylenecyclopropane nucleoside analogs requires enzymatic modification by adenosine deaminase-like protein 1",
abstract = "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.",
author = "Vollmer, {Kathryn J.} and Burns, {Anna C.} and Sauer, {Hannah E.} and Williams, {John D.} and Gloria Komazin-Meredith and Steve Cardinale and Michelle Butler and Zachary Aron and Islam Hussein and Busch, {Marc G.} and Bowlin, {Terry L.} and Gentry, {Brian G.}",
year = "2019",
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doi = "10.1128/AAC.01301-19",
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Vollmer, KJ, Burns, AC, Sauer, HE, Williams, JD, Komazin-Meredith, G, Cardinale, S, Butler, M, Aron, Z, Hussein, I, Busch, MG, Bowlin, TL & Gentry, BG 2019, 'Activation of 6-alkoxy-substituted methylenecyclopropane nucleoside analogs requires enzymatic modification by adenosine deaminase-like protein 1', Antimicrobial agents and chemotherapy, vol. 63, no. 10, e01301-19. https://doi.org/10.1128/AAC.01301-19

Activation of 6-alkoxy-substituted methylenecyclopropane nucleoside analogs requires enzymatic modification by adenosine deaminase-like protein 1. / Vollmer, Kathryn J.; Burns, Anna C.; Sauer, Hannah E.; Williams, John D.; Komazin-Meredith, Gloria; Cardinale, Steve; Butler, Michelle; Aron, Zachary; Hussein, Islam; Busch, Marc G.; Bowlin, Terry L.; Gentry, Brian G.

In: Antimicrobial agents and chemotherapy, Vol. 63, No. 10, e01301-19, 01.01.2019.

Research output: Contribution to journalArticle

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.

PY - 2019/1/1

Y1 - 2019/1/1

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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