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

2 Scopus citations

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

    Fingerprint

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

Cite this

Vollmer, K. J., Burns, A. C., Sauer, H. E., Williams, J. D., Komazin-Meredith, G., Cardinale, S., Butler, M., Aron, Z., Hussein, I., Busch, M. G., Bowlin, T. L., & Gentry, B. G. (2019). Activation of 6-alkoxy-substituted methylenecyclopropane nucleoside analogs requires enzymatic modification by adenosine deaminase-like protein 1. Antimicrobial agents and chemotherapy, 63(10), [e01301-19]. https://doi.org/10.1128/AAC.01301-19