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

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 K_m of 17.5 ± 2.4 μM and a V_max 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.