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.

Recent Advances in Molecular Mechanisms of Nucleoside Antivirals

“…This approach was also applied in the development of bemnifosbuvir (Figure 5). One of the recent and most interesting examples of 6-modified purine NAs is MBX-2168, which is a third-generation methylenecyclopropane NA designed to act against herpesviruses (Figure 25) [134]. The unique feature of herpesviruses is kinases encoded in the genome.…”

“…On the one hand, this broadens the activity spectrum since the transformation of the analog into the corresponding monophosphate can occur independently of viral enzymes. On the other hand, the existence of a metabolic pathway independent of viral kinases provides One of the recent and most interesting examples of 6-modified purine NAs is MBX-2168, which is a third-generation methylenecyclopropane NA designed to act against herpesviruses (Figure 25) [134]. The unique feature of herpesviruses is kinases encoded in the genome.…”

Antiviral Classification

“…As of September 2020, many UL30 inhibitors such as idoxuridine, brivudine, trifluridine foscarnet, aciclovir, famciclovir, valaciclovir, and penciclovir have been approved for HSV treatment. Because current compounds face the challenge of drug resistance and adverse reactions ( Vollmer et al ., 2019 ), many experimental inhibitors such as synguanol ( Vollmer et al ., 2019 ), mitoxantrone dihydrochloride ( Huang et al ., 2019 ), and PHA767491 ( Hou et al ., 2017 ) are under development. For instance, synguanol is a methylenecyclopropane nucleoside analog that competitively inhibits HSV-1 UL30 (IC 50 : 0.33 ± 0.16 µM)( Vollmer et al ., 2019 ).…”

“…Because current compounds face the challenge of drug resistance and adverse reactions ( Vollmer et al ., 2019 ), many experimental inhibitors such as synguanol ( Vollmer et al ., 2019 ), mitoxantrone dihydrochloride ( Huang et al ., 2019 ), and PHA767491 ( Hou et al ., 2017 ) are under development. For instance, synguanol is a methylenecyclopropane nucleoside analog that competitively inhibits HSV-1 UL30 (IC 50 : 0.33 ± 0.16 µM)( Vollmer et al ., 2019 ). Mitoxantrone dihydrochloride inhibits viral DNA synthesis by blocking the transcription of UL30 (IC 50 : 1.21 µM) ( Huang et al ., 2019 ).…”