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. 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.

[1]  C. Andersen,et al.  Epstein-Barr virus and its association with disease - a review of relevance to general practice , 2019, BMC Family Practice.

[2]  Kimberley A Foley,et al.  Tools for the Diagnosis of Herpes Simplex Virus 1/2: Systematic Review of Studies Published Between 2012 and 2018 , 2019, JMIR public health and surveillance.

[3]  W. Britt,et al.  New therapies for human cytomegalovirus infections. , 2018, Antiviral research.

[4]  P. Kennedy,et al.  Clinical Features of Varicella-Zoster Virus Infection , 2018, Viruses.

[5]  D. Coen,et al.  Potency and Stereoselectivity of Cyclopropavir Triphosphate Action on Human Cytomegalovirus DNA Polymerase , 2016, Antimicrobial Agents and Chemotherapy.

[6]  Gloria Komazin-Meredith,et al.  TAOK3 phosphorylates the methylenecyclopropane nucleoside MBX 2168 to its monophosphate. , 2015, Antiviral research.

[7]  H. Agut,et al.  Laboratory and Clinical Aspects of Human Herpesvirus 6 Infections , 2015, Clinical Microbiology Reviews.

[8]  J. Drach,et al.  Metabolism of Cyclopropavir and Ganciclovir in Human Cytomegalovirus-Infected Cells , 2014, Antimicrobial Agents and Chemotherapy.

[9]  S. Chou,et al.  Human Cytomegalovirus UL97 Kinase Is Involved in the Mechanism of Action of Methylenecyclopropane Analogs with 6-Ether and -Thioether Substitutions , 2013, Antimicrobial Agents and Chemotherapy.

[10]  N. Peet,et al.  Synthesis and Antiviral Activities of Methylenecyclopropane Analogs with 6-Alkoxy and 6-Alkylthio Substitutions That Exhibit Broad-Spectrum Antiviral Activity against Human Herpesviruses , 2013, Antimicrobial Agents and Chemotherapy.

[11]  Xiaohong Liu,et al.  Mutations in adenosine deaminase-like (ADAL) protein confer resistance to the antiproliferative agents N6-cyclopropyl-PMEDAP and GS-9219. , 2013, Anticancer research.

[12]  S. Chou,et al.  Cyclopropavir Susceptibility of Cytomegalovirus DNA Polymerase Mutants Selected after Antiviral Drug Exposure , 2011, Antimicrobial Agents and Chemotherapy.

[13]  R. Razonable.  Antiviral Drugs for Viruses Other Than Human Immunodeficiency Virus , 2011, Mayo Clinic Proceedings.

[14]  M. Sofia,et al.  Adenosine deaminase-like protein 1 (ADAL1): characterization and substrate specificity in the hydrolysis of N(6)- or O(6)-substituted purine or 2-aminopurine nucleoside monophosphates. , 2011, Journal of medicinal chemistry.

[15]  S. Chou,et al.  Cytomegalovirus UL97 Mutations Affecting Cyclopropavir and Ganciclovir Susceptibility , 2010, Antimicrobial Agents and Chemotherapy.

[16]  D. Coen,et al.  Stereoselective Phosphorylation of Cyclopropavir by pUL97 and Competitive Inhibition by Maribavir , 2010, Antimicrobial Agents and Chemotherapy.

[17]  M. Prichard,et al.  Phosphonate analogues of cyclopropavir phosphates and their E-isomers. Synthesis and antiviral activity. , 2009, Bioorganic & medicinal chemistry.

[18]  T. Bowlin,et al.  Preclinical Pharmacokinetic, Toxicokinetic and Toxicology Results for Cyclopropavir, a Promising New Agent for the Treatment of Beta- and Gamma-herpesviruses , 2009 .

[19]  S. Chou.  Cytomegalovirus UL97 mutations in the era of ganciclovir and maribavir , 2008, Reviews in medical virology.

[20]  K. Biron.  Antiviral drugs for cytomegalovirus diseases. , 2006, Antiviral research.

[21]  I. Votruba,et al.  N6-methyl-AMP aminohydrolase activates N6-substituted purine acyclic nucleoside phosphonates. , 2006, Biochemical pharmacology.

[22]  E. Kern,et al.  In Vitro Activity and Mechanism of Action of Methylenecyclopropane Analogs of Nucleosides against Herpesvirus Replication , 2005, Antimicrobial Agents and Chemotherapy.

[23]  E. Gullen,et al.  Synthesis and antiviral activity of (Z)- and (E)-2,2-[bis(hydroxymethyl)cyclopropylidene]methylpurines and -pyrimidines: second-generation methylenecyclopropane analogues of nucleosides. , 2004, Journal of medicinal chemistry.

[24]  S. Chou,et al.  Viral DNA polymerase mutations associated with drug resistance in human cytomegalovirus. , 2003, The Journal of infectious diseases.

[25]  S. Landolfo,et al.  The human cytomegalovirus. , 2003, Pharmacology & therapeutics.

[26]  B. Davidson,et al.  Superior cytotoxicity with ganciclovir compared with acyclovir and 1-beta-D-arabinofuranosylthymine in herpes simplex virus-thymidine kinase-expressing cells: a novel paradigm for cell killing. , 1998, Cancer research.

[27]  J B Houston,et al.  Utility of in vitro drug metabolism data in predicting in vivo metabolic clearance. , 1994, Biochemical pharmacology.

[28]  R. Midgett,et al.  Adenosine deaminase inhibitors. Synthesis and biological evaluation of C1' and nor-C1' derivatives of (+)-erythro-9-(2(S)-hydroxy-3(R)-nonyl)adenine. , 1992, Journal of medicinal chemistry.

[29]  F. Moolten.  Tumor chemosensitivity conferred by inserted herpes thymidine kinase genes: paradigm for a prospective cancer control strategy. , 1986, Cancer research.

[30]  Trachette L. Jackson,et al.  Phosphorylation of antiviral and endogenous nucleotides to di- and triphosphates by guanosine monophosphate kinase. , 2011, Biochemical pharmacology.

[31]  A. Denys.  [Antiviral drugs]. , 2011, Polski merkuriusz lekarski : organ Polskiego Towarzystwa Lekarskiego.

[32]  L. Pantanowitz,et al.  Kaposi Sarcoma Pathogenesis: A Triad of Viral Infection, Oncogenesis and Chronic Inflammation. , 2010, Translational biomedicine.

[33]  H. Balfour.  Drug therapy : Antiviral drugs , 1999 .

[34]  R. Ptak,et al.  (Z)- and (E)-2-((hydroxymethyl)cyclopropylidene)methyladenine and -guanine. New nucleoside analogues with a broad-spectrum antiviral activity. , 1998, Journal of medicinal chemistry.

[35]  C. Crumpacker.  Drug therapy : Ganciclovir , 1996 .