Acyclic guanosine analogs as inhibitors of human cytomegalovirus

Abstract

The acyclic guanosine analogs R-and S-enantiomers of 9-(3,4-dihydroxybutyl)guanine [(R)-and (S)-DHBG], 9-(4-hydroxybutyl)guanine (HBG), and 9-(2-hydroxyethoxymethyl)guanine (ACV) were examined for their effects on human cytomegalovirus (CMV) replication and on CMV DNA synthesis in cell culture as well as for their ability as triphosphates to interact with CMV DNA polymerase. Production of early CMV antigens was not affected. All analogs inhibited CMV DNA synthesis and late viral antigen synthesis. Primary CMV isolates were less susceptible to all tested analogs than was the laboratory strain CMV Ad.169. The triphosphate of ACV was the most potent inhibitor of CMV DNA polymerase, with an observed Ki of 0.0076 ,uM. The corresponding Ki values of the triphosphates of (R)-DHBG, (S)-DHBG, and HBG were 3.5, 13.0 and 0.23 ,uM, respectively. All triphosphates of the analogs given above inhibited CMV DNA polymerase in a competitive manner with respect to dGTP. The triphosphates of the analogs also inhibited reactions when the synthetic template poly(dC)oligo(dG)1218 was used, whereas no inhibition was observed with poly(dA)oligo(dT)12-18None of the triphosphate analogs supported DNA synthesis in the absence of dGTP, showing that no analog was an alternative substrate to dGTP.Several nucleoside analogs are selective inhibitors of viral replication at concentrations that do not affect host cells. Two classes of nucleoside analogs have received particular attention: 5-substituted 2'-deoxypyrimidines (3) and acyclic guanosine analogs (4). They are strongly active against herpes simplex virus (HSV) types 1 and 2 (HSV-1 and HSV-2). A general property of these analogs is postulated to be a selective phosphorylation to monophosphates by the HSV-induced thymidine kinase (TK), followed by preferential inhibition of HSV-induced DNA polymerase activity by the triphosphorylated forms (3,4,8,13,20). Human cytomegalovirus (CMV) lacks a virus-encoded TK (7, 29). Therefore, one would expect the acyclic nucleoside analogs, which do not seem to be substrates for cellular TK, to remain unphosphorylated in CMV-infected cells and thus not to be inhibitors of CMV replication. Acyclic nucleoside analogs have been reported to have lower activities against CMV than against HSV or varicella zoster virus (1, 10). Therefore, it was surprising that 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) was a much more potent inhibitor of CMV multiplication in cell culture than 9-(2-hydroxyethoxymethyl)guanine (ACV) (18, 24). The triphosphates of these analogs, DHPGTP and ACVTP, were inhibitors of the partially purified CMV DNA polymerase. ACVTP had a higher affinity than DHPGTP for the CMV DNA polymerase (19). Higher amounts of DHPGTP, however, were formed in CMV-infected cells compared with those of ACVTP (2). This probably explains the better activity of DHPG, but the enzyme which mediates monophosphorylation of DHPG still has not been defined.In this study we investigated several acyclic guanosine analogs, the R-and S-enantiomers of 9-(3,4-dihydroxybutyl...

Lobucavir is phosphorylated in human cytomegalovirus-infected and -uninfected cells and inhibits the viral DNA polymerase

“…HCMV DNA polymerase eluted from the Q-Sepharose column at approximately 150 to 250 mM NaCl and from the heparin-agarose column at approximately 500 mM NaCl. This preparation was active in 100 mM (NH 4 ) 2 SO 4 , displayed a K m for dGTP of approximately 0.2 M, and was more sensitive to phosphonoformic acid (50% inhibitory concentration [IC 50 ] ϭ 5 M) than was HeLa cell polymerase alpha (IC 50 ϭ 74 M; kindly provided by Robert Hamatake), similar to previously purified HCMV DNA polymerase preparations (29,46,54). HSV-1 TK was affinity purified by thymidine-agarose affinity chromatography from an extract of HSV-1 (KOS)infected HeLa cells (15).…”

“…5 show that LBV-TP proved to be a potent, competitive inhibitor of the HCMV DNA polymerase, with a K i of approximately 5 nM. This potency is on the order of that of acyclovir triphosphate (29,46,54) and is similar to the potency of LBV-TP against HSV DNA polymerase (13,53). Furthermore, LBV-TP is a more potent inhibitor of the HCMV DNA polymerase than of various cellular DNA polymerases: the K i for HeLa polymerase alpha is reported to be 0.22 to 14 M (13,16,21,53); the K i for polymerase gamma is 54 to 330 M (16,21); and polymerase beta is not inhibited at any concentration tested (16,21).…”

Identification and evaluation of antivirals for Rift Valley fever virus

“…While there are arbitrarily 13 functional groups available now for the nine human infectious diseases (De Clercq & Li, 2016). They are: (i) 5-substituted 2'deoxyuridine analogues (treatment for HSV (De Winter & Herdewijn, 1996), and VZV (McGuigan et al, 1999)); (ii) nucleoside analogues (for HSV (Fyfe, Keller, Furman, Miller, & Elion, 1978), HBV (van Bömmel et al, 2010), and VZV (Balzarini & McGuigan, 2002)); (iii) (nonnucleoside) pyrophosphate analogues (for HCMV (Gilbert & Boivin, 2005), and HSV (Eriksson, Öberg, & Wahren, 1982)); (iv) nucleoside reverse transcriptase inhibitors (for HIV (Group, 2008), and HBV (Margeridon-Thermet et al, 2009)); (v) nonnucleoside reverse transcriptase inhibitors (for HIV (Merluzzi, Hargrave, Labadia, Grozinger, & Skoog, 1990)); (vi) protease inhibitors (for HIV (Condra, Schleif, Blahy, & Gabryelski, 1995), and HCV (C Lin et al, 2004)); (vii) integrase inhibitors (for HIV (Pommier, Johnson, & Marchand, 2005)); (viii) entry inhibitors (for HIV (Reeves et al, 2002), HSV (Gong et al, 2002), VZV (Zhu, Gershon, Ambron, Gabel, & Gershon, 1995), and RSV (Razinkov, Huntley, Ellestad, & Krishnamurthy, 2002)); (ix) acyclic guanosine analogues (for HCMV (Wahren, Larsson, Rudén, Sundqvist, & Sølver, 1987), HSV (De Clercq et al, 2001), and VZV (Karlström, Källander, Abele, & Larsson, 1986)); (x) acyclic nucleoside phosphonate (ANP)analogues (for HIV, HCMV, and HBV(De Clercq, 2007a),); (xi) HCV NS5A and NS5B inhibitors; (xii) influenza virus inhibitors; and (xiii) immunostimulators, interferons, oligonucleotides, and antimitotic inhibitors (for HBV (Sokal et al, 1998), HCV (Manns et al, 2001), HCMV (Chapman, Thayer, Vincent, & Haigwood, 1991), and HPV (Jach, Basta, & Szczudrawa, 2003)). Unlike the viruses with available treatment, the underlying mechanisms of RVFV entry, replication, and release are poorly understood.…”

Research on the Chemical Materials of Inosine Di- and Triphosphate

“…The study on the specificity and kinetics of ATPase also employed ITP as a substrate. [6,7] At present, the general method for the synthesis of ITP and IDP was to treat ATP and ADP with sodium nitrite under acidic conditions, and the yield was around 70%. [8] Otherwise, the traditional "one-pot, three-step" and salicyl chlorophosphite methods, [9,10] could be employed to afford the target molecules in moderate yield.…”