ABSTRACT
Herpes simplex virus (HSV) types 1 and 2 thymidine kinases (TK) are responsible for phosphorylation of antiherpes acyclonucleosides such as acyclovir (ACV) and 9-(4-hydroxybutyl)guanine (HBG). Related compounds, the N2-phenyl-9-(hydroxyalkyl)guanines, are devoid of direct antiviral activity, but potently inhibit the viral TKs and block viral reactivation from latency in vivo. The similarity in structure between the acyclonucleosides and TK inhibitors raised the question of the relevance of phosphorylation of certain of the latter analogs in their mechanisms of action. Using recombinant TKs and HPLC analysis of reaction mixtures, we report that the lead TK inhibitor N2-phenyl-9 -(4-hydroxybutyl)guanine (HBPG) and its pentyl homolog (HPnPG) are excellent substrates for the enzymes, approaching the efficiency with which the natural substrate thymidine is phosphorylated, and significantly better than ACV or HBG. Other 9-hydroxyalkyl congeners are substrates for the enzymes, but with much poorer efficiency. HBPG triphosphate was a poor inhibitor of HSV DNA polymerase, the target of acyclonucleoside triphosphates, suggesting that phosphorylation of HBPG is not important in its mechanism of blocking viral reactivation in vivo. The fact that HBPG is an efficient substrate is consistent, however, with its binding mode based both on molecular modeling studies and x-ray structure of the HBPG:TK complex.
ABSTRACT
Several 2-anilino- and 2-benzylamino-3-deaza-6-oxopurines [3-deazaguanines] and selected 8-methyl and 8-aza analogs have been synthesized. 7-Substituted N(2)-(3-ethyl-4-methylphenyl)-3-deazaguanines were potent and selective inhibitors of Gram+ bacterial DNA polymerase (pol) IIIC, and 7-substituted N(2)-(3,4-dichlorobenzyl)-3-deazaguanines were potent inhibitors of both pol IIIC and pol IIIE from Gram+ bacteria, but weakly inhibited pol IIIE from Gram- bacteria. Potent enzyme inhibitors in both classes inhibited the growth of Gram+ bacteria (MICs 2.5-10µg/ml), and were inactive against the Gram- organism Escherichia coli. Several derivatives had moderate protective activity in Staphylococcus aureus-infected mice.
Subject(s)
Anti-Bacterial Agents/chemical synthesis , DNA Polymerase III/antagonists & inhibitors , Enzyme Inhibitors/chemical synthesis , Guanine/analogs & derivatives , Animals , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , DNA Polymerase III/metabolism , Enzyme Inhibitors/pharmacology , Enzyme Inhibitors/therapeutic use , Escherichia coli/drug effects , Gram-Positive Bacteria/drug effects , Gram-Positive Bacteria/enzymology , Guanine/chemistry , Guanine/pharmacology , Guanine/therapeutic use , Mice , Microbial Sensitivity Tests , Staphylococcal Infections/drug therapyABSTRACT
Herpes B virus (B virus [BV]) is a macaque herpesvirus that is occasionally transmitted to humans where it can cause rapidly ascending encephalitis that is often fatal. To understand the low susceptibility of BV to the acyclonucleosides, we have cloned, expressed, and characterized the BV thymidine kinase (TK), an enzyme that is expected to "activate" nucleoside analogs. This enzyme is similar in sequence and properties to the TK of herpes simplex virus (HSV), i.e., it has a broad substrate range and low enantioselectivity and is sensitive to inhibitors of HSV TKs. The BV enzyme phosphorylates some modified nucleosides and acyclonucleosides and l enantiomers of thymidine and related antiherpetic analogs. However, the potent anti-HSV drugs acyclovir (ACV), ganciclovir (GCV), and 5-bromovinyldeoxyuridine were poorly or not phosphorylated by the BV enzyme under the experimental conditions. The antiviral activities of a number of marketed antiherpes drugs and experimental compounds were compared against BV strains and, for comparison, HSV type 1 (HSV-1) in Vero cell cultures. For most compounds tested, BV was found to be about as sensitive as HSV-1 was. However, BV was less sensitive to ACV and GCV than HSV-1 was. The abilities of thymidine analogs and acyclonucleosides to inhibit replication of BV in Vero cell culture were not always proportional to their substrate properties for BV TK. Our studies characterize BV TK for the first time and suggest new lead compounds, e.g., 5-ethyldeoxyuridine and pencyclovir, which may be superior to ACV or GCV as treatment for this emerging infectious disease.
Subject(s)
Antiviral Agents , Herpesvirus 1, Cercopithecine/drug effects , Nucleosides , Thymidine Kinase/metabolism , Acyclovir/analogs & derivatives , Acyclovir/chemistry , Acyclovir/pharmacology , Amino Acid Sequence , Animals , Antiviral Agents/chemistry , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Chlorocebus aethiops , Deoxyuridine/analogs & derivatives , Deoxyuridine/chemistry , Deoxyuridine/pharmacology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Guanine , Herpesvirus 1, Cercopithecine/enzymology , Herpesvirus 1, Cercopithecine/genetics , Microbial Sensitivity Tests/methods , Molecular Sequence Data , Nucleosides/chemistry , Nucleosides/metabolism , Nucleosides/pharmacology , Phosphorylation , Substrate Specificity , Thymidine/analogs & derivatives , Thymidine/metabolism , Thymidine Kinase/antagonists & inhibitors , Thymidine Kinase/chemistry , Thymidine Kinase/genetics , Vero CellsABSTRACT
Derivatives of the herpes simplex thymidine kinase inhibitor HBPG [2-phenylamino-9-(4-hydroxybutyl)-6-oxopurine] have been synthesized and tested for inhibitory activity against recombinant enzymes (TK) from herpes simplex types 1 and 2 (HSV-1, HSV-2). The compounds inhibited phosphorylation of [3H]thymidine by both enzymes, but potencies differed quantitatively from those of HBPG and were generally greater for HSV-2 than HSV-1 TKs. Changes in inhibitory potency were generally consistent with the inhibitor/substrate binding site structure based on published X-ray structures of HSV-1 TK. In particular, several 9-(4-aminobutyl) analogues with bulky tertiary amino substituents were among the most potent inhibitors. Variable substrate assays showed that the most potent compound, 2-phenylamino-9-[4-(1-decahydroquinolyl)butyl]-6-oxopurine, was a competitive inhibitor, with Ki values of 0.03 and 0.005 microM against HSV-1 and HSV-2 TKs, respectively. The parent compound HBPG was uniquely active in viral infection models in mice, both against ocular HSV-2 reactivation and against HSV-1 and HSV-2 encephalitis. In assays lacking [3H]thymidine, HBPG was found to be an efficient substrate for the enzymes. The ability of the TKs to phosphorylate HBPG may relate to its antiherpetic activity in vivo.
Subject(s)
Antiviral Agents/chemical synthesis , Guanine/analogs & derivatives , Guanine/chemical synthesis , Herpesvirus 1, Human/enzymology , Herpesvirus 2, Human/enzymology , Purinones/chemical synthesis , Thymidine Kinase/antagonists & inhibitors , Animals , Antiviral Agents/metabolism , Antiviral Agents/pharmacology , Cloning, Molecular , Encephalitis, Herpes Simplex/drug therapy , Encephalitis, Herpes Simplex/virology , Eye Infections, Viral/drug therapy , Eye Infections, Viral/virology , Guanine/chemistry , Guanine/pharmacology , Herpesvirus 1, Human/drug effects , Herpesvirus 2, Human/drug effects , Mice , Phosphorylation , Purinones/metabolism , Purinones/pharmacology , Recombinant Proteins/antagonists & inhibitors , Recombinant Proteins/biosynthesis , Recombinant Proteins/isolation & purification , Structure-Activity Relationship , Thymidine Kinase/biosynthesis , Thymidine Kinase/isolation & purification , Virus Activation/drug effectsABSTRACT
7-Substituted-N(2)-(3,4-dichlorobenzyl)guanines potently and competitively inhibit DNA polymerases IIIC and IIIE from Gram(+) bacteria. Certain derivatives are also competitive inhibitors of DNA polymerase IIIE from Gram(-) bacteria.
Subject(s)
Bacterial Proteins/antagonists & inhibitors , Guanine/analogs & derivatives , Nucleic Acid Synthesis Inhibitors , Uracil/analogs & derivatives , Binding Sites , Binding, Competitive , DNA Polymerase III/antagonists & inhibitors , DNA Replication/drug effects , Gram-Negative Bacteria/enzymology , Gram-Positive Bacteria/enzymology , Guanine/chemical synthesis , Guanine/pharmacology , Humans , Kinetics , Structure-Activity Relationship , Uracil/chemical synthesis , Uracil/pharmacologyABSTRACT
Certain substituted 6-anilinouracils are potent and selective inhibitors of Gram+ bacterial DNA polymerase IIIC (pol IIIC). In addition, analogues with 3-substituents in the uracil ring have potent antibacterial activity against Gram+ organisms in culture. In an attempt to find optimal anilino substituents for pol IIIC binding and optimal 3-substituents for antibacterial activity, we have prepared several series of 3-substituted-6-aminouracils and assayed their activity against pol IIIC from Bacillus subtilis and a panel of Gram+ and Gram- bacteria in culture. The 6-(3-ethyl-4-methylanilino) group and closely related substituent patterns maximized pol IIIC inhibition potency. Among a series of 3-(substituted-butyl)-6-(3-ethyl-4-methylanilino)uracils, basic amino substituents increased pol IIIC inhibition, but decreased antibacterial activity. The most potent antibacterials were simple hydroxybutyl and methoxybutyl derivatives, and hydrophobically substituted piperidinylbutyl derivatives.