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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 μM 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.

Thymidine phosphorylase/platelet-derived endothelial cell growth factor (TP/PD-ECGF) is an enzyme involved in thymidine metabolism and homeostasis, and its catalytic activity appears to play an important role in angiogenesis. Here we describe the cloning and expression of a His-tagged human TP/PD-ECGF and its assay with uracil and thymine analogues. We present the design, synthesis, and biological evaluation of novel 6-(phenylalkylamino)uracil derivatives which, at micromolar concentrations, inhibit both catabolic and anabolic reactions of human TP in vitro. These base analogues are not converted by the enzyme into the nucleoside form, thus representing pure nonsubstrate inhibitors of the enzyme.

We recently reported the properties of the first selective inhibitors of herpes simplex virus type 1 (HSV1) uracil-DNA glycosylase (UDG), an enzyme of DNA repair that has been proposed to be required for reactivation of the virus from latency. 6-(4-Octylanilino)uracil (octAU) was the most potent inhibitor among a series of 6-(4-alkylanilino)uracils, acting in the micromolar range and without effect against human UDG. A 28.5-kDa catalytic fragment of HSV1 UDG has been crystallized in the presence of uracil, and the structure was recently solved. We have used the coordinates of this structure in order to study interaction of our inhibitors with the enzyme, and a model of binding between octAU and UDG has been derived. Starting with the optimized model, the activity of several octAU analogues was predicted, and the values compared favorably with experimental results found for the synthetic compounds. Several hydrophilic derivatives were predicted and found to be active as UDG inhibitors. These compounds will be useful to determine if UDG, like the viral thymidine kinase, is required for reactivation of HSV1 from latency in nerve cells.

5-Iodo-2'-deoxy-L-uridine (L-IdU) and (E)-5-(2-bromovinyl)-2'-deoxy-L-uridine (L-BVdU) have been prepared and found to inhibit herpes simplex virus type 1 (HSV-1) thymidine kinase (TK) with activities comparable to those of their analogs with the natural D-sugar configuration. The mechanism of inhibition is purely competitive for L-IdU (Ki = 0.24 microM) and mixed-type for L-BVdU (Ki = 0.13 microM). High performance liquid chromatographic analysis of the reaction products demonstrated that the viral enzyme phosphorylates both L-enantiomers to their corresponding monophosphates with efficiency comparable to that for D-enantiomers. Neither L-enantiomer inhibits the human cytosolic TK. In contrast to their D-enantiomers, L-IdU and L-BVdU have no effect on human thymidylate synthase, either in HeLa cells or in TK-deficient HeLa cells transformed with the HSV-1 TK gene. Both L-enantiomers (i) have no effect on HeLa cell growth, (ii) are 1000-fold less cytotoxic toward TK-deficient HeLa cells transformed with the HSV-1 TK gene than are their D-enantiomers, (iii) in contrast to their D-enantiomers, are fully resistant to hydrolysis by nucleoside phosphorylase, and, (iv) in spite of their much lower cytotoxicity, most probably due to the very low affinity of L-BVdU monophosphate and L-IdU monophosphate for thymidylate synthase, are only 1 or 2 orders of magnitude less potent than their D-enantiomers in inhibiting viral growth, with potency comparable to that of acyclovir.

L-β-Deoxythymldine (L-dT), the optical enantiomer of D-β-deoxythymidine (o-dT), and L-enantiomers of nucleoslde analogs, such as 5-iodo-2′-deoxy-L-uridine (L-ldU) and E-5-(2-bromovinyl)-2′-deoxy-L-uridine (L-BVdU), are not recognized in vitro by human cytosolic thymldine kinase (TK), but are phosphorylated by herpes simplex virus type 1 (HSV-1) TK and inhibit HSV-1 proliferation in infected cells. Here we report that: (i) L-dT is selectively phosphorylated in vivo to L-dTMP by HSV-1 TK and L-dTMP Is further phosphorylated to the di- and triphosphate forms by non-stereospecific cellular kinases; (ii) L-dTTP not only inhibits HSV-1 DNA polymerase In vitro, but also human DNA polymerases α, γ, δ and ɛ, human Immunodeficiency virus reverse transcriptase (HIV-1 RT), Escherichia coll DNA polymerase I and calf thymus terminal transferase, although DNA polymerase β was resistant; (iii) whereas DNA polymerases β, γ, ɛ and e are unable to utilize L-dTTP as a substrate, the other DNA polymerases clearly Incorporate at least one L-dTMP residue, with DNA polymerase a and HIV-1 RT able to further elongate the DNA chain by catalyzing the formation of the phosphodiester bond between the incorporated L-dTMP and an incoming L-dTTP; (iv) incorporated L-nucleotides at the 3′-OH terminus make DNA more resistant to 3′←5′ exonucleases. In conclusion, our results suggest a possible mechanism for the inhibition of viral proliferation by L-nucleosides.