acyclovir and 9-(4-fluoro-3-hydroxymethylbutyl)guanine

Nucleoside analogues, such as penciclovir, ganciclovir, acyclovir, and their fluoro-substituted derivatives, have wide utility as antivirals. Among these analogues, FHBG ((18)F-Fluorohydroxybutylguanine) is a well-validated PET (positron emission tomography) probe for monitoring reporter gene expression. To evaluate whether or not imposing rigidity into the flexible side chain of FHBG 4 could also impact its interaction, with amino acid residues within the binding site of HSV1-TK (Herpes Simplex Virus-1 Thymidine Kinase), thus influencing its cytotoxic activity. Herein, the synthesis of a new fluorinated nucleoside analogue 6 (conceived via ligand-docking studies) is reported. Agent 6 demonstrates selective activity against HeLa cells stably transfected with mutant HSV1-sr39TK and is also 47-fold more potent than FHBG.

Topics: Acyclovir; Antiviral Agents; Binding Sites; Cell Line, Tumor; Guanine; HeLa Cells; Herpesvirus 1, Human; Humans; Molecular Structure; Nucleosides; Positron-Emission Tomography; Radiopharmaceuticals; Thymidine Kinase; Viral Proteins

The herpes simplex virus type 1 thymidine kinase (HSV1-tk) gene is widely used as a suicide gene in combination with ganciclovir (GCV) and as a nuclear imaging reporter gene with an appropriate reporter probe. Wild-type HSV1-tk recognizes a variety of pyrimidine and acycloguanosine nucleoside analogs, including clinically used antiviral drugs. PET of HSV1-tk reporter gene expression will be compromised in patients receiving nucleoside-based antiviral treatment. With the use of an acycloguanosine-specific mutant of the enzyme, PET of HSV1-tk reporter gene expression can be successfully performed with acycloguanosine-based radiotracers without interference from pyrimidine-based antiviral drugs.. The levels of expression of wild-type HSV1-tk and HSV1-A167Ytk, HSV1-sr39tk, and HSV1-A167Ysr39tk mutants fused with green fluorescent protein (GFP) and transduced into U87 cells were normalized to the mean fluorescence of GFP measured by fluorescence-activated cell sorting. The levels of enzymatic activities of wild-type HSV1-tk and its mutants were compared by 2-h in vitro radiotracer uptake assays with (3)H-2'-fluoro-2'-deoxy-1-beta-d-arabinofuranosyl-5-ethyluracil ((3)H-FEAU), (3)H-pencyclovir ((3)H-PCV), and (3)H-GCV and by drug sensitivity assays. PET with (18)F-FEAU and (18)F-9-[4-fluoro-3-(hydroxymethyl)butyl]guanine ((18)F-FHBG) was performed in mice with established subcutaneous tumors, expressing wild-type HSV1-tk and its mutants, followed by tissue sampling.. FEAU accumulation was not detected in HSV1-A167Ysr39tk-expressing cells and xenografts. Lack of conversion of pyrimidine derivatives by the HSV1-A167Ysr39tk supermutant was also confirmed by a drug sensitivity assay, in which the 50% inhibitory concentrations for thymine 1-beta-d-arabinofuranoside and bromovinyldeoxyuridine were found to be similar to those in nontransduced cells. In contrast, we found that HSV1-A167Ysr39tk could readily phosphorylate (3)H-GCV at levels similar to those of wild-type HSV1-tk and HSV1-A167Ytk but showed enhanced activity with (3)H-PCV in vitro and with (18)F-FHBG in vivo.. We developed a new reporter gene, HSV1-A167Ysr39tk, which exhibits specificity and high phosphorylation activity for acycloguanosine derivatives. The resulting supermutant can be used for PET with (18)F-FHBG and suicidal gene therapy protocols with GCV in patients treated with pyrimidine-based cytotoxic drugs.

Topics: Acyclovir; Animals; Antiviral Agents; Cell Line, Tumor; Cytotoxins; Ganciclovir; Gene Expression Regulation, Viral; Genes, Reporter; Genetic Therapy; Guanine; Herpesvirus 1, Human; Humans; Mice; Mutation; Phosphorylation; Positron-Emission Tomography; Pyrimidines; Substrate Specificity; Thymidine Kinase

Synthesis and preliminary biological evaluation of 9-(4-[18F]-fluoro-3-hydroxymethylbutyl)-guanine ([18F]FHBG) is reported. 9-(4-Hydroxy-3-hydroxymethylbutyl)-guanine (penciclovir) 4 was converted to 9-[N2, O-bis-(methoxytrityl)-3-(tosylmethybutyl)]guanine 7 by treatment with methoxytrityl chloride followed by tosylation. The tosylate 7 was reacted with either tetrabutylammonium fluoride or KF in the presence of kryptofix 2.2.2. to produce the 4-fluoro-N2-O-bis-(methoxytrityl) derivative 8. Removal of the methoxytrityl groups by acidic hydrolysis produced FHBG 5. Radiolabeled product [18F]FHBG was prepared by fluorination of the tosylate 7 with [18F]KF and kryptofix 2.2.2. The labeled product was isolated by HPLC purification on a reverse-phase C18 column, and eluted at 12 min with 15% acetonitrile in water at a flow rate of 2.25 mL/min. Radiochemical yield was 8.0-22.3% with an average of 12% in 7 runs (corrected for decay). Synthesis time was 90 to 100 min including HPLC purification with radiochemical purity >99%, and average specific activity of 320 mCi/micromol. In vitro studies of the compound in HT-29 colon cancer cells revealed 18.2-fold higher uptake into transduced cells compared to control in 3 h. The agent may be useful for imaging viral infection or transfected cells in gene therapy.

Topics: Acyclovir; Antiviral Agents; Biological Transport; Colonic Neoplasms; Fluorine Radioisotopes; Genetic Therapy; Guanine; Humans; Indicators and Reagents; Molecular Structure; Radionuclide Imaging; Tumor Cells, Cultured; Virus Diseases