6-Fluoro-3-hydroxy-2-pyrazinecarboxamide (T-705) is a novel antiviral compound with broad activity against influenza virus and diverse RNA viruses. Its active metabolite, T-705-ribose-59-triphosphate (T-705-RTP), is recognized by influenza virus RNA polymerase as a substrate competing with GTP, giving inhibition of viral RNA synthesis and lethal virus mutagenesis. Which enzymes perform the activation of T-705 is unknown. We here demonstrate that human hypoxanthine guanine phosphoribosyltransferase (HGPRT) converts T-705 into its ribose-59-monophosphate (RMP) prior to formation of T-705-RTP. The anti-influenza virus activity of T-705 and T-1105 (3-hydroxy-2-pyrazinecarboxamide; the analog lacking the 6-fluoro atom) was lost in HGPRT-deficient Madin-Darby canine kidney cells. This HGPRT dependency was confirmed in human embryonic kidney 293T cells undergoing HGPRT-specific gene knockdown followed by influenza virus ribonucleoprotein reconstitution.Knockdown for adenine phosphoribosyltransferase (APRT) or nicotinamide phosphoribosyltransferase did not change the antiviral activity of T-705 and T-1105. Enzymatic assays showed that T-705 and T-1105 are poor substrates for human HGPRT having K m app values of 6.4 and 4.1 mM, respectively. Formation of the RMP metabolites by APRT was negligible, and so was the formation of the ribosylated metabolites by human purine nucleoside phosphorylase. Phosphoribosylation and antiviral activity of the 2-pyrazinecarboxamide derivatives was shown to require the presence of the 3-hydroxyl but not the 6-fluoro substituent. The crystal structure of T-705-RMP in complex with human HGPRT showed how this compound binds in the active site. Since conversion of T-705 by HGPRT appears to be inefficient, T-705-RMP prodrugs may be designed to increase the antiviral potency of this new antiviral agent.

The CCAAT/enhancer binding protein alpha is an essential transcription factor for granulocytic differentiation. Recent studies reported N- and C-terminal CEBPA mutations in approximately 7% of acute myeloid leukaemia (AML) patients. C-terminal mutations are usually in-frame and occur in the basic-leucine zipper (bZIP) domain, resulting in deficient DNA binding. Using a rapid PCR approach, we screened for bZIP mutations and determined the prognostic value of these mutations in a cohort of 277 de novo AMLs. In addition, we set out to quantify CEBPA mRNA levels by 'real-time' PCR using TaqMan technology. In-frame insertions were observed in 12 (4.3%) cases. All cases with mutations carried an intermediate-risk karyotype and all but one belonged to M1 or M2 FAB class. Further sequence analysis revealed that CEBPA C-terminal mutations are associated with frameshift mutations in the N-terminus of CEBPA. These two mutations were always found in different alleles. Event-free survival (EFS) and overall survival (OS) of patients with CEBPA mutations were significantly increased (P=0.02 and 0.03, respectively) in comparison to the patients lacking these mutations. Mutations were associated with a significantly reduced hazard ratio for death (OS: HR=0.35, P=0.04) and failure (EFS: no CR, death in CR or relapse, HR=0.37, P=0.03). This favourable hazard ratio was maintained after adjustment for cytogenetic risk, FLT3-ITD and CEBPA expression levels in multivariable analysis. In contrast, low CEBPA expression in AML with intermediate-risk karyotype (n=6) seemed to be associated with poor prognosis (not significant). By including this newly developed PCR assay, we define a subgroup of good-risk patients within the heterogeneous intermediate-risk group of AML.

Dihydropyrimidine dehydrogenase (DPD) is the initial enzyme acting in the catabolism of the widely used antineoplastic agent 5-fluorouracil (5FU). DPD deficiency is known to cause a potentially lethal toxicity following administration of 5FU. Here, we report novel genetic mechanisms underlying DPD deficiency in patients presenting with grade III/IV 5FU-associated toxicity. In one patient a genomic DPYD deletion of exons 21–23 was observed. In five patients a deep intronic mutation c.1129–5923C>G was identified creating a cryptic splice donor site. As a consequence, a 44 bp fragment corresponding to nucleotides c.1129–5967 to c.1129–5924 of intron 10 was inserted in the mature DPD mRNA. The deleterious c.1129–5923C>G mutation proved to be in cis with three intronic polymorphisms (c.483 + 18G>A, c.959–51T>G, c.680 + 139G>A) and the synonymous mutation c.1236G>A of a previously identified haplotype. Retrospective analysis of 203 cancer patients showed that the c.1129–5923C>G mutation was significantly enriched in patients with severe 5FU-associated toxicity (9.1%) compared to patients without toxicity (2.2%). In addition, a high prevalence was observed for the c.1129–5923C>G mutation in the normal Dutch (2.6%) and German (3.3%) population. Our study demonstrates that a genomic deletion affecting DPYD and a deep intronic mutation affecting pre-mRNA splicing can cause severe 5FU-associated toxicity. We conclude that screening for DPD deficiency should include a search for genomic rearrangements and aberrant splicing.Electronic supplementary materialThe online version of this article (doi:10.1007/s00439-010-0879-3) contains supplementary material, which is available to authorized users.