Nonsense mutations introduce premature termination codons and underlie 11% of genetic disease cases. High concentrations of aminoglycosides can restore gene function by eliciting premature termination codon readthrough but with low efficiency. Using a high-throughput screen, we identified compounds that potentiate readthrough by aminoglycosides at multiple nonsense alleles in yeast. Chemical optimization generated phthalimide derivative CDX5-1 with activity in human cells. Alone, CDX5-1 did not induce readthrough or increase TP53 mRNA levels in HDQ-P1 cancer cells with a homozygous TP53 nonsense mutation. However, in combination with aminoglycoside G418, it enhanced readthrough up to 180-fold over G418 alone. The combination also increased readthrough at all three nonsense codons in cancer cells with other TP53 nonsense mutations, as well as in cells from rare genetic disease patients with nonsense mutations in the CLN2, SMARCAL1 and DMD genes. These findings open up the possibility of treating patients across a spectrum of genetic diseases caused by nonsense mutations.

Harnessing genetic differences between cancerous and noncancerous cells offers a strategy for the development of new therapies. Extrapolating from yeast genetic interaction data, we used cultured human cells and siRNA to construct and evaluate a synthetic lethal interaction network comprised of chromosome instability (CIN) genes that are frequently mutated in colorectal cancer. A small number of genes in this network were found to have synthetic lethal interactions with a large number of cancer CIN genes; these genes are thus attractive targets for anticancer therapeutic development. The protein product of one highly connected gene, the flap endonuclease FEN1, was used as a target for small-molecule inhibitor screening using a newly developed fluorescence-based assay for enzyme activity. Thirteen initial hits identified through in vitro biochemical screening were tested in cells, and it was found that two compounds could selectively inhibit the proliferation of cultured cancer cells carrying inactivating mutations in CDC4, a gene frequently mutated in a variety of cancers. Inhibition of flap endonuclease activity was also found to recapitulate a genetic interaction between FEN1 and MRE11A, another gene frequently mutated in colorectal cancers, and to lead to increased endogenous DNA damage. These chemical-genetic interactions in mammalian cells validate evolutionarily conserved synthetic lethal interactions and demonstrate that a cross-species candidate gene approach is successful in identifying small-molecule inhibitors that prove effective in a cell-based cancer model.

Persons with chronic hepatitis C virus (HCV) infections of the liver are at a significantly increased risk for cirrhosis and hepatocellular carcinoma. Despite a high level of interest in this virus, the mechanisms responsible for viral persistence are poorly understood, as are many other aspects of the biology of this flavivirus (21). One question that is important to both pathogenesis and persistence is whether HCV undergoes replication in cells outside of the liver. Genomic RNA has been detected in peripheral blood mononuclear cells (PBMC) as well as liver tissue and serum or plasma from infected persons by reverse transcription (RT)-PCR (4, 17-19, 22, 30). However, although a considerable number of studies have focused on the possible presence of the virus in PBMC, many of these reports remain controversial because of uncertainty concerning the strand specificity of putative negative-strand-specific RT-PCR assays used for the detection of viral replicative intermediates (16). Nonetheless, several recent studies using well-validated and highly strand-specific RT-PCR assays have demonstrated the presence of negative-strand RNA in PBMC from infected patients (18,19,22). These studies suggest the existence of a potentially important extrahepatic site of replication for HCV, although the magnitude of the pool of virus replicating outside of the liver and the exact nature of the cell types in which this replication may occur remain unknown.Other data that indirectly support PBMC as an extrahepatic site of replication come from in vitro studies, as several lymphoblastoid cell lines appear to be permissive for HCV replication. Shimizu and colleagues (31-34) extensively characterized the replication of the virus in both B-cell (Daudi) and T-cell (HPBMa10-2 and Molt4) lines, while Kato et al. (14) and Nissen et al. (25) also described the replication of HCV in human T-cell lines (MT-2 and H9, respectively). By sequencing the hypervariable region of the E2 coding segment as well as the viral 5Ј nontranslated RNA (5ЈNTR), Nakajima et al. (23) demonstrated a change in the dominant viral quasispecies in both Daudi cells and HPBMa10-2 cells inoculated with the genotype 1a H77 strain of HCV (34). In both cell lines, a new dominant quasispecies emerged in which there were three identical nucleotide substitutions within the 5ЈNTR: G 107 3A, C 204 3A, and G 243 3A (hereafter referred to as the A-A-A variant). Quasispecies with this 5ЈNTR sequence were not detected in the original H77 serum inoculum (23). The parallel selection of the A-A-A variant in long-term cultures of HCV in two different lymphoblastoid cell lines suggests the possibility that these nucleotide substitutions may enhance the replication capacity of the virus in such cells. Thus, these 5ЈNTR substi-* Corresponding author. Mailing address:

Mutations of DNA repair pathways contribute to tumorigenesis and provide a therapeutic target for synthetic lethal interactions in tumor cells. Given that tyrosyl-DNA phosphodiesterase 1 (Tdp1) repairs stalled topoisomerase-I DNA complexes, we hypothesized that inhibition of Tdp1 has synthetic lethal effects in some cancers. To test this, we screened tumor arrays for Tdp1 expression and observed that Tdp1 is expressed in many tumors, including more than 90% of human breast tumors. Subsequent chemical screening identified putative Tdp1 inhibitors. Treatment of control human mammary epithelial cells and the breast cancer cell line MCF-7 with compound CD00509 preferentially sensitized MCF-7 cells to camptothecin and decreased cell proliferation 25% more than camptothecin treatment alone. This suggests that CD00509 specifically targeted Tdp1 in vitro, and CD00509 increased the sensitivity of wild-type murine embryonic fibroblasts (MEFs) to camptothecin to a degree comparable to that of Tdp1 −/− MEFs. In addition, consistent with poly ADPribose polymerase-1 (PARP-1) collaborating with Tdp1 in DNA repair, combined Tdp1 and PARP-1 inhibition was more detrimental to MCF-7 cells than either treatment alone, whereas the combination was not additively harmful to control mammary cells. We conclude that targeting Tdp1 in anticancer therapy preferentially enhances the sensitivity of some breast cancer cells to camptothecin and may be an effective adjuvant for breast cancer therapy.

The M2 proton channel of the influenza A virus is the target of the anti-influenza drugs amantadine and rimantadine. The effectiveness of these drugs has been dramatically limited by the rapid spread of drug resistant mutations, mainly at sites S31N, V27A and L26F in the pore of the channel. Despite progress in designing inhibitors of V27A and L26F M2, there are currently no drugs targeting these mutated channels in clinical trials. Progress in developing new drugs has been hampered by the lack of a robust assay with sufficient throughput for discovery of new active chemotypes among chemical libraries and sufficient sensitivity to provide the SAR data essential for their improvement and development as drugs. In this study we adapted a yeast growth restoration assay, in which expression of the M2 channel inhibits yeast growth and exposure to an M2 channel inhibitor restores growth, into a robust and sensitive high-throughput screen for M2 channel inhibitors. A screen of over 250,000 pure chemicals and semi-purified fractions from natural extracts identified 21 active compounds comprising amantadine, rimantadine, 13 related adamantanes and 6 non-adamantanes. Of the non-adamantanes, hexamethylene amiloride and a triazine derivative represented new M2 inhibitory chemotypes that also showed antiviral activity in a plaque reduction assay. Of particular interest is the fact that the triazine derivative was not sufficiently potent for detection as an inhibitor in the traditional two electrode voltage clamp assay for M2 channel activity, but its discovery in the yeast assay led to testing of analogues of which one was as potent as amantadine.