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The nucleoprotein (NP) of the influenza virus exists as trimers, and its tail-loop binding pocket has been suggested as a potential target for antiinfluenza therapeutics. The possibility of NP as a drug target was validated by the recent reports that nucleozin and its analogs can inhibit viral replication by inducing aggregation of NP trimers. However, these inhibitors were identified by random screening, and the binding site and inhibition mechanism are unclear. We report a rational approach to target influenza virus with a new mechanism-disruption of NP-NP interaction. Consistent with recent work, E339A, R416A, and deletion mutant Δ402-428 were unable to support viral replication in the absence of WT NP. However, only E339A and R416A could form hetero complex with WT NP, but the complex was unable to bind the RNA polymerase, leading to inhibition of viral replication. These results demonstrate the importance of the E339…R416 salt bridge in viral survival and establish the salt bridge as a sensitive antiinfluenza target. To provide further support, we showed that peptides encompassing R416 can disrupt NP-NP interaction and inhibit viral replication. Finally we performed virtual screening to target E339…R416, and some small molecules identified were shown to disrupt the formation of NP trimers and inhibit replication of WT and nucleozinresistant strains. This work provides a new approach to design antiinfluenza drugs.T he RNA-dependent RNA polymerase (RDRP) of the influenza A virus is composed of polymerase basic protein 1 (PB1), basic protein 2 (PB2), and acidic protein (PA) (1). The function of RDRP for viral replication requires association with the nucleoprotein (NP) (2) to form the ribonucleoprotein (RNP) complex. Only low resolution structures of the RNP complex are available from cryo-EM studies (2-9), whereas high resolution structures have been reported for some individual components or fragments (10-12). Crystal structures of NP indicate that it exists in trimers (13,14), with the tail-loop (residues 402-428) region playing an important role in the trimerization (Fig. 1A). Based on the structural information, it was suggested that the tail-loop binding pocket could be a target for antiinfluenza therapeutics (13,14).Disruption of the NP-NP interaction as a strategy for designing antiinfluenza drugs has been further reported. Many mutants of NP, including some tail-loop mutants, lose the ability to support the RDRP activity in reconstitution experiments (2,(15)(16)(17)(18). In addition, some of the mutants are shown to exist in monomers instead of trimers. These results support the importance of NP in the RDRP activity and viral replication, and the possibility of NP as a drug target. However, it remains to be shown that molecules capable of disrupting the NP-NP interaction would inhibit viral replication.Recently Kao et al. (19) and our group (20) reported the use of high throughput screening to identify nucleozin and its analogs as inhibitors that halt viral replication by binding to NP and causing it...

BackgroundIt is unclear whether germline breast cancer susceptibility gene mutations affect breast cancer related outcomes. We wanted to evaluate mutation patterns in 20 breast cancer susceptibility genes and correlate the mutations with clinical characteristics to determine the effects of these germline mutations on breast cancer prognosis.MethodsThe study cohort included 480 ethnic Chinese individuals in Taiwan with at least one of the six clinical risk factors for hereditary breast cancer: family history of breast or ovarian cancer, young age of onset for breast cancer, bilateral breast cancer, triple negative breast cancer, both breast and ovarian cancer, and male breast cancer. PCR-enriched amplicon-sequencing on a next generation sequencing platform was used to determine the germline DNA sequences of all exons and exon-flanking regions of the 20 genes. Protein-truncating variants were identified as pathogenic.ResultsWe detected a 13.5% carrier rate of pathogenic germline mutations, with BRCA2 being the most prevalent and the non-BRCA genes accounting for 38.5% of the mutation carriers. BRCA mutation carriers were more likely to be diagnosed of breast cancer with lymph node involvement (66.7% vs 42.6%; P = 0.011), and had significantly worse breast cancer specific outcomes. The 5-year disease-free survival was 73.3% for BRCA mutation carriers and 91.1% for non-carriers (hazard ratio for recurrence or death 2.42, 95% CI 1.29–4.53; P = 0.013). After adjusting for clinical prognostic factors, BRCA mutation remained an independent poor prognostic factor for cancer recurrence or death (adjusted hazard ratio 3.04, 95% CI 1.40–6.58; P = 0.005). Non-BRCA gene mutation carriers did not exhibit any significant difference in cancer characteristics or outcomes compared to those without detected mutations. Among the risk factors for hereditary breast cancer, the odds of detecting a germline mutation increased significantly with having bilateral breast cancer (adjusted odds ratio 3.27, 95% CI 1.64–6.51; P = 0.0008) or having more than one risk factor (odds ratio 2.07, 95% CI 1.22–3.51; P = 0.007).ConclusionsWithout prior knowledge of the mutation status, BRCA mutation carriers had more advanced breast cancer on initial diagnosis and worse cancer-related outcomes. Optimal approach to breast cancer treatment for BRCA mutation carriers warrants further investigation.Electronic supplementary materialThe online version of this article (10.1186/s12885-018-4229-5) contains supplementary material, which is available to authorized users.

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Lineshape simulations are presented for the multiple, overlapping X-band electron paramagnetic resonance (EPR) spectra in two non-heme, high-spin iron proteins: phenylalanine hydroxylase (PAH) and diferric transferrin. The aim of the calculations is to determine the fraction of iron contributing to each of the sites visible by EPR. The simulations are limited to the experimentally accessible transitions occurring at g-values greater than 1.7. In both PAH and transferrin, at least one of the iron sites is characterized by the ratio of zero-field splitting parameters, E/D, near 1/3 and a broad, asymmetric lineshape. A distribution in E/D-values is used in the simulations to account for this breadth and asymmetry. To test the E/D-distribution model, experimental X-band spectra of diferric transferrin at several salt concentrations are fit by simulation. In this test, first the low-field features arising from transitions between the lowest Kramers doublet levels are simulated using E/D-distributions for two sites. Second, parameters that provide a good fit for the lowest doublet transitions are shown also to fit the resonance near an effective g-value of 4.3 from the middle Kramers doublet transition. When applied to spectra of PAH in the resting state, the E/D-distribution approach accounts for the intensity of one of the two major species of iron. The other species is characterized by E/D = 0.032, and the spectrum of this portion of the resting enzyme may be simulated using a frequency-swept Gaussian lineshape. Spectra for the enzyme in an inhibitor-saturated state are also simulated. The simulations are consistent with previous biochemical studies that indicate that only the E/D = 0.032 form of iron participates in catalysis.