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Simple Summary The identification of new specific anti-human papillomavirus (HPV) drugs is highly needed, as HPV-induced cancers still represent a significant medical issue. The aim of this study was to analyze in more detail the therapeutic potential of a compound, Cpd12, that acts by blocking the binding between HPV E6 oncoprotein and cellular tumor suppressor p53. We demonstrated that by blocking such an interaction, driven by highly conserved residues among oncogenic HPVs, Cpd12 exhibits broad activity against cervical cancer cell lines infected by different HPV genotypes and HPV-positive head-and-neck cancer cells. Interestingly, Cpd12 also showed the ability to inhibit cancer cell migration and to increase the activity of chemotherapeutic drugs such as taxanes and topoisomerase inhibitors. These findings improve the knowledge about the in vitro efficacy of Cpd12, paving the way to preclinical studies to develop new therapeutic strategies against HPV-induced tumors. Abstract High-risk human papillomaviruses (HR-HPV) are the etiological agents of almost all cervical cancer cases and a high percentage of head-and-neck malignancies. Although HPV vaccination can reduce cancer incidence, its coverage significantly differs among countries, and, therefore, in the next decades HPV-related tumors will not likely be eradicated worldwide. Thus, the need of specific treatments persists, since no anti-HPV drug is yet available. We recently discovered a small molecule (Cpd12) able to inhibit the E6-mediated degradation of p53 through the disruption of E6/p53 binding in HPV16- and HPV18-positive cervical cancer cells. By employing several biochemical and cellular assays, here we show that Cpd12 is also active against cervical cancer cells transformed by other HR-HPV strains, such as HPV68 and HPV45, and against a HPV16-transformed head-and-neck cancer cell line, suggesting the possibility to employ Cpd12 as a targeted drug against a broad range of HPV-induced cancers. In these cancer cell lines, the antitumoral mechanism of action of Cpd12 involves p53-dependent cell cycle arrest, a senescent response, and inhibition of cancer cell migration. Finally, we show that Cpd12 can strongly synergize with taxanes and topoisomerase inhibitors, encouraging the evaluation of Cpd12 in preclinical studies for the targeted treatment of HPV-related carcinomas.

The identification and validation of new small molecules able to inhibit the replication of human cytomegalovirus (HCMV) remains a priority to develop alternatives to the currently used DNA polymerase inhibitors, which are often burdened by long-term toxicity and emergence of cross-resistance. To contribute to this advancement, here we report on the characterization of the mechanism of action of a bioactive plant-derived alkaloid, berberine (BBR), selected in a previous drug repurposing screen expressly devised to identify early inhibitors of HCMV replication. Low micromolar concentrations of BBR were confirmed to suppress the replication of different HCMV strains, including clinical isolates and strains resistant to approved DNA polymerase inhibitors. Analysis of the HCMV replication cycle in infected cells treated with BBR then revealed that the bioactive compound compromised the progression of virus cycle at a stage prior to viral DNA replication and Early (E) genes expression, but after Immediate-Early (IE) proteins expression. Mechanistic studies in fact highlighted that BBR interferes with the transactivating functions of the viral IE2 protein, thus impairing efficient E gene expression and the progression of HCMV replication cycle. Finally, the mechanism of the antiviral activity of BBR appears to be conserved among different CMVs, since BBR suppressed murine CMV (MCMV) replication and inhibited the transactivation of the prototypic MCMV E1 gene by the IE3 protein, the murine homolog of IE2. Together, these observations warrant for further experimentation to obtain proof of concept that BBR could represent an attractive candidate for alternative anti-HCMV therapeutic strategies.

Simple Summary Understanding the mechanisms of action of HPV oncoproteins is pivotal for the rationale development of anti-cancer drugs to treat HPV-related malignancies. The aim of the present study was to explore more in detail the mechanism of action of the HPV16 oncoprotein E6 that directly fosters the YAP/TAZ signaling pathway, a conserved cascade highly active in HPV-related cancers. We confirmed previous evidence about the importance of the PDZ-protein targeting in this process, highlighting here the importance of hScrib degradation, and discovered that the targeting of the Scribble module involves the dimeric form of HPV16 E6. The findings here presented extend our knowledge about the mechanism through which the oncoprotein E6 targets a PDZ-host factor to degradation in cancer cells. Abstract Human papillomavirus is the most common viral infectious agent responsible for cancer development in humans. High-risk strains are known to induce cancer through the expression of the viral oncogenes E6 and E7, yet we have only a partial understanding of the precise mechanisms of action of these viral proteins. Here we investigated the molecular mechanism through which the oncoprotein E6 alters the Hippo-YAP/TAZ pathway to trigger YAP/TAZ induction in cancer cells. By employing E6 overexpression systems combined with protein–protein interaction studies and loss-of-function approaches, we discovered that the E6-mediated targeting of hScrib, which supports YAP/TAZ upregulation, intimately requires E6 homodimerization. We show that the self-association of E6, previously reported only in vitro, takes place in the cytoplasm and, as a dimer, E6 targets the fraction of hScrib at the cell cortex for proteasomal degradation. Thus, E6 homodimerization emerges as an important event in the mechanism of E6-mediated hScrib targeting to sustain downstream YAP/TAZ upregulation, unraveling for the first time the key role of E6 homodimerization in the context of its transforming functions and thus paving the way for the possible development of E6 dimerization inhibitors.

The 4th European Seminars in Virology (EuSeV), which was focused on oncogenic and oncolytic viruses, was held in Bertinoro (Bologna), Italy, from June 10 to 12, 2016. This article summarizes the plenary lectures and aims to illustrate the main topics discussed at 4th EuSeV, which brought together knowledge and expertise in the field of oncogenic and oncolytic viruses from all over the world. The meeting was divided in two parts, "Mechanisms of Viral Oncogenesis" and "Viral Oncolysis and Immunotherapy," which were both focused on dissecting the complex and multi-factorial interplay between cancer and human viruses and on exploring new anti-cancer strategies. J. Cell. Physiol. 232: 2641-2648, 2017. © 2016 Wiley Periodicals, Inc.

International audience; Despite prophylactic vaccination campaigns, human papillomavirus (HPV)-induced cancers still represent a major medical issue for global population, thus specific anti-HPV drugs are needed. Since the ability of HPV E6 oncoprotein to promote p53 degradation is linked to tumor progression, E6 has been proposed as an ideal target for cancer treatment. Using the crystal structure of the E6/E6AP/p53 complex, we performed an in silico screening of small-molecule libraries against a highly conserved alpha-helix in the N-terminal domain of E6 involved in the E6-p53 interaction. We discovered a compound able to inhibit the E6-mediated degradation of p53 through disruption of E6-p53 binding both in vitro and in cells. This compound could restore p53 intracellular levels and transcriptional activity, reduce the viability and proliferation of HPV-positive cancer cells, and block 3D cervospheres formation. Mechanistic studies revealed that the compound anti-tumor activity mainly relies on induction of cell cycle arrest and senescence. Our data demonstrate that the disruption of the direct E6-p53 interaction can be obtained with a small-molecule compound leading to specific antitumoral activity in HPV-positive cancer cells and thus represents a new approach for anti-HPV drug development.

High-risk human papillomaviruses (HR-HPVs) are the causative agents for the onset of several epithelial cancers in humans. The deregulated expression of the viral oncoproteins E6 and E7 is the driving force sustaining the progression of malignant transformation in pre-neoplastic lesions. Targeting the viral E6 oncoprotein through inhibitory compounds can counteract the survival of cancer cells due to the reactivation of p53-mediated pathways and represents an intriguing strategy to treat HPV-associated neoplasias. Here, we describe the development of a quantitative and easy-to-perform assay to monitor the E6-mediated degradation of p53 in living cells to be used for small-molecule testing. This assay allows to unbiasedly determine whether a compound can protect p53 from the E6-mediated degradation in cells, through a simple 3-step protocol. We validated the assay by testing two small molecules, SAHA and RITA, reported to impair the E6-mediated p53 degradation. Interestingly, we observed that only SAHA efficiently rescued p53, while RITA could not provide the same degree of protection. The possibility to specifically and quantitatively monitor the ability of a selected compound to rescue p53 in a cellular context through our LumiFluo assay could represent an important step towards the successful development of anti-HPV drugs.

Persistent infection by high-risk human papillomaviruses (HPVs) is associated with the development of cervical cancer and a subset of anogenital and head and neck squamous cell carcinomas. Abnormal expression of cellular microRNAs (miRNAs) plays an important role in the development of cancer, including HPV-related tumors. In this study, we demonstrated that miR-146a-5p was down-regulated by E6 and, less efficiently, by E7 of high-risk HPV16 in keratinocytes and the presence of low levels of this miRNA in cervical carcinoma cell lines and in high-risk HPV-positive cervical specimens. Down-regulation of miR-146a-5p was mediated at least in part by the transcription repressor c-MYC, through binding sites in the miR-146a promoter. Overexpression of miR-146a-5p significantly inhibited proliferation and migration of keratinocytes and cervical cancer cells. The histone demethylase KDM2B was validated as a new direct target of miR-146a-5p and two putative binding sites for miR-146a-5p were identified in its 3'UTR sequence. Western blot analysis and immunohistochemistry showed that KDM2B was overexpressed in HPV16 E6/E7-positive keratinocytes, in cervical cancer cell lines, and in a subset of invasive cervical carcinomas and HPV-positive laryngeal squamous cell carcinomas. In these tumors, KDM2B overexpression was associated with c-MYC copy number gain. In vitro, silencing of KDM2B inhibited proliferation of cervical cancer cells. In conclusion, this study identified a novel player, the hystone demethylase KDM2B, in HPV-mediated tumorigenesis. E6 and, less efficiently, E7 of high-risk HPV16 up-regulated KDM2B expression in human keratinocytes through a pathway involving overexpression of c-MYC, which in turn downregulated miR-146a-5p.

Daclatasvir is an inhibitor of hepatitis C virus NS5A protein that is used for the therapy of chronic hepatitis. So far, published methods for analysis of daclatasvir in plasma are exclusively based on mass spectrometry, which is not always available in standard clinical laboratories. Thus, we wished to develop and validate a simple, but still reliable and sensitive high-performance liquid chromatography (HPLC) assay with UV detection for the quantification of daclatasvir, feasible for a wide-spread clinical routine use. The method consisted of solid-phase extraction of daclatasvir using Waters Oasis HLB 1cc cartridges, reversed-phase liquid chromatography with a Waters XTerra RP18 (150 mm × 4.6 mm, 3.5 μm) column and a mobile phase of ammonium acetate buffer (pH 5.0, 10 mM) and acetonitrile (56:44, v/v), and UV detection at 318 nm. This assay proved to be sensitive (lower limit of quantification of 0.05 μg/mL), linear (correlation coefficients ≥0.997), specific (no interference with various potentially co-administrated drugs), reproducible (both intra-day and inter-day coefficients of variation ≤8.9%), and accurate (deviations ranged from −2.2 to 8.0% and from −6.5 to 9.2% for intra-day and inter-day assays, respectively). The method was applied to therapeutic monitoring of patients undergoing daclatasvir therapy for hepatitis C and showed to be reliable and robust. Thus, this method provides a simple, sensitive, precise, and reproducible assay for dosing daclatasvir that can be readily adaptable to routine use by clinical laboratories with standard equipment. In addition, the stability of daclatasvir in plasma was evaluated under various conditions, including after the heating procedure required for inactivation of infectious viruses and in different light exposure conditions. These studies evidenced photo-instability of the compound under sunlight exposure over time. Thus, blood sampling and the whole handling procedure have to be performed quickly and with minimal light exposure.

Objectives:\ud Effective treatment with direct-acting antiviral drugs against hepatitis C virus (HCV) is a medical need in cirrhotic HIV–HCV co-infected patients.\ud \ud Methods:\ud This study investigated the plasma levels of daclatasvir (DCV) and ribavirin (RBV) in HIV–HCV co-infected subjects treated with DCV, sofosbuvir, and RBV. Drug concentrations were quantified using validated high-performance liquid chromatography methods with ultraviolet detection. The HCV non-structural protein 5A and non-structural protein 5B coding regions were analyzed by population-based sequencing.\ud \ud Results:\ud DCV was dosed at week 4 and at week 8 of treatment, and RBV at week 8. One patient had the lowest DCV level, corresponding to 32.7% of the overall median value of the other patients at week 4 and about 40% at week 8. The Y93H variant was detected in this subject at weeks 8, 16, and 20 of treatment, but not before treatment or at day 2, and the patient experienced virological failure. Another subject with the Y93H variant at baseline and appropriate DCV levels had HCV RNA