Fascioliasis is an infectious parasitic disease distributed globally and caused by the liver fluke Fasciola hepatica or F. gigantica. This neglected tropical disease affects both animals and humans, and it represents a latent public health problem due to the significant economic losses related to its effects on animal husbandry. For decades, triclabendazole has been the unique anti-Fasciola drug that can effectively treat this disease. However, triclabendazole resistance in fascioliasis has more recently been reported around the world, and thus, the discovery of novel drugs is an urgent need. The aim of this study was to investigate the fasciocidal properties of 400 compounds contained in the Pathogen Box. The first stage of the screening was carried out by measuring the fasciocidal activity on metacercariae at a concentration of 33 μM each compound (the standard dose). Subsequently, the activities of the most active compounds (n = 33) at their 50% inhibitory concentration (IC50) values against metacercariae were assayed, and the results showed that 13 compounds had IC50s of ≤10 μM. The second stage queried the activities of these compounds at 33 μM against adult flukes, with seven of the compounds producing high mortality rates of >50%. Four hit compounds were selected on the basis of their predicted nontoxic properties, and the IC50 values obtained for adult worms were <10 μM; thus, these compounds represented the best fasciocidal compounds tested here. A cytotoxicity assay on four types of cell lines demonstrated that three compounds were nontoxic at their most active concentration. In conclusion, three hit compounds identified in this proof-of-concept study are potential candidates in the discovery of new fasciocidal drugs. Further studies are warranted.

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

UBE3A gene encodes the ubiquitin ligase E6 associated protein (E6AP), an enzyme that catalyzes the high-risk human papilloma virus (HPV) E6-mediated ubiquitination of p53. Such modification in p53 contributes to the neoplastic progression of cells infected by HPV-16/18 that leads to cervical cancer (CC). The hijacking of E6AP also occurs by Hepatitis C virus (HCV) that targets Rb protein thus conducting to hepatocellular carcinoma (HCC). We hypothesized that certain non-synonymous (missense) mutations of E6AP drives to elevated degradation rates of either p53 or Rb by increasing its enzymatic activity. This study aimed to predict changes in: (i) functional activity in E6AP mutants, and (b) E6AP affinity to viral oncoproteins and p53/Rb. A subset of three virus-associated cancers including cervical carcinoma (CC), hepatocellular carcinoma (HCC) and oral squamous carcinoma (OSC) were analyzed. The Cancer Genome Atlas (TCGA) dataset as well as Biomuta were mined and missense mutations in human UBE3A gene were retrieved. To explore the impairment of protein function and stability across the UBE3A gene mutants, machine learning approaches (i.e. Polyphen, I-Mutant 3.0, Panther and Provean) and NetSurfP were applied. The 3D structures of E6AP variants were modelled and docking simulations with viral oncoproteins and p53/Rb were performed using PyDock. Our results showed 22 mutations in UBE3A gene that were present across CC, HCC and OSC, with most of variants (60%) appearing in HCC. UBE3A variants were different across the three tumor types analyzed. Seven of 22 UBE3A variants were predicted damaging or deleterious substitutions. Five damaging mutations affected the HECT domain responsible for the enzyme activity. Such corresponded to E743K and P850A, existing in CC, as well as M589L, E754K, and Q576*, found in HCC. Two predicted damaging mutations were located within the HCV binding site including G492V (found in CC) as well as F477C (present in OSC). No mutation involved the highly conserved LXXL domain implicated in E6 binding or the E6AP trimerization residues. The predicted E6AP-E6 complexes revealed that LXXLL domain in E6AP participates in E6 binding, both in wild-type and mutants. Interestingly, E6AP E743K mutant showed increased complex stability in comparison with wild-type. In conclusion, most of E6AP mutations (68%) existing in CC, HCC and OSC are likely to be neutral whereas some others (32%) are potentially damaging variants and likely to increase E6AP activity. E6AP-dependent ubiquitylation of p53 represents an important opportunity to discover novel entities that inhibit protein-protein interactions thus avoiding p53 degradation. Citation Format: Claudia Machicado, Maria Pia Soto. Computational study of the effects of non-synonymous mutations in human UBE3A gene present in virus-associated cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1322A.