Gonzalez-Pacanowska, D. | Search bioRxiv

bioRxiv2025

Role of the Nucleotide Excision Repair endonuclease XPF in the kinetoplastid parasite Trypanosoma brucei

The nucleotide excision repair (NER) mechanism is responsible for the removal of bulky DNA damage such as pyrimidine dimers induced by ultraviolet light. The NER pathway detects such lesions and excises the damaged strand through incisions at 5 and 3 of the damage. The 5 incision is catalyzed by a heterodimeric endonuclease composed of XPF (catalytic subunit) and ERCC1 (non-catalytic). Here, we show that the genome of Trypanosoma brucei, the causal agent of human African trypanosomiasis or sleeping sickness, codes for an XPF ortholog. RNAi silencing of TbXPF sensitizes cells to UV irradiation, thus providing evidence that NER operates in these parasites. In addition, TbXPF confers protection against intra- and inter-strand crosslinks induced by cisplatin and mitomycin C respectively. Consistent with a role in DNA repair, XPF localizes to the cell nucleus, and is found associated to nucleoplasmic and nucleolar regions. The presence of a functional NER pathway in trypanosomes suggests that in vivo, they are susceptible to undergo replication and transcription-blocking DNA damages. The results obtained with various antitumor agents provide proof of concept for the potential of NER inhibition as a means to improve antiparasitic therapies.

microbiology↗

bioRxiv2023

Establishment of a screening platform based on human coronavirus OC43 for the identification of microbial natural products with antiviral activity

Human coronaviruses (HCoVs) cause respiratory tract infections and are of great importance due to the recent severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic. Human betacoronavirus OC43 (HCoV-OC43) is an adequate surrogate for SARS-CoV-2 because it infects the human respiratory system, presents a comparable biology, and is transmitted in a similar way. Its use is advantageous since it only requires biosafety level (BSL)-2 infrastructure which minimizes costs and biosafety associated limitations. In this report, we describe a high-throughput screening (HTS) platform to identify compounds that inhibit the propagation of HCoV-OC43. Optimization of assays based on inhibition of the cytopathic effect and virus immunodetection with a specific antibody, has provided a robust methodology for the screening of a selection of microbial natural product extracts from the Fundacion MEDINA collection. Using this approach, a subset of 1280 extracts has been explored. Of these, upon hit confirmation and early LC-MS dereplication, 10 extracts were identified that contain potential new compounds. In addition, we report on the novel antiviral activity of some previously described natural products whose presence in bioactive extracts was confirmed by LC/MS analysis. IMPORTANCEThe COVID-19 pandemic has revealed the lack of effective treatments against betacoronaviruses and the urgent need for new broad-spectrum antivirals. Natural products are a valuable source of bioactive compounds with pharmaceutical potential that may lead to the discovery of new antiviral agents. Specifically, compared to conventional synthetic molecules, microbial natural extracts possess a unique and vast chemical diversity and are amenable to large-scale production. The implementation of a high-throughput screening platform using the betacoronavirus OC43 in a human cell line infection model has provided proof of concept of the approach and has allowed for the rapid and efficient evaluation of 1280 microbial extracts. The identification of several active compounds validates the potential of the platform for the search for new compounds with antiviral capacity.

microbiology↗