Biology subjects

Castillo, F.

Publications and source records attributed to Castillo, F..

4 recordsLinked to original sources

bioRxiv2025

Affinity-based selection of anti-Feline Leukaemia Virus p27 monoclonal antibodies for efficient lateral flow assay development

Classic diagnostic modalities--including RT-PCR and proviral DNA PCR--present certain drawbacks, highlighting the need of alternative diagnostic techniques, such as lateral flow immunoassays (LFIA), valued for their rapidity and sensitivity. Said desirable features depend on the efficiency of the ternary interaction wherein the antigen bridges immobilized capture and labelled detection antibodies. However, the cost and the throughput of current screening methods, for finding monoclonal antibody (mAbs) pairs efficient at establishing this type of ternary interactions, remains as a universal bottleneck for LFIA development. Traditional sandwich enzyme-linked immunosorbent assays (ELISA), although extensively employed in mAb screening procedures, is burdened by its high consumption of labelled mAbs and the potential alteration of antigen conformation upon plate immobilization, often yielding suboptimal surrogates for native interactions within the final LFIA setup. In this study, we developed an orthogonal approach integrating ELISA and Spectral Shift (SST) that jointly enable the minimisation of reagent consumption (mAbs, antigens, labelling reagents) to comprehensively profile a panel of mAbs against a bona fide antigen of Feline Leukaemia Virus FeLV p27. This facilitated the quick selection of two low nanomolar mAbs with distinct epitope recognition, for which we validated a ternary complex formation in a LFIA prototype. This strategy enables advanced, resource-efficient LFIA development for FeLV and other critical antigens, such as H5 Avian Influenza Virus or African Swine Fever Virus, in addition to major human pathogens including Helicobacter pylori or Mpox Virus, facilitating sensitive diagnostics and streamlined quality control.

biophysics↗

bioRxiv2024

Exploring the druggability of the UEV domain of human TSG101 in search for broad-spectrum antivirals

The ubiquitin E2 variant domain of TSG101 (TSG101-UEV) plays a pivotal role in protein sorting and virus budding by recognizing PTAP motifs within ubiquitinated proteins. Disruption of TSG101-UEV/PTAP interactions has emerged as a promising strategy for the development of host-oriented broad-spectrum antivirals with low susceptibility to resistance. TSG101 is a challenging target characterized by an extended and flat binding interface, low affinity for PTAP ligands, and complex binding energetics. Here, we assess the druggability of the TSG101-UEV/PTAP binding interface by searching for drug-like inhibitors and evaluating their ability to block PTAP recognition, impair budding, and inhibit viral proliferation. A discovery workflow was established combining in vitro miniaturized HTS assays and a set of cell-based activity assays including high-content bimolecular complementation, virus-like particle release measurement, and antiviral testing in live virus infection. This approach has allowed us to identify a set of chemically diverse molecules that block TSG101-UEV/PTAP binding with IC50s in the low M range, and able to disrupt the interaction between full-length TSG101 and viral proteins in human cells and inhibit viral replication. State-of-the-art molecular docking studies reveal that the active compounds exploit binding hotspots at the PTAP binding site, unlocking the full binding potential of the TSG101-UEV binding pockets. These inhibitors represent promising hits for the development of novel broad-spectrum antivirals through targeted optimization and are also valuable tools for investigating the involvement of ESCRT in the proliferation of different virus families and study the secondary effects induced by the disruption of ESCRT/virus interactions. ImportanceMany viruses rely on the interaction between TSG101 and viral proteins containing PTAP motifs for their proliferation. Here we show that these interactions can be efficiently blocked by drug-like compounds that impair budding and replication of viruses from different families. We have also provided valuable insights into the determinants of high affinity for these small molecule inhibitors that open new avenues for developing the identified candidates into broad-spectrum antivirals with low susceptibility to resistance.

molecular biology↗

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↗

bioRxiv2020

Pooling nasopharyngeal swab specimens to increase testing capacity for SARS-CoV-2

The recent emergence of SARS-CoV-2 has lead to a global pandemic of unprecedented proportions. Current diagnosis of COVID-19 relies on the detection of SARS-CoV-2 RNA by RT-PCR in upper and lower respiratory specimens. While sensitive and specific, these RT-PCR assays require considerable supplies and reagents, which are often limited during global pandemics and surge testing. Here, we show that a nasopharyngeal swab pooling strategy can detect a single positive sample in pools of up to 10 samples without sacrificing RT-PCR sensitivity and specificity. We also report that this pooling strategy can be applied to rapid, moderate complexity assays, such as the BioFire COVID-19 test. Implementing a pooling strategy can significantly increase laboratory testing capacity while simultaneously reducing turnaround times for rapid identification and isolation of positive COVID-19 cases in high risk populations.

microbiology↗