Biology subjects

Genilloud, O.

Publications and source records attributed to Genilloud, O..

10 recordsLinked to original sources

bioRxiv2026

BODIPY-Tagged β-Lactams as Selective Quenched Activity-Based Probes to Target Human Neutrophil Elastase

Activity-based probes are indispensable tools for interrogating protease function, and quenched fluorescent variants enable dynamic, real-time imaging of enzymatic activity. Despite these advances, very few quenched activity-based probes (qABPs) have been reported for serine proteases, which constitute the largest and most diverse mechanistic class of proteases. {beta}-Lactams have been extensively used to develop molecular tools and drugs designed to bind or be hydrolysed by serine-dependent bacterial enzymes. Here, we report the first monocyclic {beta}-lactam-containing qABPs for detecting serine proteases. Both the enzyme-triggered activation mechanism and intrinsic reactivity of the probes were highly dependent on the relative position of the BODIPY-FL fluorophore and quencher moiety at the {beta}-lactam core. qABPs displaying the most efficient turn-on mechanism were shown to selectively target human neutrophil elastase (HNE) in different human cell lysates. The most successful qABP was rapidly internalised and targeted HNE in U937 cells and human neutrophils. These results demonstrate the potential of the modular {beta}-lactam warhead to develop turn-on probes to track neutrophil serine proteases in live cells

pharmacology and toxicology↗

bioRxiv2025

Paenitracins, a novel family of bacitracin-type nonribosomal peptide antibiotics produced by plant-associated Paenibacillus species

The growing threat of antimicrobial resistance necessitates the discovery of novel antibiotics with activity against drug-resistant pathogens. Members of the genus Paenibacillus are a rich source of nonribosomal peptides (NRPs), including well-known antibiotics such as polymyxins, paenibacterin and tridecaptins. Here we use a targeted Mass-QL-based mass spectrometry approach to identify the NRPs produced by a collection of 227 taxonomically diverse plant-associated Paenibacillus strains, providing detailed insights into their NRP-producing potential. Using MassQL to zoom in specifically on NRPs containing basic amino acids, we discovered a novel family of bacitracins, which we designated paenitracins. The paenitracins are the first bacitracin-type peptides reported in Paenibacillus, and are distinguished from canonical bacitracins by three previously unseen amino acid substitutions. The paenitracins exhibit potent activity against Gram-positive pathogens, including vancomycin-resistant Enterococcus faecium E155. Our work provides a novel metabolomics- and genomics-guided workflow for the discovery of bioactive NRPs as a strategy to prioritize natural product chemical space and accelerate antibiotic discovery. IMPORTANCEMembers of the genus Paenibacillus play an important role in soil ecology, producing a range of important nonribosomal peptides (NRPs) that protect their eukaryotic host. A collection of plant-associated Paenibacillus spp. was analyzed for their phylogenetic and metabolic diversity. We developed a novel discovery pipeline that combines feature-based molecular networking with MassQL queries to systematically prioritize bioactive NRPs containing basic amino acids. Thus we provide a comprehensive genus-wide inventory of NRPs produced by Paenibacillus spp. We thereby identified the paenitracins, a new subfamily of bacitracins active against multidrug-resistant Gram-positive pathogens. Our pipeline enables the discovery of novel peptidic natural products to accelerate the prioritization of chemical space for antibiotics.

microbiology↗

bioRxiv2025

Discovery of a novel antibiotic class targeting the enolase of Acinetobacterbaumannii

High-throughput screening studies provide an additional approach to discovering repurposed drugs for antimicrobial treatments. In this work, we report the identification of ENOblock, an anticancer drug, as a novel antibiotic class. We computationally and experimentally validated that ENOblock synergizes with colistin, the last resort antibiotic, the colistin. Additionally, we identified enolase as the potential bacterial target for ENOblock. The in silico and in vitro antibacterial activity of ENOblock translated into potent in vivo efficacy in animal infection model. Collectively, the preclinical data support the selection of ENOblock as a promising candidate for antimicrobial development, with the potential to address the urgent threat of infections caused by Acinetobacter baumannii.

microbiology↗

bioRxiv2024

Predicting antimicrobial class specificity of small molecules using machine learning

Whilst the useful armory of antibiotic drugs is continually depleted due to the emergence of drug-resistant pathogens, the development of novel therapeutics has also slowed down. In the era of advanced computational methods, approaches like machine learning (ML) could be one potential solution to help reduce the high costs and complexity of antibiotic drug discovery and attract collaboration across organizations. In our work, we developed a large antimicrobial knowledge graph (AntiMicrobial-KG) as a repository for collecting and visualizing public in-vitro antibacterial assay. Utilizing this data, we build ML models to efficiently scan compound libraries to identify compounds with the potential to exhibit antimicrobial activity. Our strategy involved training seven classic ML models across six compound fingerprint representations, of which the Random Forest trained on the MHFP6 fingerprint outperformed, demonstrating an accuracy of 75.9% and Cohens Kappa score of 0.68. Finally, we illustrated the models applicability for predicting the antimicrobial properties of two small molecule screening libraries. Firstly, the EU-OpenScreen library was tested against a panel of Gram-positive, Gram-negative, and Fungal pathogens. Here, we unveiled that the model was able to correctly predict more than 30% of active compounds for Gram-positive, Gram-negative, and Fungal pathogens. Secondly, with the Enamine library, a commercially available HTS compound collection with claimed antibacterial properties, we predicted its antimicrobial activity and pathogen class specificity. These results may provide a means for accelerating research in AMR drug discovery efforts by carefully filtering out compounds from commercial libraries with lower chances of being active.

bioinformatics↗

bioRxiv2024

Morphological Profiling Dataset of EU-OPENSCREEN Bioactive Compounds Over Multiple Imaging Sites and Cell Lines

Morphological profiling with the Cell Painting assay has emerged as a promising method in drug discovery research. The assay captures morphological changes across various cellular compartments enabling the rapid identification of the effect of compounds. We present a comprehensive morphological profiling dataset using the carefully curated and well-annotated EU-OPENSCREEN Bioactive Compound Set. Our profiling dataset was generated across multiple imaging sites with high-throughput confocal microscopes using the Hep G2 as well as the U2 OS cell line. We employed an extensive assay optimization process to achieve high data quality across the different imaging sites. An analysis of the four replicates validates the robustness of the generated data. We compare morphological features of the different cell lines and map the profiles to activity, toxicity, and basic compound targets to further describe the dataset as well as to demonstrate the potential of this dataset to be used for mechanism of action exploration.

cell 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↗

bioRxiv2021

New from old: discovery of the novel antibiotic actinomycin L in Streptomyces sp. MBT27

Streptomycetes are major producers of bioactive natural products, including the majority of the antibiotics. While much if the low-hanging fruit has been discovered, it is predited that less than 5% of the chemical space has been mined. Here, we describe the novel actinomycins L1 and L2, which are produced by Streptomyces sp. MBT27. The molecules were discovered via metabolic analysis combined with molecular networking of cultures grown with different combinations of carbon sources. Actinomycins L1 and L2 are diastereoisomers, and the structure of actinomycin L2 was resolved using NMR and single crystal X-ray crystallography. Actinomycin L is formed via a unique spirolinkage of anthranilamide to the 4-oxoproline moiety of actinomycin X2, prior to the condensation of the actinomycin halves. Feeding anthranilamide to cultures of Streptomyces antibioticus, which has the same biosynthetic gene cluster as Streptomyces sp. MBT27 but only produces actinomycin X2, resulted in the production of actinomycin L. This shows that actinomycin L results from joining two distinct metabolic pathways, namely those for actinomycin X2 and for anthranilamide. Actinomycins L1 and L2 showed significant antimicrobial activity against Gram- positive bacteria. Our work shows how new molecules can still be identified even in the oldest of natural product families. IMPORTANCEActinomycin was the first antibiotic discovered in an actinobacterium by Selman Waksman and colleagues, as early as 1940. This period essentially marks the start of the golden era of antibiotic discovery. Over time, emerging antimicrobial resistance (AMR) and the declining success rate of antibiotic discovery resulted in the current antibiotic crisis. We surprisingly discovered that under some growth conditions, Streptomyces sp. MBT27 can produce actinomycins that are significantly different from those that have been published so far. The impact of this work is not only that we have discovered a novel molecule with very interesting chemical modifications in one of the oldest antibiotics ever described, but also that this requires the combined action of primary and secondary metabolic pathways, namely the biosynthesis of anthranilamide and of actinomycin X2, respectively. The implication of the discovery is that even the most well-studied families of natural products may still have surprises in store for us.

microbiology↗

bioRxiv2021

Identification of the 216kbp gene cluster and structure elucidation of gargantulides B and C, new complex 52-membered macrolides from Amycolatopsis sp.

Gargantulides B and C, two new and highly complex 52-membered glycosylated macrolactones, were isolated from Amycolatopsis sp. strain CA-230715 during an antibacterial screening campaign. The structures of these giant macrolides were elucidated by 2D NMR spectroscopy and shown to be related to gargantulide A, although containing additional {beta}-glucopyranose and/or -arabinofuranose monosaccharides separately attached to their backbones. Genome sequencing allowed the identification of a strikingly large 216 kbp biosynthetic gene cluster, among the largest type I PKS clusters described so far, and the proposal of a biosynthetic pathway for gargantulides A-C. Additionally, genes putatively responsible for the biosynthesis of the amino sugar {beta}-3,6-deoxy-3-methylamino glucose, reported exclusively in gargantulide macrolides, were also found in the cluster and described in this work. The absolute configurations of gargantulides B and C were assigned based on a combination of NMR and bioinformatics analysis of ketoreductase and enoylreductase domains within the multimodular type I PKS. Furthermore, the absolute stereochemistry of the related macrolide gargantulide A has now been revised and completed. Gargantulides B and C display potent antibacterial activity against a set of drug-resistant Gram-positive bacteria and moderate activity against the clinically relevant Gram-negative pathogen Acinetobacter baumannii.

microbiology↗

bioRxiv2020

One pathway, two cyclic pentapeptides: heterologous expression of BE-18257 A-C and pentaminomycins A-E from Streptomyces cacaoi CA-170360

1.The strain Streptomyces cacaoi CA-170360 produces the cyclic pentapeptides pentaminomycins A-E and BE-18257 A-C, two families of cyclopeptides synthesized by two nonribosomal peptide synthetases encoded in tandem within the same biosynthetic gene cluster. In this work, we have cloned and confirmed the heterologous expression of this biosynthetic gene cluster, demonstrating that each of the nonribosomal peptide synthetases present in the cluster is involved in the biosynthesis of each group of cyclopeptides. In addition, we discuss the involvement of a stand-alone enzyme belonging to the Penicillin Binding Protein family in the release and macrocyclization of the peptides.

molecular biology↗

bioRxiv2020

Biosynthesis and heterologous expression of cacaoidin, the first member of the lanthidin family of RiPPS

1.Cacaoidin is the first member of the new lanthidin RiPP family, a lanthipeptide produced by the strain Streptomyces cacaoi CA-170360 with unprecedented features such as an unusually high number of D-amino acids, a double methylation in the N-terminal alanine and a tyrosine residue glycosylated with a disaccharide. In this work, we describe the complete identification, cloning and heterologous expression of the cacaoidin biosynthetic gene cluster, which shows unique RiPP genes.

molecular biology↗