Terciano, D. | Search bioRxiv

bioRxiv2025

MOAST: Mechanism of Action Similarity Tool

Determining the mechanism of action (MOA) for natural products remains a significant bottleneck in drug discovery, particularly for researchers with limited computational resources or small compound libraries. Traditional approaches require screening large numbers of annotated compounds alongside unknowns, which is cost-prohibitive, or depend on complex machine learning models that need substantial computational resources and large datasets. Here, we present a dissertation chapter excerpt: MOAST (Mechanism of Action Similarity Tool), a BLAST-inspired computational workflow that addresses these limitations by providing rapid MOA hypotheses for newly screened compounds. This chapter investigates two complementary approaches: a kernel density estimation (KDE) method providing statistical significance measures and E-values for MOA class membership, and a CatBoost machine learning classifier for multi-class prediction with ranked outputs. Using cytological profiling data from HeLa and A549 cell lines, MOAST achieved 22% accuracy for the top 5 predictions among [~] 300 MOA classes, with the CatBoost classifier reaching 10% balanced accuracy--significantly better than the [~] 3% reported in literature. The tool suggests a 0.8 prediction probability threshold for trustworthy results and demonstrates robust performance across multiple feature reduction strategies. MOAST provides a practical, accessible solution that bridges traditional phenotypic screening and modern computational approaches, making MOA determination feasible for researchers with limited resources while maintaining statistical rigor and interpretability.

bioinformatics↗

bioRxiv2025

Cell Painting in activated cells illuminates phenotypic dark space and uncovers novel drug mechanisms of action.

As drug and natural product libraries expand, assays for assessing mechanisms of action (MoA) are increasingly critical. Performing cytological profiling using the Cell Painting (CP) assay enables image-based profiling of cellular states upon treatment, yet many bioactive compounds remain uncharacterized due to undetectable cellular effects under standard conditions. To address this, we combined drug dosing with cell activation using the protein kinase C (PKC) agonist phorbol myristate acetate (PMA). Profiling A549 lung cancer cells treated with 8,387 compounds at two concentrations (1 and 10 {micro}M) in both resting and PMA-activated states allowed us to detect phenotypic effects for up to 40% of all screened compounds, effectively illuminating new phenotypic "dark space". Over 1,000 compounds exhibited phenotypes exclusively under PMA activation, establishing its advantage for MoA studies. We introduce novel quality control measures for CP screens and demonstrate that integrating phenotypic signatures enhances MoA discovery. Notably, 2-methoxycinnamaldehyde clustered with glucocorticoid receptor modulators and induced nuclear translocation, emphasizing the power of this approach in uncovering novel drug mechanisms and, therefore, aiding in improving therapeutic strategies.

systems biology↗

bioRxiv2024

The polyadenylase PAPI is required for virulence plasmid maintenance in pathogenic bacteria

Many species of pathogenic bacteria harbor critical plasmid-encoded virulence factors, and yet the regulation of plasmid replication is often poorly understood despite playing a critical role in plasmid-encoded gene expression. Human pathogenic Yersinia, including the plague agent Y. pestis and its close relative Y. pseudotuberculosis, require the type III secretion system (T3SS) virulence factor to subvert host defense mechanisms and colonize host tissues. The Yersinia T3SS is encoded on the IncFII plasmid for Yersinia virulence (pYV). Several layers of gene regulation enables a large increase in expression of Yersinia T3SS genes at mammalian body temperature. Surprisingly, T3SS expression is also controlled at the level of gene dosage. The number of pYV molecules relative to the number of chromosomes per cell, referred to as plasmid copy number, increases with temperature. The ability to increase and maintain elevated pYV plasmid copy number, and therefore T3SS gene dosage, at 37{degrees}C is important for Yersinia virulence. In addition, pYV is highly stable in Yersinia at all temperatures, despite being dispensable for growth outside the host. Yet how Yersinia reinforces elevated plasmid replication and plasmid stability remains unclear. In this study, we show that the chromosomal gene pcnB encoding the polyadenylase PAP I is required for regulation of pYV plasmid copy number (PCN), maintenance of pYV in the bacterial population outside the host, robust T3SS activity, and Yersinia virulence in a mouse infection model. Likewise, pcnB/PAP I is also required for robust expression of the Shigella flexneri virulence plasmid-encoded T3SS. Furthermore, Yersinia and Shigella pcnB/PAP I is required for maintaining normal PCN of model antimicrobial resistance (AMR) plasmids whose replication is regulated by sRNA, thereby increasing antibiotic resistance by ten-fold. These data suggest that pcnB/PAP I contributes to the spread and stabilization of virulence and AMR plasmids in bacterial pathogens, and is essential in maintaining the gene dosage required to mediate plasmid-encoded traits. Importantly pcnB/PAP I has been bioinformatically identified in many species of bacteria despite being studied in only a few species to date. Our work highlights the potential importance of pcnB/PAP I in antibiotic resistance, and shows for the first time that pcnB/PAP I reinforces PCN and virulence plasmid stability in natural pathogenic hosts with a direct impact on bacterial virulence. Author SummaryMany pathogens carry extrachromosomal DNA elements known as plasmids, which encode genes that confer bacterial virulence or antimicrobial resistance (AMR). Acquisition of these plasmids by bacteria can lead to the emergence of new pathogenic traits and the spread of AMR, yet the mechanisms by which plasmids are retained in bacterial populations particularly in the absence of selective pressure remain incompletely understood. Here we show that the major bacterial polyadenylase enzyme PAP I, encoded by the pcnB gene, is critical for the human pathogen Yersinia pseudotuberculosis to maintain its native virulence plasmid as well as AMR plasmids. Very little is known about the process of polyadenylation in bacteria, or the post-transcriptional addition of adenosine residues to the 3 end of transcripts. This study represents the first demonstration that PAP I-mediated polyadenylation contributes to bacterial pathogenesis.

microbiology↗