Search bioRxiv⌕ Search

bioRxiv · 10.1101/2025.07.08.663638

Interdisciplinary Study on Drug-Induced-Phospholipidosis of Repurposing Libraries through Machine Learning and Experimental Evaluation in Different Cell Lines

Abstract

Bigger PicturePhospholipidosis is a cellular condition characterized by the excessive accumulation of phospholipids within cells, that also can be induced by medications--especially those known as cationic amphiphilic drugs. While this phenomenon is not always harmful in itself, it can interfere with how cells respond in laboratory experiments and complicate the interpretation of drug screening results, leading to potential delays or failures in the development of new therapies. As the pharmaceutical industry increasingly relies on high-throughput screening and machine learning to accelerate early-stage drug discovery, there is growing interest in identifying compounds that may cause phospholipidosis before they move too far through the development pipeline. In response to this challenge, we at the EU-Horizon HLTH Remedi4ALL project compiled and analyzed one of the most comprehensive datasets to date--over 5,000 repurposed drugs tested across multiple cell lines--to better understand which compounds are likely to induce phospholipidosis. Using this dataset, we developed a machine learning model capable of predicting the risk of phospholipidosis based on a drugs chemical structure. By validating our approach across diverse experimental settings, our goal is to provide the broader scientific and pharmaceutical communities with a practical tool to flag problematic compounds early, ultimately helping bring safer, more effective treatments to patients faster. Highlights- A comprehensive dataset for a repurposing collection, comprising 5,228 compounds, for induction of phospholipidosis in A549-ACE2 and Vero-E6 cells -A machine learning model developed using in-house experimental phospholipidosis data across multiple cell lines -Exploration of chemical features that induce phospholipidosis and investigation of the relationships underlying different cell line susceptibilities -A case study: correlation of drug-induced phospholipidosis with antiviral compound effects against SARS-CoV-2 using phenotypic screening data Phospholipidosis (PLD), a cellular adverse effect that is, among others, caused by numerous cationic amphiphilic drugs. Interest is raised within pharma discovery to predict this phenomenon, as it can impact the outcome of phenotypic cellular screens and significantly delay drug development processes. The development of accurate and validated machine learning models for predicting drug-induced PLD across different cell lines and research centers could provide a valuable early application tool for the pharmaceutical industry, potentially accelerating drug discovery and reducing the risk of late-stage failures. We report here the assembly, curation, testing and modeling of one of the largest datasets of repurposed drugs (5K+) tested for PLD induction on different cell lines. A machine-learning classification method was developed and validated to predict whether molecules are prone to induce PLD effects when applied in cell-based screens. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=187 SRC="FIGDIR/small/663638v1_ufig1.gif" ALT="Figure 1"> View larger version (67K): org.highwire.dtl.DTLVardef@1c4760org.highwire.dtl.DTLVardef@92084dorg.highwire.dtl.DTLVardef@15effbaorg.highwire.dtl.DTLVardef@1e73f4d_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical abstractC_FLOATNO C_FIG

Explore related subjects

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Kuzikov, M., Kalman, A., Reinshagen, J., Huchting, J., Qian, K., Axelsson, H., Oestling, P., Seashore-Ludlow, B., Gadiya, Y., Gribbon, P., Zaliani, A.. 2025-07-09. Interdisciplinary Study on Drug-Induced-Phospholipidosis of Repurposing Libraries through Machine Learning and Experimental Evaluation in Different Cell Lines. https://doi.org/10.1101/2025.07.08.663638

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

A G-Quadruplex Containing First Intron Harbors a Functional Polyadenylation Signal in TP53

First introns of mammalian genes are often longer than other introns and may contain regulatory elements, suggesting that they may have roles in gene regulation. We detected a previously unannotated, low abundance intronic transcript, TP53-INT1, using long-read direct RNA sequencing data. TP53-INT1 has low coding potential and terminates within the first and longest intron of TP53. The transcript structure and 3'-end were validated by independent experimental and in silico evidence. We identified a canonical polyadenylation signal upstream of the transcript termination site and demonstrated its functionality using reporter assays. Furthermore, a potential G-quadruplex (G4)-forming sequence was identified upstream of this polyadenylation signal. The G4-forming potential was confirmed using spectroscopic and thermal stability analyses. Complementing the in vitro characterizations, treatment with Pyridostatin, a G4-stabilizing drug, was associated with decreased TP53 expression. Together, these findings characterize a previously unannotated TP53 intronic transcript and reveal a local genomic context combining accessible chromatin, a functional polyadenylation signal, and a potential G4-forming sequence that may influence full-length transcription.

molecular biology↗

An architectural RNA builds a reaction crucible through molecular concentration and activated enzyme selection

Architectural RNAs (arcRNAs) scaffold membraneless organelles (MLOs), yet how they organize functional proteins remains unclear. Nuclear stress bodies (nSBs), MLOs scaffolded by HSATIII arcRNAs, incorporate SRSFs during stress. During stress recovery, nSBs function as reaction crucibles that recruit the kinase CLK1 to accelerate SRSF rephosphorylation and normalize stress-induced splicing. Here, we identify the ASAP complex as a recruitment factor that recognizes Ser341-phosphorylated, catalytically activated CLK1. HSATIII-bound SRSFs recruit ASAP, assembling a platform for CLK1 recruitment. Biochemical reconstitution reveals that this platform enhances SRSF phosphorylation by concentrating SRSFs and CLK1 while selectively recruiting activated CLK1, establishing nSB reaction crucibles as molecular concentrators and selectors. The SRSF-ASAP-CLK1 interaction network is conserved between humans and mice, suggesting that primate-specific HSATIII arcRNAs co-opted this pre-existing network to establish reaction-crucible function in nSBs. Our findings reveal how arcRNAs organize functional proteins through quantitative enrichment and qualitative selection to build biochemically active MLOs.

molecular biology↗

Adult-like thick-filament protein stoichiometry in immature human iPSC-derived cardiomyocytes

Human induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) retain immature sarcomeric isoforms, raising questions about their ability to recapitulate the protein stoichiometry of adult human myocardium. We integrated two independent quantitative proteomic datasets of iPSC-CMs with published fetal and region-resolved adult human heart proteomes to compare sarcomeric protein abundance. Although iPSC-CMs clustered with fetal heart based on their global shared proteome and retained fetal troponin I expression, key thick-filament protein ratios closely matched those of adult myocardium. Myosin heavy-to-light chain ratios approached the expected 2:4 stoichiometry, while myosin-binding protein abundance relative to myosin heavy chains was comparable across iPSC-CMs and adult cardiac regions. These findings reveal that developmental immaturity in sarcomeric isoform expression does not entail altered thick-filament protein proportions. Preservation of adult-like myosin-binding protein stoichiometry supports the use of iPSC-CMs to investigate sarcomeric diseases associated with altered protein dosage, particularly MYBPC3-associated hypertrophic cardiomyopathy.

molecular biology↗