Biology subjects

Rotili, D.

Publications and source records attributed to Rotili, D..

9 recordsLinked to original sources

bioRxiv2026

m6A RNA methylation modulates Zika virus infection by regulating serine proteases in Aedes albopictus

Epitranscriptomic RNA modifications, particularly N6-methyladenosine (mA), have emerged as important regulators of host-virus interctions. However, the role of mA in arbovirus infection within mosquito vectors remains poorly defined. Here, we characterized the mA RNA methylation machinery in Aedes albopictus C6/36 cells and examined its contribution to Zika virus (ZIKV) replication. Arbovirus infection did not significantly alter the transcriptional levels or enzymatic activity of the core mA methyltransferase components METTL3 and METTL14. In contrast, pharmacological inhibition of METTL3 markedly enhanced ZIKV replication, indicating an antiviral role for mA in mosquito cells. Transcriptome-wide analysis of C6/36 cells treated with the METTL3 inhibitor STM2457 revealed extensive changes in gene expression, including the pronounced upregulation of multiple serine proteases, particularly members of the CLIP family. Single-nucleotide-resolution mapping of mA using GLORI-sequencing showed that m6A is absent from Zika virus RNA, but readily detectable in the A. albopictus transcriptome. Data analysis defined key features of the mosquito epitranscriptome and demonstrated that mA modifications are enriched within the coding regions of serine protease transcripts, supporting their direct regulation by mA. Functionally, inhibition of serine protease activity using AEBSF resulted in a significant reduction of ZIKV replication. Together, these findings identify mA RNA methylation as a critical regulator of ZIKV infection in mosquito cells and uncover an epitranscriptomic pathway linking mA-dependent control of serine proteases to vector-virus interactions. Author SummaryMosquito-borne viruses such as Zika virus pose a major threat to global public health. Successful transmission of these viruses depends not only on infection in humans, but also on their ability to replicate efficiently inside mosquito vectors. Chemical modifications of RNA, collectively known as epitranscriptomic marks, have recently emerged as important regulators of gene expression and virus-host interactions. Among these, N6-methyladenosine (m6A) is the most abundant internal RNA modification in eukaryotic cells. While m6A has been extensively studied in mammalian systems, its role in mosquito antiviral responses remains poorly understood. In this study, we investigated how m6A RNA methylation influences Zika virus infection in mosquito cells derived from Aedes albopictus. We found that reducing m6A levels enhances viral replication, indicating that this RNA modification restricts infection in mosquito cells. Notably, Zika virus RNA itself does not contain detectable m6A modifications. Instead, m6A regulates the expression of specific mosquito genes, including a group of serine proteases that influence viral replication. Pharmacological inhibition of these proteases significantly impaired virus growth, identifying them as key downstream effectors. Our findings reveal an antiviral role for m6A in mosquito cells and uncover a previously unrecognized epitranscriptomic pathway that shapes mosquito-virus interactions. Understanding how RNA modifications regulate arbovirus infection in vectors may open new avenues for strategies aimed at limiting virus transmission.

molecular biology↗

bioRxiv2025

A cell-based degrader assessment platform facilitates discovery of functional NUDT5 PROTACs

Targeted protein degradation (TPD) via PROTACs and molecular glues holds significant therapeutic promise but demands detailed mechanistic evaluation in live cells to fully understand compound behavior and optimize efficacy. Here, we present an integrated, cell-first platform that combines a modular degradation assay with E3 ligase target engagement readouts for comprehensive assessment of TPD molecules in cells and use it to evaluate PROTACs towards NUDT5. To mimic endogenous degradation conditions and TPD amenability, we established a fusion protein expression system consisting of a lysine-free FKBP12 F36V PROTAC handle (FKBPVK0) and used a HiBiT/akaLuc dual luciferase reporter to accurately measure degradation dynamics. This set-up identified a VHL-dependent NUDT5 PROTAC, DDD2, that induced robust NUDT5 degradation, despite impaired NUDT5 binding in vitro and in cellulo, but no CRBN-dependent degraders. NUDT5 lysine availability mapping with DDD2 and FKBP12 F36V-directed PROTACs suggested that the CRL4CRBN complex is more sensitive to target lysine accessibility than CRL2VHL, which may have implications for E3 ligase choice and therapeutic resistance. CeTEAM drug biosensors were also established towards CRBN and VHL to quantitatively monitor degrader engagement in living cells and confirmed that the tested CRBN-directed NUDT5 PROTACs poorly engaged the E3. All together, this platform provides a versatile and scalable framework for TPD molecule discovery in a cellular context.

biochemistry↗

bioRxiv2024

The inhibitory effects of Remodelin on murine myoblasts differentiation

Myoblasts differentiation is a highly regulated and complex process leading to the formation of fused and aligned mature myotubes. Growing interest in the role of epigenetics in muscle differentiation has highlighted epi-modulators as crucial regulators of this process. Our in vitro study aimed to explore the potential effects of the inhibition of the acetyltransferase Nat10 on myoblasts differentiation, by using Remodelin, a Nat10 selective inhibitor. We cultivated and differentiated murine C2C12 myoblasts on ultra-compliant gelatin substrates for up to 16 days and treated them with Remodelin. A combination of morphological analyses, confocal microscopy, transcriptomic profiling (RNA-seq), quantitative proteomics and metabolomics analyses was employed to assess the impact of Nat10 inhibition on myotube formation and maturation. To evaluate the reproducibility of Remodelin effects across myogenic systems and species, L6 rat myoblasts were included as a secondary comparative model. Remodelin treatment impaired myotube organization, alignment, and structural maturation in both C2C12 and L6 cells compared to untreated controls. In C2C12 cultures, Remodelin also abolished spontaneous myotube contractility. Intersection of transcriptomics and proteomics analyses confirmed that Remodelin effectively slowed myotube formation. Overall, these results indicate that Remodelin broadly affects the regulatory networks involved in skeletal muscle differentiation.

cell biology↗

bioRxiv2024

Probing intracellular determinants of PARP inhibitor selectivity and pharmacology with CeTEAM

PARP inhibitors (PARPi) predominantly targeting PARP1 and PARP2 have revolutionized cancer therapy by selectively killing cancer cells with defective DNA repair. However, achieving PARP1 or PARP2-selective inhibitors is difficult due to their close structural homology. Selectivity profiling is typically done with purified proteins, but these lack the complexity of intracellular environments and could therefore be inaccurate. Here, we duplex PARP1 L713F-GFP and PARP2 L269A-mCherry CeTEAM drug biosensors to systematically characterize binding and cell cycle alterations of 27 PARPi at the single cell level. Our results reveal that most PARPi are generally equipotent for both PARPs, including the next-generation drug, senaparib. However, benzimidazole carboxamide (niraparib) derivatives demonstrated PARP1-selective tendencies, while pthalazinones (olaparib) favored PARP2. AZD5305, a reported PARP1-selective inhibitor with characteristics of both series, was the exception and appears [~]1600-fold more potent towards PARP1. In agreement with current understanding, we see that PARP trapping phenotypes positively correlate with PARP1/2 binding potency, while some potent binders, such as veliparib, did not - likely reflecting their allosteric influence on DNA retention. We also assessed the effect of the PARP1/2 active site component, HPF1, on intracellular PARPi binding and see that HPF1 depletion elicits slight deviations in apparent binding potency, while contributing additively to PARP-DNA trapping phenotypes. The PARP1/2 CeTEAM platform thus provides a structural roadmap for the development of selective PARPi and should facilitate the discovery of better targeted therapies. Furthermore, our results highlight that multiplexing CeTEAM biosensors and layered genetic perturbations can systematically profile determinants of intracellular drug selectivity.

biochemistry↗

bioRxiv2024

Quinoline-based compounds can inhibit diverse enzymes that act on DNA

DNA methylation, as exemplified by cytosine-C5 methylation in mammals and adenine-N6 methylation in bacteria, is a crucial epigenetic mechanism driving numerous vital biological processes. Developing non-nucleoside inhibitors to cause DNA hypomethylation is a high priority, in order to treat a variety of significant medical conditions without the toxicities associated with existing cytidine-based hypomethylating agents. In this study, we have characterized fifteen quinoline-based analogs. Notably, compounds with additions like a methylamine (9) or methylpiperazine (11) demonstrate similar low micromolar inhibitory potency against both human DNMT1 (which generates C5-methylcytosine) and Clostridioides difficile CamA (which generates N6-methyladenine). Structurally, compounds 9 and 11 specifically intercalate into CamA-bound DNA via the minor groove, adjacent to the target adenine, leading to a substantial conformational shift that moves the catalytic domain away from the DNA. This study adds to the limited examples of DNA methyltransferases being inhibited by non-nucleotide compounds through DNA intercalation, following the discovery of dicyanopyridine-based inhibitors for DNMT1. Furthermore, our study shows that some of these quinoline-based analogs inhibit other enzymes that act on DNA, such as polymerases and base excision repair glycosylases. Finally, in cancer cells compound 11 elicits DNA damage response via p53 activation. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=108 SRC="FIGDIR/small/587980v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@da2e94org.highwire.dtl.DTLVardef@27f909org.highwire.dtl.DTLVardef@4ec628org.highwire.dtl.DTLVardef@1b384df_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LISix of fifteen quinoline-based derivatives demonstrated comparable low micromolar inhibitory effects on human cytosine methyltransferase DNMT1, and the bacterial adenine methyltransferases Clostridioides difficile CamA and Caulobacter crescentus CcrM. C_LIO_LICompounds 9 and 11 were found to intercalate into a DNA substrate bound by CamA. C_LIO_LIThese quinoline-based derivatives also showed inhibitory activity against various base excision repair DNA glycosylases, and DNA and RNA polymerases. C_LIO_LICompound 11 provokes DNA damage response via p53 activation in cancer cells. C_LI

molecular biology↗

bioRxiv2023

M6A reduction relieves FUS-associated ALS granules

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease due to gradual motorneurons (MN) degeneration1. Among the processes associated to ALS pathogenesis, there is the formation of cytoplasmic inclusions produced by mutant protein aggregation, among which the RNA binding protein FUS2. In this work we show that such inclusions are significantly reduced in number and dissolve faster when the RNA m6A content is diminished as a consequence of the m6A writer METTL3 knock-down. These effects were observed both in neuronal cell lines and in iPSC-derived human motor neurons expressing mutant FUS. Importantly, stress granules formed in ALS condition showed a distinctive transcriptome with respect to control cells; interestingly, after METTL3 downregulation, it reverted to similar to control. Finally, we show that FUS inclusions are reduced also in patient-derived fibroblasts treated with STM-2457, a well characterized inhibitor of METTL3 activity, paving the way for its possible use for counteracting aggregate formation in ALS.

molecular biology↗

bioRxiv2023

Inhibition of the macrophage demethylase LSD1 reverses Leishmania amazonensis-induced transcriptomic changes and causes a decrease in parasite load

Intracellular pathogens exploit host cell functions to favor their own survival. In recent years, the subversion of epigenetic regulation has emerged as a key microbial strategy to modify host cell gene expression and evade antimicrobial immune responses. Using the protozoan parasite Leishmania as a model system, we have recently demonstrated that infection causes histone H3 hypomethylation, which is associated with the establishment of an anti-inflammatory phenotype, suggesting that host cell demethylases may play a role in the intracellular survival of these parasites. In this study, we combined pharmacological inhibition with RNA sequencing and quantitative immune-precipitation analysis to investigate the role of the macrophage lysine demethylase LSD1 (KDM1a) in Leishmania intracellular infection in vitro. Treatment of infected macrophages with validated, LSD1-specific inhibitors resulted in a significant reduction in parasite burden. We confirmed the impact of these inhibitors on LSD1 activity within macrophage nuclear extracts using an in vitro demethylase assay and established their LSD1 target engagement in situ by cellular thermal shift assay. RNA-seq analysis of infected and inhibitor-treated macrophages linked parasite killing to a partial reversion of infection-dependent expression changes, restoring the macrophage anti-microbial response and limiting cholesterol biosynthesis. While we ruled out any impact of Leishmania on LSD1 expression or localization, we uncovered significant alterations in LSD1 complex formation within infected macrophages, involving unique interactions with host cell regulatory proteins such as Rcor-1. Our study sheds important new light on the epigenetic mechanisms of macrophage immuno-metabolic subversion by intracellular Leishmania and identifies LSD1 as a potential candidate for host-directed, anti-leishmanial therapy.

cell biology↗

bioRxiv2023

An oxadiazole-based compound potentiates anti- tuberculosis treatment by increasing host resistance via zinc poisoning

Anti-tuberculosis drugs, mostly developed over 60 years ago, combined with a poorly effective vaccine, have failed to eradicate tuberculosis. More worryingly, multi-resistant strains of Mycobacterium tuberculosis are constantly emerging. Innovative strategies are thus urgently needed to improve tuberculosis treatment. Recently, host-directed therapy has emerged as a promising strategy to be used in adjunct with existing or future antibiotics, by improving innate immunity or limiting immunopathology. Here, using high content imaging, we identified novel 1,2,4-oxadiazole-based compounds, that allow human macrophages to control MTB replication. Genome-wide gene expression analysis revealed that these molecules induced zinc remobilization inside cells, resulting in bacterial zinc intoxication. More importantly, we also demonstrated that, upon treatment with these novel compounds, M. tuberculosis became even more sensitive to anti-tuberculosis drugs, in vitro and in vivo, in a mouse model of tuberculosis. Manipulation of heavy metal homeostasis holds thus great promise to be exploited to develop host-directed therapeutic interventions.

microbiology↗

bioRxiv2019

Pharmacological inhibition of lysine-specific demethylase 1 (LSD1) induces global transcriptional deregulation and ultrastructural alterations that impair viability in Schistosoma mansoni

Treatment and control of schistosomiasis still rely on only one effective drug, praziquantel (PZQ), and due to mass treatment, the increasing risk of selecting for schistosome strains that are resistant to PZQ has alerted investigators to the urgent need to develop novel therapeutic strategies. The histone-modifying enzymes (HMEs) represent promising targets for the development of epigenetic drugs against Schistosoma mansoni. In the present study, we targeted the S. mansoni lysine-specific demethylase 1 (SmLSD1), a transcriptional corepressor, using a novel and selective synthetic inhibitor, MC3935. We synthesized a novel and potent LSD1 inhibitor, MC3935, which was used to treat schistosomula or adult worms in vitro. By using cell viability assays and optical and electron microscopy, we showed that treatment with MC3935 affected parasite motility, egg-laying, tegument, and cellular organelle structures, culminating in the death of schistosomula and adult worms. In silico molecular modeling and docking analysis suggested that MC3935 binds to the catalytic pocket of SmLSD1. Western blot analysis revealed that MC3935 inhibited SmLSD1 demethylation activity of H3K4me1/2. Knockdown of SmLSD1 by RNAi recapitulated MC3935 phenotypes in adult worms. RNA-seq analysis of MC3935-treated parasites revealed significant differences in gene expression related to critical biological processes. Collectively, our findings show that SmLSD1 is a promising drug target for the treatment of schistosomiasis and strongly support the further development and in vivo testing of selective schistosome LSD1 inhibitors. Author SummarySchistosomiasis mansoni is a chronic and debilitating tropical disease caused by the helminth Schistosoma mansoni. The control and treatment of the disease rely almost exclusively on praziquantel (PZQ). Thus, there is an urgent need to search for promising protein targets to develop new drugs. Drugs that inhibit enzymes that modify the chromatin structure have been developed for a number of diseases. We and others have shown that S. mansoni epigenetic enzymes are also potential therapeutic targets. Here we evaluated the potential of the S. mansoni histone demethylase LSD1 (SmLSD1) as a drug target. We reported the synthesis of a novel and potent LSD1 inhibitor, MC3935, and show that it selectively inhibited the enzymatic activity of SmLSD1. Treatment of juvenile or adult worms with MC3935 caused severe damage to the tegument of the parasites and compromised egg production. Importantly, MC3935 proved to be highly toxic to S. mansoni, culminating in the death of juvenile or adult worms within 96 h. Transcriptomic analysis of MC3935-treated parasites revealed changes in the gene expression of hundreds of genes involved in key biological processes. Importantly, SmLSD1 contains unique sequences within its polypeptide chain that could be explored to develop a S. mansoni selective drug.

molecular biology↗