Biology subjects

Moro, S.

Publications and source records attributed to Moro, S..

8 recordsLinked to original sources

bioRxiv2026

A novel dual-chimeric surrogate virus to select, test and characterize mutants of protease and glycoprotein inhibitors.

Antivirals are life-saving medications. However, pathogens develop resistances, mitigating their effect. Resistance studies (gain-of-function, GOF) are therefore paramount before and while antivirals are used clinically to characterize resistances and ideally study how to counteract them with other antivirals. The ability to switch from one antiviral, against which the virus is resistant, to another, depends on the availability of alternative drugs. Furthermore, targeting different parts of the virus synergistically with multiple drugs decreases the odds of simultaneous resistance. However, studying resistance, cross-resistance and remaining susceptibility requires working with the actual pathogen. Especially when introducing resistances into dangerous viruses, performing GOF studies would raise safety concerns and require firm biological containment. In this study we developed a novel dual-chimeric virus encoding both SARS-CoV-2 spike glycoprotein and main protease (Mpro) based on the model virus, vesicular stomatitis virus (VSV). We applied selection pressure with spike and Mpro inhibitors, generated mutants of these viruses and determined their resistance and cross-resistance profiles by performing dose-response experiments with live and pseudotyped viruses. Additionally, we conducted in silico calculations to determine the impact of spike substitutions to the observed resistance phenotype. With the new technology presented here, we demonstrate that it is possible to carry out GOF studies more safely and highlight their importance in the future challenges of virological research.

microbiology↗

bioRxiv2025

Exomer cooperates with Ryh1RAB6 in regulating TORC2 activity

Exomer is a trans-Golgi protein complex involved in multiple biological processes, including lipid homeostasis and stress response. We have found that in fission yeast, the absence of exomer leads to a defect in the recovery of "target of rapamycin complex 2" (TORC2) activity in response to high concentrations of KCl, but not to sorbitol, which indicates that the mutants are impaired in their ability to respond to salt stress. Changes in lipid homeostasis did not suppress this defect. Ryh1RAB6 is a Rab GTPase that resides in the Golgi and endosomes and facilitates or stabilizes the interaction between TORC2 and its substrate, the AGC kinase Gad8. We have found genetic and functional interactions between exomer and Ryh1RAB6. The recovery of TORC2 activity in response to KCl requires Ryh1RAB6 and exomer, and the localization of Ryh1RAB6 is aberrant in exomer mutants. Co-immunoprecipitation experiments indicate that the interaction between TORC2 and Gad8 is weaker in the absence of exomer. We propose that exomer regulates TORC2 by facilitating appropriate Ryh1 localization.

cell biology↗

bioRxiv2024

TumFlow: An AI Model for Predicting New Anticancer Molecules

MotivationMelanoma is a severe form of skin cancer increasing globally with about 324.000 cases in 2020, making it the fifth most common cancer in the United States. Conventional drug discovery methods face limitations due to the inherently time consuming and costly. However, the emergence of artificial intelligence (AI) has opened up new possibilities. AI models can effectively simulate and evaluate the properties of a vast number of potential drug candidates, substantially reducing the time and resources required by traditional drug discovery processes. In this context, the development of AI normalizing flow models, employing machine learning techniques to create new molecular structures, holds great promise for accelerating the discovery of effective anticancer therapies. ResultsThis manuscript introduces a novel AI model, named TumFlow, aimed at generating new molecular entities with potential therapeutic value in cancer treatment. It has been trained on the comprehensive NCI-60 dataset, encompassing thousands of molecules tested across 60 tumour cell lines, with a specific emphasis on the melanoma SK-MEL-28 cell line. The model successfully generated new molecules with predicted improved efficacy in inhibiting tumour growth while being synthetically feasible. This represents a significant advancement over conventional generative models, which often produce molecules that are challenging or impossible to synthesize. Furthermore, TumFlow has also been utilized to optimize molecules known for their efficacy in clinical melanoma treatments. This led to the creation of novel molecules with a predicted enhanced likelihood of effectiveness against melanoma, currently undocumented on PubChem. Availability and Implementationhttps://github.com/drigoni/TumFlow. Supplementary informationUploaded.

bioinformatics↗

bioRxiv2023

A comprehensive study of SARS-CoV-2 main protease (Mpro) inhibitor-resistant mutants selected in a VSV-based system

Nirmatrelvir was the first protease inhibitor (PI) specifically developed against the SARS-CoV-2 main protease (3CLpro/Mpro) and licensed for clinical use. As SARS-CoV-2 continues to spread, variants resistant to nirmatrelvir and other currently available treatments are likely to arise. This study aimed to identify and characterize mutations that confer resistance to nirmatrelvir. To safely generate Mpro resistance mutations, we passaged a previously developed, chimeric vesicular stomatitis virus (VSV-Mpro) with increasing, yet suboptimal concentrations of nirmatrelvir. Using Wuhan-1 and Omicron Mpro variants, we selected a large set of mutants. Some mutations are frequently present in GISAID, suggesting their relevance in SARS-CoV-2. The resistance phenotype of a subset of mutations was characterized against clinically available PIs (nirmatrelvir and ensitrelvir) with cell-based and biochemical assays. Moreover, we showed the putative molecular mechanism of resistance based on in silico molecular modelling. These findings have implications on the development of future generation Mpro inhibitors, will help to understand SARS-CoV-2 protease-inhibitor-resistance mechanisms and show the relevance of specific mutations in the clinic, thereby informing treatment decisions. TeaserUnderstanding how SARS-CoV-2 could counter the antiviral drug nirmatrelvir and what it means for the future of COVID-19 treatment.

microbiology↗

bioRxiv2023

Mitochondrial Calcium Uptake Declines during Aging and is Directly Activated by Oleuropein to Boost Energy Metabolism and Skeletal Muscle Performance

Mitochondrial calcium (mtCa2+) uptake via the Mitochondrial Calcium Uniporter (MCU) couples the regulation of calcium homeostasis to energy production. mtCa2+ uptake is rate-limiting for mitochondrial activation during muscle contraction, but how MCU is affected during physiopathology and whether it can be stimulated therapeutically remains largely uncharacterized. By profiling human and preclinical aging of skeletal muscle, we discovered a conserved down-regulation of MCUR1 during aging that decreases mtCa2+ uptake and drives sarcopenia. Through a screen of 5000 bioactive nutrients, we identify the natural polyphenol Oleuropein as a specific MCU activator that stimulates mitochondrial respiration via binding to MICU1. Oleuropein activates mtCa2+ uptake and oxidative energy metabolism to enhance endurance and limit fatigue in vivo both in young and aged. These effects of Oleuropein are mediated by an MCU-dependent mechanism in skeletal muscle as they are lost upon muscle-specific MCU KO. Our work demonstrates that impaired mtCa2+ uptake causes mitochondrial dysfunction during aging and establishes Oleuropein as a novel nutrient that specifically targets MCU to stimulate mitochondrial bioenergetics and muscle performance.

physiology↗

bioRxiv2022

The E2F4/p130 repressor complex cooperates with oncogenic ΔNp73α to promote cell survival in human papillomavirus 38 E6/E7-transformed keratinocytes and in cancer cells

Tumor suppressor p53 and its related proteins, p63 and p73, can be synthesized as multiple isoforms lacking part of the N- or C-terminal regions. Specifically, high expression of the {Delta}Np73 isoform is notoriously associated with various human malignancies characterized by poor prognosis. This isoform is also accumulated by oncogenic viruses such as Epstein-Barr virus (EBV), as well as genus beta human papillomaviruses (HPV) that appear to be involved in carcinogenesis. To gain additional insight into {Delta}Np73 mechanisms, we have performed proteomics analyses using human keratinocytes transformed by the E6 and E7 proteins of the beta-HPV type 38 virus as an experimental model (38HK). We find that {Delta}Np73 associates with the E2F4/p130 repressor complex through a direct interaction with E2F4. This interaction is favored by the N-terminal truncation of p73 characteristic of {Delta}Np73 isoforms. Moreover, it is independent of the C-terminal splicing status, suggesting that it could represent a general feature of {Delta}Np73 isoforms (, {beta}, {gamma}, {delta}, {varepsilon}, {zeta}, {theta}, {eta}, and {eta}1). We also show that the {Delta}Np73- E2F4/p130 complex inhibits the expression of specific genes, including genes encoding for negative regulators of proliferation, both in 38HK and in HPV-negative cancer-derived cell lines. Consistently, silencing of E2F4 in 38HK and in cancer cells results in induction of senescence. In conclusion, we have identified and characterized a novel transcriptional regulatory complex that exerts pro-survival functions in transformed cells. IMPORTANCEThe TP53 gene is mutated in about 50% of human cancers. In contrast, the TP63 and TP73 genes are rarely mutated but rather expressed as {Delta}Np63 and {Delta}Np73 isoforms in a wide range of malignancies, where they act as p53 antagonists. Accumulation of {Delta}Np63 and {Delta}Np73, which is associated with chemoresistance, can result from infection by oncogenic viruses such as EBV or HPV. Our study focuses on the highly carcinogenic {Delta}Np73 isoform and uses a viral model of cellular transformation. We unveil a physical interaction between {Delta}Np73 and the E2F4/p130 complex involved in cell cycle control, which rewires the E2F4/p130 transcriptional program. Consistently, we find that E2F4 gains pro-survival functions in transformed cells expressing {Delta}Np73. This report shows, for the first time, that {Delta}Np73 isoforms acquire novel protein-protein interactions with respect to the TAp73 tumor suppressor. This situation is analogous to the gain-of-function interactions of p53 mutants supporting cellular proliferation.

cancer biology↗

bioRxiv2022

Hidden GPCR structural transitions addressed by multiple walker supervised molecular dynamics (mwSuMD)

The structural basis for the pharmacology of G protein-coupled receptors (GPCRs), the most abundant membrane proteins and the target of about 35% of approved drugs, is still a matter of intense study. What makes GPCRs challenging to study is the inherent flexibility and the metastable nature of interaction with extra-and intracellular partners that drive their effects. Here, we present a molecular dynamics (MD) adaptive sampling algorithm, namely multiple walker supervised molecular dynamics (mwSuMD), to address complex structural transitions involving GPCRs without energy input. We first report the binding and unbinding of the vasopressin peptide from its receptor V2. Successively, we present the complete transition of the glucagon-like peptide-1 receptor (GLP-1R) from inactive to active, agonist and Gs-bound state, and the GDP release from Gs. To our knowledge, this is the first time the whole sequence of events leading from an inactive GPCR to the GDP release is simulated without any energy bias. We demonstrate that mwSuMD can address complex binding processes intrinsically linked to protein dynamics out of reach of classic MD.

bioinformatics↗

bioRxiv2021

A novel conformational state for SARS-CoV-2 main protease

The SARS-CoV-2 main protease (Mpro) has a pivotal role in mediating viral genome replication and transcription of coronavirus, making it a promising target for drugs against Covid-19 pandemic. Here we present a crystal structure of Mpro disclosing new structural features of key regions of the enzyme. We show that the oxyanion loop, involved in substrate recognition and enzymatic activity, can adopt a new conformation, which is stable and significantly different from the known ones. In this new state the S1 subsite of the substrate binding region is completely reshaped and a new cavity near the S2 subsite is created. This new structural information expands the knowledge of the conformational space available to Mpro, paving the way for the design of novel classes of inhibitors specifically designed to target this unprecedented binding site conformation, thus enlarging the chemical space for urgent antiviral drugs against Covid-19 pandemic.

biochemistry↗