Biology subjects

Pellegrino, S.

Publications and source records attributed to Pellegrino, S..

9 recordsLinked to original sources

bioRxiv2026

AI-Driven Computational Design of Peptide-Based WWP1 Inhibitors as Promising Therapeutic Agents Against Breast Cancer, Including Triple-Negative Subtype

Breast cancer (BC) is the second most common noncutaneous cancer and the second leading cause of cancer-related death in women. BC is classified into three primary subtypes, with triple-negative breast cancer (TNBC) having the poorest prognosis because it lacks specific targetable markers. Preclinical studies on TNBC indicated a common occurrence of diminished tumor-suppressor activity of PTEN, activating the PI3K/AKT/mTOR signaling pathway. Notably, published studies reveal that the WWP1 enzyme plays a pivotal role in driving PTEN degradation via ubiquitination, unveiling a promising therapeutic target for treating TNBC. In the search of new WWP1 inhibitors, we used artificial intelligence (AI)-driven computational strategies for de novo design of peptide-based WWP1 inhibitors and identified a hexapeptide, termed WI23-B, which demonstrated high nanomolar binding affinity to WWP1. In TR-FRET enzymatic assays, WI23-B inhibited WWP1 activity with an IC of approximately 11 {micro}M. In MCF7 and MDA-MB-231 breast cancer cell lines, WI23-B showed promising cytotoxic efficacy, particularly in combination with the PI3K inhibitor BYL719, also when it was loaded into nanocapsules. Collectively, these findings highlight WI23-B as a promising lead peptide with potent WWP1 inhibitory activity and synergistic antiproliferative effects when combined with PI3K inhibitors. While further structural optimization is required to enhance its potency and pharmacological properties, our results provide a strong foundation for the development of next-generation WWP1 inhibitors. Such agents have the potential to reshape therapeutic strategies for BC and TNBC by enabling more effective and less toxic treatment regimens, ultimately reducing the reliance on high-dose chemotherapy and minimizing adverse effects.

cancer biology↗

bioRxiv2025

Investigation of anti-SARS CoV-2 multimeric bicyclic peptide inhibitors in a range of pre-clinical therapeutic settings

The spread of respiratory viruses, such as Influenza and SARS-CoV-2 has presented significant challenges over the last 30 years with few effective therapeutic options available to this day. Bi-cyclic peptides represent a unique, modular, modality in the antiviral armamentarium against future pandemics. This study provides a deeper evaluation of multivalent bi-cyclic (Bicycle(R)) molecule efficacy in several preclinical SARS-CoV-2 challenge settings. We explore both pre-exposure prophylaxis and post-exposure therapeutic settings via subcutaneous and intranasal routes of administration. We contextualize this further in bespoke scenarios of immune compromisation, and viral transmission. Promisingly, in all studies we observe efficacy, significantly reducing infectious viral burden at each study endpoint. These data further support candidacy of Bicycle molecules as a differentiated antiviral therapeutic class in the context of pandemic preparedness. ImportanceThe COVID-19 pandemic, triggered a rapid wave of innovation, accelerating the delivery of new vaccine technology and anti-viral treatments. In our first paper, we described the discovery and molecular optimization of Bicycle molecules as a novel drug class for the potential treatment of SARS CoV-2. Here, we have performed deeper characterization of these molecules in established animal models that simulate SARS-CoV-2 transmission, testing more convenient delivery routes, such as intra-nasal. The Bicycle molecules demonstrated positive outcomes in each of these studies and suggest that Bicycle molecules, as convenient and effective anti-viral treatments, could be an important addition to help future preparedness against new viral pandemics.

pharmacology and toxicology↗

bioRxiv2025

Complementary Volume Electron Microscopy-based approaches reveal ultrastructural changes in germline intercellular bridges of D. melanogaster

Intercellular bridges are essential to connect developing germline cells. The Drosophila melanogaster egg chamber is a powerful model system to study germline intercellular bridges, or ring canals (RCs). RCs connect the developing oocyte to supporting nurse cells, and defects in their stability or growth lead to infertility. Despite their importance, it has been technically difficult to use electron microscopy-based approaches to monitor changes in RC structure during oogenesis. Here, we describe the application of a complementary set of volume EM-based approaches to visualize ultrastructural changes in the germline RCs. The combination of array tomography (AT) and focused ion beam (FIB) scanning electron microscopy (SEM) has allowed us to gain insight into previously unappreciated aspects of RC structure. We were able to quantify differences in RC size and thickness within and between germ cell clusters at different developmental stages. Within a cluster, RC size correlates with lineage; the largest RCs were formed during the first division, and the smallest RCs were formed during the fourth mitotic division. We observed the formation of membrane interdigitations in the vicinity of RCs much earlier than previously reported, and reconstruction of a RC from a mid-stage EC provided insight into the 3D orientation of these extensive cell-cell contacts. Our imaging also revealed a novel membrane structure that appeared to line the interior of the RC lumen. Although the focus was on ultrastructural changes in the germline RCs, our dataset contains valuable details of additional cell types and structures, including the fusome, the germline stem cells and their niche, and the migrating border cells. This imaging framework could be applied to other tissues or samples that face similar technical challenges, where the small structure of interest is located within a large sample volume.

developmental biology↗

bioRxiv2023

The chemical landscape of the human ribosome at 1.67 A resolution

The ability of ribosomes to translate the genetic code into protein requires a finely tuned ion and solvent ecosystem. However, the lack of high-resolution structures has precluded accurate positioning of all the functional elements of the ribosome and limited our understanding of the specific role of ribosomal RNA chemical modifications in modulating ribosome function in health and disease. Here, using a new sample preparation methodology based on functionalised pristine graphene-coated grids, we solve the cryo-EM structure of the human large ribosomal subunit to a resolution of 1.67 [A]. The accurate assignment of water molecules, magnesium and potassium ions in our model highlights the fundamental biological role of ribosomal RNA methylation in harnessing unconventional carbon-oxygen hydrogen bonds to establish chemical interactions with the environment and fine-tune the functional interplay with tRNA. In addition, the structures of three translational inhibitors bound to the human large ribosomal subunit at better than 2 [A] resolution provide mechanistic insights into how three key druggable pockets of the ribosome are targeted and illustrate the potential of this methodology to accelerate high-throughput structure-based design of anti-cancer therapeutics.

biochemistry↗

bioRxiv2022

Multi-eGO: an in-silico lens to look into protein aggregation kinetics at atomic resolution

Protein aggregation into amyloid fibrils is the archetype of aberrant biomolecular self-assembly processes, with more than 50 diseases associated that are mostly uncurable. Understanding aggregation mechanisms is thus of fundamental importance and goes in parallel with the characterization of the structures of the transient oligomers formed in the process. Oligomers have been proven elusive to high-resolution structural techniques, while the large sizes and long-time scales typical of aggregation processes have limited, so far, the use of computational methods. To surmount these limitations, we introduce here multi-eGO, an atomistic, hybrid structure-based model, which leveraging on the knowledge of monomers conformational dynamics and of fibril structures, can efficiently capture the essential structural and kinetics aspects of protein aggregation. Multi-eGO molecular dynamics simulations can describe the aggregation kinetics of thousands of monomers. The concentration dependence of the simulated kinetics, as well as the structural features of the resulting fibrils, are in qualitative agreement with in vitro experiments on an amyloidogenic peptide of Transthyretin, a protein responsible for one of the most common cardiac amyloidosis. Multi-eGO simulations allow to observe in time and at atomic resolution the formation of primary nuclei in a sea of transient lower order oligomers, to follow their growth and the subsequent secondary nucleation events, till the maturation of multiple fibrils. Multi-eGO, combined with the many experimental techniques deployed to study protein aggregation, can provide the structural basis needed to advance the design of molecules targeting amyloidogenic diseases. Significance StatementAlzheimers and Parkinsons diseases are uncurable pathologies associated to the aberrant aggregation of specific proteins into amyloid fibrils. Understanding the mechanism leading to protein aggregation, by characterizing the structures of the oligomeric species populated in the process, would have a tremendous impact on the design of therapeutic molecules. We propose that a structure-based approach to molecular dynamics simulations can allow following at high resolution the aggregation kinetics of thousands of monomers. Having shown that simulations can describe the aggregation of a Transthyretin amyloidogenic peptide, we demonstrate how their efficiency allows acquiring a wealth of structural information. We foresee that integrating the latter with the many techniques developed to study protein aggregation will support the design of molecules to modulate amyloidogenesis.

biochemistry↗

bioRxiv2022

Cryo-EM reconstruction of the human 40S ribosomal subunit at 2.15 A resolution

The chemical modification of ribosomal RNA and proteins is critical for ribosome assembly, for protein synthesis and may drive ribosome specialization in development and disease. However, the inability to accurately visualize these modifications has limited mechanistic understanding of the role of these modifications in ribosome function. Here we report the 2.15 [A] resolution cryo-EM reconstruction of the human 40S ribosomal subunit. We directly visualize post-transcriptional modifications within the 18S rRNA and post-translational modifications at the N-termini of two ribosomal proteins. Additionally, we interpret the solvation shells in the core regions of the 40S ribosomal subunit and reveal how potassium and magnesium ions establish both universally conserved and eukaryote-specific coordination to promote the stabilization and folding of key ribosomal elements. This work provides unprecedented structural details for the human 40S ribosomal subunit that will serve as an important reference for unraveling the functional role of ribosomal RNA modifications.

biochemistry↗

bioRxiv2021

A late-stage assembly checkpoint of the human mitochondrial ribosome large subunit

The epitranscriptome plays a key regulatory role in cellular processes in health and disease, including ribosome biogenesis. Here, analysis of the human mitochondrial transcriptome shows that 2-O-methylation is limited to residues of the mitoribosomal large subunit (mtLSU) 16S mt-rRNA, modified by MRM1, MRM2, and MRM3. Ablation of MRM2 leads to a severe impairment of the oxidative phosphorylation system, caused by defective mitochondrial translation and accumulation of mtLSU assembly intermediates. Structures of these particles (2.58 [A]) present disordered RNA domains, partial occupancy of bL36m and bound MALSU1:L0R8F8:mtACP anti-association module. Additionally, we present five mtLSU assembly states with different intersubunit interface configurations. Complementation studies demonstrate that the methyltransferase activity of MRM2 is dispensable for mitoribosome biogenesis. The Drosophila melanogaster orthologue, DmMRM2, is an essential gene, with its knock-down leading to developmental arrest. This work identifies a key late-stage quality control step during mtLSU assembly, ultimately contributing to the maintenance of mitochondrial homeostasis.

molecular biology↗

bioRxiv2020

Replicated chromatin curtails 53BP1 recruitment in BRCA1-proficient and -deficient cells

DNA double-strand breaks can be repaired by two competing mechanisms, non-homologous end-joining (NHEJ) and homologous recombination (HR). Whether one or the other repair pathway is favored depends on the availability of an undamaged template DNA that allows for homology-directed repair. The tumor suppressor proteins 53BP1 and BRCA1 are considered antagonistic players in this repair pathway choice, as 53BP1 restrains DNA end resection, whereas BRCA1, together with its partner protein BARD1, displaces 53BP1 from damaged replicated chromatin and promotes HR. How cells switch from a 53BP1-dominated to a BRCA1-dominated response as they progress through the cell cycle is incompletely understood. Here we reveal, using high-throughput microscopy and applying single cell normalization to control for increased genome size as cells replicate their DNA, that 53BP1 recruitment to damaged replicated chromatin is inefficient in both BRCA1-proficient and BRCA1-deficient cells, in comparison to 53BP1 accumulation at damaged unreplicated chromatin. These findings substantiate a dual switch model from a 53BP1-dominated response in unreplicated chromatin to a BRCA1-BARD1-dominated response in replicated chromatin, in which replication-coupled dilution of 53BP1s binding mark H4K20me2 functionally cooperates with BRCA1-BARD1-mediated suppression of 53BP1 binding. More generally, we suggest that appropriate normalization of single cell data, e.g. to DNA content, provides additional layers of information, which can be critical for quantifying and interpreting cellular phenotypes.

cell biology↗

bioRxiv2019

Tuning antiviral CD8 T-cell response via proline-altered peptide ligand vaccination

Viral escape from CD8+ cytotoxic T lymphocyte responses correlates with disease progression and represents a significant challenge for vaccination. Here, we demonstrate that CD8+ T cell recognition of the naturally occurring MHC-I-restricted LCMV-associated immune escape variant Y4F is restored following vaccination with a proline-altered peptide ligand (APL). The APL increases MHC/peptide (pMHC) complex stability, rigidifies the peptide and facilitates T cell receptor (TCR) recognition through reduced entropy costs. Structural analyses of pMHC complexes before and after TCR binding, combined with biophysical analyses, revealed that although the TCR binds similarly to all complexes, the p3P modification alters the conformations of a very limited amount of specific MHC and peptide residues, facilitating efficient TCR recognition. This approach can be easily introduced in peptides restricted to other MHC alleles, and can be combined with currently available and future vaccination protocols in order to prevent viral immune escape. Author SummaryViral escape mutagenesis correlates often with disease progression and represents a major hurdle for vaccination-based therapies. Here, we have designed and developed a novel generation of altered epitopes that re-establish and enhance significantly CD8+ T cell recognition of a naturally occurring viral immune escape variant. Biophysical and structural analyses provide a clear understanding of the molecular mechanisms underlying this reestablished recognition. We believe that this approach can be implemented to currently available or novel vaccination approaches to efficiently restore T cell recognition of virus escape variants to control disease progression.

immunology↗