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Huntington’s disease (HD) is caused by a CAG-repeat expansion mutation in the Huntingtin (HTT) gene. It is characterized by progressive psychiatric and neurological symptoms in combination with a progressive movement disorder. Despite the ubiquitous expression of HTT, pathological changes occur quite selectively in the central nervous system. Since the discovery of HD more than 150 years ago, a lot of research on molecular mechanisms contributing to neurotoxicity has remained the focal point. While traditionally, the protein encoded by the HTT gene remained the cynosure for researchers and was extensively reviewed elsewhere, several studies in the last few years clearly indicated the contribution of the mutant RNA transcript to cellular dysfunction as well. In this review, we outline recent studies on RNA-mediated molecular mechanisms that are linked to cellular dysfunction in HD models. These mechanisms include mis-splicing, aberrant translation, deregulation of the miRNA machinery, deregulated RNA transport and abnormal regulation of mitochondrial RNA. Furthermore, we summarize recent therapeutical approaches targeting the mutant HTT transcript. While currently available treatments are of a palliative nature only and do not halt the disease progression, recent clinical studies provide hope that these novel RNA-targeting strategies will lead to better therapeutic approaches.
Annika Heinz; Deepti Nabariya; Sybille Krauss. Huntingtin and Its Role in Mechanisms of RNA-Mediated Toxicity. Toxins 2021, 13, 487 .
AMA StyleAnnika Heinz, Deepti Nabariya, Sybille Krauss. Huntingtin and Its Role in Mechanisms of RNA-Mediated Toxicity. Toxins. 2021; 13 (7):487.
Chicago/Turabian StyleAnnika Heinz; Deepti Nabariya; Sybille Krauss. 2021. "Huntingtin and Its Role in Mechanisms of RNA-Mediated Toxicity." Toxins 13, no. 7: 487.
Huntington’s disease (HD) is an autosomal dominant disorder caused by a CAG repeat expansion within the coding region of the Huntingtin (HTT) gene. The typical age of onset is around the age of 40 years, and patients show progressively worsening symptoms including involuntary movements as well as cognitive and psychiatric abnormalities. To date, there is no cure for HD, and currently available treatments are of palliative nature only. One especially difficult aspect of drug development is the broad range of cellular events that contribute to neurotoxicity. Both the mutant transcript and the mutant protein are associated with toxic gain-of-function mechanisms that range from transcriptional deregulation and deregulated splicing to aberrant translation, impaired intracellular trafficking, and mitochondrial dysfunction. This chapter describes different disease mechanisms caused by the mutant HTT gene product and highlights recent promising therapeutic developments.
Annika Heinz; Deepti Kailash Nabariya; Sybille Krauß. Huntington’s Disease and Neurodegeneration. Handbook of Neurotoxicity 2021, 1 -23.
AMA StyleAnnika Heinz, Deepti Kailash Nabariya, Sybille Krauß. Huntington’s Disease and Neurodegeneration. Handbook of Neurotoxicity. 2021; ():1-23.
Chicago/Turabian StyleAnnika Heinz; Deepti Kailash Nabariya; Sybille Krauß. 2021. "Huntington’s Disease and Neurodegeneration." Handbook of Neurotoxicity , no. : 1-23.
Intracellular ADP-ribosyltransferases catalyze mono- and poly-ADP-ribosylation and affect a broad range of biological processes. The mono-ADP-ribosyltransferase PARP10 is involved in signaling and DNA repair. Previous studies identified OUL35 as a selective, cell permeable inhibitor of PARP10. We have further explored the chemical space of OUL35 by synthesizing and investigating structurally related analogs. Key synthetic steps were metal-catalyzed cross-couplings and functional group modifications. We identified 4-(4-cyanophenoxy)benzamide and 3-(4-carbamoylphenoxy)benzamide as PARP10 inhibitors with distinct selectivities. Both compounds were cell permeable and interfered with PARP10 toxicity. Moreover, both revealed some inhibition of PARP2 but not PARP1, unlike clinically used PARP inhibitors, which typically inhibit both enzymes. Using crystallography and molecular modeling the binding of the compounds to different ADP-ribosyltransferases was explored regarding selectivity. Together, these studies define additional compounds that interfere with PARP10 function and thus expand our repertoire of inhibitors to further optimize selectivity and potency.
Patricia Korn; Arno Classen; Sudarshan Murthy; Riccardo Guareschi; Mirko M. Maksimainen; Barbara E. Lippok; Albert Galera‐Prat; Sven T. Sowa; Catharina Voigt; Dr. Giulia Rossetti; Dr. Lari Lehtiö; Dr. Carsten Bolm; Dr. Bernhard Lüscher. Evaluation of 3‐ and 4‐Phenoxybenzamides as Selective Inhibitors of the Mono‐ADP‐Ribosyltransferase PARP10. ChemistryOpen 2021, 1 .
AMA StylePatricia Korn, Arno Classen, Sudarshan Murthy, Riccardo Guareschi, Mirko M. Maksimainen, Barbara E. Lippok, Albert Galera‐Prat, Sven T. Sowa, Catharina Voigt, Dr. Giulia Rossetti, Dr. Lari Lehtiö, Dr. Carsten Bolm, Dr. Bernhard Lüscher. Evaluation of 3‐ and 4‐Phenoxybenzamides as Selective Inhibitors of the Mono‐ADP‐Ribosyltransferase PARP10. ChemistryOpen. 2021; ():1.
Chicago/Turabian StylePatricia Korn; Arno Classen; Sudarshan Murthy; Riccardo Guareschi; Mirko M. Maksimainen; Barbara E. Lippok; Albert Galera‐Prat; Sven T. Sowa; Catharina Voigt; Dr. Giulia Rossetti; Dr. Lari Lehtiö; Dr. Carsten Bolm; Dr. Bernhard Lüscher. 2021. "Evaluation of 3‐ and 4‐Phenoxybenzamides as Selective Inhibitors of the Mono‐ADP‐Ribosyltransferase PARP10." ChemistryOpen , no. : 1.
Aberrant RNA–protein complexes are formed in a variety of diseases. Identifying the ligands that interfere with their formation is a valuable therapeutic strategy. Molecular simulation, validated against experimental data, has recently emerged as a powerful tool to predict both the pose and energetics of such ligands. Thus, the use of molecular simulation may provide insight into aberrant molecular interactions in diseases and, from a drug design perspective, may allow for the employment of less wet lab resources than traditional in vitro compound screening approaches. With regard to basic research questions, molecular simulation can support the understanding of the exact molecular interaction and binding mode. Here, we focus on examples targeting RNA–protein complexes in neurodegenerative diseases and viral infections. These examples illustrate that the strategy is rather general and could be applied to different pharmacologically relevant approaches. We close this study by outlining one of these approaches, namely the light-controllable association of small molecules with RNA, as an emerging approach in RNA-targeting therapy.
Daria Berdnikova; Paolo Carloni; Sybille Krauß; Giulia Rossetti. Role and Perspective of Molecular Simulation-Based Investigation of RNA–Ligand Interaction: From Small Molecules and Peptides to Photoswitchable RNA Binding. Molecules 2021, 26, 3384 .
AMA StyleDaria Berdnikova, Paolo Carloni, Sybille Krauß, Giulia Rossetti. Role and Perspective of Molecular Simulation-Based Investigation of RNA–Ligand Interaction: From Small Molecules and Peptides to Photoswitchable RNA Binding. Molecules. 2021; 26 (11):3384.
Chicago/Turabian StyleDaria Berdnikova; Paolo Carloni; Sybille Krauß; Giulia Rossetti. 2021. "Role and Perspective of Molecular Simulation-Based Investigation of RNA–Ligand Interaction: From Small Molecules and Peptides to Photoswitchable RNA Binding." Molecules 26, no. 11: 3384.
A variety of enhanced sampling methods can predict free energy landscapes associated with protein/ligand binding events, characterizing in a precise way the intermolecular interactions involved. Unfortunately, these approaches are challenged by not uncommon induced fit mecchanisms. Here, we present a variant of the recently reported volume-based metadynamics (MetaD) method which describes ligand binding even when it affects protein structure. The validity of the approach is established by applying it to a substrate/enzyme complexes of pharmacological relevance: this is the mono-ADP-ribose (ADPr) in complex with mono-ADP-ribosylation hydrolases (MacroD1 and MacroD2), where induced-fit phenomena are known to be operative. The calculated binding free energies are consistent with experiments, with an absolute error less than 0.5 kcal/mol. Our simulations reveal that in all circumstances the active loops, delimiting the boundaries of the binding site, rearrange from an open to a closed conformation upon ligand binding. The calculations further provide, for the first time, the molecular basis of the experimentally observed affinity changes in ADPr binding on passing from MacroD1 to MacroD2 and all its mutants. Our study paves the way to investigate in a completely general manner ligand binding to proteins and receptors.
Qianqian Zhao; Riccardo Capelli; Paolo Carloni; Bernhard Luescher; Jinyu Li; Giulia Rossetti. An Enhanced Sampling Approach to the Induced Fit Docking Problem in Protein-Ligand Binding: the case of mono-ADP-ribosylation hydrolases inhibitors. 2021, 1 .
AMA StyleQianqian Zhao, Riccardo Capelli, Paolo Carloni, Bernhard Luescher, Jinyu Li, Giulia Rossetti. An Enhanced Sampling Approach to the Induced Fit Docking Problem in Protein-Ligand Binding: the case of mono-ADP-ribosylation hydrolases inhibitors. . 2021; ():1.
Chicago/Turabian StyleQianqian Zhao; Riccardo Capelli; Paolo Carloni; Bernhard Luescher; Jinyu Li; Giulia Rossetti. 2021. "An Enhanced Sampling Approach to the Induced Fit Docking Problem in Protein-Ligand Binding: the case of mono-ADP-ribosylation hydrolases inhibitors." , no. : 1.
Polyglutamine (polyQ) diseases, including Huntington’s disease, are characterized by an expansion of cytosine-adenine-guanine (CAG) trinucleotide repeats encoding for an uninterrupted prolonged polyQ tract. We previously identified TRMT2A as a strong modifier of polyQ-induced toxicity in an unbiased large-scale screen in Drosophila melanogaster. RNAi-mediated silencing of TRMT2A ameliorated polyQ-induced toxicity and polyQ aggregation in flies. This work aimed at identifying and validating pharmacological TRMT2A inhibitors as treatment opportunities for polyQ diseases. An in silico structure- and ligand-based lead discovery approach and computer-aided drug discovery (CADD) was used to identify TRMT2A inhibitors. Additionally, the crystal structure of one protein domain, the RNA recognition motif (RRM), was determined and Biacore experiments with the RRM were performed. The identified inhibitors were functionally validated for their potency to reduce polyQ aggregation and polyQ-induced cell death in human HEK293T cells and patient derived fibroblasts. Several candidate molecules were able to decrease cell death and ameliorate the aggregation of polyQ peptides in cultured cells comparable to the TRMT2A knockdown experiments. Among these, spermidine was identified as able to cause a decrease in the abundance of polyQ aggregates in SCA3-patient derived fibroblasts. Our work provides a first step towards a pharmacological inhibition of this enzyme and indicates TRMT2A as a viable drug target for polyQ diseases.
Michael A Margreiter; Monika Witzenberger; Yasmine Wasser; Elena Davydova; Robert Janowski; Carina Sobisch; Jonas Metz; Benedetta Poma; Oscar Paolomino-Hernandez; Jon N Shah; Jörg B Schulz; Dierk Niessing; Aaron Voigt; Giulia Rossetti. Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death. 2021, 1 .
AMA StyleMichael A Margreiter, Monika Witzenberger, Yasmine Wasser, Elena Davydova, Robert Janowski, Carina Sobisch, Jonas Metz, Benedetta Poma, Oscar Paolomino-Hernandez, Jon N Shah, Jörg B Schulz, Dierk Niessing, Aaron Voigt, Giulia Rossetti. Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death. . 2021; ():1.
Chicago/Turabian StyleMichael A Margreiter; Monika Witzenberger; Yasmine Wasser; Elena Davydova; Robert Janowski; Carina Sobisch; Jonas Metz; Benedetta Poma; Oscar Paolomino-Hernandez; Jon N Shah; Jörg B Schulz; Dierk Niessing; Aaron Voigt; Giulia Rossetti. 2021. "Small-molecule modulators of TRMT2A decrease PolyQ aggregation and PolyQ-induced cell death." , no. : 1.
The KIT D816V mutation is found in >80% of patients with systemic mastocytosis (SM) and is key to neoplastic mast cell (MC) expansion and accumulation in affected organs. Therefore, KIT D816V represents a prime therapeutic target for SM. Here, we generated a panel of patient-specific KIT D816V induced pluripotent stem cells (iPSCs) from patients with aggressive SM and mast cell leukemia to develop a patient-specific SM disease model for mechanistic and drug-discovery studies. KIT D816V iPSCs differentiated into neoplastic hematopoietic progenitor cells and MCs with patient-specific phenotypic features, thereby reflecting the heterogeneity of the disease. CRISPR/Cas9n-engineered KIT D816V human embryonic stem cells (ESCs), when differentiated into hematopoietic cells, recapitulated the phenotype observed for KIT D816V iPSC hematopoiesis. KIT D816V causes constitutive activation of the KIT tyrosine kinase receptor, and we exploited our iPSCs and ESCs to investigate new tyrosine kinase inhibitors targeting KIT D816V. Our study identified nintedanib, a US Food and Drug Administration–approved angiokinase inhibitor that targets vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and fibroblast growth factor receptor, as a novel KIT D816V inhibitor. Nintedanib selectively reduced the viability of iPSC-derived KIT D816V hematopoietic progenitor cells and MCs in the nanomolar range. Nintedanib was also active on primary samples of KIT D816V SM patients. Molecular docking studies show that nintedanib binds to the adenosine triphosphate binding pocket of inactive KIT D816V. Our results suggest nintedanib as a new drug candidate for KIT D816V–targeted therapy of advanced SM.
Marcelo A. Szymanski Toledo; Malrun Gatz; Stephanie Sontag; Karoline Veronika Gleixner; Gregor Eisenwort; Kristina Feldberg; Ahmed Emad Ibrahim Hamouda; Frederick Kluge; Riccardo Guareschi; Giulia Rossetti; Antonio Salvatore Sechi; Olli M. J. Dufva; Satu M. Mustjoki; Angela Maurer; Herdit M. Schüler; Roman Goetzke; Till Braunschweig; Anne Kaiser; Jens Peter Panse; Mohamad Jawhar; Andreas Reiter; Frank Hilberg; Peter Ettmayer; Wolfgang Wagner; Steffen Koschmieder; Tim H. Brümmendorf; Peter Valent; Nicolas Chatain; Martin Zenke. Nintedanib targets KIT D816V neoplastic cells derived from induced pluripotent stem cells of systemic mastocytosis. Blood 2021, 137, 2070 -2084.
AMA StyleMarcelo A. Szymanski Toledo, Malrun Gatz, Stephanie Sontag, Karoline Veronika Gleixner, Gregor Eisenwort, Kristina Feldberg, Ahmed Emad Ibrahim Hamouda, Frederick Kluge, Riccardo Guareschi, Giulia Rossetti, Antonio Salvatore Sechi, Olli M. J. Dufva, Satu M. Mustjoki, Angela Maurer, Herdit M. Schüler, Roman Goetzke, Till Braunschweig, Anne Kaiser, Jens Peter Panse, Mohamad Jawhar, Andreas Reiter, Frank Hilberg, Peter Ettmayer, Wolfgang Wagner, Steffen Koschmieder, Tim H. Brümmendorf, Peter Valent, Nicolas Chatain, Martin Zenke. Nintedanib targets KIT D816V neoplastic cells derived from induced pluripotent stem cells of systemic mastocytosis. Blood. 2021; 137 (15):2070-2084.
Chicago/Turabian StyleMarcelo A. Szymanski Toledo; Malrun Gatz; Stephanie Sontag; Karoline Veronika Gleixner; Gregor Eisenwort; Kristina Feldberg; Ahmed Emad Ibrahim Hamouda; Frederick Kluge; Riccardo Guareschi; Giulia Rossetti; Antonio Salvatore Sechi; Olli M. J. Dufva; Satu M. Mustjoki; Angela Maurer; Herdit M. Schüler; Roman Goetzke; Till Braunschweig; Anne Kaiser; Jens Peter Panse; Mohamad Jawhar; Andreas Reiter; Frank Hilberg; Peter Ettmayer; Wolfgang Wagner; Steffen Koschmieder; Tim H. Brümmendorf; Peter Valent; Nicolas Chatain; Martin Zenke. 2021. "Nintedanib targets KIT D816V neoplastic cells derived from induced pluripotent stem cells of systemic mastocytosis." Blood 137, no. 15: 2070-2084.
Molecular simulations and molecular docking are widely used tools to investigate ligand/target interactions and in drug design. High‐performance computing (HPC) is boosting both the accuracy and predictive power of these approaches. With the advent of exascale computing, HPC may become standardly applied in many drug design campaigns and pharmacological applications. This review discusses how innovative HPC algorithms and hardware are being exploited in current simulations and docking codes, pointing also at some of the limitations of these approaches. The focus is on technical aspects which might not be all that familiar to the computational pharmacologist. This article is categorized under: Software > Molecular Modeling Software > Simulation Methods Structure and Mechanism > Computational Biochemistry and Biophysics
Viacheslav Bolnykh; Giulia Rossetti; Ursula Rothlisberger; Paolo Carloni. Expanding the boundaries of ligand–target modeling by exascale calculations. WIREs Computational Molecular Science 2021, e1535 .
AMA StyleViacheslav Bolnykh, Giulia Rossetti, Ursula Rothlisberger, Paolo Carloni. Expanding the boundaries of ligand–target modeling by exascale calculations. WIREs Computational Molecular Science. 2021; ():e1535.
Chicago/Turabian StyleViacheslav Bolnykh; Giulia Rossetti; Ursula Rothlisberger; Paolo Carloni. 2021. "Expanding the boundaries of ligand–target modeling by exascale calculations." WIREs Computational Molecular Science , no. : e1535.
The SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of ∼30 000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing ∼200 virtual screenings of compound libraries on selected protein structures, we redefine the protein’s druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites.
Jonas Gossen; Simone Albani; Anton Hanke; Benjamin P. Joseph; Cathrine Bergh; Maria Kuzikov; Elisa Costanzi; Candida Manelfi; Paola Storici; Philip Gribbon; Andrea R. Beccari; Carmine Talarico; Francesca Spyrakis; Erik Lindahl; Andrea Zaliani; Paolo Carloni; Rebecca C. Wade; Francesco Musiani; Daria B. Kokh; Giulia Rossetti. A Blueprint for High Affinity SARS-CoV-2 Mpro Inhibitors from Activity-Based Compound Library Screening Guided by Analysis of Protein Dynamics. ACS Pharmacology & Translational Science 2021, 4, 1079 -1095.
AMA StyleJonas Gossen, Simone Albani, Anton Hanke, Benjamin P. Joseph, Cathrine Bergh, Maria Kuzikov, Elisa Costanzi, Candida Manelfi, Paola Storici, Philip Gribbon, Andrea R. Beccari, Carmine Talarico, Francesca Spyrakis, Erik Lindahl, Andrea Zaliani, Paolo Carloni, Rebecca C. Wade, Francesco Musiani, Daria B. Kokh, Giulia Rossetti. A Blueprint for High Affinity SARS-CoV-2 Mpro Inhibitors from Activity-Based Compound Library Screening Guided by Analysis of Protein Dynamics. ACS Pharmacology & Translational Science. 2021; 4 (3):1079-1095.
Chicago/Turabian StyleJonas Gossen; Simone Albani; Anton Hanke; Benjamin P. Joseph; Cathrine Bergh; Maria Kuzikov; Elisa Costanzi; Candida Manelfi; Paola Storici; Philip Gribbon; Andrea R. Beccari; Carmine Talarico; Francesca Spyrakis; Erik Lindahl; Andrea Zaliani; Paolo Carloni; Rebecca C. Wade; Francesco Musiani; Daria B. Kokh; Giulia Rossetti. 2021. "A Blueprint for High Affinity SARS-CoV-2 Mpro Inhibitors from Activity-Based Compound Library Screening Guided by Analysis of Protein Dynamics." ACS Pharmacology & Translational Science 4, no. 3: 1079-1095.
Flavonoids are plant bioactives that are recognized as hormone-like polyphenols because of their similarity to the endogenous sex steroids 17β-estradiol and testosterone, and to their estrogen- and androgen-like activity. Most efforts to verify flavonoid binding to nuclear receptors (NRs) and explain their action have been focused on ERα, while less attention has been paid to other nuclear and non-nuclear membrane androgen and estrogen receptors. Here, we investigate six flavonoids (apigenin, genistein, luteolin, naringenin, quercetin, and resveratrol) that are widely present in fruits and vegetables, and often used as replacement therapy in menopause. We performed comparative computational docking simulations to predict their capability of binding nuclear receptors ERα, ERβ, ERRβ, ERRγ, androgen receptor (AR), and its variant ART877A and membrane receptors for androgens, i.e., ZIP9, GPRC6A, OXER1, TRPM8, and estrogens, i.e., G Protein-Coupled Estrogen Receptor (GPER). In agreement with data reported in literature, our results suggest that these flavonoids show a relevant degree of complementarity with both estrogen and androgen NR binding sites, likely triggering genomic-mediated effects. It is noteworthy that reliable protein–ligand complexes and estimated interaction energies were also obtained for some suggested estrogen and androgen membrane receptors, indicating that flavonoids could also exert non-genomic actions. Further investigations are needed to clarify flavonoid multiple genomic and non-genomic effects. Caution in their administration could be necessary, until the safe assumption of these natural molecules that are largely present in food is assured.
Giulia D’Arrigo; Eleonora Gianquinto; Giulia Rossetti; Gabriele Cruciani; Stefano Lorenzetti; Francesca Spyrakis. Binding of Androgen- and Estrogen-Like Flavonoids to Their Cognate (Non)Nuclear Receptors: A Comparison by Computational Prediction. Molecules 2021, 26, 1613 .
AMA StyleGiulia D’Arrigo, Eleonora Gianquinto, Giulia Rossetti, Gabriele Cruciani, Stefano Lorenzetti, Francesca Spyrakis. Binding of Androgen- and Estrogen-Like Flavonoids to Their Cognate (Non)Nuclear Receptors: A Comparison by Computational Prediction. Molecules. 2021; 26 (6):1613.
Chicago/Turabian StyleGiulia D’Arrigo; Eleonora Gianquinto; Giulia Rossetti; Gabriele Cruciani; Stefano Lorenzetti; Francesca Spyrakis. 2021. "Binding of Androgen- and Estrogen-Like Flavonoids to Their Cognate (Non)Nuclear Receptors: A Comparison by Computational Prediction." Molecules 26, no. 6: 1613.
Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro and have identified 62 additional compounds with IC50 values below 1 μM and profiled their selectivity toward chymotrypsin and 3CL-Pro from the Middle East respiratory syndrome virus. A subset of eight inhibitors showed anticytopathic effect in a Vero-E6 cell line, and the compounds thioguanosine and MG-132 were analyzed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Å, showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity.
Maria Kuzikov; Elisa Costanzi; Jeanette Reinshagen; Francesca Esposito; Laura Vangeel; Markus Wolf; Bernhard Ellinger; Carsten Claussen; Gerd Geisslinger; Angela Corona; Daniela Iaconis; Carmine Talarico; Candida Manelfi; Rolando Cannalire; Giulia Rossetti; Jonas Gossen; Simone Albani; Francesco Musiani; Katja Herzog; Yang Ye; Barbara Giabbai; Nicola Demitri; Dirk Jochmans; Steven De Jonghe; Jasper Rymenants; Vincenzo Summa; Enzo Tramontano; Andrea R. Beccari; Pieter Leyssen; Paola Storici; Johan Neyts; Philip Gribbon; Andrea Zaliani. Identification of Inhibitors of SARS-CoV-2 3CL-Pro Enzymatic Activity Using a Small Molecule in Vitro Repurposing Screen. ACS Pharmacology & Translational Science 2021, 4, 1096 -1110.
AMA StyleMaria Kuzikov, Elisa Costanzi, Jeanette Reinshagen, Francesca Esposito, Laura Vangeel, Markus Wolf, Bernhard Ellinger, Carsten Claussen, Gerd Geisslinger, Angela Corona, Daniela Iaconis, Carmine Talarico, Candida Manelfi, Rolando Cannalire, Giulia Rossetti, Jonas Gossen, Simone Albani, Francesco Musiani, Katja Herzog, Yang Ye, Barbara Giabbai, Nicola Demitri, Dirk Jochmans, Steven De Jonghe, Jasper Rymenants, Vincenzo Summa, Enzo Tramontano, Andrea R. Beccari, Pieter Leyssen, Paola Storici, Johan Neyts, Philip Gribbon, Andrea Zaliani. Identification of Inhibitors of SARS-CoV-2 3CL-Pro Enzymatic Activity Using a Small Molecule in Vitro Repurposing Screen. ACS Pharmacology & Translational Science. 2021; 4 (3):1096-1110.
Chicago/Turabian StyleMaria Kuzikov; Elisa Costanzi; Jeanette Reinshagen; Francesca Esposito; Laura Vangeel; Markus Wolf; Bernhard Ellinger; Carsten Claussen; Gerd Geisslinger; Angela Corona; Daniela Iaconis; Carmine Talarico; Candida Manelfi; Rolando Cannalire; Giulia Rossetti; Jonas Gossen; Simone Albani; Francesco Musiani; Katja Herzog; Yang Ye; Barbara Giabbai; Nicola Demitri; Dirk Jochmans; Steven De Jonghe; Jasper Rymenants; Vincenzo Summa; Enzo Tramontano; Andrea R. Beccari; Pieter Leyssen; Paola Storici; Johan Neyts; Philip Gribbon; Andrea Zaliani. 2021. "Identification of Inhibitors of SARS-CoV-2 3CL-Pro Enzymatic Activity Using a Small Molecule in Vitro Repurposing Screen." ACS Pharmacology & Translational Science 4, no. 3: 1096-1110.
The translocator protein (TSPO) is a 18kDa transmembrane protein, ubiquitously present in human mitochondria. It is overexpressed in tumor cells and at the sites of neuroinflammation, thus representing an important biomarker, as well as a promising drug target. In mammalian TSPO, there are cholesterol–binding motifs, as well as a binding cavity able to accommodate different chemical compounds. Given the lack of structural information for the human protein, we built a model of human (h) TSPO in the apo state and in complex with PK11195, a molecule routinely used in positron emission tomography (PET) for imaging of neuroinflammatory sites. To better understand the interactions of PK11195 and cholesterol with this pharmacologically relevant protein, we ran molecular dynamics simulations of the apo and holo proteins embedded in a model membrane. We found that: (i) PK11195 stabilizes hTSPO structural fold; (ii) PK11195 might enter in the binding site through transmembrane helices I and II of hTSPO; (iii) PK11195 reduces the frequency of cholesterol binding to the lower, N–terminal part of hTSPO in the inner membrane leaflet, while this impact is less pronounced for the upper, C–terminal part in the outer membrane leaflet, where the ligand binding site is located; (iv) very interestingly, cholesterol most frequently binds simultaneously to the so-called CRAC and CARC regions in TM V in the free form (residues L150–X–Y152–X(3)–R156 and R135–X(2)–Y138–X(2)–L141, respectively). However, when the protein is in complex with PK11195, cholesterol binds equally frequently to the CRAC–resembling motif that we observed in TM I (residues L17–X(2)–F20–X(3)–R24) and to CRAC in TM V. We expect that the CRAC–like motif in TM I will be of interest in future experimental investigations. Thus, our MD simulations provide insight into the structural features of hTSPO and the previously unknown interplay between PK11195 and cholesterol interactions with this pharmacologically relevant protein.
Hien Lai; Alejandro Giorgetti; Giulia Rossetti; Toan Nguyen; Paolo Carloni; Agata Kranjc. The Interplay of Cholesterol and Ligand Binding in hTSPO from Classical Molecular Dynamics Simulations. Molecules 2021, 26, 1250 .
AMA StyleHien Lai, Alejandro Giorgetti, Giulia Rossetti, Toan Nguyen, Paolo Carloni, Agata Kranjc. The Interplay of Cholesterol and Ligand Binding in hTSPO from Classical Molecular Dynamics Simulations. Molecules. 2021; 26 (5):1250.
Chicago/Turabian StyleHien Lai; Alejandro Giorgetti; Giulia Rossetti; Toan Nguyen; Paolo Carloni; Agata Kranjc. 2021. "The Interplay of Cholesterol and Ligand Binding in hTSPO from Classical Molecular Dynamics Simulations." Molecules 26, no. 5: 1250.
The 3CL-Protease appears to be a very promising medicinal target to develop anti-SARS-CoV-2 agents. The availability of resolved structures allows structure-based computational approaches to be carried out even though the lack of known inhibitors prevents a proper validation of the performed simulations. The innovative idea of the study is to exploit known inhibitors of SARS-CoV 3CL-Pro as a training set to perform and validate multiple virtual screening campaigns. Docking simulations using four different programs (Fred, Glide, LiGen, and PLANTS) were performed investigating the role of both multiple binding modes (by binding space) and multiple isomers/states (by developing the corresponding isomeric space). The computed docking scores were used to develop consensus models, which allow an in-depth comparison of the resulting performances. On average, the reached performances revealed the different sensitivity to isomeric differences and multiple binding modes between the four docking engines. In detail, Glide and LiGen are the tools that best benefit from isomeric and binding space, respectively, while Fred is the most insensitive program. The obtained results emphasize the fruitful role of combining various docking tools to optimize the predictive performances. Taken together, the performed simulations allowed the rational development of highly performing virtual screening workflows, which could be further optimized by considering different 3CL-Pro structures and, more importantly, by including true SARS-CoV-2 3CL-Pro inhibitors (as learning set) when available.
Candida Manelfi; Jonas Gossen; Silvia Gervasoni; Carmine Talarico; Simone Albani; Benjamin Philipp; Francesco Musiani; Giulio Vistoli; Giulia Rossetti; Andrea Beccari; Alessandro Pedretti. Combining Different Docking Engines and Consensus Strategies to Design and Validate Optimized Virtual Screening Protocols for the SARS-CoV-2 3CL Protease. Molecules 2021, 26, 797 .
AMA StyleCandida Manelfi, Jonas Gossen, Silvia Gervasoni, Carmine Talarico, Simone Albani, Benjamin Philipp, Francesco Musiani, Giulio Vistoli, Giulia Rossetti, Andrea Beccari, Alessandro Pedretti. Combining Different Docking Engines and Consensus Strategies to Design and Validate Optimized Virtual Screening Protocols for the SARS-CoV-2 3CL Protease. Molecules. 2021; 26 (4):797.
Chicago/Turabian StyleCandida Manelfi; Jonas Gossen; Silvia Gervasoni; Carmine Talarico; Simone Albani; Benjamin Philipp; Francesco Musiani; Giulio Vistoli; Giulia Rossetti; Andrea Beccari; Alessandro Pedretti. 2021. "Combining Different Docking Engines and Consensus Strategies to Design and Validate Optimized Virtual Screening Protocols for the SARS-CoV-2 3CL Protease." Molecules 26, no. 4: 797.
Compound repurposing is an important strategy for the identification of effective treatment options against SARS-CoV-2 infection and COVID-19 disease. In this regard, SARS-CoV-2 main protease (3CL-Pro), also termed M-Pro, is an attractive drug target as it plays a central role in viral replication by processing the viral polyproteins pp1a and pp1ab at multiple distinct cleavage sites. We here report the results of a repurposing program involving 8.7 K compounds containing marketed drugs, clinical and preclinical candidates, and small molecules regarded as safe in humans. We confirmed previously reported inhibitors of 3CL-Pro, and have identified 62 additional compounds with IC50 values below 1 μM and profiled their selectivity towards Chymotrypsin and 3CL-Pro from the MERS virus. A subset of 8 inhibitors showed anti-cytopathic effect in a Vero-E6 cell line and the compounds thioguanosine and MG-132 were analysed for their predicted binding characteristics to SARS-CoV-2 3CL-Pro. The X-ray crystal structure of the complex of myricetin and SARS-Cov-2 3CL-Pro was solved at a resolution of 1.77 Å, showing that myricetin is covalently bound to the catalytic Cys145 and therefore inhibiting its enzymatic activity. Graphical abstract Abstract Figure. Workflow for identification and profiling of inhibitors of SARS-CoV-2 3CL-Pro using a large scale repurposing and bioactive compound collection (rhs). Primary assay principle based on quenched FRET peptide substrate of SARS-CoV-2 3CL-Pro (lhs). Inhibiting compounds reduce fluorescence signal relative to DMSO controls. Hit profiling using X-ray.
Maria Kuzikov; Elisa Costanzi; Jeanette Reinshagen; Francesca Esposito; Laura Vangeel; Markus Wolf; Bernhard Ellinger; Carsten Claussen; Gerd Geisslinger; Angela Corona; Daniela Iaconis; Carmine Talarico; Candida Manelfi; Rolando Cannalire; Giulia Rossetti; Jonas Gossen; Simone Albani; Francesco Musiani; Katja Herzog; Yang Ye; Barbara Giabbai; Nicola Demitri; Dirk Jochmans; Steven De Jonghe; Jasper Rymenants; Vincenzo Summa; Enzo Tramontano; Andrea Rosario Beccari; Pieter Leyssen; Paola Storici; Johan Neyts; Philip Gribbon; Andrea Zaliani. Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen. 2020, 1 .
AMA StyleMaria Kuzikov, Elisa Costanzi, Jeanette Reinshagen, Francesca Esposito, Laura Vangeel, Markus Wolf, Bernhard Ellinger, Carsten Claussen, Gerd Geisslinger, Angela Corona, Daniela Iaconis, Carmine Talarico, Candida Manelfi, Rolando Cannalire, Giulia Rossetti, Jonas Gossen, Simone Albani, Francesco Musiani, Katja Herzog, Yang Ye, Barbara Giabbai, Nicola Demitri, Dirk Jochmans, Steven De Jonghe, Jasper Rymenants, Vincenzo Summa, Enzo Tramontano, Andrea Rosario Beccari, Pieter Leyssen, Paola Storici, Johan Neyts, Philip Gribbon, Andrea Zaliani. Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen. . 2020; ():1.
Chicago/Turabian StyleMaria Kuzikov; Elisa Costanzi; Jeanette Reinshagen; Francesca Esposito; Laura Vangeel; Markus Wolf; Bernhard Ellinger; Carsten Claussen; Gerd Geisslinger; Angela Corona; Daniela Iaconis; Carmine Talarico; Candida Manelfi; Rolando Cannalire; Giulia Rossetti; Jonas Gossen; Simone Albani; Francesco Musiani; Katja Herzog; Yang Ye; Barbara Giabbai; Nicola Demitri; Dirk Jochmans; Steven De Jonghe; Jasper Rymenants; Vincenzo Summa; Enzo Tramontano; Andrea Rosario Beccari; Pieter Leyssen; Paola Storici; Johan Neyts; Philip Gribbon; Andrea Zaliani. 2020. "Identification of inhibitors of SARS-CoV-2 3CL-Pro enzymatic activity using a small molecule in-vitro repurposing screen." , no. : 1.
Advances in coarse-grained molecular dynamics (CGMD) simulations have extended the use of computational studies on biological macromolecules and their complexes, as well as the interactions of membrane protein and lipid complexes at a reduced level of representation, allowing longer and larger molecular dynamics simulations. Here, we present a computational platform dedicated to the preparation, running, and analysis of CGMD simulations. The platform is built on a completely revisited version of our Martini coarsE gRained MembrAne proteIn Dynamics (MERMAID) web server, and it integrates this with other three dedicated services. In its current version, the platform expands the existing implementation of the Martini force field for membrane proteins to also allow the simulation of soluble proteins using the Martini and the SIRAH force fields. Moreover, it offers an automated protocol for carrying out the backmapping of the coarse-grained description of the system into an atomistic one.
Alessandro Marchetto; Zeineb Chaib; Carlo Rossi; Rui Ribeiro; Sergio Pantano; Giulia Rossetti; Alejandro Giorgetti. CGMD Platform: Integrated Web Servers for the Preparation, Running, and Analysis of Coarse-Grained Molecular Dynamics Simulations. Molecules 2020, 25, 5934 .
AMA StyleAlessandro Marchetto, Zeineb Chaib, Carlo Rossi, Rui Ribeiro, Sergio Pantano, Giulia Rossetti, Alejandro Giorgetti. CGMD Platform: Integrated Web Servers for the Preparation, Running, and Analysis of Coarse-Grained Molecular Dynamics Simulations. Molecules. 2020; 25 (24):5934.
Chicago/Turabian StyleAlessandro Marchetto; Zeineb Chaib; Carlo Rossi; Rui Ribeiro; Sergio Pantano; Giulia Rossetti; Alejandro Giorgetti. 2020. "CGMD Platform: Integrated Web Servers for the Preparation, Running, and Analysis of Coarse-Grained Molecular Dynamics Simulations." Molecules 25, no. 24: 5934.
The SARS-CoV-2 coronavirus outbreak continues to spread at a rapid rate worldwide. The main protease (Mpro) is an attractive target for anti-COVID-19 agents. Unfortunately, unexpected difficulties have been encountered in the design of specific inhibitors. Here, by analyzing an ensemble of ~30,000 SARS-CoV-2 Mpro conformations from crystallographic studies and molecular simulations, we show that small structural variations in the binding site dramatically impact ligand binding properties. Hence, traditional druggability indices fail to adequately discriminate between highly and poorly druggable conformations of the binding site. By performing ~200 virtual screenings of compound libraries on selected protein structures, we redefine the protein’s druggability as the consensus chemical space arising from the multiple conformations of the binding site formed upon ligand binding. This procedure revealed a unique SARS-CoV-2 Mpro blueprint that led to a definition of a specific structure-based pharmacophore. The latter explains the poor transferability of potent SARS-CoV Mpro inhibitors to SARS-CoV-2 Mpro, despite the identical sequences of the active sites. Importantly, application of the pharmacophore predicted novel high affinity inhibitors of SARS-CoV-2 Mpro, that were validated by in vitro assays performed here and by a newly solved X-ray crystal structure. These results provide a strong basis for effective rational drug design campaigns against SARS-CoV-2 Mpro and a new computational approach to screen protein targets with malleable binding sites.
Jonas Gossen; Simone Albani; Anton Hanke; Benjamin P. Joseph; Cathrine Bergh; Maria Kuzikov; Elisa Costanzi; Candida Manelfi; Paola Storici; Philip Gribbon; Andrea R. Beccari; Carmine Talarico; Francesca Spyrakis; Erik Lindahl; Andrea Zaliani; Paolo Carloni; Rebecca C. Wade; Francesco Musiani; Daria B. Kokh; Giulia Rossetti. A blueprint for high affinity SARS-CoV-2 Mpro inhibitors from activity-based compound library screening guided by analysis of protein dynamics. 2020, 1 .
AMA StyleJonas Gossen, Simone Albani, Anton Hanke, Benjamin P. Joseph, Cathrine Bergh, Maria Kuzikov, Elisa Costanzi, Candida Manelfi, Paola Storici, Philip Gribbon, Andrea R. Beccari, Carmine Talarico, Francesca Spyrakis, Erik Lindahl, Andrea Zaliani, Paolo Carloni, Rebecca C. Wade, Francesco Musiani, Daria B. Kokh, Giulia Rossetti. A blueprint for high affinity SARS-CoV-2 Mpro inhibitors from activity-based compound library screening guided by analysis of protein dynamics. . 2020; ():1.
Chicago/Turabian StyleJonas Gossen; Simone Albani; Anton Hanke; Benjamin P. Joseph; Cathrine Bergh; Maria Kuzikov; Elisa Costanzi; Candida Manelfi; Paola Storici; Philip Gribbon; Andrea R. Beccari; Carmine Talarico; Francesca Spyrakis; Erik Lindahl; Andrea Zaliani; Paolo Carloni; Rebecca C. Wade; Francesco Musiani; Daria B. Kokh; Giulia Rossetti. 2020. "A blueprint for high affinity SARS-CoV-2 Mpro inhibitors from activity-based compound library screening guided by analysis of protein dynamics." , no. : 1.
The translocator protein (TSPO) is a transmembrane protein present across the three domains of life. Its functional quaternary structure consists of one or more subunits. In mice, the dimer-to-monomer equilibrium is shifted in vitro towards the monomer by adding cholesterol, a natural component of mammalian membranes. Here, we present a coarse-grained molecular dynamics study on the mouse protein in the presence of a physiological content and of an excess of cholesterol. The latter turns out to weaken the interfaces of the dimer by clusterizing mostly at the inter-monomeric space and pushing the contact residues apart. It also increases the compactness and the rigidity of the monomer. These two factors might play a role for the experimentally observed incremented stability of the monomeric form with increased content of cholesterol. Comparison with simulations on bacterial proteins suggests that the effect of cholesterol is much less pronounced for the latter than for the mouse protein.
Zeineb Si Chaib; Alessandro Marchetto; Klevia Dishnica; Paolo Carloni; Alejandro Giorgetti; Giulia Rossetti. Impact of Cholesterol on the Stability of Monomeric and Dimeric Forms of the Translocator Protein TSPO: A Molecular Simulation Study. Molecules 2020, 25, 4299 .
AMA StyleZeineb Si Chaib, Alessandro Marchetto, Klevia Dishnica, Paolo Carloni, Alejandro Giorgetti, Giulia Rossetti. Impact of Cholesterol on the Stability of Monomeric and Dimeric Forms of the Translocator Protein TSPO: A Molecular Simulation Study. Molecules. 2020; 25 (18):4299.
Chicago/Turabian StyleZeineb Si Chaib; Alessandro Marchetto; Klevia Dishnica; Paolo Carloni; Alejandro Giorgetti; Giulia Rossetti. 2020. "Impact of Cholesterol on the Stability of Monomeric and Dimeric Forms of the Translocator Protein TSPO: A Molecular Simulation Study." Molecules 25, no. 18: 4299.
The translocator protein (TSPO) is a transmembrane protein present in the three domains of life. Its functional quaternary structure consists of one or more subunits. In mouse, the dimer-to-monomer equilibrium is shifted in vitro towards the monomer by adding cholesterol, a natural component of mammalian membranes. Here, we present a coarse-grained molecular dynamics study on the mouse protein in the presence of a physiological content and of an excess of cholesterol. The latter turns out to weaken the interfaces of the dimer by clusterizing mostly at the inter-monomeric space and pushing the contact residues apart. It also increases the compactness and the rigidity of the monomer. These two factors might play a role for the experimentally observed incremented stability of the monomeric form with increased content of cholesterol. Comparison with simulations on bacterial proteins suggests that the effect of cholesterol is much less pronounced for the latter than for the mouse protein.
Zeineb Si Chaib; Alessandro Marchetto; Klevia Dishnica; Paolo Carloni; Alejandro Giorgetti; Giulia Rossetti. Impact of Cholesterol on the Stability of Monomeric and Dimeric Forms of the Translocator Protein TSPO: a Molecular Simulation Study. 2020, 1 .
AMA StyleZeineb Si Chaib, Alessandro Marchetto, Klevia Dishnica, Paolo Carloni, Alejandro Giorgetti, Giulia Rossetti. Impact of Cholesterol on the Stability of Monomeric and Dimeric Forms of the Translocator Protein TSPO: a Molecular Simulation Study. . 2020; ():1.
Chicago/Turabian StyleZeineb Si Chaib; Alessandro Marchetto; Klevia Dishnica; Paolo Carloni; Alejandro Giorgetti; Giulia Rossetti. 2020. "Impact of Cholesterol on the Stability of Monomeric and Dimeric Forms of the Translocator Protein TSPO: a Molecular Simulation Study." , no. : 1.
Recent studies suggest that Tyr-39 might play a critical role for both the normal function and the pathological dysfunction of α-synuclein (αS), an intrinsically disordered protein involved in Parkinson’s disease. We perform here a comparative analysis between the structural features of human αS and its Y39A, Y39F, and Y39L variants. By the combined application of site-directed mutagenesis, biophysical techniques, and enhanced sampling molecular simulations, we show that removing aromatic functionality at position 39 of monomeric αS leads to protein variants populating more compact conformations, conserving its disordered nature and secondary structure propensities. Contrasting with the subtle changes induced by mutations on the protein structure, removing aromaticity at position 39 impacts strongly on the interaction of αS with the potent amyloid inhibitor phthalocyanine tetrasulfonate (PcTS). Our findings further support the role of Tyr-39 in forming essential inter and intramolecular contacts that might have important repercussions for the function and the dysfunction of αS.
Oscar Palomino-Hernandez; Fiamma A. Buratti; Pamela S. Sacco; Giulia Rossetti; Paolo Carloni; Claudio O. Fernandez. Role of Tyr-39 for the Structural Features of α-Synuclein and for the Interaction with a Strong Modulator of Its Amyloid Assembly. International Journal of Molecular Sciences 2020, 21, 5061 .
AMA StyleOscar Palomino-Hernandez, Fiamma A. Buratti, Pamela S. Sacco, Giulia Rossetti, Paolo Carloni, Claudio O. Fernandez. Role of Tyr-39 for the Structural Features of α-Synuclein and for the Interaction with a Strong Modulator of Its Amyloid Assembly. International Journal of Molecular Sciences. 2020; 21 (14):5061.
Chicago/Turabian StyleOscar Palomino-Hernandez; Fiamma A. Buratti; Pamela S. Sacco; Giulia Rossetti; Paolo Carloni; Claudio O. Fernandez. 2020. "Role of Tyr-39 for the Structural Features of α-Synuclein and for the Interaction with a Strong Modulator of Its Amyloid Assembly." International Journal of Molecular Sciences 21, no. 14: 5061.
Novel therapeutic approaches are being developed to tackle neurodegenerative diseases, due to the lack of efficiency of the known druggable targets. For Huntington's disease, a promising approach is the regulation of the RNA product. This target would allow for a selective and effective inhibition of the toxic effects exerted by the final nucleic product and the coded protein. In this review, the current state of the art of RNA regulation is discussed, with a brief but insightful view on novel plausible targets. After this, an emphasis on successful computational and experimental approaches tailored in modeling and regulating RNA aberrant behavior are extensively presented. Finally, the application and limitations of current computational methods are discussed, and possible avenues for improvement are outlined.
Oscar Palomino‐Hernandez; Michael A. Margreiter; Jun.‐Prof. Dr. Dr. Giulia Rossetti. Challenges in RNA Regulation in Huntington's Disease: Insights from Computational Studies. Israel Journal of Chemistry 2020, 60, 681 -693.
AMA StyleOscar Palomino‐Hernandez, Michael A. Margreiter, Jun.‐Prof. Dr. Dr. Giulia Rossetti. Challenges in RNA Regulation in Huntington's Disease: Insights from Computational Studies. Israel Journal of Chemistry. 2020; 60 (7):681-693.
Chicago/Turabian StyleOscar Palomino‐Hernandez; Michael A. Margreiter; Jun.‐Prof. Dr. Dr. Giulia Rossetti. 2020. "Challenges in RNA Regulation in Huntington's Disease: Insights from Computational Studies." Israel Journal of Chemistry 60, no. 7: 681-693.