Interrelationship between miRNA and splicing factors in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers because of diagnosis at late stage and inherent/acquired chemoresistance. Recent advances in genomic profiling and biology of this disease have not yet been translated to a relevant improvement in terms of disease management and patient’s survival. However, new possibilities for treatment may emerge from studies on key epigenetic factors. Deregulation of microRNA (miRNA) dependent gene expression and mRNA splicing are epigenetic processes that modulate the protein repertoire at the transcriptional level. These processes affect all aspects of PDAC pathogenesis and have great potential to unravel new therapeutic targets and/or biomarkers. Remarkably, several studies showed that they actually interact with each other in influencing PDAC progression. Some splicing factors directly interact with specific miRNAs and either facilitate or inhibit their expression, such as Rbfox2, which cleaves the well-known oncogenic miRNA miR-21. Conversely, miR-15a-5p and miR-25-3p significantly downregulate the splicing factor hnRNPA1 which acts also as a tumour suppressor gene and is involved in processing of miR-18a, which in turn, is a negative regulator of KRAS expression. Therefore, this review describes the interaction between splicing and miRNA, as well as bioinformatic tools to explore the effect of splicing modulation towards miRNA profiles, in order to exploit this interplay for the development of innovative treatments. Targeting aberrant splicing and deregulated miRNA, alone or in combination, may hopefully provide novel therapeutic approaches to fight the complex biology and the common treatment recalcitrance of PDAC

Progettazione e sintesi di nuovi derivati 4-chinazolinonici potenziali inibitori della diidrofolato reduttasi

I chinazolinoni sono composti eterociclici azotati che, insieme alle chinazoline, rappresentano degli importanti farmacofori in possesso di un ampio spettro di proprietà biologiche tra cui quella antitumorale. Recentemente sono stati riportati in letteratura dei derivati 4-chinazolinonici in grado di inibire in vitro l’enzima diidrofolato reduttasi (DHFR) con IC50 comprese tra 0.4 e 1.0 µM [1]. Allo scopo di progettare la sintesi di nuovi potenziali inibitori della DHFR, è stato condotto uno studio di modellistica molecolare considerando tale enzima come biotarget. Tale studio ha portato alla selezione di 42 nuovi derivati 4-chinazolinonici (Figura 1). Attualmente, sono stati sintetizzati 20 dei 42 nuovi derivati 4-chinazolinonici, che sono stati saggiati preliminarmente sulla linea cellulare K562. Il derivato più attivo ha mostrato una IC50 di 18 µM. Sono in corso saggi enzimatici per valutare in vitro l’inibizione dell’enzima DHFR. Bibliografia 1 Al-Omary F.A.M.; et al, Bioorganic & Medicinal Chemistry, 2010, 18, 2849

Sintesi di un isostero del 3,5-dimetil-6-fenil-8-(trifluorometil)-5,6-diidropirazolo[3,4-f][1,2,3,5]tetrazepin-4(3H)-one (CF3-TZP) con potenziale attività biologica

In un precedente lavoro abbiamo mostrato i risultati relativi alla sintesi ed all’attività biologica del CF3-TZP 1[1] (Figura 1). Le attività antiproliferativa e apoptotica del composto 1 sono state testate su differenti linee cellulari, HL60 sensibili, HL60-R (MDR), K562 e K562-R (resistenti al Gleevec®), mostrando un profilo di attività biologica similare sulle cellule sensibili e resistenti nel range di 21-40 µM per l’IC50 e 36-62 µM per l’AC50. L’analisi citofluorimetrica sulle K562 sensibili ha indicato che il composto 1 determina un arresto dose-dipendente del ciclo cellulare in fase G0-G1 nelle prime 24 h di trattamento, mentre nelle successive 24 h si è notato una riduzione del picco G0-G1 ed un incremento del picco apoptotico subG0-G1. Gli incoraggianti risultati biologici ci hanno spinto a continuare gli studi su questa tipologia di molecole sintetizzando l’isostero 2 (Figura 1) attraverso una lunga via di sintesi (15 steps). Attualmente, sono in corso i saggi biologici per valutare le attività antiproliferativa e apoptotica. Bibliografia 1 Maggio, B.; et al, Eur. J. Med. Chem., 2008, 43, 120

Pirrolomicine che inibiscono la Sortasi A nelle infezioni sostenute da batteri Gram-positivi

La Sortasi A è un enzima di membrana responsabile dell’ancoraggio delle proteine di superficie sulla parete cellulare dei batteri Gram-positivi. Essa è considerata un interessante obiettivo per lo sviluppo di nuovi farmaci anti-infettivi che mirino ad interferire con importanti meccanismi di virulenza Gram-positivi. In un precedente lavoro abbiamo indagato sull’attività antistafilococcica e antibiofilm di alcune Pirrolomicine naturali e sintetiche, composti pirrolici polialogenati attivi su patogeni Gram-positivi, alle concentrazioni di 1.5 e 0.045 µg/mL. I risultati biologici hanno mostrato percentuali di inibizione di biofilm comprese tra 50-80% [1]. Allo scopo di indagare sul loro meccanismo d’azione sono stati condotti studi di modellistica molecolare e saggi di inibizione in vitro sull’enzima Sortasi A (Figura 1). I risultati ottenuti indicano che la Sortasi A potrebbe essere il bersaglio sul quale le Pirrolomicine agiscono, con IC50 comprese tra 130-250 µM, nell’inibizione della formazione di biofilm. Bibliografia 1 Schillaci, D.; et al, Biofouling, 2010, 26, 433

Event reconstruction for KM3NeT/ORCA using convolutional neural networks

The KM3NeT research infrastructure is currently under construction at two locations in the Mediterranean Sea. The KM3NeT/ORCA water-Cherenkov neutrino de tector off the French coast will instrument several megatons of seawater with photosensors. Its main objective is the determination of the neutrino mass ordering. This work aims at demonstrating the general applicability of deep convolutional neural networks to neutrino telescopes, using simulated datasets for the KM3NeT/ORCA detector as an example. To this end, the networks are employed to achieve reconstruction and classification tasks that constitute an alternative to the analysis pipeline presented for KM3NeT/ORCA in the KM3NeT Letter of Intent. They are used to infer event reconstruction estimates for the energy, the direction, and the interaction point of incident neutrinos. The spatial distribution of Cherenkov light generated by charged particles induced in neutrino interactions is classified as shower-or track-like, and the main background processes associated with the detection of atmospheric neutrinos are recognized. Performance comparisons to machine-learning classification and maximum-likelihood reconstruction algorithms previously developed for KM3NeT/ORCA are provided. It is shown that this application of deep convolutional neural networks to simulated datasets for a large-volume neutrino telescope yields competitive reconstruction results and performance improvements with respect to classical approaches

Event reconstruction for KM3NeT/ORCA using convolutional neural networks

The KM3NeT research infrastructure is currently under construction at two locations in the Mediterranean Sea. The KM3NeT/ORCA water-Cherenkov neutrino detector off the French coast will instrument several megatons of seawater with photosensors. Its main objective is the determination of the neutrino mass ordering. This work aims at demonstrating the general applicability of deep convolutional neural networks to neutrino telescopes, using simulated datasets for the KM3NeT/ORCA detector as an example. To this end, the networks are employed to achieve reconstruction and classification tasks that constitute an alternative to the analysis pipeline presented for KM3NeT/ORCA in the KM3NeT Letter of Intent. They are used to infer event reconstruction estimates for the energy, the direction, and the interaction point of incident neutrinos. The spatial distribution of Cherenkov light generated by charged particles induced in neutrino interactions is classified as shower- or track-like, and the main background processes associated with the detection of atmospheric neutrinos are recognized. Performance comparisons to machine-learning classification and maximum-likelihood reconstruction algorithms previously developed for KM3NeT/ORCA are provided. It is shown that this application of deep convolutional neural networks to simulated datasets for a large-volume neutrino telescope yields competitive reconstruction results and performance improvements with respect to classical approaches

DESIGN OF SF3B1 SUBUNIT MODULATORS OF THE SF3B SPLICEOSOME COMPLEX

The subject of this dissertation is the search for new therapeutic strategies for pancreatic cancer and aims to implement a Drug Discovery process for the rational design and synthesis of molecules active in the modulation of pathways related to the regulation of pre-mRNA splicing process. This research project is the result of a joint PhD between the University of Palermo, Italy, and the Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands. It integrates complementary skills in pharmaceutical chemistry and translational cancer research with a special focus on the rational design of new anticancer compounds potentially active on SF3B1 (Splicing Factor 3B subunit 1), an effective therapeutic target involved in the pre-mRNA splicing process. Furthermore, SF3B1 is a frequently mutated gene in haematological malignancies and some solid tumors, including pancreatic cancer. Specifically, pancreatic ductal adenocarcinoma (PDAC) is the most common type of exocrine pancreatic cancer with a highly poor prognosis and an increasing incidence. The urgent need to find effective therapeutic strategies for the treatment of this disease prompted us to evaluate the antitumor activity of these compounds on preclinical models of PDAC. Based on the interesting anticancer properties described for the imidazothiadiazole nucleus as a scaffold for the development of pharmacologically active derivatives, we were encouraged to continue with the same approach. A preliminary computational study led us to the realization of a common pharmacophore that was used for the virtual screening of an in-house molecular library and commercially available molecular libraries, in order to identify the molecular scaffold of interest and then select the most promising molecules that were tested in vitro on PDAC cell lines. We also tested for the first time two splicing modulators targeting SF3B1, in order to identify new potential therapeutic targets for the treatment of PDAC. Furthermore, the status of the transmembrane protein hENT-1 (human Equilibrative Nucleoside Transporter-1) as a potential biomarker in PDAC is explored in detail in the present Thesis. In conclusion, the need to find new diagnostic biomarkers and the interesting antiproliferative activity previously shown by imidazothiadiazole compounds prompted us to extend the evaluation of the biological activity in vitro concerning a new class of imidazothiadiazole compounds

Design of SF3B1 subunit modulators of the SF3B spliceosome complex

The subject of this dissertation is the search for new therapeutic strategies for pancreatic cancer and aims to implement a Drug Discovery process for the rational design and synthesis of molecules active in the modulation of pathways related to the regulation of pre-mRNA splicing process. This research project is the result of a joint PhD between the University of Palermo, Italy, and the Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands. It integrates complementary skills in pharmaceutical chemistry and translational cancer research with a special focus on the rational design of new anticancer compounds potentially active on SF3B1 (Splicing Factor 3B subunit 1), an effective therapeutic target involved in the pre-mRNA splicing process. Furthermore, SF3B1 is a frequently mutated gene in haematological malignancies and some solid tumors, including pancreatic cancer. Specifically, pancreatic ductal adenocarcinoma (PDAC) is the most common type of exocrine pancreatic cancer with a highly poor prognosis and an increasing incidence. The urgent need to find effective therapeutic strategies for the treatment of this disease prompted us to evaluate the antitumor activity of these compounds on preclinical models of PDAC. Based on the interesting anticancer properties described for the imidazothiadiazole nucleus as a scaffold for the development of pharmacologically active derivatives, we were encouraged to continue with the same approach. A preliminary computational study led us to the realization of a common pharmacophore that was used for the virtual screening of an in-house molecular library and commercially available molecular libraries, in order to identify the molecular scaffold of interest and then select the most promising molecules that were tested in vitro on PDAC cell lines. We also tested for the first time two splicing modulators targeting SF3B1, in order to identify new potential therapeutic targets for the treatment of PDAC. Furthermore, the status of the transmembrane protein hENT-1 (human Equilibrative Nucleoside Transporter-1) as a potential biomarker in PDAC is explored in detail in the present Thesis. In conclusion, the need to find new diagnostic biomarkers and the interesting antiproliferative activity previously shown by imidazothiadiazole compounds prompted us to extend the evaluation of the biological activity in vitro concerning a new class of imidazothiadiazole compounds

Design of SF3B1 subunit modulators of the SF3B spliceosome complex

The subject of this dissertation is the search for new therapeutic strategies for pancreatic cancer and aims to implement a Drug Discovery process for the rational design and synthesis of molecules active in the modulation of pathways related to the regulation of pre-mRNA splicing process. This research project is the result of a joint PhD between the University of Palermo, Italy, and the Department of Medical Oncology, VU University Medical Center, Amsterdam, The Netherlands. It integrates complementary skills in pharmaceutical chemistry and translational cancer research with a special focus on the rational design of new anticancer compounds potentially active on SF3B1 (Splicing Factor 3B subunit 1), an effective therapeutic target involved in the pre-mRNA splicing process. Furthermore, SF3B1 is a frequently mutated gene in haematological malignancies and some solid tumors, including pancreatic cancer. Specifically, pancreatic ductal adenocarcinoma (PDAC) is the most common type of exocrine pancreatic cancer with a highly poor prognosis and an increasing incidence. The urgent need to find effective therapeutic strategies for the treatment of this disease prompted us to evaluate the antitumor activity of these compounds on preclinical models of PDAC. Based on the interesting anticancer properties described for the imidazothiadiazole nucleus as a scaffold for the development of pharmacologically active derivatives, we were encouraged to continue with the same approach. A preliminary computational study led us to the realization of a common pharmacophore that was used for the virtual screening of an in-house molecular library and commercially available molecular libraries, in order to identify the molecular scaffold of interest and then select the most promising molecules that were tested in vitro on PDAC cell lines. We also tested for the first time two splicing modulators targeting SF3B1, in order to identify new potential therapeutic targets for the treatment of PDAC. Furthermore, the status of the transmembrane protein hENT-1 (human Equilibrative Nucleoside Transporter-1) as a potential biomarker in PDAC is explored in detail in the present Thesis. In conclusion, the need to find new diagnostic biomarkers and the interesting antiproliferative activity previously shown by imidazothiadiazole compounds prompted us to extend the evaluation of the biological activity in vitro concerning a new class of imidazothiadiazole compounds