Cellular senescence can be defined as a specific cell state triggered by a variety of stimuli and physiological pathways, characterized by a prolonged and usually irreversible cell cycle arrest, transcriptional changes, acquisition of a bioactive secretome that impacts the nearby tissue, known as the senescence-associated secretory phenotype (SASP), macromolecular damage and deregulated metabolism. Senescence is one of the hallmarks of aging and aging related diseases, together with genomic instability, telomere attrition, epigenetic alteration, loss of proteostasis, mitochondrial dysfunction, stem cell exhaustion and altered intercellular communication. Senescent cells not only accumulate during aging but also amass at the pathological sites of various disorders and across numerous chronic diseases. Among these we can mention cancer, cardiovascular disease, fibrosis, diabetes and neurological disorders. Lately, diverse approaches have been attempted to avoid the formation of senescent cells and the inflammatory consequences of their accumulation. In particular, researchers have taken into examination and tested senomorphic molecules, aiming to prevent or decelerate the aging process and cellular senescence. Conversely, senolytics have recently emerged as agents capable of selectively eradicate senescent cells and induce apoptosis, thereby diminishing their presence in specific organs. Although this has the potential to promote cellular healthspan, several senolytics with different targets have been identified, but they often pose safety and specificity challenges. Our research aims to identify and characterize novel HSP90-inhibitors with senolytic characteristics. Heat shock protein 90 (Hsp90) is a molecular chaperone which protects polypeptides against numerous types of stress and is a protein under epigenetic control that plays a critical role in senescent cells survival. There are four Hsp90 homologues: cytosolic Hsp90α, Hsp90β, organ-specific Grp94 (glucose-regulated protein found in the endoplasmic reticulum), and TRAP1 (tumour necrosis factor (TNF) receptor-associated protein 1 located in the mitochondrial matrix). We used a combination of structure-based and ligand-based virtual screening approaches to identify new, optimized HSP90α- inhibitors with minimal cytotoxicity. Two of them, K4 and K5, exhibited senolytic activity in vitro without any observable cytotoxicity in non-senescent cells. Proliferation and mortality assays conducted on various tumour cell lines derived from hormone-driven cancers revealed that these drugs induce cytostatic effects rather than cytotoxic. Our findings suggest that K5 display senolytic properties and may be leveraged with tamoxifen to stimulate cell death. Indeed, the identification and characterization of these inhibitors could be useful in several applications, among them, hormone-driven cancers, to prevent tumour recurrence and even to catalyse tumour regression.
La senescenza cellulare può essere definita come uno specifico stato provocato da diversi stimoli e processi fisiologici, caratterizzati da un prolungato e solitamente irreversibile arresto del ciclo cellulare, cambiamenti trascrizionali, acquisizione di un secretoma bioattivo che coinvolge il tessuto circostante, noto come fenotipo secretorio associato alla senescenza (SASP), danni a livello di macromolecole e metabolismo deregolato. La senescenza è uno dei segni distintivi dell’invecchiamento e delle malattie associate all’invecchiamento, insieme all’instabilità genomica, l’accorciamento telomerico, alterazioni dell’epigenoma, perdita di proteostasi, alterazioni metaboliche, disfunzioni mitocondriali, etc. Queste cellule non solo vengono accumulate durante l’invecchiamento ma anche all’interno di siti patologici in seguito a vari disordini e numerose malattie croniche. Tra quest’ultime vi sono il cancro, malattie cardiovascolari, fibrosi, diabete e disordini neurologici. Recentemente, sono stati tentati diversi approcci per evitare sia lo sviluppo di cellule senescenti che le conseguenze infiammatorie legate al loro accumulo. In particolare sono state prese in esame e testate delle molecole definite senomorfiche, indirizzate a impedire o rallentare il processo di invecchiamento e senescenza cellulare. Al contrario, i cosiddetti senolitici sono emersi ultimamente come agenti in grado di eradicare selettivamente cellule senescenti e indurre apoptosi, diminuendo quindi la loro presenza in organi specifici. Nonostante questi abbiano il potenziale di promuovere uno stato di salute cellulare, sono stati identificati molti senolitici con target differenti con problematiche relative a sicurezza e specificità. La heat shock protein 90 (Hsp90) è uno chaperone molecolare, il quale protegge i polipeptidi da numerosi tipi di stress ed è una proteina sotto controllo epigenetico che gioca un ruolo critico nella sopravvivenza delle cellule senescenti. Esistono quattro omologhi di HSP90: Hsp90α citosolico, Hsp90β, Grp94 organo-specifico (reticolo endoplasmatico e TRAP1 (matrice mitocondriale). Abbiamo utilizzato una combinazione di approcci di screening virtuale basati su la struttura della proteina e dei suoi ligandi per identificare ed ottimizzare nuovi inibitori di HSP90α aventi citotossicità minima. Due di questi, K4 e K5, hanno mostrato un’attività senolitica in vitro con scarsa citotossicità in cellule non senescenti. Abbiamo condotto saggi di proliferazione e mortalità su varie linee cellulari tumorali derivanti da tumori ormono-dipendenti che hanno rivelato come questi composti inducano effetti citostatici piuttosto che citotossici. Le nostre scoperte suggeriscono che K5 mostri proprietà senolitiche e possa essere sfruttato in combinazione con il tamoxifene per stimolare la morte cellulare. Infatti, l’identificazione e la caratterizzazione di questi inibitori può essere utile in svariate applicazioni, tra le quali i tumori ormono-dipendenti, per prevenirne la ricorrenza e/o per favorirne la regressione.
Caratterizzazione di nuove small-molecules con proprietà senolitiche
CIGALA FULGOSI, PIETRO
2022/2023
Abstract
Cellular senescence can be defined as a specific cell state triggered by a variety of stimuli and physiological pathways, characterized by a prolonged and usually irreversible cell cycle arrest, transcriptional changes, acquisition of a bioactive secretome that impacts the nearby tissue, known as the senescence-associated secretory phenotype (SASP), macromolecular damage and deregulated metabolism. Senescence is one of the hallmarks of aging and aging related diseases, together with genomic instability, telomere attrition, epigenetic alteration, loss of proteostasis, mitochondrial dysfunction, stem cell exhaustion and altered intercellular communication. Senescent cells not only accumulate during aging but also amass at the pathological sites of various disorders and across numerous chronic diseases. Among these we can mention cancer, cardiovascular disease, fibrosis, diabetes and neurological disorders. Lately, diverse approaches have been attempted to avoid the formation of senescent cells and the inflammatory consequences of their accumulation. In particular, researchers have taken into examination and tested senomorphic molecules, aiming to prevent or decelerate the aging process and cellular senescence. Conversely, senolytics have recently emerged as agents capable of selectively eradicate senescent cells and induce apoptosis, thereby diminishing their presence in specific organs. Although this has the potential to promote cellular healthspan, several senolytics with different targets have been identified, but they often pose safety and specificity challenges. Our research aims to identify and characterize novel HSP90-inhibitors with senolytic characteristics. Heat shock protein 90 (Hsp90) is a molecular chaperone which protects polypeptides against numerous types of stress and is a protein under epigenetic control that plays a critical role in senescent cells survival. There are four Hsp90 homologues: cytosolic Hsp90α, Hsp90β, organ-specific Grp94 (glucose-regulated protein found in the endoplasmic reticulum), and TRAP1 (tumour necrosis factor (TNF) receptor-associated protein 1 located in the mitochondrial matrix). We used a combination of structure-based and ligand-based virtual screening approaches to identify new, optimized HSP90α- inhibitors with minimal cytotoxicity. Two of them, K4 and K5, exhibited senolytic activity in vitro without any observable cytotoxicity in non-senescent cells. Proliferation and mortality assays conducted on various tumour cell lines derived from hormone-driven cancers revealed that these drugs induce cytostatic effects rather than cytotoxic. Our findings suggest that K5 display senolytic properties and may be leveraged with tamoxifen to stimulate cell death. Indeed, the identification and characterization of these inhibitors could be useful in several applications, among them, hormone-driven cancers, to prevent tumour recurrence and even to catalyse tumour regression.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/16854