28 research outputs found
Studio e caratterizzazione di nuovi farmaci molecolari nel trattamento dei tumori dipendenti dalla via oncogenica di Hedgehog.
La via di trasduzione del segnale di Hedgehog (Hh) gioca un ruolo cruciale nello sviluppo e nella tumorigenesi, nonchè nella proliferazione e mantenimento delle cellule staminali tumorali (CSCs). L’attivazione di questa via del segnale avviene in seguito al legame del ligando Hh con il recettore inibitorio Patched1 (Ptch1). Tale interazione rimuove l’effetto inibitorio che Ptch1 esercita su un altro recettore, Smoothened (Smo), che rappresenta il regolatore positivo chiave nella trasduzione del segnale. Questo evento consente il rilascio dei fattori trascrizionali zinc-finger della famiglia Gli (Gli1, Gli2 e Gli3), permettendone la traslocazione nucleare e la conseguente attività trascrizionale.
Un'aberrante attivazione del signaling è responsabile dell'insorgenza di diversi tipi di tumore tra i quali il medulloblastoma, il rabdomiosarcoma, il carcinoma a cellule basali e molti altri. La carcinogenesi Hh dipendente è legata ad alterazioni genetiche e/o molecolari a carico dei componenti più importanti della via, quali ad esempio mutazioni attivanti del recettore Smo, inattivanti il recettore Ptch1 o il regolatore SuFu, traslocazioni di Gli1 o amplificazioni di Gli1 o Gli2, iperattivazione di Gli1 attraverso meccanismi non canonici.
Dato il ruolo fondamentale nella tumorigenesi e nel mantenimento delle nicchie di cellule staminali tumorali, la via del segnale di Hh rappresenta oggi un attraente bersaglio terapeutico nel cancro. Negli ultimi anni la maggior parte delle ricerche sono state focalizzate sullo sviluppo di farmaci in grado di bloccare l’effetto attivatorio di Smo. Tra questi, il vismodegib (GDC-0449/Eridevige®) è stato il primo inibitore di Hh approvato dalla FDA nel 2012 per il trattamento del carcinoma a cellule basali. Tuttavia, diversi studi hanno portato alla luce alcuni limiti degli inibitori sin qui individuati, legati in particolare alla scarsa selettività , allo sviluppo di farmaco resistenza e all’attivazione dell’effettore finale della pathway, Gli1, mediata da altre vie oncogeniche. Queste evidenze sollevano la necessità di identificare nuovi e più efficaci inibitori di Hh in grado di vincere la farmaco resistenza e contrastare la crescita del tumore.
Al fine di raggiungere questo obiettivo, abbiamo condotto uno studio innovativo di tipo computazionale finalizzato all’individuazione, caratterizzazione e ottimizzazione di nuove molecole in grado di bloccare il potenziale oncogenico di Hh. Attraverso uno screening in silico di una libreria di piccole molecole di origine naturale, sintetica o semi-sintetica e sfruttando la struttura cristallografica dei componenti chiave di nostro interesse, il recettore Smo e il fattore di trascrizione Gli1, abbiamo identificato nuovi efficaci inibitori della via di Hh. In particolare, abbiamo individuato la prima molecola in grado di impedire l’attività trascrizionale di Gli1, grazie alla sua capacità di legare direttamente questo fattore di trascrizione ed inibirne il legame con il DNA. L’efficacia di tali molecole nel bloccare la proliferazione cellulare e quindi la crescita del tumore, è stata comprovata da test effettuati, in vitro ed in vivo, su modelli tumorali caratterizzati da un’aberrante attivazione del signaling di Hh. I nostri risultati svelano il potenziale di queste molecole come nuovi e promettenti approcci terapeutici per la cura dei tumori associati alla via di Hh
Targeting GLI factors to inhibit the Hedgehog pathway
Hedgehog (Hh) signaling has emerged in recent years as an attractive target for anticancer therapy because its aberrant activation is implicated in several cancers. Major progress has been made in the development of SMOOTHENED (SMO) antagonists, although they have shown several limitations due to downstream SMO pathway activation or the occurrence of drug-resistant SMO mutations. Recently, particular interest has been elicited by the identification of molecules able to hit glioma-associated oncogene (GLI) factors, the final effectors of the Hh pathway, which provide a valid tool to overcome anti-SMO resistance. Here, we review results achieved in developing GLI antagonists, explaining their mechanisms of action and highlighting their therapeutic potential. We also underline the relevance of structural details in their discovery and optimization
Synergistic inhibition of the Hedgehog pathway by newly designed Smo and Gli antagonists bearing the isoflavone scaffold
Aberrant activation of the Hedgehog (Hh) pathway is responsible for the onset and progression of several malignancies. Small molecules able to block the pathway at the upstream receptor Smoothened (Smo) or the downstream effector Gli1 have thus emerged recently as valuable anticancer agents. Here, we have designed, synthesized, and tested new Hh inhibitors taking advantage by the highly versatile and privileged isoflavone scaffold. The introduction of specific substitutions on the isoflavone's ring B allowed the identification of molecules targeting preferentially Smo or Gli1. Biological assays coupled with molecular modeling corroborated the design strategy, and provided new insights into the mechanism of action of these molecules. The combined administration of two different isoflavones behaving as Smo and Gli antagonists, respectively, in primary medulloblastoma (MB) cells highlighted the synergistic effects of these agents, thus paving the way to further and innovative strategies for the pharmacological inhibition of Hh signaling
Inhibition of Hedgehog-dependent tumors and cancer stem cells by a newly identified naturally occurring chemotype
Hedgehog (Hh) inhibitors have emerged as valid tools in the treatment of a wide range of cancers. Indeed, aberrant activation of the Hh pathway occurring either by ligand-dependent or -independent mechanisms is a key driver in tumorigenesis. The smoothened (Smo) receptor is one of the main upstream transducers of the Hh signaling and is a validated target for the development of anticancer compounds, as underlined by the FDA-approved Smo antagonist Vismodegib (GDC-0449/Erivedge) for the treatment of basal cell carcinoma. However, Smo mutations that confer constitutive activity and drug resistance have emerged during treatment with Vismodegib. For this reason, the development of new effective Hh inhibitors represents a major challenge for cancer therapy. Natural products have always represented a unique source of lead structures in drug discovery, and in recent years have been used to modulate the Hh pathway at multiple levels. Here, starting from an in house library of natural compounds and their derivatives, we discovered novel chemotypes of Hh inhibitors by mean of virtual screening against the crystallographic structure of Smo. Hh functional based assay identified the chalcone derivative 12 as the most effective Hh inhibitor within the test set. The chalcone 12 binds the Smo receptor and promotes the displacement of Bodipy-Cyclopamine in both Smo WT and drug-resistant Smo mutant. Our molecule stands as a promising Smo antagonist able to specifically impair the growth of Hh-dependent tumor cells in vitro and in vivo and medulloblastoma stem-like cells and potentially overcome the associated drug resistance
MK-4101 - a potent inhibitor of the hedgehog pathway - is highly active against medulloblastoma and basal cell carcinoma
Aberrant activation of the Hedgehog (Hh) signaling pathway is implicated in the pathogenesis of many cancers, including medulloblastoma and basal cell carcinoma (BCC). In this study, using neonatally irradiated Ptch1+/- mice as a model of Hh-dependent tumors, we investigated the in vivo effects of MK-4101, a novel SMO antagonist, for treatment of medulloblastoma and BCC. Results clearly demonstrated a robust antitumor activity of MK-4101, achieved through the inhibition of proliferation and induction of extensive apoptosis in tumor cells. Of note, beside antitumor activity on transplanted tumors, MK-4101 was highly efficacious against primary medulloblastoma and BCC developing in the cerebellum and skin of Ptch1+/- mice. By identifying the changes induced by MK-4101 in gene expression profiles in tumors, we also elucidated the mechanism of action of this novel, orally administrable compound. MK-4101 targets the Hh pathway in tumor cells, showing the maximum inhibitory effect on Gli1. MK-4101 also induced deregulation of cell cycle and block of DNA replication in tumors. Members of the IGF and Wnt signaling pathways, were among the most highly deregulated genes by MK-4101, suggesting that the interplay among Hh, IGF and Wnt is crucial in Hh-dependent tumorigenesis. Altogether, the results of this preclinical study support a therapeutic opportunity for MK-4101 in the treatment of Hh-driven cancers, also providing useful information for combination therapy with drugs targeting pathways cooperating with Hh oncogenic activity
Itch/β-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis
Suppressor of Fused (SuFu), a tumour suppressor mutated in medulloblastoma, is a central player of Hh signalling, a pathway crucial for development and deregulated in cancer. Although the control of Gli transcription factors by SuFu is critical in Hh signalling, our understanding of the mechanism regulating this key event remains limited. Here, we show that the Itch/β-arrestin2 complex binds SuFu and induces its Lys63-linked polyubiquitylation without affecting its stability. This process increases the association of SuFu with Gli3, promoting the conversion of Gli3 into a repressor, which keeps Hh signalling off. Activation of Hh signalling antagonises the Itch-dependent polyubiquitylation of SuFu. Notably, different SuFu mutations occurring in medulloblastoma patients are insensitive to Itch activity, thus leading to deregulated Hh signalling and enhancing medulloblastoma cell growth. Our findings uncover mechanisms controlling the tumour suppressive functions of SuFu and reveal that their alterations are implicated in medulloblastoma tumorigenesis
Itch/β-arrestin2-dependent non-proteolytic ubiquitylation of SuFu controls Hedgehog signalling and medulloblastoma tumorigenesis
Suppressor of Fused (SuFu), a tumour suppressor mutated in medulloblastoma, is a central player of Hh signalling, a pathway crucial for development and deregulated in cancer. Although the control of Gli transcription factors by SuFu is critical in Hh signalling, our understanding of the mechanism regulating this key event remains limited. Here, we show that the Itch/β-arrestin2 complex binds SuFu and induces its Lys63-linked polyubiquitylation without affecting its stability. This process increases the association of SuFu with Gli3, promoting the conversion of Gli3 into a repressor, which keeps Hh signalling off. Activation of Hh signalling antagonises the Itch-dependent polyubiquitylation of SuFu. Notably, different SuFu mutations occurring in medulloblastoma patients are insensitive to Itch activity, thus leading to deregulated Hh signalling and enhancing medulloblastoma cell growth. Our findings uncover mechanisms controlling the tumour suppressive functions of SuFu and reveal that their alterations are implicated in medulloblastoma tumorigenesis
Gli1/DNA interaction is a druggable target for Hedgehog-dependent tumors
Hedgehog signaling is essential for tissue development and stemness, and its deregulation has been observed in many tumors. Aberrant activation of Hedgehog signaling is the result of genetic mutations of pathway components or other Smo-dependent or independent mechanisms, all triggering the downstream effector Gli1. For this reason, understanding the poorly elucidated mechanism of Gli1-mediated transcription allows to identify novel molecules blocking the pathway at a downstream level, representing a critical goal in tumor biology. Here, we clarify the structural requirements of the pathway effector Gli1 for binding to DNA and identify Glabrescione B as the first small molecule binding to Gli1 zinc finger and impairing Gli1 activity by interfering with its interaction with DNA. Remarkably, as a consequence of its robust inhibitory effect on Gli1 activity, Glabrescione B inhibited the growth of Hedgehog-dependent tumor cells in vitro and in vivo as well as the self-renewal ability and clonogenicity of tumor-derived stem cells. The identification of the structural requirements of Gli1/DNA interaction highlights their relevance for pharmacologic interference of Gli signaling
New Indole Tubulin Assembly Inhibitors Cause Stable Arrest of Mitotic Progression, Enhanced Stimulation of Natural Killer Cell Cytotoxic Activity, and Repression of Hedgehog-Dependent Cancer
We designed 39 new 2-phenylindole derivatives as potential anticancer agents bearing the 3,4,5-trimethoxyphenyl moiety with a sulfur, ketone, or methylene bridging group at position 3 of the indole and with halogen or methoxy substituent(s) at positions 4-7. Compounds 33 and 44 strongly inhibited the growth of the P-glycoprotein-overexpressing multi-drug-resistant cell lines NCI/ADR-RES and Messa/Dx5. At 10 nM, 33 and 44 stimulated the cytotoxic activity of NK cells. At 20-50 nM, 33 and 44 arrested >80% of HeLa cells in the G2/M phase of the cell cycle, with stable arrest of mitotic progression. Cell cycle arrest was followed by cell death. Indoles 33, 44, and 81 showed strong inhibition of the SAG-induced Hedgehog signaling activation in NIH3T3 Shh-Light II cells with IC50 values of 19, 72, and 38 nM, respectively. Compounds of this class potently inhibited tubulin polymerization and cancer cell growth, including stimulation of natural killer cell cytotoxic activity and repression of Hedgehog-dependent cancer
New pyrrole derivatives with potent tubulin polymerization inhibiting activity as anticancer agents including hedgehog-dependent cancer
We synthesized 3-aroyl-1-arylpyrrole (ARAP) derivatives as potential anticancer agents having different substituents at the pendant 1-phenyl ring. Both the 1-phenyl ring and 3-(3,4,5-trimethoxyphenyl)carbonyl moieties were mandatory to achieve potent inhibition of tubulin polymerization, binding of colchicine to tubulin, and cancer cell growth. ARAP 22 showed strong inhibition of the P-glycoprotein-overexpressing NCI-ADR-RES and Messa/Dx5MDR cell lines. Compounds 22 and 27 suppressed in vitro the Hedgehog signaling pathway, strongly reducing luciferase activity in SAG treated NIH3T3 Shh-Light II cells, and inhibited the growth of medulloblastoma D283 cells at nanomolar concentrations. ARAPs 22 and 27 represent a new potent class of tubulin polymerization and cancer cell growth inhibitors with the potential to inhibit the Hedgehog signaling pathway