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1.
Chem Sci ; 15(16): 6168-6177, 2024 Apr 24.
Artigo em Inglês | MEDLINE | ID: mdl-38665538

RESUMO

A stimuli-sensitive linker is one of the indispensable components of prodrugs for cancer therapy as it covalently binds the drug and releases it upon external stimulation at the tumour site. Quinone methide elimination has been widely used as the key transformation to release drugs based on their nucleofugacity. The usual approach is to bind the drug to the linker as a carbamate and release it as a free amine after a self-immolative 1,6-elimination. Although this approach is very efficient, it is limited to amines (as carbamates), alcohols or phenols (as carbonates) or other acidic functional groups. We report here a self-immolative spacer capable of directly linking and releasing amines, phenols, thiols, sulfonamides and carboxyamides after a reductive stimulus. The spacer is based on the structure of (5-nitro-2-pyrrolyl)methanol (NPYM-OH), which was used for the direct alkylation of the functional groups mentioned above. The spacer is metabolically stable and has three indispensable sites for bioconjugation: the bioresponsive trigger, the conjugated 1,6 self-immolative system and a third arm suitable for conjugation with a carrier or other modifiers. Release was achieved by selective reduction of the nitro group over Fe/Pd nanoparticles (NPs) in a micellar aqueous environment (H2O/TPGS-750-M), or by NADH mediated nitroreductase activation. A DFT study demonstrates that, during the 1,6 elimination, the transition state formed from 5-aminopyrrole has a lower activation energy compared to other 5-membered heterocycles or p-aminobenzyl derivatives. The NPYM scaffold was validated by late-stage functionalisation of approved drugs such as celecoxib, colchicine, vorinostat or ciprofloxacin. A hypoxia-activated NPYM-based prodrug (HAP) derived from HDAC inhibitor ST7612AA1 was also produced, which was active in cancer cells under hypoxic conditions.

2.
Pharmacol Res ; 195: 106858, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37473878

RESUMO

Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.


Assuntos
Neoplasias , Proteína GLI1 em Dedos de Zinco , Proteína Gli2 com Dedos de Zinco , Animais , Humanos , Camundongos , Proteínas Hedgehog/metabolismo , Fatores de Transcrição Kruppel-Like/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Proteína GLI1 em Dedos de Zinco/antagonistas & inibidores , Proteína Gli2 com Dedos de Zinco/antagonistas & inibidores , Neoplasias/tratamento farmacológico , Neoplasias/patologia
3.
Chem Commun (Camb) ; 58(75): 10532-10535, 2022 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-36043993

RESUMO

We report a new 1-6 self-immolative, traceless crosslinker derived from the natural product gallic acid. The linker acts through a pH-dependent mechanism for drug release. This 5-(hydroxymethyl)pyrogallol orthoester derivative (HMPO) was stable for 24 hours at pH values of 7.4 and 6.6 and in plasma, releasing molecules bound to the hydroxymethyl moiety under acid-dependent stimuli at pH 5.5. The linker was non-toxic and was used for the conjugation of Doxorubicin (Doxo) or Combretastatin A4 with Cetuximab. The ADCs formed showed their pH responsivity reducing cell viability of A431 and A549 cancer cells better than Cetuximab alone.


Assuntos
Produtos Biológicos , Imunoconjugados , Linhagem Celular Tumoral , Cetuximab/farmacologia , Doxorrubicina/farmacologia , Ácido Gálico/farmacologia , Concentração de Íons de Hidrogênio , Imunoconjugados/química , Imunoconjugados/farmacologia , Pirogalol
4.
Chem Commun (Camb) ; 57(7): 867-870, 2021 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-33433550

RESUMO

Antitumor hydroxamates SAHA and Dacinostat have been linked to cetuximab and trastuzumab through a non-cleavable linker based on the p-mercaptobenzyl alcohol structure. These antibody drug conjugates (ADCs) were able to inhibit HDAC in several tumour cell lines. The cetuximab based ADCs block human lung adenocarcinoma cell proliferation, demonstrating that bioconjugation with antibodies is a suitable approach for targeted therapy based on hydroxamic acid-containing drugs. This work also shows that ADC-based delivery might be used to overcome the classical pharmacokinetic problems of hydroxamic acids.


Assuntos
Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/química , Imunoconjugados/química , Células A549 , Proliferação de Células/efeitos dos fármacos , Cetuximab/química , Inibidores de Histona Desacetilases/metabolismo , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/metabolismo , Ácidos Hidroxâmicos/farmacologia , Imunoconjugados/metabolismo , Trastuzumab/química
5.
ACS Med Chem Lett ; 11(5): 832-838, 2020 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-32435392

RESUMO

Starting from known GLI1 inhibitors, a pharmacophore-based virtual screening approach was applied to databases of commercially available compounds with the aim of identifying new GLI1 modulators. As a result, three different chemical scaffolds emerged that were characterized by a significant ability to reduce the transcriptional activity of the endogenous Hedgehog-GLI pathway and GLI1 protein level in murine NIH3T3 cells. They also showed a micromolar antiproliferative activity in human melanoma (A375) and medulloblastoma (DAOY) cell lines, without cytotoxicity in non-neoplastic mammary epithelial cells.

6.
Front Oncol ; 9: 1534, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-32039017

RESUMO

Targeted therapy using monoclonal antibodies conjugated to toxins is gaining space in the treatment of cancer. Here, we report the anti-tumor effect of a new antibody drug conjugate (ADC) delivering a HDAC inhibitor to ErbB2+ solid tumors. Trastuzumab was partially reduced with tris [2-carboxyethyl] phosphine (TCEP) and conjugated to ST7464AA1, the active form of the prodrug HDAC inhibitor ST7612AA1, through a maleimide-thiol linker to obtain the Antibody Drug Conjugate (ADC) ST8176AA1. The average drug/antibody ratio (DAR) was 4.5 as measured by hydrophobic interaction chromatography (HIC). Binding of ST8176AA1 to ErbB2 receptor and internalization in tumor cells were investigated by enzyme-linked immunosorbent assay (ELISA), surface plasmon resonance (SPR), cytofluorimetry, and High Content Screening (HCS) Imaging. The biological activity of the ADC was evaluated in vitro and in vivo by measuring cell proliferation/cell cycle, apoptosis/DNA damage, tubulin, and histone acetylation and modulation of Epithelial/Mesenchymal Transition (EMT) markers. Receptor binding and internalization of ST8176AA1 were confirmed to be similar to trastuzumab. Higher anti-tumor activity of ST8176AA1 compared to trastuzumab was observed in vitro in tumor cell lines. Such higher activity correlated with increased acetylation of histones and alfa-tubulin as a consequence of HDAC inhibitor-mediated epigenetic modulation that also induced increased expression of ErbB2 and estrogen receptor in triple negative breast cancer cells. Consistently with in vitro data, ST8176AA1 exhibited higher tumor growth inhibition than trastuzumab in xenograft models of ovary and colon carcinoma and in two patient-derived xenograft (PDX) models of pancreatic carcinoma. Immunohistochemistry analysis of tumor masses showed lower expression of the proliferation marker Ki67 and higher expression of cleaved caspase-3 in mice treated with the ADC compared to those treated with trastuzumab and results correlated with increased acetylation of both histones and tubulin. Collectively, present data indicate that ADC ST8176AA1 can target epigenetic modulation to ErbB2+ tumors. Interestingly, the amount of HDACi estimated to be delivered at the ST8176AA1 effective dose would correspond to ~1/1,000 of ST7612AA1 effective dose. Therefore, ST8176AA1 is an attractive new therapeutic candidate because it exhibits increased anti-tumor potency compared to trastuzumab by exerting epigenetic modulation at a much safer dose compared to standard HDACi-based therapeutic protocols.

7.
Chem Sci ; 9(31): 6490-6496, 2018 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-30288233

RESUMO

We describe here two novel antibody-drug conjugates loaded with the HDAC inhibitor ST7612AA1 (IC50 equal to 0.07 µM on NCI-H460 cells), a thiol-based molecule with a moderate toxicity in vivo. Two payloads were prepared using cleavable and non-cleavable linkers. After anchoring to cetuximab through amide bond with lysines, the resulting HDAC inhibitor-antibody conjugates showed ability to recognize EGFR and efficient internalization in tumor cells. Both ADCs induced sensible increment of histones 3 and 4 and alpha-tubulin acetylation. Animal models of human solid tumors showed high anti-tumor efficacy of the conjugates without the toxicity generally observed with traditional ADCs delivering highly potent cytotoxic drugs. These compounds, the first ADCs charged with not highly cytotoxic warheads, are potentially suitable for epigenetic modulation, extending the ADC strategy to the targeted delivery of HDAC inhibitors with many possible therapeutic applications beyond cancer.

8.
Cell Death Dis ; 9(2): 142, 2018 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-29396391

RESUMO

Aberrant activation of the Hedgehog (HH) signaling is a critical driver in tumorigenesis. The Smoothened (SMO) receptor is one of the major upstream transducers of the HH pathway and a target for the development of anticancer agents. The SMO inhibitor Vismodegib (GDC-0449/Erivedge) has been approved for treatment of basal cell carcinoma. However, the emergence of resistance during Vismodegib treatment and the occurrence of numerous side effects limit its use. Our group has recently discovered and developed novel and potent SMO inhibitors based on acylguanidine or acylthiourea scaffolds. Here, we show that the two acylguanidine analogs, compound (1) and its novel fluoride derivative (2), strongly reduce growth and self-renewal of melanoma cells, inhibiting the level of the HH signaling target GLI1 in a dose-dependent manner. Both compounds induce apoptosis and DNA damage through the ATR/CHK1 axis. Mechanistically, they prevent G2 to M cell cycle transition, and induce signs of mitotic aberrations ultimately leading to mitotic catastrophe. In a melanoma xenograft mouse model, systemic treatment with 1 produced a remarkable inhibition of tumor growth without body weight loss in mice. Our data highlight a novel route for cell death induction by SMO inhibitors and support their use in therapeutic approaches for melanoma and, possibly, other types of cancer with active HH signaling.


Assuntos
Replicação do DNA/efeitos dos fármacos , Guanidinas/farmacologia , Proteínas Hedgehog/metabolismo , Melanoma/patologia , Mitose/efeitos dos fármacos , Transdução de Sinais , Estresse Fisiológico , Proteína GLI1 em Dedos de Zinco/metabolismo , Animais , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular , Sobrevivência Celular/efeitos dos fármacos , Dano ao DNA , Feminino , Humanos , Concentração Inibidora 50 , Camundongos Nus , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Receptor Smoothened/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
9.
J Cell Physiol ; 233(6): 4961-4971, 2018 06.
Artigo em Inglês | MEDLINE | ID: mdl-29215700

RESUMO

Osteosarcoma (OS) is an ultra-rare highly malignant tumor of the skeletal system affecting mainly children and young adults and it is characterized by an extremely aggressive clinical course. OS patients are currently treated with chemotherapy and complete surgical resection of cancer tissue. However, resistance to chemotherapy and the recurrence of disease, as pulmonary metastasis, remain the two greatest challenges in the management, and treatment of this tumor. For these reasons, it is of primary interest to find alternative therapeutic strategies for OS. Dysregulated Hedgehog signalling is involved in the development of various types of cancers including OS. It has also been implicated in tumor/stromal interaction and cancer stem cell biology, and therefore presents a novel therapeutic strategy for cancer treatment. In our work, we tested the activity of five potent Smoothened (SMO) inhibitors, four acylguanidine and one acylthiourea derivatives, against an OS cell line. We found that almost all our compounds were able to inhibit OS cells proliferation and to reduce Gli1 protein levels. Our results also indicated that SMO inhibition in OS cells by such compounds, induces apoptosis with a nanomolar potency. These findings suggest that inactivation of SMO may be a useful approach to the treatment of patients with OS.


Assuntos
Antineoplásicos/farmacologia , Neoplasias Ósseas/tratamento farmacológico , Guanidinas/farmacologia , Osteossarcoma/tratamento farmacológico , Receptor Smoothened/antagonistas & inibidores , Tioureia/farmacologia , Acilação , Apoptose/efeitos dos fármacos , Neoplasias Ósseas/metabolismo , Neoplasias Ósseas/patologia , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Osteossarcoma/metabolismo , Osteossarcoma/patologia , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/metabolismo , Tioureia/análogos & derivados , Células Tumorais Cultivadas , Proteína GLI1 em Dedos de Zinco/metabolismo
10.
J Cell Physiol ; 232(11): 3103-3111, 2017 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-28019670

RESUMO

Alkaptonuria (AKU) is an ultra-rare genetic disease, in which the accumulation of a toxic metabolite, homogentisic acid (HGA) leads to the systemic development of ochronotic aggregates. These aggregates cause severe complications mainly at the level of joints with extensive degradation of the articular cartilage. Primary cilia have been demonstrated to play an essential role in development and the maintenance of articular cartilage homeostasis, through their involvement in mechanosignaling and Hedgehog signaling pathways. Hedgehog signaling has been demonstrated to be activated in osteoarthritis (OA) and to drive cartilage degeneration in vivo. The numerous similarities between OA and AKU suggest that primary cilia Hedgehog signaling may also be altered in AKU. Thus, we characterized an AKU cellular model in which healthy chondrocytes were treated with HGA (66 µM) to replicate AKU cartilage pathology. We investigated the degree of activation of the Hedgehog signaling pathway and how treatment with inhibitors of the receptor Smoothened (Smo) influenced Hedgehog activation and primary cilia structure. The results obtained in this work provide a further step in the comprehension of the pathophysiological features of AKU, suggesting a potential therapeutic approach to modulate AKU cartilage degradation processes through manipulation of the Hedgehog pathway.


Assuntos
Alcaptonúria/induzido quimicamente , Anilidas/farmacologia , Condrócitos/efeitos dos fármacos , Proteínas Hedgehog/metabolismo , Ácido Homogentísico/toxicidade , Piridinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened/antagonistas & inibidores , Alcaloides de Veratrum/farmacologia , Alcaptonúria/metabolismo , Alcaptonúria/patologia , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Cílios/efeitos dos fármacos , Cílios/metabolismo , Cílios/patologia , Relação Dose-Resposta a Droga , Humanos , Hiperpigmentação/induzido quimicamente , Hiperpigmentação/metabolismo , Receptor Smoothened/metabolismo , Proteína GLI1 em Dedos de Zinco/metabolismo
11.
Curr Top Med Chem ; 17(4): 441-459, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-27558677

RESUMO

BACKGROUND: Synthesis, computational study and biological evaluation of peptidomimetic analogues of FR235222 (3), a natural immunosuppressant and HDAC inhibitor, have been reported. These new compounds, bearing α-hydroxyketone moiety, as more stable zinc binding group (ZBG), were evaluated in vitro as HDAC inhibitors against the human HDACs isoforms 1-9 and in cellular antiproliferative assays on U937 human leukemia cell line. The 1,4-benzodiazepin-2,5-dione (BDZ), capping group and the natural ZBG, (S,R)-2-amino-9-hydroxy-8-oxodecanoic acid (Ahoda), were evaluated in order to probe HDAC inhibition and/or paralogue selectivity. Some of the new derivatives showed an interesting activity against a number of HDAC isozymes. The observed activity profile was rationalized by a computational assisted SAR study, in order to understand how the BDZ classes interact with the enzyme into the catalytic pocket. Despite its poor solubility, compound 17b showed significant antiproliferative profile and HDAC inhibition activity. RESULT: In order to assess how the solubility issue could have affected the biological outcome, bioassay conditions were reproduced and quantification of precipitated particulate material was evaluated by turbidimetric and NMR studies together with physicochemical descriptors prediction. Thus, BDZ 17b has been chosen to be promising lead compounds for further optimization, in order to elucidate molecule- enzyme surface recognition.


Assuntos
Benzodiazepinas/química , Inibidores de Histona Desacetilases/farmacologia , Peptídeos Cíclicos/química , Benzodiazepinas/farmacologia , Inibidores de Histona Desacetilases/química , Humanos , Espectroscopia de Ressonância Magnética , Peptídeos Cíclicos/farmacologia , Solubilidade , Relação Estrutura-Atividade
12.
Sci Rep ; 6: 23479, 2016 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-27010359

RESUMO

Hedgehog (Hh) is a critical regulator of adipogenesis. Extracellular vesicles are natural Hh carriers, as illustrated by activated/apoptotic lymphocytes specifically shedding microparticles (MP) bearing the morphogen (MP(Hh+)). We show that MP(Hh+) inhibit adipocyte differentiation and orientate mesenchymal stem cells towards a pro-osteogenic program. Despite a Smoothened (Smo)-dependency, MP(Hh+) anti-adipogenic effects do not activate a canonical Hh signalling pathway in contrast to those elicited either by the Smo agonist SAG or recombinant Sonic Hedgehog. The Smo agonist GSA-10 recapitulates many of the hallmarks of MP(Hh+) anti-adipogenic effects. The adipogenesis blockade induced by MP(Hh+) and GSA-10 was abolished by the Smo antagonist LDE225. We further elucidate a Smo/Lkb1/Ampk axis as the non-canonical Hh pathway used by MP(Hh+) and GSA-10 to inhibit adipocyte differentiation. Our results highlight for the first time the ability of Hh-enriched MP to signal via a non-canonical pathway opening new perspectives to modulate fat development.


Assuntos
Adipócitos/citologia , Diferenciação Celular/fisiologia , Proteínas Hedgehog/fisiologia , Células 3T3-L1 , Animais , Células Cultivadas , Proteínas Hedgehog/metabolismo , Humanos , Células-Tronco Mesenquimais/citologia , Camundongos , Fatores de Transcrição/metabolismo
13.
PLoS One ; 11(3): e0149919, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26934052

RESUMO

The most relevant therapeutic approaches to treat CML rely on the administration of tyrosine kinase inhibitors (TKIs) like Imatinib, which are able to counteract the activity of Bcr-Abl protein increasing patient's life expectancy and survival. Unfortunately, there are some issues TKIs are not able to address; first of all TKIs are not so effective in increasing survival of patients in blast crisis, second they are not able to eradicate leukemic stem cells (LSC) which represent the major cause of disease relapse, and third patients often develop resistance to TKIs due to mutations in the drug binding site. For all these reasons it's of primary interest to find alternative strategies to treat CML. Literature shows that Hedgehog signaling pathway is involved in LSC maintenance, and pharmacological inhibition of Smoothened (SMO), one of the key molecules of the pathway, has been demonstrated to reduce Bcr-Abl positive bone marrow cells and LSC. Consequently, targeting SMO could be a promising way to develop a new treatment strategy for CML overcoming the limitations of current therapies. In our work we have tested some compounds able to inhibit SMO, and among them MRT92 appears to be a very potent SMO antagonist. We found that almost all our compounds were able to reduce Gli1 protein levels in K-562 and in KU-812 CML cell lines. Furthermore, they were also able to increase Gli1 and SMO RNA levels, and to reduce cell proliferation and induce apoptosis/autophagy in both the tested cell lines. Finally, we demonstrated that our compounds were able to modulate the expression of some miRNAs related to Hedgehog pathway such as miR-324-5p and miR-326. Being Hedgehog pathway deeply implicated in the mechanisms of CML we may conclude that it could be a good therapeutic target for CML and our compounds seem to be promising antagonists of such pathway.


Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Inibidores de Proteínas Quinases/farmacologia , Autofagia/efeitos dos fármacos , Crise Blástica/metabolismo , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Proteínas de Fusão bcr-abl/metabolismo , Proteínas Hedgehog/metabolismo , Humanos , Mesilato de Imatinib/farmacologia , Células K562 , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened
14.
FASEB J ; 29(5): 1817-29, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25636740

RESUMO

The Smoothened (Smo) receptor, a member of class F G protein-coupled receptors, is the main transducer of the Hedgehog (Hh) signaling pathway implicated in a wide range of developmental and adult processes. Smo is the target of anticancer drugs that bind to a long and narrow cavity in the 7-transmembrane (7TM) domain. X-ray structures of human Smo (hSmo) bound to several ligands have revealed 2 types of 7TM-directed antagonists: those binding mostly to extracellular loops (site 1, e.g., LY2940680) and those penetrating deeply in the 7TM cavity (site 2, e.g., SANT-1). Here we report the development of the acylguanidine MRT-92, which displays subnanomolar antagonist activity against Smo in various Hh cell-based assays. MRT-92 inhibits rodent cerebellar granule cell proliferation induced by Hh pathway activation through pharmacologic (half maximal inhibitory concentration [IC50] = 0.4 nM) or genetic manipulation. Using [(3)H]MRT-92 (Kd = 0.3 nM for hSmo), we created a comprehensive framework for the interaction of small molecule modulators with hSmo and for understanding chemoresistance linked to hSmo mutations. Guided by molecular docking and site-directed mutagenesis data, our work convincingly confirms that MRT-92 simultaneously recognized and occupied both sites 1 and 2. Our data demonstrate the existence of a third type of Smo antagonists, those entirely filling the Smo binding cavity from the upper extracellular part to the lower cytoplasmic-proximal subpocket. Our studies should help design novel potent Smo antagonists and more effective therapeutic strategies for treating Hh-linked cancers and associated chemoresistance.


Assuntos
Antineoplásicos/farmacologia , Membrana Celular/metabolismo , Neoplasias Cerebelares/metabolismo , Guanidinas/farmacologia , Proteínas Hedgehog/antagonistas & inibidores , Meduloblastoma/metabolismo , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Adulto , Animais , Sítios de Ligação , Western Blotting , Membrana Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Neoplasias Cerebelares/tratamento farmacológico , Neoplasias Cerebelares/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteínas Hedgehog/metabolismo , Humanos , Técnicas Imunoenzimáticas , Meduloblastoma/tratamento farmacológico , Meduloblastoma/patologia , Camundongos , Simulação de Acoplamento Molecular , Mutagênese Sítio-Dirigida , Mutação/genética , Ligação Proteica , Conformação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor Smoothened
15.
J Med Chem ; 57(20): 8358-77, 2014 Oct 23.
Artigo em Inglês | MEDLINE | ID: mdl-25233084

RESUMO

A systematic study of medicinal chemistry aimed at identifying a new generation of HDAC inhibitors, through the introduction of a thiol zinc-binding group (ZBG) and of an amide-lactam in the ω-position of the polyethylene chain of the vorinostat scaffold, allowed the selection of a new class of potent pan-HDAC inhibitors (pan-HDACis). Simple, highly versatile, and efficient synthetic approaches were used to synthesize a library of these new derivatives, which were then submitted to a screening for HDAC inhibition as well as to a preliminary in vitro assessment of their antiproliferative activity. Molecular docking into HDAC crystal structures suggested a binding mode for these thiol derivatives consistent with the stereoselectivity observed upon insertion of amide-lactam substituents in the ω-position. ST7612AA1 (117), selected as a drug candidate for further development, showed an in vitro activity in the nanomolar range associated with a remarkable in vivo antitumor activity, highly competitive with the most potent HDAC inhibitors, currently under clinical trials. A preliminary study of PK and metabolism is also illustrated.


Assuntos
Anilidas/farmacologia , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Pirrolidinonas/farmacologia , Administração Oral , Anilidas/administração & dosagem , Animais , Antineoplásicos/farmacologia , Técnicas de Química Sintética , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Células HCT116 , Inibidores de Histona Desacetilases/administração & dosagem , Inibidores de Histona Desacetilases/síntese química , Histona Desacetilases/química , Histonas/metabolismo , Humanos , Camundongos Nus , Simulação de Acoplamento Molecular , Pirrolidinonas/administração & dosagem , Proteínas Repressoras/química , Tubulina (Proteína)/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
16.
Stem Cell Reports ; 3(2): 312-23, 2014 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-25254344

RESUMO

In the adult brain, self-renewal is essential for the persistence of neural stem cells (NSCs) throughout life, but its regulation is still poorly understood. One NSC can give birth to two NSCs or one NSC and one transient progenitor. A correct balance is necessary for the maintenance of germinal areas, and understanding the molecular mechanisms underlying NSC division mode is clearly important. Here, we report a function of the Sonic Hedgehog (SHH) receptor Patched in the direct control of long-term NSC self-renewal in the subependymal zone. We show that genetic conditional activation of SHH signaling in adult NSCs leads to their expansion and the depletion of their direct progeny. These phenotypes are associated in vitro with an increase in NSC symmetric division in a process involving NOTCH signaling. Together, our results demonstrate a tight control of adult neurogenesis and NSC renewal driven by Patched.


Assuntos
Proteínas Hedgehog/metabolismo , Células-Tronco Neurais/citologia , Sistema X-AG de Transporte de Aminoácidos/genética , Animais , Encéfalo/metabolismo , Proliferação de Células/efeitos dos fármacos , Proteínas Hedgehog/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Neurais/metabolismo , Neurogênese , Receptores Patched , Receptores de Superfície Celular/antagonistas & inibidores , Receptores de Superfície Celular/genética , Receptores de Superfície Celular/metabolismo , Receptores Notch/metabolismo , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Transdução de Sinais , Tamoxifeno/farmacologia , Regulação para Cima , Proteína GLI1 em Dedos de Zinco
17.
J Med Chem ; 57(6): 2258-74, 2014 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-24588105

RESUMO

Ruthenium catalyzed 1,3-cycloaddition (click chemistry) of an azido moiety installed on dihydroxycumene scaffold with differently substituted aryl propiolates gave a new family of 1,4,5-trisubstituted triazole carboxylic acid derivatives that showed high affinity toward Hsp90 associated with cell proliferation inhibition, both in nanomolar range. The 1,5 arrangement of the resorcinol, the aryl moieties, and the presence of an alkyl (secondary) amide in position 4 of the triazole ring were essential to get high activity. Docking simulations suggested that the triazoles penetrate the Hsp90 ATP binding site. Some 1,4,5-trisubstituted triazole carboxamides induced dramatic depletion of the examined client proteins and a very strong increase in the expression levels of the chaperone Hsp70. In vitro metabolic stability and in vivo preliminary studies on selected compounds have shown promising results comparable to the potent Hsp90 inhibitor NVP-AUY922. One of them, (compound 18, SST0287CL1) was selected for further investigation as the most promising drug candidate.


Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Triazóis/síntese química , Triazóis/farmacologia , Animais , Antineoplásicos/metabolismo , Western Blotting , Catálise , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Biologia Computacional , Citometria de Fluxo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Indicadores e Reagentes , Isoxazóis/síntese química , Isoxazóis/farmacologia , Camundongos , Modelos Moleculares , Ligação Proteica , Resorcinóis/síntese química , Resorcinóis/farmacologia , Rutênio , Espectrometria de Fluorescência , Relação Estrutura-Atividade , Difração de Raios X , Ensaios Antitumorais Modelo de Xenoenxerto
18.
Bioorg Med Chem Lett ; 24(1): 61-4, 2014 Jan 01.
Artigo em Inglês | MEDLINE | ID: mdl-24345446

RESUMO

A series of SAHA-like molecules were prepared introducing different lactam-carboxyamides in position 7 of the suberoylanilide skeleton. The activity against different HDAC isoforms was tested and the data compared with the corresponding linear products, without substituent in position 7. In general, this modification provided an effective reinforcement of in vitro activity. While the lactam size or the CO/NH group orientation did not strongly influence the inhibition, the contemporary modification of the suberoylamide fragment gave vary active variants in the lactam series, with compound 28 (ST8078AA1) that showed IC50 values between 2 and 10nM against all Class I HDAC isoforms, demonstrating it to be a large spectrum pan-inhibitor. This strong affinity with HDAC was also confirmed by the value of IC50=0.5µM against H460 cells, ranking 28 as one of the most potent HDAC inhibitors described so far.


Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Ácidos Hidroxâmicos/farmacologia , Lactamas/química , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/síntese química , Ácidos Hidroxâmicos/química , Estrutura Molecular , Relação Estrutura-Atividade
19.
Mol Pharmacol ; 83(5): 1020-9, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23448715

RESUMO

Activation of the Smoothened (Smo) receptor mediates Hedgehog (Hh) signaling. Hh inhibitors are in clinical trials for cancer, and small-molecule Smo agonists may have therapeutic interests in regenerative medicine. Here, we have generated and validated a pharmacophoric model for Smo agonists and used this model for the virtual screening of a library of commercially available compounds. Among the 20 top-scoring ligands, we have identified and characterized a novel quinolinecarboxamide derivative, propyl 4-(1-hexyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxamido) benzoate, (GSA-10), as a Smo agonist. GSA-10 fits to the agonist pharmacophoric model with two hydrogen bond acceptor groups and four hydrophobic regions. Using pharmacological, biochemical, and molecular approaches, we provide compelling evidence that GSA-10 acts at Smo to promote the differentiation of multipotent mesenchymal progenitor cells into osteoblasts. However, this molecule does not display the hallmarks of reference Smo agonists. Remarkably, GSA-10 does not recognize the classic bodipy-cyclopamine binding site. Its effect on cell differentiation is inhibited by Smo antagonists, such as MRT-83, SANT-1, LDE225, and M25 in the nanomolar range, by GDC-0449 in the micromolar range, but not by cyclopamine and CUR61414. Thus, GSA-10 allows the pharmacological characterization of a novel Smo active site, which is notably not targeted to the primary cilium and strongly potentiated by forskolin and cholera toxin. GSA-10 belongs to a new class of Smo agonists and will be helpful for dissecting Hh mechanism of action, with important implications in physiology and in therapy.


Assuntos
Quinolinas/farmacologia , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Benzoatos/farmacologia , Sítios de Ligação/efeitos dos fármacos , Receptores de Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , AMP Cíclico/metabolismo , Cicloexilaminas/farmacologia , Células HEK293 , Proteínas Hedgehog/metabolismo , Humanos , Ligantes , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas , Receptor Smoothened , Tiofenos/farmacologia , Fatores de Transcrição/metabolismo , Proteínas Wnt/metabolismo , Proteína GLI1 em Dedos de Zinco
20.
J Med Chem ; 55(4): 1559-71, 2012 Feb 23.
Artigo em Inglês | MEDLINE | ID: mdl-22268551

RESUMO

The Smoothened (Smo) receptor is the major transducer of the Hedgehog (Hh) signaling pathway. On the basis of the structure of the acylthiourea Smo antagonist (MRT-10), a number of different series of analogous compounds were prepared by ligand-based structural optimization. The acylthioureas, originally identified as actives, were converted into the corresponding acylureas or acylguanidines. In each series, similar structural trends delivered potent compounds with IC(50) values in the nanomolar range with respect to the inhibition of the Hh signaling pathway in various cell-based assays and of BODIPY-cyclopamine binding to human Smo. The similarity of their biological activities, in spite of discrete structural differences, may reveal the existence of hydrogen-bonding interactions between the ligands and the receptor pocket. Biological potency of compounds 61, 72, and 86 (MRT-83) were comparable to those of the clinical candidate GDC-0449. These findings suggest that these original molecules will help delineate Smo and Hh functions and can be developed as potential anticancer agents.


Assuntos
Antineoplásicos/síntese química , Guanidinas/síntese química , Proteínas Hedgehog/antagonistas & inibidores , Receptores Acoplados a Proteínas G/antagonistas & inibidores , Ureia/análogos & derivados , Ureia/síntese química , Animais , Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Cerebelo/citologia , Guanidinas/química , Guanidinas/farmacologia , Humanos , Ligação de Hidrogênio , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Camundongos , Modelos Moleculares , Osteoblastos/citologia , Osteoblastos/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/efeitos dos fármacos , Ratos , Transdução de Sinais , Receptor Smoothened , Relação Estrutura-Atividade , Tioureia/análogos & derivados , Tioureia/química , Tioureia/farmacologia , Ureia/farmacologia
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