Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 8 de 8
Filtrar
1.
Nat Chem Biol ; 17(11): 1157-1167, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34675414

RESUMO

Bivalent proteolysis-targeting chimeras (PROTACs) drive protein degradation by simultaneously binding a target protein and an E3 ligase and forming a productive ternary complex. We hypothesized that increasing binding valency within a PROTAC could enhance degradation. Here, we designed trivalent PROTACs consisting of a bivalent bromo and extra terminal (BET) inhibitor and an E3 ligand tethered via a branched linker. We identified von Hippel-Lindau (VHL)-based SIM1 as a low picomolar BET degrader with preference for bromodomain containing 2 (BRD2). Compared to bivalent PROTACs, SIM1 showed more sustained and higher degradation efficacy, which led to more potent anticancer activity. Mechanistically, SIM1 simultaneously engages with high avidity both BET bromodomains in a cis intramolecular fashion and forms a 1:1:1 ternary complex with VHL, exhibiting positive cooperativity and high cellular stability with prolonged residence time. Collectively, our data along with favorable in vivo pharmacokinetics demonstrate that augmenting the binding valency of proximity-induced modalities can be an enabling strategy for advancing functional outcomes.


Assuntos
Ubiquitina-Proteína Ligases/metabolismo , Humanos , Proteólise
2.
Croat Med J ; 60(3): 201-211, 2019 Jun 13.
Artigo em Inglês | MEDLINE | ID: mdl-31187947

RESUMO

AIM: To assess whether an adenoviral vector carrying the bone morphogenetic protein genes (Ad.BMP-2) can transduce human muscle tissue and direct it toward osteogenic differentiation within one hour. METHODS: This in vitro study, performed at the Department of Molecular Biology, Faculty of Science, Zagreb from 2012 to 2017, used human muscle tissue samples collected during anterior cruciate ligament reconstructions performed in St Catherine Hospital, Zabok. Samples from 28 patients were transduced with adenoviral vector carrying firefly luciferase cDNA (Ad.luc) by using different doses and times of transduction, and with addition of positive ions for transduction enhancement. The optimized protocol was further tested on muscle samples from three new patients, which were transduced with Ad.BMP-2. Released bone morphogenetic protein 2 (BMP-2) levels in osteogenic medium were measured every three days during a period of 21 days. Expression of osteogenic markers was measured at day 14 and 21. After 21 days of cultivation, muscle tissue was immunohistochemically stained for collagen type I detection (COL-I). RESULTS: The new transduction protocol was established using 108 plaque-forming units (P<0.001) as an optimal dose of adenoviral vector and 30 minutes (P<0.001) as an optimal contact time. Positive ions did not enhance transduction. Samples transduced with Ad.BMP-2 according to the optimized protocol showed enhanced expression of osteogenic markers (P<0.050), BMP-2 (P<0.001), and COL I. CONCLUSION: This study confirms that Ad.BMP-2 can transduce human muscle tissue and direct it toward osteogenic differentiation within 30 minutes.


Assuntos
Proteína Morfogenética Óssea 2/genética , Proteína Morfogenética Óssea 2/metabolismo , Diferenciação Celular/genética , Músculo Esquelético/fisiologia , Osteogênese/genética , Transdução Genética , Adenoviridae , Adolescente , Adulto , Células Cultivadas , Melhoramento Genético , Vetores Genéticos , Humanos , Pessoa de Meia-Idade , Tendões/fisiologia , Adulto Jovem
3.
ACS Chem Biol ; 19(7): 1484-1494, 2024 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-38958654

RESUMO

Targeted protein degradation has recently emerged as a novel option in drug discovery. Natural protein half-life is expected to affect the efficacy of degrading agents, but to what extent it influences target protein degradation has not been systematically explored. Using simple mathematical modeling of protein degradation, we find that the natural half-life of a target protein has a dramatic effect on the level of protein degradation induced by a degrader agent which can pose significant hurdles to screening efforts. Moreover, we show that upon screening for degraders of short-lived proteins, agents that stall protein synthesis, such as GSPT1 degraders and generally cytotoxic compounds, deceptively appear as protein-degrading agents. This is exemplified by the disappearance of short-lived proteins such as MCL1 and MDM2 upon GSPT1 degradation and upon treatment with cytotoxic agents such as doxorubicin. These findings have implications for target selection as well as for the type of control experiments required to conclude that a novel agent works as a bona fide targeted protein degrader.


Assuntos
Proteólise , Humanos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Meia-Vida , Doxorrubicina/farmacologia , Doxorrubicina/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas/metabolismo , Proteínas/química
4.
Science ; 385(6715): 1338-1347, 2024 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-39298590

RESUMO

Mutations in the Kirsten rat sarcoma viral oncogene homolog (KRAS) protein are highly prevalent in cancer. However, small-molecule concepts that address oncogenic KRAS alleles remain elusive beyond replacing glycine at position 12 with cysteine (G12C), which is clinically drugged through covalent inhibitors. Guided by biophysical and structural studies of ternary complexes, we designed a heterobifunctional small molecule that potently degrades 13 out of 17 of the most prevalent oncogenic KRAS alleles. Compared with inhibition, KRAS degradation results in more profound and sustained pathway modulation across a broad range of KRAS mutant cell lines, killing cancer cells while sparing models without genetic KRAS aberrations. Pharmacological degradation of oncogenic KRAS was tolerated and led to tumor regression in vivo. Together, these findings unveil a new path toward addressing KRAS-driven cancers with small-molecule degraders.


Assuntos
Antineoplásicos , Neoplasias , Quimera de Direcionamento de Proteólise , Proteínas Proto-Oncogênicas p21(ras) , Animais , Humanos , Camundongos , Alelos , Antineoplásicos/química , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Mutação , Neoplasias/tratamento farmacológico , Neoplasias/genética , Proteólise , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/química , Bibliotecas de Moléculas Pequenas/química , Bibliotecas de Moléculas Pequenas/farmacologia , Bibliotecas de Moléculas Pequenas/uso terapêutico , Quimera de Direcionamento de Proteólise/química , Quimera de Direcionamento de Proteólise/farmacologia , Quimera de Direcionamento de Proteólise/uso terapêutico
5.
Nat Commun ; 13(1): 5969, 2022 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-36216795

RESUMO

Targeted protein degradation offers an alternative modality to classical inhibition and holds the promise of addressing previously undruggable targets to provide novel therapeutic options for patients. Heterobifunctional molecules co-recruit a target protein and an E3 ligase, resulting in ubiquitylation and proteosome-dependent degradation of the target. In the clinic, the oral route of administration is the option of choice but has only been achieved so far by CRBN- recruiting bifunctional degrader molecules. We aimed to achieve orally bioavailable molecules that selectively degrade the BAF Chromatin Remodelling complex ATPase SMARCA2 over its closely related paralogue SMARCA4, to allow in vivo evaluation of the synthetic lethality concept of SMARCA2 dependency in SMARCA4-deficient cancers. Here we outline structure- and property-guided approaches that led to orally bioavailable VHL-recruiting degraders. Our tool compound, ACBI2, shows selective degradation of SMARCA2 over SMARCA4 in ex vivo human whole blood assays and in vivo efficacy in SMARCA4-deficient cancer models. This study demonstrates the feasibility for broadening the E3 ligase and physicochemical space that can be utilised for achieving oral efficacy with bifunctional molecules.


Assuntos
Adenosina Trifosfatases , Fatores de Transcrição , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , DNA Helicases/genética , DNA Helicases/metabolismo , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteólise , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismo
6.
Cell Death Differ ; 27(8): 2417-2432, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32081986

RESUMO

Second generation TRAIL-based therapeutics, combined with sensitising co-treatments, have recently entered clinical trials. However, reliable response predictors for optimal patient selection are not yet available. Here, we demonstrate that a novel and translationally relevant hexavalent TRAIL receptor agonist, IZI1551, in combination with Birinapant, a clinically tested IAP antagonist, efficiently induces cell death in various melanoma models, and that responsiveness can be predicted by combining pathway analysis, data-driven modelling and pattern recognition. Across a panel of 16 melanoma cell lines, responsiveness to IZI1551/Birinapant was heterogeneous, with complete resistance and pronounced synergies observed. Expression patterns of TRAIL pathway regulators allowed us to develop a combinatorial marker that predicts potent cell killing with high accuracy. IZI1551/Birinapant responsiveness could be predicted not only for cell lines, but also for 3D tumour cell spheroids and for cells directly isolated from patient melanoma metastases (80-100% prediction accuracies). Mathematical parameter reduction identified 11 proteins crucial to ensure prediction accuracy, with x-linked inhibitor of apoptosis protein (XIAP) and procaspase-3 scoring highest, and Bcl-2 family members strongly represented. Applied to expression data of a cohort of n = 365 metastatic melanoma patients in a proof of concept in silico trial, the predictor suggested that IZI1551/Birinapant responsiveness could be expected for up to 30% of patient tumours. Overall, response frequencies in melanoma models were very encouraging, and the capability to predict melanoma sensitivity to combinations of latest generation TRAIL-based therapeutics and IAP antagonists can address the need for patient selection strategies in clinical trials based on these novel drugs.


Assuntos
Proteínas Inibidoras de Apoptose/antagonistas & inibidores , Reconhecimento Automatizado de Padrão , Ligante Indutor de Apoptose Relacionado a TNF/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dipeptídeos/farmacologia , Humanos , Indóis/farmacologia , Proteínas Inibidoras de Apoptose/metabolismo , Metástase Neoplásica
7.
Cell Death Differ ; 26(8): 1365-1378, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-30323272

RESUMO

Malignant melanoma is a highly aggressive form of skin cancer responsible for the majority of skin cancer-related deaths. Recent insight into the heterogeneous nature of melanoma suggests more personalised treatments may be necessary to overcome drug resistance and improve patient care. To this end, reliable molecular signatures that can accurately predict treatment responsiveness need to be identified. In this study, we applied multiplex phosphoproteomic profiling across a panel of 24 melanoma cell lines with different disease-relevant mutations, to predict responsiveness to MEK inhibitor trametinib. Supported by multivariate statistical analysis and multidimensional pattern recognition algorithms, the responsiveness of individual cell lines to trametinib could be predicted with high accuracy (83% correct predictions), independent of mutation status. We also successfully employed this approach to case specifically predict whether individual melanoma cell lines could be sensitised to trametinib. Our predictions identified that combining MEK inhibition with selective targeting of c-JUN and/or FAK, using siRNA-based depletion or pharmacological inhibitors, sensitised resistant cell lines and significantly enhanced treatment efficacy. Our study indicates that multiplex proteomic analyses coupled with pattern recognition approaches could assist in personalising trametinib-based treatment decisions in the future.


Assuntos
Antineoplásicos/farmacologia , Melanoma/tratamento farmacológico , Piridonas/farmacologia , Pirimidinonas/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Melanoma/metabolismo , Melanoma/patologia
8.
Oncol Res ; 25(9): 1489-1494, 2017 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-28337955

RESUMO

Antagonists of inhibitors of apoptosis proteins (IAPs), alone or in combination with genotoxic therapeutics, have been shown to efficiently induce cell death in various solid tumors. The IAP antagonist birinapant is currently being tested in phase II clinical trials. We herein aimed to investigate the antitumor efficacy of dacarbazine in vitro, both as a single agent and in combination with birinapant, in melanoma cell lines. Covering clinically relevant drug concentration ranges, we conducted a total of 5,400 measurements in a panel of 12 human melanoma cell lines representing different stages of disease progression. Surprisingly, functionally relevant synergies or response potentiation in combination treatments was not observed, and only one cell line modestly responded to birinapant single treatment (approximately 16% cell death). Although we did not study the underlying resistance mechanisms or more complex in vivo scenarios in which dacarbazine/birinapant response synergies may still possibly manifest, our findings are nevertheless noteworthy because IAP antagonists were demonstrated to strongly enhance responses to DNA-damaging agents in cell lines of other cancer types under comparable experimental conditions in vitro.


Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Dipeptídeos/farmacologia , Indóis/farmacologia , Melanoma/tratamento farmacológico , Neoplasias Cutâneas/tratamento farmacológico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Dacarbazina/administração & dosagem , Dipeptídeos/administração & dosagem , Humanos , Indóis/administração & dosagem , Melanoma/patologia , Mutação , Neoplasias Cutâneas/patologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA