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1.
Ann Hematol ; 100(8): 2023-2029, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34110462

RESUMO

Resistance remains the major clinical challenge for the therapy of Philadelphia chromosome-positive (Ph+) leukemia. With the exception of ponatinib, all approved tyrosine kinase inhibitors (TKIs) are unable to inhibit the common "gatekeeper" mutation T315I. Here we investigated the therapeutic potential of crizotinib, a TKI approved for targeting ALK and ROS1 in non-small cell lung cancer patients, which inhibited also the ABL1 kinase in cell-free systems, for the treatment of advanced and therapy-resistant Ph+ leukemia. By inhibiting the BCR-ABL1 kinase, crizotinib efficiently suppressed growth of Ph+ cells without affecting growth of Ph- cells. It was also active in Ph+ patient-derived long-term cultures (PD-LTCs) independently of the responsiveness/resistance to other TKIs. The efficacy of crizotinib was confirmed in vivo in syngeneic mouse models of BCR-ABL1- or BCR-ABL1T315I-driven chronic myeloid leukemia-like disease and in BCR-ABL1-driven acute lymphoblastic leukemia (ALL). Although crizotinib binds to the ATP-binding site, it also allosterically affected the myristol binding pocket, the binding site of GNF2 and asciminib (former ABL001). Therefore, crizotinib has a seemingly unique double mechanism of action, on the ATP-binding site and on the myristoylation binding pocket. These findings strongly suggest the clinical evaluation of crizotinib for the treatment of advanced and therapy-resistant Ph+ leukemia.


Assuntos
Antineoplásicos/farmacologia , Crizotinibe/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Humanos , Células Jurkat , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/metabolismo , Camundongos , Mutação/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo
2.
Protein Sci ; 33(1): e4840, 2024 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-37984441

RESUMO

Autophagy is a highly conserved cellular process that allows degradation of large macromolecules. p62/SQSTM1 is a key adaptor protein that interacts both with material to be degraded and with LC3 at the autophagosome, enabling degradation of cargos such as protein aggregates, lipid droplets and damaged organelles by selective autophagy. Dysregulation of autophagy contributes to the pathogenesis of many diseases. In this study, we investigated if the interaction of p62/SQSTM1 with LC3B could be regulated. We purified full-length p62/SQSTM1 and established an in vitro assay that measures the interaction with LC3B. We used the assay to determine the role of the different domains of p62/SQSTM1 in the interaction with LC3B. We identified a mechanism of regulation of p62/SQSTM1 where the ZZ and the PB1 domains regulate the exposure of the LIR-sequence to enable or inhibit the interaction with LC3B. A mutation to mimic the phosphorylation of a site on the ZZ domain leads to increased interaction with LC3B. Also, a small compound that binds to the ZZ domain enhances interaction with LC3B. Dysregulation of these mechanisms in p62/SQSTM1 could have implications for diseases where autophagy is affected. In conclusion, our study highlights the regulated nature of p62/SQSTM1 and its ability to modulate the interaction with LC3B through a LIR-sequence Accessibility Mechanism (LAM). Furthermore, our findings suggest the potential for pharmacological modulation of the exposure of LIR, paving the way for future therapeutic strategies.


Assuntos
Autofagossomos , Proteínas Associadas aos Microtúbulos , Autofagossomos/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Autofagia/genética
3.
J Med Chem ; 64(7): 3720-3746, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33769048

RESUMO

Autophagy is the common name for a number of lysosome-based degradation pathways of cytosolic cargos. The key components of autophagy are members of Atg8 family proteins involved in almost all steps of the process, from autophagosome formation to their selective fusion with lysosomes. In this study, we show that the homologous members of the human Atg8 family proteins, LC3A and LC3B, are druggable by a small molecule inhibitor novobiocin. Structure-activity relationship (SAR) studies of the 4-hydroxy coumarin core scaffold were performed, supported by a crystal structure of the LC3A dihydronovobiocin complex. The study reports the first nonpeptide inhibitors for these protein interaction targets and will lay the foundation for the development of more potent chemical probes for the Atg8 protein family which may also find applications for the development of autophagy-mediated degraders (AUTACs).


Assuntos
4-Hidroxicumarinas/farmacologia , Autofagia/efeitos dos fármacos , Proteínas Associadas aos Microtúbulos/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteína Sequestossoma-1/metabolismo , 4-Hidroxicumarinas/síntese química , 4-Hidroxicumarinas/metabolismo , Células HEK293 , Humanos , Ligantes , Estrutura Molecular , Novobiocina/química , Relação Estrutura-Atividade
4.
Front Immunol ; 9: 1656, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30072997

RESUMO

BACKGROUND: A significant amount of common variable immunodeficiency (CVID) patients manifest with autoimmunity. Particularly, autoimmune thrombocytopenia (AITP) is commonly seen. Intravenous immunoglobulins (IVIG) are an established treatment option for both, CVID and AITP. Nonetheless, due to fewer systemic side effects, immunoglobulins are increasingly applied subcutaneously (SCIG). OBJECTIVE: To compare the efficacy and safety of IVIG and SCIG treatment in patients with both CVID and clinical relevant thrombocytopenia in the prevention of AITP bouts. METHODS: Patients with both CVID and AITP were enrolled at the Centre for Chronic Immunodeficiency in Freiburg, Germany and at the Royal Free Hospital, London, UK. Clinical and laboratory features of patients were collected and analyzed. RESULTS: This retrospective study recruited 61 adult patients between 19 and 71 years of age who had a diagnosis of CVID and at least one bout of thrombocytopenia defined as a platelet count of <50,000/µl if bleeding episodes occurred, or a platelet count of <20,000/µl without bleeding. Thirty patients received immunoglobulin through IVIG, and 31 patients were on SCIG replacement. One patient of the IVIG-group was excluded, because of a diffuse large B-cell lymphoma. We did not find a higher occurrence of thrombocytopenic events in CVID patients who received SCIG, compared to CVID patients who had IVIG, but we identified a low IgG through level as a risk factor for AITP bouts. CONCLUSION: SCIG is at least as safe as IVIG for patients with CVID and concomitant AITP. However, an IgG through level under 7 g/l is a key factor for the development of AITP.

5.
ACS Chem Biol ; 13(8): 1921-1931, 2018 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-29927572

RESUMO

The Polo-like kinases (Plks) are an evolutionary conserved family of Ser/Thr protein kinases that possess, in addition to the classical kinase domain at the N-terminus, a C-terminal polo-box domain (PBD) that binds to phosphorylated proteins and modulates the kinase activity and its localization. Plk1, which regulates the formation of the mitotic spindle, has emerged as a validated drug target for the treatment of cancer, because it is required for numerous types of cancer cells but not for the cell division in noncancer cells. Here, we employed chemical biology methods to investigate the allosteric communication between the PBD and the catalytic domain of Plk1. We identified small compounds that bind to the catalytic domain and inhibit or enhance the interaction of Plk1 with the phosphorylated peptide PoloBoxtide in vitro. In cells, two new allosteric Plk1 inhibitors affected the proliferation of cancer cells in culture and the cell cycle but had distinct phenotypic effects on spindle formation. Both compounds inhibited Plk1 signaling, indicating that they specifically act on Plk1 in cultured cells.


Assuntos
Proteínas de Ciclo Celular/agonistas , Proteínas de Ciclo Celular/antagonistas & inibidores , Ativadores de Enzimas/química , Inibidores de Proteínas Quinases/química , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/agonistas , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/química , Regulação Alostérica/efeitos dos fármacos , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Domínio Catalítico , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Centrossomo/metabolismo , Ativadores de Enzimas/farmacologia , Pontos de Checagem da Fase G2 do Ciclo Celular/efeitos dos fármacos , Células HeLa , Humanos , Cinetocoros/metabolismo , Oligopeptídeos/química , Fosfopeptídeos/química , Fosfopeptídeos/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/química , Proteínas Proto-Oncogênicas/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Spodoptera/química , Quinase 1 Polo-Like
6.
Cell Adh Migr ; 5(3): 215-9, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21339704

RESUMO

Cell adhesion is an important part of many complex biological processes. It plays crucial roles in cancer, development, and maintenance of stem cell compartment. The measurement of adhesion under experimental conditions might provide important information for cell biology. There are several protocols to measure adhesion, usually based on washing or shaking to remove non-adherent cells. Here, we describe a quantification method based on gravitational force to measure adhesion in a 96-well format. Non-adherent cells are separated and only vital cells are quantified with a colorimetric assay. As example we provide the quantification of cell-cell interaction with blocking function antibodies for CD44, an N-cadherin antagonists and the stromal cell derived factor-1 alpha (SDF-1). This method facilitates fast and reliable measurement of cell adhesion in multiwell format for screening assays.


Assuntos
Adesão Celular/fisiologia , Adesão Celular/genética , Linhagem Celular , Linhagem Celular Tumoral , Quimiocina CXCL12/metabolismo , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/metabolismo , Humanos , Receptores de Hialuronatos/metabolismo , Células Jurkat/citologia
7.
Stem Cell Res ; 4(2): 129-39, 2010 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-20116358

RESUMO

Specific cell-cell junctions between hematopoietic stem cells (HSC) and their niche have been shown to regulate stem cell function. N-cadherin was suggested to play a central role in this process, whereas other studies indicated that it did not play an essential role in the murine model. We have analyzed the role of N-cadherin for interaction between hematopoietic progenitor cells (HPC) and supportive mesenchymal stromal cells (MSC) in a human-human setting. Expression of N-cadherin and of cadherin-11 (osteoblast cadherin) was analyzed in HPC by quantitative RT-PCR, Western blot, and flow cytometry. N-cadherin and cadherin-11 were expressed in HPC at a moderate level, whereas they were not detectable in differentiated cells. Confocal laser scanning microscopy revealed that N-cadherin and beta-catenin are colocalized at the junction of HPC and MSC. siRNA knockdown of N-cadherin or cadherin-11 as well as treatment with the blocking function antibody decreased adhesive interaction of HPC to MSC. Furthermore, knockdown of N-cadherin or blocking function antibody impaired maintenance of long-term culture-initiating cells (LTC-IC) on coculture of HPC and MSC. These results indicate that N-cadherin is involved in the bidirectional interaction of human HPC with their cellular determinants in the niche.


Assuntos
Caderinas/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células-Tronco Mesenquimais/metabolismo , Western Blotting , Caderinas/genética , Células Cultivadas , Citometria de Fluxo , Humanos , Reação em Cadeia da Polimerase Via Transcriptase Reversa
8.
Exp Hematol ; 37(12): 1423-34, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19819294

RESUMO

OBJECTIVE: Animal models have provided evidence for the existence of leukemia stem cells (LSC). However, prospective isolation of human LSC from patients with acute myeloid leukemia (AML), as well as the assessment of their clinical significance, has remained a major challenge. MATERIALS AND METHODS: We have studied the functional characteristics of a subset of leukemia cells that expressed CD34 and high aldehyde dehydrogenase activity (ALDH(br)), which was freshly isolated from the mononuclear cells at the time of diagnosis from the marrow of 68 consecutive patients suffering from AML. RESULTS: The percentage of ALDH(br) cells ranged from 0.01% to 16.0% with a median of 0.5%. Compared to their counterparts with low aldehyde dehydrogenase activity from the same individual patients, the ALDH(br) population showed a significantly higher affinity to human mesenchymal stromal cells (n=12; p<0.01), a more than twofold higher proportion of slow-dividing and quiescent cells (n=4; p<0.05), higher numbers of long-term culture-initiating cell colonies in vitro (n=25; p<0.01), and an enhanced engraftment in the nonobese diabetic/severe combined immunodeficient mouse model (n=3; p<0.05). Above all, we found that the frequency of ALDH(br) cells correlated significantly with diminished survival probability (p=0.025) and with adverse cytogenetic factors (p<0.05). CONCLUSION: A small proportion of leukemia cells derived from the marrow of patients with AML were ALDH(br) and CD34(+). They demonstrated functional characteristics of LSC and high percentages of these cells among the leukemia cells correlated significantly with poor clinical outcome.


Assuntos
Aldeído Desidrogenase/metabolismo , Leucemia Mieloide/enzimologia , Células-Tronco Neoplásicas/enzimologia , Doença Aguda , Adulto , Idoso , Aldeído Desidrogenase/genética , Animais , Antígenos CD34/metabolismo , Feminino , Citometria de Fluxo , Humanos , Leucemia Experimental/enzimologia , Leucemia Experimental/patologia , Leucemia Mieloide/sangue , Leucemia Mieloide/patologia , Masculino , Mesoderma/patologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Células-Tronco Neoplásicas/patologia , Prognóstico , Células Estromais/enzimologia , Células Estromais/patologia , Análise de Sobrevida , Transplante Heterólogo , Células Tumorais Cultivadas , Adulto Jovem
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