Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 1.522
Filtrar
1.
PLoS One ; 16(12): e0261227, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34962936

RESUMO

We examined keratin aggregate formation and the possible mechanisms involved. With this aim, we observed the effect that different ratios between mutant and wild-type keratins expressed in cultured keratinocytes may have on aggregate formation in vitro, as well as how keratin aggregate formation affects the mechanical properties of cells at the cell cortex. To this end we prepared clones with expression rates as close as possible to 25%, 50% and 100% of the EGFP-K14 proteins (either WT or R125P and V270M mutants). Our results showed that only in the case of the 25% EGFP-K14 R125P mutant significant differences could be seen. Namely, we observed in this case the largest accumulation of keratin aggregates and a significant reduction in cell stiffness. To gain insight into the possible mechanisms behind this observation, we extended our previous mathematical model of keratin dynamics by implementing a more complex reaction network that considers the coexistence of wild-type and mutant keratins in the cell. The new model, consisting of a set of coupled, non-linear, ordinary differential equations, allowed us to draw conclusions regarding the relative amounts of intermediate filaments and aggregates in cells, and suggested that aggregate formation by asymmetric binding between wild-type and mutant keratins could explain the data obtained on cells grown in culture.


Assuntos
Queratinócitos/metabolismo , Queratinas/química , Proteínas Mutantes/química , Agregados Proteicos , Linhagem Celular , Simulação por Computador , Corantes Fluorescentes/química , Proteínas de Fluorescência Verde/metabolismo , Humanos , Queratinócitos/efeitos dos fármacos , Modelos Biológicos , Inibidores de Proteassoma/farmacologia , Agregados Proteicos/efeitos dos fármacos
2.
Cells ; 10(12)2021 12 11.
Artigo em Inglês | MEDLINE | ID: mdl-34944009

RESUMO

The proteasome increases its activity at the onset of sperm capacitation due to the action of the SACY/PRKACA pathway; this increase is required for capacitation to progress. PRKA activity also increases and remains high during capacitation. However, intracellular levels of cAMP decrease in this process. Our goal was to evaluate the role of the proteasome in regulating PRKA activity once capacitation has started. Viable human sperm were incubated in the presence and absence of epoxomicin or with 0.1% DMSO. The activity of PRKA; the phosphorylation pattern of PRKA substrates (pPRKAs); and the expression of PRKAR1, PRKAR2, and AKAP3 were evaluated by Western blot. The localization of pPRKAs, PRKAR1, PRKAR2, and AKAP3 was evaluated by immunofluorescence. Treatment with epoxomicin changed the localization and phosphorylation pattern and decreased the percentage of pPRKAs-positive sperm. PRKA activity significantly increased at 1 min of capacitation and remained high throughout the incubation. However, epoxomicin treatment significantly decreased PRKA activity after 30 min. In addition, PRKAR1 and AKAP3 were degraded by the proteasome but with a different temporal kinetic. Our results suggest that PRKAR1 is the target of PRKA regulation by the proteasome.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Capacitação Espermática/fisiologia , Proteínas de Ancoragem à Quinase A/metabolismo , Adulto , Humanos , Fosforilação/efeitos dos fármacos , Inibidores de Proteassoma/farmacologia , Proteólise/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Capacitação Espermática/efeitos dos fármacos , Frações Subcelulares/metabolismo , Especificidade por Substrato/efeitos dos fármacos , Adulto Jovem
3.
Cells ; 10(12)2021 12 06.
Artigo em Inglês | MEDLINE | ID: mdl-34943940

RESUMO

Constitutive- and immunoproteasomes are part of the ubiquitin-proteasome system (UPS), which is responsible for the protein homeostasis. Selective inhibition of the immunoproteasome offers opportunities for the treatment of numerous diseases, including inflammation, autoimmune diseases, and hematologic malignancies. Although several inhibitors have been reported, selective nonpeptidic inhibitors are sparse. Here, we describe two series of compounds that target both proteasomes. First, benzoxazole-2-carbonitriles as fragment-sized covalent immunoproteasome inhibitors are reported. Systematic substituent scans around the fragment core of benzoxazole-2-carbonitrile led to compounds with single digit micromolar inhibition of the ß5i subunit. Experimental and computational reactivity studies revealed that the substituents do not affect the covalent reactivity of the carbonitrile warhead, but mainly influence the non-covalent recognition. Considering the small size of the inhibitors, this finding emphasizes the importance of the non-covalent recognition step in the covalent mechanism of action. As a follow-up series, bidentate inhibitors are disclosed, in which electrophilic heterocyclic fragments, i.e., 2-vinylthiazole, benzoxazole-2-carbonitrile, and benzimidazole-2-carbonitrile were linked to threonine-targeting (R)-boroleucine moieties. These compounds were designed to bind both the Thr1 and ß5i-subunit-specific residue Cys48. However, inhibitory activities against (immuno)proteasome subunits showed that bidentate compounds inhibit the ß5, ß5i, ß1, and ß1i subunits with submicromolar to low-micromolar IC50 values. Inhibitory assays against unrelated enzymes showed that compounds from both series are selective for proteasomes. The presented nonpeptidic and covalent derivatives are suitable hit compounds for the development of either ß5i-selective immunoproteasome inhibitors or compounds targeting multiple subunits of both proteasomes.


Assuntos
Cisteína/química , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Treonina/química , Ubiquitina/química , Doenças Autoimunes/imunologia , Doenças Autoimunes/patologia , Benzoxazóis/química , Benzoxazóis/farmacologia , Química Computacional , Cisteína/imunologia , Neoplasias Hematológicas/imunologia , Neoplasias Hematológicas/patologia , Humanos , Inflamação/imunologia , Inflamação/patologia , Modelos Moleculares , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/imunologia , Inibidores de Proteassoma/química , Inibidores de Proteassoma/farmacologia , Subunidades Proteicas/química , Subunidades Proteicas/imunologia , Relação Estrutura-Atividade , Treonina/imunologia , Ubiquitina/imunologia
4.
Cells ; 10(12)2021 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-34944095

RESUMO

Cell-mediated immunity is driven by antigenic peptide presentation on major histocompatibility complex (MHC) molecules. Specialized proteasome complexes called immunoproteasomes process viral, bacterial, and tumor antigens for presentation on MHC class I molecules, which can induce CD8 T cells to mount effective immune responses. Immunoproteasomes are distinguished by three subunits that alter the catalytic activity of the proteasome and are inducible by inflammatory stimuli such as interferon-γ (IFN-γ). This inducible activity places them in central roles in cancer, autoimmunity, and inflammation. While accelerated proteasomal degradation is an important tumorigenic mechanism deployed by several cancers, there is some ambiguity regarding the role of immunoproteasome induction in neoplastic transformation. Understanding the mechanistic and functional relevance of the immunoproteasome provides essential insights into developing targeted therapies, including overcoming resistance to standard proteasome inhibition and immunomodulation of the tumor microenvironment. In this review, we discuss the roles of the immunoproteasome in different cancers.


Assuntos
Neoplasias/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Subunidades Proteicas/imunologia , Animais , Humanos , Modelos Biológicos , Neoplasias/tratamento farmacológico , Neoplasias/patologia , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Subunidades Proteicas/química
5.
Biomolecules ; 11(12)2021 11 30.
Artigo em Inglês | MEDLINE | ID: mdl-34944433

RESUMO

The proteasome system is a large and complex molecular machinery responsible for the degradation of misfolded, damaged, and redundant cellular proteins. When proteasome function is impaired, unwanted proteins accumulate, which can lead to several diseases including age-related and neurodegenerative diseases. Enhancing proteasome-mediated substrate degradation with small molecules may therefore be a valuable strategy for the treatment of various neurodegenerative diseases such as Parkinson's, Alzheimer's, and Huntington's diseases. In this review, we discuss the structure of proteasome and how proteasome's proteolytic activity is associated with aging and various neurodegenerative diseases. We also summarize various classes of compounds that are capable of enhancing, directly or indirectly, proteasome-mediated protein degradation.


Assuntos
Envelhecimento/metabolismo , Doenças Neurodegenerativas/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Bibliotecas de Moléculas Pequenas/farmacologia , Humanos , Inibidores de Proteassoma/farmacologia , Dobramento de Proteína , Proteólise/efeitos dos fármacos
6.
J Biomed Sci ; 28(1): 75, 2021 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-34758826

RESUMO

BACKGROUND: A new strategy, particularly a novel combination, for immunotherapy in microsatellite stable metastatic colorectal cancer (mCRC) treatment needs to be formulated. Studies on the interferon-γ (IFN-γ)/ Janus kinase (JAK)/ signal transducer and activator of transcription (STAT)1 pathway provide new directions in this regard. METHODS: Our study applies three colon cancer cell lines, including microsatellite stable (MSS) cell lines, which are SW480 and SW620, and microsatellite instability-high (MSI-H) cell line, which is DLD-1. We compared the expressions of immune surface markers on colon cancer cells in response to IFN-γ. We elucidated these mechanisms, which involved the upregulation of immune surface markers. Furthermore, we examined real-world clinical samples using the PerkinElmer Opal multiplex system and NanoString analysis. RESULTS: We established that the baseline expression of major histocompatibility complex (MHC) class I alleles and programmed death-ligand 1 (PD-L1) were generally low in cell line models. The immune surface markers were significantly increased after IFN-γ stimulation on SW480 but were notably unresponsive on the SW620 cell line. We discovered that STAT1 and phosphorylated STAT1 (pSTAT1) were downregulated in the SW620 cell line. We verified that the STAT1/pSTAT1 could be restored through the application of proteasome inhibitors, especially bortezomib. The expression of MHC class I as downstream signals of STAT1 was also up-regulated by proteasome inhibitors. The similar results were reproduced in DLD-1 cell line, which was also initially unresponsive to IFN-γ. In real-world samples of patients with mCRC, we found that higher STAT1 expression in tumor cells was strongly indicative of a highly immunogenic microenvironment, with significantly higher expression levels of MHC class I and PD-L1, not only on tumor cells but also on non-tumor cells. Furthermore, tumor infiltrating lymphocytes (TILs) were increased in the positive-STAT1 group. Through NanoString analysis, we confirmed that the mRNA expressions of IFN-γ, human leukocyte antigen (HLA)-A, HLA-E, and HLA-G were also significantly higher in the positive-STAT1 group than those in the negative-STAT1 group. CONCLUSION: Our study provides a novel rationale for the addition of bortezomib, a proteasome inhibitor, into new immunotherapy combinations.


Assuntos
Neoplasias do Colo/fisiopatologia , Expressão Gênica/efeitos dos fármacos , Genes MHC Classe I/efeitos dos fármacos , Antígenos de Histocompatibilidade Classe I/genética , Inibidores de Proteassoma/farmacologia , Fator de Transcrição STAT1/genética , Transdução de Sinais/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Fator de Transcrição STAT1/metabolismo
7.
Int J Mol Sci ; 22(19)2021 Sep 23.
Artigo em Inglês | MEDLINE | ID: mdl-34638592

RESUMO

The development of neuropathy and of mood alterations is frequent after chemotherapy. These complications, independent from the antitumoral mechanism, are interconnected due to an overlapping in their processing pathways and a common neuroinflammatory condition. This study aims to verify whether in mice the treatment with the proteasome inhibitor bortezomib (BTZ), at a protocol capable of inducing painful neuropathy, is associated with anxiety, depression and supraspinal neuroinflammation. We also verify if the therapeutic treatment with the antagonist of the prokineticin (PK) system PC1, which is known to contrast pain and neuroinflammation, can prevent mood alterations. Mice were treated with BTZ (0.4 mg/kg three times/week for 4 weeks); mechanical allodynia and locomotor activity were evaluated over time while anxiety (dark light and marble burying test), depression (sucrose preference and swimming test) and supraspinal neuroinflammation were checked at the end of the protocol. BTZ treated neuropathic mice develop anxiety and depression. The presence of mood alterations is related to the presence of neuroinflammation and PK system activation in prefrontal cortex, hippocampus and hypothalamus with high levels of PK2 and PKR2 receptor, IL-6 and TNF-α, TLR4 and an upregulation of glial markers. PC1 treatment, counteracting pain, prevented the development of supraspinal inflammation and depression-like behavior in BTZ mice.


Assuntos
Afeto/efeitos dos fármacos , Bortezomib/farmacologia , Inibidores de Proteassoma/farmacologia , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismo , Animais , Ansiedade/tratamento farmacológico , Ansiedade/metabolismo , Biomarcadores/metabolismo , Citocinas/metabolismo , Depressão/tratamento farmacológico , Depressão/metabolismo , Modelos Animais de Doenças , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Locomoção/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dor/tratamento farmacológico , Dor/metabolismo , Doenças do Sistema Nervoso Periférico/tratamento farmacológico , Doenças do Sistema Nervoso Periférico/metabolismo , Córtex Pré-Frontal/efeitos dos fármacos , Córtex Pré-Frontal/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Regulação para Cima/efeitos dos fármacos
8.
Zhong Nan Da Xue Xue Bao Yi Xue Ban ; 46(8): 900-908, 2021 Aug 28.
Artigo em Inglês, Chinês | MEDLINE | ID: mdl-34565737

RESUMO

Multiple myeloma (MM) is a highly heterogeneous malignant plasma cell disease. Proteasome inhibitors (PIs) are the first line of medicine for MM. Bortezomib, ixazomib, and carfilzomib are also widely used for MM. Marizomib, oprozomib, and KZR-616 are in clinical trials. However, the drug resistance of PIs in MM is still a problem. The mechanisms for PIs resistance to MM include ubiquitin-proteasome pathway, autophagy lysosome pathway, endoplasmic reticulum stress pathway, cell survival signal pathway, exosome-mediated resistance, and bone marrow microenvironment-mediated resistance.


Assuntos
Mieloma Múltiplo , Inibidores de Proteassoma , Autofagia , Medula Óssea , Bortezomib , Humanos , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Microambiente Tumoral
9.
Molecules ; 26(18)2021 Sep 14.
Artigo em Inglês | MEDLINE | ID: mdl-34577052

RESUMO

Multiple myeloma is an incurable plasma cell neoplastic disease representing about 10-15% of all haematological malignancies diagnosed in developed countries. Proteasome is a key player in multiple myeloma and proteasome inhibitors are the current first-line of treatment. However, these are associated with limited clinical efficacy due to acquired resistance. One of the solutions to overcome this problem is a polypharmacology approach, namely combination therapy and multitargeting drugs. Several polypharmacology avenues are currently being explored. The simultaneous inhibition of EZH2 and Proteasome 20S remains to be investigated, despite the encouraging evidence of therapeutic synergy between the two. Therefore, we sought to bridge this gap by proposing a holistic in silico strategy to find new dual-target inhibitors. First, we assessed the characteristics of both pockets and compared the chemical space of EZH2 and Proteasome 20S inhibitors, to establish the feasibility of dual targeting. This was followed by molecular docking calculations performed on EZH2 and Proteasome 20S inhibitors from ChEMBL 25, from which we derived a predictive model to propose new EZH2 inhibitors among Proteasome 20S compounds, and vice versa, which yielded two dual-inhibitor hits. Complementarily, we built a machine learning QSAR model for each target but realised their application to our data is very limited as each dataset occupies a different region of chemical space. We finally proceeded with molecular dynamics simulations of the two docking hits against the two targets. Overall, we concluded that one of the hit compounds is particularly promising as a dual-inhibitor candidate exhibiting extensive hydrogen bonding with both targets. Furthermore, this work serves as a framework for how to rationally approach a dual-targeting drug discovery project, from the selection of the targets to the prediction of new hit compounds.


Assuntos
Descoberta de Drogas , Mieloma Múltiplo , Linhagem Celular Tumoral , Humanos , Simulação de Acoplamento Molecular , Proteínas Oncogênicas , Inibidores de Proteassoma/farmacologia
10.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34548400

RESUMO

The Plasmodium falciparum proteasome is a potential antimalarial drug target. We have identified a series of amino-amide boronates that are potent and specific inhibitors of the P. falciparum 20S proteasome (Pf20S) ß5 active site and that exhibit fast-acting antimalarial activity. They selectively inhibit the growth of P. falciparum compared with a human cell line and exhibit high potency against field isolates of P. falciparum and Plasmodium vivax They have a low propensity for development of resistance and possess liver stage and transmission-blocking activity. Exemplar compounds, MPI-5 and MPI-13, show potent activity against P. falciparum infections in a SCID mouse model with an oral dosing regimen that is well tolerated. We show that MPI-5 binds more strongly to Pf20S than to human constitutive 20S (Hs20Sc). Comparison of the cryo-electron microscopy (EM) structures of Pf20S and Hs20Sc in complex with MPI-5 and Pf20S in complex with the clinically used anti-cancer agent, bortezomib, reveal differences in binding modes that help to explain the selectivity. Together, this work provides insights into the 20S proteasome in P. falciparum, underpinning the design of potent and selective antimalarial proteasome inhibitors.


Assuntos
Compostos de Boro/farmacologia , Malária Falciparum/tratamento farmacológico , Plasmodium falciparum/efeitos dos fármacos , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Administração Oral , Animais , Compostos de Boro/administração & dosagem , Compostos de Boro/química , Domínio Catalítico , Humanos , Malária Falciparum/enzimologia , Malária Falciparum/parasitologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Moleculares , Plasmodium falciparum/enzimologia , Inibidores de Proteassoma/administração & dosagem , Inibidores de Proteassoma/química
11.
FEBS Lett ; 595(18): 2383-2394, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34358326

RESUMO

Maintenance of the proteome (proteostasis) is essential for cellular homeostasis and prevents cytotoxic stress responses that arise from protein misfolding. However, little is known about how different types of misfolded proteins impact homeostasis, especially when protein degradation pathways are compromised. We examined the effects of misfolded protein expression on yeast growth by characterizing a suite of substrates possessing the same aggregation-prone domain but engaging different quality control pathways. We discovered that treatment with a proteasome inhibitor was more toxic in yeast expressing misfolded membrane proteins, and this growth defect was mirrored in yeast lacking a proteasome-specific transcription factor, Rpn4p. These results highlight weaknesses in the proteostasis network's ability to handle the stress arising from an accumulation of misfolded membrane proteins.


Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/classificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Processos de Crescimento Celular/efeitos dos fármacos , Citoplasma/metabolismo , Proteínas de Ligação a DNA/deficiência , Degradação Associada com o Retículo Endoplasmático , Proteínas de Choque Térmico/metabolismo , Nucleotídeos/metabolismo , Inibidores de Proteassoma/farmacologia , Ligação Proteica , Domínios Proteicos , Proteólise , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/química , Fatores de Transcrição/deficiência
12.
Phytother Res ; 35(10): 5781-5794, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34363252

RESUMO

Cinnamon contains bioactive substances with diverse medicinal properties. We investigated the anticancer potential of abundant monophenols from cinnamon, namely, cinnamaldehyde, cinnamic acid, and eugenol, by hypothesizing that they possess proteasome inhibitory activities capable of suppressing cancer cell proliferation and inducing apoptosis. This hypothesis was tested by evaluating proteasome inhibitory activities of the compounds, and assessing downstream molecular and cellular events that are known to be impacted by proteasome inhibitors. The cinnamon compounds inhibited the catalytic activities of the proteasome in prostate cancer cells, but not in normal cells. Treatment with cinnamon compounds or the synthetic proteasome inhibitor MG132 upregulated p27 and IkBα proteins, and downregulated FoxM1 and angiogenic markers. These molecular events were associated with the decreased proliferation of prostate cancer cells. Treatment with cinnamon compounds or MG132 upregulated the expression of genes associated with endoplasmic reticulum (ER) stress/unfolded protein response (BIP, PERK, CHOP, and XBP1(S)). Furthermore, cinnamon compounds or MG132 upregulated the expression of genes required for the assembly of the caspase-8 activation platform in autophagosomes (LC3B, ATG5, p62, and Beclin1). The autophagy inhibitor, 3-methyladenine, blocked the compounds-mediated activation of caspase-8 and its downstream target caspase-3. In conclusion, proteasome inhibition by aromatic monophenols from cinnamon inhibits proliferation and leads to the death of prostate cancer cells by autophagy-dependent apoptosis.


Assuntos
Neoplasias da Próstata , Inibidores de Proteassoma , Apoptose , Linhagem Celular Tumoral , Cinnamomum zeylanicum , Estresse do Retículo Endoplasmático , Proteína Forkhead Box M1 , Humanos , Casca de Planta , Neoplasias da Próstata/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma , Inibidores de Proteassoma/farmacologia
13.
Biol Aujourdhui ; 215(1-2): 1-23, 2021.
Artigo em Francês | MEDLINE | ID: mdl-34397372

RESUMO

The proteasome is the central component of the adaptable ubiquitin proteasome system (UPS) discovered in the 1980's. It sustains protein homeostasis (proteostasis) under a large variety of physiological and pathological conditions. Its dysregulation has been often associated to various human diseases. Its potential regulation by modulators has emerged as promising avenue to develop treatments of various pathologies. The FDA approval in 2003 of the proteasome inhibitor bortezomib to treat multiple myeloma, then mantle lymphoma in 2006, has considerably increased the clinical interest of proteasome inhibition. Second-generation proteasome inhibitors (carfilzomib and ixazomib) have been approved to overcome bortezomib resistance and improved toxicity profile and route of administration. Selective inhibition of immunoproteasome is a promising approach towards the development of immunomodulatory drugs. The design of these drugs relies greatly on the elucidation of high-resolution structures of the targeted proteasomes. The ATPase-dependent 26S proteasome (2.4 MDa) consists of a 20S proteolytic core and one or two 19S regulatory particles. The 20S core contains three types of catalytic sites. In recent years, due to technical advances especially in atomic cryo-electron microscopy, significant progress has been made in the understanding of 26S proteasome structure and its dynamics. Stepwise conformational changes of the 19S particle induced by ATP hydrolysis lead to substrate translocation, 20S pore opening and processive protein degradation by the 20S proteolytic subunits (2ß1, 2ß2 and 2ß5). A large variety of structurally different inhibitors, both natural products or synthetic compounds targeting immuno- and constitutive proteasomes, has been discovered. The latest advances in this drug discovery are presented. Knowledge about structures, inhibition mechanism and detailed biological regulations of proteasomes can guide strategies for the development of next-generation inhibitors to treat human diseases, especially cancers, immune disorders and pathogen infections. Proteasome activators are also potentially applicable to the reduction of proteotoxic stresses in neurodegeneration and aging.


Assuntos
Preparações Farmacêuticas , Complexo de Endopeptidases do Proteassoma , Microscopia Crioeletrônica , Descoberta de Drogas , Humanos , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico
14.
Int J Mol Sci ; 22(16)2021 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-34445660

RESUMO

Bortezomib (BTZ) has demonstrated its efficacy in several hematological disorders and has been associated with thrombocytopenia. There is controversy about the effect of BTZ on human platelets, so we set out to determine its effect on various types of platelet samples. Human platelets were investigated in platelet-rich plasma (PRP) and as gel-filtered platelets (GFPs). Mitochondrial inner membrane potential depolarization and phosphatidylserine (PS) and P-selectin expression levels were studied by flow cytometry, while thrombin generation was measured by a fluorescent method. In PRP, BTZ caused negligible PS expression after 60 min of treatment. However, in GFPs, PS expression was dose- and time-dependently increased in the BTZ-treated groups, as was P-selectin. The percentage of depolarized cells was also higher after BTZ pretreatment at both time points. Peak thrombin and velocity index increased significantly even with the lowest BTZ concentration (p = 0.0019; p = 0.0032) whereas time to peak and start tail parameters decreased (p = 0.0007; p = 0.0034). The difference between PRP and GFP results can be attributed to the presence of plasma proteins in PRP, as the PS-stimulating effect of BTZ could be attenuated by supplementing GFPs with purified human albumin. Overall, BTZ induces a procoagulant platelet phenotype in an experimental setting devoid of plasma proteins.


Assuntos
Apoptose , Coagulação Sanguínea/efeitos dos fármacos , Plaquetas/patologia , Bortezomib/farmacologia , Selectina-P/metabolismo , Ativação Plaquetária , Inibidores de Proteassoma/farmacologia , Antineoplásicos/farmacologia , Plaquetas/efeitos dos fármacos , Humanos , Selectina-P/genética
15.
J Med Chem ; 64(14): 10230-10245, 2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34228444

RESUMO

Proteasomes are broadly expressed key components of the ubiquitin-dependent protein degradation pathway containing catalytically active subunits (ß1, ß2, and ß5). LMP7 (ß5i) is a subunit of the immunoproteasome, an inducible isoform that is predominantly expressed in hematopoietic cells. Clinically effective pan-proteasome inhibitors for the treatment of multiple myeloma (MM) nonselectively target LMP7 and other subunits of the constitutive proteasome and immunoproteasome with comparable potency, which can limit the therapeutic applicability of these drugs. Here, we describe the discovery and structure-based hit optimization of novel amido boronic acids, which selectively inhibit LMP7 while sparing all other subunits. The exploitation of structural differences between the proteasome subunits culminated in the identification of the highly potent, exquisitely selective, and orally available LMP7 inhibitor 50 (M3258). Based on the strong antitumor activity observed with M3258 in MM models and a favorable preclinical data package, a phase I clinical trial was initiated in relapsed/refractory MM patients.


Assuntos
Descoberta de Drogas , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Relação Dose-Resposta a Droga , Humanos , Estrutura Molecular , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/química , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade
16.
J Med Chem ; 64(15): 10934-10950, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34309393

RESUMO

Previously, we reported that immunoproteasome (iP)-targeting linear peptide epoxyketones improve cognitive function in mouse models of Alzheimer's disease (AD) in a manner independent of amyloid ß. However, these compounds' clinical prospect for AD is limited due to potential issues, such as poor brain penetration and metabolic instability. Here, we report the development of iP-selective macrocyclic peptide epoxyketones prepared by a ring-closing metathesis reaction between two terminal alkenes attached at the P2 and P3/P4 positions of linear counterparts. We show that a lead macrocyclic compound DB-60 (20) effectively inhibits the catalytic activity of iP in ABCB1-overexpressing cells (IC50: 105 nM) and has metabolic stability superior to its linear counterpart. DB-60 (20) also lowered the serum levels of IL-1α and ameliorated cognitive deficits in Tg2576 mice. The results collectively suggest that macrocyclic peptide epoxyketones have improved CNS drug properties than their linear counterparts and offer promising potential as an AD drug candidate.


Assuntos
Doença de Alzheimer/tratamento farmacológico , Compostos Macrocíclicos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Doença de Alzheimer/metabolismo , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Compostos Macrocíclicos/síntese química , Compostos Macrocíclicos/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Estrutura Molecular , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/química , Relação Estrutura-Atividade
17.
Molecules ; 26(13)2021 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-34279386

RESUMO

The selective inhibition of immunoproteasome is a valuable strategy to treat autoimmune, inflammatory diseases, and hematologic malignancies. Recently, a new series of amide derivatives as non-covalent inhibitors of the ß1i subunit with Ki values in the low/submicromolar ranges have been identified. Here, we investigated the binding mechanism of the most potent and selective inhibitor, N-benzyl-2-(2-oxopyridin-1(2H)-yl)propanamide (1), to elucidate the steps from the ligand entrance into the binding pocket to the ligand-induced conformational changes. We carried out a total of 400 ns of MD-binding analyses, followed by 200 ns of plain MD. The trajectories clustering allowed identifying three representative poses evidencing new key interactions with Phe31 and Lys33 together in a flipped orientation of a representative pose. Further, Binding Pose MetaDynamics (BPMD) studies were performed to evaluate the binding stability, comparing 1 with four other inhibitors of the ß1i subunit: N-benzyl-2-(2-oxopyridin-1(2H)-yl)acetamide (2), N-cyclohexyl-3-(2-oxopyridin-1(2H)-yl)propenamide (3), N-butyl-3-(2-oxopyridin-1(2H)-yl)propanamide (4), and (S)-2-(2-oxopyridin-1(2H)-yl)-N,4-diphenylbutanamide (5). The obtained results in terms of free binding energy were consistent with the experimental values of inhibition, confirming 1 as a lead compound of this series. The adopted methods provided a full dynamic description of the binding events, and the information obtained could be exploited for the rational design of new and more active inhibitors.


Assuntos
Simulação de Acoplamento Molecular , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/farmacologia , Sítios de Ligação , Dipeptídeos/química , Dipeptídeos/farmacologia , Simulação de Dinâmica Molecular , Oligopeptídeos/química , Oligopeptídeos/farmacologia , Compostos de Organossilício/química , Compostos de Organossilício/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Ligação Proteica
18.
Cells ; 10(7)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206607

RESUMO

The ubiquitin-proteasome system (UPS) is a central part of protein homeostasis, degrading not only misfolded or oxidized proteins but also proteins with essential functions. The fact that a healthy hematopoietic system relies on the regulation of protein homeostasis and that alterations in the UPS can lead to malignant transformation makes the UPS an attractive therapeutic target for the treatment of hematologic malignancies. Herein, inhibitors of the proteasome, the last and most important component of the UPS enzymatic cascade, have been approved for the treatment of these malignancies. However, their use has been associated with side effects, drug resistance, and relapse. Inhibitors of the immunoproteasome, a proteasomal variant constitutively expressed in the cells of hematopoietic origin, could potentially overcome the encountered problems of non-selective proteasome inhibition. Immunoproteasome inhibitors have demonstrated their efficacy and safety against inflammatory and autoimmune diseases, even though their development for the treatment of hematologic malignancies is still in the early phases. Various immunoproteasome inhibitors have shown promising preliminary results in pre-clinical studies, and one inhibitor is currently being investigated in clinical trials for the treatment of multiple myeloma. Here, we will review data on immunoproteasome function and inhibition in hematopoietic cells and hematologic cancers.


Assuntos
Neoplasias Hematológicas/enzimologia , Neoplasias Hematológicas/imunologia , Hematopoese/imunologia , Complexo de Endopeptidases do Proteassoma/imunologia , Neoplasias Hematológicas/tratamento farmacológico , Hematopoese/efeitos dos fármacos , Humanos , Complexo de Endopeptidases do Proteassoma/genética , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Transdução de Sinais/efeitos dos fármacos
19.
Int J Mol Sci ; 22(14)2021 Jul 06.
Artigo em Inglês | MEDLINE | ID: mdl-34298888

RESUMO

We recently demonstrated that chemical proteasome inhibition induced inner retinal degeneration, supporting the pivotal roles of the ubiquitin-proteasome system in retinal structural integrity maintenance. In this study, using beclin1-heterozygous (Becn1-Het) mice with autophagic dysfunction, we tested our hypothesis that autophagy could be a compensatory retinal protective mechanism for proteasomal impairment. Despite the reduced number of autophagosome, the ocular tissue morphology and intraocular pressure were normal. Surprisingly, Becn1-Het mice experienced the same extent of retinal degeneration as was observed in wild-type mice, following an intravitreal injection of a chemical proteasome inhibitor. Similarly, these mice equally responded to other chemical insults, including endoplasmic reticulum stress inducer, N-methyl-D-aspartate, and lipopolysaccharide. Interestingly, in cultured neuroblastoma cells, we found that the mammalian target of rapamycin-independent autophagy activators, lithium chloride and rilmenidine, rescued these cells against proteasome inhibition-induced death. These results suggest that Becn1-mediated autophagy is not an effective intrinsic protective mechanism for retinal damage induced by insults, including impaired proteasomal activity; furthermore, autophagic activation beyond normal levels is required to alleviate the cytotoxic effect of proteasomal inhibition. Further studies are underway to delineate the precise roles of different forms of autophagy, and investigate the effects of their activation in rescuing retinal neurons under various pathological conditions.


Assuntos
Autofagia/fisiologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Retina/metabolismo , Degeneração Retiniana/metabolismo , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Autofagia/efeitos dos fármacos , Proteína Beclina-1/metabolismo , Morte Celular/efeitos dos fármacos , Morte Celular/fisiologia , Linhagem Celular Tumoral , Estresse do Retículo Endoplasmático/fisiologia , Humanos , Camundongos , Retina/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo
20.
Cells ; 10(5)2021 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-34066177

RESUMO

Proteasomes are intracellular structures responsible for protein degradation. The 20S proteasome is a core catalytic element of the proteasome assembly. Variations of catalytic subunits generate different forms of 20S proteasomes including immunoproteasomes (iPs), which are present mostly in the immune cells. Certain cells of the immune system are primary targets of retroviruses. It has been shown that several viral proteins directly affect proteasome functionality, while inhibition of proteasome activity with broad specificity proteasome inhibitors stimulates viral transduction. Here we specifically addressed the role of the immunoproteasomes during early stages of viral transduction and investigated the effects of specific immunoproteasome inhibition and activation prior to infection using a panel of cell lines. Inhibition of iPs in hematopoietic cells with immunoproteasome-specific inhibitor ONX-0914 resulted in increased infection by VSV-G pseudotyped lentiviruses. Moreover, a tendency for increased infection of cloned cells with endogenously decreased proteasome activity was revealed. Conversely, activation of iPs by IFN-γ markedly reduced the viral infectivity, which was rescued upon simultaneous immunoproteasome inhibition. Our results indicate that immunoproteasome activity might be determinative for the cellular antiretroviral resistance at least for the cells with high iP content. Finally, therapeutic application of immunoproteasome inhibitors might promote retroviral infection of cells in vivo.


Assuntos
Células-Tronco Hematopoéticas/efeitos dos fármacos , Lentivirus , Complexo de Endopeptidases do Proteassoma/imunologia , Antirretrovirais/farmacologia , Bortezomib/farmacologia , Linhagem Celular , Citocinas/metabolismo , Células HEK293 , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , Células HL-60 , Células-Tronco Hematopoéticas/citologia , Células-Tronco Hematopoéticas/imunologia , Humanos , Oligopeptídeos/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Retroviridae , Células THP-1 , Células U937
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...