RESUMO
BACKGROUND: Loss of P-glycoprotein (P-gp) at the blood-brain barrier contributes to amyloid-ß (Aß) brain accumulation in Alzheimer's disease (AD). Using transgenic human amyloid precursor protein (hAPP)-overexpressing mice (Tg2576), we previously showed that Aß triggers P-gp loss by activating the ubiquitin-proteasome pathway, which leads to P-gp degradation. Furthermore, we showed that inhibiting the ubiquitin-activating enzyme (E1) prevents P-gp loss and lowers Aß accumulation in the brain of hAPP mice. Based on these data, we hypothesized that repurposing the FDA-approved proteasome inhibitor, bortezomib (Velcade®; BTZ), protects blood-brain barrier P-gp from degradation in hAPP mice in vivo. METHODS: We treated hAPP mice with the proteasome inhibitor BTZ or a combination of BTZ with the P-gp inhibitor cyclosporin A (CSA) for 2 weeks. Vehicle-treated wild-type (WT) mice were used as a reference for normal P-gp protein expression and transport activity. In addition, we used the opioid receptor agonist loperamide as a P-gp substrate in tail flick assays to indirectly assess P-gp transport activity at the blood-brain barrier in vivo. We also determined P-gp protein expression by Western blotting, measured P-gp transport activity levels in isolated brain capillaries with live cell confocal imaging and assessed Aß plasma and brain levels with ELISA. RESULTS: We found that 2-week BTZ treatment of hAPP mice restored P-gp protein expression and transport activity in brain capillaries to levels found in WT mice. We also observed that hAPP mice displayed significant loperamide-induced central antinociception compared to WT mice indicating impaired P-gp transport activity at the blood-brain barrier of hAPP mice in vivo. Furthermore, BTZ treatment prevented loperamide-induced antinociception suggesting BTZ protected P-gp loss in hAPP mice. Further, BTZ-treated hAPP mice had lower Aß40 and Aß42 brain levels compared to vehicle-treated hAPP mice. CONCLUSIONS: Our data indicate that BTZ protects P-gp from proteasomal degradation in hAPP mice, which helps to reduce Aß brain levels. Our data suggest that the proteasome system could be exploited for a novel therapeutic strategy in AD, particularly since increasing Aß transport across the blood-brain barrier may prove an effective treatment for patients.
Assuntos
Doença de Alzheimer , Humanos , Camundongos , Animais , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Barreira Hematoencefálica/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Complexo de Endopeptidases do Proteassoma/uso terapêutico , Loperamida/metabolismo , Loperamida/farmacologia , Loperamida/uso terapêutico , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Inibidores de Proteassoma/metabolismo , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/metabolismo , Encéfalo/metabolismo , Camundongos Transgênicos , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismoRESUMO
Tribbles related homolog 1 (TRIB1) contributes to lipid and glucose homeostasis by facilitating the degradation of cognate cargos by the proteasome. In view of the key metabolic role of TRIB1 and the impact of proteasome inhibition on hepatic function, we continue our exploration of TRIB1 regulation in two commonly used human hepatocyte models, transformed cell lines HuH-7 and HepG2. In both models, proteasome inhibitors potently upregulated both endogenous and recombinant TRIB1 mRNA and protein levels. Increased transcript abundance was unaffected by MAPK inhibitors while ER stress was a weaker inducer. Suppressing proteasome function via PSMB3 silencing was sufficient to increase TRIB1 mRNA expression. ATF3 was required to sustain basal TRIB1 expression and support maximal induction. Despite increasing TRIB1 protein abundance and stabilizing bulk ubiquitylation, proteasome inhibition delayed but did not prevent TRIB1 loss upon translation block. Immunoprecipitation experiments indicated that TRIB1 was not ubiquitylated in response to proteasome inhibition. A control bona fide proteasome substrate revealed that high doses of proteasome inhibitors resulted in incomplete proteasome inhibition. Cytoplasm retained TRIB1 was unstable, suggesting that TRIB1 lability is regulated prior to its nuclear import. N-terminal deletion and substitutions were insufficient to stabilize TRIB1. These findings identify transcriptional regulation as a prominent mechanism increasing TRIB1 abundance in transformed hepatocyte cell lines in response to proteasome inhibition and provide evidence of an inhibitor resistant proteasome activity responsible for TRIB1 degradation.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Serina-Treonina Quinases , Humanos , Hepatócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , RNA Mensageiro/genéticaRESUMO
Eponemycin is an α,ß-epoxyketone natural product that inhibits the proteasome via covalent interaction of the epoxyketone warhead with catalytic N-terminal threonine residues. The epoxyketone warhead is biosynthesized from a ß-ketoacid substrate by EpnF, a recently identified flavin-dependent acyl-CoA dehydrogenase-like enyzme. Herein, we report biochemical characterization of EpnF kinetics and substrate scope using a series of synthetic ß-ketoacid substrates. These studies indicate that epoxide formation likely occurs prior to other tailoring reactions in the biosynthetic pathway, and have led to the identification of novel epoxyketone analogues with potent anticancer activity.
Assuntos
Antineoplásicos , Inibidores de Proteassoma , Inibidores de Proteassoma/metabolismo , Antineoplásicos/farmacologia , Amidas/química , Serina/químicaRESUMO
Methylmercury (MeHg), an environmental pollutant, disrupts and impairs cellular function. MeHg binds to various cellular proteins, causing dysfunction and misfolding, which are considered underlying causes of MeHg toxicity. The p62 protein, also termed SQSTM1, is a ubiquitin-binding protein that targets ubiquitinated substrates to undergo autophagy and plays a key role in ameliorating MeHg toxicity. p62 also delivers ubiquitinated substrates to proteasomes. However, the role of these degradation systems in mitigating MeHg toxicity remains unknown. Herein, we explored the impact of the proteasome inhibitor MG132 on MeHg toxicity and examined the toxicity of co-treatment with MG132 and MeHg in p62KO mouse embryonic fibroblasts (MEFs) by analyzing cell viability, immunoblotting, mRNA levels, immunofluorescence, and the mercury content. The proteasome inhibitor MG132 enhanced MeHg-induced cytotoxicity while reducing intracellular mercury levels in MEFs. Co-treatment with MG132 and MeHg markedly increased levels of p62 and ubiquitinated proteins. Furthermore, co-treatment with MG132 and MeHg reduced p62KO MEF viability compared to that of wild-type MEFs. Our findings suggest that the proteasome participates in mitigating MeHg cytotoxicity, while p62 may play an important role in transporting MeHg-induced ubiquitinated proteins to the proteasome, as well as in autophagy. Collectively, these results imply that p62, and proteasome, and autophagy are vital for cytoprotection against MeHg toxicity.
Assuntos
Mercúrio , Compostos de Metilmercúrio , Animais , Camundongos , Autofagia , Fibroblastos , Mercúrio/metabolismo , Compostos de Metilmercúrio/metabolismo , Compostos de Metilmercúrio/toxicidade , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/metabolismo , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismo , Proteínas Ubiquitinadas/metabolismo , Intoxicação por Mercúrio/tratamento farmacológico , Intoxicação por Mercúrio/prevenção & controleRESUMO
The antimalarial activity of the frontline drug artemisinin involves generation of reactive oxygen species (ROS) leading to oxidative damage of parasite proteins. To achieve homeostasis and maintain protein quality control in the overwhelmed parasite, the ubiquitin-proteasome system kicks in. Even though molecular markers for artemisinin resistance like pfkelch13 have been identified, the intricate network of mechanisms driving resistance remains to be elucidated. Here, we report a forward genetic screening strategy that enables a broader identification of genetic factors responsible for altering sensitivity to dihydroartemisinin (DHA) and a proteasome inhibitor, bortezomib (BTZ). Using a library of isogenic piggyBac mutants in P. falciparum, we defined phenotype-genotype associations influencing drug responses and highlighted shared mechanisms between the two processes, which mainly included proteasome-mediated degradation and the lipid metabolism genes. Additional transcriptomic analysis of a DHA/BTZ-sensitive piggyBac mutant showed it is possible to find differences between the two response mechanisms on the specific components for regulation of the exportome. Our results provide further insight into the molecular mechanisms of antimalarial drug resistance. IMPORTANCE Malaria control is seriously threatened by the emergence and spread of Plasmodium falciparum resistance to the leading antimalarial, artemisinin. The potent killing activity of artemisinin results from oxidative damage unleashed by free heme activation released by hemoglobin digestion. Although the ubiquitin-proteasome system is considered critical for parasite survival of this toxicity, the diverse genetic changes linked to artemisinin resistance are complex and, so far, have not included the ubiquitin-proteasome system. In this study, we use a systematic forward genetic approach by screening a library of P. falciparum random piggyBac mutants to decipher the genetic factors driving malaria parasite responses to the oxidative stress caused by antimalarial drugs. This study compares phenotype-genotype associations influencing dihydroartemisinin responses with the proteasome inhibitor bortezomib to delineate the role of ubiquitin-proteasome system. Our study highlights shared and unique pathways from the complex array of molecular processes critical for P. falciparum survival resulting from the oxidative damage of artemisinin.
Assuntos
Antimaláricos , Artemisininas , Malária Falciparum , Malária , Humanos , Plasmodium falciparum , Antimaláricos/farmacologia , Antimaláricos/uso terapêutico , Bortezomib/farmacologia , Bortezomib/metabolismo , Bortezomib/uso terapêutico , Metabolismo dos Lipídeos , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Complexo de Endopeptidases do Proteassoma/farmacologia , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Proteínas de Protozoários/genética , Artemisininas/farmacologia , Malária Falciparum/tratamento farmacológico , Resistência a Medicamentos/genética , Ubiquitina/metabolismoRESUMO
We have previously shown that proteasome inhibitor bortezomib stabilizes p53 in stem and progenitor cells within gastrointestinal tissues. Here, we characterize the effect of bortezomib treatment on primary and secondary lymphoid tissues in mice. We find that bortezomib stabilizes p53 in significant fractions of hematopoietic stem and progenitor cells in the bone marrow, including common lymphoid and myeloid progenitors, granulocyte-monocyte progenitors, and dendritic cell progenitors. The stabilization of p53 is also observed in multipotent progenitors and hematopoietic stem cells, albeit at lower frequencies. In the thymus, bortezomib stabilizes p53 in CD4-CD8- T cells. Although there is less p53 stabilization in secondary lymphoid organs, cells in the germinal center of the spleen and Peyer's patch accumulate p53 in response to bortezomib. Bortezomib induces the upregulation of p53 target genes and p53 dependent/independent apoptosis in the bone marrow and thymus, suggesting that cells in these organs are robustly affected by proteasome inhibition. Comparative analysis of cell percentages in the bone marrow indicates expanded stem and multipotent progenitor pools in p53R172H mutant mice compared with p53 wild-type mice, suggesting a critical role for p53 in regulating the development and maturation of hematopoietic cells in the bone marrow. We propose that progenitors along the hematopoietic differentiation pathway express relatively high levels of p53 protein, which under steady-state conditions is constantly degraded by Mdm2 E3 ligase; however, these cells rapidly respond to stress to regulate stem cell renewal and consequently maintain the genomic integrity of hematopoietic stem/progenitor cell populations.
Assuntos
Inibidores de Proteassoma , Proteína Supressora de Tumor p53 , Camundongos , Animais , Bortezomib/farmacologia , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Células Progenitoras Mieloides/metabolismo , Camundongos Endogâmicos C57BLRESUMO
MG-132, an aldehyde-based peptide proteasome inhibitor (PI) that binds to the proteasome and reversibly inhibits proteasome activity, has been widely used in experimental research. However, it is not clear whether MG-132 has anti-inflammatory effects on liver injury. The molecular mechanism of the anti-inflammatory effect of the PI MG-132 on Con A-induced acute liver injury (ALI) mice was investigated by ELISA, HE, q RT-PCR, and IHC. The results showed that the serum activities of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and TNF-α and IL-6 contents of mice in the high and medium dose groups were reduced compared with those in the ALI group. The superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) levels in liver tissues were significantly increased, and the malondialdehyde (MDA) content was decreased. The pathological sections of mice in the ALI group showed typical ALI manifestations such as significant central venous stasis of liver tissues, cell swelling, and inflammatory cell infiltration. The pathological damage of liver tissues was relieved significantly in the three dose groups, especially in the high-dose group. The transcriptional level of TLR4/NF-κB pathway key factors mRNA was significantly reduced, and the expression of TLR4 and NF-κB P65 protein in liver tissues was significantly and positively correlated with the contents of TNF-α and IL-1ß (p < 0.01). Our findings suggest that MG-132 can alleviate the inflammatory response to Con A-induced ALI and exert a hepatoprotective effect, and its anti-inflammatory effect is related to the inhibition of TLR4/NF-κB signaling pathway activation.
Assuntos
NF-kappa B , Inibidores de Proteassoma , Camundongos , Animais , NF-kappa B/metabolismo , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Fator de Necrose Tumoral alfa/metabolismo , Receptor 4 Toll-Like/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Fígado/metabolismo , Anti-Inflamatórios/farmacologiaRESUMO
Advances in DNA sequencing technology and bioinformatics have revealed the enormous potential of microbes to produce structurally complex specialized metabolites with diverse uses in medicine and agriculture. However, these molecules typically require structural modification to optimize them for application, which can be difficult using synthetic chemistry. Bioengineering offers a complementary approach to structural modification but is often hampered by genetic intractability and requires a thorough understanding of biosynthetic gene function. Expression of specialized metabolite biosynthetic gene clusters (BGCs) in heterologous hosts can surmount these problems. However, current approaches to BGC cloning and manipulation are inefficient, lack fidelity, and can be prohibitively expensive. Here, we report a yeast-based platform that exploits transformation-associated recombination (TAR) for high efficiency capture and parallelized manipulation of BGCs. As a proof of concept, we clone, heterologously express and genetically analyze BGCs for the structurally related nonribosomal peptides eponemycin and TMC-86A, clarifying remaining ambiguities in the biosynthesis of these important proteasome inhibitors. Our results show that the eponemycin BGC also directs the production of TMC-86A and reveal contrasting mechanisms for initiating the assembly of these two metabolites. Moreover, our data shed light on the mechanisms for biosynthesis and incorporation of 4,5-dehydro-l-leucine (dhL), an unusual nonproteinogenic amino acid incorporated into both TMC-86A and eponemycin.
Assuntos
Inibidores de Proteassoma , Saccharomyces cerevisiae , Inibidores de Proteassoma/química , Inibidores de Proteassoma/metabolismo , Sequência de Bases , Saccharomyces cerevisiae/genética , Família MultigênicaRESUMO
Parkinson's disease (PD) is characterised by the progressive loss of midbrain dopaminergic neurons and the presence of aggregated α-synuclein (α-syn). Pericytes and microglia, two non-neuronal cells contain α-syn in the human brain, however, their role in disease processes is poorly understood. Pericytes, found surrounding the capillaries in the brain are important for maintaining the blood-brain barrier, controlling blood flow and mediating inflammation. In this study, primary human brain pericytes and microglia were exposed to two different α-synuclein aggregates. Inflammatory responses were assessed using immunocytochemistry, cytometric bead arrays and proteome profiler cytokine array kits. Fixed flow cytometry was used to investigate the uptake and subsequent degradation of α-syn in pericytes. We found that the two α-syn aggregates are devoid of inflammatory and cytotoxic actions on human brain derived pericytes and microglia. Although α-syn did not induce an inflammatory response, pericytes efficiently take up and degrade α-syn through the lysosomal pathway but not the ubiquitin-proteasome system. Furthermore, when pericytes were exposed the ubiquitin proteasome inhibitor-MG132 and α-syn aggregates, there was profound cytotoxicity through the production of reactive oxygen species resulting in apoptosis. These results suggest that the observed accumulation of α-syn in pericytes in human PD brains likely plays a role in PD pathogenesis, perhaps by causing cerebrovascular instability, under conditions of cellular stress.
Assuntos
Doença de Parkinson , alfa-Sinucleína , Apoptose , Citocinas/metabolismo , Humanos , Doença de Parkinson/metabolismo , Pericitos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Proteoma/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Ubiquitina/metabolismo , alfa-Sinucleína/metabolismoRESUMO
BACKGROUND: Lung cancer is a leading cause of cancer-related deaths, primarily as a result of metastases. In this metastasis, the epithelial-to-mesenchymal transition (EMT) is essential. Interaction with the cancer cell microenvironment is primarily dependent on M1- and M2-polarized macrophage. METHODS AND RESULTS: In this study, we revealed the EMT-associated activity of M1, M2a and M2c macrophages in A549 lung cancer cells. We established a co-culture model of A549 lung cancer cells utilizing THP-1-derived M1/M2 polarised macrophages to explore the involvement of macrophages in the immune response, apoptosis, and EMT in lung cancer. Although multiple polarising agents are routinely used for M1 and M2 conversion, we assessed a new possible polarising agent, hydrocortisone. CONCLUSIONS: M1 increased A549 cell sensitivity to proteasome inhibitors and decreased A549 cell viability by inducing apoptosis. EMT was induced in the presence of M2c macrophages in A549 cells by the levels of vimentin, fibronectin, E-cadherin, NF-kB, CCL-17. We also revealed the antiproliferative effects of bortezomib and ixazomib on A549 cells in both 2D and 3D cultures. Our findings could help develop an immunotherapeutic strategy by shedding light on a previously undiscovered part of the EMT pathway. Furthermore, additional investigation may reveal that polarising tumour-associated macrophages to M1 and eliminating the M2a or particularly the M2c subtype are effective anti-cancer strategies.
Assuntos
Transição Epitelial-Mesenquimal , Neoplasias Pulmonares , Humanos , Hidrocortisona/metabolismo , Imunoterapia , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Macrófagos/metabolismo , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Microambiente TumoralRESUMO
Nitazoxanide and related thiazolides are a novel class of anti-infectious agents against protozoan parasites, bacteria and viruses. In recent years, it is demonstrated that thiazolides can also induce cell cycle arrest and apoptotic cell death in cancer cells. Due to their fast proliferating nature, cancer cells highly depend on the proteasome system to remove aberrant proteins. Many of these aberrant proteins are regulators of cell cycle progression and apoptosis, such as the cyclins, BCL2 family members and nuclear factor of κB (NF-κB). Here, we demonstrate at both molecular and cellular levels that the 20S proteasome is a direct target of NTZ and related thiazolides. By concurrently inhibiting the multiple catalytic subunits of 20S proteasome, NTZ promotes cell cycle arrest and triggers cell death in colon cancer cells, either directly or as a sensitizer to other anti-tumor agents, especially doxorubicin. We further show that the binding mode of NTZ in the ß5 subunit of the 20S proteasome is different from that of bortezomib and other existing proteasome inhibitors. These findings provide new insights in the design of novel small molecular proteasome inhibitors as anti-tumor agents suitable for solid tumor treatment in an oral dosing form.
Assuntos
Antineoplásicos/metabolismo , Morte Celular/efeitos dos fármacos , Sistemas de Liberação de Medicamentos/métodos , Nitrocompostos/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/metabolismo , Tiazóis/metabolismo , Animais , Antineoplásicos/administração & dosagem , Células CACO-2 , Morte Celular/fisiologia , Relação Dose-Resposta a Droga , Células HCT116 , Células HEK293 , Células HeLa , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Nitrocompostos/administração & dosagem , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/administração & dosagem , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Tiazóis/administração & dosagemRESUMO
Ubiquitin/26 S proteasome system(UPS) is one of the main ways to regulate the degradation of proteins in plants, and plays an important role in physiological processes such as secondary metabolism and plant hormone signal transduction. As indicated recently, UPS is involved in plant-microbe interactions, and presumably regulates arbuscular mycorrhizal symbiosis to affect its effects. This study investigated the effects of interaction between Cbz-leu-leu-leucinal(MG132) and the mycorrhiza on the growth and effective components of Salvia miltiorrhiza by inoculation with Glomus intraradices and spraying MG132 solution. The results showed that the inoculation with G. intraradices could promote the growth of S. miltiorrhiza, increase the accumulation of effective components in the aerial and underground parts, and decrease the relative expression level of JMT. Additionally, MG132 could strengthen the growth-promoting effect of G. intraradices. As compared with the control group, the inoculation with G. intraradices could significantly increase aerial and underground fresh weights by 267% and 95%, respectively, under the treatment with MG132 spraying, while under the MG132 spraying-free condition, the increase was 195% and 32%, respectively. Meanwhile, MG132 spraying could enhance the promotion of mycorrhizal fungi on the accumulation of active components of S. miltiorrhiza. On the other hand, regardless of inoculation with G. intraradices or not, MG132 treatment could promote the root division of S. miltiorrhiza, reduce the content of effective components in the aerial parts, and increase the content in the underground part. The inoculation with G. intraradices could alleviate the inhibitory effect of MG132 on the accumulation of effective components in the aerial part of S. miltiorrhiza. The results show that arbuscular mycorrhizal fungi(AMF) can promote the growth of S. miltiorrhiza and the accumulation of effective components, and MG132 treatment can strengthen such promotion effect, which lays a foundation for the application of MG132 in the mycorrhizal cultivation of S. miltiorrhiza in the future.
Assuntos
Micorrizas , Salvia miltiorrhiza , Micorrizas/fisiologia , Inibidores de Proteassoma/metabolismo , Ubiquitina/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Raízes de Plantas , Simbiose/fisiologiaRESUMO
Tuberculosis (TB) has been recently declared as a health emergency because of sporadic increase in Multidrug-resistant Tuberculosis (MDR-TB) problem throughout the world. TB causing bacteria, Mycobacterium tuberculosis has become resistant to the first line of treatment along with second line of treatment and drugs, which are accessible to us. Thus, there is an urgent need of identification of key targets and development of potential therapeutic approach(s), which can overcome the Mycobacterium tuberculosis complications. In the present study, Mycobacterium tuberculosis proteasome has been taken as a potential target as it is one of the key regulatory proteins in Mycobacterium tuberculosis propagation. Further, a library of 400 compounds (small molecule) from Medicines for Malaria Venture (MMV) were screened against the target (proteasome) using molecular docking and simulation approach, and selected lead compounds were validated in in vitro model. In this study, we have identified two potent small molecules from the MMV Pathogen Box library, MMV019838 and MMV687146 with -9.8 kcal/mol and -8.7 kcal/mol binding energy respectively, which actively interact with the catalytic domain/active domain of Mycobacterium tuberculosis proteasome and inhibit the Mycobacterium tuberculosis growth in in vitro culture. Furthermore, the molecular docking and simulation study of MMV019838 and MMV687146 with proteasome show strong and stable interaction with Mycobacterium tuberculosis compared to human proteasome and show no cytotoxicity effect. A better understanding of proteasome inhibition in Mycobacterium tuberculosis in in vitro and in vivo model would eventually allow us to understand the molecular mechanism(s) and discover a novel and potent therapeutic agent against Tuberculosis. Active efflux of drugs mediated by efflux pumps that confer drug resistance is one of the mechanisms developed by bacteria to counter the adverse effects of antibiotics and chemicals. Efflux pump activity was tested for a specific compound MMV019838 which was showing good in silico results than MIC values.Communicated by Ramaswamy H. Sarma.
Assuntos
Mycobacterium tuberculosis , Tuberculose Resistente a Múltiplos Medicamentos , Antituberculosos/química , Humanos , Simulação de Acoplamento Molecular , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Tuberculose Resistente a Múltiplos Medicamentos/tratamento farmacológicoRESUMO
There is an urgent need for new treatments for visceral leishmaniasis (VL), a parasitic infection which impacts heavily large areas of East Africa, Asia, and South America. We previously reported on the discovery of GSK3494245/DDD01305143 (1) as a preclinical candidate for VL and, herein, we report on the medicinal chemistry program that led to its identification. A hit from a phenotypic screen was optimized to give a compound with in vivo efficacy, which was hampered by poor solubility and genotoxicity. The work on the original scaffold failed to lead to developable compounds, so an extensive scaffold-hopping exercise involving medicinal chemistry design, in silico profiling, and subsequent synthesis was utilized, leading to the preclinical candidate. The compound was shown to act via proteasome inhibition, and we report on the modeling of different scaffolds into a cryo-EM structure and the impact this has on our understanding of the series' structure-activity relationships.
Assuntos
Desenho de Fármacos , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Proteínas de Protozoários/metabolismo , Animais , Antiprotozoários/química , Antiprotozoários/metabolismo , Antiprotozoários/farmacologia , Antiprotozoários/uso terapêutico , Sítios de Ligação , Linhagem Celular , Avaliação Pré-Clínica de Medicamentos , Meia-Vida , Humanos , Leishmania donovani/efeitos dos fármacos , Leishmania donovani/metabolismo , Leishmaniose Visceral/tratamento farmacológico , Leishmaniose Visceral/parasitologia , Camundongos , Simulação de Dinâmica Molecular , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Inibidores de Proteassoma/uso terapêutico , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , Proteínas de Protozoários/química , Piridinas/química , Piridinas/metabolismo , Piridinas/farmacologia , Piridinas/uso terapêutico , Solubilidade , Relação Estrutura-AtividadeRESUMO
Proteasomes contribute to maintaining protein homeostasis and their inhibition is beneficial in certain types of cancer and in autoimmune diseases. However, the inhibition of the proteasomes in healthy cells leads to unwanted side-effects and significant effort has been made to identify inhibitors specific for the immunoproteasome, especially to treat diseases which manifest increased levels and activity of this proteasome isoform. Here, we report our efforts to discover fragment-sized inhibitors of the human immunoproteasome. The screening of an in-house library of structurally diverse fragments resulted in the identification of benzo[d]oxazole-2(3H)-thiones, benzo[d]thiazole-2(3H)-thiones, benzo[d]imidazole-2(3H)-thiones, and 1-methylbenzo[d]imidazole-2(3H)-thiones (with a general term benzoXazole-2(3H)-thiones) as inhibitors of the chymotrypsin-like (ß5i) subunit of the immunoproteasome. A subsequent structure-activity relationship study provided us with an insight regarding growing vectors. Binding to the ß5i subunit was shown and selectivity against the ß5 subunit of the constitutive proteasome was determined. Thorough characterization of these compounds suggested that they inhibit the immunoproteasome by forming a disulfide bond with the Cys48 available specifically in the ß5i active site. To obtain fragments with biologically more tractable covalent interactions, we performed a warhead scan, which yielded benzoXazole-2-carbonitriles as promising starting points for the development of selective immunoproteasome inhibitors with non-peptidic scaffolds.
Assuntos
Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Avaliação Pré-Clínica de Medicamentos , Humanos , Concentração Inibidora 50 , Oxazóis/química , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/metabolismo , Subunidades Proteicas/antagonistas & inibidores , Subunidades Proteicas/metabolismo , Relação Estrutura-Atividade , Tiazóis/química , Tionas/químicaRESUMO
Inhibitors of the proteasome have been extensively studied for their applications in the treatment of human diseases such as hematologic malignancies, autoimmune disorders, and viral infections. Many of the proteasome inhibitors reported in the literature target the non-primed site of proteasome's substrate binding pocket. In this study, we designed, synthesized and characterized a series of novel α-keto phenylamide derivatives aimed at both the primed and non-primed sites of the proteasome. In these derivatives, different substituted phenyl groups at the head group targeting the primed site were incorporated in order to investigate their structure-activity relationship and optimize the potency of α-keto phenylamides. In addition, the biological effects of modifications at the cap moiety, P1, P2 and P3 side chain positions were explored. Many derivatives displayed highly potent biological activities in proteasome inhibition and anticancer activity against a panel of six cancer cell lines, which were further rationalized by molecular modeling analyses. Furthermore, a representative α-ketoamide derivative was tested and found to be active in inhibiting the cellular infection of SARS-CoV-2 which causes the COVID-19 pandemic. These results demonstrate that this new class of α-ketoamide derivatives are potent anticancer agents and provide experimental evidence of the anti-SARS-CoV-2 effect by one of them, thus suggesting a possible new lead to develop antiviral therapeutics for COVID-19.
Assuntos
Amidas/farmacologia , Antineoplásicos/farmacologia , Antivirais/farmacologia , Cetonas/farmacologia , Inibidores de Proteassoma/farmacologia , SARS-CoV-2/efeitos dos fármacos , Amidas/síntese química , Amidas/metabolismo , Antineoplásicos/síntese química , Antineoplásicos/metabolismo , Antivirais/síntese química , Antivirais/metabolismo , Sítios de Ligação , Calpaína/química , Calpaína/metabolismo , Linhagem Celular Tumoral , Proteases 3C de Coronavírus/química , Proteases 3C de Coronavírus/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Cetonas/síntese química , Cetonas/metabolismo , Testes de Sensibilidade Microbiana , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Estrutura Molecular , Complexo de Endopeptidases do Proteassoma/química , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/síntese química , Inibidores de Proteassoma/metabolismo , Ligação Proteica , Relação Estrutura-AtividadeRESUMO
The glidobactin-like natural products (GLNPs) glidobactinâ A and cepafunginâ I have been reported to be potent proteasome inhibitors and are regarded as promising candidates for anticancer drug development. Their biosynthetic gene cluster (BGC) plu1881-1877 is present in entomopathogenic Photorhabdus laumondii but silent under standard laboratory conditions. Here we show the largest subset of GLNPs, which are produced and identified after activation of the silent BGC in the native host and following heterologous expression of the BGC in Escherichia coli. Their chemical diversity results from a relaxed substrate specificity and flexible product release in the assembly line of GLNPs. Crystal structure analysis of the yeast proteasome in complex with new GLNPs suggests that the degree of unsaturation and the length of the aliphatic tail are critical for their bioactivity. The results in this study provide the basis to engineer the BGC for the generation of new GLNPs and to optimize these natural products resulting in potential drugs for cancer therapy.
Assuntos
Photorhabdus/genética , Inibidores de Proteassoma/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Desenho de Fármacos , Escherichia coli/metabolismo , Família Multigênica/genética , Peptídeos Cíclicos/biossíntese , Peptídeos Cíclicos/química , Photorhabdus/metabolismo , Inibidores de Proteassoma/química , Relação Estrutura-AtividadeRESUMO
BACKGROUND: The ubiquitin-proteasome pathway is involved in almost all cellular processes (cell cycle, gene transcription and translation, cell survival and apoptosis, cell metabolism and protein quality control) mainly through the specific degradation of the majority of intracellular proteins (>80%) or partial processing of transcription factors (e.g., NF-κB). A growing amount of evidence now indicates that epigenetic changes are also regulated by the ubiquitin-proteasome pathway. Recent studies indicate that epigenetic regulations are equally crucial for almost all biological processes as well as for pathological conditions such as tumorigenesis, as compared to non-epigenetic control mechanisms (i.e., genetic alterations or classical signal transduction pathways). OBJECTIVE: Here, we reviewed the recent work highlighting the interaction of the ubiquitin-proteasome pathway components (e.g., ubiquitin, E1, E2 and E3 enzymes and 26S proteasome) with epigenetic regulators (histone deacetylases, histone acetyltransferases and DNA methyltransferases). RESULTS: Alterations in the regulation of the ubiquitin-proteasome pathway have been discovered in many pathological conditions. For example, a 2- to 32-fold increase in proteasomal activity and/or subunits has been noted in primary breast cancer cells. Although proteasome inhibitors have been successfully applied in the treatment of hematological malignancies (e.g., multiple myeloma), the clinical efficacy of the proteasomal inhibition is limited in solid cancers. Interestingly, recent studies show that the ubiquitin-proteasome and epigenetic pathways intersect in a number of ways through the regulation of epigenetic marks (i.e., acetylation, methylation and ubiquitylation). CONCLUSION: It is therefore believed that novel treatment strategies involving new generation ubiquitinproteasome pathway inhibitors combined with DNA methyltransferase, histone deacetylase or histone acetyltransferase inhibitors may produce more effective results with fewer adverse effects in cancer treatment as compared to standard chemotherapeutics in hematological as well as solid cancers.
Assuntos
Epigênese Genética/efeitos dos fármacos , Neoplasias/tratamento farmacológico , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/química , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Ubiquitina/metabolismo , Acetilação , Compostos de Boro/farmacologia , Bortezomib/química , Bortezomib/farmacologia , Metilases de Modificação do DNA/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Glicina/análogos & derivados , Glicina/farmacologia , Histona Acetiltransferases/antagonistas & inibidores , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/farmacologia , Metilação , NF-kappa B/metabolismo , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Transdução de Sinais , Compostos de Terfenil/farmacologia , Ubiquitinação , Ácido Valproico/farmacologiaRESUMO
Protein homeostasis is essential for life in eukaryotes. Organisms respond to proteotoxic stress by activating heat shock transcription factors (HSFs), which play important roles in cytoprotection, longevity and development. Of six human HSFs, HSF1 acts as a proteostasis guardian regulating stress-induced transcriptional responses, whereas HSF2 has a critical role in development, in particular of brain and reproductive organs. Unlike HSF1, that is a stable protein constitutively expressed, HSF2 is a labile protein and its expression varies in different tissues; however, the mechanisms regulating HSF2 expression remain poorly understood. Herein we demonstrate that the proteasome inhibitor anticancer drug bortezomib (Velcade), at clinically relevant concentrations, triggers de novo HSF2 mRNA transcription in different types of cancers via HSF1 activation. Similar results were obtained with next-generation proteasome inhibitors ixazomib and carfilzomib, indicating that induction of HSF2 expression is a general response to proteasome dysfunction. HSF2-promoter analysis, electrophoretic mobility shift assays, and chromatin immunoprecipitation studies unexpectedly revealed that HSF1 is recruited to a heat shock element located at 1.397 bp upstream from the transcription start site in the HSF2-promoter. More importantly, we found that HSF1 is critical for HSF2 gene transcription during proteasome dysfunction, representing an interesting example of transcription factor involved in controlling the expression of members of the same family. Moreover, bortezomib-induced HSF2 was found to localize in the nucleus, interact with HSF1, and participate in bortezomib-mediated control of cancer cell migration. The results shed light on HSF2-expression regulation, revealing a novel level of HSF1/HSF2 interplay that may lead to advances in pharmacological modulation of these fundamental transcription factors.
Assuntos
Fatores de Transcrição de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Fatores de Transcrição/metabolismo , Compostos de Boro/química , Compostos de Boro/metabolismo , Bortezomib/química , Bortezomib/metabolismo , Bortezomib/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Núcleo Celular/metabolismo , Ensaio de Desvio de Mobilidade Eletroforética , Glicina/análogos & derivados , Glicina/química , Glicina/metabolismo , Fatores de Transcrição de Choque Térmico/antagonistas & inibidores , Fatores de Transcrição de Choque Térmico/genética , Proteínas de Choque Térmico/antagonistas & inibidores , Proteínas de Choque Térmico/genética , Humanos , Regiões Promotoras Genéticas , Complexo de Endopeptidases do Proteassoma/química , Inibidores de Proteassoma/química , Inibidores de Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Interferência de RNA , RNA Mensageiro/metabolismo , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Sítio de Iniciação de Transcrição , Transcrição GênicaRESUMO
Regulating protein production and degradation is critical to maintaining cellular homeostasis. The proteasome is a key player in keeping proteins at the proper levels. However, proteasome activity can be altered in certain disease states, such as blood cancers and neurodegenerative diseases. Cancers often exhibit enhanced proteasomal activity, as protein synthesis is increased in these cells compared with normal cells. Conversely, neurodegenerative diseases are characterized by protein accumulation, leading to reduced proteasome activity. As a result, the proteasome has emerged as a target for therapeutic intervention. The potential of the proteasome as a therapeutic target has come from studies involving chemical stimulators and inhibitors, and the development of a suite of assays and probes that can be used to monitor proteasome activity with purified enzyme and in live cells.