RESUMO
Angiotensin-converting enzyme (ACE) metabolizes a number of important peptides participating in blood pressure regulation and vascular remodeling. Elevated ACE expression in tissues (which is generally reflected by blood ACE levels) is associated with an increased risk of cardiovascular diseases. Elevated blood ACE is also a marker for granulomatous diseases. Decreased blood ACE activity is becoming a new risk factor for Alzheimer's disease. We applied our novel approach-ACE phenotyping-to characterize pairs of tissues (lung, heart, lymph nodes) and serum ACE in 50 patients. ACE phenotyping includes (1) measurement of ACE activity with two substrates (ZPHL and HHL); (2) calculation of the ratio of hydrolysis of these substrates (ZPHL/HHL ratio); (3) determination of ACE immunoreactive protein levels using mAbs to ACE; and (4) ACE conformation with a set of mAbs to ACE. The ACE phenotyping approach in screening format with special attention to outliers, combined with analysis of sequencing data, allowed us to identify patient with a unique ACE phenotype related to decreased ability of inhibition of ACE activity by albumin, likely due to competition with high CCL18 in this patient for binding to ACE. We also confirmed recently discovered gender differences in sialylation of some glycosylation sites of ACE. ACE phenotyping is a promising new approach for the identification of ACE phenotype outliers with potential clinical significance, making it useful for screening in a personalized medicine approach.
RESUMO
We hypothesized that subjects with heterozygous loss-of-function (LoF) ACE mutations are at risk for Alzheimer's disease because amyloid Aß42, a primary component of the protein aggregates that accumulate in the brains of AD patients, is cleaved by ACE (angiotensin I-converting enzyme). Thus, decreased ACE activity in the brain, either due to genetic mutation or the effects of ACE inhibitors, could be a risk factor for AD. To explore this hypothesis in the current study, existing SNP databases were analyzed for LoF ACE mutations using four predicting tools, including PolyPhen-2, and compared with the topology of known ACE mutations already associated with AD. The combined frequency of >400 of these LoF-damaging ACE mutations in the general population is quite significant-up to 5%-comparable to the frequency of AD in the population > 70 y.o., which indicates that the contribution of low ACE in the development of AD could be under appreciated. Our analysis suggests several mechanisms by which ACE mutations may be associated with Alzheimer's disease. Systematic analysis of blood ACE levels in patients with all ACE mutations is likely to have clinical significance because available sequencing data will help detect persons with increased risk of late-onset Alzheimer's disease. Patients with transport-deficient ACE mutations (about 20% of damaging ACE mutations) may benefit from preventive or therapeutic treatment with a combination of chemical and pharmacological (e.g., centrally acting ACE inhibitors) chaperones and proteosome inhibitors to restore impaired surface ACE expression, as was shown previously by our group for another transport-deficient ACE mutation-Q1069R.
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Only praziquantel is available for treating schistosomiasis, a disease affecting more than 200 million people. Praziquantel-resistant worms have been selected for in the lab and low cure rates from mass drug administration programs suggest that resistance is evolving in the field. Thioredoxin glutathione reductase (TGR) is essential for schistosome survival and a validated drug target. TGR inhibitors identified to date are irreversible and/or covalent inhibitors with unacceptable off-target effects. In this work, we identify noncovalent TGR inhibitors with efficacy against schistosome infections in mice, meeting the criteria for lead progression indicated by WHO. Comparisons with previous in vivo studies with praziquantel suggests that these inhibitors outperform the drug of choice for schistosomiasis against juvenile worms.
Assuntos
Esquistossomose , Esquistossomicidas , Animais , Camundongos , Esquistossomicidas/farmacologia , Esquistossomicidas/uso terapêutico , Praziquantel/farmacologia , Schistosoma , NADH NADPH Oxirredutases/farmacologia , NADH NADPH Oxirredutases/uso terapêutico , Schistosoma mansoniRESUMO
Hepatitis B virus (HBV) is a worldwide health problem without curative treatments. Investigation of the regulation of HBV biosynthesis by class I and II histone deacetylases (HDACs) demonstrated that catalytically active HDAC5 upregulates HBV biosynthesis. HDAC5 expression increased both the stability and splicing of the HBV 3.5 kb RNA without altering the translational efficiency of the viral pregenomic or spliced 2.2 kb RNAs. Together, these observations point to a broader role of HDAC5 in regulating RNA splicing and transcript stability while specifically identifying a potentially novel approach toward antiviral HBV therapeutic development.
Assuntos
Genoma Viral , Vírus da Hepatite B/metabolismo , Hepatite B/virologia , Histona Desacetilases/metabolismo , Estabilidade de RNA , RNA Viral/biossíntese , RNA Viral/química , Regulação Viral da Expressão Gênica , Células Hep G2 , Vírus da Hepatite B/genética , Histona Desacetilases/genética , Humanos , Transcrição Gênica , Replicação ViralRESUMO
Members of the FAD/NAD-linked reductase family are recognized as crucial targets in drug development for cancers, inflammatory disorders, and infectious diseases. However, individual FAD/NAD reductases are difficult to inhibit in a selective manner with off-target inhibition reducing usefulness of identified compounds. Thioredoxin glutathione reductase (TGR), a high molecular weight thioredoxin reductase-like enzyme, has emerged as a promising drug target for the treatment of schistosomiasis, a parasitosis afflicting more than 200 million people. Taking advantage of small molecules selected from a high-throughput screen and using X-ray crystallography, functional assays, and docking studies, we identify a critical secondary site of the enzyme. Compounds binding at this site interfere with well-known and conserved conformational changes associated with NADPH reduction, acting as a doorstop for cofactor entry. They selectively inhibit TGR from Schistosoma mansoni and are active against parasites in culture. Since many members of the FAD/NAD-linked reductase family have similar catalytic mechanisms, the unique mechanism of inhibition identified in this study for TGR broadly opens new routes to selectively inhibit homologous enzymes of central importance in numerous diseases.
Assuntos
Anti-Helmínticos/farmacologia , Inibidores Enzimáticos/farmacologia , Complexos Multienzimáticos/antagonistas & inibidores , NADH NADPH Oxirredutases/antagonistas & inibidores , NADP/metabolismo , Schistosoma mansoni/efeitos dos fármacos , Schistosoma mansoni/enzimologia , Esquistossomose mansoni/parasitologia , Animais , Anti-Helmínticos/química , Cristalografia por Raios X , Descoberta de Drogas , Inibidores Enzimáticos/química , Humanos , Camundongos , Modelos Moleculares , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , NADH NADPH Oxirredutases/química , NADH NADPH Oxirredutases/metabolismo , Schistosoma mansoni/química , Schistosoma mansoni/metabolismo , Esquistossomose mansoni/tratamento farmacológicoRESUMO
Hydrogen sulfide is produced from l-cysteine by the action of both cystathionine γ-lyase (CSE) and cystathionine ß-synthase (CBS) and increasingly has been found to play a profound regulatory role in a range of physiological processes. Mounting evidence suggests that upregulation of hydrogen sulfide biosynthesis occurs in several disease states, including rheumatoid arthritis, hypertension, ischemic injury, and sleep-disordered breathing. In addition to being critical tools in our understanding of hydrogen sulfide biology, inhibitors of CSE hold therapeutic potential for the treatment of diseases in which increased levels of this gasotransmitter play a role. We describe the discovery and development of a novel series of potent CSE inhibitors that show increased activity over the benchmark inhibitor and, importantly, display high selectivity for CSE versus CBS.
RESUMO
Histone deacetylases (HDACs) are promising drug targets for a variety of therapeutic applications. Herein we describe the design, synthesis, biological evaluation in cellular models of cancer, and preliminary drug metabolism and pharmacokinetic studies (DMPK) of a series of secondary and tertiary N-substituted 7-aminoheptanohydroxamic acid-based HDAC inhibitors. Introduction of an amino group with one or two surface binding groups (SBGs) yielded a successful strategy to develop novel and potent HDAC inhibitors. The secondary amines were found to be generally more potent than the corresponding tertiary amines. Docking studies suggested that the SBGs of tertiary amines cannot be favorably accommodated at the gorge region of the binding site. The secondary amines with naphthalen-2-ylmethyl, 5-phenylthiophen-2-ylmethyl, and 1H-indol-2-ylmethyl (2 j) substituents exhibited the highest potency against classâ I HDACs: HDAC1 IC50 39-61â nm, HDAC2 IC50 260-690â nm, HDAC3 IC50 25-68â nm, and HDAC8 IC50 320-620â nm. The cytotoxicity of a representative set of secondary and tertiary N-substituted 7-aminoheptanoic acid hydroxyamide-based inhibitors against HT-29, SH-SY5Y, and MCF-7 cancer cells correlated with their inhibition of HDAC1, 2, and 3 and was found to be similar to or better than that of suberoylanilide hydroxamic acid (SAHA). Compounds in this series increased the acetylation of histones H3 and H4 in a time-dependent manner. DMPK studies indicated that secondary amine 2 j is metabolically stable and has plasma and brain concentrations >23- and >1.6-fold higher than the IC50 value for classâ I HDACs, respectively. Overall, the secondary and tertiary N-substituted 7-aminoheptanoic acid hydroxyamide-based inhibitors exhibit excellent lead- and drug-like properties and therapeutic capacity for cancer applications.
Assuntos
Aminas/farmacologia , Antineoplásicos/farmacologia , Desenho de Fármacos , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Aminas/síntese química , Aminas/química , Antineoplásicos/síntese química , Antineoplásicos/química , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cristalografia por Raios X , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Humanos , Modelos Moleculares , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The mechanism of action of histone deacetylase inhibitors (HDACi) is mainly attributed to the inhibition of the deacetylase catalytic activity for their histone substrates. In this study, we analyzed the abundance of class I HDACs in the cytosolic, nuclear soluble and chromatin bound cellular fractions in breast cancer cells after HDACi treatment. We found that potent N-hydroxy propenamide-based HDACi induced a concentration dependent decrease in the HDAC1 associated with chromatin and a lasting concomitant increase in cytoplasmic HDAC1 while maintaining total protein expression. No such change occurred with HDAC2 or 8, however, an increase in cytoplasmic non-phosphorylated HDAC3 was also observed. The subcellular re-equilibration of HDAC1 was subsequent to the accumulation of acetylated histones and might be cell cycle dependent. This study suggests that the biological activity of a subset of N-hydroxy propenamide-based HDACi may stem from direct competition with histone substrates of HDACs as well as from spatial separation from their substrates in the nucleus and/or change in post-translational modification status of HDACs.
Assuntos
Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Acetilação/efeitos dos fármacos , Citosol/efeitos dos fármacos , Citosol/enzimologia , Inibidores de Histona Desacetilases/química , Histonas/metabolismo , Humanos , Células MCF-7 , Microscopia Confocal , Mitógenos/farmacologia , Modelos Biológicos , Fosforilação/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/enzimologiaRESUMO
Histone deacetylase (HDAC) catalytic activity is regulated by formation of co-regulator complexes and post-translational modification. Whether these mechanisms are transformed in cancer and how this affects the binding and selectivity of HDAC inhibitors (HDACis) is unclear. In this study, we developed a method that identified a 3- to 16-fold increase in HDACi selectivity for HDAC3 in triple-negative breast cancer (TNBC) cells in comparison with luminal subtypes that was not predicted by current practice measurements with recombinant proteins. We found this increase was caused by c-Jun N-terminal kinase (JNK) phosphorylation of HDAC3, was independent of HDAC3 complex composition or subcellular localization, and was associated with a 5-fold increase in HDAC3 enzymatic activity. This study points to HDAC3 and the JNK axes as targets in TNBC, highlights how HDAC phosphorylation affects HDACi binding and selectivity, and outlines a method to identify changes in individual HDAC isoforms catalytic activity, applicable to any disease state.
Assuntos
Histona Desacetilases/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Linhagem Celular Tumoral , Feminino , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/metabolismo , Histona Desacetilases/química , Histona Desacetilases/genética , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/metabolismo , Isoenzimas/química , Isoenzimas/genética , Isoenzimas/metabolismo , Células MCF-7 , Fosforilação , Ligação Proteica , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Neoplasias de Mama Triplo Negativas/metabolismo , Neoplasias de Mama Triplo Negativas/patologiaRESUMO
Histone deacetylase 8 (HDAC8) is a promising drug target for multiple therapeutic applications. Here, we describe the modeling, design, synthesis, and biological evaluation of a novel series of C1-substituted tetrahydroisoquinoline (TIQ)-based HDAC8 inhibitors. Minimization of entropic loss upon ligand binding and use of the unique HDAC8 "open" conformation of the binding site yielded a successful strategy for improvement of both HDAC8 potency and selectivity. The TIQ-based 3g and 3n exhibited the highest 82 and 55 nM HDAC8 potency and 330- and 135-fold selectivity over HDAC1, respectively. Selectivity over other class I isoforms was comparable or better, whereas inhibition of HDAC6, a class II HDAC isoform, was below 50% at 10 µM. The cytotoxicity of 3g and 3n was evaluated in neuroblastoma cell lines, and 3n displayed concentration-dependent cytotoxicity similar to or better than that of PCI-34051. The selectivity of 3g and 3n was confirmed in SH-SY5Y cells as both did not increase the acetylation of histone H3 and α-tubulin. Discovery of the novel TIQ chemotype paves the way for the development of HDAC8 selective inhibitors for therapeutic applications.
RESUMO
Androgen deprivation therapy in prostate cancer is extremely effective; however, due to the continuous expression and/or mutagenesis of androgen receptor (AR), the resistance to antihormonal therapy is a natural progression. Consequently, targeting the AR for degradation offers an alternate approach to overcome this resistance in prostate cancer. In this study, we demonstrate that carnosic acid, a benzenediol diterpene, binds the ligand-binding domain of the AR and degrades the AR via endoplasmic reticulum (ER) stress-mediated proteasomal degradative pathway. In vitro, carnosic acid treatment induced degradation of AR and decreased expression of prostate-specific antigen in human prostate cancer cell lines LNCaP and 22Rv1. Carnosic acid also promoted the expression of ER proteins including BiP and CHOP in a dose-dependent manner. Downregulation of CHOP by small interfering RNA somewhat restored expression of AR suggesting that AR degradation is dependent on ER stress pathway. Future studies will need to evaluate other aspects of the unfolded protein response pathway to characterize the regulation of AR degradation. Furthermore, cotreating cells individually with carnosic acid and proteasome inhibitor (MG-132) and carnosic acid and an ER stress modulator (salubrinal) restored protein levels of AR, suggesting that AR degradation is mediated by ER stress-dependent proteasomal degradation pathway. Degradation of AR and induction of CHOP protein were also evident in vivo along with a 53% reduction in growth of xenograft prostate cancer tumors. In addition, carnosic acid-induced ER stress in prostate cancer cells but not in normal prostate epithelial cells procured from patient biopsies. In conclusion, these data suggest that molecules such as carnosic acid could be further evaluated and optimized as a potential therapeutic alternative to target AR in prostate cancer.
Assuntos
Abietanos/metabolismo , Antígeno Prostático Específico/genética , Neoplasias da Próstata/tratamento farmacológico , Receptores Androgênicos/biossíntese , Fator de Transcrição CHOP/biossíntese , Abietanos/administração & dosagem , Animais , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Cinamatos/administração & dosagem , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leupeptinas/administração & dosagem , Masculino , Camundongos , Antígeno Prostático Específico/biossíntese , Antígeno Prostático Específico/metabolismo , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Proteólise/efeitos dos fármacos , Tioureia/administração & dosagem , Tioureia/análogos & derivados , Fator de Transcrição CHOP/genética , Resposta a Proteínas não Dobradas/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
SCOPE: Androgen receptor (AR) has been a target of prostate cancer for nearly seven decades. In the last several years there has been an interest in identifying compounds that promote degradation of the androgen receptor. In the present study, gartanin, an isoprentylated xanthone in the mangosteen fruit, was evaluated for enhancing AR degradation, and inducing the unfolded protein response pathway. METHODS AND RESULTS: The interaction of gartanin with the ligand-binding domain was characterized using a fluorescence polarization cell-free assay and cell-based FRET assay. Western blot analysis identified modulation of ER stress markers (BiP, PERK, IRE1, and CHOP) along with androgen receptor degradation. A computation simulation was performed to identify possible orientations of gartanin with the ligand-binding domain. Utilizing a cell-free and cell-based FRET assays gartanin was found to interact with the ligand-binding domain through a solely antagonist interaction. Interestingly, inhibition of CHOP, a critical component of the ER stress pathway, was observed to stabilize AR. CONCLUSIONS: Gartanin is an isoprenylated xanthone that promotes AR degradation with evidence suggesting this process is critically regulated by the unfolded protein response pathway.
Assuntos
Frutas/química , Garcinia mangostana/química , Receptores Androgênicos/metabolismo , Xantonas/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Estresse do Retículo Endoplasmático/efeitos dos fármacos , Estresse do Retículo Endoplasmático/genética , Endorribonucleases/genética , Endorribonucleases/metabolismo , Humanos , Concentração Inibidora 50 , Masculino , Modelos Moleculares , Estrutura Molecular , Neoplasias da Próstata/tratamento farmacológico , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Androgênicos/genética , Fator de Transcrição CHOP/genética , Fator de Transcrição CHOP/metabolismo , eIF-2 Quinase/genética , eIF-2 Quinase/metabolismoRESUMO
Alzheimer's disease, one of the most important brain pathologies associated with neurodegenerative processes, is related to overactivation of calpain-mediated proteolysis. Previous data showed a compelling efficacy of calpain inhibition against abnormal synaptic plasticity and memory produced by the excess of amyloid-ß, a distinctive marker of the disease. Moreover, a beneficial effect of calpain inhibitors in Alzheimer's disease is predictable by the occurrence of calpain hyperactivation leading to impairment of memory-related pathways following abnormal calcium influxes that might ensue independently of amyloid-ß elevation. However, molecules currently available as effective calpain inhibitors lack adequate selectivity. This work is aimed at characterizing the efficacy of a novel class of epoxide-based inhibitors, synthesized to display improved selectivity and potency towards calpain 1 compared to the prototype epoxide-based generic calpain inhibitor E64. Both functional and preliminary toxicological investigations proved the efficacy, potency, and safety of the novel and selective calpain inhibitors NYC438 and NYC488 as possible therapeutics against the disease.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Glicoproteínas/uso terapêutico , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animais , Inibidores de Cisteína Proteinase/farmacologia , Inibidores de Cisteína Proteinase/uso terapêutico , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Medo/efeitos dos fármacos , Glicoproteínas/química , Glicoproteínas/farmacologia , Hipocampo/citologia , Humanos , Técnicas In Vitro , Potenciação de Longa Duração/efeitos dos fármacos , Potenciação de Longa Duração/genética , Aprendizagem em Labirinto/efeitos dos fármacos , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos ICR , Camundongos Transgênicos , Mutação/genética , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/metabolismo , Presenilina-1/genética , Espectrina/metabolismoRESUMO
Breast cancer remains a significant cause of death in women, and few therapeutic options exist for estrogen receptor negative (ER (-)) cancers. Epigenetic reactivation of target genes using histone deacetylase (HDAC) inhibitors has been proposed in ER (-) cancers to resensitize to therapy using selective estrogen receptor modulators (SERMs) that are effective in ER (+) cancer treatment. Based upon preliminary studies in ER (+) and ER (-) breast cancer cells treated with combinations of HDAC inhibitors and SERMs, hybrid drugs, termed SERMostats, were designed with computational guidance. Assay for inhibition of four typeâ I HDAC isoforms and antagonism of estrogenic activity in two cell lines yielded a SERMostat with 1-3â µM potency across all targets. The superior hybrid caused significant cell death in ER (-) human breast cancer cells and elicited cell death at the same concentration as the parent SERM in combination treatment and at an earlier time point.
Assuntos
Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/patologia , Inibidores de Histona Desacetilases/farmacologia , Moduladores Seletivos de Receptor Estrogênico/farmacologia , Antineoplásicos/síntese química , Antineoplásicos/química , Neoplasias da Mama/enzimologia , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Histona Desacetilases/síntese química , Inibidores de Histona Desacetilases/química , Histona Desacetilases/metabolismo , Humanos , Isoenzimas/antagonistas & inibidores , Isoenzimas/metabolismo , Células MCF-7 , Estrutura Molecular , Moduladores Seletivos de Receptor Estrogênico/síntese química , Moduladores Seletivos de Receptor Estrogênico/química , Relação Estrutura-AtividadeRESUMO
OBJECTIVE: Progesterone (P4) plays a central role in women's health. Synthetic progestins are used clinically in hormone replacement therapy (HRT), oral contraceptives, and for the treatment of endometriosis and infertility. Unfortunately, synthetic progestins are associated with side effects, including cardiovascular disease and breast cancer. Botanical dietary supplements are widely consumed for the alleviation of a variety of gynecological issues, but very few studies have characterized natural compounds in terms of their ability to bind to and activate progesterone receptors (PR). Kaempferol is a flavonoid that functions as a non-steroidal selective progesterone receptor modulator (SPRM) in vitro. This study investigated the molecular and physiological effects of kaempferol in the ovariectomized rat uteri. METHODS: Since genistein is a phytoestrogen that was previously demonstrated to increase uterine weight and proliferation, the ability of kaempferol to block genistein action in the uterus was investigated. Analyses of proliferation, steroid receptor expression, and induction of well-established PR-regulated targets Areg and Hand2 were completed using histological analysis and qPCR gene induction experiments. In addition, kaempferol in silico binding analysis was completed for PR. The activation of estrogen and androgen receptor signalling was determined in vitro. RESULTS: Molecular docking analysis confirmed that kaempferol adopts poses that are consistent with occupying the ligand-binding pocket of PRA. Kaempferol induced expression of PR regulated transcriptional targets in the ovariectomized rat uteri, including Hand2 and Areg. Consistent with progesterone-l ke activity, kaempferol attenuated genistein-induced uterine luminal epithelial proliferation without increasing uterine weight. Kaempferol signalled without down regulating PR expression in vitro and in vivo and without activating estrogen and androgen receptors. CONCLUSION: Taken together, these data suggest that kaempferol is a unique natural PR modulator that activates PR signaling in vitro and in vivo without triggering PR degradation.
RESUMO
A novel series of HDAC8 inhibitors without a zinc-chelating hydroxamic acid moiety is reported. Photoaffinity labeling and molecular modeling studies suggest that these ligands are likely to bind in an 'upside-down' fashion in a secondary binding site proximal to the main catalytic site. The most potent ligand in the series exhibits an IC(50) of 28 µM for HDAC8 and is found to inhibit the deacetylation of H4 but not α-tubulin in SH-SY5Y cell line.
Assuntos
Quelantes , Histona Desacetilases/metabolismo , Proteínas Repressoras/metabolismo , Sítios de Ligação , Western Blotting , Linhagem Celular , Linhagem Celular Tumoral , Quelantes/síntese química , Quelantes/metabolismo , Quelantes/farmacologia , Histona Desacetilases/química , Humanos , Ácidos Hidroxâmicos/química , Concentração Inibidora 50 , Ligantes , Estrutura Molecular , Ligação Proteica/efeitos dos fármacos , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/química , Zinco/químicaRESUMO
With 2 to 3% of the worldwide population chronically infected, hepatitis C virus (HCV) infection continues to be a major health care burden. Unfortunately, current interferon-based treatment options are not effective in all patients and are associated with significant side effects. Consequently, there is an ongoing need to identify and develop new anti-HCV therapies. Toward this goal, we previously developed a cell-based HCV infection assay for antiviral compound screening based on a low-multiplicity-of-infection approach that uniquely allows for the identification of antiviral compounds that target cell culture-derived HCV (HCVcc) at any step of the viral infection cycle. Using this assay, here we report the screening of the NCI Diversity Set II library, containing 1,974 synthesized chemical compounds, and the identification of compounds with specific anti-HCV activity. In combination with toxicity counterscreening, we identified 30 hits from the compound library, 13 of which showed reproducible and dose-dependent inhibition of HCV with mean therapeutic indices (50% cytotoxic concentration [CC(50)]/50% effective concentration [EC(50)]) of greater than 6. Using HCV pseudotype and replicon systems of multiple HCV genotypes, as well as infectious HCVcc-based assembly and secretion analysis, we determined that different compounds within this group of candidate inhibitors target different steps of viral infection. The compounds identified not only will serve as biological probes to study and further dissect the biology of viral infection but also should facilitate the development of new anti-HCV therapeutic treatments.
Assuntos
Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Hepatite C/tratamento farmacológico , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Análise por Conglomerados , Relação Dose-Resposta a Droga , Transferência Ressonante de Energia de Fluorescência , Hepatite C/virologia , Ensaios de Triagem em Larga Escala , Humanos , Indicadores e Reagentes , RNA Viral/genética , RNA Viral/isolamento & purificação , Reação em Cadeia da Polimerase em Tempo Real , Retroviridae/genética , Infecções por Retroviridae/genética , Infecções por Retroviridae/virologia , Bibliotecas de Moléculas Pequenas , Relação Estrutura-AtividadeRESUMO
The design, modeling, synthesis, biological evaluation of a novel series of photoreactive benzamide probes for class I HDAC isoforms is reported. The probes are potent and selective for HDAC1 and 2 and are efficient in crosslinking to HDAC2 as demonstrated by photolabeling experiments. The probes exhibit a time-dependent inhibition of class I HDACs. The inhibitory activities of the probes were influenced by the positioning of the aryl and alkyl azido groups necessary for photocrosslinking and attachment of the biotin tag. The probes inhibited the deacetylation of H4 in MDA-MB-231 cell line, indicating that they are cell permeable and target the nuclear HDACs.
Assuntos
Marcadores de Afinidade/química , Benzamidas/química , Desenho de Fármacos , Histona Desacetilase 2/química , Inibidores de Histona Desacetilases/síntese química , Modelos Moleculares , Biotina/química , Domínio Catalítico , Linhagem Celular Tumoral , Histona Desacetilase 1/química , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/metabolismo , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/metabolismo , Humanos , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismoRESUMO
The design, synthesis, docking, and biological evaluation of novel potent HDAC3 and HDAC8 isoxazole- and pyrazole-based diazide probes suitable for binding ensemble profiling with photoaffinity labeling (BEProFL) experiments in cells is described. Both the isoxazole- and pyrazole-based probes exhibit low nanomolar inhibitory activity against HDAC3 and HDAC8, respectively. The pyrazole-based probe 3f appears to be one of the most active HDAC8 inhibitors reported in the literature with an IC(50) of 17 nM. Our docking studies suggest that unlike the isoxazole-based ligands the pyrazole-based ligands are flexible enough to occupy the second binding site of HDAC8. Probes/inhibitors 2b, 3a, 3c, and 3f exerted the antiproliferative and neuroprotective activities at micromolar concentrations through inhibition of nuclear HDACs, indicating that they are cell permeable and the presence of an azide or a diazide group does not interfere with the neuroprotection properties, or enhance cellular cytotoxicity, or affect cell permeability.
Assuntos
Azidas/síntese química , Inibidores de Histona Desacetilases/síntese química , Histona Desacetilases/química , Ácidos Hidroxâmicos/síntese química , Isoxazóis/síntese química , Modelos Moleculares , Marcadores de Fotoafinidade/síntese química , Pirazóis/síntese química , Antineoplásicos/síntese química , Antineoplásicos/química , Antineoplásicos/farmacologia , Azidas/química , Azidas/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores de Histona Desacetilases/química , Inibidores de Histona Desacetilases/farmacologia , Histona Desacetilases/metabolismo , Humanos , Ácidos Hidroxâmicos/química , Ácidos Hidroxâmicos/farmacologia , Isoxazóis/química , Isoxazóis/farmacologia , Inibidores de Metaloproteinases de Matriz , Metaloproteinases da Matriz/química , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/química , Fármacos Neuroprotetores/farmacologia , Marcadores de Fotoafinidade/química , Marcadores de Fotoafinidade/farmacologia , Ligação Proteica , Pirazóis/química , Pirazóis/farmacologia , Proteínas Repressoras/química , Proteínas Repressoras/metabolismo , Relação Estrutura-AtividadeRESUMO
Previously, we identified five active phosphatidylinositol ether lipid analogues (PIAs) that target the pleckstrin homology domain of Akt and selectively induce apoptosis in cancer cells with high levels of Akt activity. To examine specificity, PIAs were screened against a panel of 29 purified kinases. No kinase was inhibited, but one isoform of p38, p38alpha, was uniformly activated 2-fold. Molecular modeling of p38alpha revealed the presence of two regions that could interact with PIAs, one in the activation loop and a heretofore unappreciated region in the upper lobe that resembles a pleckstrin homology domain. In cells, two phases of activation were observed, an early phase that was independent of the upstream kinase MKK3/6 and inhibited by the p38 inhibitor SB203580 and a latter phase that was coincident with MKK3/6 activation. In short term xenograft experiments that employed immunohistochemistry and immunoblotting, PIA administration increased phosphorylation of p38 but not MKK3/6 in tumors in a statistically significant manner. Although PIAs rapidly activated p38 with similar time and dose dependence as Akt inhibition, p38 activation and Akt inhibition were independent events induced by PIAs. Using SB203580 or p38alpha(-/-) cells, we showed that p38alpha is not required for PIA-induced apoptosis but is required for H(2)O(2)- and anisomycin-induced apoptosis. Nonetheless, activation of p38a contributes to PIA-induced apoptosis, because reconstitution of p38a into p38alpha(-/-) cells increased apoptosis. These studies indicate that p38alpha is activated by PIAs through a novel mechanism and show that p38alpha activation contributes to PIA-induced cell death. Independent modulation of Akt and p38alpha could account for the profound cytotoxicity of PIAs.