RESUMEN
50 years ago, Vincent Allfrey and colleagues discovered that lymphocyte activation triggers massive acetylation of chromatin. However, the molecular mechanisms driving epigenetic accessibility are still unknown. We here show that stimulated lymphocytes decondense chromatin by three differentially regulated steps. First, chromatin is repositioned away from the nuclear periphery in response to global acetylation. Second, histone nanodomain clusters decompact into mononucleosome fibers through a mechanism that requires Myc and continual energy input. Single-molecule imaging shows that this step lowers transcription factor residence time and non-specific collisions during sampling for DNA targets. Third, chromatin interactions shift from long range to predominantly short range, and CTCF-mediated loops and contact domains double in numbers. This architectural change facilitates cognate promoter-enhancer contacts and also requires Myc and continual ATP production. Our results thus define the nature and transcriptional impact of chromatin decondensation and reveal an unexpected role for Myc in the establishment of nuclear topology in mammalian cells.
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Linfocitos B/metabolismo , Ciclo Celular , Núcleo Celular/metabolismo , Ensamble y Desensamble de Cromatina , Cromatina/metabolismo , Histonas/metabolismo , Activación de Linfocitos , Proteínas Proto-Oncogénicas c-myc/metabolismo , Acetilcoenzima A/metabolismo , Acetilación , Adenosina Trifosfato/metabolismo , Animales , Linfocitos B/inmunología , Línea Celular , Cromatina/química , Cromatina/genética , Metilación de ADN , Epigénesis Genética , Genotipo , Histonas/química , Inmunidad Humoral , Metilación , Ratones Endogámicos C57BL , Ratones Noqueados , Conformación de Ácido Nucleico , Fenotipo , Dominios y Motivos de Interacción de Proteínas , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-myc/química , Proteínas Proto-Oncogénicas c-myc/genética , Imagen Individual de Molécula , Relación Estructura-Actividad , Factores de Tiempo , Transcripción GenéticaRESUMEN
We recently reported on small-molecule inhibitors of the GroES/GroEL chaperone system as potential antibiotics against Escherichia coli and the ESKAPE pathogens but were unable to establish GroES/GroEL as the cellular target, leading to cell death. In this study, using two of our most potent bis-sulfonamido-2-phenylbenzoxazoles (PBZs), we established the binding site of the PBZ molecules using cryo-EM and found that GroEL was the cellular target responsible for the mode of action. Cryo-EM revealed that PBZ1587 binds at the GroEL ring-ring interface (RRI). A cellular reporter assay confirmed that PBZ1587 engaged GroEL in cells, but cellular rescue experiments showed potential off-target effects. This prompted us to explore a closely related analogue, PBZ1038, which is also bound to the RRI. Biochemical characterization showed potent inhibition of Gram-negative chaperonins but much lower potency of chaperonin from a Gram-positive organism, Enterococcus faecium. A cellular reporter assay showed that PBZ1038 also engaged GroEL in cells and that the cytotoxic phenotype could be rescued by a chromosomal copy of E. faecium GroEL/GroES or by expressing a recalcitrant RRI mutant. These data argue that PBZ1038's antimicrobial action is exerted through inhibition of GroES/GroEL, validating this chaperone system as an antibiotic target.
Asunto(s)
Antibacterianos , Chaperonina 10 , Escherichia coli , Antibacterianos/farmacología , Antibacterianos/química , Antibacterianos/síntesis química , Chaperonina 10/metabolismo , Chaperonina 10/antagonistas & inhibidores , Chaperonina 10/química , Escherichia coli/efectos de los fármacos , Chaperonina 60/metabolismo , Chaperonina 60/antagonistas & inhibidores , Chaperonina 60/química , Benzoxazoles/química , Benzoxazoles/farmacología , Benzoxazoles/síntesis química , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/antagonistas & inhibidores , Proteínas de Escherichia coli/químicaRESUMEN
BACKGROUND: Patients with locally advanced head and neck squamous cell cancer (HNSCC) are treated with surgery followed by adjuvant (chemo) radiotherapy or definitive chemoradiation, but recurrence rates are high. Immune checkpoint blockade improves survival in patients with recurrent/metastatic HNSCC; however, the role of chemo-immunotherapy in the curative setting is not established. METHODS: This phase 2, single-arm, multicenter study evaluated neoadjuvant chemo-immunotherapy with carboplatin, nab-paclitaxel, and durvalumab in patients with resectable locally advanced HNSCC. The primary end point was a hypothesized pathologic complete response rate of 50%. After chemo-immunotherapy and surgical resection, patients received study-defined, pathologic risk adapted adjuvant therapy consisting of either durvalumab alone (low risk), involved field radiation plus weekly cisplatin and durvalumab (intermediate risk), or standard chemoradiation plus durvalumab (high risk). RESULTS: Between December 2017 and November 2021, 39 subjects were enrolled at three centers. Oral cavity was the most common primary site (69%). A total of 35 of 39 subjects underwent planned surgical resection; one subject had a delay in surgery due to treatment-related toxicity. The most common treatment-related adverse events were cytopenias, fatigue, and nausea. Post treatment imaging demonstrated an objective response rate of 57%. Pathologic complete response and major pathologic response were achieved in 29% and 49% of subjects who underwent planned surgery, respectively. The 1-year progression-free survival was 83.8% (95% confidence interval, 67.4%-92.4%). CONCLUSIONS: Neoadjuvant carboplatin, nab-paclitaxel, and durvalumab before surgical resection of HNSCC were safe and feasible. Although the primary end point was not met, encouraging rates of pathologic complete response and clinical to pathologic downstaging were observed.
RESUMEN
GroES/GroEL is the only bacterial chaperone essential under all conditions, making it a potential antibiotic target. Rationally targeting ESKAPE GroES/GroEL as an antibiotic strategy necessitates studying their structure and function. Herein, we outline the structural similarities between Escherichia coli and ESKAPE GroES/GroEL and identify significant differences in intra- and inter-ring cooperativity, required in the refolding cycle of client polypeptides. Previously, we observed that one-half of ESKAPE GroES/GroEL family members could not support cell viability when each was individually expressed in GroES/GroEL-deficient E. coli cells. Cell viability was found to be dependent on the allosteric compatibility between ESKAPE and E. coli subunits within mixed (E. coli and ESKAPE) tetradecameric GroEL complexes. Interestingly, differences in allostery did not necessarily result in differences in refolding rate for a given homotetradecameric chaperonin. Characterization of ESKAPE GroEL allostery, ATPase, and refolding rates in this study will serve to inform future studies focused on inhibitor design and mechanism of action studies.
Asunto(s)
Sitio Alostérico , Proteínas de Escherichia coli/química , Proteínas de Choque Térmico/química , Adenosina Difosfato/metabolismo , Adenosina Trifosfato/metabolismo , Regulación Alostérica , Chaperonina 10/química , Chaperonina 10/genética , Chaperonina 10/metabolismo , Escherichia coli , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Subunidades de Proteína/química , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismoRESUMEN
While many studies have established the importance of protein homeostasis in tumor progression, little effort has been made to examine the therapeutic potential of targeting the HSP60 chaperonin system. In healthy cells, HSP60 is localized to the mitochondrial matrix; however, emerging evidence indicates HSP60 can be over-expressed and mis-localized to the cytosol of cancer cells, which is hypothesized to promote tumor cell survival and proliferation. This opens a potential avenue to selectively target the aberrant HSP60 in the cytosol as a chemotherapeutic strategy. In the present work, we examined a series of bis-aryl-α,ß-unsaturated ketone (ABK) HSP60 inhibitors for their ability to selectively target cancerous vs non-cancerous colon and intestine cells. We found that lead analogs inhibited migration and clonogenicity of cancer cells, with cytotoxicity correlating with the level of aberrant HSP60 in the cytosol.
RESUMEN
Lymphoepithelioma-like carcinoma of the bladder (LELC-B) is a rare subtype of urothelial carcinoma consisting of undifferentiated epithelial cells within a dense inflammatory cell infiltrate. We set out to molecularly characterize LELC-B through RNA expression profiling as well as immunohistochemistry (IHC) to understand its underlying biology. Sixteen cases of LELC-B were identified at Johns Hopkins University. RNA sequencing was performed on 14 cases. IHC staining for programmed cell death ligand 1 (PD-L1) and mismatch repair proteins MutL homolog 1 (MLH1), MutS homolog 2 (MSH2), MSH6, and PMS1 homolog, mismatch repair system component 2 (PMS2) was performed. Transcriptomic profiling of LELC-B showed that they are enriched in a basal-like phenotype, with 12 of 14 LELC-B cases correlating to the basal centroid of the bladder cancer analysis of subtypes by gene expression 47 (BASE47) predictive analysis of microarrays (PAM) classifier. Gene signature analysis confirmed the lymphocyte infiltration profile consistent with the histomorphology. LELC-B lacked features to explain the robust lymphocytic infiltrate, such as loss of mismatch repair protein expression or expression of Epstein-Barr virus transcripts. Nonetheless, PD-L1 IHC was positive in 93% of LELC cases. Our study demonstrates that LELC-B tumors are enriched in a basal-like molecular subtype and share a high level of immune infiltration and PD-L1 expression, similar to basal tumors. The basal-like phenotype is consistent with the known sensitivity of LELC-B to chemotherapy and suggests that immune checkpoint therapy should be explored in this rare disease.
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Adenocarcinoma/patología , Biomarcadores de Tumor/genética , Carcinoma Basocelular/patología , Carcinoma de Células Transicionales/patología , Neoplasias de la Vejiga Urinaria/patología , Adenocarcinoma/genética , Adenocarcinoma/virología , Carcinoma Basocelular/genética , Carcinoma Basocelular/virología , Carcinoma de Células Transicionales/genética , Carcinoma de Células Transicionales/virología , Infecciones por Virus de Epstein-Barr/complicaciones , Infecciones por Virus de Epstein-Barr/virología , Perfilación de la Expresión Génica , Herpesvirus Humano 4/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Células Tumorales Cultivadas , Neoplasias de la Vejiga Urinaria/genética , Neoplasias de la Vejiga Urinaria/virologíaRESUMEN
The ASXL1 and SRSF2 mutations in AML are frequently found in patients with preexisting myeloid malignancies and are individually associated with poor outcomes. In this multi-institutional retrospective analysis, we assessed the genetic features and clinical outcomes of 43 patients with ASXL1mut SRSF2mut AML and compared outcomes to patients with either ASXL1 (n = 57) or SRSF2 (n = 70) mutations. Twenty-six (60%) had secondary-AML (s-AML). Variant allele fractions suggested that SRSF2 mutations preceded ASXL1 mutational events. Median overall survival (OS) was 7.0 months (95% CI:3.8,15.3) and was significantly longer in patients with de novo vs s-AML (15.3 vs 6.4 months, respectively; P = .04 on adjusted analysis). Compared to ASXL1mut SRSF2wt and ASXL1wt SRSF2mut , co-mutated patients had a 1.4 and 1.6 times increase in the probability of death, respectively (P = .049), with a trend towards inferior OS (median OS = 7.0 vs 11.5 vs 10.9 months, respectively; P = .10). Multivariable analysis suggests this difference in OS is attributable to the high proportion of s-AML patients in the co-mutated cohort (60% vs 32% and 23%, respectively). Although this study is limited by the retrospective data collection and the relatively small sample size, these data suggest that ASXL1mut SRSF2mut AML is a distinct subgroup of AML frequently associated with s-AML and differs from ASXL1mut SRSF2wt /ASXL1wt SRSF2mut with respect to etiology and leukemogenesis.
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Leucemia Mieloide Aguda/genética , Mutación , Proteínas Represoras/genética , Factores de Empalme Serina-Arginina/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Transformación Celular Neoplásica/genética , Cocarcinogénesis/genética , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Estimación de Kaplan-Meier , Leucemia Mieloide Aguda/clasificación , Leucemia Mieloide Aguda/mortalidad , Masculino , Persona de Mediana Edad , Pronóstico , Proteínas Represoras/fisiología , Estudios Retrospectivos , Factores de Empalme Serina-Arginina/fisiologíaRESUMEN
Over the past few decades, an increasing variety of molecular chaperones have been investigated for their role in tumorigenesis and as potential chemotherapeutic targets; however, the 60 kDa Heat Shock Protein (HSP60), along with its HSP10 co-chaperone, have received little attention in this regard. In the present study, we investigated two series of our previously developed inhibitors of the bacterial homolog of HSP60/10, called GroEL/ES, for their selective cytotoxicity to cancerous over non-cancerous colorectal cells. We further developed a third "hybrid" series of analogs to identify new candidates with superior properties than the two parent scaffolds. Using a series of well-established HSP60/10 biochemical screens and cell-viability assays, we identified 24 inhibitors (14%) that exhibited > 3-fold selectivity for targeting colorectal cancer over non-cancerous cells. Notably, cell viability EC50 results correlated with the relative expression of HSP60 in the mitochondria, suggesting a potential for this HSP60-targeting chemotherapeutic strategy as emerging evidence indicates that HSP60 is up-regulated in colorectal cancer tumors. Further examination of five lead candidates indicated their ability to inhibit the clonogenicity and migration of colorectal cancer cells. These promising results are the most thorough analysis and first reported instance of HSP60/10 inhibitors being able to selectively target colorectal cancer cells and highlight the potential of the HSP60/10 chaperonin system as a viable chemotherapeutic target.
Asunto(s)
Antineoplásicos/farmacología , Benzoxazoles/farmacología , Chaperonina 10/antagonistas & inhibidores , Chaperonina 60/antagonistas & inhibidores , Neoplasias Colorrectales/tratamiento farmacológico , Salicilanilidas/farmacología , Antineoplásicos/síntesis química , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Benzoxazoles/síntesis química , Benzoxazoles/química , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Estructura Molecular , Salicilanilidas/síntesis química , Salicilanilidas/química , Relación Estructura-Actividad , Células Tumorales CultivadasRESUMEN
Activated PI3 kinase delta syndrome (APDS) is a combined immunodeficiency characterized by recurrent sinopulmonary infections, increased risk of herpesvirus infections, lymphoproliferation, autoimmunity, and increased risk of lymphoid malignancies. Gain-of-function mutations in PIK3CD and PIK3R1 result in increased phosphoinositide-3-kinase-delta activity which causes hyperactivation of lymphocytes and abnormal development and activation of T and B cells. Cytopenias are the most common autoimmune process occurring in patients with APDS and typically occur as a later manifestation of the disease. Here we present a female patient with an early autoimmune hemolytic anemia, hepatosplenomegaly, and frequent infections presenting in infancy, followed by development of significant lymphadenopathy before her diagnosis with APDS type 1. She had significant improvement in her infectious history with immunoglobulin replacement, and control of autoimmune hemolytic anemia with initiation of sirolimus after her diagnosis with APDS type 1. We utilize this case to review the literature on APDS and present the novel finding of early-onset autoimmune disease in the setting of APDS. Autoimmune cytopenias are seen in many primary immunodeficiencies, and workup of autoimmune cytopenias in young patients should include evaluation for underlying immune disorder.
Asunto(s)
Anemia Hemolítica Autoinmune/patología , Enfermedades de Inmunodeficiencia Primaria/complicaciones , Adulto , Anemia Hemolítica Autoinmune/etiología , Fosfatidilinositol 3-Quinasa Clase I , Femenino , Humanos , Pronóstico , Adulto JovenRESUMEN
Mutation in leucine-rich repeat kinase 2 (LRRK2) is a common cause of familial Parkinson's disease (PD). Recently, we showed that a disease-associated mutation R1441H rendered the GTPase domain of LRRK2 catalytically less active and thereby trapping it in a more persistently "on" conformation. However, the mechanism involved and characteristics of this on conformation remained unknown. Here, we report that the Ras of complex protein (ROC) domain of LRRK2 exists in a dynamic dimer-monomer equilibrium that is oppositely driven by GDP and GTP binding. We also observed that the PD-associated mutations at residue 1441 impair this dynamic and shift the conformation of ROC to a GTP-bound-like monomeric conformation. Moreover, we show that residue Arg-1441 is critical for regulating the conformational dynamics of ROC. In summary, our results reveal that the PD-associated substitutions at Arg-1441 of LRRK2 alter monomer-dimer dynamics and thereby trap its GTPase domain in an activated state.
Asunto(s)
Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina , Mutación Missense , Enfermedad de Parkinson , Multimerización de Proteína , Sustitución de Aminoácidos , Guanosina Difosfato/química , Guanosina Difosfato/genética , Guanosina Trifosfato/química , Guanosina Trifosfato/genética , Células HEK293 , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/química , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Enfermedad de Parkinson/enzimología , Enfermedad de Parkinson/genética , Dominios ProteicosRESUMEN
In two previous studies, we identified compound 1 as a moderate GroEL/ES inhibitor with weak to moderate antibacterial activity against Gram-positive and Gram-negative bacteria including Bacillus subtilis, methicillin-resistant Staphylococcus aureus, Klebsiella pneumonia, Acinetobacter baumannii, and SM101 Escherichia coli (which has a compromised lipopolysaccharide biosynthetic pathway making bacteria more permeable to drugs). Extending from those studies, we developed two series of analogs with key substructures resembling those of known antibacterials, nitroxoline (hydroxyquinoline moiety) and nifuroxazide/nitrofurantoin (bis-cyclic-N-acylhydrazone scaffolds). Through biochemical and cell-based assays, we identified potent GroEL/ES inhibitors that selectively blocked E. faecium, S. aureus, and E. coli proliferation with low cytotoxicity to human colon and intestine cells in vitro. Initially, only the hydroxyquinoline-bearing analogs were found to be potent inhibitors in our GroEL/ES-mediated substrate refolding assays; however, subsequent testing in the presence of an E. coli nitroreductase (NfsB) in situ indicated that metabolites of the nitrofuran-bearing analogs were potent GroEL/ES inhibitor pro-drugs. Consequently, this study has identified a new target of nitrofuran-containing drugs, and is the first reported instance of such a unique class of GroEL/ES chaperonin inhibitors. The intriguing results presented herein provide impetus for expanded studies to validate inhibitor mechanisms and optimize this antibacterial class using the respective GroEL/ES chaperonin systems and nitroreductases from E. coli and the ESKAPE bacteria.
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Antibacterianos/farmacología , Chaperonina 60/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Nitrofuranos/farmacología , Profármacos/farmacología , Antibacterianos/síntesis química , Antibacterianos/química , Chaperonina 60/metabolismo , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Bacterias Gramnegativas/metabolismo , Bacterias Grampositivas/metabolismo , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Nitrofuranos/síntesis química , Nitrofuranos/química , Profármacos/síntesis química , Profármacos/química , Relación Estructura-ActividadRESUMEN
Immune memory is a defining characteristic of adaptive immunity, but recent work has shown that the activation of innate immunity can also improve responsiveness in subsequent exposures. This has been coined "trained immunity" and diverges with the perception that the innate immune system is primitive, non-specific, and reacts to novel and recurrent antigen exposures similarly. The "exposome" is the cumulative exposures (diet, exercise, environmental exposure, vaccination, genetics, etc.) an individual has experienced and provides a mechanism for the establishment of immune training or immunotolerance. It is becoming increasingly clear that trained immunity constitutes a delicate balance between the dose, duration, and order of exposures. Upon innate stimuli, trained immunity or tolerance is shaped by epigenetic and metabolic changes that alter hematopoietic stem cell lineage commitment and responses to infection. Due to the immunomodulatory role of the exposome, understanding innate immune training is critical for understanding why some individuals exhibit protective phenotypes while closely related individuals may experience immunotolerant effects (e.g., the order of exposure can result in completely divergent immune responses). Research on the exposome and trained immunity may be leveraged to identify key factors for improving vaccination development, altering inflammatory disease development, and introducing potential new prophylactic treatments, especially for diseases such as COVID-19, which is currently a major health issue for the world. Furthermore, continued exposome research may prevent many deleterious effects caused by immunotolerance that frequently result in host morbidity or mortality.
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Betacoronavirus/inmunología , Linaje de la Célula/inmunología , Infecciones por Coronavirus/inmunología , Inmunidad Innata/inmunología , Memoria Inmunológica/inmunología , Neumonía Viral/inmunología , COVID-19 , Metilación de ADN/genética , Células Dendríticas/inmunología , Exposoma , Código de Histonas/genética , Humanos , Tolerancia Inmunológica/inmunología , Células Asesinas Naturales/inmunología , Macrófagos/inmunología , Pandemias , SARS-CoV-2RESUMEN
All living organisms contain a unique class of molecular chaperones called 60â¯kDa heat shock proteins (HSP60 - also known as GroEL in bacteria). While some organisms contain more than one HSP60 or GroEL isoform, at least one isoform has always proven to be essential. Because of this, we have been investigating targeting HSP60 and GroEL chaperonin systems as an antibiotic strategy. Our initial studies focused on applying this antibiotic strategy for treating African sleeping sickness (caused by Trypanosoma brucei parasites) and drug-resistant bacterial infections (in particular Methicillin-resistant Staphylococcus aureus - MRSA). Intriguingly, during our studies we found that three known antibiotics - suramin, closantel, and rafoxanide - were potent inhibitors of bacterial GroEL and human HSP60 chaperonin systems. These findings prompted us to explore what other approved drugs, natural products, and known bioactive molecules might also inhibit HSP60 and GroEL chaperonin systems. Initial high-throughput screening of 3680 approved drugs, natural products, and known bioactives identified 161 hit inhibitors of the Escherichia coli GroEL chaperonin system (4.3% hit rate). From a purchased subset of 60 hits, 29 compounds (48%) re-confirmed as selective GroEL inhibitors in our assays, all of which were nearly equipotent against human HSP60. These findings illuminate the notion that targeting chaperonin systems might be a more common occurrence than we previously appreciated. Future studies are needed to determine if the in vivo modes of action of these approved drugs, natural products, and known bioactive molecules are related to GroEL and HSP60 inhibition.
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Productos Biológicos/química , Chaperonina 10/metabolismo , Chaperonina 60/metabolismo , Rafoxanida/química , Salicilanilidas/química , Suramina/química , Productos Biológicos/metabolismo , Chaperonina 10/antagonistas & inhibidores , Chaperonina 60/antagonistas & inhibidores , Escherichia coli/metabolismo , Humanos , Concentración 50 Inhibidora , Pliegue de Proteína , Rafoxanida/metabolismo , Salicilanilidas/metabolismo , Suramina/metabolismoRESUMEN
Current treatments for Mycobacterium tuberculosis infections require long and complicated regimens that can lead to patient non-compliance, increasing incidences of antibiotic-resistant strains, and lack of efficacy against latent stages of disease. Thus, new therapeutics are needed to improve tuberculosis standard of care. One strategy is to target protein homeostasis pathways by inhibiting molecular chaperones such as GroEL/ES (HSP60/10) chaperonin systems. M. tuberculosis has two GroEL homologs: GroEL1 is not essential but is important for cytokine-dependent granuloma formation, while GroEL2 is essential for survival and likely functions as the canonical housekeeping chaperonin for folding proteins. Another strategy is to target the protein tyrosine phosphatase B (PtpB) virulence factor that M. tuberculosis secretes into host cells to help evade immune responses. In the present study, we have identified a series of GroEL/ES inhibitors that inhibit M. tuberculosis growth in liquid culture and biochemical function of PtpB in vitro. With further optimization, such dual-targeting GroEL/ES and PtpB inhibitors could be effective against all stages of tuberculosis - actively replicating bacteria, bacteria evading host cell immune responses, and granuloma formation in latent disease - which would be a significant advance to augment current therapeutics that primarily target actively replicating bacteria.
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Chaperonina 60/uso terapéutico , Mycobacterium tuberculosis/patogenicidad , Tuberculosis/tratamiento farmacológico , Proteínas Bacterianas/metabolismo , Chaperonina 60/farmacología , Humanos , Modelos Moleculares , PolifarmacologíaRESUMEN
Glucose-regulated protein 78 (GRP78) is the ER resident 70â¯kDa heat shock protein 70 (HSP70) and has been hypothesized to be a therapeutic target for various forms of cancer due to its role in mitigating proteotoxic stress in the ER, its elevated expression in some cancers, and the correlation between high levels for GRP78 and a poor prognosis. Herein we report the development and use of a high throughput fluorescence polarization-based peptide binding assay as an initial step toward the discovery and development of GRP78 inhibitors. This assay was used in a pilot screen to discover the anti-infective agent, hexachlorophene, as an inhibitor of GRP78. Through biochemical characterization we show that hexachlorophene is a competitive inhibitor of the GRP78-peptide interaction. Biological investigations showed that this molecule induces the unfolded protein response, induces autophagy, and leads to apoptosis in a colon carcinoma cell model, which is known to be sensitive to GRP78 inhibition.
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Proteínas HSP70 de Choque Térmico/efectos de los fármacos , Hexaclorofeno/uso terapéutico , Ensayos Analíticos de Alto Rendimiento/métodos , Chaperón BiP del Retículo Endoplásmico , Hexaclorofeno/farmacología , HumanosRESUMEN
Rate-limiting dissociation of the tetrameric protein transthyretin (TTR), followed by monomer misfolding and misassembly, appears to cause degenerative diseases in humans known as the transthyretin amyloidoses, based on human genetic, biochemical and pharmacologic evidence. Small molecules that bind to the generally unoccupied thyroxine binding pockets in the native TTR tetramer kinetically stabilize the tetramer, slowing subunit dissociation proportional to the extent that the molecules stabilize the native state over the dissociative transition state-thereby inhibiting amyloidogenesis. Herein, we use previously reported structure-activity relationship data to develop two semi-quantitative algorithms for identifying the structures of potent and selective transthyretin kinetic stabilizers/amyloidogenesis inhibitors. The viability of these prediction algorithms, in particular the more robust in silico docking model, is perhaps best validated by the clinical success of tafamidis, the first-in-class drug approved in Europe, Japan, South America, and elsewhere for treating transthyretin aggregation-associated familial amyloid polyneuropathy. Tafamidis is also being evaluated in a fully-enrolled placebo-controlled clinical trial for its efficacy against TTR cardiomyopathy. These prediction algorithms will be useful for identifying second generation TTR kinetic stabilizers, should these be needed to ameliorate the central nervous system or ophthalmologic pathology caused by TTR aggregation in organs not accessed by oral tafamidis administration.
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Neuropatías Amiloides Familiares/tratamiento farmacológico , Diseño de Fármacos , Prealbúmina/metabolismo , Estabilidad Proteica/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Neuropatías Amiloides Familiares/metabolismo , Simulación por Computador , Humanos , Simulación del Acoplamiento Molecular , Prealbúmina/química , Multimerización de Proteína/efectos de los fármacosRESUMEN
Nucleosomes are the basic packaging units of chromatin, modulating accessibility of regulatory proteins to DNA and thus influencing eukaryotic gene regulation. Elaborate chromatin remodelling mechanisms have evolved that govern nucleosome organization at promoters, regulatory elements, and other functional regions in the genome. Analyses of chromatin landscape have uncovered a variety of mechanisms, including DNA sequence preferences, that can influence nucleosome positions. To identify major determinants of nucleosome organization in the human genome, we used deep sequencing to map nucleosome positions in three primary human cell types and in vitro. A majority of the genome showed substantial flexibility of nucleosome positions, whereas a small fraction showed reproducibly positioned nucleosomes. Certain sites that position in vitro can anchor the formation of nucleosomal arrays that have cell type-specific spacing in vivo. Our results unveil an interplay of sequence-based nucleosome preferences and non-nucleosomal factors in determining nucleosome organization within mammalian cells.
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Ensamble y Desensamble de Cromatina/fisiología , Regulación de la Expresión Génica , Nucleosomas/metabolismo , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Células Cultivadas , Genoma Humano/genética , Granulocitos/metabolismo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Nucleasa Microcócica/metabolismo , Nucleosomas/química , Nucleosomas/genética , Especificidad de Órganos , Transcripción GenéticaAsunto(s)
Anemia Aplásica/genética , Cirrosis Hepática/genética , Hígado/patología , Telomerasa/genética , Acortamiento del Telómero , Adulto , Anemia Aplásica/diagnóstico , Biopsia , Análisis Mutacional de ADN , Humanos , Hígado/citología , Hígado/diagnóstico por imagen , Cirrosis Hepática/diagnóstico , Cirrosis Hepática/patología , Masculino , Mutación Missense , Síndrome , Telomerasa/metabolismo , Telómero/metabolismo , Tomografía Computarizada por Rayos XRESUMEN
Trypanosoma brucei are protozoan parasites that cause African sleeping sickness in humans (also known as Human African Trypanosomiasis-HAT). Without treatment, T. brucei infections are fatal. There is an urgent need for new therapeutic strategies as current drugs are toxic, have complex treatment regimens, and are becoming less effective owing to rising antibiotic resistance in parasites. We hypothesize that targeting the HSP60/10 chaperonin systems in T. brucei is a viable anti-trypanosomal strategy as parasites rely on these stress response elements for their development and survival. We recently discovered several hundred inhibitors of the prototypical HSP60/10 chaperonin system from Escherichia coli, termed GroEL/ES. One of the most potent GroEL/ES inhibitors we discovered was compound 1. While examining the PubChem database, we found that a related analog, 2e-p, exhibited cytotoxicity to Leishmania major promastigotes, which are trypanosomatids highly related to Trypanosoma brucei. Through initial counter-screening, we found that compounds 1 and 2e-p were also cytotoxic to Trypanosoma brucei parasites (EC50=7.9 and 3.1µM, respectively). These encouraging initial results prompted us to develop a library of inhibitor analogs and examine their anti-parasitic potential in vitro. Of the 49 new chaperonin inhibitors developed, 39% exhibit greater cytotoxicity to T. brucei parasites than parent compound 1. While many analogs exhibit moderate cytotoxicity to human liver and kidney cells, we identified molecular substructures to pursue for further medicinal chemistry optimization to increase the therapeutic windows of this novel class of chaperonin-targeting anti-parasitic candidates. An intriguing finding from this study is that suramin, the first-line drug for treating early stage T. brucei infections, is also a potent inhibitor of GroEL/ES and HSP60/10 chaperonin systems.
Asunto(s)
Antiprotozoarios/farmacología , Chaperonina 10/efectos de los fármacos , Chaperonina 60/efectos de los fármacos , Trypanosoma brucei brucei/metabolismo , Tripanosomiasis Africana/tratamiento farmacológico , Animales , Antiprotozoarios/uso terapéutico , Evaluación Preclínica de Medicamentos , Ensayos Analíticos de Alto Rendimiento , HumanosRESUMEN
We recently reported results from a high-throughput screening effort that identified 235 inhibitors of the Escherichia coli GroEL/ES chaperonin system [Bioorg. Med. Chem. Lett.2014, 24, 786]. As the GroEL/ES chaperonin system is essential for growth under all conditions, we reasoned that targeting GroEL/ES with small molecule inhibitors could be a viable antibacterial strategy. Extending from our initial screen, we report here the antibacterial activities of 22 GroEL/ES inhibitors against a panel of Gram-positive and Gram-negative bacteria, including E. coli, Bacillus subtilis, Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter cloacae. GroEL/ES inhibitors were more effective at blocking the proliferation of Gram-positive bacteria, in particular S. aureus, where lead compounds exhibited antibiotic effects from the low-µM to mid-nM range. While several compounds inhibited the human HSP60/10 refolding cycle, some were able to selectively target the bacterial GroEL/ES system. Despite inhibiting HSP60/10, many compounds exhibited low to no cytotoxicity against human liver and kidney cell lines. Two lead candidates emerged from the panel, compounds 8 and 18, that exhibit >50-fold selectivity for inhibiting S. aureus growth compared to liver or kidney cell cytotoxicity. Compounds 8 and 18 inhibited drug-sensitive and methicillin-resistant S. aureus strains with potencies comparable to vancomycin, daptomycin, and streptomycin, and are promising candidates to explore for validating the GroEL/ES chaperonin system as a viable antibiotic target.