RESUMEN
Patients with cystic fibrosis (CF) have a significantly higher risk of acquiring nontuberculous mycobacteria infections, predominantly due to Mycobacterium abscessus, than the healthy population. Because M. abscessus infections are a major cause of clinical decline and morbidity in CF patients, improving treatment and the detection of this mycobacterium in the context of a polymicrobial culture represents a critical component to better manage patient care. We report here the synthesis of fluorescent Dansyl derivatives of four active cyclipostins and cyclophostin analogues (CyCs) and provide new insights regarding the CyC's lack of activity against Gram-negative and Gram-positive bacteria, and above all into their mode of action against intramacrophagic M. abscessus cells. Our results pointed out that the intracellularly active CyC accumulate in acidic compartments within macrophage cells, that this accumulation appears to be essential for their delivery to mycobacteria-containing phagosomes, and consequently, for their antimicrobial effect against intracellular replicating M. abscessus, and that modification of such intracellular localization via disruption of endolysosomal pH strongly affects the CyC accumulation and efficacy. Moreover, we discovered that these fluorescent compounds could become efficient probes to specifically label mycobacterial species with high sensitivity, including M. abscessus in the presence several other pathogens like Pseudomonas aeruginosa and Staphylococcus aureus. Collectively, all present and previous data emphasized the therapeutic potential of unlabeled CyCs and the attractiveness of the fluorescent CyC as a potential new efficient diagnostic tool to be exploited in future diagnostic developments against mycobacterial-related infections, especially against M. abscessus.
RESUMEN
AIM: Thoracoscopically placed internal traction sutures (ITS) for the initial management of long gap oesophageal atresia (LGOA), not amenable to primary anastomosis, was first described in 2015. Here we describe our experience using ITS both thoracoscopically and at thoracotomy where the gap between upper and lower oesophagus is too wide for primary anastomosis. METHOD: The case notes of all infants treated with ITS for oesophageal atresia (01/10/2015 to 01/12/2019) were reviewed. Gaps considered too wide for an anastomosis had ITS placed between the two pouches as described by Patkowski in 2015. All patients were gastrostomy fed. Patients returned to theatre with an expectation to complete the anastomosis or re-tighten the traction sutures. RESULTS: Seven patients (4 OA, 1 OA with proximal fistula, 2 OA/distal TOF) median birthweight 2.28 kg (1.2-3.6 kg) were managed using ITS. Median gap length 4.5 (3-9) vertebral bodies. ITS were placed thoracoscopically in 5 cases and at thoracotomy in 2 at median 46 days (1-120) old. In all cases, ITS was associated with significant intra-thoracic adhesions. Five patients leaked from the traction sutures. Four patients had a delayed primary anastomosis performed at thoracotomy and 3 required a cervical oesophagostomy. The median length of stay was 159 days (98-282). All patients started thoracoscopically eventually required thoracotomy. CONCLUSION: The use of ITS in our department was associated with significant complications, particularly intra-thoracic leaks and adhesions. In our hands ITS did not improve the feasibility of thoracoscopic repair for LGOA and has been abandoned by us. LEVEL OF EVIDENCE: Level IV Case Series.
Asunto(s)
Atresia Esofágica , Fístula Traqueoesofágica , Anastomosis Quirúrgica , Atresia Esofágica/cirugía , Humanos , Lactante , Suturas , Fístula Traqueoesofágica/cirugía , TracciónRESUMEN
Not available.
Asunto(s)
Mieloma Múltiple , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Humanos , Factores Inmunológicos , Mieloma Múltiple/tratamiento farmacológico , Mieloma Múltiple/genéticaRESUMEN
Twelve new Cyclophostin and Cyclipostins analogues (CyC19-30) were synthesized, thus extending our series to 38 CyCs. Their antibacterial activities were evaluated against four pathogenic mycobacteria (Mycobacterium abscessus, Mycobacterium marinum, Mycobacterium bovis BCG, and Mycobacterium tuberculosis) and two Gram negative bacteria. The CyCs displayed very low toxicity toward host cells and were only active against mycobacteria. Importantly, several CyCs were active against extracellular M. abscessus (CyC17/CyC18ß/CyC25/CyC26) or intramacrophage residing mycobacteria (CyC7(α,ß)/CyC8(α,ß)) with minimal inhibitory concentrations (MIC50) values comparable to or better than those of amikacin or imipenem, respectively. An activity-based protein profiling combined with mass spectrometry allowed identification of the potential target enzymes of CyC17/CyC26, mostly being involved in lipid metabolism and/or in cell wall biosynthesis. Overall, these results strengthen the selective activity of the CyCs against mycobacteria, including the most drug-resistant M. abscessus, through the cumulative inhibition of a large number of Ser- and Cys-enzymes participating in key physiological processes.
Asunto(s)
Antibacterianos/síntesis química , Bacterias/crecimiento & desarrollo , Compuestos Organofosforados/síntesis química , Antibacterianos/química , Antibacterianos/farmacología , Bacterias/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Viabilidad Microbiana/efectos de los fármacos , Estructura Molecular , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium abscessus/crecimiento & desarrollo , Mycobacterium bovis/efectos de los fármacos , Mycobacterium bovis/crecimiento & desarrollo , Mycobacterium marinum/efectos de los fármacos , Mycobacterium marinum/crecimiento & desarrollo , Mycobacterium tuberculosis/efectos de los fármacos , Mycobacterium tuberculosis/crecimiento & desarrollo , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacologíaRESUMEN
With the high number of patients infected by tuberculosis and the sharp increase of drug-resistant tuberculosis cases, developing new drugs to fight this disease has become increasingly urgent. In this context, analogs of the naturally occurring enolphosphates Cyclipostins and Cyclophostin (CyC analogs) offer new therapeutic opportunities. The CyC analogs display potent activity both in vitro and in infected macrophages against several pathogenic mycobacteria including Mycobacterium tuberculosis and Mycobacterium abscessus. Interestingly, these CyC inhibitors target several enzymes with active-site serine or cysteine residues that play key roles in mycobacterial lipid and cell wall metabolism. Among them, TesA, a putative thioesterase involved in the synthesis of phthiocerol dimycocerosates (PDIMs) and phenolic glycolipids (PGLs), has been identified. These two lipids (PDIM and PGL) are non-covalently bound to the outer cell wall in several human pathogenic mycobacteria and are important virulence factors. Herein, we used biochemical and structural approaches to validate TesA as an effective pharmacological target of the CyC analogs. We confirmed both thioesterase and esterase activities of TesA, and showed that the most active inhibitor CyC17 binds covalently to the catalytic Ser104 residue leading to a total loss of enzyme activity. These data were supported by the X-ray structure, obtained at a 2.6-Å resolution, of a complex in which CyC17 is bound to TesA. Our study provides evidence that CyC17 inhibits the activity of TesA, thus paving the way to a new strategy for impairing the PDIM and PGL biosynthesis, potentially decreasing the virulence of associated mycobacterial species.
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Glucolípidos/metabolismo , Mycobacterium tuberculosis/enzimología , Compuestos Organofosforados/farmacología , Tioléster Hidrolasas/química , Sitios de Unión , Dominio Catalítico/efectos de los fármacos , Pared Celular/metabolismo , Cristalografía por Rayos X , Inhibidores Enzimáticos , Lípidos , Mycobacterium tuberculosis/química , Tioléster Hidrolasas/antagonistas & inhibidores , Tioléster Hidrolasas/metabolismo , Factores de Virulencia/metabolismoRESUMEN
The progression of mycobacterial diseases requires the development of new therapeutics. This study evaluated the efficacy and selectivity of a panel of Cyclophostin and Cyclipostins analogues (CyCs) against various bacteria and mycobacteria. The activity 26 CyCs was first assayed by the agar plate method. Compounds exhibiting 50-100% growth inhibition were then selected to determine their minimum inhibitory concentrations (MICs) by the resazurin microtiter assay (REMA). The best drug candidate was further tested against clinical mycobacterial isolates and bacteria responsible for nosocomial infections, including 6 Gram-negative bacteria, 5 Gram-positive bacteria, 29 rapid-growing mycobacteria belonging to the Mycobacterium chelonae-abscessus clade and 3 slow-growing mycobacteria (Mycobacterium marinum, Mycobacterium bovis BCG and Mycobacterium tuberculosis). Among the 26 CyCs tested, 10 were active and their inhibitory activity was exclusively restricted to mycobacteria. The best candidate (CyC17) was further tested against 26 clinical strains and showed high selectivity for mycobacteria, with MICs (<2-40 µg/mL) comparable with those of most classical antimicrobials used to treat M. abscessus infections. Together, these results support the fact that such CyCs represent a new family of potent and selective inhibitors against mycobacteria. This is of particular interest for future chemotherapeutic developments against mycobacterial-associated infections, especially against M. abscessus, the most drug-resistant mycobacterial species.
Asunto(s)
Antibacterianos/uso terapéutico , Infecciones por Mycobacterium/tratamiento farmacológico , Mycobacterium abscessus/efectos de los fármacos , Mycobacterium bovis/efectos de los fármacos , Mycobacterium marinum/efectos de los fármacos , Mycobacterium tuberculosis/efectos de los fármacos , Compuestos Organofosforados/uso terapéutico , Infección Hospitalaria/tratamiento farmacológico , Infección Hospitalaria/microbiología , Farmacorresistencia Bacteriana Múltiple , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones por Mycobacterium/microbiología , Mycobacterium abscessus/aislamiento & purificación , Mycobacterium bovis/aislamiento & purificación , Mycobacterium marinum/aislamiento & purificación , Mycobacterium tuberculosis/aislamiento & purificaciónRESUMEN
A new class of Cyclophostin and Cyclipostins (CyC) analogs have been investigated against Mycobacterium tuberculosis H37Rv (M. tb) grown either in broth medium or inside macrophages. Our compounds displayed a diversity of action by acting either on extracellular M. tb bacterial growth only, or both intracellularly on infected macrophages as well as extracellularly on bacterial growth with very low toxicity towards host macrophages. Among the eight potential CyCs identified, CyC 17 exhibited the best extracellular antitubercular activity (MIC50 = 500 nM). This compound was selected and further used in a competitive labelling/enrichment assay against the activity-based probe Desthiobiotin-FP in order to identify its putative target(s). This approach, combined with mass spectrometry, identified 23 potential candidates, most of them being serine or cysteine enzymes involved in M. tb lipid metabolism and/or in cell wall biosynthesis. Among them, Ag85A, CaeA and HsaD, have previously been reported as essential for in vitro growth of M. tb and/or survival and persistence in macrophages. Overall, our findings support the assumption that CyC 17 may thus represent a novel class of multi-target inhibitor leading to the arrest of M. tb growth through a cumulative inhibition of a large number of Ser- and Cys-containing enzymes participating in important physiological processes.
Asunto(s)
Antituberculosos , Macrófagos/microbiología , Mycobacterium tuberculosis/crecimiento & desarrollo , Compuestos Organofosforados , Tuberculosis/tratamiento farmacológico , Antituberculosos/química , Antituberculosos/farmacología , Humanos , Macrófagos/metabolismo , Macrófagos/patología , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Tuberculosis/metabolismo , Tuberculosis/patologíaRESUMEN
BET inhibitors (BETi) target bromodomain-containing proteins and are currently being evaluated as anti-cancer agents. We find that maximal therapeutic effects of BETi in a Myc-driven B cell lymphoma model required an intact host immune system. Genome-wide analysis of the BETi-induced transcriptional response identified the immune checkpoint ligand Cd274 (Pd-l1) as a Myc-independent, BETi target-gene. BETi directly repressed constitutively expressed and interferon-gamma (IFN-γ) induced CD274 expression across different human and mouse tumor cell lines and primary patient samples. Mechanistically, BETi decreased Brd4 occupancy at the Cd274 locus without any change in Myc occupancy, resulting in transcriptional pausing and rapid loss of Cd274 mRNA production. Finally, targeted inhibition of the PD-1/PD-L1 axis by combining anti-PD-1 antibodies and the BETi JQ1 caused synergistic responses in mice bearing Myc-driven lymphomas. Our data uncover an interaction between BETi and the PD-1/PD-L1 immune-checkpoint and provide mechanistic insight into the transcriptional regulation of CD274.
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Desoxirribonucleasas de Localización Especificada Tipo II/inmunología , Sistema Inmunológico/inmunología , Proteínas Nucleares/inmunología , Receptor de Muerte Celular Programada 1/inmunología , Factores de Transcripción/inmunología , Animales , Antígeno B7-H1/inmunología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica/inmunología , Humanos , Interferón gamma/inmunología , Ligandos , Linfoma de Células B/inmunología , Ratones , Proteínas Proto-Oncogénicas c-myc/inmunología , ARN Mensajero/inmunología , Transcripción Genética/inmunologíaRESUMEN
The Eµ-Myc mouse is an extensively used model of MYC driven malignancy; however to date there has only been partial characterization of MYC co-operative mutations leading to spontaneous lymphomagenesis. Here we sequence spontaneously arising Eµ-Myc lymphomas to define transgene architecture, somatic mutations, and structural alterations. We identify frequent disruptive mutations in the PRC1-like component and BCL6-corepressor gene Bcor. Moreover, we find unexpected concomitant multigenic lesions involving Cdkn2a loss and other cancer genes including Nras, Kras and Bcor. These findings challenge the assumed two-hit model of Eµ-Myc lymphoma and demonstrate a functional in vivo role for Bcor in suppressing tumorigenesis.
Asunto(s)
Linfocitos B/metabolismo , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B/genética , Mutación , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Represoras/genética , Alelos , Animales , Linfocitos B/inmunología , Linfocitos B/patología , Sistemas CRISPR-Cas , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/inmunología , Modelos Animales de Enfermedad , Edición Génica , Frecuencia de los Genes , Janus Quinasa 2/genética , Janus Quinasa 2/inmunología , Linfoma de Células B/inmunología , Linfoma de Células B/patología , Ratones , Ratones Transgénicos , Proteínas Proto-Oncogénicas c-myc/inmunología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/inmunología , Proteínas Represoras/inmunología , Factor de Transcripción STAT5/genética , Factor de Transcripción STAT5/inmunología , Transcriptoma , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/inmunología , Secuenciación Completa del GenomaRESUMEN
Targeting BET bromodomain proteins using small molecules is an emerging anticancer strategy with clinical evaluation of at least six inhibitors now underway. Although MYC downregulation was initially proposed as a key mechanistic property of BET inhibitors, recent evidence suggests that additional antitumor activities are important. Using the Eµ-Myc model of B-cell lymphoma, we demonstrate that BET inhibition with JQ1 is a potent inducer of p53-independent apoptosis that occurs in the absence of effects on Myc gene expression. JQ1 skews the expression of proapoptotic (Bim) and antiapoptotic (BCL-2/BCL-xL) BCL-2 family members to directly engage the mitochondrial apoptotic pathway. Consistent with this, Bim knockout or Bcl-2 overexpression inhibited apoptosis induction by JQ1. We identified lymphomas that were either intrinsically resistant to JQ1-mediated death or acquired resistance following in vivo exposure. Strikingly, in both instances BCL-2 was strongly upregulated and was concomitant with activation of RAS pathways. Eµ-Myc lymphomas engineered to express activated Nras upregulated BCL-2 and acquired a JQ1 resistance phenotype. These studies provide important information on mechanisms of apoptosis induction and resistance to BET-inhibition, while providing further rationale for the translation of BET inhibitors in aggressive B-cell lymphomas. Mol Cancer Ther; 15(9); 2030-41. ©2016 AACR.
Asunto(s)
Apoptosis/genética , Azepinas/farmacología , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B/genética , Proteínas Proto-Oncogénicas c-bcl-2/genética , Triazoles/farmacología , Animales , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Resistencia a Antineoplásicos/genética , Genes myc , Humanos , Linfoma de Células B/metabolismo , Linfoma de Células B/mortalidad , Linfoma de Células B/patología , Ratones , Familia de Multigenes , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Transducción de Señal/efectos de los fármacos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
New monocyclic phosphate, phosphonate and difluorophosphonate analogs of the natural AChE inhibitor cyclophostin were synthesized and their activity toward human AChE examined. Surprisingly, the phosphate, phosphonate, and difluorophosphonate analogs all showed diminished activity when compared with the natural product.
Asunto(s)
Inhibidores de la Colinesterasa/química , Inhibidores de la Colinesterasa/farmacología , Compuestos Organofosforados/química , Compuestos Organofosforados/farmacología , Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/síntesis química , Halogenación , Humanos , Organofosfatos/síntesis química , Organofosfatos/química , Organofosfatos/farmacología , Organofosfonatos/síntesis química , Organofosfonatos/química , Organofosfonatos/farmacología , Compuestos Organofosforados/síntesis químicaRESUMEN
Using the semiempirical method PM7, an attempt has been made to quantify the error in prediction of the in vivo structure of proteins relative to X-ray structures. Three important contributory factors are the experimental limitations of X-ray structures, the difference between the crystal and solution environments, and the errors due to PM7. The geometries of 19 proteins from the Protein Data Bank that had small R values, that is, high accuracy structures, were optimized and the resulting drop in heat of formation was calculated. Analysis of the changes showed that about 10% of this decrease in heat of formation was caused by faults in PM7, the balance being attributable to the X-ray structure and the difference between the crystal and solution environments. A previously unknown fault in PM7 was revealed during tests to validate the geometries generated using PM7. Clashscores generated by the Molprobity molecular mechanics structure validation program showed that PM7 was predicting unrealistically close contacts between nonbonding atoms in regions where the local geometry is dominated by very weak noncovalent interactions. The origin of this fault was traced to an underestimation of the core-core repulsion between atoms at distances smaller than the equilibrium distance.
Asunto(s)
Biología Computacional/métodos , Modelos Moleculares , Conformación Proteica , Proteínas/química , Cristalografía por Rayos X , Bases de Datos de Proteínas , Reproducibilidad de los Resultados , TermodinámicaRESUMEN
Cyclipostins are bicyclic lipophilic phosphate natural products. We report here that synthesized individual diastereomers of cyclipostins P and R have nanomolar IC50s toward hormone sensitive lipase (HSL). The less potent diastereomers of these compounds have 10-fold weaker IC50s. The monocyclic phosphate analog of cyclipostin P is nearly as potent as the bicyclic natural product. Bicyclic phosphonate analogs of both cyclipostins exhibit IC50s similar to those of the weaker diastereomer phosphates (about 400nM). The monocyclic phosphonate analog of cyclipostin P has similar potency. A series of monocyclic phosphonate analogs in which a hydrophobic tail extends from the lactone side of the ring are considerably poorer inhibitors, with IC50s around 50µM. Finally cyclophostin, a related natural product inhibitor of acetylcholinesterase (AChE) that lacks the hydrocarbon tail of cyclipostins, is not active against HSL. These results indicate a critical SAR for these compounds, the hydrophobic tail. The smaller lactone ring is not critical to activity, a similarity shared with cyclophostin and AChE. The HSL kinetics of inhibition for the cyclipostin P trans diastereomer were examined in detail. The reaction is irreversible with a KI of 40nM and a rate constant for inactivation of 0.2min(-1). These results are similar to those observed for cyclophostin and AChE.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Compuestos Organofosforados/farmacología , Esterol Esterasa/antagonistas & inhibidores , Animales , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/química , Concentración 50 Inhibidora , Compuestos Organofosforados/química , Ratas , Células Sf9 , EstereoisomerismoRESUMEN
An accurate model of three-dimensional protein structure is important in a variety of fields such as structure-based drug design and mechanistic studies of enzymatic reactions. While the entries in the Protein Data Bank ( http://www.pdb.org ) provide valuable information about protein structures, a small fraction of the PDB structures were found to contain anomalies not reported in the PDB file. The semiempirical PM7 method in MOPAC2012 was used for identifying anomalously short hydrogen bonds, C-Hâ¯O/C-Hâ¯N interactions, non-bonding close contacts, and unrealistic covalent bond lengths in recently published Protein Data Bank files. It was also used to generate new structures with these faults removed. When the semiempirical models were compared to those of PDB_REDO (http://www.cmbi.ru.nl/pdb_redo/), the clashscores, as defined by MolProbity ( http://molprobity.biochem.duke.edu/), were better in about 50% of the structures. The semiempirical models also had a lower root-mean-square-deviation value in nearly all cases than those from PDB_REDO, indicative of a better conservation of the tertiary structure. Finally, the semiempirical models were found to have lower clashscores than the initial PDB file in all but one case. Because this approach maintains as much of the original tertiary structure as possible while improving anomalous interactions, it should be useful to theoreticians, experimentalists, and crystallographers investigating the structure and function of proteins.
Asunto(s)
Bases de Datos de Proteínas , Modelos Moleculares , Proteínas/química , Enlace de Hidrógeno , Conformación ProteicaRESUMEN
N-methyl-2-pyrrolidone (NMP) is a common solvent and drug vehicle. We discovered unexpected antineoplastic and immunomodulatory activity of NMP in a cMYC-driven myeloma model. Coincident to this, NMP was identified as an acetyllysine mimetic and candidate bromodomain ligand. Accordingly, NMP-treated cells demonstrated transcriptional overlap with BET-bromodomain inhibition, including downregulation of cMYC and IRF4. NMP's immunomodulatory activity occurred at sub-BET inhibitory concentrations, and, despite phenotypic similarities to lenalidomide, its antimyeloma activity was independent of the IMiD targets cereblon and Ikaros-1/3. Thus, low-affinity yet broad-spectrum bromodomain inhibition by NMP mediates biologically potent, cereblon-independent immunomodulation and at higher doses targets malignant cells directly via BET antagonism. These data reveal that NMP is a functional acetyllysine mimetic with pleotropic antimyeloma and immunomodulatory activities. Our studies highlight the potential therapeutic benefits of NMP, the consequences of current human NMP exposures, and the need for reassessment of scientific literature where NMP was used as an "inert" drug-delivery vehicle.
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Antineoplásicos/farmacología , Factores Inmunológicos/farmacología , Mieloma Múltiple/tratamiento farmacológico , Pirrolidinonas/farmacología , Animales , Diferenciación Celular/efectos de los fármacos , Células Cultivadas , Humanos , Ratones , Ratones Endogámicos C57BL , Mieloma Múltiple/inmunología , Mieloma Múltiple/metabolismo , Mieloma Múltiple/patología , Transducción de Señal/efectos de los fármacos , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Epigenetic modifying enzymes such as histone deacetylases (HDACs), p300, and PRMT1 are recruited by AML1/ETO, the pathogenic protein for t(8;21) acute myeloid leukemia (AML), providing a strong molecular rationale for targeting these enzymes to treat this disease. Although early phase clinical assessment indicated that treatment with HDAC inhibitors (HDACis) may be effective in t(8;21) AML patients, rigorous preclinical studies to identify the molecular and biological events that may determine therapeutic responses have not been performed. Using an AML mouse model driven by expression of AML1/ETO9a (A/E9a), we demonstrated that treatment of mice bearing t(8;21) AML with the HDACi panobinostat caused a robust antileukemic response that did not require functional p53 nor activation of conventional apoptotic pathways. Panobinostat triggered terminal myeloid differentiation via proteasomal degradation of A/E9a. Importantly, conditional A/E9a deletion phenocopied the effects of panobinostat and other HDACis, indicating that destabilization of A/E9a is critical for the antileukemic activity of these agents.
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Antineoplásicos/uso terapéutico , Diferenciación Celular/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Ácidos Hidroxámicos/uso terapéutico , Indoles/uso terapéutico , Leucemia Mieloide Aguda/tratamiento farmacológico , Animales , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Cromosomas Humanos Par 21/genética , Cromosomas Humanos Par 8/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Modelos Animales de Enfermedad , Embrión de Mamíferos , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Fusión Oncogénica/genética , Panobinostat , Proteína 1 Compañera de Translocación de RUNX1 , Translocación GenéticaRESUMEN
Pharmacological strategies capable of directly targeting MYC are elusive. Previous studies have shown that MYC-driven lymphomagenesis is associated with mammalian target of rapamycin (mTOR) activation and a MYC-evoked DNA damage response (DDR) transduced by phosphatidylinositol-3-kinase (PI3K)-related kinases (DNA-PK, ATM, and ATR). Here we report that BEZ235, a multitargeted pan-PI3K/dual-mTOR inhibitor, potently killed primary Myc-driven B-cell lymphomas and human cell lines bearing IG-cMYC translocations. Using pharmacologic and genetic dissection of PI3K/mTOR signaling, dual DDR/mTORC1 inhibition was identified as a key mediator of apoptosis. Moreover, apoptosis was initiated at drug concentrations insufficient to antagonize PI3K/mTORC2-regulated AKT phosphorylation. p53-independent induction of the proapoptotic BH3-only protein BMF was identified as a mechanism by which dual DDR/mTORC1 inhibition caused lymphoma cell death. BEZ235 treatment induced apoptotic tumor regressions in vivo that correlated with suppression of mTORC1-regulated substrates and reduced H2AX phosphorylation and also with feedback phosphorylation of AKT. These mechanistic studies hold important implications for the use of multitargeted PI3K inhibitors in the treatment of hematologic malignancies. In particular, the newly elucidated role of PI3K-related DDR kinases in response to PI3K inhibitors offers a novel therapeutic opportunity for the treatment of hematologic malignancies with an MYC-driven DDR.
Asunto(s)
Apoptosis/efectos de los fármacos , Imidazoles/farmacología , Linfoma de Células B/prevención & control , Quinolinas/farmacología , Animales , Western Blotting , Línea Celular , Daño del ADN/efectos de los fármacos , Receptor con Dominio Discoidina 1 , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Histonas/metabolismo , Humanos , Linfoma de Células B/genética , Linfoma de Células B/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Complejos Multiproteicos/antagonistas & inhibidores , Complejos Multiproteicos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación/efectos de los fármacos , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Tirosina Quinasas Receptoras/antagonistas & inhibidores , Proteínas Tirosina Quinasas Receptoras/metabolismo , Análisis de Supervivencia , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , Células Tumorales CultivadasRESUMEN
A new series of customizable diastereomeric cis- and trans-monocyclic enol-phosphonate analogs to Cyclophostin and Cyclipostins were synthesized. Their potencies and mechanisms of inhibition toward six representative lipolytic enzymes belonging to distinct lipase families were examined. With mammalian gastric and pancreatic lipases no inhibition occurred with any of the compounds tested. Conversely, Fusarium solani Cutinase and lipases from Mycobacterium tuberculosis (Rv0183 and LipY) were all fully inactivated. The best inhibitors displayed a cis conformation (H and OMe) and exhibited higher inhibitory activities than the lipase inhibitor Orlistat toward the same enzymes. Our results have revealed that chemical group at the γ-carbon of the phosphonate ring strongly impacts the inhibitory efficiency, leading to a significant improvement in selectivity toward a target lipase over another. The powerful and selective inhibition of microbial (fungal and mycobacterial) lipases suggests that these seven-membered monocyclic enol-phosphonates should provide useful leads for the development of novel and highly selective antimicrobial agents.
Asunto(s)
Hidrolasas de Éster Carboxílico/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Inhibidores Enzimáticos/farmacología , Organofosfonatos/química , Compuestos Organofosforados/química , Hidrolasas de Éster Carboxílico/metabolismo , Fusarium/enzimología , Cinética , Lipasa/antagonistas & inhibidores , Lipasa/metabolismo , Estructura Molecular , Mycobacterium tuberculosis/enzimología , Relación Estructura-ActividadRESUMEN
Histone deacetylase inhibitor (HDACI)-induced thrombocytopenia (TCP) is a major dose-limiting toxicity of this new class of drugs. Using preclinical models to study the molecular and biologic events that underpin this effect of HDACI, we found that C57BL/6 mice treated with both the HDAC1/2-selective HDACI romidepsin and the pan-HDACI panobinostat developed significant TCP. HDACI-induced TCP was not due to myelosuppression or reduced platelet lifespan, but to decreased platelet release from megakaryocytes. Cultured primary murine megakaryocytes showed reductions in proplatelet extensions after HDACI exposure and a dose-dependent increase in the phosphorylation of myosin light chain 2 (MLC2). Phosphorylation of MLC to phospho-MLC (pMLC) and subsequent proplatelet formation in megakaryocytes is regulated by the Rho-GTPase proteins Rac1, CDC42, and RhoA. Primary mouse megakaryocytes and the human megakaryoblastic cell line Meg-01 showed reductions in Rac1, CDC42, and RhoA protein levels after treatment with HDACIs. We were able to overcome HDACI-induced TCP by administering the mouse-specific thrombopoietin (TPO) mimetic AMP-4, which improved platelet numbers to levels similar to untreated controls. Our report provides the first detailed account of the molecular and biologic processes involved in HDACI-mediated TCP. Moreover, our preclinical studies provide evidence that dose-limiting TCP induced by HDACIs may be circumvented using a TPO mimetic.