RESUMEN
Mutations in isocitrate dehydrogenase 1 (IDH1) drive most low-grade gliomas and secondary glioblastomas and many chondrosarcomas and acute myeloid leukemia cases. Most tumor-relevant IDH1 mutations are deficient in the normal oxidization of isocitrate to α-ketoglutarate (αKG), but gain the neomorphic activity of reducing αKG to D-2-hydroxyglutarate (D2HG), which drives tumorigenesis. We found previously that IDH1 mutants exhibit one of two reactivities: deficient αKG and moderate D2HG production (including commonly observed R132H and R132C) or moderate αKG and high D2HG production (R132Q). Here, we identify a third type of reactivity, deficient αKG and high D2HG production (R132L). We show that R132Q IDH1 has unique structural features and distinct reactivities towards mutant IDH1 inhibitors. Biochemical and cell-based assays demonstrate that while most tumor-relevant mutations were effectively inhibited by mutant IDH1 inhibitors, R132Q IDH1 had up to a 16 300-fold increase in IC50 versus R132H IDH1. Only compounds that inhibited wild-type (WT) IDH1 were effective against R132Q. This suggests that patients with a R132Q mutation may have a poor response to mutant IDH1 therapies. Molecular dynamics simulations revealed that near the NADP+/NADPH-binding site in R132Q IDH1, a pair of α-helices switches between conformations that are more wild-type-like or more mutant-like, highlighting mechanisms for preserved WT activity. Dihedral angle changes in the dimer interface and buried surface area charges highlight possible mechanisms for loss of inhibitor affinity against R132Q. This work provides a platform for predicting a patient's therapeutic response and identifies a potential resistance mutation that may arise upon treatment with mutant IDH inhibitors.
Asunto(s)
Carcinogénesis/genética , Carcinogénesis/metabolismo , Isocitrato Deshidrogenasa/genética , Isocitrato Deshidrogenasa/metabolismo , Mutación/fisiología , Sitios de Unión/fisiología , Células HEK293 , Células HeLa , Humanos , Isocitrato Deshidrogenasa/química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Mutations in isocitrate dehydrogenase 1 (IDH1) impart a neomorphic reaction that produces the oncometabolite D-2-hydroxyglutarate (D2HG), which can inhibit DNA and histone demethylases to drive tumorigenesis via epigenetic changes. Though heterozygous point mutations in patients primarily affect residue R132, there are myriad D2HG-producing mutants that display unique catalytic efficiency of D2HG production. Here, we show that catalytic efficiency of D2HG production is greater in IDH1 R132Q than R132H mutants, and expression of IDH1 R132Q in cellular and mouse xenograft models leads to higher D2HG concentrations in cells, tumors, and sera compared to R132H-expressing models. Reduced representation bisulfite sequencing (RRBS) analysis of xenograft tumors shows expression of IDH1 R132Q relative to R132H leads to hypermethylation patterns in pathways associated with DNA damage. Transcriptome analysis indicates that the IDH1 R132Q mutation has a more aggressive pro-tumor phenotype, with members of EGFR, Wnt, and PI3K signaling pathways differentially expressed, perhaps through non-epigenetic routes. Together, these data suggest that the catalytic efficiency of IDH1 mutants modulate D2HG levels in cellular and in vivo models, resulting in unique epigenetic and transcriptomic consequences where higher D2HG levels appear to be associated with more aggressive tumors.
RESUMEN
PURPOSE: The current pilot, single-blind, randomized controlled trial (RCT) examined the feasibility of remotely-delivered and supported aerobic walking exercise training compared with an active control condition on cognitive processing speed (CPS) in 19 fully-ambulatory persons with multiple sclerosis (pwMS) who were pre-screened for impaired CPS. METHODS: Feasibility was assessed in the domains of process (e.g., recruitment), resource (e.g., monetary costs), management (e.g., time requirements), and scientific outcomes (i.e., treatment effect). Fully-ambulatory, but CPS-impaired pwMS were randomly assigned into either 16-weeks of home-based aerobic walking exercise or home-based stretching and range-of-motion activities. Both conditions involved delivery of informational newsletters and one-on-one, online video chats with a behavior coach. Participants across both conditions tracked their activity using highly accurate wearable motion sensors. Treatment-blinded assessors administered the Symbol Digit Modalities Test (SDMT) remotely before and after the 16-week study period. RESULTS: The study was cost-effective, accessible, and acceptable. The intervention further was safe. Adherence and compliance rates across both conditions exceeded 80%. There was an overall moderate effect for change in SDMT score between the conditions (d = 0.42). The intervention was associated with a 4.8-point improvement in SDMT scores (d = 0.70; 10% increase) compared with a 1-point improvement for the control condition (d = 0.09; 2% increase). CONCLUSIONS: This remotely-delivered and supported aerobic walking exercise training intervention was safe and feasible for fully-ambulatory, CPS-impaired pwMS. The pattern of results, including the promising effects on CPS, support the design and implementation of an appropriately-powered RCT on this approach for managing CPS impairment in a large MS sample.
Asunto(s)
Esclerosis Múltiple , Velocidad de Procesamiento , Humanos , Estudios de Factibilidad , Ejercicio Físico , Esclerosis Múltiple/complicaciones , Esclerosis Múltiple/terapia , Esclerosis Múltiple/psicología , Caminata , Terapia por Ejercicio/métodosRESUMEN
RATIONALE: Mast cells and neutrophils are key contributors to the pathophysiological inflammatory processes that underpin asthma and chronic obstructive pulmonary disease, partly through the release of noxious serine proteases, including cathepsin G (Cat G) and chymase. From this standpoint, a dual inhibitor of neutrophil Cat G and mast cell chymase could protect against these disease-related inflammatory responses. OBJECTIVES: We examined the antiinflammatory pharmacology of RWJ-355871, a dual inhibitor of Cat G and chymase, in animal models of inflammation that evince pathophysiological pathways relevant to asthma and chronic obstructive pulmonary disease to determine the therapeutic potential of this compound. METHODS: In an ovalbumin (OVA)-sensitized rat model, RWJ-355871 was administered to block the mast-cell-mediated increase in paw volume caused by OVA injection. In a sheep asthma model, antigen-induced airway responses were assessed with and without aerosol treatment with RWJ-355871. In a murine tobacco-smoke model of airway inflammation, the effect of RWJ-355871 on smoke-induced neutrophilia was determined. MEASUREMENTS AND MAIN RESULTS: Intravenous treatment of OVA-sensitized rats with RWJ-355871 provided dose-dependent reduction in the increase in rat paw volume. In allergic sheep, aerosol pretreatment with RWJ-355871 showed dose-dependent inhibition of the antigen-induced early response, late response, and post-antigen-induced airway hyperreponsiveness. In tobacco-smoke-exposed mice, nebulized RWJ-355871 significantly reduced the smoke-induced neutrophilia from the levels observed in untreated mice. CONCLUSIONS: The preclinical antiinflammatory effects of RWJ-355871 in these animal models of inflammation indicate that this dual inhibitor may have therapeutic utility for treating airway inflammatory diseases involving mechanisms that depend on Cat G and/or chymase.
Asunto(s)
Catepsina G/antagonistas & inhibidores , Quimasas/antagonistas & inhibidores , Enfermedades Pulmonares/enzimología , Organofosfonatos/uso terapéutico , Piperidinas/uso terapéutico , Enfermedad Pulmonar Obstructiva Crónica/enzimología , Animales , Biomarcadores/metabolismo , Líquido del Lavado Bronquioalveolar/química , Catepsina G/metabolismo , Quimasas/metabolismo , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Ensayo de Inmunoadsorción Enzimática , Femenino , Inyecciones Intravenosas , Enfermedades Pulmonares/tratamiento farmacológico , Ratones , Organofosfonatos/administración & dosificación , Piperidinas/administración & dosificación , Enfermedad Pulmonar Obstructiva Crónica/tratamiento farmacológico , Ratas , Ovinos , Resultado del TratamientoRESUMEN
Zika virus (ZIKV), an emerging flavivirus that causes neurodevelopmental impairment to fetuses and has been linked to Guillain-Barré syndrome continues to threaten global health due to the absence of targeted prophylaxis or treatment. Nucleoside analogues are good examples of efficient anti-viral inhibitors, and prodrug strategies using phosphate masking groups (ProTides) have been employed to improve the bioavailability of ribonucleoside analogues. Here, we synthesized and tested a small library of 13 ProTides against ZIKV in human neural stem cells. Strong activity was observed for 2'-C-methyluridine and 2'-C-ethynyluridine ProTides with an aryloxyl phosphoramidate masking group. Substitution of a 2-(methylthio) ethyl phosphoramidate for the aryloxyl phosphoramidate ProTide group of 2'-C-methyluridine completely abolished antiviral activity of the compound. The aryloxyl phosphoramidate ProTide of 2'-C-methyluridine outperformed the hepatitis C virus (HCV) drug sofosbuvir in suppression of viral titers and protection from cytopathic effect, while the former compound's triphosphate active metabolite was better incorporated by purified ZIKV NS5 polymerase over time. These findings suggest both a nucleobase and ProTide group bias for the anti-ZIKV activity of nucleoside analogue ProTides in a disease-relevant cell model.
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Antivirales/farmacología , Células-Madre Neurales/virología , Nucleósidos/análogos & derivados , Nucleósidos/farmacología , Virus Zika/efectos de los fármacos , Descubrimiento de Drogas , Humanos , Profármacos/farmacología , Bibliotecas de Moléculas PequeñasRESUMEN
AIMS: Bombesin receptors (BB receptors) and bombesin related peptides are expressed in the lower urinary tract of rodents. Here we investigated whether in vivo activation of BB receptors can contract the urinary bladder and facilitate micturition in sham rats and in a diabetic rat model of voiding dysfunction. MATERIAL AND METHODS: In vivo cystometry experiments were performed in adult female Sprague-Dawley rats under urethane anesthesia. Diabetes was induced by streptozotocin (STZ; 65mg/kg, i.p.) injection. Experiments were performed 9 and 20weeks post STZ-treatment. Drugs included neuromedin B (NMB; BB1 receptor preferring agonist), and gastrin-releasing peptide (GRP; BB2 receptor preferring agonist). KEY FINDINGS: NMB and GRP (0.01-100µg/kg in sham rats; 0.1-300µg/kg in STZ-treated rats, i.v.) increased micturition frequency, bladder contraction amplitude and area under the curve dose dependently in both sham and STZ-treated rats. In addition, NMB (3, 10µg/kg i.v.) triggered voiding in >80% of STZ-treated rats when the bladder was filled to a sub-threshold voiding volume. NMB and GRP increased mean arterial pressure and heart rate at the highest doses, 100 and 300µg/kg. SIGNIFICANCE: Activation of bombesin receptors facilitated neurogenic bladder contractions in vivo. Single applications of agonists enhanced or triggered voiding in sham rats as well as in the STZ-treated rat model of diabetic voiding dysfunction. These results suggest that BB receptors may be targeted for drug development for conditions associated with poor detrusor contraction such as an underactive bladder condition.
Asunto(s)
Diabetes Mellitus Experimental/fisiopatología , Receptores de Bombesina/fisiología , Vejiga Urinaria/efectos de los fármacos , Animales , Diabetes Mellitus Experimental/metabolismo , Evaluación Preclínica de Medicamentos , Femenino , Péptido Liberador de Gastrina/farmacología , Contracción Muscular , Neuroquinina B/análogos & derivados , Neuroquinina B/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Bombesina/agonistas , Vejiga Urinaria/metabolismo , Vejiga Urinaria/fisiopatología , Micción/efectos de los fármacosRESUMEN
AIMS: To determine if the muscarinic agonist, bethanechol, inhibits the non-cholinergic, atropine-resistant (i.e. putatively purinergic) component of naturally occurring (i.e. reflexogenic) bladder contractions in vivo in the rat, as previously described in vitro. Our second aim was to determine if elevation of endogenous acetylcholine (ACh) with distigmine, an acetylcholine esterase (AChE) inhibitor, could also inhibit non-cholinergic component of reflexogenic bladder contractions. MAIN METHODS: Cystometry was performed in urethane anesthetized adult female Sprague Dawley rats. The nonselective muscarinic receptor (mAChR) antagonist, atropine, was administered intravenously (i.v.) before and after i.v. administration of the non-selective mAChR agonist, bethanechol, the AChE inhibitor, distigmine or the neurokinin receptor 2 agonist, [ßAla(8)]-Neurokinin A(4-10). Intermicturition interval (IMI), bladder contraction amplitude (BCA), postvoid bladder pressure (PVBP), and voiding efficiency (VE) were measured. KEY FINDINGS: Atropine (0.4 mg/kg; n=11 rats) delivered as the first drug, had insignificant effects on BCA (~15% reduction) or PVBP (~15% increase) and weakly reduced IMI and VE by ~40% (p<0.05) relative to vehicle. Bethanechol and distigmine on their own produced excitatory effects on bladder activity, consistent with mAChR activation. Unexpectedly atropine, administered after bethanechol or after distigmine but not after [ßAla(8)]-Neurokinin A(4-10), completely blocked bladder activity for 3-10 min. Partial recovery of bladder activity occurred after that time, but BCA, IMI, and VE remained significantly reduced and PVBP remained significantly increased. SIGNIFICANCE: Activation of mAChRs by an exogenous agonist or elevation of endogenous ACh levels by an AChE inhibitor inhibits the non-cholinergic, atropine-resistant, component of reflexogenic bladder contractions in vivo.
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Betanecol/farmacología , Agonistas Muscarínicos/farmacología , Contracción Muscular/efectos de los fármacos , Receptores Muscarínicos/metabolismo , Vejiga Urinaria/efectos de los fármacos , Acetilcolina/metabolismo , Animales , Atropina/farmacología , Inhibidores de la Colinesterasa/farmacología , Femenino , Antagonistas Muscarínicos/farmacología , Compuestos de Piridinio/farmacología , Ratas , Ratas Sprague-Dawley , Vejiga Urinaria/fisiologíaRESUMEN
AIMS: Bombesin receptors (BB receptors) and/or bombesin related peptides are expressed in the lower urinary tract, though their function and distribution in different species is largely unknown. This study examines whether BB receptor agonists can contract bladder smooth muscle in rats, mice, pigs and humans. METHODS: Bladder strips were placed in tissue baths for in vitro contractility. Neuronally evoked contractions were elicited using electric field stimulation (EFS). Effects of the BB receptor agonists, neuromedin B (NMB; BB1 receptor agonist) and gastrin-releasing peptide (GRP; BB2 receptor agonist) on baseline tone and EFS-induced contractions were monitored. RESULTS: In rat and human bladder strips, NMB and GRP (10(-11)-10(-6)M) increased EFS-induced contractions in a concentration dependent manner. In these species, NMB and GRP also increased baseline tension. In mouse and pig bladder strips, NMB and GRP (10(-8)-3×10(-6)M) had no effects on either parameter. CONCLUSIONS: These data suggest that bombesin receptors BB receptor 1 and/or BB receptor 2 increase bladder contractions in rat and human. The site of action of these receptors may be pre- and/or post-synaptic, increasing release of transmitters or enhancing smooth muscle excitability, respectively. Thus, BB1 receptor and/or BB2 receptor may offer therapeutic targets for voiding dysfunction associated with impaired bladder contractility; however, species differences must be considered when studying these receptors. Part of this work was published in an abstract form at the SFN meeting New Orleans, 2012.
Asunto(s)
Receptores de Bombesina/metabolismo , Vejiga Urinaria/fisiología , Adulto , Anciano , Animales , Estimulación Eléctrica , Femenino , Péptido Liberador de Gastrina/farmacología , Humanos , Técnicas In Vitro , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neuroquinina B/análogos & derivados , Neuroquinina B/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Bombesina/agonistas , Porcinos , Vejiga Urinaria/efectos de los fármacos , Vejiga Urinaria/metabolismoRESUMEN
Time- and vehicle-related variability of bladder and urethral rhabdosphincter (URS) activity as well as cardiorespiratory and blood chemistry values were examined in the acetic acid-induced bladder irritation model in α-chloralose-anesthetized female cats. Additionally, bladder and urethra were evaluated histologically using Mason trichrome and toluidine blue staining. Urodynamic, cardiovascular and respiratory parameters were collected during intravesical saline infusion followed by acetic acid (0.5%) to irritate the bladder. One hour after starting acetic acid infusion, a protocol consisting of a cystometrogram, continuous infusion-induced rhythmic voiding contractions, and a 5 min "quiet period" (bladder emptied without infusion) was precisely repeated every 30 minutes. Administration of vehicle (saline i.v.) occurred 15 minutes after starting each of the first 7 cystometrograms and duloxetine (1mg/kg i.v.) after the 8(th). Acetic acid infusion into the bladder increased URS-EMG activity, bladder contraction frequency, and decreased contraction amplitude and capacity, compared to saline. Bladder activity and URS activity stabilized within 1 and 2 hours, respectively. Duloxetine administration significantly decreased bladder contraction frequency and increased URS-EMG activity to levels similar to previous reports. Cardiorespiratory parameters and blood gas levels remained consistent throughout the experiment. The epithelium of the bladder and urethra were greatly damaged and edema and infiltration of neutrophils in the lamina propria of urethra were observed. These data provide an ample evaluation of the health of the animals, stability of voiding function and appropriateness of the model for testing drugs designed to evaluate lower urinary tract as well as cardiovascular and respiratory systems function.
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Anestesia/métodos , Cloralosa/farmacología , Enfermedades de la Vejiga Urinaria/fisiopatología , Vejiga Urinaria/fisiopatología , Ácido Acético , Inhibidores de Captación Adrenérgica/administración & dosificación , Inhibidores de Captación Adrenérgica/farmacología , Animales , Presión Sanguínea/efectos de los fármacos , Gatos , Cloralosa/administración & dosificación , Modelos Animales de Enfermedad , Clorhidrato de Duloxetina , Electromiografía , Femenino , Frecuencia Cardíaca/efectos de los fármacos , Humanos , Hipnóticos y Sedantes/administración & dosificación , Hipnóticos y Sedantes/farmacología , Contracción Muscular/efectos de los fármacos , Respiración/efectos de los fármacos , Tiofenos/administración & dosificación , Tiofenos/farmacología , Factores de Tiempo , Uretra/efectos de los fármacos , Uretra/fisiopatología , Vejiga Urinaria/efectos de los fármacos , Enfermedades de la Vejiga Urinaria/inducido químicamente , Micción/efectos de los fármacosRESUMEN
This study evaluated the effects of a 5-HT4 agonist, cisapride, on neuronally evoked smooth muscle responses in bladder, urethra and ileum and compared these effects with those of an acetylcholinesterase inhibitor, distigmine. Electrical field stimulation (EFS) was applied to human bladder and ileum smooth muscle strips from human organ transplant donors and to urethral strips from prostatectomy patients, to evoke neuronally mediated smooth muscle responses. EFS induced contractions in bladder and mixed responses, consisting of contractions and relaxations, in urethra and ileum. Relaxations were mediated by nitric oxide while contractions were partially cholinergic (i.e. atropine sensitive). This atropine sensitive component amounted to~95% in bladder and ~75% in ileum, and it was enhanced by distigmine in a concentration dependent manner (0.1-3 µM; ~100-600% increase in bladder and ~50-250% increase in ileum). Cisapride (0.0003-1 µM) also enhanced bladder contractions (~75-100% increase) but had no effect on urethral contractions or relaxations, and modestly enhanced ileum contractions (~10-40% increase). Facilitatory effects of cisapride were reversed by the specific 5-HT4 receptor antagonist, SB-203186 (3 µM), but were resistant to repeated washing in the bladder. These data indicate that 5-HT4 receptor agonists enhanced EFS-induced contractions in bladder and ileum without an effect on urethra and suggest that it may be possible to enhance bladder activity with a dose of cisapride that is at, or below, those producing gastrointestinal (GI) effects. Although distigmine's maximal facilitation of bladder and GI tract function was greater than that of cisapride, at clinically relevant concentrations cisapride showed much greater efficacy.
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Cisaprida/farmacología , Potenciales Evocados/efectos de los fármacos , Íleon/fisiología , Contracción Muscular/fisiología , Neuronas/fisiología , Receptores de Serotonina 5-HT4/fisiología , Agonistas del Receptor de Serotonina 5-HT4/farmacología , Uretra/fisiología , Adulto , Anciano , Estimulación Eléctrica/métodos , Potenciales Evocados/fisiología , Femenino , Humanos , Íleon/efectos de los fármacos , Masculino , Persona de Mediana Edad , Contracción Muscular/efectos de los fármacos , Músculo Liso/efectos de los fármacos , Músculo Liso/fisiología , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Uretra/efectos de los fármacos , Vejiga UrinariaRESUMEN
Certain leukocytes release serine proteases that sustain inflammatory processes and cause disease conditions, such as asthma and chronic obstructive pulmonary disease. We identified beta-ketophosphonate 1 (JNJ-10311795; RWJ-355871) as a novel, potent dual inhibitor of neutrophil cathepsin G (K(i) = 38 nm) and mast cell chymase (K(i) = 2.3 nm). The x-ray crystal structures of 1 complexed with human cathepsin G (1.85 A) and human chymase (1.90 A) reveal the molecular basis of the dual inhibition. Ligand 1 occupies the S(1) and S(2) subsites of cathepsin G and chymase similarly, with the 2-naphthyl in S(1), the 1-naphthyl in S(2), and the phosphonate group in a complex network of hydrogen bonds. Surprisingly, however, the carboxamido-N-(naphthalene-2-carboxyl)piperidine group is found to bind in two distinct conformations. In cathepsin G, this group occupies the hydrophobic S(3)/S(4) subsites, whereas in chymase, it does not; rather, it folds onto the 1-naphthyl group of the inhibitor itself. Compound 1 exhibited noteworthy anti-inflammatory activity in rats for glycogen-induced peritonitis and lipopolysaccharide-induced airway inflammation. In addition to a marked reduction in neutrophil influx, 1 reversed increases in inflammatory mediators interleukin-1alpha, interleukin-1beta, tissue necrosis factor-alpha, and monocyte chemotactic protein-1 in the glycogen model and reversed increases in airway nitric oxide levels in the lipopolysaccharide model. These findings demonstrate that it is possible to inhibit both cathepsin G and chymase with a single molecule and suggest an exciting opportunity in the treatment of asthma and chronic obstructive pulmonary disease.