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1.
J Org Chem ; 85(19): 12067-12079, 2020 10 02.
Artículo en Inglés | MEDLINE | ID: mdl-32809816

RESUMEN

The synthesis of a series of 2-amidomethylated pyridines (3-8) was investigated, starting from 4-chloro-3-fluoropyridine. Kinetic deprotonation at -75 °C followed by reaction with DMF gave 2-formyl-4-chloro-3-fluoropyridine 10 regioselectively, which was converted to 2-aminomethyl analogue 1 via sulfinamide 2. Alternatively, Minisci-type amidomethylation under Ag+/persulfate or photoredox-mediated conditions using a series of amino acid derivatives gave (3-8, 19, and 34) in 30-74% yield and isomer ratios in the range 6.7:1 to >50:1. The latter methods gave overall yields similar to that of the deprotonation approach, but were shorter and more amenable to scale-up. In particular, N-Boc analogue 8 was obtained in a single step. The amidomethylations of another six 3-fluoropyridines under the photoredox conditions were briefly examined.

2.
EMBO J ; 39(4): e102723, 2020 02 17.
Artículo en Inglés | MEDLINE | ID: mdl-31880004

RESUMEN

Cilia serve as cellular antennae that translate sensory information into physiological responses. In the sperm flagellum, a single chemoattractant molecule can trigger a Ca2+ rise that controls motility. The mechanisms underlying such ultra-sensitivity are ill-defined. Here, we determine by mass spectrometry the copy number of nineteen chemosensory signaling proteins in sperm flagella from the sea urchin Arbacia punctulata. Proteins are up to 1,000-fold more abundant than the free cellular messengers cAMP, cGMP, H+ , and Ca2+ . Opto-chemical techniques show that high protein concentrations kinetically compartmentalize the flagellum: Within milliseconds, cGMP is relayed from the receptor guanylate cyclase to a cGMP-gated channel that serves as a perfect chemo-electrical transducer. cGMP is rapidly hydrolyzed, possibly via "substrate channeling" from the channel to the phosphodiesterase PDE5. The channel/PDE5 tandem encodes cGMP turnover rates rather than concentrations. The rate-detection mechanism allows continuous stimulus sampling over a wide dynamic range. The textbook notion of signal amplification-few enzyme molecules process many messenger molecules-does not hold for sperm flagella. Instead, high protein concentrations ascertain messenger detection. Similar mechanisms may occur in other small compartments like primary cilia or dendritic spines.


Asunto(s)
Arbacia/fisiología , Quimiotaxis , Proteómica , Transducción de Señal , Animales , Arbacia/ultraestructura , Calcio/metabolismo , Cilios/fisiología , Cilios/ultraestructura , GMP Cíclico/metabolismo , Tomografía con Microscopio Electrónico , Flagelos/fisiología , Flagelos/ultraestructura , Guanilato Ciclasa/metabolismo , Masculino , Espectrometría de Masas , Espermatozoides/fisiología , Espermatozoides/ultraestructura
3.
Br J Pharmacol ; 175(15): 3144-3161, 2018 08.
Artículo en Inglés | MEDLINE | ID: mdl-29723408

RESUMEN

BACKGROUND AND PURPOSE: Sperm from many species share the sperm-specific Ca2+ channel CatSper that controls the intracellular Ca2+ concentration and, thereby, the swimming behaviour. A growing body of evidence suggests that the mechanisms controlling the activity of CatSper and its role during fertilization differ among species. A lack of suitable pharmacological tools has hampered the elucidation of the function of CatSper. Known inhibitors of CatSper exhibit considerable side effects and also inhibit Slo3, the principal K+ channel of mammalian sperm. The compound RU1968 was reported to suppress Ca2+ signaling in human sperm by an unknown mechanism. Here, we examined the action of RU1968 on CatSper in sperm from humans, mice, and sea urchins. EXPERIMENTAL APPROACH: We resynthesized RU1968 and studied its action on sperm from humans, mice, and the sea urchin Arbacia punctulata by Ca2+ fluorimetry, single-cell Ca2+ imaging, electrophysiology, opto-chemistry, and motility analysis. KEY RESULTS: RU1968 inhibited CatSper in sperm from invertebrates and mammals. The compound lacked toxic side effects in human sperm, did not affect mouse Slo3, and inhibited human Slo3 with about 15-fold lower potency than CatSper. Moreover, in human sperm, RU1968 mimicked CatSper dysfunction and suppressed motility responses evoked by progesterone, an oviductal steroid known to activate CatSper. Finally, RU1968 abolished CatSper-mediated chemotactic navigation in sea urchin sperm. CONCLUSION AND IMPLICATIONS: We propose RU1968 as a novel tool to elucidate the function of CatSper channels in sperm across species.


Asunto(s)
Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio/fisiología , Pregnatrienos/farmacología , Espermatozoides/efectos de los fármacos , Animales , Calcio/metabolismo , Células HEK293 , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Erizos de Mar , Espermatozoides/fisiología
5.
ACS Chem Biol ; 12(12): 2952-2957, 2017 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-29024582

RESUMEN

The voltage-gated proton channel Hv1 is expressed in various human cell types, including macrophages, epithelial cells, and sperm. Hv1 opening leads to proton efflux that alkalizes the cytosol. Here, we describe light-activated Hv1 inhibitors (photoswitches) that allow controlling its activity with high spatiotemporal precision. The photoswitches comprise a light-sensitive azobenzene moiety and 2-guanidinobenzimidazole (2GBI), a known Hv1 inhibitor. In the dark, photoGBI inhibits heterologously expressed Hv1 channels. Blue light, which isomerizes the azobenzene group from trans to cis conformation, releases inhibition. We demonstrate photocontrol of native proton currents in human macrophages and sperm using photoGBI, underlining their use as valuable optochemical tools to study the function of Hv1 channels.


Asunto(s)
Regulación de la Expresión Génica/efectos de la radiación , Canales Iónicos/metabolismo , Animales , Humanos , Canales Iónicos/química , Luz , Oocitos/metabolismo , Análisis Espectral , Xenopus/metabolismo
6.
J Biol Chem ; 292(15): 6177-6189, 2017 04 14.
Artículo en Inglés | MEDLINE | ID: mdl-28258214

RESUMEN

The lysosomal acid ß-glucosidase GBA1 and the non-lysosomal ß-glucosidase GBA2 degrade glucosylceramide (GlcCer) to glucose and ceramide in different cellular compartments. Loss of GBA2 activity and the resulting accumulation of GlcCer results in male infertility, whereas mutations in the GBA1 gene and loss of GBA1 activity cause the lipid-storage disorder Gaucher disease. However, the role of GBA2 in Gaucher disease pathology and its relationship to GBA1 is not well understood. Here, we report a GBA1-dependent down-regulation of GBA2 activity in patients with Gaucher disease. Using an experimental approach combining cell biology, biochemistry, and mass spectrometry, we show that sphingosine, the cytotoxic metabolite accumulating in Gaucher cells through the action of GBA2, directly binds to GBA2 and inhibits its activity. We propose a negative feedback loop, in which sphingosine inhibits GBA2 activity in Gaucher cells, preventing further sphingosine accumulation and, thereby, cytotoxicity. Our findings add a new chapter to the understanding of the complex molecular mechanism underlying Gaucher disease and the regulation of ß-glucosidase activity in general.


Asunto(s)
Regulación hacia Abajo , Enfermedad de Gaucher/enzimología , Regulación Enzimológica de la Expresión Génica , Modelos Biológicos , Esfingosina/metabolismo , beta-Glucosidasa/biosíntesis , Animales , Línea Celular , Enfermedad de Gaucher/genética , Glucosilceramidasa , Glucosilceramidas/genética , Glucosilceramidas/metabolismo , Humanos , Masculino , Ratones , Esfingosina/genética , beta-Glucosidasa/genética
7.
J Neurosci ; 35(46): 15240-53, 2015 Nov 18.
Artículo en Inglés | MEDLINE | ID: mdl-26586813

RESUMEN

Dendritic voltage-gated ion channels profoundly shape the integrative properties of neuronal dendrites. In epilepsy, numerous changes in dendritic ion channels have been described, all of them due to either their altered transcription or phosphorylation. In pilocarpine-treated chronically epileptic rats, we describe a novel mechanism that causes an increased proximal dendritic persistent Na(+) current (INaP). We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of dendritic INaP is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic INaP causes augmented dendritic summation of excitatory inputs. These results establish a novel post-transcriptional modification of ion channels in chronic epilepsy and may provide a novel avenue for treatment of temporal lobe epilepsy. SIGNIFICANCE STATEMENT: In this paper, we describe a novel mechanism that causes increased dendritic persistent Na(+) current. We demonstrate using a combination of electrophysiology and molecular approaches that the upregulation of persistent Na(+) currents is due to a relief from polyamine-dependent inhibition. The polyamine deficit in hippocampal neurons is likely caused by an upregulation of the degrading enzyme spermidine/spermine acetyltransferase. Multiphoton glutamate uncaging experiments revealed that the increase in dendritic persistent Na current causes augmented dendritic summation of excitatory inputs. We believe that these results establish a novel post-transcriptional modification of ion channels in chronic epilepsy.


Asunto(s)
Región CA1 Hipocampal/patología , Dendritas/fisiología , Regulación hacia Abajo/fisiología , Canales de Sodio/fisiología , Espermina/metabolismo , Estado Epiléptico/patología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/genética , Análisis de Varianza , Animales , Dendritas/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación hacia Abajo/efectos de los fármacos , Humanos , Técnicas In Vitro , Masculino , Agonistas Muscarínicos/toxicidad , Pilocarpina/toxicidad , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/efectos de los fármacos , Estadísticas no Paramétricas , Estado Epiléptico/inducido químicamente , Sinaptofisina/metabolismo , Tetrodotoxina/farmacología , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiología
8.
Nat Commun ; 6: 7985, 2015 Aug 17.
Artículo en Inglés | MEDLINE | ID: mdl-26278469

RESUMEN

Sperm require a sense of direction to locate the egg for fertilization. They follow gradients of chemical and physical cues provided by the egg or the oviduct. However, the principles underlying three-dimensional (3D) navigation in chemical landscapes are unknown. Here using holographic microscopy and optochemical techniques, we track sea urchin sperm navigating in 3D chemoattractant gradients. Sperm sense gradients on two timescales, which produces two different steering responses. A periodic component, resulting from the helical swimming, gradually aligns the helix towards the gradient. When incremental path corrections fail and sperm get off course, a sharp turning manoeuvre puts sperm back on track. Turning results from an 'off' Ca(2+) response signifying a chemoattractant stimulation decrease and, thereby, a drop in cyclic GMP concentration and membrane voltage. These findings highlight the computational sophistication by which sperm sample gradients for deterministic klinotaxis. We provide a conceptual and technical framework for studying microswimmers in 3D chemical landscapes.


Asunto(s)
Arbacia/fisiología , Factores Quimiotácticos/farmacología , Quimiotaxis/fisiología , Espermatozoides/efectos de los fármacos , Espermatozoides/fisiología , Animales , Masculino
9.
Bioorg Med Chem ; 20(21): 6523-32, 2012 Nov 01.
Artículo en Inglés | MEDLINE | ID: mdl-23000293

RESUMEN

Supramolecular self-assembly of amyloidogenic peptides is closely associated with numerous pathological conditions. For instance, Alzheimer´s disease (AD) is characterized by abundant amyloid plaques originating from the proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. Compounds named γ-secretase modulators (GSMs) can shift the substrate cleavage specificity of γ-secretase toward the production of non-amyloidogenic, shorter Aß fragments. Herein, we describe the synthesis of highly potent acidic GSMs, equipped with a photoreactive diazirine moiety for photoaffinity labeling. The probes labeled the N-terminal fragment of presenilin (the catalytic subunit of γ-secretase), supporting a mode of action involving binding to γ-secretase. This fundamental step toward the elucidation of the molecular mechanism governing the GSM-induced shift in γ-secretase proteolytic specificity should pave the way for the development of improved drugs against AD.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/metabolismo , Azirinas/química , Azirinas/farmacología , Animales , Azirinas/síntesis química , Azirinas/efectos de la radiación , Células CHO , Cricetinae , Relación Dosis-Respuesta a Droga , Modelos Moleculares , Estructura Molecular , Procesos Fotoquímicos/efectos de la radiación , Relación Estructura-Actividad
10.
PLoS One ; 7(1): e30484, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22238696

RESUMEN

The intramembrane-cleaving protease γ-secretase catalyzes the last step in the generation of toxic amyloid-ß (Aß) peptides and is a principal therapeutic target in Alzheimer's disease. Both preclinical and clinical studies have demonstrated that inhibition of γ-secretase is associated with prohibitive side effects due to suppression of Notch processing and signaling. Potentially safer are γ-secretase modulators (GSMs), which are small molecules that selectively lower generation of the highly amyloidogenic Aß42 peptides but spare Notch processing. GSMs with nanomolar potency and favorable pharmacological properties have been described, but the molecular mechanism of GSMs remains uncertain and both the substrate amyloid precursor protein (APP) and subunits of the γ-secretase complex have been proposed as the molecular target of GSMs. We have generated a potent photo-probe based on an acidic GSM that lowers Aß42 generation with an IC(50) of 290 nM in cellular assays. By combining in vivo photo-crosslinking with affinity purification, we demonstrated that this probe binds the N-terminal fragment of presenilin (PSEN), the catalytic subunit of the γ-secretase complex, in living cells. Labeling was not observed for APP or any of the other γ-secretase subunits. Binding was readily competed by structurally divergent acidic and non-acidic GSMs suggesting a shared mode of action. These findings indicate that potent acidic GSMs target presenilin to modulate the enzymatic activity of the γ-secretase complex.


Asunto(s)
Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Células/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Presenilinas/antagonistas & inhibidores , Presenilinas/fisiología , Animales , Antiinflamatorios no Esteroideos/farmacología , Células CHO , Células/metabolismo , Células Cultivadas , Cricetinae , Cricetulus , Evaluación Preclínica de Medicamentos , Células HEK293 , Humanos , Concentración 50 Inhibidora , Modelos Biológicos , Terapia Molecular Dirigida
11.
Curr Neuropharmacol ; 9(4): 598-622, 2011 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-22798753

RESUMEN

Comprehensive evidence supports that oligomerization and accumulation of amyloidogenic Aß42 peptides in brain is crucial in the pathogenesis of both familial and sporadic forms of Alzheimer's disease. Imaging studies indicate that the buildup of Aß begins many years before the onset of clinical symptoms, and that subsequent neurodegeneration and cognitive decline may proceed independently of Aß. This implies the necessity for early intervention in cognitively normal individuals with therapeutic strategies that prioritize safety. The aspartyl protease γ-secretase catalyses the last step in the cellular generation of Aß42 peptides, and is a principal target for anti-amyloidogenic intervention strategies. Due to the essential role of γ-secretase in the NOTCH signaling pathway, overt mechanism-based toxicity has been observed with the first generation of γ-secretase inhibitors, and safety of this approach has been questioned. However, two new classes of small molecules, γ-secretase modulators (GSMs) and NOTCH-sparing γ-secretase inhibitors, have revitalized γ-secretase as a drug target in AD. GSMs are small molecules that cause a product shift from Aß42 towards shorter and less toxic Ab peptides. Importantly, GSMs spare other physiologically important substrates of the γ-secretase complex like NOTCH. Recently, GSMs with nanomolar potency and favorable in vivo properties have been described. In this review, we summarize the knowledge about the unusual proteolytic activity of γ-secretase, and the chemical biology, molecular mechanisms and clinical perspective of compounds that target the γ-secretase complex, with a particular focus on GSMs.

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