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1.
Ann Neurol ; 81(5): 677-689, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28380698

RESUMEN

OBJECTIVE: To comprehensively describe the new syndrome of myoclonus epilepsy and ataxia due to potassium channel mutation (MEAK), including cellular electrophysiological characterization of observed clinical improvement with fever. METHODS: We analyzed clinical, electroclinical, and neuroimaging data for 20 patients with MEAK due to recurrent KCNC1 p.R320H mutation. In vitro electrophysiological studies were conducted using whole cell patch-clamp to explore biophysical properties of wild-type and mutant KV 3.1 channels. RESULTS: Symptoms began at between 3 and 15 years of age (median = 9.5), with progressively severe myoclonus and rare tonic-clonic seizures. Ataxia was present early, but quickly became overshadowed by myoclonus; 10 patients were wheelchair-bound by their late teenage years. Mild cognitive decline occurred in half. Early death was not observed. Electroencephalogram (EEG) showed generalized spike and polyspike wave discharges, with documented photosensitivity in most. Polygraphic EEG-electromyographic studies demonstrated a cortical origin for myoclonus and striking coactivation of agonist and antagonist muscles. Magnetic resonance imaging revealed symmetrical cerebellar atrophy, which appeared progressive, and a prominent corpus callosum. Unexpectedly, transient clinical improvement with fever was noted in 6 patients. To explore this, we performed high-temperature in vitro recordings. At elevated temperatures, there was a robust leftward shift in activation of wild-type KV 3.1, increasing channel availability. INTERPRETATION: MEAK has a relatively homogeneous presentation, resembling Unverricht-Lundborg disease, despite the genetic and biological basis being quite different. A remarkable improvement with fever may be explained by the temperature-dependent leftward shift in activation of wild-type KV 3.1 subunit-containing channels, which would counter the loss of function observed for mutant channels, highlighting KCNC1 as a potential target for precision therapeutics. Ann Neurol 2017;81:677-689.


Asunto(s)
Ataxia , Disfunción Cognitiva/etiología , Epilepsias Mioclónicas , Calor , Canales de Potasio Shaw/metabolismo , Adolescente , Adulto , Edad de Inicio , Ataxia/complicaciones , Ataxia/diagnóstico por imagen , Ataxia/genética , Ataxia/fisiopatología , Electroencefalografía , Epilepsias Mioclónicas/complicaciones , Epilepsias Mioclónicas/diagnóstico por imagen , Epilepsias Mioclónicas/genética , Epilepsias Mioclónicas/fisiopatología , Femenino , Células HEK293 , Humanos , Imagen por Resonancia Magnética , Masculino , Persona de Mediana Edad , Mutación , Linaje , Canales de Potasio Shaw/genética , Síndrome , Adulto Joven
2.
Bioorg Med Chem ; 18(14): 5129-36, 2010 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-20579888

RESUMEN

We synthesized new tropolone derivatives substituted with cyclic amines: piperidine, piperazine or pyrrolidine. The most active anti-helicase compound (IC50=3.4 microM), 3,5,7-tri[(4'-methylpiperazin-1'-yl)methyl]tropolone (2), inhibited RNA replication by 50% at 46.9 microM (EC50) and exhibited the lowest cytotoxicity (CC50)>1 mM resulting in a selectivity index (SI=CC50/EC50)>21. The most efficient replication inhibitor, 3,5,7-tri[(4'-methylpiperidin-1'-yl)methyl]tropolone (6), inhibited RNA replication with an EC50 of 32.0 microM and a SI value of 17.4, whereas 3,5,7-tri[(3'-methylpiperidin-1'-yl)methyl]tropolone (7) exhibited a slightly lower activity with an EC50 of 35.6 microM and a SI of 9.8.


Asunto(s)
Antivirales/química , Antivirales/farmacología , ADN Helicasas/metabolismo , Hepacivirus/efectos de los fármacos , Tropolona/análogos & derivados , Tropolona/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/síntesis química , Línea Celular Tumoral , ADN Helicasas/antagonistas & inhibidores , ADN Helicasas/química , Sinergismo Farmacológico , Hepacivirus/enzimología , Hepatitis C/tratamiento farmacológico , Humanos , Interferón gamma/farmacología , Modelos Moleculares , Mutación , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/química , ARN Helicasas/metabolismo , ARN Viral/antagonistas & inhibidores , ARN Viral/metabolismo , Ribavirina/farmacología , Tropolona/síntesis química , Proteínas no Estructurales Virales/antagonistas & inhibidores , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo
3.
Acta Biochim Pol ; 55(1): 57-66, 2008.
Artículo en Inglés | MEDLINE | ID: mdl-18213401

RESUMEN

The non-structural protein 3 (NS3) of Hepatitis C virus (HCV) is a bifunctional enzyme with RNA-dependent NTPase/RNA helicase and serine protease activities, and thus represents a promising target for anti-HCV therapy. These functions are performed by two distinct moieties; the N-terminal protease domain and the C-terminal helicase domain that further folds into three structural subdomains. To obtain lower molecular mass proteins suitable for nuclear magnetic resonance studies of helicase-inhibitor complexes, helicase domains 1, 2, and 1+2 devoid of a hydrophobic beta-loop were overexpressed and purified. Circular dichroism studies were carried out to confirm the secondary structure content and to determine thermodynamic parameters describing the stability of the proteins. Both thermal and GuHCl-induced unfolding experiments confirmed the multidomain organization of the helicase. The unfolding transition observed for domain 1+2 was in agreement with the model of two well-resolved successive steps corresponding to the independent unfolding of domains 1 and 2, respectively. In the case of the full-length helicase, the presence of domain 3 remarkably changed the transition profile, leading to fast and irreversible transformation of partially unfolded protein.


Asunto(s)
Hepacivirus/genética , Proteínas no Estructurales Virales/química , Dicroismo Circular , Clonación Molecular , Calor , Espectroscopía de Resonancia Magnética , Conformación Molecular , Conformación Proteica , Desnaturalización Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , ARN Helicasas/química , Termodinámica
4.
Bioorg Med Chem ; 16(19): 8846-52, 2008 Oct 01.
Artículo en Inglés | MEDLINE | ID: mdl-18801660

RESUMEN

A new class of compounds--acridone derivatives--was tested using the direct fluorometric helicase activity assay to determine the inhibitory properties of the derivatives towards the NS3 helicase of Hepatitis C virus (HCV). The compounds were also tested as putative transcription inhibitors of in vitro transcription based on the DNA-dependent T7 RNA polymerase. Most of the acridone derivatives tested were transcription inhibitors; however, only four of them inhibited the NS3 helicase at low concentrations (IC(50) from 3 microM to 20 microM) and were therefore selected for further studies on the mechanism of inhibition. The acridone derivatives probably act via intercalation into double-stranded nucleic acids but they may also interact directly with viral enzymes. Selected carboxamides were tested in the subgenomic HCV replicon system. Two of the compounds: N-(pyridin-4-yl)-amide and N-(pyridin-2-yl)-amide of acridone-4-carboxylic acid are efficient RNA replication inhibitors with selectivity indexes of 19.4 and 40.5, respectively, proving that the acridone derivatives may be regarded as potential antiviral agents.


Asunto(s)
Acridonas/farmacología , Antivirales/farmacología , Ácidos Carboxílicos/farmacología , Inhibidores Enzimáticos/farmacología , Hepacivirus/efectos de los fármacos , Virus ARN/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Acridonas/síntesis química , Antivirales/síntesis química , Ácidos Carboxílicos/síntesis química , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Inhibidores Enzimáticos/síntesis química , Fluorometría , Hepacivirus/enzimología , Concentración 50 Inhibidora , ARN Helicasas/antagonistas & inhibidores , ARN Helicasas/metabolismo , Relación Estructura-Actividad
5.
FEBS Lett ; 567(2-3): 253-8, 2004 Jun 04.
Artículo en Inglés | MEDLINE | ID: mdl-15178332

RESUMEN

The non-structural protein 3 (NS3) of hepatitis C virus (HCV) is a highly promising target for anti-HCV therapy because of its multiple enzymatic activities, such as RNA-stimulated nucleoside triphosphatase, RNA helicase and serine protease. The helicase domain of NS3 as well as domain 2 of the helicase were expressed in a baculovirus system to obtain in high yield active proteins for prospective studies of complexes of the helicase with its inhibitors. A novel direct fluorometric test of helicase activity with a quenched DNA substrate, 3' labeled with a Cy3 dye and 5' labeled with a Black Hole Quencher, was developed and optimal reaction conditions established. This test based on fluorescence resonance energy transfer is simple and fast. It allows for direct measurements of enzyme activity, circumventing laborious and complicated radioactive techniques that are poorly reproducible. The results obtained encourage us to propose this new fluorescent assay as a method enabling high throughput screening of anti-helicase compounds.


Asunto(s)
Fluorometría/métodos , Hepacivirus/enzimología , Proteínas no Estructurales Virales/metabolismo , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Baculoviridae/genética , Dominio Catalítico , Línea Celular , Magnesio/química , Magnesio/metabolismo , Manganeso/química , Manganeso/metabolismo , Oligonucleótidos/genética , Oligonucleótidos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera/citología , Especificidad por Sustrato , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética
7.
J Med Chem ; 53(8): 3117-26, 2010 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-20337460

RESUMEN

A new goup of acridone derivatives, obtained by reaction of acridone-4-carboxylic acid derivatives with aromatic amines, was tested to determine the inhibitory properties toward the NS3 helicase of hepatitis C virus (HCV). Six compounds inhibited the NS3 helicase at low concentrations (IC(50) from 1.5 to 20 microM). The acridone derivatives probably act via intercalation into double-stranded nucleic acids with a strong specificity for double-stranded RNA, although an interaction with the enzyme cannot be excluded. Testing in the subgenomic HCV replicon system revealed that compounds 10 and 13 are efficient RNA replication inhibitors, with EC(50) of 3.5 and 1 microM and therapeutic indexes of >28 and 20, respectively. Compound 16, with EC(50) < 1 microM and TI > 1000, is extremely specific and practically noncytotoxic at the concentrations tested, proving that the acridone derivatives may be regarded as potential antiviral agents. Although the mechanism of action of 16 in the replicon system remains unclear, it is the key lead compound for further development of anti-HCV drugs.


Asunto(s)
Acridonas/síntesis química , Antivirales/síntesis química , Hepacivirus/efectos de los fármacos , ARN Helicasas/antagonistas & inhibidores , Proteínas no Estructurales Virales/antagonistas & inhibidores , Acridonas/farmacología , Acridonas/toxicidad , Antivirales/farmacología , Antivirales/toxicidad , Línea Celular Tumoral , Farmacorresistencia Viral , Hepacivirus/genética , Hepacivirus/fisiología , Humanos , Modelos Moleculares , Mutación , ARN Viral/genética , Replicón , Relación Estructura-Actividad , Replicación Viral/efectos de los fármacos
8.
Biol Chem ; 390(4): 351-60, 2009 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-19199832

RESUMEN

Hepatitis C virus (HCV) infections represent one of the major and still unresolved health problems because current therapy is effective in only 50-80% of cases, depending on viral genotype. A large group of amidinoanthracyclines, with decreased acute toxicity and cardiotoxicity compared to the parent antibiotics, was tested in a high-throughput fluorometric HCV helicase assay. Here, we report the selection of more than 50 potent inhibitors of helicase activity that inhibit the enzyme with IC(50) values in the range of 0.03- 10 mum; four of these compounds are the most potent inhibitors of helicase activity described in the literature. The activity of these inhibitors is highly dependent on their chemical structure, mainly on the substituent at the amidino carbon atom and on the orientation of the hydroxyl group at the 4 inch position of the daunosamine moiety. The most effective compounds act not solely via intercalation into the double-stranded DNA substrate, but also compete with the enzyme for access to the substrate, impeding formation of the active helicase complex. Selected amidinoanthracyclines were tested in the subgenomic HCV replicon system. These experiments confirmed the antiviral activity of two selected inhibitors (EC(50) values below 0.2 mum with selectivity indices of 19 and 33) and proved that they may be considered as potential anti-HCV drugs.


Asunto(s)
Amidas/química , Antraciclinas/química , Antraciclinas/farmacología , Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Amidas/metabolismo , Antraciclinas/metabolismo , Línea Celular Tumoral , Humanos , Concentración 50 Inhibidora
9.
Antimicrob Agents Chemother ; 52(2): 393-401, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18039921

RESUMEN

Hepatitis C virus (HCV) chronic infections represent one of the major and still unresolved health problems because of low efficiency and high cost of current therapy. Therefore, our studies centered on a viral protein, the NS3 helicase, whose activity is indispensable for replication of the viral RNA, and on its peptide inhibitor that corresponds to a highly conserved arginine-rich sequence of domain 2 of the helicase. The NS3 peptide (p14) was expressed in bacteria. Its 50% inhibitory activity in a fluorometric helicase assay corresponded to 725 nM, while the ATPase activity of NS3 was not affected. Nuclear magnetic resonance (NMR) studies of peptide-protein interactions using the relaxation filtering technique revealed that p14 binds directly to the full-length helicase and its separately expressed domain 1 but not to domain 2. Changes in the NMR chemical shift of backbone amide nuclei ((1)H and (15)N) of domain 1 or p14, measured during complex formation, were used to identify the principal amino acids of both domain 1 and the peptide engaged in their interaction. In the proposed interplay model, p14 contacts the clefts between domains 1 and 2, as well as between domains 1 and 3, preventing substrate binding. This interaction is strongly supported by cross-linking experiments, as well as by kinetic studies performed using a fluorometric assay. The antiviral activity of p14 was tested in a subgenomic HCV replicon assay that showed that the peptide at micromolar concentrations can reduce HCV RNA replication.


Asunto(s)
Antivirales/farmacología , Hepacivirus/efectos de los fármacos , Péptidos/farmacología , ARN Helicasas/antagonistas & inhibidores , Replicón/efectos de los fármacos , Proteínas no Estructurales Virales/química , Secuencia de Aminoácidos , Antivirales/química , Línea Celular Tumoral , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Hepacivirus/enzimología , Hepacivirus/genética , Humanos , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Péptidos/química , ARN Helicasas/química , ARN Viral/metabolismo , Replicación Viral/efectos de los fármacos
10.
Biochem Biophys Res Commun ; 341(2): 641-7, 2006 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-16438939

RESUMEN

Hepatitis C virus (HCV) is considered one of the most dangerous pathogens since about 3% of the world population is HCV-infected and the virus is a major cause of hepatitis, cirrhosis, and liver carcinoma. A need for a more efficient therapy prompted us to investigate new class of compounds, such as tropolone derivatives that possess antiviral, antibacterial, and antifungal activities. To synthesize bromo- and morpholinomethyl-analogues of tropolone, the previously reported methods were modified. The influence of new derivatives on the activity of the helicase and NTP-ase of HCV was investigated. The most potent inhibitory effect in the fluorometric helicase assay was exerted by 3,7-dibromo-5-morpholinomethyltropolone, for which the IC50 value was at low micromolar range. All the morpholino-derivatives had inhibitory activities higher than those of the non-modified analogues. Low toxicity in a yeast-based toxicity assay indicates that these compounds could be further modified to develop potent inhibitors of the HCV helicase and of viral replication.


Asunto(s)
Antivirales/farmacología , Hepacivirus/metabolismo , Hepatitis C/tratamiento farmacológico , Tropolona/análogos & derivados , Adenosina Trifosfatasas/química , Adenosina Trifosfato/metabolismo , ADN/química , Relación Dosis-Respuesta a Droga , Fluorometría/métodos , Humanos , Concentración 50 Inhibidora , Espectroscopía de Resonancia Magnética/métodos , Modelos Químicos , Espectrometría de Masa por Ionización de Electrospray/métodos , Temperatura , Tropolona/síntesis química , Tropolona/farmacología , Proteínas no Estructurales Virales/metabolismo , Replicación Viral/efectos de los fármacos
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