RESUMEN
Botulinum neurotoxin A (BoNT) is being shown to have anticancer action as a potential adjuvant treatment. The transient receptor potential (TRP) melastatin 2 (TRPM2) stimulator action of BoNT was reported in glioblastoma cells, but not in colorectal cancer (HT29) cells. By activating TRPM2, we evaluated the impacts of BoNT and oxaliplatin (OXA) incubations on oxidant and apoptotic values within the HT29 cells. Control, BoNT (5 IU for 24 h), OXA (50 µM for 24 h) and their combinations were induced. We found that TRPM2 protein is upregulated and mediates enhanced BoNT and OXA-induced Ca2+ entry in cells as compared to control cells. The increase of free reactive oxygen species (ROS), but the decrease of glutathione is the main ROS responsible for TRPM2 activation on H29 exposure to oxidative stress. BoNT and OXA-mediated Ca2+ entry through TRPM2 stimulation in response to H2 O2 results in mitochondrial Ca2+ overload, followed by mitochondrial membrane depolarization, apoptosis and caspase-3/-8/-9, although they were diminished in the TRPM2 antagonist groups (N-(p-amylcinnamoyl)anthranilic acid and carvacrol). In conclusion, by increasing the susceptibility of HT29 tumour cells to oxidative stress and apoptosis, the combined administration of BoNT and OXA via the targeting of TRPM2 may offer a different approach to kill the tumour cells.
Asunto(s)
Toxinas Botulínicas Tipo A , Neoplasias Colorrectales , Canales Catiónicos TRPM , Humanos , Oxaliplatino/farmacología , Especies Reactivas de Oxígeno/metabolismo , Toxinas Botulínicas Tipo A/metabolismo , Regulación hacia Arriba , Canales Catiónicos TRPM/genética , Canales Catiónicos TRPM/metabolismo , Muerte Celular , Estrés Oxidativo/fisiología , Apoptosis/fisiología , Neoplasias Colorrectales/tratamiento farmacológico , Calcio/metabolismoRESUMEN
CASO CLÍNICO: Mujer de 38 años con diplopía y endotropía. Limitación total de la abducción en AO al explorar las versiones, que se normalizan al explorar el reflejo de los ojos de muñeca. Es diagnosticada de espasmo del reflejo de cerca (ERC) y tratada con inyecciones repetidas de Botox en rectos medios, resolviéndose temporalmente el espasmo. DISCUSIÓN: El ERC se caracteriza por miosis, seudomiopía y convergencia que producen diplopía, visión borrosa, cefalea y endotropía variable, progresiva e intermitente. Se puede confundir con una paresia bilateral del vi nervio. El tratamiento con inyecciones repetidas de bótox puede ser efectivo en algunos casos
CLINICAL CASE: A 38-year old female with diplopia and esotropia, with limitation of ocular abduction in both eyes, with full abduction after doll's head rotation also being observed. She was diagnosed with spasm of the near reflex. Treatment with injections of botulinum toxin in both medial rectus has temporally resolved the convergence spasm. DISCUSSION: Near reflex spasm is characterized as miosis, pseudomyopia, and convergent strabismus that lead to diplopia, blurred vision, headache, and variable, progressive, and intermittent esotropia. As the spasm worsens there will be limited ocular versions and ductions simulating a sixth nerve palsy. Botulinum toxin may be effective in some cases
Asunto(s)
Femenino , Humanos , Espasmo/patología , Toxinas Botulínicas Tipo A/farmacología , Toxinas Botulínicas Tipo A/administración & dosificación , Esotropía/diagnóstico , Esotropía/patología , Esotropía/congénito , Esotropía/metabolismo , Enfermedad de Meniere/inducido químicamente , Enfermedad de Meniere/enfermería , Espasmo/genética , Toxinas Botulínicas Tipo A , Toxinas Botulínicas Tipo A/metabolismo , Esotropía/complicaciones , Esotropía/enfermería , Esotropía/cirugía , Enfermedad de Meniere/metabolismo , Enfermedad de Meniere/patologíaRESUMEN
The neuroactive peptide calcitonin-gene related peptide (CGRP) is known to act at efferent synapses and their targets in hair cell organs, including the cochlea and lateral line. CGRP is also expressed in vestibular efferent neurons as well as a number of central vestibular neurons. Although CGRP-null (-/-) mice demonstrate a significant reduction in cochlear nerve sound-evoked activity compared with wild-type mice, it is unknown whether and how the loss of CGRP influence vestibular system function. Vestibular function was assessed by quantifying the vestibulo-ocular reflex (VOR) in alert mice. The loss of CGRP in (-/-) mice was associated with a reduction of the VOR gain of ≈50% without a concomitant change in phase. Using immunohistochemistry, we confirmed that, although CGRP staining was absent in the vestibular end-organs of null (-/-) mice, cholinergic staining appeared normal, suggesting that the overall gross development of vestibular efferent innervation was unaltered. We further confirmed that the observed deficit in vestibular function of null (-/-) mice was not the result of nontargeted effects at the level of the extraocular motor neurons and/or their innervation of extraocular muscles. Analysis of the relationship between vestibular quick phase amplitude and peak velocity revealed that extraocular motor function was unchanged, and immunohistochemistry revealed no abnormalities in motor endplates. Together, our findings show that the neurotransmitter CGRP plays a key role in ensuring VOR efficacy.
Asunto(s)
Péptido Relacionado con Gen de Calcitonina/deficiencia , Reflejo Vestibuloocular/genética , Análisis de Varianza , Animales , Toxinas Botulínicas Tipo A/metabolismo , Calbindina 2/metabolismo , Péptido Relacionado con Gen de Calcitonina/genética , Colina O-Acetiltransferasa/metabolismo , Movimientos Oculares/genética , Femenino , Regulación de la Expresión Génica/genética , Masculino , Ratones , Ratones Noqueados , Miosina VIIa , Miosinas/metabolismo , Vestíbulo del Laberinto/metabolismoRESUMEN
A novel method for applying high-throughput docking to challenging metalloenzyme targets is described. The method utilizes information-based virtual transformation of library carboxylates to hydroxamic acids prior to docking, followed by compound acquisition, one-pot (two steps) chemical synthesis and in vitro screening. In two experiments targeting the botulinum neurotoxin serotype A metalloprotease light chain, hit rates of 32% and 18% were observed.
Asunto(s)
Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Inhibidores de Proteasas/química , Sitios de Unión , Toxinas Botulínicas Tipo A/metabolismo , Ácidos Carboxílicos/química , Dominio Catalítico , Química Farmacéutica , Diseño de Fármacos , Evaluación Preclínica de Medicamentos , Ácidos Hidroxámicos/química , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/síntesis química , Inhibidores de Proteasas/metabolismo , Unión Proteica , Serotipificación , Bibliotecas de Moléculas Pequeñas/síntesis química , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/metabolismo , Relación Estructura-ActividadRESUMEN
Botulinum neurotoxins (BoNTs) are the most lethal biotoxins known to mankind and are responsible for the neuroparalytic disease botulism. Current treatments for botulinum poisoning are all protein based and thus have a limited window of treatment opportunity. Inhibition of the BoNT light chain protease (LC) has emerged as a therapeutic strategy for the treatment of botulism as it may provide an effective post exposure remedy. Using a combination of crystallographic and modeling studies a series of hydroxamates derived from 1-adamantylacetohydroxamic acid (3a) were prepared. From this group of compounds, an improved potency of about 17-fold was observed for two derivatives. Detailed mechanistic studies on these structures revealed a competitive inhibition model, with a K(i)=27 nM, which makes these compounds some of the most potent small molecule, non-peptidic BoNT/A LC inhibitors reported to date.
Asunto(s)
Adamantano/química , Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Ácidos Hidroxámicos/química , Inhibidores de Proteasas/síntesis química , Animales , Sitios de Unión , Toxinas Botulínicas Tipo A/metabolismo , Dominio Catalítico , Células Cultivadas , Cristalografía por Rayos X , Evaluación Preclínica de Medicamentos , Ácidos Hidroxámicos/síntesis química , Ácidos Hidroxámicos/toxicidad , Cinética , Simulación del Acoplamiento Molecular , Inhibidores de Proteasas/química , Inhibidores de Proteasas/toxicidad , Ratas , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/metabolismo , Relación Estructura-ActividadRESUMEN
Objetivo. Determinar la eficacia de la estimulación eléctrica neuromuscular (EENM) asociada a la toxina botulínica (TB) tipo A en la función motora, capacidad funcional y espasticidad de la mano y la muñeca en pacientes con ictus crónico. Pacientes y métodos. Ensayo clinico randomizado controlado en 25 pacientes con ictus de más de 6 meses de evolución. Tras infiltración de 200 unidades de TB en la musculatura flexora de muñeca y dedos, los participantes se asignaron en dos grupos: EENM en músculos extensores (grupo 1), electroestimulación placebo (grupo 2). Evaluación a las 4 y 16 semanas de la infiltración mediante diferentes escalas de función motora (Fugl-Meyer Motor Assessment [FMA], Medical Research Council Scale [MRC], Motricity Index for Motor Impairment after stroke and dynamometry), capacidad funcional de miembro superior (Chedoke Arm and Hand Activity Inventory [CAHAI], Box and Block Test [BBT]) y tono muscular (escala modificada de Ashworth [MAS]). Resultados. A las 4 semanas, se observa mejoría significativa en: FMA 2,43 (DE 4,08) en el componente para el MS, MRC 0,22 (DE 0,42), CAHAI 4,21 y BBT 1,47 (DE 3,3), así como una reducción de la MAS 0,96 (DE 0,88) en muñeca y 1,17 (DE 1,37) en dedos. A los 4 meses se mantuvo la mejoría en la función motora respecto a los valores basales, pero no de la espasticidad. No se observaron diferencias en ninguna de las variables de función motora y capacidad funcional del MS entre los dos grupos. Conclusiones. La infiltración de TB tipo A en la espasticidad flexora distal del MS mejora la función motora, capacidad funcional y espasticidad en el ictus crónico, pero añadir EENM de la musculatura extensora de la muñeca y dedos no muestra beneficios adicionales (AU)
Objective. To evaluate the efficacy of the Neuromuscular Electrical Stimulation (NMES) associated to Botulinum Toxin (TB) type A in motor function, functional capacity and spasticity in chronic post-stroke upper limb (UL) spasticity. Patients and methods. A randomized controlled clinical trial was performed in 25 ictus patients with more than 6 months evolution. After infiltration of 200 units of TB in the flexor muscle of the finger and wrist muscles, the patients were assigned to two groups: NMES in extensor muscles (group 1) and sham-stimulation (group 2). Patients were assessed at weeks 4/16 post-injection by standard motor function scales (Fugl-Meyer Motor Assessment (FMA), Medical Research Council Scale (MRC), Motricity Index for Motor Impairment after stroke and dynamometry), UL functional capacity (Chedoke Arm and Hand Activity Inventory (CAHAI), Box and Block Test (BBT) and muscular tone (Modified Ashworth Scale (MAS)). Results. At week 4, significant improvement was observed in FMA 2.4 (SD 4.1) in UL component, MRC 0.2 (SD 0.4), CAHAI 4.2 and BBT 1.5 (SD 3.3), as well as a reduction in MAS 1.0 (SD 0.9) in the wrist and 1.2 (SD 1.4) in the fingers. At month 4, this effect was maintained in motor function but not in spasticity. No significant differences were observed in UL motor function, functional capacity between the groups. Conclusions. TB type A injection in UL distal flexor spasticity enhances motor function, functional capacity and spasticity in chronic stroke patients. Adding a program of NMES for the extensor wrist/fingers muscles does not provide any additional benefits (AU)
Asunto(s)
Humanos , Masculino , Femenino , Toxinas Botulínicas Tipo A/administración & dosificación , Toxinas Botulínicas Tipo A/uso terapéutico , Estimulación Eléctrica Transcutánea del Nervio/métodos , Accidente Cerebrovascular/rehabilitación , Espasticidad Muscular/rehabilitación , Toxinas Botulínicas Tipo A/metabolismo , Estimulación Eléctrica Transcutánea del Nervio/tendencias , Estimulación Eléctrica Transcutánea del NervioRESUMEN
The current treatment of botulism is to administer animal-derived antitoxin, which frequently causes severe adverse reactions in the recipients. In this study, a heavy chain antibody fragment (VH/V(H)H) phage display library was constructed by amplification of the immunoglobulin genes of a nonimmune camel, Camelus dromedarius, using primers specific to human VH gene segments. A recombinant light chain of type A botulinum toxin, BoTxA/LC, with zinc endoprotease activity was used in phage bio-panning to select phage clones displaying BoTxA/LC-bound VH/V(H)H. Soluble VH/V(H)H were produced and purified from 10 VH/V(H)H phagemid-transformed E. coli clones. Complementary determining regions (CDRs) and immunoglobulin frameworks (FRs) of the 10 camel VH/V(H)H-deduced amino acid sequences were determined. FR2 sequences of two clones showed a hallmark of camel V(H)H, i.e. (F/Y)(42)E(49)R(50)(G/F)(52). The remaining eight clones had an FR2 amino acid tetrad of conventional VH, i.e. V(42)G(49)L(50)W(52). V(H)H of one clone (V(H)H17) neutralized the SNAP25 hydrolytic activity of BoTxA/LC, whereas mouse polyclonal anti-BoTxA/LC did not have such activity. Mimotope sequences of V(H)H17 matched with the 194-206 amino acid residues of BoTxA/LC, which are located near the S'1 subsite of the catalytic cleft of the enzyme. Molecular docking revealed that CDR3 of the V(H)H17 bound to epitope in the toxin enzymatic cleft. Therefore, the BoTxA/LC neutralization by the V(H)H17 should be due to the V(H)H insertion into the enzymatic cleft of the toxin, which is usually inaccessible to a conventional antibody molecule. This antibody fragment warrants further development as a therapeutic agent for botulism.
Asunto(s)
Anticuerpos/química , Toxinas Botulínicas Tipo A/metabolismo , Metaloproteasas/química , Zinc/química , Secuencia de Aminoácidos , Animales , Camelus , Humanos , Región Variable de Inmunoglobulina/química , Masculino , Ratones , Ratones Endogámicos ICR , Datos de Secuencia Molecular , Biblioteca de Péptidos , Conformación Proteica , Proteínas Recombinantes/química , Homología de Secuencia de AminoácidoRESUMEN
Botulinum neurotoxin serotype A is the most lethal of all known toxins. Here, we report the crystal structure, along with SAR data, of the zinc metalloprotease domain of BoNT/A bound to a potent peptidomimetic inhibitor (K(i)=41 nM) that resembles the local sequence of the SNAP-25 substrate. Surprisingly, the inhibitor adopts a helical conformation around the cleavage site, in contrast to the extended conformation of the native substrate. The backbone of the inhibitor's P1 residue displaces the putative catalytic water molecule and concomitantly interacts with the "proton shuttle" E224. This mechanism of inhibition is aided by residue contacts in the conserved S1' pocket of the substrate binding cleft and by the induction of new hydrophobic pockets, which are not present in the apo form, especially for the P2' residue of the inhibitor. Our inhibitor is specific for BoNT/A as it does not inhibit other BoNT serotypes or thermolysin.
Asunto(s)
Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Fragmentos de Péptidos/química , Fragmentos de Péptidos/farmacología , Proteína 25 Asociada a Sinaptosomas/química , Secuencia de Aminoácidos , Sitios de Unión , Biomimética , Toxinas Botulínicas Tipo A/química , Toxinas Botulínicas Tipo A/metabolismo , Evaluación Preclínica de Medicamentos , Modelos Biológicos , Modelos Moleculares , Datos de Secuencia Molecular , Fragmentos de Péptidos/metabolismo , Unión Proteica , Conformación Proteica , Especificidad por Sustrato , Proteína 25 Asociada a Sinaptosomas/metabolismoRESUMEN
AIM: Botulinum neurotoxins (BoNT) abort the process of neurotransmitter release at presynaptic motor nerve terminals, causing muscle paralysis. The ability of botulinum toxin to produce its effect is dependent on the ability of the light chain to cleave the SNARE proteins associated with transmitter release. Translocation of the light chain protease through the heavy chain-formed channel is a pivotal step in the intoxication process. Toosendanin (TSN), a triterpenoid derivative extracted from a Chinese traditional medicine, has been demonstrated to be an effective cure for experimental botulism. This study was designed to explore the antibotulismic mechanisms of toosendanin. METHODS: The inside-out single-channel recording patch-clamp technique was used to record the BoNT/A-induced currents in the presence and absence of TSN. RESULTS: Channel formation was delayed and the sizes of the channels were reduced in the TSN-treated PC12 cell membrane. CONCLUSION: The antibotulismic effect of TSN might occur via interference with toxin translocation.
Asunto(s)
Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Membrana Celular/fisiología , Medicamentos Herbarios Chinos/farmacología , Canales Iónicos/efectos de los fármacos , Animales , Toxinas Botulínicas Tipo A/metabolismo , Membrana Celular/efectos de los fármacos , Medicamentos Herbarios Chinos/aislamiento & purificación , Melia/química , Células PC12 , Plantas Medicinales/química , Transporte de Proteínas , RatasRESUMEN
We investigated the inhibitory effect of stinging nettle leaf extract on the protease activity of botulinum neurotoxin type A and B light chains. The nettle leaf infusion was fractionated and HPLC-based enzymatic assays were performed to determine the capacity of each fraction to inhibit the protease activity of botulinum neurotoxin type A and B light chains. Assay results demonstrated that a water-soluble fraction obtained from the nettle leaf infusion inhibited type A, but did not inhibit type B light chain protease activity. The inhibition mode of water soluble fraction against protease activity of type A light chain was analyzed and found to be a non-competitive.
Asunto(s)
Toxinas Botulínicas Tipo A/metabolismo , Péptido Hidrolasas/metabolismo , Inhibidores de Proteasas/farmacología , Urtica dioica/química , Extractos Vegetales/farmacología , Hojas de la Planta/químicaRESUMEN
Toosendanin (TSN), a triterpenoid derivative extracted from Chinese traditional medicine, has been demonstrated to be an effective cure for experimental botulism. This study is designed to explore its antibotulismic mechanism by Western blotting. The results showed that TSN incubation did not change the electrophoresis pattern and the amounts of synaptosomal-associated protein of 25 kDa (SNAP-25), syntaxin and synaptobrevin/vesicle-associated membrane protein in rat cerebral synaptosomes, but made the synaptosomes completely resistant to botulinum neurotoxin A (BoNT/A)-mediated cleavage of SNAP-25. After binding of BoNT/A to synaptosomes, TSN still partially antagonized the toxin-mediated cleavage of SNAP-25. However, TSN-incubated synaptosomal membrane fraction did not resist the cleavage of SNAP-25 by the light chain of BoNT/A. It is suggested that the antibotulismic effect of TSN results from blocking the toxin's approach to its enzymatic substrate.
Asunto(s)
Toxinas Botulínicas Tipo A/antagonistas & inhibidores , Toxinas Botulínicas Tipo A/metabolismo , Corteza Cerebral/metabolismo , Medicamentos Herbarios Chinos/farmacología , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sinaptosomas/metabolismo , Proteínas de Transporte Vesicular , Animales , Western Blotting , Toxinas Botulínicas Tipo A/química , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Corteza Cerebral/efectos de los fármacos , Ditiotreitol/farmacología , Electroforesis en Gel de Poliacrilamida , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Endogámicos , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Oxidación-Reducción , Proteínas Qa-SNARE , Proteínas R-SNARE , Ratas , Ratas Sprague-Dawley , Proteínas SNARE , Proteína 25 Asociada a Sinaptosomas , Sinaptosomas/efectos de los fármacosRESUMEN
The seven botulinum neurotoxins (BoNTs) are zinc metalloproteases that cleave neuronal proteins involved in neurotransmitter release and are among the most toxic natural products known. High-throughput BoNT assays are needed for use in antibotulinum drug discovery and to characterize BoNT protease activities. Compared to other proteases, BoNTs exhibit unusually stringent substrate requirements with respect to amino acid sequences and polypeptide lengths. Nonetheless, we have devised a strategy for development of fluorigenic BoNT protease assays, based on earlier structure-function studies, that has proven successful for three of the seven serotypes: A, B, and F. In synthetic peptide substrates, the P(1) and P(3)' residues were substituted with 2,4-dinitrophenyl-lysine and S-(N-[4-methyl-7-dimethylamino-coumarin-3-yl]-carboxamidomethyl)-cysteine, respectively. By monitoring the BoNT-catalyzed increase in fluorescence over time, initial hydrolysis rates could be obtained in 1 to 2 min when BoNT concentrations were 60 ng/ml (about 1 nM) or higher. Each BoNT cleaved its fluorigenic substrate at the same location as in the neuronal target protein, and kinetic constants indicated that the substrates were selective and efficient. The fluorigenic assay for BoNT B was used to characterize a new competitive inhibitor of BoNT B protease activity with a K(i) value of 4 micro M. In addition to real-time activity measurements, toxin concentration determinations, and kinetic studies, the BoNT substrates described herein may be directly incorporated into automated high-throughput assay systems to screen large numbers of compounds for potential antibotulinum drugs.