RESUMEN
Small molecules directly targeting the voltage-gated sodium channel (VGSC) NaV1.7 have not been clinically successful. We reported that preventing the addition of a small ubiquitin-like modifier onto the NaV1.7-interacting cytosolic collapsin response mediator protein 2 (CRMP2) blocked NaV1.7 function and was antinociceptive in rodent models of neuropathic pain. Here, we discovered a CRMP2 regulatory sequence (CRS) unique to NaV1.7 that is essential for this regulatory coupling. CRMP2 preferentially bound to the NaV1.7 CRS over other NaV isoforms. Substitution of the NaV1.7 CRS with the homologous domains from the other eight VGSC isoforms decreased NaV1.7 currents. A cell-penetrant decoy peptide corresponding to the NaV1.7-CRS reduced NaV1.7 currents and trafficking, decreased presynaptic NaV1.7 expression, reduced spinal CGRP release, and reversed nerve injury-induced mechanical allodynia. Importantly, the NaV1.7-CRS peptide did not produce motor impairment, nor did it alter physiological pain sensation, which is essential for survival. As a proof-of-concept for a NaV1.7 -targeted gene therapy, we packaged a plasmid encoding the NaV1.7-CRS in an AAV virus. Treatment with this virus reduced NaV1.7 function in both rodent and rhesus macaque sensory neurons. This gene therapy reversed and prevented mechanical allodynia in a model of nerve injury and reversed mechanical and cold allodynia in a model of chemotherapy-induced peripheral neuropathy. These findings support the conclusion that the CRS domain is a targetable region for the treatment of chronic neuropathic pain.
Asunto(s)
Dolor Crónico , Neuralgia , Animales , Hiperalgesia/inducido químicamente , Dolor Crónico/genética , Dolor Crónico/terapia , Macaca mulatta/metabolismo , Neuralgia/genética , Neuralgia/terapia , Canal de Sodio Activado por Voltaje NAV1.7/genética , Canal de Sodio Activado por Voltaje NAV1.7/metabolismo , Ganglios Espinales/metabolismo , Canal de Sodio Activado por Voltaje NAV1.8RESUMEN
Transmembrane Cav2.2 (N-type) voltage-gated calcium channels are genetically and pharmacologically validated, clinically relevant pain targets. Clinical block of Cav2.2 (e.g., with Prialt/Ziconotide) or indirect modulation [e.g., with gabapentinoids such as Gabapentin (GBP)] mitigates chronic pain but is encumbered by side effects and abuse liability. The cytosolic auxiliary subunit collapsin response mediator protein 2 (CRMP2) targets Cav2.2 to the sensory neuron membrane and regulates their function via an intrinsically disordered motif. A CRMP2-derived peptide (CBD3) uncouples the Cav2.2-CRMP2 interaction to inhibit calcium influx, transmitter release, and pain. We developed and applied a molecular dynamics approach to identify the A1R2 dipeptide in CBD3 as the anchoring Cav2.2 motif and designed pharmacophore models to screen 27 million compounds on the open-access server ZincPharmer. Of 200 curated hits, 77 compounds were assessed using depolarization-evoked calcium influx in rat dorsal root ganglion neurons. Nine small molecules were tested electrophysiologically, while one (CBD3063) was also evaluated biochemically and behaviorally. CBD3063 uncoupled Cav2.2 from CRMP2, reduced membrane Cav2.2 expression and Ca2+ currents, decreased neurotransmission, reduced fiber photometry-based calcium responses in response to mechanical stimulation, and reversed neuropathic and inflammatory pain across sexes in two different species without changes in sensory, sedative, depressive, and cognitive behaviors. CBD3063 is a selective, first-in-class, CRMP2-based peptidomimetic small molecule, which allosterically regulates Cav2.2 to achieve analgesia and pain relief without negative side effect profiles. In summary, CBD3063 could potentially be a more effective alternative to GBP for pain relief.
Asunto(s)
Dolor Crónico , Peptidomiméticos , Ratas , Animales , Dolor Crónico/tratamiento farmacológico , Dolor Crónico/metabolismo , Ratas Sprague-Dawley , Peptidomiméticos/farmacología , Calcio/metabolismo , Canales de Calcio Tipo N/genética , Canales de Calcio Tipo N/metabolismo , Células Receptoras Sensoriales/metabolismo , Ganglios Espinales/metabolismoRESUMEN
The 13 Hsp70 proteins in humans act on unique sets of substrates with diversity often being attributed to J-domain-containing protein (Hsp40 or JDP) cofactors. We were therefore surprised to find drastically different binding affinities for Hsp70-peptide substrates, leading us to probe substrate specificity among the 8 canonical Hsp70s from humans. We used peptide arrays to characterize Hsp70 binding and then mined these data using machine learning to develop an algorithm for isoform-specific prediction of Hsp70 binding sequences. The results of this algorithm revealed recognition patterns not predicted based on local sequence alignments. We then showed that none of the human isoforms can complement heat-shocked DnaK knockout Escherichia coli cells. However, chimeric Hsp70s consisting of the human nucleotide-binding domain and the substrate-binding domain of DnaK complement during heat shock, providing further evidence in vivo of the divergent function of the Hsp70 substrate-binding domains. We also demonstrated that the differences in heat shock complementation among the chimeras are not due to loss of DnaJ binding. Although we do not exclude JDPs as additional specificity factors, our data demonstrate substrate specificity among the Hsp70s, which has important implications for inhibitor development in cancer and neurodegeneration.
Asunto(s)
Proteínas de Escherichia coli , Proteínas de Choque Térmico , Humanos , Proteínas de Choque Térmico/metabolismo , Proteínas de Escherichia coli/química , Sitios de Unión , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas del Choque Térmico HSP40/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Péptidos/metabolismo , Unión ProteicaRESUMEN
The Ras-like GTPases RalA and RalB are important drivers of tumour growth and metastasis. Chemicals that block Ral function would be valuable as research tools and for cancer therapeutics. Here we used protein structure analysis and virtual screening to identify drug-like molecules that bind to a site on the GDP-bound form of Ral. The compounds RBC6, RBC8 and RBC10 inhibited the binding of Ral to its effector RALBP1, as well as inhibiting Ral-mediated cell spreading of murine embryonic fibroblasts and anchorage-independent growth of human cancer cell lines. The binding of the RBC8 derivative BQU57 to RalB was confirmed by isothermal titration calorimetry, surface plasmon resonance and (1)H-(15)N transverse relaxation-optimized spectroscopy (TROSY) NMR spectroscopy. RBC8 and BQU57 show selectivity for Ral relative to the GTPases Ras and RhoA and inhibit tumour xenograft growth to a similar extent to the depletion of Ral using RNA interference. Our results show the utility of structure-based discovery for the development of therapeutics for Ral-dependent cancers.
Asunto(s)
Ensayos de Selección de Medicamentos Antitumorales , Terapia Molecular Dirigida , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacología , Proteínas de Unión al GTP ral/antagonistas & inhibidores , Transportadoras de Casetes de Unión a ATP/metabolismo , Animales , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Simulación por Computador , Femenino , Proteínas Activadoras de GTPasa/metabolismo , Humanos , Ratones , Modelos Moleculares , Neoplasias/tratamiento farmacológico , Neoplasias/enzimología , Neoplasias/metabolismo , Neoplasias/patología , Unión Proteica/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Especificidad por Sustrato , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas de Unión al GTP ral/química , Proteínas de Unión al GTP ral/metabolismo , Proteínas ras/metabolismoRESUMEN
Voltage-gated sodium channels are crucial determinants of neuronal excitability and signaling. Trafficking of the voltage-gated sodium channel NaV1.7 is dysregulated in neuropathic pain. We identify a trafficking program for NaV1.7 driven by hierarchical interactions with posttranslationally modified versions of the binding partner collapsin response mediator protein 2 (CRMP2). The binding described between CRMP2 and NaV1.7 was enhanced by conjugation of CRMP2 with small ubiquitin-like modifier (SUMO) and further controlled by the phosphorylation status of CRMP2. We determined that CRMP2 SUMOylation is enhanced by prior phosphorylation by cyclin-dependent kinase 5 and antagonized by Fyn phosphorylation. As a consequence of CRMP2 loss of SUMOylation and binding to NaV1.7, the channel displays decreased membrane localization and current density, and reduces neuronal excitability. Preventing CRMP2 SUMOylation with a SUMO-impaired CRMP2-K374A mutant triggered NaV1.7 internalization in a clathrin-dependent manner involving the E3 ubiquitin ligase Nedd4-2 (neural precursor cell expressed developmentally down-regulated protein 4) and endocytosis adaptor proteins Numb and epidermal growth factor receptor pathway substrate 15. Collectively, our work shows that diverse modifications of CRMP2 cross-talk to control NaV1.7 activity and illustrate a general principle for regulation of NaV1.7.
Asunto(s)
Péptidos y Proteínas de Señalización Intercelular/fisiología , Canal de Sodio Activado por Voltaje NAV1.7/fisiología , Proteínas del Tejido Nervioso/fisiología , Procesamiento Proteico-Postraduccional , Animales , Línea Celular , Membrana Celular/metabolismo , Endocitosis , Endosomas/metabolismo , Células HEK293 , Humanos , Masculino , Neuronas/metabolismo , Dolor/genética , Dolor/metabolismo , Técnicas de Placa-Clamp , Fosforilación , Transporte de Proteínas , Ratas , Ratas Sprague-DawleyRESUMEN
Glycogen is a glucose polymer that contains minor amounts of covalently attached phosphate. Hyperphosphorylation is deleterious to glycogen structure and can lead to Lafora disease. Recently, it was demonstrated that glycogen synthase catalyzes glucose-phosphate transfer in addition to its characteristic glucose transfer reaction. Glucose-1,2-cyclic-phosphate (GCP) was proposed to be formed from UDP-Glc breakdown and subsequently transferred, thus providing a source of phosphate found in glycogen. To gain further insight into the molecular basis for glucose-phosphate transfer, two structures of yeast glycogen synthase were determined; a 3.0-Å resolution structure of the complex with UMP/GCP and a 2.8-Å resolution structure of the complex with UDP/glucose. Structural superposition of the complexes revealed that the bound ligands and most active site residues are positioned similarly, consistent with the use of a common transfer mechanism for both reactions. The N-terminal domain of the UDP-glucose complex was found to be 13.3° more closed compared with a UDP complex. However, the UMP · GCP complex was 4.8° less closed than the glucose complex, which may explain the low efficiency of GCP transfer. Modeling of either α- or ß-glucose or a mixture of both anomers can account for the observed electron density of the UDP-glucose complex. NMR studies of UDP-Glc hydrolysis by yeast glycogen synthase were used to verify the stereochemistry of the product, and they also showed synchronous GCP accumulation. The similarities in the active sites of glycogen synthase and glycogen phosphorylase support the idea of a common catalytic mechanism in GT-B enzymes independent of the specific reaction catalyzed.
Asunto(s)
Glucógeno Sintasa/metabolismo , Glucógeno/química , Modelos Moleculares , Fosfatos/química , Cristalografía , Glucógeno/metabolismo , Glucógeno Sintasa/química , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Mutagénesis , Fosfatos/metabolismoRESUMEN
Although hippocampal neurons are well-distinguished by the morphological characteristics of their dendrites and their structural plasticity, the mechanisms involved in regulating their neurite initiation, dendrite growth, network formation and remodeling are still largely unknown, in part because the key molecules involved remain elusive. Identifying new dendrite-active cues could uncover unknown molecular mechanisms that would add significant understanding to the field and possibly lead to the development of novel neuroprotective therapy because these neurons are impaired in many neuropsychiatric disorders. In our previous studies, we deleted the gene encoding CRMP3 in mice and identified the protein as a new endogenous signaling molecule that shapes diverse features of the hippocampal pyramidal dendrites without affecting axon morphology. We also found that CRMP3 protects dendrites against dystrophy induced by prion peptide PrP(106-126). Here, we report that CRMP3 has a profound influence on neurite initiation and dendrite growth of hippocampal neurons in vitro. Our deletional mapping revealed that the C-terminus of CRMP3 probably harbors its dendritogenic capacity and supports an active transport mechanism. By contrast, overexpression of the C-terminal truncated CRMP3 phenocopied the effect of CRMP3 gene deletion with inhibition of neurite initiation or decrease in dendrite complexity, depending on the stage of cell development. In addition, this mutant inhibited the activity of CRMP3, in a similar manner to siRNA. Voltage-gated calcium channel inhibitors prevented CRMP3-induced dendritic growth and somatic Ca(2+) influx in CRMP3-overexpressing neurons was augmented largely via L-type channels. These results support a link between CRMP3-mediated Ca(2+) influx and CRMP3-mediated dendritic growth in hippocampal neurons.
Asunto(s)
Canales de Calcio/metabolismo , Dendritas/metabolismo , Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuritas/metabolismo , Animales , Canales de Calcio/fisiología , Dendritas/fisiología , Hipocampo/fisiología , Ratones , Morfogénesis , Proteínas del Tejido Nervioso/genética , Transducción de Señal , TransfecciónRESUMEN
We have successfully expressed and purified active human glycogen synthase-1 (hGYS1). Successful production of the recombinant hGYS1 protein was achieved by co-expression of hGYS1 and rabbit glycogenin (rGYG1) using the MultiBac baculovirus expression system (BEVS). Functional measurements of activity ratios of hGYS1 in the absence and presence of glucose-6-phosphate and treatment with phosphatase indicate that the expressed protein is heavily phosphorylated. We used mass spectrometry to further characterize the sites of phosphorylation, which include most of the known regulatory phosphorylation sites, as well as several sites unique to the insect cell over-expression. Obtaining large quantities of functional hGYS1 will be invaluable for future structural studies as well as detailed studies on the effects on specific sites of phosphorylation.
Asunto(s)
Glucógeno Sintasa/genética , Glucógeno Sintasa/aislamiento & purificación , Animales , Línea Celular , Glucosiltransferasas/genética , Glucosiltransferasas/metabolismo , Glucógeno Sintasa/metabolismo , Glicoproteínas/genética , Glicoproteínas/metabolismo , Humanos , Concentración de Iones de Hidrógeno , Insectos/citología , Fosforilación , Conejos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The uPAR·uPA protein-protein interaction (PPI) is involved in signaling and proteolytic events that promote tumor invasion and metastasis. A previous study had identified 4 (IPR-803) from computational screening of a commercial chemical library and shown that the compound inhibited uPAR·uPA PPI in competition biochemical assays and invasion cellular studies. Here, we synthesize 4 to evaluate in vivo pharmacokinetic (PK) and efficacy studies in a murine breast cancer metastasis model. First, we show, using fluorescence polarization and saturation transfer difference (STD) NMR, that 4 binds directly to uPAR with sub-micromolar affinity of 0.2 µM. We show that 4 blocks invasion of breast MDA-MB-231, and inhibits matrix metalloproteinase (MMP) breakdown of the extracellular matrix (ECM). Derivatives of 4 also inhibited MMP activity and blocked invasion in a concentration-dependent manner. Compound 4 also impaired MDA-MB-231 cell adhesion and migration. Extensive in vivo PK studies in NOD-SCID mice revealed a half-life of nearly 5h and peak concentration of 5 µM. Similar levels of the inhibitor were detected in tumor tissue up to 10h. Female NSG mice inoculated with highly malignant TMD-MDA-MB-231 in their mammary fat pads showed that 4 impaired metastasis to the lungs with only four of the treated mice showing severe or marked metastasis compared to ten for the untreated mice. Compound 4 is a promising template for the development of compounds with enhanced PK parameters and greater efficacy.
Asunto(s)
Antineoplásicos/uso terapéutico , Neoplasias de la Mama/tratamiento farmacológico , Metástasis de la Neoplasia/tratamiento farmacológico , Mapas de Interacción de Proteínas/efectos de los fármacos , Receptores del Activador de Plasminógeno Tipo Uroquinasa/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/uso terapéutico , Activador de Plasminógeno de Tipo Uroquinasa/antagonistas & inhibidores , Animales , Antineoplásicos/química , Antineoplásicos/farmacocinética , Antineoplásicos/farmacología , Mama/efectos de los fármacos , Mama/metabolismo , Mama/patología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Femenino , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Invasividad Neoplásica/patología , Metástasis de la Neoplasia/patología , Receptores del Activador de Plasminógeno Tipo Uroquinasa/metabolismo , Bibliotecas de Moléculas Pequeñas/química , Bibliotecas de Moléculas Pequeñas/farmacocinética , Bibliotecas de Moléculas Pequeñas/farmacología , Activador de Plasminógeno de Tipo Uroquinasa/metabolismoRESUMEN
Human aldehyde dehydrogenases (ALDHs) comprise a family of 17 homologous enzymes that metabolize different biogenic and exogenic aldehydes. To date, there are relatively few general ALDH inhibitors that can be used to probe the contribution of this class of enzymes to particular metabolic pathways. Here, we report the discovery of a general class of ALDH inhibitors with a common mechanism of action. The combined data from kinetic studies, mass spectrometric measurements, and crystallographic analyses demonstrate that these inhibitors undergo an enzyme-mediated ß-elimination reaction generating a vinyl ketone intermediate that covalently modifies the active site cysteine residue present in these enzymes. The studies described here can provide the basis for rational approach to design ALDH isoenzyme-specific inhibitors as research tools and perhaps as drugs, to address diseases such as cancer where increased ALDH activity is associated with a cellular phenotype.
Asunto(s)
Aldehído Deshidrogenasa/antagonistas & inhibidores , Aldehído Deshidrogenasa/química , Aldehído Deshidrogenasa/metabolismo , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Familia de Aldehído Deshidrogenasa 1 , Aldehído Deshidrogenasa Mitocondrial , Aldehídos/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Ciclofosfamida/análogos & derivados , Ciclofosfamida/farmacología , Humanos , Cinética , Espectrometría de Masas , Estructura Molecular , Estructura Secundaria de Proteína , Retinal-DeshidrogenasaRESUMEN
BACKGROUND: The ubiquity of protein-protein interactions in biological signaling offers ample opportunities for therapeutic intervention. We previously identified a peptide, designated CBD3, that suppressed inflammatory and neuropathic behavioral hypersensitivity in rodents by inhibiting the ability of collapsin response mediator protein 2 (CRMP-2) to bind to N-type voltage-activated calcium channels (CaV2.2) [Brittain et al. Nature Medicine 17:822-829 (2011)]. RESULTS AND DISCUSSION: Here, we utilized SPOTScan analysis to identify an optimized variation of the CBD3 peptide (CBD3A6K) that bound with greater affinity to Ca²âº channels. Molecular dynamics simulations demonstrated that the CBD3A6K peptide was more stable and less prone to the unfolding observed with the parent CBD3 peptide. This mutant peptide, conjugated to the cell penetrating motif of the HIV transduction domain protein TAT, exhibited greater anti-nociception in a rodent model of AIDS therapy-induced peripheral neuropathy when compared to the parent TAT-CBD3 peptide. Remarkably, intraperitoneal administration of TAT-CBD3A6K produced none of the minor side effects (i.e. tail kinking, body contortion) observed with the parent peptide. Interestingly, excitability of dissociated small diameter sensory neurons isolated from rats was also reduced by TAT-CBD3A6K peptide suggesting that suppression of excitability may be due to inhibition of T- and R-type Ca²âº channels. TAT-CBD3A6K had no effect on depolarization-evoked calcitonin gene related peptide (CGRP) release compared to vehicle control. CONCLUSIONS: Collectively, these results establish TAT-CBD3A6K as a peptide therapeutic with greater efficacy in an AIDS therapy-induced model of peripheral neuropathy than its parent peptide, TAT-CBD3. Structural modifications of the CBD3 scaffold peptide may result in peptides with selectivity against a particular subset of voltage-gated calcium channels resulting in a multipharmacology of action on the target.
Asunto(s)
Síndrome de Inmunodeficiencia Adquirida/terapia , Canales de Calcio Tipo N/metabolismo , Péptidos y Proteínas de Señalización Intercelular/química , Proteínas del Tejido Nervioso/química , Nocicepción , Nociceptores/metabolismo , Péptidos/uso terapéutico , Enfermedades del Sistema Nervioso Periférico/tratamiento farmacológico , Síndrome de Inmunodeficiencia Adquirida/complicaciones , Secuencia de Aminoácidos , Animales , Separación Celular , Modelos Animales de Enfermedad , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Hiperalgesia/complicaciones , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/patología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Simulación de Dinámica Molecular , Datos de Secuencia Molecular , Mutagénesis/genética , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Neurotransmisores/metabolismo , Nocicepción/efectos de los fármacos , Nociceptores/efectos de los fármacos , Nociceptores/patología , Péptidos/química , Péptidos/metabolismo , Péptidos/farmacología , Enfermedades del Sistema Nervioso Periférico/etiología , Unión Proteica/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Productos del Gen tat del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
The urokinase receptor (uPAR) serves as a docking site to the serine protease urokinase-type plasminogen activator (uPA) to promote extracellular matrix (ECM) degradation and tumor invasion and metastasis. Previously, we had reported a small molecule inhibitor of the uPAR·uPA interaction that emerged from structure-based virtual screening. Here, we measure the affinity of a large number of derivatives from commercial sources. Synthesis of additional compounds was carried out to probe the role of various groups on the parent compound. Extensive structure-based computational studies suggested a binding mode for these compounds that led to a structure-activity relationship study. Cellular studies in non-small cell lung cancer (NSCLC) cell lines that include A549, H460 and H1299 showed that compounds blocked invasion, migration and adhesion. The effects on invasion of active compounds were consistent with their inhibition of uPA and MMP proteolytic activity. These compounds showed weak cytotoxicity consistent with the confined role of uPAR to metastasis.
Asunto(s)
Antineoplásicos/farmacología , Benzoatos/farmacología , Diseño de Fármacos , Lectinas de Unión a Manosa/antagonistas & inhibidores , Glicoproteínas de Membrana/antagonistas & inhibidores , Simulación de Dinámica Molecular , Piperidinas/farmacología , Receptores de Superficie Celular/antagonistas & inhibidores , Antineoplásicos/síntesis química , Antineoplásicos/química , Benzoatos/síntesis química , Benzoatos/química , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Ensayos de Selección de Medicamentos Antitumorales , Humanos , Lectinas de Unión a Manosa/aislamiento & purificación , Lectinas de Unión a Manosa/metabolismo , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/metabolismo , Estructura Molecular , Peso Molecular , Piperidinas/síntesis química , Piperidinas/química , Receptores de Superficie Celular/aislamiento & purificación , Receptores de Superficie Celular/metabolismo , Relación Estructura-ActividadRESUMEN
Pontocerebellar Hypoplasia 1B (PCH1B) is a severe autosomal recessive neurological disorder that is associated with mutations in the exosome complex component RRP40 (EXOSC3) gene. We generated and characterized an iPSC line from an individual with PCH1B that harbors a recessive homozygous c.395 A > C mutation in EXOSC3 and a family matched control from the probands unaffected mother. Each iPSC line presents with normal morphology and karyotype and express high levels of pluripotent markers. UAZTi009-A and UAZTi011-A are capable of directed differentiation and can be used as a vital experimental tool to study the development of PCH1B.
Asunto(s)
Complejo Multienzimático de Ribonucleasas del Exosoma , Proteínas de Unión al ARN , Humanos , Mutación/genética , Células Madre Pluripotentes Inducidas , Línea CelularRESUMEN
Neurodegeneration is a progressive deterioration of neural structures leading to cognitive or motor impairment of the affected patient. There is still no effective therapy for any of the most common neurodegenerative diseases (NDs) such as Alzheimer's or Parkinson's disease. Although NDs exhibit distinct clinical characteristics, many are characterized by the accumulation of misfolded proteins or peptide fragments in the brain and/or spinal cord. The presence of similar inclusion bodies in patients with diverse NDs provides a rationale for developing therapies directed at overlapping disease mechanisms. A novel targeting strategy involves the use of aptamers for therapeutic development. Aptamers are short nucleic acid ligands able to recognize molecular targets with high specificity and high affinity. Despite the fact that several academic groups have shown that aptamers have the potential to be used in therapeutic and diagnostic applications, their clinical translation is still limited. In this study, we describe aptamers that have been developed against proteins relevant to NDs, including prion protein and amyloid beta (Aß), cell surface receptors and other cytoplasmic proteins. This review also describes advances in the application of these aptamers in imaging, protein detection, and protein quantification, and it provides insights about their accelerated clinical use for disease diagnosis and therapy.
Asunto(s)
Aptámeros de Nucleótidos , Priones , Péptidos beta-Amiloides/genética , Aptámeros de Nucleótidos/química , Aptámeros de Nucleótidos/genética , Aptámeros de Nucleótidos/uso terapéutico , Humanos , Ligandos , Fragmentos de PéptidosRESUMEN
Neurodegenerative diseases represent a formidable challenge to global health. As advances in other areas of medicine grant healthy living into later decades of life, aging diseases such as Alzheimer's disease (AD) and other neurodegenerative disorders can diminish the quality of these additional years, owed largely to the lack of efficacious treatments and the absence of durable cures. Alzheimer's disease prevalence is predicted to more than double in the next 30 years, affecting nearly 15 million Americans, with AD-associated costs exceeding $1 billion by 2050. Delaying onset of AD and other neurodegenerative diseases is critical to improving the quality of life for patients and reducing the burden of disease on caregivers and healthcare systems. Significant progress has been made to model disease pathogenesis and identify points of therapeutic intervention. While some researchers have contributed to our understanding of the proteins and pathways that drive biological dysfunction in disease using in vitro and in vivo models, others have provided mathematical, biophysical, and computational technologies to identify potential therapeutic compounds using in silico modeling. The most exciting phase of the drug discovery process is now: by applying a target-directed approach that leverages the strengths of multiple techniques and validates lead hits using Drosophila as an animal model of disease, we are on the fast-track to identifying novel therapeutics to restore health to those impacted by neurodegenerative disease.
RESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease with no cure or effective treatment in which TAR DNA Binding Protein of 43 kDa (TDP-43) abnormally accumulates into misfolded protein aggregates in affected neurons. It is widely accepted that protein misfolding and aggregation promotes proteotoxic stress. The molecular chaperones are a primary line of defense against proteotoxic stress, and there has been long-standing interest in understanding the relationship between chaperones and aggregated protein in ALS. Of particular interest are the heat shock protein of 70 kDa (Hsp70) family of chaperones. However, defining which of the 13 human Hsp70 isoforms is critical for ALS has presented many challenges. To gain insight into the specific Hsp70 that modulates TDP-43, we investigated the relationship between TDP-43 and the Hsp70s using proximity-dependent biotin identification (BioID) and discovered several Hsp70 isoforms associated with TDP-43 in the nucleus, raising the possibility of an interaction with native TDP-43. We further found that HspA5 bound specifically to the RNA-binding domain of TDP-43 using recombinantly expressed proteins. Moreover, in a Drosophila strain that mimics ALS upon TDP-43 expression, the mRNA levels of the HspA5 homologue (Hsc70.3) were significantly increased. Similarly we observed upregulation of HspA5 in prefrontal cortex neurons from human ALS patients. Finally, overexpression of HspA5 in Drosophila rescued TDP-43-induced toxicity, suggesting that upregulation of HspA5 may have a compensatory role in ALS pathobiology.
Asunto(s)
Esclerosis Amiotrófica Lateral , Enfermedades Neurodegenerativas , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proteínas de Unión al ADN/metabolismo , Drosophila/metabolismo , Chaperón BiP del Retículo Endoplásmico , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Chaperonas MolecularesRESUMEN
Cwc21 (complexed with Cef1 protein 21) is a 135 amino acid yeast protein that shares homology with the N-terminal domain of human SRm300/SRRM2, a large serine/arginine-repeat protein shown previously to associate with the splicing coactivator and 3'-end processing stimulatory factor, SRm160. Proteomic analysis of spliceosomal complexes has suggested a role for Cwc21 and SRm300 at the core of the spliceosome. However, specific functions for these proteins have remained elusive. In this report, we employ quantitative genetic interaction mapping, mass spectrometry of tandem affinity-purified complexes, and microarray profiling to investigate genetic, physical, and functional interactions involving Cwc21. Combined data from these assays support multiple roles for Cwc21 in the formation and function of splicing complexes. Consistent with a role for Cwc21 at the core of the spliceosome, we observe strong genetic, physical, and functional interactions with Isy1, a protein previously implicated in the first catalytic step of splicing and splicing fidelity. Together, the results suggest multiple functions for Cwc21/SRm300 in the splicing process, including an important role in the activation of splicing in association with Isy1.
Asunto(s)
Proteínas Portadoras/metabolismo , Empalme del ARN , Proteínas de Unión al ARN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Empalmosomas/metabolismo , Proteínas Portadoras/genética , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Unión Proteica , Proteómica , ARN Nuclear Pequeño/genética , Proteínas de Unión al ARN/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMEN
We assess the performance of our previously reported structure-based support vector machine target-specific scoring function across 41 targets, 40 among them from the Directory of Useful Decoys (DUD). The area under the curve of receiver operating characteristic plots (ROC-AUC) revealed that scoring with SVM-SP resulted in consistently better enrichment over all target families, outperforming Glide and other scoring functions, most notably among kinases. In addition, SVM-SP performance showed little variation among protein classes, exhibited excellent performance in a test case using a homology model, and in some cases showed high enrichment even with few structures used to train a model. We put SVM-SP to the test by virtual screening 1125 compounds against two kinases, EGFR and CaMKII. Among the top 25 EGFR compounds, three compounds (1-3) inhibited kinase activity in vitro with IC50 of 58, 2, and 10 µM. In cell cultures, compounds 1-3 inhibited nonsmall cell lung carcinoma (H1299) cancer cell proliferation with similar IC50 values for compound 3. For CaMKII, one compound inhibited kinase activity in a dose-dependent manner among 20 tested with an IC50 of 48 µM. These results are encouraging given that our in-house library consists of compounds that emerged from virtual screening of other targets with pockets that are different from typical ATP binding sites found in kinases. In light of the importance of kinases in chemical biology, these findings could have implications in future efforts to identify chemical probes of kinases within the human kinome.
Asunto(s)
Inteligencia Artificial , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Descubrimiento de Drogas/métodos , Receptores ErbB/antagonistas & inhibidores , Bibliotecas de Moléculas Pequeñas/farmacología , Algoritmos , Sitios de Unión , Carcinoma de Pulmón de Células no Pequeñas/enzimología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Humanos , Concentración 50 Inhibidora , Neoplasias Pulmonares/enzimología , Modelos Moleculares , Relación Estructura-Actividad Cuantitativa , Curva ROCRESUMEN
Tar DNA binding (TDP)-43 proteinopathy, typically described as cytoplasmic accumulation of highly modified and misfolded TDP-43 molecules, is characteristic of several neurodegenerative diseases such as Amyotrophic Lateral Sclerosis (ALS) and limbic-predominant age-related TDP-43 encephalopathy (LATE). TDP-43 proposed proteinopathies include homeostatic imbalance between nuclear and cytoplasmic localization, aggregation of ubiquitinated and hyper-phosphorylated TDP-43, and an increase in protein truncation of cytoplasmic TDP-43. Given the therapeutic interest of targeting TDP-43, this review focuses on the current landscape of strategies, ranging from biologics to small molecules, that directly target TDP-43. Antibodies, peptides and compounds have been designed or found to recognize specific TDP-43 sequences but alleviate TDP-43 toxicity through different mechanisms. While two antibodies described here were able to induce degradation of pathological TDP-43, the peptides and small molecules were primarily designed to reduce aggregation of TDP-43. Furthermore, we discuss promising emerging therapeutic targets.
RESUMEN
RNA targeting has gained traction over the past decade. It has become clear that dysregulation of RNA can be linked to many diseases, leading to a need for new scaffolds recognizing RNA specifically. Long noncoding RNAs are emerging as key controllers of gene expression and potential therapeutic targets. However, traditional targeting methods have overwhelmingly been focused on proteins. In this study, we used a protein computational tool and found several possible targetable pockets in a structurally characterized long noncoding RNA, MALAT1. Screening against those identified pockets revealed several hit compounds. We tested the binding of those compounds to MALAT1 RNA and tRNA as a negative control, using SPR. While several compounds were nonspecific binders, others were able to recognize MALAT1 specifically. One of them, MTC07, has an apparent affinity of 400.2 ± 14.4 µM. Although it has weak affinity, MTC07 is the first compound targeting MALAT1 originating from in silico docking.