Moduladores del sensor neuronal de calcio DREAM: herramientas farmacológicas para el estudio de su interactoma y su aplicación en la enfermedad de Huntington

La enfermedad de Huntington (EH) se caracteriza por una combinación de alteraciones motoras, psiquiátricas y cognitivas. En la actualidad, se encuentra entre las patologías neurodegenerativas carentes de un tratamiento que incida de manera efectiva en su generación y desarrollo. Hasta el momento, los tratamientos existentes sólo han logrado paliar los síntomas, sin incidir en la causa de los mismos. Por tanto, la búsqueda de nuevas estrategias que ayuden a elucidar las bases moleculares de esta enfermedad y sirvan como punto de partida para el desarrollo de nuevos fármacos, es crucial. En este sentido, esta Memoria se ha orientado al estudio de una estrategia novedosa mediante la búsqueda de moléculas pequeñas que modulen las interacciones proteína-proteína del sensor neuronal de calcio DREAM involucradas en la enfermedad de Huntington. La finalidad es aportar conocimiento en los procesos implicados en la generación y progresión de esta patología. DREAM, también conocida como calsenilina o KChIP3, es una proteína de unión a calcio de la superfamilia de los sensores neuronales de calcio con características estructurales de tipo manos-EF. La unión de calcio a los dominios mano- EF de DREAM, origina cambios estructurales que afectan a su capacidad de unión a sitios específicos del ADN y a la interacción con otras proteínas, modificando sus funciones biológicas. A nivel del sistema nervioso, la acumulación excesiva de Ca2+ se ha asociado a diversas enfermedades neurodegenerativas, entre las que se encuentra la EH..

Small Molecules as Dream Modulators: New Avenues for the Search of Drugs for Neurodegenerative Diseases

Trabajo presentado en el 9th drug Design and Medicinal Chemistry, celebrado en Berlín (Alemania) del 05 al 06 de mayo de 2015.Altered neuronal calcium homeostasis and early compensatory changes in transcriptional programs are common features of many neurodegenerative pathologies including Alzheimer¿s disease, Down syndrome and Huntington¿s disease. DREAM (Downstream Regulatory Element Antagonist Modulator), also known as calsenilin or KChIP-3 (potassium channel interacting protein-3), is a multifunctional calcium binding protein that controls the expression level and/or the activity of several proteins related to calcium homeostasis, neuronal excitability and neuronal survival. This protein is widely expressed in the brain and, depending on the cell type and physiological conditions, shows multiple subcellular localizations, in the nucleus, cytosol or cell membrane. The interest in DREAM is based on its key role in the regulation of intracellular calcium levels. As a calcium-dependent transcriptional repressor, DREAM is a master regulator of activity-dependent gene expression and controls genes important for calcium homeostasis such as the sodium/calcium exchanger-3 (NCX3), IP3R and L-type calcium channels. As an auxiliary protein in the plasma membrane, DREAM interacts with and regulates the gating of Kv4 potassium channels, L- and T-type voltage-dependent calcium channels and NMDA receptors. These findings suggest that DREAM could be a novel and versatile target for therapeutic intervention in neurodegeneration and that molecules able to bind to DREAM and block its physiological functions could be candidates for drugs to treat neurodegenerative diseases. Moreover, up to now, only two DREAM-binding molecules have been identified. In this communication we report the rational design and the synthesis of novel DREAM-binding molecules and their effects on the modulation of DREAM/protein interactions

IQM-PC332, a Novel DREAM Ligand with Antinociceptive Effect on Peripheral Nerve Injury-Induced Pain

Neuropathic pain is a form of chronic pain arising from damage of the neural cells that sense, transmit or process sensory information. Given its growing prevalence and common refractoriness to conventional analgesics, the development of new drugs with pain relief effects constitutes a prominent clinical need. In this respect, drugs that reduce activity of sensory neurons by modulating ion channels hold the promise to become effective analgesics. Here, we evaluated the mechanical antinociceptive effect of IQM-PC332, a novel ligand of the multifunctional protein downstream regulatory element antagonist modulator (DREAM) in rats subjected to chronic constriction injury of the sciatic nerve as a model of neuropathic pain. IQM-PC332 administered by intraplantar (0.01–10 µg) or intraperitoneal (0.02–1 µg/kg) injection reduced mechanical sensitivity by ≈100% of the maximum possible effect, with ED50 of 0.27 ± 0.05 µg and 0.09 ± 0.01 µg/kg, respectively. Perforated-patch whole-cell recordings in isolated dorsal root ganglion (DRG) neurons showed that IQM-PC332 (1 and 10 µM) reduced ionic currents through voltage-gated K+ channels responsible for A-type potassium currents, low, T-type, and high voltage-activated Ca2+ channels, and transient receptor potential vanilloid-1 (TRPV1) channels. Furthermore, IQM-PC332 (1 µM) reduced electrically evoked action potentials in DRG neurons from neuropathic animals. It is suggested that by modulating multiple DREAM–ion channel signaling complexes, IQM-PC332 may serve a lead compound of novel multimodal analgesics

Activating transcription factor 6 derepression mediates neuroprotection in Huntington disease

Deregulated protein and Ca2+ homeostasis underlie synaptic dysfunction and neurodegeneration in Huntington disease (HD); however, the factors that disrupt homeostasis are not fully understood. Here, we determined that expression of downstream regulatory element antagonist modulator (DREAM), a multifunctional Ca2+-binding protein, is reduced in murine in vivo and in vitro HD models and in HD patients. DREAM downregulation was observed early after birth and was associated with endogenous neuroprotection. In the R6/2 mouse HD model, induced DREAM haplodeficiency or blockade of DREAM activity by chronic administration of the drug repaglinide delayed onset of motor dysfunction, reduced striatal atrophy, and prolonged life span. DREAM-related neuroprotection was linked to an interaction between DREAM and the unfolded protein response (UPR) sensor activating transcription factor 6 (ATF6). Repaglinide blocked this interaction and enhanced ATF6 processing and nuclear accumulation of transcriptionally active ATF6, improving prosurvival UPR function in striatal neurons. Together, our results identify a role for DREAM silencing in the activation of ATF6 signaling, which promotes early neuroprotection in HDThis work was funded by the Instituto de Salud Carlos III/CIBERNED (to J.R. Naranjo, B. Mellström, and A. Rábano), FISS-RIC RD12/0042/0019 (to C. Valenzuela), Madrid regional government/Neurodegmodels (to J.R. Naranjo), MINECO grants SAF2010-21784 and SAF2014-53412-R (to J.R. Naranjo), SAF2012-32209 (to M. Gutierrez-Rodriguez), SAF2010-14916 and SAF2013-45800-R (to C. Valenzuela), and a grant from the Swedish Research Council (J.Y. Li

Identification of IQM-266, a Novel DREAM Ligand That Modulates KV4 Currents

Downstream Regulatory Element Antagonist Modulator (DREAM)/KChIP3/calsenilin is a neuronal calcium sensor (NCS) with multiple functions, including the regulation of A-type outward potassium currents (IA). This effect is mediated by the interaction between DREAM and KV4 potassium channels and it has been shown that small molecules that bind to DREAM modify channel function. A-type outward potassium current (IA) is responsible of the fast repolarization of neuron action potentials and frequency of firing. Using surface plasmon resonance (SPR) assays and electrophysiological recordings of KV4.3/DREAM channels, we have identified IQM-266 as a DREAM ligand. IQM-266 inhibited the KV4.3/DREAM current in a concentration-, voltage-, and time-dependent-manner. By decreasing the peak current and slowing the inactivation kinetics, IQM-266 led to an increase in the transmembrane charge (QKV4.3/DREAM) at a certain range of concentrations. The slowing of the recovery process and the increase of the inactivation from the closed-state inactivation degree are consistent with a preferential binding of IQM-266 to a pre-activated closed state of KV4.3/DREAM channels. Finally, in rat dorsal root ganglion neurons, IQM-266 inhibited the peak amplitude and slowed the inactivation of IA. Overall, the results presented here identify IQM-266 as a new chemical tool that might allow a better understanding of DREAM physiological role as well as modulation of neuronal IA in pathological processes

Caracterización electrofisiológica de nuevos moduladores selectivos de KChIP3

Resumen del trabajo presentado a la 24ª edición de la Reunión de Farmacólogos de la Comunidad de Madrid (Farmadrid).DREAM, también denominada calsenilina o KChIP3, es una proteína sensora de calcio que regula diferentes genes tras su unión a los sitios DRE del DNA. Fuera del núcleo, DREAM regula diversas funciones celulares mediante su unión a múltiples proteínas, una de las cuales es el canal Kv4.3. Los canales Kv4.3 generan, tras su unión a diferentes subunidades moduladoras, una corriente rápida y transitoria de salida de potasio. En el corazón, los canales Kv4.3 se unen a las subunidades KChIP2 generando la Ito1, que es una de las principales corrientes repolarizantes cardiacas. En neuronas, se asocian a KChIP1 o KChIP3 y generan la IA, que está implicada en funciones muy especializadas como el aprendizaje, la memoria y el comportamiento. En este trabajo, describimos los efectos electrofisiológicos sobre canales Kv4.3 y Kv4.3+KChIP3 expresados en células CHO, de nuevos compuestos diseñados para unirse a KChIP3 con alta afinidad. PC342 inhibió en mayor grado Kv4.3 que Kv4.3+KChIP3, mientras que PC332 y PC358 produjeron una mayor inhibición de la corriente Kv4.3+KChIP3. El bloqueo de los canales inducido por PC332 y PC358 fue tiempo- y voltaje-dependiente, consistente con una unión preferencial a un estado pre-abierto. Así pues, PC332 y PC358 podrían representar un buen punto de partida para el desarrollo de nuevos fármacos que modulen selectivamente la actividad eléctrica neuronal en diferentes poblaciones de neuronas.Peer Reviewe

Effects of CL888 on Kv4.3, Kv4.3/KChip2c and Kv4.3/KChiP3 channels

Resumen del póster presentado al 58th Annual Meeting of the Biophysical Society, celebrado en San Francisco-California (US) del 15 al 19 de febrero de 2014.Kv4.3 generates the transient outward current that plays an essential role in shaping the early phase of the cardiac action potential. These channels are regulated by a family of calcium-binding proteins called KChIPs. Four members of this family have been cloned. KChIP3 and KChIP2c bind to Kv4.3 in the endoplasmic reticulum and facilitate trafficking to the membrane and regulate the channel gating. CL888 induces alteration or disruption in the binding between KChIP1 and Kv4.3, modulating the function of the complex. The aim of our study was to determine the effects of CL888 on Kv4.3/KChIP3 and Kv4.3/KChIP2 complex, as well as on Kv4.3 alone. KChIP3, KChiP2 and Kv4.3 channels were expressed in CHO cells. Currents were recorded using the whole-cell patch-clamp technique. Block of Kv4.3/KChIP3 channels induced by CL888 was concentration dependent with an IC50 of 23 nM, whereas Kv4.3 and Kv4.3/KChIP2c was not. At 100 nM, degree of block ranged according to: Kv4.3/KChIP3>Kv4.3/KChIP2c>Kv4.3. In all 3 channel complexes, CL888 accelerated the inactivation kinetics of the current by decreasing the slow time constant (τs). Thus, for Kv4.3/KChIP3 the τs value changed from 20.6±1.6 ms to 17.0±1.5 ms (n=5, P<0.05); for Kv4.3, from 19.7±1.6 ms to 10.8±1.5 ms (n=4, P<0.05); and for Kv4.3/KChIP2, from 38.1±5.8 ms to 23.9±4.0 ms (n=5, P<0.05). CL888 did not affect the voltage dependency of steady-state inactivation of Kv4.3/KChIP3 channels. However, it accelerated their recovery from inactivation kinetics (τre=62.0±12.1 ms vs. 49.8±9.8 ms, n=6, P<0.05). We conclude that CL888 binds to Kv4.3 channels and modulates the gating and the kinetics of these channels. However, Kv4.3/KChIP3 complex results to be more sensitive than Kv4.3 and Kv4.3/KChIP2c. Therefore, the sensitivity of Kv4.3 channels to CL888-like drugs will vary depending of the associated regulatory subunits.Granted by SAF2010-14916 and FIS-RIC RD12/0042/0019.Peer Reviewe

Electrophysiological effects of IQM-266 on KV1.5 channels

Resumen del trabajo presentado al VII Congreso Red Española Canales Iónicos, celebrado en Cáceres del 15 al 17 de mayo de 2019.The outward potassium current IKur is the main responsible of the atrial repolarization process and it is generated by the activation of KV1.5 channels, widely expressed in human atria. It is known that mutations in KCNA5 gene, which induce both gain- and loss-of-function in KV1.5 channel, enhance atrial fibrillation susceptibility. Thus, these channels represent a pharmacological target for the development of antiarrhythmic drugs useful in the treatment of supraventricular arrhythmias. KV1.5 channels assembly with several regulatory subunits such as KVβ and KChIPs (KV Channel Interacting Proteins). It has been described that KChIP2 physically interacts with KV1.5 and reduces KV1.5 cell surface expression levels. Our research group has demonstrated that IQM-266 inhibits the current generated by the activation of KV4.3 and KV4.3/KChIP2, being the effects more marked when KChIP2 is present. The aim of the present study is to analyze the effects of IQM-266 on KV1.5 channels. In order to achieve these objectives, HEK293 cells transiently expressing KV1.5 were used. Currents were recorded using the whole-cell configuration of the patch-clamp technique. The effects of IQM-266 on KV1.5 currents were concentration-dependent with an IC50 of 11 μM (n=24). Block induced by IQM-266 (20 μM) sharply increased within the membrane voltage range of the channel activation, arising a maximum degree of block at +20 mV that remained constant at more positive membrane potentials. This compound at 20 μM produced a timedependent block, inducing a: 1) faster inactivation (τ = 305.8±47.6 ms vs. 168.0±13.4 ms, in the absence and in the presence of IQM-266, respectively, n=9, p<0.01), and 2) slower deactivation kinetics, increasing the contribution of the slow component of deactivation to the total process (0.36±0.05 vs. 0.57±0.05, in the absence and in the presence of IQM-266, respectively, n=11, p<0.01). These results are consistent with an open channel block mechanism. Finally, IQM-266 (20 μM) enhanced the degree of use-dependent block of the current (25.6±2.7% vs. 77.6±4.9%, in the absence and in the presence of IQM-266, n=5, p<0.001). These phenomenon was explained by a slowing of the recovery process in the presence of the compound (991.1±131.8 ms vs. 5132.8±763.6 ms in the absence and in the presence of IQM-266, respectively, n=4, p<0.05). Thus, IQM-266 is able to bind KV1.5 channels, although with less affinity than that shown in KV4.3/KChIP2 complexes.Supported by SAF2016-75021-R and CSIC PIE201820E104 to CV, BFU2015-67284-R and CSIC PIE201880E109 to MGR.Peer reviewe

Targeting the dream protein: new avenues for the search of drugs for neurodegenerative diseases

Resumen del póster presentado al III SEQT Summer School: "Medicinal Chemistry in Drug Discovery: The Pharma Perspective", celebrado en Tres Cantos (Madrid) del 25 al 27 de junio de 2013.Altered neuronal calcium homeostasis and early compensatory changes in transcriptional programs are common features of many neurodegenerative pathologies including Alzheimer’s disease (AD), Down syndrome (DS) and Huntington’s disease (HD). DREAM (Downstream Regulatory Element Antagonist Modulator), also known as calsenilin or KChIP-3 (potasium channel interacting protein-3), is a multifunctional Ca2+ binding protein that controls the expression level and/or the activity of several proteins related to Ca2+ homeostasis, neuronal excitability and neuronal survival. This protein is widely expressed in the brain and, depending on the cell type and physiological conditions, shows multiple subcellular localizations, in the nucleus, cytosol or cell membrane. Initially, the interest in DREAM was based on its key role in the regulation of intracelular Ca2+ levels. An early reduction in DREAM levels is found in the pre-symptomatic phase of several neurodegenerative mouse models, including AD, DS and HD. These data support the idea that an early down regulation of the DREAM level in neurons during the pre-symptomatic phase of the AD, DS and HD might be part of its neuroprotective mechanism. These findings suggest that DREAM could be a novel and versatile target for therapeutic intervention in neurodegeneration and that molecules able to bind to DREAM and block its physiological functions could be candidates of drugs to treat neurodegenerative diseases. Up to know, low molecular weight molecules have not been described able to interact with DREAM and to modulate its action. In this communication we report the rational design, the synthesis and the biological evaluation of novel DREAM-binding molecules.Peer Reviewe

Electrophysiological characterization of new selective KChIP3 modulators

Resumen del póster presentado a la 5ª Reunión de la Red Española de Canales Iónicos: "Present and future in ion channel research", celebrada en Barcelona (España) del 4 al 6 de octubre de 2015.DREAM, also named calsenilin or KChIP3, is a neuronal calcium sensor that regulates multiple genes after binding to DRE sites in the nucleus. Outside nucleus, DREAM regulates diverse cellular functions by binding to several proteins, one of them the Kv4.3 channel. Kv4.3 channels, after assembling with different modulatory subunits, generate a fast and transient outward potassium current. In the heart, Kv4.3 binds with KChIP2 subunits, generating the Ito1, one of the main repolarizing cardiac currents. In neurons, they associate to KChIP1 or KChIP3 and generate the IA, which is implicated in very specialized functions as learning, memory and behavior. Here, we describe the electrophysiological effects of new compounds designed to bind with high affinity to KChIP3, in Kv4.3 and Kv4.3+KChIP3 channels expressed in CHO cells. PC342 inhibited Kv4.3 and Kv4.3+KChIP3 to a similar extent, whereas PC332 and PC358 inhibited Kv4.3+KChIP3 to a greater extent. Block induced by PC332 was time- and voltage-dependent, consistent with binding to a closed-active state. Therefore, PC332 and PC358 may represent good compounds to develop new drugs that selectively modulate the neuronal electric activity in distinct neuronal populations.Peer Reviewe