Synthesis and pharmacological evaluation of novel non-nucleotide purine derivatives as P2X7 antagonists for the treatment of neuroinflammation

The ATP-gated P2X7 purinergic receptor (P2X7) is involved in the pathogenesis of many neurodegenerative diseases (NDDs). Several P2X7 antagonists have been developed, though none of them reached clinical trials for this indication. In this work, we designed and synthesized novel blood-brain barrier (BBB)-permeable derivatives as potential P2X7 antagonists. They comprise purine or xanthine cores linked to an aryl group through different short spacers. Compounds were tested in YO-PRO-1 uptake assays and intracellular calcium dynamics in a human P2X7-expressing HEK293 cell line, two-electrode voltage-clamp recordings in Xenopus laevis oocytes, and in interleukin 1β release assays in mouse peritoneal macrophages. BBB permeability was assessed by parallel artificial membrane permeability assays and P-glycoprotein ATPase activity. Dichloroarylpurinylethanones featured a certain P2X7 blockade, being compound 6 (2-(6-chloro-9H-purin-9-yl)-1-(2,4-dichlorophenyl)ethan-1-one), named ITH15004, the most potent, selective, and BBB-permeable antagonist. Compound 6 can be considered as a first non-nucleotide purine hit for future drug optimizationsThis work has been supported by the following grants: EU Horizon 2020 Research and Innovation Program under Marie Skłodowska-Curie, Grant Agreement N. 766124 to AGG and AN, and Ministerio de Economía y Competitividad, Spain, Grant Number SAF2016-78892R to AGG; Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) Project-ID: 335447717, SFB 1328 (TP15) to A.N.; Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain, Grant Numbers PI16/01041 and PI19/01724 (Cofunded by FEDER) to CdlR; Instituto de Salud Carlos III, Ministerio de Economía y Competitividad, Spain, Grant Numbers PI16/00735 and PI19/00082 (Co-funded by FEDER) to J.E

TLR4-pathway impairs synaptic number and cerebrovascular functions through astrocyte activation following traumatic brain injury

Background and purpose: Activation of astrocytes contributes to synaptic remodelling, tissue repair and neuronal survival following traumatic brain injury (TBI). The mechanisms by which these cells interact to resident/infiltrated inflammatory cells to rewire neuronal networks and repair brain functions remain poorly understood. Here, we explored how TLR4-induced astrocyte activation modified synapses and cerebrovascular integrity following TBI. Experimental approach: To determine how functional astrocyte alterations induced by activation of TLR4 pathway in inflammatory cells regulate synapses and neurovascular integrity after TBI, we used pharmacology, genetic approaches, live calcium imaging, immunofluorescence, flow cytometry, blood-brain barrier (BBB) integrity assessment and molecular and behavioural methods. Key results: Shortly after a TBI, there is a recruitment of excitable and reactive astrocytes mediated by TLR4 pathway activation with detrimental effects on post-synaptic density-95 (PSD-95)/vesicular glutamate transporter 1 (VGLUT1) synaptic puncta, BBB integrity and neurological outcome. Pharmacological blockage of the TLR4 pathway with resatorvid (TAK-242) partially reversed many of the observed effects. Synapses and BBB recovery after resatorvid administration were not observed in IP3 R2-/- mice, indicating that effects of TLR4 inhibition depend on the subsequent astrocyte activation. In addition, TBI increased the astrocytic-protein thrombospondin-1 necessary to induce a synaptic recovery in a sub-acute phase. Conclusions and implications: Our data demonstrate that TLR4-mediated signalling, most probably through microglia and/or infiltrated monocyte-astrocyte communication, plays a crucial role in the TBI pathophysiology and that its inhibition prevents synaptic loss and BBB damage accelerating tissue recovery/repair, which might represent a therapeutic potential in CNS injuries and disorders.This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) (Programa Miguel Servet II Grants CPII19/00005;PI16/00735; PI19/00082 to JE; and PI18/00357 to DC, partiallyfunded by FEDER - European Union ‘Una manera de hacer Europa’) and Fundación Mutua Madrileña to JE; European Union's Horizon2020 research and innovation programme under the H2020 MarieSkłodowska-Curie Actions grant agreement no. 794926 and StopFuga de Cerebros Roche Pharma to JMR; and Ministerio de Ciencia e Innovación RTI2018-094887-B-I00 and RYC-2016-20414 to MN andRYC2019-026870-I to JMR. DC, MCO, VVS and EFL are hired bySESCAM

TLR4-pathway impairs synaptic number and cerebrovascular functions through astrocyte activation following traumatic brain injury.

Background and purpose: Activation of astrocytes contributes to synaptic remodelling, tissue repair and neuronal survival following traumatic brain injury (TBI). The mechanisms by which these cells interact to resident/infiltrated inflammatory cells to rewire neuronal networks and repair brain functions remain poorly understood. Here, we explored how TLR4-induced astrocyte activation modified synapses and cerebrovascular integrity following TBI. Experimental approach: To determine how functional astrocyte alterations induced by activation of TLR4 pathway in inflammatory cells regulate synapses and neurovascular integrity after TBI, we used pharmacology, genetic approaches, live calcium imaging, immunofluorescence, flow cytometry, blood-brain barrier (BBB) integrity assessment and molecular and behavioural methods. Key results: Shortly after a TBI, there is a recruitment of excitable and reactive astrocytes mediated by TLR4 pathway activation with detrimental effects on post-synaptic density-95 (PSD-95)/vesicular glutamate transporter 1 (VGLUT1) synaptic puncta, BBB integrity and neurological outcome. Pharmacological blockage of the TLR4 pathway with resatorvid (TAK-242) partially reversed many of the observed effects. Synapses and BBB recovery after resatorvid administration were not observed in IP3 R2-/- mice, indicating that effects of TLR4 inhibition depend on the subsequent astrocyte activation. In addition, TBI increased the astrocytic-protein thrombospondin-1 necessary to induce a synaptic recovery in a sub-acute phase. Conclusions and implications: Our data demonstrate that TLR4-mediated signalling, most probably through microglia and/or infiltrated monocyte-astrocyte communication, plays a crucial role in the TBI pathophysiology and that its inhibition prevents synaptic loss and BBB damage accelerating tissue recovery/repair, which might represent a therapeutic potential in CNS injuries and disorders.This work was supported by grants from the Instituto de Salud Carlos III (ISCIII) (Programa Miguel Servet II Grants CPII19/00005;PI16/00735; PI19/00082 to JE; and PI18/00357 to DC, partiallyfunded by FEDER - European Union ‘Una manera de hacer Europa’) and Fundación Mutua Madrileña to JE; European Union's Horizon2020 research and innovation programme under the H2020 MarieSkłodowska-Curie Actions grant agreement no. 794926 and StopFuga de Cerebros Roche Pharma to JMR; and Ministerio de Ciencia e Innovación RTI2018-094887-B-I00 and RYC-2016-20414 to MN andRYC2019-026870-I to JMR. DC, MCO, VVS and EFL are hired bySESCAM

Synthesis and Pharmacological Evaluation of New N-Sulfonylureas as NLRP3 Inflammasome Inhibitors: Identification of a Hit Compound to Treat Gout

NLRP3 is involved in the pathophysiology of several inflammatory diseases. Therefore, there is high current interest in the clinical development of new NLRP3 inflammasome small inhibitors to treat these diseases. Novel N-sulfonylureas were obtained by the replacement of the hexahydroindacene moiety of the previously described NLRP3 inhibitor MCC950. These new derivatives show moderate to high potency in inhibiting IL-1β release in vitro. The greatest effect was observed for compound 4b, which was similar to MCC950. Moreover, compound 4b was able to reduce caspase-1 activation, oligomerization of ASC, and therefore, IL-1β processing. Additional in silico predictions confirmed the safety profile of compound 4b, and in vitro studies in AML12 hepatic cells confirmed the absence of toxicological effects. Finally, we evaluated in vivo anti-inflammatory properties of compound 4b, which showed a significant anti-inflammatory effect and reduced mechanical hyperalgesia at 3 and 10 mg/kg (i.p.) in an in vivo mouse model of gout.J.E. thanks Fondo de Investigaciones Sanitarias (ISCIII/ FEDER) (Programa Miguel Servet: CP19/00005 and PI19/ 00082) and Fundación Mutua Madrileñ a. D.D.-I. thanks the Spanish Ministry of Science, Innovation, and Universities for predoctoral FPU grant

Serum amyloid a1/toll-like receptor-4 Axis, an important link between inflammation and outcome of TBI patients

Traumatic brain injury (TBI) is one of the leading causes of mortality and disability world-wide without any validated biomarker or set of biomarkers to help the diagnosis and evaluation of the evolution/prognosis of TBI patients. To achieve this aim, a deeper knowledge of the biochemical and pathophysiological processes triggered after the trauma is essential. Here, we identified the serum amyloid A1 protein-Toll-like receptor 4 (SAA1-TLR4) axis as an important link between inflammation and the outcome of TBI patients. Using serum and mRNA from white blood cells (WBC) of TBI patients, we found a positive correlation between serum SAA1 levels and injury severity, as well as with the 6-month outcome of TBI patients. SAA1 levels also correlate with the presence of TLR4 mRNA in WBC. In vitro, we found that SAA1 contributes to inflammation via TLR4 activation that releases inflammatory cytokines, which in turn increases SAA1 levels, establishing a positive proinflammatory loop. In vivo, post-TBI treatment with the TLR4-antagonist TAK242 reduces SAA1 levels, improves neurobehavioral outcome, and prevents blood–brain barrier disruption. Our data support further evaluation of (i) post-TBI treatment in the presence of TLR4 inhibition for limiting TBI-induced damage and (ii) SAA1-TLR4 as a biomarker of injury progression in TBI patientsThis work was supported by grants from Fundación Mutua Madrileña and Fondo de Investigaciones Sanitarias (FIS) (ISCIII/FEDER) (Programa Miguel Servet CP14/00008; CPII19/00005; PI16/00735; PI19/00082) to JE, RYC2019-026870-I to JMR and PI18/01387 to A

Nuevas estrategias farmacológicas dirigidas a la inhibición del inflamasoma NLRP3

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Farmacología. Fecha de Lectura: 24-05-2022Esta Tesis tiene embargado el acceso al texto completo hasta el 24-11-2023La respuesta inflamatoria desencadenada por el inflamasoma NLRP3 es necesaria para eliminar al agente infeccioso o resolver el daño tisular que la origina. Esta respuesta debe ser controlada en el tiempo y detenida una vez que el estímulo causante de la inflamación se ha resuelto. La activación exacerbada del inflamasoma contribuye a la cronificación de esta respuesta y a la progresión de numerosas patologías con un componente inflamatorio, incluyendo enfermedades cardiovasculares, del sistema nervioso central o patologías metabólicas, entre otras. Actualmente existen terapias basadas en fármacos biológicos que neutralizan la citoquina IL-1β, producida por NLRP3, o que inhiben su respuesta, bloqueando así la cascada proinflamatoria que esta citoquina desencadena. Estos fármacos son efectivos para algunas de las patologías inflamatorias en cuyo desarrollo está implicado el inflamasoma NLRP3. Sin embargo, presentan importantes limitaciones basadas en los efectos inmunosupresores que generan, en la falta de acción sobre otros componentes de la ruta del inflamasoma y en el modo de administración. Estos motivos hacen que el desarrollo de fármacos para el tratamiento de patologías inflamatorias se dirija hacia la inhibición selectiva del inflamasoma NLRP3. En esta tesis doctoral hemos explorado dos estrategias dirigidas a la inhibición del inflamasoma NLRP3. La primera de ellas ha consistido en estudiar el intercambiador Na+/Ca2+ mitocondrial (NCLX) como una posible diana para inhibir el inflamasoma. La inhibición farmacológica de NCLX mediante el compuesto selectivo ITH12575 ha demostrado reducir el ensamblaje y activación del inflamasoma NLRP3 in vitro en macrófagos derivados de médula ósea (BMDM), e in vivo en un modelo de gota aguda en ratón. ITH12575 ha reducido tanto la inflamación como los niveles proteicos de los componentes del inflamasoma en la pata de los animales sometidos al modelo. La segunda estrategia ha consistido en la inhibición directa de NLRP3 a través de nuevos compuestos N-sulfonilureas obtenidos en base a la estructura del conocido inhibidor del inflamasoma NLRP3 MCC950. Entre los nuevos compuestos, la molécula 4b ha resultado ser un potente inhibidor de NLRP3, mostrando una EC50 muy similar a la del MCC950. Además, la molécula 4b ha demostrado reducir la inflamación y el dolor inflamatorio in vivo en un modelo de gota aguda en ratón. Teniendo en cuenta estos resultados, proponemos al inhibidor de NCLX, ITH12575, y a la nueva N-sulfonilurea, el compuesto 4b, como nuevas moléculas inhibidoras del inflamasoma NLRP3. Además, reforzamos la hipótesis de la inhibición de NLRP3 como diana para el tratamiento de los ataques de gotaLa investigación que se presenta ha sido financiada por proyectos del Instituto de Salud Carlos III (ISCIII) PI16/00735 y PI19/00082 (investigador principal Dr. Javier Egea), por el proyecto de la Agencia Estatal de Investigación RTI2018-094203-B-I00 (investigador principal Dr. Antonio Martínez Ruiz) y por la ayuda de la Comunidad de Madrid para la contratación de investigadores PEJD-2019-PRE/BMD-17028, cofinanciados por los fondos FEDER y FSE de la Unión Europe

Activation of NLRP3 Is Required for a Functional and Beneficial Microglia Response after Brain Trauma

Despite the numerous research studies on traumatic brain injury (TBI), many physiopathologic mechanisms remain unknown. TBI is a complex process, in which neuroinflammation and glial cells play an important role in exerting a functional immune and damage-repair response. The activation of the NLRP3 inflammasome is one of the first steps to initiate neuroinflammation and so its regulation is essential. Using a closed-head injury model and a pharmacological (MCC950; 3 mg/kg, pre- and post-injury) and genetical approach (NLRP3 knockout (KO) mice), we defined the transcriptional and behavioral profiles 24 h after TBI. Wild-type (WT) mice showed a strong pro-inflammatory response, with increased expression of inflammasome components, microglia and astrocytes markers, and cytokines. There was no difference in the IL1β production between WT and KO, nor compensatory mechanisms of other inflammasomes. However, some microglia and astrocyte markers were overexpressed in KO mice, resulting in an exacerbated cytokine expression. Pretreatment with MCC950 replicated the behavioral and blood–brain barrier results observed in KO mice and its administration 1 h after the lesion improved the damage. These findings highlight the importance of NLRP3 time-dependent activation and its role in the fine regulation of glial response

Dérivés de N-sulfonylurées et utilisation thérapeutique de ces derniers

[EN] The present invention relates to a series of N-sulfonlyurea derivative compounds, compounds of general formula (I), with pharmacological inhibitory activity of the NLRP3 inflammasome. The use of the compounds of general formula (I) as agents for treating preferably human diseases that involve an inflammatory process, preferably involving what are known as inflammasomes, preferably of the NLRP3 type, is claimed. These diseases include neurodegenerative diseases, such as Alzheimer's and Parkinson's disease, inflammatory diseases, metabolic diseases, autoimmune diseases and generally any pathology caused by impaired biological functions in which the NLRP3 inflammasome is involved. The present invention has been developed in the field of pharmaceutical industry.[ES] La presente invención se refiere a una serie de compuestos derivados de N-sulfonilureas, compuestos de fórmula general (I), con actividad farmacológica inhibidora del inflamasoma NLRP3. Se reivindica el uso de los compuestos referidos con la fórmula general (I) como agentes para el tratamiento de enfermedades preferentemente humanas que cursan con proceso inflamatorio, preferentemente mediado por lo que se conoce como inflamasoma, preferentemente del tipo denominado NLRP3. Entre estas enfermedades se encuentran las denominadas neurodegenerativas, como el Alzheimer y el Parkinson, las enfermedades inflamatorias, la enfermedades metabólicas, las autoinmunes y, en general, cualquier patología producida por la alteración de las funciones biológicas donde está implicado el inflamasoma NLRP3. La presente invención se puede enmarcar en el campo de la industria farmacéutica.[FR] La présente invention se rapporte à une série de composés dérivés de N-sulfonylurées, de composés de formule générale (I), ayant une activité pharmacologique inhibitrice de l'inflammasome NLRP3. L'invention porte également sur l'utilisation desdits composés ayant la formule générale (I) en tant qu'agents pour le traitement de maladies de préférence humaines qui peuvent accompagner un processus inflammatoire, de préférence médié par ce qu'on appelle l'inflammasome, de préférence du type appelé NLRP3. Parmi ces maladies on trouve les maladies neurodégénératives, comme la maladie d'Alzheimer et la maladie de Parkinson, les maladies inflammatoires, les maladies métaboliques, les maladies autoimmunes et, en général, toute pathologie produite par l'altération des fonctions biologiques dans lesquelles est impliqué l'inflammasome NLRP3. La présente invention peut se situer dans le domaine de l'industrie pharmaceutique.Peer reviewedFundación para la Investigación Biomédica del Hospital de La Princesa, Consejo Superior de Investigaciones CientíficasA1 Solicitud de patente con informe sobre el estado de la técnic

N-sulfonylurea derivatives and their therapeutic use

[ES] Derivados de n-sulfonilureas y su uso terapéutico. La presente invención se refiere a una serie de compuestos derivados de N-sufoniureas, compuestos de fórmula general (1), con actividad farmacológica inhibidora del inflamasoma NLRP3. {IMAGEN-01} Se reivindica el uso de los compuestos referidos con la fórmula general (I) como agentes para el tratamiento de enfermedades preferentemente humanas que cursan con proceso inflamatorio, preferentemente mediado por lo que se conoce como inflamasoma, preferentemente del tipo denominado NLRP3. Entre estas enfermedades se encuentran las denominadas neurodegenerativas, como el Alzheimer y el Parkinson, las enfermedades inflamatorias, las enfermedades metabólicas, las autoinmunes y, en general, cualquier patología producida por la alteración de las funciones biológicas donde está implicado el inflamasoma NLRP3. La presente invención se puede enmarcar en el campo de la industria farmacéutica.[EN] Derivatives of n-sulfonylureas and their therapeutic use. The present invention relates to a series of compounds derived from N-suffoniureas, compounds of general formula (1), with pharmacological activity that inhibits the NLRP3 inflammasome. {IMAGE-01} The use of the compounds referred to with the general formula (I) is claimed as agents for the treatment of preferably human diseases that involve an inflammatory process, preferably mediated by what is known as an inflammasome, preferably of the type called NLRP3. Among these diseases are the so-called neurodegenerative diseases, such as Alzheimer's and Parkinson's, inflammatory diseases, metabolic diseases, autoimmune diseases and, in general, any pathology caused by the alteration of biological functions where the NLRP3 inflammasome is involved. The present invention can be framed in the field of the pharmaceutical industry.Peer reviewedFundación para la Investigación Biomédica del Hospital de La Princesa, Consejo Superior de Investigaciones CientíficasA1 Solicitud de patente con informe sobre el estado de la técnic

Time-dependent dual effect of NLRP3 inflammasome in brain ischaemia

Background Inflammasomes are cytosolic multiprotein complexes which, upon assembly, activate the maturation and secretion of the inflammatory cytokines IL-1 beta and IL-18. However, participation of the NLRP3 inflammasome in ischaemic stroke remains controversial. Our aims were to determine the role of NLRP3 in brain ischaemia, and explore the mechanism involved in the potential protective effect of the neurovascular unit. Methods WT and NLRP3 knock-out mice were subjected to ischaemia by middle cerebral artery occlusion (60 min) with or without treatment with MCC950 at different time points post-stroke. Brain injury was measured histologically with 2,3,5-triphenyltetrazolium chloride (TTC) staining. Results We identified a time-dependent dual effect of NLRP3. While neither the pre-treatment with MCC950 nor the genetic approach (NLRP3 KO) proved to be neuroprotective, post-reperfusion treatment with MCC950 significantly reduced the infarct volume in a dose-dependent manner. Importantly, MCC950 improved the neuro-motor function and reduced the expression of different pro-inflammatory cytokines (IL-1 beta and TNF-alpha), NLRP3 inflammasome components (NLRP3 and pro-caspase-1), protease expression (MMP9), and endothelial adhesion molecules (ICAM and VCAM). We observed a marked protection of the blood-brain barrier (BBB), which was also reflected in the recovery of the tight junction proteins (ZO-1 and Claudin-5). Additionally, MCC950 produced a reduction of the CCL2 chemokine in blood serum and in brain tissue, which lead to a reduction in the immune cell infiltration. Conclusions These findings suggest that post-reperfusion NLRP3 inhibition may be an effective acute therapy for protecting the blood-brain barrier in cerebral ischaemia with potential clinical translation