Clinical mutations in the TERT and TERC genes coding for telomerase components induced oxidative stress, DNA damage at telomeres and cell apoptosis besides decreased telomerase activity.

Telomeres are nucleoprotein structures at the end of chromosomes that maintain their integrity. Mutations in genes coding for proteins involved in telomere protection and elongation produce diseases such as dyskeratosis congenita or idiopathic pulmonary fibrosis known as telomeropathies. These diseases are characterized by premature telomere shortening, increased DNA damage and oxidative stress. Genetic diagnosis of telomeropathy patients has identified mutations in the genes TERT and TERC coding for telomerase components but the functional consequences of many of these mutations still have to be experimentally demonstrated. The activity of twelve TERT and five TERC mutants, five of them identified in Spanish patients, has been analyzed. TERT and TERC mutants were expressed in VA-13 human cells that express low telomerase levels and the activity induced was analyzed. The production of reactive oxygen species, DNA oxidation and TRF2 association at telomeres, DNA damage response and cell apoptosis were determined. Most mutations presented decreased telomerase activity, as compared to wild-type TERT and TERC. In addition, the expression of several TERT and TERC mutants induced oxidative stress, DNA oxidation, DNA damage, decreased recruitment of the shelterin component TRF2 to telomeres and increased apoptosis. These observations might indicate that the increase in DNA damage and oxidative stress observed in cells from telomeropathy patients is dependent on their TERT or TERC mutations. Therefore, analysis of the effect of TERT and TERC mutations of unknown function on DNA damage and oxidative stress could be of great utility to determine the possible pathogenicity of these variants.

Extracellular Kir2.1C122Y Mutant Upsets Kir2.1-PIP2 Bonds and Is Arrhythmogenic in Andersen-Tawil Syndrome.

BACKGROUND: Andersen-Tawil syndrome type 1 is a rare heritable disease caused by mutations in the gene coding the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys (cysteine)122-to-Cys154 disulfide bond in the channel structure is crucial for proper folding but has not been associated with correct channel function at the membrane. We evaluated whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing its open state. METHODS: We identified a Kir2.1 loss-of-function mutation (c.366 A>T; p.Cys122Tyr) in an ATS1 family. To investigate its pathophysiological implications, we generated an AAV9-mediated cardiac-specific mouse model expressing the Kir2.1C122Y variant. We employed a multidisciplinary approach, integrating patch clamping and intracardiac stimulation, molecular biology techniques, molecular dynamics, and bioluminescence resonance energy transfer experiments. RESULTS: Kir2.1C122Y mice recapitulated the ECG features of ATS1 independently of sex, including corrected QT prolongation, conduction defects, and increased arrhythmia susceptibility. Isolated Kir2.1C122Y cardiomyocytes showed significantly reduced inwardly rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking. Molecular dynamics predicted that the C122Y mutation provoked a conformational change over the 2000-ns simulation, characterized by a greater loss of hydrogen bonds between Kir2.1 and phosphatidylinositol 4,5-bisphosphate than wild type (WT). Therefore, the phosphatidylinositol 4,5-bisphosphate-binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch clamping, the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing phosphatidylinositol 4,5-bisphosphate concentrations. In addition, the Kir2.1C122Y mutation resulted in channelosome degradation, demonstrating temporal instability of both Kir2.1 and NaV1.5 proteins. CONCLUSIONS: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential for the channel function. We demonstrate that breaking disulfide bonds in the extracellular domain disrupts phosphatidylinositol 4,5-bisphosphate-dependent regulation, leading to channel dysfunction and defects in Kir2.1 energetic stability. The mutation also alters functional expression of the NaV1.5 channel and ultimately leads to conduction disturbances and life-threatening arrhythmia characteristic of Andersen-Tawil syndrome type 1.

Exchangeable Self-Assembled Lanthanide Antennas for PLIM Microscopy.

Lanthanides have unique photoluminescence (PL) emission properties, including very long PL lifetimes. This makes them ideal for biological imaging applications, especially using PL lifetime imaging microscopy (PLIM). PLIM is an inherently multidimensional technique with exceptional advantages for quantitative biological imaging. Unfortunately, due to the required prolonged acquisitions times, photobleaching of lanthanide PL emission currently constitutes one of the main drawbacks of PLIM. In this study, we report a small aqueous-soluble, lanthanide antenna, 8-methoxy-2-oxo-1,2,4,5-tetrahydrocyclopenta[de]quinoline-3-phosphonic acid, PAnt, specifically designed to dynamically interact with lanthanide ions, serving as exchangeable dye aimed at mitigating photobleaching in PLIM microscopy in cellulo. Thus, self-assembled lanthanide complexes that may be photobleached during image acquisition are continuously replenished by intact lanthanide antennas from a large reservoir. Remarkably, our self-assembled lanthanide complex clearly demonstrated a significant reduction of PL photobleaching when compared to well-established lanthanide cryptates, used for bioimaging. This concept of exchangeable lanthanide antennas opens new possibilities for quantitative PLIM bioimaging.

Psychogeriatric care in times of COVID. Lessons learned and proposals for similar situations.

The objective is to describe the problems related to outpatient psychogeriatric care in the context of the SARS-CoV-2 pandemic, as well as the proposed and implemented solutions for optimizing care for elderly people with mental disorders during the pandemic, that can also be applied in emerging similar situations in the future.

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.

Modulation of KV4.3-KChIP2 Channels by IQM-266: Role of DPP6 and KCNE2.

The transient outward potassium current (Itof) is generated by the activation of KV4 channels assembled with KChIP2 and other accessory subunits (DPP6 and KCNE2). To test the hypothesis that these subunits modify the channel pharmacology, we analyzed the electrophysiological effects of (3-(2-(3-phenoxyphenyl)acetamido)-2-naphthoic acid) (IQM-266), a new KChIP2 ligand, on the currents generated by KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 channels. CHO cells were transiently transfected with cDNAs codifying for different proteins (KV4.3/KChIP2, KV4.3/KChIP2/DPP6 or KV4.3/KChIP2/KCNE2), and the potassium currents were recorded using the whole-cell patch-clamp technique. IQM-266 decreased the maximum peak of KV4.3/KChIP2, KV4.3/KChIP2/DPP6 and KV4.3/KChIP2/KCNE2 currents, slowing their time course of inactivation in a concentration-, voltage-, time- and use-dependent manner. IQM-266 produced an increase in the charge in KV4.3/KChIP2 channels that was intensified when DPP6 was present and abolished in the presence of KCNE2. IQM-266 induced an activation unblocking effect during the application of trains of pulses to cells expressing KV4.3/KChIP2 and KV4.3/KChIP2/KCNE2, but not in KV4.3/KChIP2/DPP6 channels. Overall, all these results are consistent with a preferential IQM-266 binding to an active closed state of Kv4.3/KChIP2 and Kv4.3/KChIP2/KCNE2 channels, whereas in the presence of DPP6, IQM-266 binds preferentially to an inactivated state. In conclusion, DPP6 and KCNE2 modify the pharmacological response of KV4.3/KChIP2 channels to IQM-266.

Pharmacological Approaches for the Modulation of the Potassium Channel KV4.x and KChIPs.

Ion channels are macromolecular complexes present in the plasma membrane and intracellular organelles of cells. Dysfunction of ion channels results in a group of disorders named channelopathies, which represent an extraordinary challenge for study and treatment. In this review, we will focus on voltage-gated potassium channels (KV), specifically on the KV4-family. The activation of these channels generates outward currents operating at subthreshold membrane potentials as recorded from myocardial cells (ITO, transient outward current) and from the somata of hippocampal neurons (ISA). In the heart, KV4 dysfunctions are related to Brugada syndrome, atrial fibrillation, hypertrophy, and heart failure. In hippocampus, KV4.x channelopathies are linked to schizophrenia, epilepsy, and Alzheimer's disease. KV4.x channels need to assemble with other accessory subunits (ß) to fully reproduce the ITO and ISA currents. ß Subunits affect channel gating and/or the traffic to the plasma membrane, and their dysfunctions may influence channel pharmacology. Among KV4 regulatory subunits, this review aims to analyze the KV4/KChIPs interaction and the effect of small molecule KChIP ligands in the A-type currents generated by the modulation of the KV4/KChIP channel complex. Knowledge gained from structural and functional studies using activators or inhibitors of the potassium current mediated by KV4/KChIPs will better help understand the underlying mechanism involving KV4-mediated-channelopathies, establishing the foundations for drug discovery, and hence their treatments.

Exploring the Phe-Gly dipeptide-derived piperazinone scaffold in the search for antagonists of the thrombin receptor PAR1.

A series of Phe-Gly dipeptide-derived piperazinones containing an aromatic urea moiety and a basic amino acid has been synthesized and evaluated as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cells. The synthetic strategy involves coupling of a protected basic amino acid benzyl amide to 1,2- and 1,2,4-substituted-piperazinone derivatives, through a carbonylmethyl group at the N1-position, followed by formation of an aromatic urea at the exocyclic moiety linked at the C2 position of the piperazine ring and removal of protecting groups. None of the compounds showed activity in the biological evaluation.

Extracellular cysteine disulfide bond break at Cys122 disrupts PIP2-dependent Kir2.1 channel function and leads to arrhythmias in Andersen-Tawil Syndrome.

Background: Andersen-Tawil Syndrome Type 1 (ATS1) is a rare heritable disease caused by mutations in the strong inwardly rectifying K+ channel Kir2.1. The extracellular Cys122-to-Cys154 disulfide bond in the Kir2.1 channel structure is crucial for proper folding, but has not been associated with correct channel function at the membrane. We tested whether a human mutation at the Cys122-to-Cys154 disulfide bridge leads to Kir2.1 channel dysfunction and arrhythmias by reorganizing the overall Kir2.1 channel structure and destabilizing the open state of the channel. Methods and Results: We identified a Kir2.1 loss-of-function mutation in Cys122 (c.366 A>T; p.Cys122Tyr) in a family with ATS1. To study the consequences of this mutation on Kir2.1 function we generated a cardiac specific mouse model expressing the Kir2.1C122Y mutation. Kir2.1C122Y animals recapitulated the abnormal ECG features of ATS1, like QT prolongation, conduction defects, and increased arrhythmia susceptibility. Kir2.1C122Y mouse cardiomyocytes showed significantly reduced inward rectifier K+ (IK1) and inward Na+ (INa) current densities independently of normal trafficking ability and localization at the sarcolemma and the sarcoplasmic reticulum. Kir2.1C122Y formed heterotetramers with wildtype (WT) subunits. However, molecular dynamic modeling predicted that the Cys122-to-Cys154 disulfide-bond break induced by the C122Y mutation provoked a conformational change over the 2000 ns simulation, characterized by larger loss of the hydrogen bonds between Kir2.1 and phosphatidylinositol-4,5-bisphosphate (PIP2) than WT. Therefore, consistent with the inability of Kir2.1C122Y channels to bind directly to PIP2 in bioluminescence resonance energy transfer experiments, the PIP2 binding pocket was destabilized, resulting in a lower conductance state compared with WT. Accordingly, on inside-out patch-clamping the C122Y mutation significantly blunted Kir2.1 sensitivity to increasing PIP2 concentrations. Conclusion: The extracellular Cys122-to-Cys154 disulfide bond in the tridimensional Kir2.1 channel structure is essential to channel function. We demonstrated that ATS1 mutations that break disulfide bonds in the extracellular domain disrupt PIP2-dependent regulation, leading to channel dysfunction and life-threatening arrhythmias.

SARS-CoV-2 Fears Green: The Chlorophyll Catabolite Pheophorbide A Is a Potent Antiviral.

SARS-CoV-2 pandemic is having devastating consequences worldwide. Although vaccination advances at good pace, effectiveness against emerging variants is unpredictable. The virus has displayed a remarkable resistance to treatments and no drugs have been proved fully effective against COVID-19. Thus, despite the international efforts, there is still an urgent need for new potent and safe antivirals against SARS-CoV-2. Here, we exploited the enormous potential of plant metabolism using the bryophyte Marchantia polymorpha L. and identified a potent SARS-CoV-2 antiviral, following a bioactivity-guided fractionation and mass-spectrometry approach. We found that the chlorophyll derivative Pheophorbide a (PheoA), a porphyrin compound similar to animal Protoporphyrin IX, has an extraordinary antiviral activity against SARS-CoV-2, preventing infection of cultured monkey and human cells, without noticeable cytotoxicity. We also show that PheoA targets the viral particle, interfering with its infectivity in a dose- and time-dependent manner. Besides SARS-CoV-2, PheoA also displayed a broad-spectrum antiviral activity against enveloped RNA viral pathogens such as HCV, West Nile, and other coronaviruses. Our results indicate that PheoA displays a remarkable potency and a satisfactory therapeutic index, which together with its previous use in photoactivable cancer therapy in humans, suggest that it may be considered as a potential candidate for antiviral therapy against SARS-CoV-2.

Targeting the neuronal calcium sensor DREAM with small-molecules for Huntington's disease treatment.

DREAM, a neuronal calcium sensor protein, has multiple cellular roles including the regulation of Ca2+ and protein homeostasis. We recently showed that reduced DREAM expression or blockade of DREAM activity by repaglinide is neuroprotective in Huntington's disease (HD). Here we used structure-based drug design to guide the identification of IQM-PC330, which was more potent and had longer lasting effects than repaglinide to inhibit DREAM in cellular and in vivo HD models. We disclosed and validated an unexplored ligand binding site, showing Tyr118 and Tyr130 as critical residues for binding and modulation of DREAM activity. IQM-PC330 binding de-repressed c-fos gene expression, silenced the DREAM effect on KV4.3 channel gating and blocked the ATF6/DREAM interaction. Our results validate DREAM as a valuable target and propose more effective molecules for HD treatment.

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 (I A). 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 (I A) 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 ( Q K V 4.3 / 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 I A. 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 I A in pathological processes.

Atención psicogeriátrica en tiempos de COVID. Lecciones aprendidas y propuestas para situaciones similares / Psychogeriatric care in times of Covid. Lessons learned and proposals for similar situations

Introducción: El objetivo es describir los problemas relacionados con la atención psicogeriátrica ambulatoria en elcontexto de la pandemia SARS-CoV-2, así como las soluciones propuestas e implementadas para optimizar la atencióna las personas mayores con trastornos mentales durante lapandemia, que también pueden aplicarse en situaciones similares emergentes en el futuro.Metodología. Se recogió información sobre prestaciónde asistencia sanitaria y problemas clínicos en la práctica psicogeriátrica durante un año de la pandemia de COVID-19como base para propuestas de actuación por consenso depsiquiatras expertos en psicogeriatría. Entorno: servicios deatención psicogeriátrica ambulatoria en la Comunidad deMadrid.Resultados. Se identificaron ocho temas relacionadoscon las dificultades en la prestación de la atención psicogeriátrica (acceso a los servicios, adherencia al tratamiento, derivaciones y contacto, continuidad de cuidados, aislamiento,residencias de ancianos y pruebas de laboratorio) y se llegóa un acuerdo sobre 14 posibles soluciones. Además, se identificaron 7 problemas clínicos de especial relevancia (duelo,sueño, tratamientos psicofarmacológicos, deterioros físico,cognitivo-conductual y social, y violencia) y se propusieron17 posibles soluciones.Conclusiones. La pandemia de SARS-CoV-2 supone unelevado riesgo vital para la población geriátrica. Medidascomo el confinamiento y la limitación de contactos, ejercenun riesgo directo para la salud mental y un riesgo indirectodebido a las mayores dificultades para acceder a la atenciónpsicogeriátrica. Es necesario detectar estas situaciones e implementar cambios en la manera de proporcionar atención aesta población altamente vulnerable. Proponemos una seriede posibles soluciones a las situaciones problemáticas detectadas que pueden ser de ayuda en diferentes contextos deatención psicogeriátrica. (AU)

Introduction: The objective is to describe the problemsrelated to outpatient psychogeriatric care in the contextof the SARS-CoV-2 pandemic, as well as the proposed andimplemented solutions for optimizing care for elderly peoplewith mental disorders during the pandemic, that can also beapplied in emerging similar situations in the future.Methods. Data on healthcare provision and clinical problemsin psychogeriatric practice over the course of one year ofthe COVID-19 pandemic were collected as the basis forproposals for action by a consensus of psychiatrists expertin psychogeriatrics. Setting: Outpatient psychogeriatric careservices in the Madrid region, Spain. Results. Eight topics relating to difficulties in the provisionof psychogeriatric care were identified (access to services,treatment adherence, referrals and contact, continuity ofcare, isolation, nursing homes and laboratory tests) andagreement was reached on 14 possible solutions. In addition,7 clinical problems of particular relevance were identified(bereavement, sleep, psychopharmacological treatments,physical, cognitive-behavioural and social deterioration, andviolence) and 17 possible solutions proposed.Conclusions. The SARS-CoV-2 pandemic poses a highrisk to life for the geriatric population. Measures suchas lockdowns and limiting contacts, exert a direct risk tomental health and an indirect risk due to greater difficultiesin accessing psychogeriatric care. It is necessary to detectthese situations and implement changes in how care isprovided to this highly vulnerable population. We proposea series of possible solutions to the problematic situationsdetected that may be helpful in a variety of psychogeriatriccare contexts. (AU)

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 HD.

From Multiple PAR1 Receptor/Protein Interactions to their Multiple Therapeutic Implications.

PAR1, member of the family of protease-activated receptors, is a GPCR whose activation requires a proteolytic cleavage at its extracellular N-terminus to unveil a tethered activating ligand. Although thrombin is the main activator of this receptor, diverse other proteases can also activate and disarm PAR1. Besides, tethered activating ligand-based peptides (PAR-APs) can also activate the receptor. PAR1 mainly signals via G proteins but, it can also signal using ß-arrestin pathways and by transactivation of other receptors. This complex PAR1 interactome is completed with the receptor desensitization, trafficking, and degradation. PAR1 has shown species-, cellular-, and physiological or pathological state-dependent specificity. This review try to give an overview on the complex PAR1 interactome, its therapeutic impact upon the cardiovascular, immune and nervous systems, inflammation and cancer, as well as, on its modulation with agonists and antagonists.

Synthesis, conformational analysis, and cytotoxicity of conformationally constrained aplidine and tamandarin A analogues incorporating a spirolactam beta-turn mimetic.

With the aim of studying the contribution of the beta II turn conformation at the side chain of didemnins to the bioactive conformation responsible for their antitumoral activity, conformationally restricted analogues of aplidine and tamandarin A, where the side chain dipeptide Pro8-N-Me-d-Leu7 is replaced with the spirolactam beta II turn mimetic (5R)-7-[(1R)-1-carbonyl-3-methylbutyl]-6-oxo-1,7-diazaspiro[4.4]nonane, were prepared. Additionally, restricted analogues, where the aplidine (pyruvyl9) or tamandarin A [(S)-Lac9] acyl groups are replaced with the isobutyryl, Boc, and 2-methylacryloyl groups, were also prepared. These structural modifications were detrimental to cytotoxic activity, leading to a decrease of 1-2 orders of magnitude with respect to that exhibited by aplidine and tamandarin A. The conformational analysis of one of these spirolactam aplidine analogues, by NMR and molecular modeling methods, showed that the conformational restriction caused by the spirolactam does not produce significant changes in the overall conformation of aplidine, apart from preferentially stabilizing the trans rotamer at the pyruvyl9-spirolactam amide bond, whereas in aplidine both cis and trans rotamers at the pyruvyl9-Pro8 amide bond are more or less equally stabilized. These results seem to indicate a preference for the cis form at that amide bond in the bioactive conformation of aplidine. The significant influence of this cis/trans isomerism upon the cytotoxicity suggests a possible participation of a peptidylprolyl cis/trans isomerase in the mechanism of action of aplidine.

Highly functionalized 2-oxopiperazine-based peptidomimetics: an approach to PAR1 antagonists.

A series of pseudodipeptide-based chiral 1,3,4,5-tetrasubstituted-2-oxopiperazines has been designed and synthesized as potential PAR1 antagonists. These highly functionalized piperazines were synthesized from aromatic and basic amino acid derived Ψ[CH(CN)NH]pseudodipeptides through a four step pathway that involves reduction of the cyano group to build the 2-oxopiperazine ring, followed by selective functionalization at the N4-, N1-positions, and at the exocyclic moiety at position C5. This regioselective functionalization required the fine tuning of reaction conditions. All new compounds were screened as inhibitors of human platelet aggregation induced by the PAR1 agonist SFLLRN and as cytotoxic agents in human cancer cell lines. Some of the compounds displayed moderate PAR1 antagonist activity, while, others were cytotoxic at µM concentration. No correlation was observed between both types of activities.

Thrombin-activated receptors: promising targets for cancer therapy?

In addition to the key role of thrombin in blood coagulation, this multifunctional serine protease activates platelets and regulates the behavior of other cells through G-protein coupled protease activated receptors (PARs). PAR-1 is the principal thrombin-activated receptor involved in platelet aggregation and in endothelial and tumor cell proliferation. PAR-1 is overexpressed in invasive and metastatic tumors and the expression levels directly correlate with the degree of invasiveness of the cancer. In an attempt to give some insight into the perspectives of targeting PAR-1 in cancer and angiogenesis, this review provides an overview on the thrombin/PAR-1 interaction, receptor activation, signaling, desensitization and dysregulation mechanisms in relation to these diseases. A central aspect of this review is that directed to summarize the approaches that have been followed to the search of PAR-1 antagonists, illustrating with some significant examples. Attention is called to the scarce data concerning the effects of these antagonists on anticancer assay models.