Sondas químicas para el estudio del sistema endocannabinoide endógeno

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Ciencias Químicas, Departamento de Química Orgánica I, leída el 27-10-2015La química biológica nació hace dos décadas con objeto de estudiar la interfase entre la química y la biología, utilizando para ello herramientas capaces de interrogar los distintos sistemas biológicos, facilitando así la comprensión de los mismos.1-3 Sin embargo, existen sistemas biológicos de gran relevancia cuyo estudio no ha sido abordado hasta ahora. Uno de ellos es el sistema cannabinoide endógeno (endogenous cannabinoid system, ECS), que durante los últimos años ha sido relacionado con diversas funciones fisiológicas a nivel tanto central como periférico.4-7 Sin embargo, no existe ningún fármaco en el mercado a día de hoy dirigido a la modulación de este sistema. En este contexto, una mayor comprensión de algunos de los aspectos de la biología del ECS, tales como la localización de los receptores de cannabinoides (cannabinoid receptors, CBRs) a nivel celular, su localización a nivel subcelular, la existencia de otras dianas distintas de los receptores conocidos y molecularmente caracterizados CB1R y CB2R, y el estudio de otros ligandos de los CBRs, podrían permitir una aplicación terapéutica real de este sistema. Así, los objetivos generales del presente trabajo de investigación son: i) el desarrollo de sondas para el estudio de la expresión de los CBRs en el sistema inmune, ii) el diseño y síntesis de herramientas para explorar las diferencias entre el CB1R de la membrana plasmática y el mitocondrial, iii) el descubrimiento de las dianas adicionales de los agonistas sintéticos HU210 y HU308, y iv) la identificación de las dianas terapéuticas del producto de origen natural honokiol...Chemical biology emerged two decades ago aimed at the study of the interface between chemistry and biology, producing a great improvement in our understanding of complex biological systems by means of the development of chemical tools able to interrogate them.1-3 In spite of these advances, there are relevant systems that hold great therapeutic promise but which have not been studied up to date. One of these cases is the endogenous cannabinoid system (ECS), which has been involved in almost every central and peripheral function.4-7 However, there is no drug in the market targeting this system so far. In this context, it seems clear that there is a crucial need to understand some aspects of the biology of the ECS in a deeper manner. Among them, the cellular location of cannabinoid receptors (CBRs), as well as their localization within the cell, the existence of other cannabinoid targets different from the molecularly characterized CB1R and CB2R, and the study of interesting cannabinoid-related ligands, might help to exploit the therapeutic potential of the ECS. Thus, the overall objectives for the present work are: i) the development of probes to study the expression of CBRs in the immune system, ii) the design and synthesis of tools to explore the differences between plasma and mitochondrial CB1Rs, iii) the discovery of the off-targets of the synthetic CBR agonists HU210 and HU308, and iv) the identification of the binding proteins of the natural product honokiol...Depto. de Química OrgánicaFac. de Ciencias QuímicasTRUEunpu

The antimalarial screening landscape-looking beyond the asexual blood stage.

In recent years, the research agenda to tackle global morbidity and mortality from malaria disease has shifted towards innovation, in the hope that efforts at the frontiers of scientific research may re-invigorate gains made towards eradication. Discovery of new antimalarial drugs with novel chemotypes or modes of action lie at the heart of these efforts. There is a particular interest in drug candidates that target stages of the malaria parasite lifecycle beyond the symptomatic asexual blood stages. This is especially important given the spectre of emerging drug resistance to all current frontline antimalarials. One approach gaining increased interest is the potential of designing novel drugs that target parasite passage from infected individual to feeding mosquito and back again. Action of such therapeutics is geared much more at the population level rather than just concerned with the infected individual. The search for novel drugs active against these stages has been helped by improvements to in vitro culture of transmission and pre-erythrocytic parasite lifecycle stages, robotic automation and high content imaging, methodologies that permit the high-throughput screening (HTS) of compound libraries for drug discovery. Here, we review recent advances in the antimalarial screening landscape, focussed on transmission blocking as a key aim for drug-treatment campaigns of the future

Structure-Activity Relationship Studies of a Novel Class of Transmission Blocking Antimalarials Targeting Male Gametes.

Malaria is still a leading cause of mortality among children in the developing world, and despite the immense progress made in reducing the global burden, further efforts are needed if eradication is to be achieved. In this context, targeting transmission is widely recognized as a necessary intervention toward that goal. After carrying out a screen to discover new transmission-blocking agents, herein we report our medicinal chemistry efforts to study the potential of the most robust hit, DDD01035881, as a male-gamete targeted compound. We reveal key structural features for the activity of this series and identify analogues with greater potency and improved metabolic stability. We believe this study lays the groundwork for further development of this series as a transmission blocking agent

Effects of hospital facilities on patient outcomes after cancer surgery: an international, prospective, observational study

Background Early death after cancer surgery is higher in low-income and middle-income countries (LMICs) compared with in high-income countries, yet the impact of facility characteristics on early postoperative outcomes is unknown. The aim of this study was to examine the association between hospital infrastructure, resource availability, and processes on early outcomes after cancer surgery worldwide.Methods A multimethods analysis was performed as part of the GlobalSurg 3 study-a multicentre, international, prospective cohort study of patients who had surgery for breast, colorectal, or gastric cancer. The primary outcomes were 30-day mortality and 30-day major complication rates. Potentially beneficial hospital facilities were identified by variable selection to select those associated with 30-day mortality. Adjusted outcomes were determined using generalised estimating equations to account for patient characteristics and country-income group, with population stratification by hospital.Findings Between April 1, 2018, and April 23, 2019, facility-level data were collected for 9685 patients across 238 hospitals in 66 countries (91 hospitals in 20 high-income countries; 57 hospitals in 19 upper-middle-income countries; and 90 hospitals in 27 low-income to lower-middle-income countries). The availability of five hospital facilities was inversely associated with mortality: ultrasound, CT scanner, critical care unit, opioid analgesia, and oncologist. After adjustment for case-mix and country income group, hospitals with three or fewer of these facilities (62 hospitals, 1294 patients) had higher mortality compared with those with four or five (adjusted odds ratio [OR] 3.85 [95% CI 2.58-5.75]; p<0.0001), with excess mortality predominantly explained by a limited capacity to rescue following the development of major complications (63.0% vs 82.7%; OR 0.35 [0.23-0.53]; p<0.0001). Across LMICs, improvements in hospital facilities would prevent one to three deaths for every 100 patients undergoing surgery for cancer.Interpretation Hospitals with higher levels of infrastructure and resources have better outcomes after cancer surgery, independent of country income. Without urgent strengthening of hospital infrastructure and resources, the reductions in cancer-associated mortality associated with improved access will not be realised

A Fluorescent Probe to Unravel Functional Features of Cannabinoid Receptor CB1 in Human Blood and Tonsil Immune System Cells

The human endogenous cannabinoid system (ECS) regulates key physiological processes and alterations in its signaling pathways, and endocannabinoid levels are associated with diseases such as neurological and neuropsychiatric conditions, cancer, pain and inflammation, obesity, and metabolic and different immune related disorders. Immune system cells express the G-protein coupled cannabinoid receptor 1 (CB1), but its functional role has not been fully understood, likely due to the lack of appropriate tools. The availability of novel tools to investigate the role of CB1 in immune regulation might contribute to identify CB1 as a potential novel therapeutic target or biomarker for many diseases. Herein, we report the development and validation of the first fluorescent small molecule probe to directly visualize and quantify CB1 in blood and tonsil immune cells by flow cytometry and confocal microscopy. We coupled the cannabinoid agonist HU210 to the fluorescent tag Alexa Fluor 488, generating a fluorescent probe with high affinity for CB1 and selectivity over CB2. We validate HU210-Alexa488 for the rapid, simultaneous, and reproducible identification of CB1 in human monocytes, T cells, and B cells by multiplexed flow cytometry. This probe is also suitable for the direct visualization of CB1 in tonsil tissues, allowing the in vivo identification of tonsil CB1-expressing T and B cells. This study provides the first fluorescent chemical tool to investigate CB1 expression and function in human blood and tonsil immune cells, which might well pave the way to unravel essential features of CB1 in different immune and ECS-related diseases

A novel class of sulphonamides potently block malaria transmission by targeting a Plasmodium vacuole membrane protein.

Phenotypic cell-based screens are critical tools for discovering candidate drugs for development, yet identification of the cellular target and mode of action of a candidate drug is often lacking. Using an imaging-based screen, we recently discovered an N-[(4-hydroxychroman-4-yl)methyl]-sulphonamide (N-4HCS) compound, DDD01035881, that blocks male gamete formation in the malaria parasite life cycle and subsequent transmission of the parasite to the mosquito with nanomolar activity. To identify the target(s) of DDD01035881, and of the N-4HCS class of compounds more broadly, we synthesised a photoactivatable derivative, probe 2. Photoaffinity labelling of probe 2 coupled with mass spectrometry identified the 16 kDa Plasmodium falciparum parasitophorous vacuole membrane protein Pfs16 as a potential parasite target. Complementary methods including cellular thermal shift assays confirmed that the parent molecule DDD01035881 stabilised Pfs16 in lysates from activated mature gametocytes. Combined with high-resolution, fluorescence and electron microscopy data, which demonstrated that parasites inhibited with N-4HCS compounds phenocopy the targeted deletion of Pfs16 in gametocytes, these data implicate Pfs16 as a likely target of DDD01035881. This finding establishes N-4HCS compounds as being flexible and effective starting candidates from which transmission-blocking antimalarials can be developed in the future