Inhibitors of the Plasminogen/Plasmin system for the treatment of Traumatic Brain Injury

La plasmina és un enzim proteolític responsable de la degradació de la fibrina, la proteïna estructural dels coàguls sanguinis. És activada a partir del zimogen plasminogen per l’activador tissular del plasminogen (tPA). La degradació de fibrina, o fibrinòlisi, és un procés natural que actua com a mecanisme de control per la hemostàsia i ajuda a mantenir la adequada fluïdesa de la sang. No obstant, hi ha situacions en que la fibrinòlisi es veu afectada, el que pot resultar en un sagnat excessiu, a més d’altres complicacions. Durant un traumatisme cerebral, es pot produir una activació excessiva de plasmina, que està correlacionada amb un increment en la permeabilitat de la barrera Hematoencefàlica (BHE). La inhibició de la plasmina o de la seva activació és per tant una estratègia interessant per minimitzar els danys a la BHE sota aquestes condicions hiperfibrinolítiques. Un nou compost anomenat LTI-6 amb elevada activitat antifibrinolítica va ser descobert en col·laboració amb l’empresa Alxerion Biotech. Aquest compost combina una piperidina, un 1,2,3-triazol i una 1,3,4- oxadiazolona. Els assaigs en sang i plasma van confirmar la seva activitat antifibrinolítica, amb una potència equivalent a l’àcid tranexàmic (TXA). Després d’estudiar la seva afinitat envers possibles receptors biològics, el mecanisme d’acció proposat per LTI-6 és la interacció amb els centres d’unió a lisina presents al plasminogen. Després de proposar diverses modificacions estructurals, un total de deu nous compostos vas ser sintetitzats i estudiats tant in vitro com in silico. La substitució de piperidina per amines lineals va reduir l’activitat global de la molècula. Per contra, substituir la 1,3,4-oxadiazolona per una 1,2,4-oxadiazolona va millorar l’activitat degut a un major nombre de ponts d’hidrogen. A més, modificar el 1,2,3-triazol per un 1,2,4-triazol va produir molècules sense activitat mesurable. Els compostos amb major activitat antifibrinolítica van ser estudiats en un model in vitro de la BHE sota condicions hiperfibrinolítiques. Concentracions elevades de plasminogen i tPA van incrementar la permeabilitat, a més de reduir l’expressió de proteïnes d’unió estreta. La presència dels nous compostos va demostrar tenir un efecte protector, ja que va esmorteir parcialment el dany sobre la BHE. Aquest treball obra la porta a desenvolupar futurs fàrmacs basats en aquests derivats de 1,2,3-triazol per a dues possibles aplicacions: com a nou fàrmac antifibrinolític que substitueixi al TXA, i com a agent protector de la BHE durant un traumatisme cerebral.La plasmina es una encima proteolítica responsable de la degradación de la fibrina, la proteína estructural de los coágulos sanguíneos. Es activada a partir del zimógeno plasminógeno por el activador de tisular del plasminógeno (tPA). La degradación de fibrina, o fibrinolisis, es un proceso natural que actúa como mecanismo de control para la hemostasis y ayuda a mantener la adecuada fluidez de la sangre. Sin embargo, hay situaciones en que la fibrinolisis se ve alterada, lo que puede resultar en excesivo sangrado y otras complicaciones. Durante un traumatismo cerebral, se puede producir una excesiva activación de plasmina que está correlacionada con un incremento en la permeabilidad de la barrera hematoencefálica (BHE). La inhibición de la plasmina o de su activación es en consecuencia una estrategia interesante para minimizar los daños a la BHE bajo dichas condiciones hiperfibrinolíticas. Un nuevo compuesto llamado LTI-6 con alta actividad antifibrinolítica fue descubierto en colaboración con la empresa Alxerion Biotech. Este compuesto combina una piperidina, un 1,2,3-triazol y una 1,3,4- oxadiazolona. Ensayos en sangre y en plasma confirmaron su actividad antifibrinolítica, con una potencia equivalente al ácido tranexámico (TXA). Tras estudiar su afinidad hacia posibles receptores biológicos, el mecanismo de acción propuesto para LTI-6 es la interacción con los centros de unión a lisina presentes en el plasminógeno. Tras propuesta de varias modificaciones estructurales, un total de diez nuevos compuestos fueron sintetizados i estudiados tanto in vitro como in silico. Sustituir la piperidina por aminas lineales disminuyó la actividad global. Por el contrario, la sustitución de la 1,3,4-oxadiazolona por 1,2,4-oxadiazolona mejoró la actividad debido a un mayor número de puentes de hidrógeno. Además, modificar el 1,2,3-triazol por un 1,2,4-triazol generó moléculas sin actividad detectable. Los compuestos con mayor actividad antifibrinolítica fueron estudiados en un modelo in vitro de BHE bajo condiciones hiperfibrinolíticas. Concentraciones elevadas de plasminógeno y tPA incrementaron la permeabilidad, además de reducir la expresión de proteínas de unión estrecha. La presencia de los nuevos compuestos demostró tener un efecto protector, ya que amortiguó parcialmente el daño a la BHE. Este trabajo abre la puerta a desarrollar futuros fármacos basados en estos derivados de 1,2,3-triazol para dos aplicaciones: como nuevo compuesto antifibrinolítico que sustituta al TXA, y como agente protector de la BHE durante un traumatismo cerebral.Plasmin is a proteolytic enzyme responsible for the degradation of fibrin, the structural protein of blood clots. It is activated from the zymogen plasminogen by tissue plasminogen activator (tPA). The degradation of fibrin, or fibrinolysis, is a natural process which constitutes a control mechanism for hemostasis and helps to ensure blood fluidity. However, there are situations in which the fibrinolytic system is dysregulated, which can result into excessive blood loss and other complications. During traumatic brain injury (TBI), an excessive activation of plasmin has been linked to an increased permeability of the blood-brain barrier (BBB). The inhibition of plasmin or plasmin activation is therefore an interesting strategy to minimize the damage to the BBB under these hyperfibrinolytic conditions during TBI. A novel compound named LTI-6 with high antifibrinolytic activity was discovered in collaboration with the start-up company Alxerion Biotech. This compound combined a piperidine, a 1,2,3-triazole and a 1,3,4- oxadiazolone ring. Blood and plasma assays confirmed its antifibrinolytic activity, with an equivalent potency to the gold standard tranexamic acid (TXA). After studying affinity towards different possible targets, the proposed mechanism of action for LTI-6 is an interaction with the lysine binding sites of plasminogen, hindering its activation into plasmin. After proposing different chemical modifications, a total of ten new compounds were synthesized and studied both in vitro and in silico. Substitution of the piperidine for linear amines hindered the overall activity. In contrast, substituting the 1,3,4-oxadiazolone for a 1,2,4-oxadiazole improved the activity due to a higher number of H-bonds. In addition, modifying the 1,2,3-triazole for a 1,2,4-triazole provided molecules with no detectable activity. The compounds with higher antifibrinolytic activity were studied in a hyperfibrinolytic BBB in vitro model. High concentrations of plasminogen and tPA caused an increased permeability, as well as a reduction of tight junction protein expression. The presence of the new compounds proved to have a protective effect, partially reducing the damage to the BBB. This work opens the door to develop future drugs based on these 1,2,3-triazole derivatives for two purposes: as a novel antifibrinolytic agent to substitute TXA, and as a potential BBB protective agent during TBI

In silico strategies on prion pathogenic conversion and inhibition from PrPC -PrPSc

Published ArticleTo date, various therapeutic strategies identified numerous anti-prion compounds and antibodies that stabilize PrPC, block the conversion of PrPC-PrPSc and increased effect on PrPSc clearance. However, no suitable drug has been identified clinically so far due to the poor oral absorption, low blood-brain-barrier [BBB] penetration, and high toxicity. Although some of the drugs were proven to be effective in prion-infected cell culture and whole animal models, none of them increased the rate of survival compared to placebo. Areas covered: In this review, the authors highlight the importance of in silico approaches like molecular docking, virtual screening, pharmacophore analysis, molecular dynamics, QSAR, CoMFA and CoMSIA applied to detect molecular mechanisms of prion inhibition and conversion from PrPC-PrPSc. Expert opinion: Several in silico approaches combined with experimental studies have provided many structural and functional clues on the stability and physiological activity of prion mutants. Further, various studies of in silico and in vivo approaches were also shown to identify several new small organic anti-scrapie compounds to decrease the accumulation of PrPres in cell culture, inhibit the aggregation of a PrPC peptide, and possess pharmacokinetic characteristics that confirm the drug-likeness of these compounds

Computational Approaches For Designing Protein/inhibitor Complexes And Membrane Protein Variants

Drug discovery of small-molecule protein inhibitors is a vast enterprise that involves several scientific disciplines (i.e. genomics, cell biology, x-ray crystallography, chemistry, computer science, statistics), with each discipline focusing on a particular aspect of the process. In this thesis, I use computational and experimental approaches to explore the most fundamental aspect of drug discovery: the molecular interactions of small-molecules inhibitors with proteins. In Part I (Chapters I and II), I describe how computational docking approaches can be used to identify structurally diverse molecules that can inhibit multiple protein targets in the brain. I illustrate this approach using the examples of microtubule-stabilizing agents and inhibitors of cyclooxygenase(COX)-I and 5-lipoxygenase (5-LOX). In Part II (Chapters III and IV), I focus on membrane proteins, which are notoriously difficult to work with due to their low natural abundances, low yields for heterologous over expression, and propensities toward aggregation. I describe a general approach for designing water-soluble variants of membrane proteins, for the purpose of developing cell-free, label-free, detergent-free, solution-phase studies of protein structure and small-molecule binding. I illustrate this approach through the design of a water-soluble variant of the membrane protein Smoothened, wsSMO. This wsSMO stands to serve as a first-step towards developing membrane protein analogs of this important signaling protein and drug target

Cholinesterase Research

This collection of 10 papers includes original as well as review articles focused on the cholinesterase structural aspects, drug design and development of novel cholinesterase ligands, but also contains papers focused on the natural compounds and their effect on the cholinergic system and unexplored effects of donepezil

Binding free energies of small-molecules in phospholipid membranes: aminoacids, serotonin and melatonin

Free energy barriers associated to the binding of small-molecules at phospholipid zwitterionic membranes have been computed at 323 K for a variety of species: tryptophan, histidine, tyrosine, serotonin and melatonin bound to a model membrane formed by di-palmitoyl-phosphatidyl-choline lipids inside aqueous sodium chloride solution. We have computed the radial distribution functions of all species for a variety of membrane and water related sites and extracted potentials of mean force through the reversible work theorem. In all cases but histidine, the molecular probes are able to either be fully solvated by water or be embedded into the interface of the membrane. Our results indicate that binding of all species to water corresponds to free energy barriers of heights between 0.2 and 1.75 kcal/mol. Free energy barriers of association of small-molecules to lipid chains range between 0.6 and 3.1 kcal/mol and show different characteristics: all species but histidine are most likely bound to oxygens belonging to the phosphate and to the glycerol groups. Histidine shows a clear preference to be fully solvated by water whereas the aqueous solvation of serotonin is the less likely case of them all. No free permeation through the membrane of any small-molecule has been observed during the time span of the simulation experiments.Preprin