RESUMO
Across all kingdoms of life, cells store energy in a specialized organelle, the lipid droplet. In general, it consists of a hydrophobic core of triglycerides and steryl esters surrounded by only one leaflet derived from the endoplasmic reticulum membrane to which a specific set of proteins is bound. We have chosen the unicellular organism Dictyostelium discoideum to establish kinetics of lipid droplet formation and degradation and to further identify the lipid constituents and proteins of lipid droplets. Here, we show that the lipid composition is similar to what is found in mammalian lipid droplets. In addition, phospholipids preferentially consist of mainly saturated fatty acids, whereas neutral lipids are enriched in unsaturated fatty acids. Among the novel protein components are LdpA, a protein specific to Dictyostelium, and Net4, which has strong homologies to mammalian DUF829/Tmem53/NET4 that was previously only known as a constituent of the mammalian nuclear envelope. The proteins analyzed so far appear to move from the endoplasmic reticulum to the lipid droplets, supporting the concept that lipid droplets are formed on this membrane.
Assuntos
Dictyostelium/metabolismo , Fosfolipídeos/metabolismo , Proteínas de Protozoários/metabolismo , Sequência de Aminoácidos , Retículo Endoplasmático/metabolismo , Dados de Sequência Molecular , Fosfolipídeos/química , Transporte Proteico , Proteínas de Protozoários/químicaRESUMO
LYS006 is a novel, highly potent and selective, new-generation leukotriene A4 hydrolase (LTA4H) inhibitor in clinical development for the treatment of neutrophil-driven inflammatory diseases. We describe the complex pharmacokinetic to pharmacodynamic (PD) relationship in blood, plasma, and skin of LYS006-treated nonclinical species and healthy human participants. In a randomized first in human study, participants were exposed to single ascending doses up to 100 mg and multiple ascending doses up to 80 mg b.i.d.. LYS006 showed rapid absorption, overall dose proportional plasma exposure and nonlinear blood to plasma distribution caused by saturable target binding. The compound efficiently inhibited LTB4 production in human blood and skin blister cells, leading to greater than 90% predose target inhibition from day 1 after treatment initiation at doses of 20 mg b.i.d. and above. Slow re-distribution from target expressing cells resulted in a long terminal half-life and a long-lasting PD effect in ex vivo stimulated blood and skin cells despite low plasma exposures. LYS006 was well-tolerated and demonstrated a favorable safety profile up to highest doses tested, without any dose-limiting toxicity. This supported further clinical development in phase II studies in predominantly neutrophil-driven inflammatory conditions, such as hidradenitis suppurativa, inflammatory acne, and ulcerative colitis.
Assuntos
Epóxido Hidrolases , Plasma , Humanos , Neutrófilos , PeleRESUMO
IntroductionLeukotriene A4 hydrolase (LTA4H) is the final and rate limiting enzyme regulating the biosynthesis of leukotriene B4 (LTB4), a pro-inflammatory lipid mediator implicated in a large number of inflammatory pathologies. Inhibition of LTA4H not only prevents LTB4 biosynthesis but also induces a lipid mediator class-switch within the 5-lipoxygenase pathway, elevating biosynthesis of the anti-inflammatory lipid mediator Lipoxin A4. Ample preclinical evidence advocates LTA4H as attractive drug target for the treatment of chronic inflammatory diseases.Areas coveredThis review covers details about the biochemistry of LTA4H and describes its role in regulating pro- and anti-inflammatory mediator generation. It summarizes recent efforts in medicinal chemistry toward novel LTA4H inhibitors, recent clinical trials testing LTA4H inhibitors in pulmonary inflammatory diseases, and potential reasons for the discontinuation of former development programs.Expert opinionGiven the prominent role of LTB4 in initiating and perpetuating inflammation, LTA4H remains an appealing drug target. The reason former attempts targeting this enzyme have not met with success in the clinic can be attributed to compound-specific liabilities of first-generation inhibitors and/or choice of target indications to test this mode of action. A new generation of highly potent and selective LTA4H inhibitors is currently undergoing clinical testing in indications with a strong link to LTB4 biology.
Assuntos
Inibidores Enzimáticos , Epóxido Hidrolases , Anti-Inflamatórios/farmacologia , Descoberta de Drogas , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/metabolismo , HumanosRESUMO
RATIONAL: Homeostasis of vascular barriers depends upon sphingosine 1-phosphate (S1P) signaling via the S1P1 receptor. Accordingly, S1P1 competitive antagonism is known to reduce vascular barrier integrity with still unclear pathophysiological consequences. This was explored in the present study using NIBR-0213, a potent and selective S1P1 competitive antagonist. RESULTS: NIBR-0213 was tolerated at the efficacious oral dose of 30 mg/kg BID in the rat adjuvant-induced arthritis (AiA) model, with no sign of labored breathing. However, it induced dose-dependent acute vascular pulmonary leakage and pleural effusion that fully resolved within 3-4 days, as evidenced by MRI monitoring. At the supra-maximal oral dose of 300 mg/kg QD, NIBR-0213 impaired lung function (with increased breathing rate and reduced tidal volume) within the first 24 hrs. Two weeks of NIBR-0213 oral dosing at 30, 100 and 300 mg/kg QD induced moderate pulmonary changes, characterized by alveolar wall thickening, macrophage accumulation, fibrosis, micro-hemorrhage, edema and necrosis. In addition to this picture of chronic inflammation, perivascular edema and myofiber degeneration observed in the heart were also indicative of vascular leakage and its consequences. CONCLUSIONS: Overall, these observations suggest that, in the rat, the lung is the main target organ for the S1P1 competitive antagonism-induced acute vascular leakage, which appears first as transient and asymptomatic but could lead, upon chronic dosing, to lung remodeling with functional impairments. Hence, this not only raises the question of organ specificity in the homeostasis of vascular barriers, but also provides insight into the pre-clinical evaluation of a potential safety window for S1P1 competitive antagonists as drug candidates.
Assuntos
Compostos de Anilina/farmacologia , Artrite Experimental/fisiopatologia , Permeabilidade Capilar/efeitos dos fármacos , Dipeptídeos/farmacologia , Inflamação/fisiopatologia , Lisofosfolipídeos/metabolismo , Receptores de Lisoesfingolipídeo/antagonistas & inibidores , Esfingosina/análogos & derivados , Adjuvantes Imunológicos/toxicidade , Animais , Artrite Experimental/induzido quimicamente , Artrite Experimental/tratamento farmacológico , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/patologia , Homeostase/efeitos dos fármacos , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Pulmão/efeitos dos fármacos , Pulmão/patologia , Masculino , Ratos , Ratos Endogâmicos Lew , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Esfingosina/metabolismoRESUMO
Long-chain fatty-acyl-coenzyme A synthetases activate fatty acids for anabolic or catabolic metabolism. They often localize to more than one organelle within eukaryotic cells. Dictyostelium contains two of these proteins, FcsA and FcsB with the latter being targeted to the membrane of the endoplasmic reticulum by virtue of an N-terminal signal sequence and from there appears to move on to peroxisomes. Deletion of this signal favors the peripheral association of the protein with the mitochondrial surface instead. A strain lacking the activity of the FcsB enzyme was constructed by homologous recombination. It has a severe deficiency in the phagocytic uptake of particles, which can be partially alleviated by a peroxisomally targeted, soluble FcsA enzyme. It is, however, not rescued by expressing FcsA in the cytoplasm or targeting it to the ER, indicating that peroxisomal ß-oxidation is important for phagocytosis. In a fcsA(-)/B(-) double mutant phagocytosis efficiency is similar to fcsB(-) cells. However, unlike the single mutants, the fcsA(-)/B(-) strain is delayed in morphogenesis, but forms viable spores, albeit within a small fruiting body. This developmental defect is also seen in other mutants affecting peroxisomal enzymes involved in ß-oxidation and the glyoxylate cycle.
Assuntos
Coenzima A Ligases/metabolismo , Dictyostelium/metabolismo , Retículo Endoplasmático/enzimologia , Peroxissomos/enzimologia , Fagocitose , Animais , Retículo Endoplasmático/metabolismo , Isoenzimas/metabolismo , Peroxissomos/metabolismoRESUMO
Lysozymes are bacteria-degrading enzymes and play a major role in the immune defense of animals. In free-living protozoa, lysozyme-like proteins are involved in the digestion of phagocytosed bacteria. Here, we purified a protein with lysozyme activity from Dictyostelium amoebae, which constitutes the founding member, a novel class of lysozymes. By tagging the protein with green fluorescent protein or the Myc epitope, a new type of lysozyme-containing vesicle was identified that was devoid of other known lysosomal enzymes. The most highly expressed isoform, encoded by the alyA gene, was knocked out by homologous recombination. The mutant cells had greatly reduced enzymatic activity and grew inefficiently when bacteria were the sole food source. Over time the mutant gained the ability to internalize bacteria more efficiently, so that the defect in digestion was compensated by increased uptake of food particles.