RESUMO
BACKGROUND: 5-LO is an important enzyme involved in the biosynthesis of leukotrienes, which are lipid mediators of immune and inflammation responses, with important roles in respiratory disease, cardiovascular disease, immune responses and certain types of cancer. Therefore, this enzyme has been investigated as a potential target for the treatment of these pathophysiological conditions. RESULTS: 5-LO inhibitory potential was investigated in intact polymorphonuclear leukocytes, a cell-free assay, in human whole blood and rodent cells to both elucidate structure-activity relationships and in vitro pharmacological evaluation. Chemical modifications for lead optimization via straight forward synthesis was used to combine small polar groups, which led to a suitable candidate (IC50 [polymorphonuclear leukocytes] = 1.15 µM, IC50 [S100] = 0.29 µM) with desired in vitro biopharmaceutical profiles in terms of solubility (451.9 µg/ml) and intrinsic clearance without demonstrating any cytotoxicity. CONCLUSION: Compound 9l is a novel, potent and selective 5-LO inhibitor with favorable preclinical drug-like properties.
Assuntos
Araquidonato 5-Lipoxigenase/química , Imidazóis/química , Inibidores de Lipoxigenase/química , Piridinas/química , Animais , Araquidonato 5-Lipoxigenase/metabolismo , Linhagem Celular , Análise Mutacional de DNA , Avaliação Pré-Clínica de Medicamentos , Humanos , Imidazóis/toxicidade , Inibidores de Lipoxigenase/síntese química , Inibidores de Lipoxigenase/toxicidade , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/metabolismo , Piridinas/síntese química , Piridinas/toxicidade , Salmonella/efeitos dos fármacos , Salmonella/genética , Solubilidade , Relação Estrutura-AtividadeRESUMO
Current research leads to the assumption that drugs affecting more than one target could result in a more efficient treatment of diseases and fewer safety concerns. Administration of drugs inhibiting only one branch of the arachidonic acid cascade is usually accompanied by side effects. We therefore designed and synthesized a library of hybrid molecules incorporating an imidazo[1,2-a]pyridine and an urea moiety as novel soluble epoxide hydrolase (sEH)/5-lipoxygenase (5-LO) dual inhibitors. Evaluation of the compounds was accomplished by in vitro testing using recombinant enzyme assays.
Assuntos
Araquidonato 5-Lipoxigenase/química , Epóxido Hidrolases/antagonistas & inibidores , Imidazóis/síntese química , Inibidores de Lipoxigenase/síntese química , Piridinas/síntese química , Ureia/análogos & derivados , Ureia/síntese química , Epóxido Hidrolases/química , Humanos , Imidazóis/química , Inibidores de Lipoxigenase/química , Piridinas/química , Proteínas Recombinantes/química , Relação Estrutura-Atividade , Ureia/químicaRESUMO
A novel class of 5-lipoxygenase (5-LO) inhibitors characterized by a central imidazo[1,2-a]pyridine scaffold, a cyclohexyl moiety and an aromatic system, is presented. This scaffold was identified in a virtual screening study and exhibits promising inhibitory potential on the 5-LO. Here, we investigate the structure-activity relationships of this compound class. With N-cyclohexyl-6-methyl-2-(4-morpholinophenyl)imidazo[1,2-a]pyridine-3-amine (14), we identified a potent 5-LO inhibitor (IC(50)=0.16µM (intact cells) and 0.1µM (cell-free)), which may possess potential as an effective lead compound intervening with inflammatory diseases and certain types of cancer.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/farmacologia , Piridinas/química , Piridinas/farmacologia , Anti-Inflamatórios/síntese química , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/enzimologia , Inibidores de Lipoxigenase/síntese química , Piridinas/síntese química , Relação Estrutura-AtividadeRESUMO
Dual-target inhibitors gained increased attention in the past years. A novel in silico approach was employed for the discovery of dual 5-lipoxygenase/soluble epoxide hydrolase inhibitors. The ligand-based approach uses excessive pharmacophore elucidation and pharmacophore alignment in conjunction with shape-based scoring. The virtual screening results were verified in vitro, leading to nine novel inhibitors including a dual-target compound.
RESUMO
5-Lipoxygenase (5-LO) is a crucial enzyme of the arachidonic acid (AA) cascade and catalyzes the formation of bioactive leukotrienes (LTs) which are involved in inflammatory diseases and allergic reactions. The pathophysiological effects of LTs are considered to be prevented by 5-LO inhibitors. In this study we present cyclohexyl-[6-methyl-2-(4-morpholin-4-yl-phenyl)-imidazo[1,2-a]pyridin-3-yl]-amine (EP6), a novel imidazo[1,2-a]pyridine based compound and its characterization in several in vitro assays. EP6 suppresses 5-LO activity in intact polymorphonuclear leukocytes with an IC(50) value of 0.16µM and exhibits full inhibitory potency in cell free assays (IC(50) value of 0.05µM for purified 5-LO). The efficacy of EP6 was not affected by the redox tone or the concentration of exogenous AA, characteristic drawbacks known for the class of nonredox-type 5-LO inhibitors. Furthermore, EP6 suppressed 5-LO activity independently of the cell stimulus or the activation pathway of 5-LO contrary to what is known for some nonredox-type inhibitors. Using molecular modeling and site-directed mutagenesis studies, we were able to derive a feasible binding region within the C2-like domain of 5-LO that can serve as a new starting point for optimization and development of new 5-LO inhibitors targeting this site. EP6 has promising effects on cell viability of tumor cells without mutagenic activity. Hence the drug may possess potential for intervention with inflammatory and allergic diseases and certain types of cancer including leukemia.
Assuntos
Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/metabolismo , Piridinas/química , Piridinas/metabolismo , Regulação Alostérica/efeitos dos fármacos , Animais , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Células HeLa , Humanos , Imidazóis/química , Imidazóis/metabolismo , Inibidores de Lipoxigenase/farmacologia , Camundongos , Oxirredução/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Piridinas/farmacologia , Ovinos , Células U937RESUMO
U73122 which was originally identified as a phospholipase C inhibitor represents a potent direct inhibitor of purified 5-lipoxygenase (5-LO) with an IC50 value of 30 nM. 5-LO catalyzes the conversion of arachidonic acid (AA) into leukotrienes which represent mediators involved in inflammatory and allergic reactions and in host defense reactions against microorganisms. Since the efficient inhibition of the human 5-LO enzyme depended on the thiol reactivity of the maleinimide group of U73122, we used this property to identify cysteine residues in the 5-LO protein that are important for 5-LO inhibition by U73122. We found by MALDI-MS that U73122 covalently binds to cysteine residues 99, 159, 248, 264, 416 and 449. Mutation of Cys416 to serine strongly reduces inhibition of 5-LO by U73122 and the additional mutation of three cysteines close to Cys416 further impairs 5-LO inhibition by the compound. Wash out experiments with U73122 and 5-LO indicated an irreversible binding of U73122. Together, our data suggest that the area around Cys416 which is close to the proposed AA entry channel to the active site is an interesting target for the development of new 5-LO inhibitors.
Assuntos
Araquidonato 5-Lipoxigenase/metabolismo , Cisteína/metabolismo , Estrenos/farmacologia , Pirrolidinonas/farmacologia , Adulto , Animais , Araquidonato 5-Lipoxigenase/química , Ácido Araquidônico/farmacologia , Estrenos/química , Células HeLa , Humanos , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/farmacologia , Camundongos , Modelos Moleculares , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/metabolismo , Ligação Proteica/efeitos dos fármacos , Estrutura Secundária de Proteína , Pirrolidinonas/química , Homologia de Sequência de Aminoácidos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Compostos de Sulfidrila/metabolismoAssuntos
Araquidonato 5-Lipoxigenase/metabolismo , Desenho de Fármacos , Inibidores de Lipoxigenase/química , Inibidores de Lipoxigenase/farmacologia , Linhagem Celular Tumoral , Humanos , L-Lactato Desidrogenase/metabolismo , Modelos Moleculares , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologiaRESUMO
Experimental results are presented for 180 in silico designed octapeptide sequences and their stabilizing effects on the major histocompatibility class I molecule H-2K(b). Peptide sequence design was accomplished by a combination of an ant colony optimization algorithm with artificial neural network classifiers. Experimental tests yielded nine H-2K(b) stabilizing and 171 nonstabilizing peptides. 28 among the nonstabilizing octapeptides contain canonical motif residues known to be favorable for MHC I stabilization. For characterization of the area covered by stabilizing and non-stabilizing octapeptides in sequence space, we visualized the distribution of 100,603 octapeptides using a self-organizing map. The experimental results present evidence that the canonical sequence motives of the SYFPEITHI database on their own are insufficient for predicting MHC I protein stabilization.
RESUMO
Sulindac is a non-selective inhibitor of cyclooxygenases (COX) used to treat inflammation and pain. Additionally, non-COX targets may account for the drug's chemo-preventive efficacy against colorectal cancer and reduced gastrointestinal toxicity. Here, we demonstrate that the pharmacologically active metabolite of sulindac, sulindac sulfide (SSi), targets 5-lipoxygenase (5-LO), the key enzyme in the biosynthesis of proinflammatory leukotrienes (LTs). SSi inhibited 5-LO in ionophore A23187- and LPS/fMLP-stimulated human polymorphonuclear leukocytes (IC(50) approximately 8-10 microM). Importantly, SSi efficiently suppressed 5-LO in human whole blood at clinically relevant plasma levels (IC(50) = 18.7 microM). SSi was 5-LO-selective as no inhibition of related lipoxygenases (12-LO, 15-LO) was observed. The sulindac prodrug and the other metabolite, sulindac sulfone (SSo), failed to inhibit 5-LO. Mechanistic analysis demonstrated that SSi directly suppresses 5-LO with an IC(50) of 20 muM. Together, these findings may provide a novel molecular basis to explain the COX-independent pharmacological effects of sulindac under therapy.
Assuntos
Anti-Inflamatórios/farmacologia , Inibidores de Lipoxigenase , Sulindaco/análogos & derivados , Proteínas Ativadoras de 5-Lipoxigenase , Anti-Inflamatórios/uso terapêutico , Araquidonato 5-Lipoxigenase/metabolismo , Sangue/efeitos dos fármacos , Sangue/metabolismo , Proteínas de Transporte/antagonistas & inibidores , Proteínas de Transporte/metabolismo , Sistema Livre de Células/efeitos dos fármacos , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos , Humanos , Inibidores de Lipoxigenase/farmacologia , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Modelos Biológicos , Neutrófilos/efeitos dos fármacos , Neutrófilos/enzimologia , Neutrófilos/metabolismo , Concentração Osmolar , Transporte Proteico/efeitos dos fármacos , Sulindaco/farmacologia , Sulindaco/uso terapêuticoRESUMO
Ant Colony Optimization (ACO) is a meta-heuristic that utilizes a computational analogue of ant trail pheromones to solve combinatorial optimization problems. The size of the ant colony and the representation of the ants' pheromone trails is unique referring to the given optimization problem. In the present study, we employed ACO to generate novel peptides that stabilize MHC I protein on the plasma membrane of a murine lymphoma cell line. A jury of feedforward neural network classifiers served as fitness function for peptide design by ACO. Bioactive murine MHC I H-2K(b) stabilizing as well as nonstabilizing octapeptides were designed, synthesized and tested. These peptides reveal residue motifs that are relevant for MHC I receptor binding. We demonstrate how the performance of the implemented ACO algorithm depends on the colony size and the size of the search space. The actual peptide design process by ACO constitutes a search path in sequence space that can be visualized as trajectories on a self-organizing map (SOM). By projecting the sequence space on a SOM we visualize the convergence of the different solutions that emerge during the optimization process in sequence space. The SOM representation reveals attractors in sequence space for MHC I binding peptides. The combination of ACO and SOM enables systematic peptide optimization. This technique allows for the rational design of various types of bioactive peptides with minimal experimental effort. Here, we demonstrate its successful application to the design of MHC-I binding and nonbinding peptides which exhibit substantial bioactivity in a cell-based assay.