RESUMO
Twenty 2-(4-alkyloxyphenyl)-imidazolines and 2-(4-alkyloxyphenyl)-imidazoles were synthesized, with the former being synthesized in two steps by using MW and ultrasonication energy, resulting in good to excellent yields. Imidazoles were obtained in moderate yields by oxidizing imidazolines with MnO2 and MW energy. In response to the urgent need to treat neglected tropical diseases, a set of 2-(4-alkyloxyphenyl)- imidazolines and imidazoles was tested in vitro on Leishmania mexicana and Trypanosoma cruzi. The leishmanicidal activity of ten compounds was evaluated, showing an IC50 < 10 µg/mL. Among these compounds, 27-31 were the most active, with IC50 values < 1 µg/mL (similar to the reference drugs). In the evaluation on epimastigotes of T. cruzi, only 30 and 36 reached an IC50 < 1 µg/mL, showing better inhibition than both reference drugs. However, compounds 29, 33, and 35 also demonstrated attractive trypanocidal activities, with IC50 values < 10 µg/mL, similar to the values for benznidazole and nifurtimox.
Assuntos
Antiprotozoários , Doença de Chagas , Imidazolinas , Leishmania mexicana , Trypanosoma cruzi , Humanos , Imidazóis/farmacologia , Compostos de Manganês , Óxidos , Antiprotozoários/farmacologiaRESUMO
Renal fibrosis is a contributor to chronic kidney disease and an important predictor of long-term prognosis. We developed a dual soluble epoxide hydrolase inhibitor-PPAR-γ agonist (sEHi/PPAR-γ), RB394, and investigated its ability to attenuate renal fibrosis in a mouse unilateral ureteral obstruction (UUO) model. RB394 efficacy was compared to an sEH inhibitor (sEHi), a PPAR-γ agonist rosiglitazone (Rosi), or their combination (sEHi + Rosi). All interventional treatments were administrated in drinking water 3 days after UUO induction surgery and continued for 7 days. UUO mice developed renal fibrosis with higher collagen formation and RB394 significantly attenuated fibrosis (P < 0.05). Renal expression of α-smooth muscle actin (α-SMA) was elevated in UUO mice and all treatments except sEHi significantly attenuated renal α-SMA expression. Renal mRNA expression fibrotic and fibrosis regulators were higher in UUO mice and RB394 and sEHi + Rosi treatments attenuated their expression. Renal inflammation was evident in UUO mice with increased infiltration of CD45 and F4/80 positive cells. RB394 and sEHi + Rosi treatments attenuated renal inflammation in UUO mice. UUO mice had renal tubular and vascular injury. Renal tubular and vascular injuries were attenuated to a greater extent by RB394 and sEHi + Rosi than sEHi or Rosi treatment alone. Renal mRNA expression of oxidative stress markers were significantly higher in UUO mice (P < 0.05). RB394 and sEHi + Rosi attenuated expression of oxidative stress markers to a greater extent than other interventional treatments (P < 0.05). These findings demonstrate that RB394 can attenuate renal fibrosis by reducing renal inflammation, oxidative stress, tubular injury, and vascular injury. In conclusion, RB394 demonstrates exciting potential as a therapeutic for renal fibrosis and chronic kidney disease.
Assuntos
Epóxido Hidrolases/antagonistas & inibidores , Fibrose/prevenção & controle , Nefropatias/prevenção & controle , PPAR gama/agonistas , Obstrução Ureteral/complicações , Animais , Modelos Animais de Doenças , Fibrose/etiologia , Fibrose/metabolismo , Fibrose/patologia , Nefropatias/etiologia , Nefropatias/metabolismo , Nefropatias/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
Multitarget anti-inflammatory drugs interfering with the arachidonic acid cascade exhibit superior efficacy. In this study, a prototype dual inhibitor of soluble epoxide hydrolase (sEH) and LTA4 hydrolase (LTA4H) with submicromolar activity toward both targets has been designed and synthesized. Preliminary structure-activity relationship studies were performed to identify optimal substitution patterns. X-ray structure analysis of a promising dual inhibitor in complex with sEH, as well as molecular docking with LTA4H provided a rationale for further optimization. Hereby, scaffold extension was successfully applied to yield potent dual sEH/LTA4H inhibitors. The spectrum of pro- and anti-inflammatory lipid mediators was evaluated in M1 and M2 macrophages, stimulated with LPS, and incubated with the most promising compound 14. The effect of 14 on the inflammatory lipid mediator profile characterizes dual sEH/LTA4H inhibitors as an interesting option for future anti-inflammatory agent investigations.
RESUMO
1-(1-Propionylpiperidin-4-yl)-3-(4-(trifluoromethoxy)phenyl)urea (TPPU) is a potent soluble epoxide hydrolase (sEH) inhibitor that is used extensively in research for modulating inflammation and protecting against hypertension, neuropathic pain, and neurodegeneration. Despite its wide use in various animal disease models, the metabolism of TPPU has not been well-studied. A broader understanding of its metabolism is critical for determining contributions of metabolites to the overall safety and effectiveness of TPPU. Herein, we describe the identification of TPPU metabolites using LC-MS/MS strategies. Four metabolites of TPPU (M1-M4) were identified from rat urine by a sensitive and specific LC-MS/MS method with double precursor ion scans. Their structures were further supported by LC-MS/MS comparison with synthesized standards. Metabolites M1 and M2 were formed from hydroxylation on a propionyl group of TPPU; M3 was formed by amide hydrolysis of the 1-propionylpiperdinyl group on TPPU; and M4 was formed by further oxidation of the hydroxylated metabolite M2. Interestingly, the predicted α-keto amide metabolite and 4-(trifluoromethoxy)aniline (metabolite from urea cleavage) were not detected by the LC-MRM-MS method. This indicates that if formed, the two potential metabolites represent <0.01% of TPPU metabolism. Species differences in the formation of these four identified metabolites was assessed using liver S9 fractions from dog, monkey, rat, mouse, and human. M1, M2, and M3 were generated in liver S9 fractions from all species, and higher amounts of M3 were generated in monkey S9 fractions compared to other species. In addition, rat and human S9 metabolism showed the highest species similarity based on the quantities of each metabolite. The presence of all four metabolites were confirmed in vivo in rats over 72-h post single oral dose of TPPU. Urine and feces were major routes for TPPU excretion. M1, M4 and parent drug were detected as major substances, and M2 and M3 were minor substances. In blood, M1 accounted for ~9.6% of the total TPPU-related exposure, while metabolites M2, M3, and M4 accounted for <0.4%. All four metabolites were potent inhibitors of human sEH but were less potent than the parent TPPU. In conclusion, TPPU is metabolized via oxidation and amide hydrolysis without apparent breakdown of the urea. The aniline metabolites were not observed either in vitro or in vivo. Our findings increase the confidence in the ability to translate preclinical PK of TPPU in rats to humans and facilitates the potential clinical development of TPPU and other sEH inhibitors.
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With the increasing antibiotic resistance of bacterial strains, alternative methods for infection control are in high demand. Quorum sensing (QS) is the bacterial communication system based on small molecules. QS is enables bacterial biofilm formation and pathogenic development. The interruption of QS has become a target for drug discovery, but remains in the early experimental phase. In this study, we synthesized a set of six compounds based on a scaffold (alkyl-quinoxalin-2(1H)-one), new in the anti-QS of Gram-negative bacteria Aeromonas caviae Sch3. By quantifying biofilm formation, we were able to monitor the effect of these compounds from concentrations of 1 to 100 µM. Significant reduction in biofilm formation was achieved by 3-hexylylquinoxalin-2(1H)-one (11), 3-hexylylquinoxalin-2(1H)-one-6-carboxylic acid (12), and 3-heptylylquinoxalin-2(1H)-one-6-carboxylic acid (14), ranging from 11% to 59% inhibition of the biofilm. This pilot study contributes to the development of anti-QS compounds to overcome the clinical challenge of resistant bacteria strains.
Assuntos
Aeromonas caviae/efeitos dos fármacos , Antibacterianos/química , Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Quinoxalinas/química , Quinoxalinas/farmacologia , Percepção de Quorum/efeitos dos fármacos , Aeromonas caviae/crescimento & desenvolvimento , Antibacterianos/síntese química , Técnicas de Química Sintética , Desenho de Fármacos , Espectroscopia de Ressonância Magnética , Quinoxalinas/síntese químicaRESUMO
AIMS/HYPOTHESIS: The metabolic syndrome is a cluster of risk correlates that can progress to type 2 diabetes. The present study aims to evaluate a novel molecule with a dual action against the metabolic syndrome and type 2 diabetes. METHODS: We developed and tested a novel dual modulator, RB394, which acts as a soluble epoxide hydrolase (sEH) inhibitor and a peroxisome proliferator-activated receptor-γ (PPAR-γ) agonist in rat models of the metabolic syndrome-the obese spontaneously hypertensive (SHROB) rat and the obese diabetic Zucker fatty/spontaneously hypertensive heart failure F1 hybrid (ZSF1) rat. In SHROB rats we studied the ability of RB394 to prevent metabolic syndrome phenotypes, while in ZSF1 obese diabetic rats we compared RB394 with the ACE inhibitor enalapril in the treatment of type 2 diabetes and associated comorbid conditions. RB394 (10 mg/kg daily) and enalapril (10 mg/kg daily) were administered orally for 8 weeks. RESULTS: RB394 blunted the development of hypertension, insulin resistance, hyperlipidaemia and kidney injury in SHROB rats and reduced fasting blood glucose and HbA1c, improved glucose tolerance, reduced blood pressure and improved lipid profiles in obese ZSF1 rats. A reduction in liver fibrosis and hepatosteatosis was evident in RB394-treated obese ZSF1 rats. Unlike RB394, enalapril did not demonstrate any positive effects in relation to diabetes, hyperlipidaemia or liver dysfunction in obese ZSF1 rats. RB394 ameliorated diabetic nephropathy by reducing renal interstitial fibrosis and renal tubular and glomerular injury in obese diabetic ZSF1 rats. Intriguingly, enalapril demonstrated a weaker action against diabetic nephropathy in obese ZSF1 rats. CONCLUSIONS/INTERPRETATION: These findings demonstrate that a novel sHE inhibitor/PPAR-γ agonist molecule targets multiple risk factors of the metabolic syndrome and is a glucose-lowering agent with a strong ability to treat diabetic complications.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , PPAR gama/agonistas , Animais , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/fisiopatologia , Modelos Animais de Doenças , Enalapril/farmacologia , Fígado Gorduroso/tratamento farmacológico , Fígado Gorduroso/patologia , Teste de Tolerância a Glucose , Hipertensão/tratamento farmacológico , Resistência à Insulina , Glomérulos Renais/patologia , Cirrose Hepática/patologia , Masculino , Síndrome Metabólica/tratamento farmacológico , Camundongos , Camundongos Endogâmicos C57BL , Obesidade/fisiopatologia , Ratos , Ratos ZuckerRESUMO
Inspired by previously discovered enhanced analgesic efficacy between soluble epoxide hydrolase (sEH) and phosphodiesterase 4 (PDE4) inhibitors, we designed, synthesized and characterized 21 novel sEH/PDE4 dual inhibitors. The best of these displayed good efficacy in in vitro assays. Further pharmacokinetic studies of a subset of four selected compounds led to the identification of a bioavailable dual inhibitor N-(4-methoxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide (MPPA). In a lipopolysaccharide induced inflammatory pain rat model, MPPA rapidly increased in the blood ( Tmax = 30 min; Cmax = 460 nM) after oral administration of 3 mg/kg and reduced inflammatory pain with rapid onset of action correlating with blood levels over a time course of 4 h. Additionally, MPPA does not alter self-motivated exploration of rats with inflammatory pain or the withdrawal latency in control rats.
Assuntos
Analgésicos/uso terapêutico , Epóxido Hidrolases/antagonistas & inibidores , Dor/tratamento farmacológico , Inibidores da Fosfodiesterase 4/uso terapêutico , Piperidinas/uso terapêutico , Administração Oral , Analgésicos/administração & dosagem , Analgésicos/síntese química , Analgésicos/farmacocinética , Animais , Células HEK293 , Humanos , Inflamação/induzido quimicamente , Inflamação/complicações , Lipopolissacarídeos , Masculino , Estrutura Molecular , Dor/etiologia , Inibidores da Fosfodiesterase 4/administração & dosagem , Inibidores da Fosfodiesterase 4/síntese química , Inibidores da Fosfodiesterase 4/farmacocinética , Piperidinas/administração & dosagem , Piperidinas/síntese química , Piperidinas/farmacocinética , Ratos Sprague-DawleyRESUMO
AIMS: 5-Lipoxygenase (5-LO) is the key enzyme of leukotriene (LT) biosynthesis and is critically involved in a number of inflammatory diseases such as arthritis, gout, bronchial asthma, atherosclerosis, and cancer. Because 5-LO contains critical nucleophilic amino acids, which are sensitive to electrophilic modifications, we determined the consequences of a drug-mediated intracellular release of nitric oxide (NO) on 5-LO product formation by human granulocytes and on 5-LO-dependent pulmonary inflammation in vivo. RESULTS: Clinically relevant concentrations of NO-releasing nonsteroidal anti-inflammatory drugs and other agents releasing NO intracellularly suppress 5-LO product synthesis in isolated human granulocytes via direct S-nitrosylation of 5-LO at the catalytically important cysteines 416 and 418. Furthermore, suppression of 5-LO product formation was observed in ionophore-stimulated human whole blood and in an animal model of pulmonary inflammation. INNOVATION: Here, we report for the first time that drugs releasing NO intracellularly are efficient 5-LO inhibitors in vitro and in vivo at least equivalent to approved 5-LO inhibitors. CONCLUSION: Our findings provide a novel mechanistic strategy for the development of a new class of drugs suppressing LT biosynthesis by site-directed nitrosylation. The results may also help to better understand the well-recognized anti-inflammatory clinically relevant actions of NO-releasing drugs. Furthermore, our study describes in detail a novel molecular mode of action of NO. Rebound Track: This work was rejected during standard peer review and rescued by Rebound Peer Review (Antioxid Redox Signal 16: 293-296, 2012) with the following serving as open reviewers: Angel Lanas, Hartmut Kühn, Joan Clària, Orina Belton. Antioxid. Redox Signal. 28, 1265-1285.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Antagonistas de Leucotrienos/farmacologia , Leucotrienos/metabolismo , Inibidores de Lipoxigenase/farmacologia , Óxido Nítrico/farmacologia , Animais , Aspirina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Relação Estrutura-AtividadeRESUMO
The arachidonic acid cascade is a key player in inflammation, and numerous well-established drugs interfere with this pathway. Previous studies have suggested that simultaneous inhibition of 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH) results in synergistic anti-inflammatory effects. In this study, a novel prototype of a dual 5-LO/sEH inhibitor KM55 was rationally designed and synthesized. KM55 was evaluated in enzyme activity assays with recombinant enzymes. Furthermore, activity of KM55 in human whole blood and endothelial cells was investigated. KM55 potently inhibited both enzymes in vitro and attenuated the formation of leukotrienes in human whole blood. KM55 was also tested in a cell function-based assay. The compound significantly inhibited the LPS-induced adhesion of leukocytes to endothelial cells by blocking leukocyte activation.
Assuntos
Anti-Inflamatórios/farmacologia , Araquidonato 5-Lipoxigenase/metabolismo , Ácido Araquidônico/metabolismo , Epóxido Hidrolases/antagonistas & inibidores , Hidrocarbonetos Fluorados/farmacologia , Inibidores de Lipoxigenase/síntese química , Inibidores de Lipoxigenase/farmacologia , Ureia/análogos & derivados , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Hidrocarbonetos Fluorados/síntese química , Hidrocarbonetos Fluorados/química , Inflamação/tratamento farmacológico , Leucócitos/metabolismo , Leucotrienos/biossíntese , Lipopolissacarídeos , Inibidores de Lipoxigenase/química , Ureia/síntese química , Ureia/química , Ureia/farmacologiaRESUMO
Metabolic syndrome (MetS) is a multifactorial disease cluster that consists of dyslipidemia, cardiovascular disease, type 2 diabetes mellitus, and obesity. MetS patients are strongly exposed to polypharmacy; however, the number of pharmacological compounds required for MetS treatment can be reduced by the application of multitarget compounds. This study describes the design of dual-target ligands that target soluble epoxide hydrolase (sEH) and the peroxisome proliferator-activated receptor type γ (PPARγ). Simultaneous modulation of sEH and PPARγ can improve diabetic conditions and hypertension at once. N-Benzylbenzamide derivatives were determined to fit a merged sEH/PPARγ pharmacophore, and structure-activity relationship studies were performed on both targets, resulting in a submicromolar (sEH IC50 = 0.3 µM/PPARγ EC50 = 0.3 µM) modulator 14c. In vitro and in vivo evaluations revealed good ADME properties qualifying 14c as a pharmacological tool compound for long-term animal models of MetS.
Assuntos
Benzamidas/síntese química , Benzamidas/farmacologia , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Síndrome Metabólica/tratamento farmacológico , PPAR gama/efeitos dos fármacos , Células 3T3 , Administração Oral , Animais , Benzamidas/farmacocinética , Células COS , Chlorocebus aethiops , Diabetes Mellitus Tipo 2/tratamento farmacológico , Desenho de Fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/farmacocinética , Humanos , Hipertensão/tratamento farmacológico , Técnicas In Vitro , Camundongos , Microssomos Hepáticos/metabolismo , Ratos , Relação Estrutura-AtividadeRESUMO
Resolution of acute inflammation is an active process coordinated by proresolving lipid mediators (SPMs) such as lipoxins (LXs) and resolvins (Rvs), which are formed by the concerted action of 2 lipoxygenases (LOs). Because the exact molecular mechanisms of SPM biosynthesis are not completely understood, we aimed to investigate LX and D-type Rv formation in human leukocytes and HEK293T cells overexpressing leukotriene (LT) pathway enzymes. Activity assays in precursor (15-hydroxyeicosatetraenoic acids, 17-HDoHE)-treated granulocytes [polymorphonuclear leukocytes (PMNLs)] showed a strict dependence of LXA4/RvD1 biosynthesis on cell integrity, and incubation with recombinant human 5-LO did not lead to LX or Rv formation. Pharmacologic inhibition of 5-LO activating protein (FLAP) by MK-886 inhibited LXA4/RvD1 biosynthesis in precursor-treated PMNLs (drug concentration causing 50% inhibition â¼ 0.3/0.2 µM), as did knockdown of the enzyme in MM6 cells, and precursor-treated HEK293T overexpressing 5-LO produced high amounts of LXA4 only in the presence of FLAP. In addition, inhibition of cytosolic phospholipase A2α (cPLA2α) interfered with LXA4/RvD1 formation from exogenous precursors in PMNLs. Furthermore, inhibition of the LT synthases LTA4 hydrolase and LTC4 synthase in PMNL/platelet coincubations augmented LXA4 levels. These findings show that several enzymes known to be involved in the biosynthesis of proinflammatory LTs, such as FLAP and cPLA2α, also contribute to LX and Rv formation.
Assuntos
Proteínas Ativadoras de 5-Lipoxigenase/metabolismo , Ácidos Docosa-Hexaenoicos/biossíntese , Lipoxinas/biossíntese , Araquidonato 5-Lipoxigenase/metabolismo , Linhagem Celular Tumoral , Citosol/enzimologia , Fosfolipases A2 do Grupo IV/metabolismo , Células HEK293 , Humanos , Indóis/farmacologia , Macrófagos/enzimologia , Macrófagos/metabolismo , Neutrófilos/efeitos dos fármacos , Proteínas Recombinantes/metabolismoRESUMO
The pharmacophore concept is commonly employed in virtual screening for hit identification. A pharmacophore is generally defined as the three-dimensional arrangement of the structural and physicochemical features of a compound responsible for its affinity to a pharmacological target. Given a number of active ligands binding to a particular target in the same manner, it can reasonably be assumed that they have some shared features, a common pharmacophore. We present a growing neural gas (GNG)-based approach for the extraction of the relevant features which we called PENG (pharmacophore elucidation by neural gas). Results of retrospective validation indicate an acceptable quality of the generated models. Additionally a prospective virtual screening for leukotriene A4 hydrolase (LTA4H) inhibitors was performed. LTA4H is a bifunctional zinc metalloprotease which displays both epoxide hydrolase and aminopeptidase activity. We could show that the PENG approach is able to predict the binding mode of the ligand by X-ray crystallography. Furthermore, we identified a novel chemotype of LTA4H inhibitors.
Assuntos
Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Epóxido Hidrolases/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Redes Neurais de Computação , Algoritmos , Aminopeptidases/química , Benchmarking , Cristalografia por Raios X , Epóxido Hidrolases/química , Humanos , Ligantes , Modelos Moleculares , Estudos Prospectivos , Ligação Proteica , Reprodutibilidade dos Testes , TermodinâmicaRESUMO
Design of multitarget drugs and polypharmacological compounds has become popular during the past decade. However, the main approach to design such compounds is to link two selective ligands via a flexible linker. Although such chimeric ligands often have reasonable potency in vitro, the in vivo efficacy is low due to high molecular weight, low ligand efficiency, and poor pharmacokinetic profile. We developed an unprecedented in silico approach for fragment-based design of multitarget ligands. It relies on superposition of the chemical spaces related to the affinity on single targets represented by self-organizing maps. We used this approach for screening of molecular fragments, which bind to the enzymes 5-lipoxygenase (5-LO) and soluble epoxide hydrolase (sEH). Using STD-NMR and activity-based assays, we were able to identify fragments binding to both targets. Furthermore, we were able to expand one of the fragments to a potent dual inhibitor bearing a reasonable molecular weight (MW = 446) and high affinity to both targets (IC50 of 0.03 µM toward 5-LO and 0.17 µM toward sEH).
RESUMO
Metabolic syndrome is a complex condition which often requires the use of multiple medications as a treatment. The resulting problems of polypharmacy are increase in side effects, drug-drug interactions, and its high economic cost. Development of multitarget compounds is a promising strategy to avoid the complications arising from administration of multiple drugs. Modulators of peroxisome proliferator-activated receptors (PPARs) are established agents in the treatment of dyslipidaemia, hyperglycaemia, and insulin resistance. Inhibitors of soluble epoxide hydrolase (sEH) are under evaluation for their use in cardiovascular diseases. In the present study, a series of dual sEH/PPAR modulators containing a pyrrole acidic headgroup and a urea pharmacophore were designed, synthesized, and evaluated in vitro using recombinant enzyme and cell-based assays. Compounds with different activity profiles were obtained which could be used in the treatment of metabolic syndrome.
Assuntos
Epóxido Hidrolases/síntese química , Receptores Ativados por Proliferador de Peroxissomo/química , Cromatografia Líquida de Alta Pressão , Interações Medicamentosas , Epóxido Hidrolases/química , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , SolubilidadeRESUMO
The photocycle of channelrhodopsin-2 is investigated in a comprehensive study by ultrafast absorption and fluorescence spectroscopy as well as flash photolysis in the visible spectral range. The ultrafast techniques reveal an excited-state decay mechanism analogous to that of the archaeal bacteriorhodopsin and sensory rhodopsin II from Natronomonas pharaonis. After a fast vibrational relaxation of the excited-state population with 150 fs its decay with mainly 400 fs is observed. Hereby, both the initial all-trans retinal ground state and the 13-cis-retinal K photoproduct are populated. The reaction proceeds with a 2.7 ps component assigned to cooling processes. Small spectral shifts are observed on a 200 ps timescale. They are attributed to conformational rearrangements in the retinal binding pocket. The subsequent dynamics progresses with the formation of an M-like intermediate (7 and 120 µs), which decays into red-shifted states within 3 ms. Ground-state recovery including channel closing and reisomerization of the retinal chromophore occurs in a triexponential manner (6 ms, 33 ms, 3.4 s). To learn more about the energy barriers between the different photocycle intermediates, temperature-dependent flash photolysis measurements are performed between 10 and 30°C. The first five time constants decrease with increasing temperature. The calculated thermodynamic parameters indicate that the closing mechanism is controlled by large negative entropy changes. The last time constant is temperature independent, which demonstrates that the photocycle is most likely completed by a series of individual steps recovering the initial structure.