Inhibitor Report for: Methylene-blue

Neurodegenerative protein misfolding diseases, including prionopathies, share the common feature of accumulating specific misfolded proteins, with a molecular mechanism closely related. Misfolded prion protein (PrP) generates soluble oligomers that, in turn, aggregate into amyloid fibers. Preventing the formation of these entities, crucially associated with the neurotoxic and/or infectious properties of the resulting abnormal PrP, represents an attractive therapeutic strategy to ameliorate prionopathies. We focused our attention into methylene blue (MB), a well-characterized drug, which is under study against Alzheimer's disease and other neurodegenerative disorders. Here, we have undertaken an in vitro study on the effects of MB on oligomerization and fibrillization of human, ovine and murine PrP. We demonstrated that MB affects the kinetics of PrP oligomerization and reduces the amount of oligomer of about 30%, in a pH-dependent manner, by using SLS and DSC methodologies. Moreover, TEM images showed that MB completely suppresses fiber formation at a PrP:MB molar ratio of 1:2. Finally, NMR revealed a direct interaction between PrP and MB, which was mapped on a surface cleft including a fibrillogenic region of the protein. Our results allowed to surmise a mechanism of action in which the MB binding to PrP surface markedly interferes with the pathway towards oligomers and fibres. Therefore MB could be considered as a general anti-aggregation compound, acting against proteinopathies.

The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a approximately 8 degrees C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.

We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (ICa) and the muscarinic activated K+ current (IK,ACh) in rat ventricular and atrial myocytes, respectively, and on the binding of [3H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 microM Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 microM) on beta-adrenergic stimulation of ICa with isoprenaline (Iso, 10 nM or 1 microM). However, Mblue had no effect on the basal ICa or on the stimulation of ICa by Iso in the absence of ACh. The activation of IK,ACh by 3 microM ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of IK,ACh by either guanosine-5'-O-(3-thio)triphosphate or guanosine-5'-(beta,gamma-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscarinic activation of IK,ACh in a dose-dependent manner. The specific binding of [3H]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperazine derivatives behaved like competitive antagonists of [3H]QNB binding, exhibiting equilibrium dissociation constant (Ki) values of 187 nM for Mblue and 366 nM for CPZ. In conclusion, Mblue exerts antimuscarinic effects on ICa and IK,ACh in rat cardiac myocytes that are best explained by the binding of Mblue to the M2 subtype of muscarinic receptors. This property probably contributes to the antimuscarinic effect of the putative guanylyl cyclase inhibitor reported in previous studies.

Methylene blue (MB) is reported to possess diverse pharmacological actions and is attracting increasing attention for the treatment of neurodegenerative disorders such as Alzheimer's disease. Among the pharmacological actions of MB, is the significant inhibition of acetylcholinesterase (AChE) and butyrylcholinesterase (BCHE). These activities may, at least in part, underlie MB's beneficial effects in Alzheimer's disease. MB is metabolized to yield N-demethylated products of which azure B, the monodemethyl metabolite, is the predominant species. Azure B has been shown to be pharmacologically active and also possesses a variety of biological actions. Azure B therefore may contribute to the pharmacological profile of MB. Based on these considerations, the present study investigates the possibility that azure B may, similar to MB, act as an inhibitor of human AChE and BCHE. The results document that azure B inhibits AChE and BCHE with IC50 values of 0.486muM and 1.99muM, respectively. The results further show that azure B inhibits AChE and BCHE reversibly, and that the modes of inhibition are most likely competitive. Although the AChE and BCHE inhibitory activities of azure B are twofold and fivefold, respectively, less potent than those recorded for MB [IC50(AChE)=0.214muM; IC50(BCHE)=0.389muM] under identical conditions, azure B may be a contributor to MB's in vivo activation of the cholinergic system and beneficial effects in Alzheimer's disease.

Neurodegenerative protein misfolding diseases, including prionopathies, share the common feature of accumulating specific misfolded proteins, with a molecular mechanism closely related. Misfolded prion protein (PrP) generates soluble oligomers that, in turn, aggregate into amyloid fibers. Preventing the formation of these entities, crucially associated with the neurotoxic and/or infectious properties of the resulting abnormal PrP, represents an attractive therapeutic strategy to ameliorate prionopathies. We focused our attention into methylene blue (MB), a well-characterized drug, which is under study against Alzheimer's disease and other neurodegenerative disorders. Here, we have undertaken an in vitro study on the effects of MB on oligomerization and fibrillization of human, ovine and murine PrP. We demonstrated that MB affects the kinetics of PrP oligomerization and reduces the amount of oligomer of about 30%, in a pH-dependent manner, by using SLS and DSC methodologies. Moreover, TEM images showed that MB completely suppresses fiber formation at a PrP:MB molar ratio of 1:2. Finally, NMR revealed a direct interaction between PrP and MB, which was mapped on a surface cleft including a fibrillogenic region of the protein. Our results allowed to surmise a mechanism of action in which the MB binding to PrP surface markedly interferes with the pathway towards oligomers and fibres. Therefore MB could be considered as a general anti-aggregation compound, acting against proteinopathies.

In this study, the binding points of MethB and two structurally-related cationic phenoxazine dyes [meldola blue (MB) and nile blue (NB)] to human butyrylcholinesterase (BChE) were investigated by molecular docking and site directed mutagenesis. The comparative inhibitory effects of MethB, MB and NB on recombinant wild type BChE and six human BChE mutants were spectrophotometrically studied. Kinetic analyses yielded the following information: MethB and MB were found to cause nonlinear inhibition of all recombinant BChEs except Y332A, compatible with a multi-site binding model. On the other hand, MethB and MB caused linear mixed inhibition of Y332A mutant, compatible with a single binding mode. Comparing the inhibitory effects in aspect of Ki values with recombinant wild type BChE (Ki=0.042 muM), MethB was found to be approximately 30, 80 and 270-fold less effective as an inhibitor of Y332A, F329A and T120F, respectively. NB caused nonlinear inhibition of all recombinant BChEs. The inhibitory effect of NB on Y332A mutant was approximately 370-fold lower, compared to recombinant wild type BChE (Ki=0.006 muM). Considering both kinetic and molecular docking results together, it was concluded that threonine 120, phenylalanine 329 and tyrosine 332 are critical amino acids in binding of cationic phenoxazine/phenothiazine structured ligands to human BChE.

The photosensitizer, methylene blue (MB), generates singlet oxygen ((1)O2) that irreversibly inhibits Torpedo californica acetylcholinesterase (TcAChE). In the dark MB inhibits reversibly, binding being accompanied by a bathochromic shift that can be used to show its displacement by other reversible inhibitors binding to the catalytic 'anionic' subsite (CAS), the peripheral 'anionic' subsite (PAS), or bridging them. Data concerning both reversible and irreversible inhibition are here reviewed. MB protects TcAChE from thermal denaturation, and differential scanning calorimetry reveals a approximately 8 degrees C increase in the denaturation temperature. The crystal structure of the MB/TcAChE complex reveals a single MB stacked against W279 in the PAS, pointing down the gorge towards the CAS. The intrinsic fluorescence of the irreversibly inhibited enzyme displays new emission bands that can be ascribed to N'-formylkynurenine (NFK); this was indeed confirmed using anti-NFK antibodies. Mass spectroscopy revealed that two Trp residues, Trp84 in the CAS, and Trp279 in the PAS, were the only Trp residues, out of a total of 14, significantly modified by photo-oxidation, both being converted to NFK. In the presence of competitive inhibitors that displace MB from the gorge, their modification is completely prevented. Thus, photo-oxidative damage caused by MB involves targeted release of (1)O2 by the bound photosensitizer within the aqueous milieu of the active-site gorge.

Methylene blue, 3, 7-bis(dimethylamino)-phenothiazin-5-ium chloride, is a reversible inhibitor of human butyrylcholinesterase (BChE) in the absence of light. In the presence of light and oxygen, methylene blue promotes irreversible inhibition of human BChE as a function of time, requiring 3 h irradiation to inhibit 95% activity. Inactivation was accompanied by a progressive loss of Coomassie-stained protein bands on native and denaturing polyacrylamide gels, suggesting backbone fragmentation. Aggregation was not detected. MALDI-TOF/TOF mass spectrometry identified oxidized tryptophan (W52, 56, 231, 376, 412, 490, 522), oxidized methionine (M81, 144, 302, 532, 554, 555), oxidized histidine (H214), oxidized proline (P230), oxidized cysteine (C519) and oxidized serine (S215). A 20 min irradiation in the presence of methylene blue resulted in 17% loss of BChE activity, suggesting that BChE is relatively resistant to methylene blue-catalyzed photoinactivation and that therefore this process could be used to sterilize BChE preparations.

The photosensitizer methylene blue MB generates singlet oxygen that irreversibly inhibits Torpedo californica acetylcholinesterase TcAChE In the dark it inhibits reversibly Binding is accompanied by a bathochromic absorption shift used to demonstrate displacement by other acetylcholinesterase inhibitors interacting with the catalytic anionic subsite CAS the peripheral anionic subsite PAS or bridging them MB is a noncompetitive inhibitor of TcAChE competing with reversible inhibitors directed at both anionic subsites but a single site is involved in inhibition MB also quenches TcAChE's intrinsic fluorescence It binds to TcAChE covalently inhibited by a small organophosphate OP but not an OP containing a bulky pyrene Differential scanning calorimetry shows an 8 degrees increase in the denaturation temperature of the MB/TcAChE complex relative to native TcAChE and a less than twofold increase in cooperativity of the transition The crystal structure reveals a single MB stacked against Trp279 in the PAS oriented down the gorge toward the CAS it is plausible that irreversible inhibition is associated with photooxidation of this residue and others within the active-site gorge The kinetic and spectroscopic data showing that inhibitors binding at the CAS can impede binding of MB are reconciled by docking studies showing that the conformation adopted by Phe330 midway down the gorge in the MB/TcAChE crystal structure precludes simultaneous binding of a second MB at the CAS Conversely binding of ligands at the CAS dislodges MB from its preferred locus at the PAS The data presented demonstrate that TcAChE is a valuable model for understanding the molecular basis of local photooxidative damage.

The principal role of AChE (acetylcholinesterase) is termination of impulse transmission at cholinergic synapses by rapid hydrolysis of the neurotransmitter acetylcholine. The active site of AChE is near the bottom of a long and narrow gorge lined with aromatic residues. It contains a CAS (catalytic 'anionic' subsite) and a second PAS (peripheral 'anionic' site), the gorge mouth, both of which bind acetylcholine via pi-cation interactions, primarily with two conserved tryptophan residues. It was shown previously that generation of 1O2 by illumination of MB (Methylene Blue) causes irreversible inactivation of TcAChE (Torpedo californica AChE), and suggested that photo-oxidation of tryptophan residues might be responsible. In the present study, structural modification of the TcAChE tryptophan residues induced by MB-sensitized oxidation was investigated using anti-N-formylkynurenine antibodies and MS. From these analyses, we determined that N-formylkynurenine derivatives were specifically produced from Trp84 and Trp279, present at the CAS and PAS respectively. Peptides containing these two oxidized tryptophan residues were not detected when the competitive inhibitors, edrophonium and propidium (which should displace MB from the gorge) were present during illumination, in agreement with their efficient protection against the MB-induced photo-inactivation. Thus the bound MB elicited selective action of 1O2 on the tryptophan residues facing on to the water-filled active-site gorge. The findings of the present study thus demonstrate the localized action and high specificity of MB-sensitized photo-oxidation of TcAChE, as well as the value of this enzyme as a model system for studying the mechanism of action and specificity of photosensitizing agents.

The kinetic effects of a selection of triarylmethane, phenoxazine and phenothiazine dyes (pararosaniline (PR), malachite green (MG), methyl green (MeG); meldola blue (MB), nile blue (NB), nile red (NR); methylene blue (MethB)) and of ethopropazine on horse serum butyrylcholinesterase were studied spectrophotometrically at 25( degrees )C in 50mM MOPS buffer, pH 8, using butyrylthiocholine as substrate. PR, MeG, MB and ethopropazine acted as linear mixed type inhibitors of the enzyme, with respective K(i) values of 4.5+/-0.50 microM, 0.41+/-0.007 microM, 0.44+/-0.086 microM and 0.050+/-0.0074 microM. MG, NB, MethB and NR caused complex, nonlinear inhibition pointing to cooperative binding at two sites. Intrinsic K' values ( identical with[I](2)(0.5) extrapolated to [S]=0) for MG, NB, NR and MethB were 0.20+/-0.096 microM, 0.0018+/-0.0015 microM, 0.92+/-0.23 microM and 0.23+/-0.08 microM. NB stood out as a potent inhibitor effective at nM levels. Comparison of inhibitory effects on horse and human serum butyrylcholinesterases suggested that the two enzymes must have distinct microstructural features.

Two cationic phenoxazine dyes, meldola blue (MB) and nile blue (NB), and the structurally related phenothiazine, methylene blue (MethB), were found to act as complex inhibitors of human plasma cholinesterase (butyrylcholinesterase, BChE). Studied at 25 degrees C, in 100mM MOPS buffer (pH 8.0), with butyrylthiocholine as substrate, the kinetic pattern of inhibition indicated cooperative I binding at 2 sites. Intrinsic K' values ( identical with[I](0.5)(2) extrapolated to [S]=0) for MB, NB and MethB were 0.64+/-0.05, 0.085+/-0.026 and 0.42+/-0.04 microM, respectively. Under the same experimental conditions the dyes acted as single-occupancy, hyperbolic-mixed inhibitors of electric eel acetylcholinesterase (AChE), with K(i)=0.035+/-0.010, 0.026+/-0.0034 and 0.017+/-0.0063 microM (for MB, NB, MethB); alpha (coefficient of competitive interaction)=1.8-2.4 and beta (coefficient of noncompetitive interaction)=0.15-0.28. The complexity of the BChE inhibitory effect of phenoxazine/phenothiazine dyes contrasted with that of conventional ChE inhibitors which cause single-occupancy (n=1), competitive or mixed inhibition in both AChE and BChE and signaled novel modes of ligand interaction at (or remote from) the active site gorge of the latter enzyme.

We studied the mechanism of action of methylene blue (Mblue), a putative guanylyl cyclase inhibitor, on the L-type calcium current (ICa) and the muscarinic activated K+ current (IK,ACh) in rat ventricular and atrial myocytes, respectively, and on the binding of [3H]quinuclidinyl benzylate in rat ventricular membranes. Superfusion, but not internal dialysis, with 30 microM Mblue antagonized the inhibitory effect of acetylcholine (ACh, 1 microM) on beta-adrenergic stimulation of ICa with isoprenaline (Iso, 10 nM or 1 microM). However, Mblue had no effect on the basal ICa or on the stimulation of ICa by Iso in the absence of ACh. The activation of IK,ACh by 3 microM ACh was also antagonized by Mblue in a dose-dependent manner. In contrast, Mblue had no effect on the activation of IK,ACh by either guanosine-5'-O-(3-thio)triphosphate or guanosine-5'-(beta,gamma-imido)triphosphate. Chlorpromazine (CPZ), a piperazine derivative like Mblue, also inhibited the muscarinic activation of IK,ACh in a dose-dependent manner. The specific binding of [3H]QNB, a muscarinic ligand, to rat ventricular membranes was displaced in a dose-dependent manner by Mblue and CPZ. The piperazine derivatives behaved like competitive antagonists of [3H]QNB binding, exhibiting equilibrium dissociation constant (Ki) values of 187 nM for Mblue and 366 nM for CPZ. In conclusion, Mblue exerts antimuscarinic effects on ICa and IK,ACh in rat cardiac myocytes that are best explained by the binding of Mblue to the M2 subtype of muscarinic receptors. This property probably contributes to the antimuscarinic effect of the putative guanylyl cyclase inhibitor reported in previous studies.

1. The inhibitory effects of methylene blue (MB) on different types of cholinesterases and [3H]-N-methylscopolamine ([3H]-NMS) binding to muscarinic receptors were studied. 2. Human plasma from young healthy male volunteers, purified human pseudocholinesterase and purified bovine true acetylcholinesterase were incubated with acetylcholine and increasing concentrations of MB (0.1-100 mumol l-1) in the presence of the pH-indicator m-nitrophenol for 30 min at 25 degrees C. The amount of acetic acid produced by the enzymatic hydrolysis of acetylcholine was determined photometrically. 3. Rat cardiac left ventricle homogenate was incubated with [3H]-NMS and with increasing concentrations of MB (0.1 mmol l-1 mumol l-1) at 37 degrees C for 20 min. THe binding of [3H]-NMS to the homogenate was quantified by a standard liquid scintillation technique. 4. MB inhibited the esterase activity of human plasma, human pseudocholinesterase and bovine acetylcholinesterase concentration-dependently with IC50 values of 1.05 +/- 0.05 mumol l-1, 5.32 +/- 0.36 mumol l-1 and 0.42 +/- 0.09 mumol l-1, respectively. MB induced complete inhibition of the esterase activity of human plasma and human pseudocholinesterase, whereas bovine acetylcholinesterase was maximally inhibited by 73 +/- 3.3%. 5. MB was able to inhibit specific [3H]-NMS binding to rat cardiac left ventricle homogenate completely with an IC50 value of 0.77 +/- 0.03 mumol l-1, which resulted in a Ki value for MB of 0.58 +/- 0.02 mumol l-1. 6. In conclusion, MB may be considered as a cholinesterase inhibitor with additional, relevant affinity for muscarinic binding sites at concentrations at which MB is used for investigations into the endothelial system. In our opinion these interactions between MB and the cholinergic system invalidate the use of MB as a tool for the investigation of the L-arginine-NO-pathway, in particular when muscarinic receptor stimulation is involved.

Methylene blue, at high concentrations, interferes with the estimation of methaemoglobin using the IL 282 CO-oximeter: the dye does not interfere with the method of Evelyn & Malloy for determination of methaemoglobin. In beagle bitches methylene blue causes both methaemoglobinogenesis and methaemoglobin reduction, the effect of the former being to delay the decline of methaemoglobin levels, when methylene blue is used to reverse the methaemoglobinaemia produced by sodium nitrite.

The kinetics of the inhibition of the acetylcholine-esterase by methylene blue has been studied using cow erythrocytes as enzyme source. Methylene blue is a true competitive inhibitor and behaves in a similar way found previously for other quaternary ammonium bases. The optimum acetylcholine concentration, characteristic of this esterase, is changed to higher concentration in the presence of the dye. The higher inhibitor concentration the higher is the optimum acetylcholine concentration. Great differences in degree of inhibition are obtained when the action of the dye on the acetylcholine-esterase is compared with that on the serum cholinesterase. Evaluating the inhibition as function of methylene blue concentration the dissociation constant (KI ) of the inhibitor-enzyme complex was found to be 5.7 X 10-7. A higher value of KI is obtained when the enzyme solution has been incubated with the inhibitor before mixed with the substrate.