Binding Of Indomethacin Research Articles

Detailed Experimental and In Silico Investigation of Indomethacin Binding with Human Serum Albumin Considering Primary and Secondary Binding Sites

The interaction of indomethacin with human serum albumin (HSA) has been studied here considering the primary and secondary binding sites. The Stern-Volmer plots were linear in the lower concentration range of indomethacin while a downward curvature was observed in the higher concentration range, suggesting the presence of more than one binding site for indomethacin inside HSA due to which the microenvironment of the fluorophore changed slightly and some of its fraction was not accessible to the quencher. The Stern-Volmer quenching constants (KSV) for the primary and secondary sites were calculated from the two linear portions of the Stern-Volmer plots. There was around a two-fold decrease in the quenching constants for the low-affinity site as compared to the primary binding site. The interaction takes place via a static quenching mechanism and the KSV decreases at both primary and secondary sites upon increasing the temperature. The binding constants were also evaluated, which show strong binding at the primary site and fair binding at the secondary site. The binding was thermodynamically favorable with the liberation of heat and the ordering of the system. In principle, hydrogen bonding and Van der Waals forces were involved in the binding at the primary site while the low-affinity site interacted through hydrophobic forces only. The competitive binding was also evaluated using warfarin, ibuprofen, hemin, and a warfarin + hemin combination as site markers. The binding profile remained unchanged in the presence of ibuprofen, whereas it decreased in the presence of both warfarin and hemin with a straight line in the Stern-Volmer plots. The reduction in the binding was at a maximum when both warfarin and hemin were present simultaneously with the downward curvature in the Stern-Volmer plots at higher concentrations of indomethacin. The secondary structure of HSA also changes slightly in the presence of higher concentrations of indomethacin. Molecular dynamics simulations were performed at the primary and secondary binding sites of HSA which are drug site 1 (located in the subdomain IIA of the protein) and the hemin binding site (located in subdomain IB), respectively. From the results obtained from molecular docking and MD simulation, the indomethacin molecule showed more binding affinity towards drug site 1 followed by the other two sites.

Experimental and in silico investigation on the interaction of indomethacin with bovine serum albumin: Effect of sodium dodecyl sulfate surfactant monomers on the binding

This paper describes the interaction of indomethacin with bovine serum albumin (BSA) using experimental and computational approaches. Inner filter effect (IFE) had significantly impacted the fluorescence results as uncorrected data shown the considerable involvement of tyrosine residues which became very low after correcting the data with the former, thus, only the tryptophan residues were found to be associated with fluorescence quenching. Analyses of the corrected data suggested that there was 1:1 strong binding between BSA and indomethacin that taken place via static quenching mechanism. The binding was exothermic in nature and energetically favorable with ordering of the system. The binding of indomethacin remained almost unchanged in presence of low amount of sodium dodecyl sulfate (SDS) surfactants monomers while it significantly diminished when the concentration of SDS monomers increased. Low concentrations of indomethacin didn’t affect the secondary structure of BSA, however, high concentrations led to the partial unfolding of the protein. Molecular docking along with molecular dynamics simulation results explained that this drug molecule occupied new binding site with strong binding affinity of −8.35 kcal/mol, which is oriented in the interface between domain 1 and domain 2. The interaction was facilitated by both hydrophobic forces as well as hydrogen bonding.

Interaction of indomethacin with calf thymus DNA: a multi-spectroscopic, thermodynamic and molecular modelling approach.

Indomethacin belongs to the acetic acid derivative class of non-steroidal anti-inflammatory drugs with diverse pharmacological and biological activities. Understanding the mechanism of interaction of drugs with possible target and off-target biomolecules can prove useful in the development of a rational drug designing system. In this paper, we have attempted to ascertain the mode of binding of indomethacin with calf thymus DNA (Ct-DNA) through various biophysical techniques and in silico molecular docking. Analysis of the UV-visible absorbance spectra and fluorescence emission profile of indomethacin upon addition of Ct-DNA indicates the formation of a drug-DNA complex. UV-visible absorbance and steady state fluorescence experiments revealed a binding constant on the order of 103 L mol-1, which is consistent with those of well-known groove binders. Competitive displacement studies with ethidium bromide, acridine orange and Hoechst 33258 further suggested that indomethacin binds to the minor groove of the Ct-DNA. The above observations were further confirmed by KI induced quenching experiments, DNA melting studies, CD spectral analysis and viscosity measurements. The thermodynamic parameters like spontaneous free energy (ΔG < 0) and large favourable enthalpy (ΔH < 0) obtained from isothermal calorimetry indicated the involvement of hydrogen bonding and van der Waals forces in the binding process. Molecular docking further corroborated the experimental results.

Interaction of Human Serum Albumin with Indomethacin: Spectroscopic and Molecular Modeling Studies

In this work, the interaction between indomethacin (IM) and human serum albumin (HSA) under simulative physiological conditions was investigated by the methods of fluorescence spectroscopy, circular dichroism (CD) spectroscopy, and molecular modeling. The experiment results showed that the fluorescence quenching of HSA by IM was a result of the formation of an IM–HSA complex and the corresponding association constants (Ka) between IM and HSA at four different temperatures were determined according to the modified Stern–Volmer equation. The resulting thermodynamic parameters ΔG, ΔH, and ΔS at different temperatures indicate that the hydrophobic force plays a major role for IM–HSA association, but hydrogen bonds also could not be excluded. A molecular modeling study further confirmed the binding mode and indicated that the binding of IM to HSA primarily takes place in sub-domain IIA (site I). The conformational investigation showed that the presence of IM decreased the α-helical content of HSA and induced slight unfolding of the polypeptides of protein, which confirmed that some microenvironmental and conformational changes occur for HSA molecules.

Indomethacin Serves as a Potential Inhibitor of Protein Phosphatases

Background/Aims: We have shown that indomethacin has the potential to activate Ca2+/ calmodulin-dependent protein kinase II (CaMKII), regardless of cyclooxygenase (COX) inhibition. To understand the underlying mechanism, the present study investigated the effect of indomethacin on protein phosphatases such as protein phosphatase 1 (PP1), protein phosphatase 2A (PP2A), and protein tyrosine phosphatase 1B (PTP1B). Methods: Activity of CaMKII was assayed in cultured rat hippocampal neurons and under the cell-free conditions. Activities of protein phosphatases were monitored under the cell-free conditions. Indomethacin binding assay was carried out using a fluorescein-conjugated indomethacin. Results: Indomethacin enhanced CaMKII activity in cultured rat hippocampal neurons, that is abolished the CaMKII inhibitor KN-93. In the cell-free assay, no CaMKII activation was obtained with indomethacin, but indomethacin otherwise inhibited PP1 in a concentration (10 µM-1 mM)-dependent manner, the maximum reaching 70% of basal levels. This indicates that indomethacin indirectly activates CaMKII due to PP1 inhibition. Likewise, indomethacin still inhibited PP2A and PTP1B in a concentration (10 µM-1 mM)-dependent manner, reaching 80 and 10% of basal levels at 1 mM, respectively. In the indomethacin binding assay, indomethacin bound to all the investigated protein phosphatases. Conclusion: The results of the present study indicate that indomethacin inhibits PP1, PP2A, and PTP1B, possibly through its direct binding and that the inhibitory effect of indomethacin on PP1 could cause indirect CaMKII activation. This may represent the novel indomethacin action.

Novel Indomethacin Action: Selective and Direct Activation of Protein Kinase C-ε

Background/Aims: In our earlier study, indomethacin potentiated α7 acetylcholine (ACh) receptor responses by activating protein kinase C (PKC). The present study was conducted to gain further insight into the indomethacin action on PKC. Methods: PKC activity was assayed in PC-12 cells or under the cell-free conditions. PKC-ε was knocked-down using the siRNA to silence the PKC-ε-targeted gene. A fluorescein-conjugated indomethacin was synthesized to examine the interaction of indomethacin with PKC-ε. Results: In the in situ PKC assay, indomethacin activated PKC in PC-12 cells in a concentration (1-100 µM)-dependent manner, and the activation was suppressed by knocking-down PKC-ε. In the cell-free PKC assay, indomethacin (100 µM) activated PKC-ε in the absence of diacylglycerol, phosphatidylserine, and calcium, but other PKC isozymes such as α, βΙ, βΙΙ, γ, δ, ι, and ζ were not activated. In the indomethacin binding assay using a fluorescent-conjugated indomethacin on blue native-polyacrylamide gel electrophoresis (blue native-PAGE), a fluorescent signal was detected at the site consistent with PKC-ε protein and the signal was attenuated by adding non-conjugated indomethacin or eliminated by pretreatment with non-conjugated indomethacin. Conclusion: The results of the present study show that indomethacin has the potential to selectively activate PKC-ε through its direct binding, independently of cyclooxygenase (COX) inhibition.

A comparison of crystallographic and DFT optimized geometries on two taxane diterpenoids and docking studies with phospholipase A2

Structure optimization of two taxane diterpenoids I and II has been carried out using GAUSSIAN03 program with B3LYP/6-31G** basis set. The structures were fully optimized without any geometric constraints. Coordinates found in crystallographic study were used as input to the GAUSSIAN. The results show that the conformations of taxoid cores are almost same as were observed from crystallography, while the conformations of the substituents are slightly different. Thus, taxoid cores are robust enough to maintain their conformations in gaseous state while the conformations of substituents may vary depending upon the reaction condition. To understand the mode of binding, the docking studies of both compounds have been carried out with Russell’s viper phospholipase A2 (vPLA2) as target using induced fit docking. The docking results show that regarding the interactions and energy for indomethacin binding with vPLA2, compound I shows the best results.

Peroxide-induced radical formation at TYR385 and TYR504 in human PGHS-1

Prostaglandin H synthase isoforms 1 and -2 (PGHS-1 and -2) react with peroxide to form a radical on Tyr385 that initiates the cyclooxygenase catalysis. The tyrosyl radical EPR signals of PGHS-1 and -2 change over time and are altered by cyclooxygenase inhibitor binding. We characterized the tyrosyl radical dynamics using wild type human PGHS-1 (hPGHS-1) and its Y504F, Y385F, and Y385F/Y504F mutants to determine whether the radical EPR signal changes involve Tyr504 radical formation, Tyr385 radical phenyl ring rotation, or both. Reaction of hPGHS-1 with peroxide produced a wide singlet, whereas its Y504F mutant produced only a wide doublet signal, assigned to the Tyr385 radical. The cyclooxygenase specific activity and K(M) value for arachidonate of hPGHS-1 were not affected by the Y504F mutation, but the peroxidase specific activity and the K(M) value for peroxide were increased. The Y385F and Y385F/Y504F mutants retained only a small fraction of the peroxidase activity; the former had a much-reduced yield of peroxide-induced radical and the latter essentially none. After binding of indomethacin, a cyclooxygenase inhibitor, hPGHS-1 produced a narrow singlet but the Y504F mutant did not form a tyrosyl radical. These results indicate that peroxide-induced radicals form on Tyr385 and Tyr504 of hPGHS-1, with radical primarily on Tyr504 in the wild type protein; indomethacin binding prevented radical formation on Tyr385 but allowed radical formation on Tyr504. Thus, hPGHS-1 and -2 have different distributions of peroxide-derived radical between Tyr385 and Tyr504. Y504F mutants in both hPGHS-1 and -2 significantly decreased the cyclooxygenase activation efficiency, indicating that formation of the Tyr504 radical is functionally important for both isoforms.

Structural Basis for Catalytic and Inhibitory Mechanisms of Human Prostaglandin Reductase PTGR2

PTGR2 catalyzes an NADPH-dependent reduction of the conjugated alpha,beta-unsaturated double bond of 15-keto-PGE(2), a key step in terminal inactivation of prostaglandins and suppression of PPARgamma-mediated adipocyte differentiation. Selective inhibition of PTGR2 may contribute to the improvement of insulin sensitivity with fewer side effects. PTGR2 belongs to the medium-chain dehydrogenase/reductase superfamily. The crystal structures reported here reveal features of the NADPH binding-induced conformational change in a LID motif and a polyproline type II helix which are critical for the reaction. Mutation of Tyr64 and Tyr259 significantly reduces the rate of catalysis but increases the affinity to substrate, confirming the structural observations. Besides targeting cyclooxygenase, indomethacin also inhibits PTGR2 with a binding mode similar to that of 15-keto-PGE(2). The LID motif becomes highly disordered upon the binding of indomethacin, indicating plasticity of the active site. This study has implications for the rational design of inhibitors of PTGR2.

Effect of lysine modification on the conformation and indomethacin binding properties of human serum albumin

In order to study the involvement of lysine residues of human serum albumin (HSA) in the binding of indomethacin, HSA was treated with different molar excess of acetic anhydride, succinic anhydride and O-methylisourea which resulted in differently modified preparations: 30%, 62% and 87% acetylated, 20%, 34%, 64% and 78% succinylated and 21%, 43% and 86% guanidinated HSAs. All the preparations were found to be homogeneous with respect to charge as well as size as judged by polyacrylamide gel electrophoresis and gel filtration on a Seralose-6B column. Hydrodynamic and circular dichroic results showed that pronounced conformational changes (both tertiary and secondary structures) were induced in the maximally acetylated (87%) and succinylated (78%) preparations. On the other hand, guanidinated preparations showed no expansion in the hydrodynamic volume. The percent decrease in α-helical content was 34% for 87% acetylated, 31% for 78% succinylated and 10% for 86% guanidinated HSAs. A significant increase in the values of Stokes radii and frictional ratios (from 3.43 nm and 1.29 for native HSA to 4.07 nm and 1.52 for 87% acetylated and 4.35 nm and 1.60 for 78% succinylated HSAs, respectively) was also noticed in these highly modified preparations. Fluorescence quench titration results obtained at pH 7.4 and ionic strength 0.15 showed that only 54.1% and 64.7% binding of indomethacin at 4:1 drug/protein molar ratio was retained by 87% acetylated and 78% succinylated HSAs, respectively, as compared to 91% retention in binding in 86% guanidinated preparation. No reversal in the binding of drug to 87% acetylated and 78% succinylated HSA preparations was observed on increasing the ionic strength to 1.0. Therefore, it seems that one or two critical lysine residue(s) that can form salt linkage with the carboxyl group of indomethacin, was (were) probably modified in these preparations. A small decrease in the binding of drug to the guanidinated preparation also confirms the involvement of positive charge, probably contributed by lysine residue(s), in the binding of indomethacin to HSA.

Molecular basis of indomethacin-human serum albumin interaction.

Studies on the strength and extent of binding of the non-steroidal anti-inflammatory drug indomethacin to human serum albumin (HSA) have provided conflicting results. In the present work, the serum-binding of indomethacin was studied in 55 mM sodium phosphate buffer (pH 7.0) at 28 degrees C, by using a fluorescence quench titration technique. The interaction of indomethacin with human serum albumin has been studied as a function of temperature, ionic strength and pH. The results suggest that electrostatic interaction plays a major role in the binding. The possible role of lysine residues in this interaction was studied by modifying exposed and buried lysine residues of HSA with potassium cyanate and studying indomethacin binding with the modified HSA. The data suggest that the interaction takes place via a salt bridge formation between the carboxylate group of indomethacin and a buried lysine residue of HSA. A technique involving fluorescence enhancement of bilirubin upon its interaction with HSA was used to study its displacement by indomethacin. The displacement, although apparently competitive in nature, was not strong suggesting that the primary sites of interaction of bilirubin and indomethacin are different.

Protein binding of some nonsteroidal anti-inflammatory drugs studied by high-performance liquid affinity chromatography

The protein binding of indomethacin, sulindak and diclofenac sodium is studied in the presence of some competitors: phenylbutazon and diazepam. A high-performance liquid affinity chromatography based on chiral stationary phases with immobilized human serum albumin is used. The competition of the markers and the drugs for two major high- and low-affinity binding sites is investigated. Using a mathematical procedure proposed by the same authors in a previous work the affinity constants of the binding drugs and markers for both types of site are calculated. An analogous behaviour is established for the three drugs—they have nearly the same affinity for the primary binding sites marked by phenylbutazon and diazepam and only one type of low-affinity site (diazepam-binding sites) is involved in binding. That can be explained assuming an overlapping sites.

Lactoperoxidase-catalysed oxidation of indomethacin, a nonsteroidal antiinflammatory drug, through the formation of a free radical

Lactoperoxidase (LPO, EC 1.11.1.7; donor-H 2O 2 oxidoreductase) catalyses the oxidation of indomethacin, a nonsteroidal antiinflammatory drug by H 2O 2 as measured by time-dependent decay of indomethacin extinction at 280 nm and concurrent appearance of stable oxidation product(s) at 412 nm. From a plot of log V max against varying pH of indomethacin oxidation, involvement of an ionizable group of the enzyme having pka = 5.7 could be ascertained for controlling the oxidation process. Spectral studies revealed that LPO-compound II oxidises indomethacin through one-electron transfer and is reduced to the native ferric state as shown by its spectral shift from 430 nm to 412 nm through an isosbestic point at 421 nm. The one-electron oxidation product is a nitrogen-centered free radical detected as a 5,5-dimethyl-l-pyrroline N-oxide (DMPO) adduct ( a N = 15 G, a H β = 16 G) in electron spin resonance spectroscopy. The free radical is scavenged by reaction with O 2 as shown by O 2 consumption sensitive to the free-radical trap, DMPO. Binding studies by optical difference spectroscopy indicate that indomethacin binds to LPO with an apparent K D value of 24.5 μM. The free energy change, ΔG′, for the binding is −26.3 KJ mol −1, suggesting that the interaction is favourable for oxidation. Indomethacin binding remains unaltered by a change of pH from 5.25 to 7.5, presumably because of hydrophobic interaction. The binding is competitive with resorcinol, an aromatic electron donor, showing the K D value to be as high as 100 μM. We suggest that indomethacin interacts at the aromatic donor binding site and is oxidised by one-electron transfer by LPO catalytic intermediates to stable oxidation product(s) through the formation of a free radical. BIOCHEM PHARMACOL 52;8:1169–1175, 1996.

Distribution of indomethacin in human milk and estimation of its milk to plasma ratio in vitro.

1. The distribution of indomethacin in fat and protein fractions of colostrum and mature milk as well as its milk to plasma drug concentration ratio (M/P ratio) were determined in vitro. 2. The extent of plasma protein binding of indomethacin (5-20 microg ml(-1)) was > or = 99.6%. The protein binding of indomethacin in colostrum was 46.0% at pH 7.4. The lower protein content of mature milk compared with colostrum was associated with a significant decrease in the extent of drug protein binding (46 +/- 1.93 to 35 +/- 1.0 s.e. mean). Protein binding was also decreased significantly in 8% fat mature milk (20.3 +/- 2.4 s.e mean) but was constant over the pH range 7.4 to 6.8. 3. About 40% of indomethacin added to milk was associated with the fatty layer. The indomethacin M/P ratio determined by equilibrium dialysis was less than 0.01. Hence the maximum infant daily dose was estimated to be 0.006 mg kg(-1). 4. Our results indicate that indomethacin transfers to milk by simple diffusion according to its physicochemical properties, and that treatment with indomethacin is not a contraindication to breast feeding.

Protein binding of indomethacin in human cerebrospinal fluid

The binding of the non-steroidal anti-inflammatory drug indomethacin to proteins in human cerebrospinal fluid (CSF), drawn during lumbar puncture from 10 patients affected by lumbosciatica, was measured by equilibrium dialysis and spectrofluorimetry. Similar binding studies on human serum albumin solutions (0.5 and 1 g/L) were performed using the same techniques. The mean binding percentage of indomethacin determined by equilibrium dialysis was 40%. The results obtained by both techniques allowed us to conclude that the binding of indomethacin in CSF was essentially due to albumin.

Plasma and cerebrospinal fluid concentrations of indomethacin in humans

Plasma and cerebrospinal fluid (CSF) concentrations of indomethacin have been determined in 52 patients hospitalized for nerve-root compression pain. Samples of blood and CSF were collected at the same time in each subject, 0.5 to 12 h after a single intramuscular injection of 50 mg indomethacin. Analgesic effect was assessed by the absolute and percentage variation in Huskisson's visual analogue scale between dosing and sampling. According to its high lipid solubility, indomethacin rapidly crossed the blood-brain barrier, being detected in CSF 0.5 h after administration. After attainment of equilibrium within 2 h, the CSF level exceeded the free plasma level. Since the drug was extensively bound to serum albumin (99.7 +/- 0.1%), this phenomenon may represent a slight degree of binding of indomethacin in CSF. The analgesic activity was not related to either the plasma or CSF concentration of indomethacin.

Evidence for the existence of a specific binding site for indomethacin on bovine vesicular gland microsomes.

Using bovine vesicular gland microsomes and [14C]indomethacin we demonstrated the presence of a specific binding site for nonsteroidal anti-inflammatory drugs. Specific binding of [14C]indomethacin to microsomes was rapid, with most of the ligand bound by 2 min at 4 degrees C. In routine binding assays the incubation temperature was maintained at 4 degrees C, because the maximal specific binding was obtained. Specific [14C]indomethacin binding appeared to increase linearly with increasing protein concentration over the range of 0.1-1.0 mg of microsomal protein. Specific binding was saturable and Scatchard analysis of binding data showed a single class of binding sites with a dissociation constant (Kd) of 3.8 microM and a maximal number of binding sites (Bmax) of about 1272 pmol/mg of protein. When these binding data were plotted according to the Hill equation, a straight line was obtained with a Hill coefficient of 1.0. Structural specificity of the nonsteroidal anti-inflammatory drug site was studied with diclofenac, arylpropionic acids (ketoprofen and indoprofen), and aspirin. Diclofenac and arylpropionic derivatives were able to compete with [14C]indomethacin for binding to microsomes, while aspirin was a weak inhibitor.

Effect of alpha-fetoprotrein and indomethacin on arachidonic acid metabolism in P388D1 macrophages : Role of leuxotrienes

Alpha-fetoprotein (AFP),is able to modify arachidonic acid metabolism In P388DI macrophage - like cells. AFP has been shown to induce mobilization of arachidonic acid and consequently an enhanced production of leukotrienes.The action of AFP on the cyclooxygenase pathway appears, however, more complex, as this protein simultaneously decreases the total amount of cyclooxygenase products and raises the production of PGE2, 6-keto-PGF1α and to a lesser extent of thromboxane B2. On the other hand, indomethacin abolishes the synthesis of PGE2 but has no effect on 6-keto-PGFlα and thromboxane B2. Indomethacin used in combination with AFP partly loses its inhibiting effect on PGE2 synthesis and paradoxically leads to a “superactivation” of P388D1 cells demonstrated by a very high arachidonic acid mobilization, an enhancement of both leukotriene synthesis and cyclooxygenase activity. Evidence for a binding of indomethacin to AFP was obtained that partly explains these results. In addition, it is shown that the effects of AFP and AFP+Indomethacin on cyclooxygenase activity might be explained by the endogenous synthesis of leukotrienes.

On the binding of cinmetacin and indomethacin to human serum albumin.

The binding of two non-steroidal anti-inflammatory drugs, indomethacin and cinmetacin, to human serum albumin was studied by dynamic dialysis at 37 degrees C and pH 7.4. Cinmetacin is bound more than indomethacin. The affinity constant for the primary binding site is 4.28 X 10(6) M-1 for cinmetacin and 1.4 X 10(6) M-1 for indomethacin. The protein binding of indomethacin is decreased in the presence of cinmetacin.

Interaction of indomethacin with adult human albumin and neonatal serum.

The binding of indomethacin to albumin was investigated at 37 degrees C, pH 7.4. The first stoichiometric binding constant is 2.5 X 10(5) M-1. Indomethacin utilizes both the bilirubin and diazepam binding functions equally. The effect on bilirubin binding to albumin is negligible at therapeutic indomethacin blood levels. Oleic acid does not affect indomethacin binding until the oleate/albumin molar ratio exceeds 2. A method for measuring the reserve albumin equivalent for binding indomethacin is described. Measurement of this reserve binding equivalent in 33 neonatal serum samples shows no significant correlation of the reserve binding to the albumin level. This methodology may be useful in studying the variable response of infants with patent ductus arteriosus to indomethacin.