Nile Red is a fluorescent allosteric substrate of cytochrome P450 3A4

Cytochrome P450 3A4 (CYP3A4) plays a critical role in the metabolism of many drugs. CYP3A4 exhibits extraordinary substrate promiscuity and unusual allosteric kinetics. In addition, many CYPs catalyze sequential oxidations on a single substrate, but in most cases, mechanistic details of these processes are not well-established. As a result, in vivo clearance of many drugs and their metabolites is difficult to predict on the basis of the complex in vitro kinetics, and new in vitro probes are required to understand these behaviors. The near-IR fluorescent probe Nile Red, which has strong solvatochromic behavior, was investigated as a probe of allostery and sequential metabolism with CYP3A4. Nile Red binds with apparent Kd values of 0.05 and 2.3 muM, based on a sigmoidal dependence of heme spin state on Nile Red concentration, where the first equivalent of Nile Red increased the high-spin fraction by only 13% of the total change at saturation. Mass spectrometry analysis indicates that Nile Red is metabolized sequentially by CYP3A4 to the N-monoethyl and N-desethyl products, confirming that the immediate vicinity of the heme iron is one binding site. In the presence of CYP3A4, steady-state fluorescence emission and excitation spectra, as well as excited-state lifetimes at varying Nile Red concentrations, indicate a high-affinity site that modulates the fluorescent properties of Nile Red. The Nile Red binding site is competitively eliminated by itraconazole, which is a high-affinity ligand known to coordinate to the heme iron. Together, the data suggest that Nile Red binds to the active site with high affinity ( approximately 50 nM), where it is desolvated in a low-dielectric environment. In addition, Nile Red is sequentially oxidized at rates comparable to or faster than those of other in vitro probes, which emphasizes its utility in the further examination of this important kinetic phenomenon in vitro.     

Effect of ethanol on spectral binding, inhibition, and activity of CYP3A4 with an antiretroviral drug nelfinavir

Cytochrome P450 3A4 (CYP3A4) is the most abundant CYP enzyme in the liver and metabolizes approximately 50% of the drugs, including antiretrovirals. Although CYP3A4 induction by ethanol and impact of CYP3A4 on drug metabolism and toxicity is known, CYP3A4-ethanol physical interaction and its impact on drug binding, inhibition, or metabolism is not known. Therefore, we studied the effect of ethanol on binding and inhibition of CYP3A4 with a representative protease inhibitor, nelfinavir, followed by the effect of alcohol on nelfinavir metabolism. Our initial results showed that methanol, ethanol, isopropanol, isobutanol, and isoamyl alcohol bind in the active site of CYP3A4 and exhibit type I spectra. Among these alcohol compounds, ethanol showed the lowest K_D (5.9 ± 0.34 mM), suggesting its strong binding affinity with CYP3A4. Ethanol (20 mM) decreased the K_D of nelfinavir by >5-fold (0.041 ± 0.007 vs. 0.227 ± 0.038 μM). Similarly, 20 mM ethanol decreased the IC₅₀ of nelfinavir by >3-fold (2.6 ± 0.5 vs. 8.3 ± 3.1 μM). These results suggest that ethanol facilitates binding of nelfinavir with CYP3A4. Furthermore, we performed nelfinavir metabolism using LCMS. Although ethanol did not alter k_cat, it decreased the K_m of nelfinavir, suggesting a decrease in catalytic efficiency (k_cat/K_m). This is an important finding because alcoholism is prevalent in HIV-1-infected persons and alcohol is shown to decrease the response to antiretroviral therapy.     

Wavelength dependence of the fluorescence emission under conditions of open and closed Photosystem II reaction centres in the green alga Chlorella sorokiniana

The fluorescence emission characteristics of the photosynthetic apparatus under conditions of open (F₀) and closed (F_M) Photosystem II reaction centres have been investigated under steady state conditions and by monitoring the decay lifetimes of the excited state, in vivo, in the green alga Chlorella sorokiniana. The results indicate a marked wavelength dependence of the ratio of the variable fluorescence, F_V = F_M − F₀, over F_M, a parameter that is often employed to estimate the maximal quantum efficiency of Photosystem II. The maximal value of the F_V/F_M ratio is observed between 660 and 680 nm and the minimal in the 690–730 nm region. It is possible to attribute the spectral variation of F_V/F_M principally to the contribution of Photosystem I fluorescence emission at room temperature. Moreover, the analysis of the excited state lifetime at F₀ and F_M indicates only a small wavelength dependence of Photosystem II trapping efficiency in vivo.     

Synthesis and properties of Asante Calcium Red—A novel family of long excitation wavelength calcium indicators

Although many synthetic calcium indicators are available, a search for compounds with improved characteristics continues. Here, we describe the synthesis and properties of Asante Calcium Red-1 (ACR-1) and its low affinity derivative (ACR-1-LA) created by linking BAPTA to seminaphthofluorescein. The indicators combine a visible light (450–540 nm) excitation with deep-red fluorescence (640 nm). Upon Ca²⁺ binding, the indicators raise their fluorescence with longer excitation wavelengths producing higher responses. Although the changes occur without any spectral shifts, it is possible to ratio Ca²⁺-dependent (640 nm) and quasi-independent (530 nm) emission when using visible (<490 nm) or multiphoton (∼780 nm) excitation. Therefore, both probes can be used as single wavelength or, less dynamic, ratiometric indicators. Long indicator emission might allow easy [Ca²⁺]_i measurement in GFP expressing cells. The indicators bind Ca²⁺ with either high (K_d = 0.49 ± 0.07 μM; ACR-1) or low affinity (K_d = 6.65 ± 0.13 μM; ACR-1-LA). Chelating Zn²⁺ (K_d = 0.38 ± 0.02 nM) or Mg²⁺ (K_d ∼ 5 mM) slightly raises and binding Co²⁺ quenches dye fluorescence. New indicators are somewhat pH-sensitive (pK_a = 6.31 ± 0.07), but fairly resistant to bleaching. The probes are rather dim, which combined with low AM ester loading efficiency, might complicate in situ imaging. Despite potential drawbacks, ACR-1 and ACR-1-LA are promising new calcium indicators.     

Abacavir and warfarin modulate allosterically kinetics of NO dissociation from ferrous nitrosylated human serum heme-albumin

Human serum albumin (HSA) participates to heme scavenging, in turn HSA-heme binds gaseous diatomic ligands at the heme-Fe-atom. Here, the effect of abacavir and warfarin on denitrosylation kinetics of HSA-heme-Fe(II)-NO (i.e., k_off) is reported. In the absence of drugs, the value of k_off is (1.3 ± 0.2) × 10⁻⁴ s⁻¹. Abacavir and warfarin facilitate NO dissociation from HSA-heme-Fe(II)-NO, the k_off value increases to (8.6 ± 0.9) × 10⁻⁴ s⁻¹. From the dependence of k_off on the drug concentration, values of the dissociation equilibrium constant for the abacavir and warfarin binding to HSA-heme-Fe(II)-NO (i.e., K = (1.2 ± 0.2) × 10⁻³ M and (6.2 ± 0.7) × 10⁻⁵ M, respectively) were determined. The increase of k_off values reflects the stabilization of the basic form of HSA-heme-Fe by ligands (e.g., abacavir and warfarin) that bind to Sudlow’s site I. This event parallels the stabilization of the six-coordinate derivative of the HSA-heme-Fe(II)-NO atom. Present data highlight the allosteric modulation of HSA-heme-Fe(II) reactivity by heterotropic effectors.     

Inhibition of human drug metabolizing cytochrome P450 enzymes by plant isoquinoline alkaloids

The human cytochrome P450 (CYP) enzymes play a major role in the metabolism of endobiotics and numerous xenobiotics including drugs. Therefore it is the standard procedure to test new drug candidates for interactions with CYP enzymes during the preclinical development phase. The purpose of this study was to determine in vitro CYP inhibition potencies of a set of isoquinoline alkaloids to gain insight into interactions of novel chemical structures with CYP enzymes. These alkaloids (n = 36) consist of compounds isolated from the Papaveraceae family (n = 20), synthetic analogs (n = 15), and one commercial compound. Their inhibitory activity was determined towards all principal human drug metabolizing CYP enzymes: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4. All alkaloids were assayed in vitro in a 96-well plate format using pro-fluorescent probe substrates and recombinant human CYP enzymes. Many of these alkaloids inhibited the CYP3A4 form, with 30/36 alkaloids inhibiting CYP3A4 with at least moderate potency (IC₅₀ < 10 μM) and 15/36 inhibiting CYP3A4 potently (IC₅₀ < 1 μM). Among them corydine, parfumine and 8-methyl-2,3,10,11-tetraethoxyberbine were potent and selective inhibitors for CYP3A4. CYP2D6 was inhibited with at least moderate potency by 26/34 alkaloids. CYP2C19 was inhibited by 15/36 alkaloids at least moderate potently, whereas CYP1A2, CYP2B6, CYP2C8, and CYP2C9 were inhibited to a lesser degree. CYP2A6 was not significantly inhibited by any of the alkaloids. The results provide initial structure-activity information about the interaction of isoquinoline alkaloids with major human xenobiotic-metabolizing CYP enzymes, and illustrate potential novel structures as CYP form-selective inhibitors.     

The tryptophan photoproduct 6-formylindolo[3,2-b]carbazole (FICZ) binds multiple AHRs and induces multiple CYP1 genes via AHR2 in zebrafish

The tryptophan photooxidation product 6-formylindolo[3,2-b]carbazole (FICZ) has been proposed as a physiological ligand for the mammalian aryl hydrocarbon receptor (AHR), which it binds with high-affinity, inducing expression of cytochrome P450 1A1 (CYP1A1). We investigated whether the response to FICZ is evolutionarily conserved in vertebrates by measuring FICZ binding to two zebrafish AHRs (AHR1B and AHR2) and its ability to induce zebrafish CYP1 genes (CYP1A, CYP1B1, CYP1C1, CYP1C2, and CYP1D1) in vivo. Exposure of zebrafish embryos (48 h-post-fertilization; hpf) to 10 nM FICZ for 6 h caused strong induction of CYP1A mRNA and a statistically significant but modest induction of CYP1B1 and CYP1C1. Neither CYP1C2 nor CYP1D1 expression was induced by FICZ under the conditions of dose, time or developmental stage examined here. CYP1A induction was significantly greater after 6 h than after 12 h of exposure to FICZ, suggesting a rapid degradation of inducer. The 6-h EC₅₀ values for induction of CYP1A and CYP1B1 by FICZ were 0.6 and 0.5 nM compared to 72-h EC₅₀ values of 2.3 and 2.7 nM for PCB126, indicating that in zebrafish embryos FICZ is a more potent inducer than PCB126. FICZ at 10 nM was able to completely displace binding of 2,3,7,8-tetrachloro-1,6[³H]-dibenzo-p-dioxin to in vitro-expressed zebrafish AHR2 and AHR1B. Inhibition of AHR2 translation in zebrafish embryos by an AHR2-specific morpholino antisense oligonucleotide decreased the induction of CYP1A and CYP1B1 by FICZ and by PCB126. Together, these results demonstrate that FICZ is a potent AHR agonist in zebrafish, inducing expression of multiple CYP1 genes largely through AHR2. Evolutionary conservation of the response to FICZ is consistent with a possible role as an endogenous signaling molecule acting through the AHR.     

Discovery of Novel Allosteric Effectors Based on the Predicted Allosteric Sites for Escherichia coli D-3-Phosphoglycerate Dehydrogenase

D-3-phosphoglycerate dehydrogenase (PGDH) from Escherichia coli catalyzes the first critical step in serine biosynthesis, and can be allosterically inhibited by serine. In a previous study, we developed a computational method for allosteric site prediction using a coarse-grained two-state Gō Model and perturbation. Two potential allosteric sites were predicted for E. coli PGDH, one close to the active site and the nucleotide binding site (Site I) and the other near the regulatory domain (Site II). In the present study, we discovered allosteric inhibitors and activators based on site I, using a high-throughput virtual screen, and followed by using surface plasmon resonance (SPR) to eliminate false positives. Compounds 1 and 2 demonstrated a low-concentration activation and high-concentration inhibition phenomenon, with IC₅₀ values of 34.8 and 58.0 µM in enzymatic bioassays, respectively, comparable to that of the endogenous allosteric effector, L-serine. For its activation activity, compound 2 exhibited an AC₅₀ value of 34.7 nM. The novel allosteric site discovered in PGDH was L-serine- and substrate-independent. Enzyme kinetics studies showed that these compounds influenced K_m, k_cat, and k_cat/K_m. We have also performed structure-activity relationship studies to discover high potency allosteric effectors. Compound 2-2, an analog of compound 2, showed the best in vitro activity with an IC₅₀ of 22.3 µM. Compounds targeting this site can be used as new chemical probes to study metabolic regulation in E. coli. Our study not only identified a novel allosteric site and effectors for PGDH, but also provided a general strategy for designing new regulators for metabolic enzymes.     

Somatostatin: A Novel Substrate and a Modulator of Insulin-Degrading Enzyme Activity

Insulin-degrading enzyme (IDE) is an interesting pharmacological target for Alzheimer's disease (AD), since it hydrolyzes β-amyloid, producing non-neurotoxic fragments. It has also been shown that the somatostatin level reduction is a pathological feature of AD and that it regulates the neprilysin activity toward β-amyloid.In this work, we report for the first time that IDE is able to hydrolyze somatostatin [k_cat (s^− 1) = 0.38 (± 0.05); K_m (M) = 7.5 (± 0.9) × 10^− 6] at the Phe6-Phe7 amino acid bond. On the other hand, somatostatin modulates IDE activity, enhancing the enzymatic cleavage of a novel fluorogenic β-amyloid through a decrease of the K_m toward this substrate, which corresponds to the 10-25 amino acid sequence of the Aβ(1-40). Circular dichroism spectroscopy and surface plasmon resonance imaging experiments show that somatostatin binding to IDE brings about a concentration-dependent structural change of the secondary and tertiary structure(s) of the enzyme, revealing two possible binding sites. The higher affinity binding site disappears upon inactivation of IDE by ethylenediaminetetraacetic acid, which chelates the catalytic Zn²⁺ ion. As a whole, these features suggest that the modulatory effect is due to an allosteric mechanism: somatostatin binding to the active site of one IDE subunit (where somatostatin is cleaved) induces an enhancement of IDE proteolytic activity toward fluorogenic β-amyloid by another subunit. Therefore, this investigation on IDE-somatostatin interaction contributes to a more exhaustive knowledge about the functional and structural aspects of IDE and its pathophysiological implications in the amyloid deposition and somatostatin homeostasis in the brain.     

An allosteric circuit in caspase-1

Structural studies of caspase-1 reveal that the dimeric thiol protease can exist in two states: in an on-state, when the active site is occupied, or in an off-state, when the active site is empty or when the enzyme is bound by a synthetic allosteric ligand at the dimer interface ∼ 15 Å from the active site. A network of 21 hydrogen bonds from nine side chains connecting the active and allosteric sites change partners when going between the on-state and the off-state. Alanine-scanning mutagenesis of these nine side chains shows that only two of them—Arg286 and Glu390, which form a salt bridge—have major effects, causing 100- to 200-fold reductions in catalytic efficiency (k_cat/K_m). Two neighbors, Ser332 and Ser339, have minor effects, causing 4- to 7-fold reductions. A more detailed mutational analysis reveals that the enzyme is especially sensitive to substitutions of the salt bridge: even a homologous R286K substitution causes a 150-fold reduction in k_cat/K_m. X-ray crystal structures of these variants suggest the importance of both the salt bridge interaction and the coordination of solvent water molecules near the allosteric binding pocket. Thus, only a small subset of side chains from the larger hydrogen bonding network is critical for activity. These form a contiguous set of interactions that run from one active site through the allosteric site at the dimer interface and onto the second active site. This subset constitutes a functional allosteric circuit or “hot wire” that promotes site-to-site coupling.     

Remarkable chiral and luminescent properties of novel Yb(III) and Eu(III) complexes containing BINAPO ligand

Lanthanide complexes are of great importance for their prospective applications in wide range of science and technology. Chiral lanthanide complexes can constitute stereo-discriminating probes in biological media, owing to the luminescent properties of the rare-earth ions. Sensitized emission with narrow bandwidth, having fast radiation rate and high emission quantum efficiency are the main perspective for synthesizing the complexes. Attention has been given on remarkable chirality with high dissymmetry factors (g = Δε_ext/ε_max) of the complexes. For this purpose, beta-diketonato ligands with chiral BINAPO (1,1′-binapthyl phosphine oxide) ligand were chosen to achieve the goal. The complexes [Ln(TFN)₃(S-BINAPO)](TFN = 4,4,4-trifluoro-1(2-napthyl)-1,3-butanedione), [Ln(HFT)₃(S-BINAPO)] (HFT = 4,4,5,5,6,6,6-heptafluoro-1-(2-thienyl)-1,3-hexanedione) and [Ln(HFA)₃(S-BINAPO)](hfa = hexafluoroacetylacetonate) (where Ln = Yb, Eu) were synthesized. The complex, [Eu(TFN)₃(S-BINAPO)] gives strong red emission at 615 nm with narrow emission band (<10 nm) when excited by 465 nm light with quantum efficiency 86%. The dissymmetry factors (g = Δε_ext/ε_max) corresponding to the ⁷F₁ → ⁵D₀ transition at 590 nm is 0.091 for [Eu(TFN)₃(S-BINAPO)] and for [Yb(hfa)₃(S-BINAPO)](hfa = hexafluoroacetylacetonate) corresponding to the ²F_7/2 → ²F_5/2 transitions is 0.12, are among the largest values for both Eu and Yb complexes to date, respectively. The Eu complexes, [Eu(HFT)₃(S-BINAPO)] and [Eu(TFN)₃(S-BINAPO)] are found to be spontaneously emissive, showing bright red emission, when placed in sunlight or even in the laboratory when light is switched on.     

Allosteric Regulation of the Human and Mouse Deoxyribonucleotide Triphosphohydrolase Sterile α-Motif/Histidine-Aspartate Domain-containing Protein 1 (SAMHD1)

The deoxyribonucleotide triphosphohydrolase SAMHD1 restricts lentiviral infection by depleting the dNTPs required for viral DNA synthesis. In cultured human fibroblasts SAMHD1 is expressed maximally during quiescence preventing accumulation of dNTPs outside S phase. siRNA silencing of SAMHD1 increases dNTP pools, stops cycling human cells in G₁, and blocks DNA replication. Surprisingly, knock-out of the mouse gene does not affect the well being of the animals. dNTPs are both substrates and allosteric effectors for SAMHD1. In the crystal structure each subunit of the homotetrameric protein contains one substrate-binding site and two nonidentical effector-binding sites, site 1 binding dGTP, site 2 dGTP or dATP. Here we compare allosteric properties of pure recombinant human and mouse SAMHD1. Both enzymes are activated 3–4-fold by allosteric effectors. We propose that in quiescent cells where SAMHD1 is maximally expressed GTP binds to site 1 with very high affinity, stabilizing site 2 of the tetrameric structure. Any canonical dNTP can bind to site 2 and activate SAMHD1, but in cells only dATP or dTTP are present at sufficient concentrations. The apparent K_m for dATP at site 2 is ∼10 μm for mouse and 1 μm for human SAMHD1, for dTTP the corresponding values are 50 and 2 μm. Tetrameric SAMHD1 is activated for the hydrolysis of any dNTP only after binding of a dNTP to site 2. The lower K_m constants for human SAMHD1 induce activation at lower cellular concentrations of dNTPs thereby limiting the size of dNTP pools more efficiently in quiescent human cells.     

Synthesis, crystal structure and solid state photoluminescence of [Pt(trpy)(CCPh)]SbF6 (trpy=2,2′:6′,2″-terpyridine)

The synthesis and characterisation of an orange polymorph of [Pt(trpy)(CCPh)]SbF₆ is described where trpy = 2,2′:6′,2″-terpyridine. An X-ray crystal structure determination at 293 K reveals that the cations are planar and stacked head-to-tail with alternating Pt?Pt distances of 3.604(1) and 4.412(1) Å. The perpendicular distances between successive cation planes are constant along the stack at a value of 3.33 Å. Crystal structure determinations at 240 and 200 K show that reducing the temperature to 200 K has no significant effect on the cation arrangement. However, below 200 K there is a phase change that we have not been able to characterise, but which has an effect on the solid state photoluminescence exhibited by [Pt(trpy)(CCPh)]SbF₆. Thus, whereas at temperatures of ?200 K, a broad peak with two components at ca. 566 and 597 nm is observed, below 200 K a longer wavelength peak develops that red-shifts as the temperature is lowered [λ(em)_max = 637 nm at 80 K]. We assign the ?200 K emission as ³MLLCT in origin, since the X-ray data show that ligand-ligand (LL) and not metal-metal (MM) interactions are important at T ? 200 K. On the other hand, the long wavelength emission observed below 200 K is typical of ³MMLCT emission, suggesting that the phase change leads to d_z²(Pt)-d_z²(Pt) orbital interactions. Of particular interest is that the cation exhibits ³MLCT emission in dichloromethane that maximizes at 619 nm, i.e., the high temperature solid state emission occurs at a shorter wavelength, an unexpected result since intermolecular interactions in the solid usually cause the emission to occur at longer wavelengths. A possible explanation for this unexpected result is given.     

Cytochrome P450 6M2 from the malaria vector Anopheles gambiae metabolizes pyrethroids: Sequential metabolism of deltamethrin revealed

Resistance to pyrethroid insecticides in the malaria vector Anopheles gambiae is a major threat to malaria control programmes. Cytochome P450-mediated detoxification is an important resistance mechanism. CYP6M2 is over-expressed in wild populations of permethrin resistant A. gambiae but its role in detoxification is not clear. CYP6M2 was expressed in Escherichia coli and a structural model was produced to examine its role in pyrethroid metabolism. Both permethrin and deltamethrin were metabolized. Rates were enhanced by A. gambiae cytochrome b₅ with kinetic parameters of K_M = 11 ± 1 ??M and k_cat = 6.1 ± 0.4 per min for permethrin (1:1 cis-trans) and K_M = 2.0 ± 0.3 ??M and k_cat = 1.2 ± 0.1 per min for deltamethrin. Mass spectrometry and NMR analysis identified 4′-hydroxy deltamethrin and hydroxymethyl deltamethrin as major and minor deltamethrin metabolites respectively. Secondary breakdown products included cyano(3-hydroxyphenyl)methyl deltamethrate and deltamethric acid. CYP6M2 was most highly transcribed in the midgut and Malpighian tubules of adult A. gambiae, consistent with a role in detoxification. Our data indicates that CYP6M2 plays an important role in metabolic resistance to pyrethroids and thus an important target for the design of new tools to combat malaria.     

A shuffled CYP2C library with a high degree of structural integrity and functional versatility

Cytochrome P450 (CYP) enzymes involved in mammalian xenobiotic metabolism are attractive targets for the engineering of biocatalysts since they have broad and overlapping substrate and reaction substrate specificities. In this report, a library of chimeric mutants was prepared from CYP2C8, CYP2C9, CYP2C18 and CYP2C19 by DNA family shuffling. Twelve randomly selected clones were fully sequenced and showed 9 ± 2 crossovers and 1.5 ± 0.5 spontaneous mutations per ∼1.5 kbp open reading frame. CYP hemoprotein expression was observed in 50% (microaerobic culture) to 54% (aerobic culture) of clones. The functional diversity of the library was assessed using three luminogenic substrates, diclofenac and indole as probe substrates. A random sample of 26 clones revealed two clones with activity towards luciferin ME, one towards luciferin H and five towards diclofenac 4′-hydroxylation. One mutant showed activity towards all three substrates. Of 96 clones screened on solid media, one showed elevated indigo production compared to the parental forms. Turnover rates for luciferin ME and H metabolism by CYP2C9 and mutants were at least one order of magnitude higher in experiments with membranes compared to whole cells, consistent with impaired product egress from cells. Apparent K_m values were increased in whole cell incubations with luciferin H suggesting impaired access of the substrate to the active site of the enzymes in whole cells. Finally screening with a panel of CYP2C ligands using CYP2C9 or active mutants revealed different patterns of inhibition and heteroactivation of metabolism of luciferin analogs.     

Is o-carborane photoluminescent?

At 77 K in the solid state and in ethanol glasses, o-carborane (1,2-C₂B₁₀H₁₂) shows a relatively intense (? ∼ 10⁻³ at λ_exc = 260 nm) structured photoluminescence (λ_max = 441 nm).In agreement with the rather slow decay of this emission (τ ∼ 4 s) it is suggested to be a phosphorescence. While it appears to be a genuine property of o-carborane an unknown impurity as origin of this luminescence is not completely excluded.     

Ibuprofen modulates allosterically NO dissociation from ferrous nitrosylated human serum heme-albumin by binding to three sites

Human serum albumin (HSA) is a monomeric allosteric protein. Here, the effect of ibuprofen on denitrosylation kinetics (k_off) and spectroscopic properties of HSA-heme-Fe(II)-NO is reported. The k_off value increases from (1.4 ± 0.2) × 10⁻⁴ s⁻¹, in the absence of the drug, to (9.5 ± 1.2) × 10⁻³ s⁻¹, in the presence of 1.0 × 10⁻² M ibuprofen, at pH 7.0 and 10.0 °C. From the dependence of k_off on the drug concentration, values of the dissociation equilibrium constants for ibuprofen binding to HSA-heme-Fe(II)-NO (K₁ = (3.1 ± 0.4) × 10⁻⁷ M, K₂ = (1.7 ± 0.2) × 10⁻⁴ M, and K₃ = (2.2 ± 0.2) × 10⁻³ M) were determined. The K₃ value corresponds to the value of the dissociation equilibrium constant for ibuprofen binding to HSA-heme-Fe(II)-NO determined by monitoring drug-dependent absorbance spectroscopic changes (H = (2.6 ± 0.3) × 10⁻³ M). Present data indicate that ibuprofen binds to the FA3-FA4 cleft (Sudlow’s site II), to the FA6 site, and possibly to the FA2 pocket, inducing the hexa-coordination of HSA-heme-Fe(II)-NO and triggering the heme-ligand dissociation kinetics.     

A new blue luminescent dichlorido-bridged dinuclear copper(II) complex with DNA binding and cytotoxic activities: Synthesis, structure and DFT studies

A blue luminescent dichlorido-bridged dinuclear copper(II) (S = 1/2) complex, [Cu^II₂(HL)₂(μ-Cl)₂]·2H₂O, 1a was synthesized with the 1:1 reaction of the acyclic tridentate salicylaldehyde 2-pyridyl hydrazone ligand, HL, 1. The complex 1a displays multiple bands in the visible region (400-470 nm). The association constant (K_ass, UV-Vis) was found to be 1.186 × 10⁴ for 1a at 298 K. The copper(II)-copper(III) oxidation potential lies near 0.32 V versus Ag/AgCl electrode. On excitation at 390 nm, the ligand 1 strongly emits at 444 nm due to an intraligand ¹(π-π^∗) transition. Upon complexation with copper(II) the emission peak is slightly red shifted (λ_ex 390 nm, λ_em 450 nm, F/F₀ 0.81) with little quenching. Molecular structure of 1a (Cu···Cu 3.523 Å) has been determined by single crystal X-ray diffraction studies. DFT and TDDFT calculations strongly support the spectral behavior of the ligand and the complex. The complex 1a exhibits a strong interaction towards DNA as revealed from the K_b (intrinsic binding constant) 2.05 × 10⁴ M⁻¹ and K_sv (Stern-Volmer quenching constant) 2.47 values. The complex exhibits cytotoxic effect and the LD₅₀ value for HeLa cells was calculated as 5.44 μM at which the cell cycle was arrested at G₂/M phase.