Studies on interaction between Vitamin B12 and human serum albumin

The binding reaction between Vitamin B12 (B12, cyanocobalamin) and human serum albumin (HSA) was investigated by fluorescence quenching, UV–vis absorption and circular dichroism (CD) spectroscopy. Under simulative physiological conditions, fluorescence quenching data revealed that the quenching constants (K_sv) are 3.99 × 10⁴, 4.33 × 10⁴, 4.76 × 10⁴ and 5.16 × 10⁴ M⁻¹ at 292, 298, 304 and 310 K, respectively. The number of binding sites, n is almost constant around 1.0. On the basis of the results of fluorescence quenching the mechanism of the interaction of B12 with HSA has been found to be a dynamic quenching procedure. Thermodynamic parameters ΔH^Θ = −13.38 kJ mol⁻¹, ΔS^Θ = 66.73 J mol⁻¹ K⁻¹ were calculated based on the binding constant. These suggested that the binding reaction was enthalpy and entropy driven, and the electrostatic interaction played major role in stabilizing the reversible complex. The binding distance r = 5.5 nm between HSA and B12 was obtained according to Förster theory of energy transfer. The effect of B12 on the conformation of HSA was analyzed by synchronous fluorescence and CD spectroscopy. Synchronous spectra indicated that the polarity around the tryptophan (Trp214) residues of HSA was decreased and its hydrophobicity was increased; however, the α-helix content of the protein was predominant in the secondary structure but the CD spectra indicated that B12 induced minor conformational changes of HSA.     

Interaction of kaempferol with human serum albumin: A fluorescence and circular dichroism study

The interaction of kaempferol (kaemp), a natural flavonoid to which antioxidative, anti-inflammatory and cardio-protective biological activities have been attributed, with human serum albumin (HSA), the main in vivo transporter of exogenous substances, was investigated by steady-state, synchronous fluorescence and circular dichroism spectroscopies. The binding constant, K, and number of binding sites, n, were computed using literature models that showed satisfactory agreement and revealed a strong interaction (K ∼ 3.5 × 10⁵ M⁻¹, n ∼ 1). The binding process was investigated at temperatures in the range 298–313 K, allowing for the evaluation of the thermodynamic parameters, which indicate the occurrence of hydrogen bonding interactions. The distance between kaemp and the tryptophan residue of HSA was estimated at 2.7 nm using Förster's theory of nonradiative resonance energy transfer. Using circular dichroism we evidenced some degree of HSA defolding upon binding.     

Characterization of interaction and the effect of carbamazepine on the structure of human serum albumin

The binding of carbamazepine (CBZ) to human serum albumin (HSA) was investigated under simulative physiological conditions. In this study, intrinsic fluorescence of tryptophan-214 in HSA was monitored upon the addition of CBZ. Binding constant of CBZ–HSA was calculated by the remarkable static quenching effect of CBZ and found to be (2.081 ± 0.023) × 10⁴ M⁻¹. The fluorimetric results revealed that the hydrophobic interaction was a predominant intermolecular force for stabilizing the complex, which is also in agreement with the results obtained from voltammetric approach. Three site probes, warfarin, ibuprofen and digitoxin, were employed in fluorescence displacement experiments to locate the exact binding site for CBZ in HSA. The alteration in secondary structure of protein in the presence of CBZ was confirmed by the evidences from circular dichroism and FT-IR spectroscopy. Further, the distance r between donor (Trp-214) and acceptor (CBZ) was obtained according to fluorescence resonance energy transfer (FRET).     

Interaction of sanguinarine and its dihydroderivative with the Na/K-ATPase. Complex view on the old problem

The effects of sanguinarine (SG) and its metabolite dihydrosanguinarine (DHSG) on Na⁺/K⁺-ATPase were investigated using fluorescence spectroscopy. The results showed that the enzyme in E1 conformation can bind both charged and neutral (pseudobase) forms of SG with a K_D = 7.2 ± 2.0 μM or 11.7 ± 0.9 μM, while the enzyme in E2 conformation binds only the charged form of SG with a K_D = 4.7 ± 1.1 μM. Fluorescence quenching experiments suggest that the binding site in E1 conformation is located on the surface of the enzyme for both forms but the binding site in E2 conformation is protected from the solvent. We found no evidence for interaction of Na⁺/K⁺-ATPase and DHSG. This implies that any in vivo effect of SG attributable to inhibition of Na⁺/K⁺-ATPase can be considered only prior to SG → DHSG transformation in the gastro-intestinal tract and/or blood. Hence, Na⁺/K⁺-ATPase inhibition will be effective in SG topical application but its duration will be very limited in SG oral or parenteral administration.     

Affinity and translocation relationships via hPEPT1 of H-Xaa-Ser-OH dipeptides: Evaluation of H-Phe-Ser-OH as a pro-moiety for ibuprofen and benzoic acid prodrugs

The intestinal di/tri-peptide transporter 1 (hPEPT1) has been suggested as a drug delivery target for peptide-based prodrugs. The aim of the study was to synthesize a series of 11 serine-containing dipeptides (H-X_aa-Ser-OH) and to investigate the relationship between binding to and transport via hPEPT1. An additional aim was to design a dipeptide which could serve as a pro-moiety for prodrugs targeted to hPEPT1. X_aa was chosen from the 20 proteogenic amino acids. The dipeptides were synthesized using solid phase peptide synthesis. The K_i-values of H-X_aa-Ser-OH dipeptides for hPEPT1 in MDCK/hPEPT1 cells ranged from 0.14 mM (log IC₅₀ = −0.85 ± 0.06) for H-Tyr-Ser-OH to 0.89 mM (log IC₅₀ = −0.09 ± 0.02) for H-Gly-Ser-OH, as measured in a competition assay with [¹⁴C]Gly-Sar. The dipeptides were translocated via hPEPT1 with K_m-values in the range of 0.20 (log IC₅₀ = −0.69 ± 0.04) for H-Met-Ser-OH to 1.04 (log IC₅₀ = 0.02 ± 0.04) mM for H-Gly-Ser-OH. The relationship between ligand and transportate properties indicated that the initial binding of the ligand to hPEPT1 is the major determinant for translocation of the investigated dipeptides. H-Phe-Ser-OH was selected as a pro-moiety, and two prodrugs were synthesized, i.e. H-Phe-Ser(Ibuprofyl)-OH and H-Phe-Ser(Bz)-OH. Both H-Phe-Ser(Ibuprofyl)-OH and H-Phe-Ser(Bz)-OH had high affinity for hPEPT1 with K_i-values of 0.07 mM (log IC₅₀ = −0.92 ± 0.12) and 0.12 mM (log IC₅₀ = −1.17 ± 0.40), respectively. However, none of the prodrugs were translocated via hPEPT1. This indicated that the coupling of the drug compounds to the peptide backbone did not decrease transporter binding, but abolished translocation, and that high affinity of prodrugs does not necessarily translate into favourable permeation properties.     

Improved photostability and reduced skin permeation of tretinoin: Development of a semisolid nanomedicine

The aims of this work were to increase the photostability and to reduce the skin permeation of tretinoin through nanoencapsulation. Tretinoin is widely used in the topical treatment of various dermatological diseases such as acne, psoriasis, skin cancer, and photoaging. Tretinoin-loaded lipid-core polymeric nanocapsules were prepared by interfacial deposition of a preformed polymer. Carbopol hydrogels containing nanoencapsulated tretinoin presented a pH value of 6.08 ± 0.14, a drug content of 0.52 ± 0.01 mg g⁻¹, pseudoplastic rheological behavior, and higher spreadability than a marketed formulation. Hydrogels containing nanoencapsulated tretinoin demonstrated a lower photodegradation (24.17 ± 3.49%) than the formulation containing the non-encapsulated drug (68.64 ± 2.92%) after 8 h of ultraviolet A irradiation. The half-life of the former was seven times higher than the latter. There was a decrease in the skin permeability coefficient of the drug by nanoencapsulation, independently of the dosage form. The liquid suspension and the semisolid form provided K_p = 0.31 ± 0.15 and K_p = 0.33 ± 0.01 cm s⁻¹, respectively (p ? 0.05), while the samples containing non-encapsulated tretinoin showed K_p = 1.80 ± 0.27 and K_p = 0.73 ± 0.12 cm s⁻¹ for tretinoin solution and hydrogel, respectively. Lag time was increased two times by nanoencapsulation, meaning that the drug is retained for a longer time on the skin surface.     

Preferential binding of insecticide phorate with sub-domain IIA of human serum albumin induces protein damage and its toxicological significance

Phorate, an organophosphorus insecticide is known for its adverse effects on acetylcholinesterase, and other neuronal and pulmonary activities. Most likely, the toxicity of drugs/agrochemicals is modulated through cellular distribution bound to plasma proteins. Therefore, the in vitro interaction of phorate with human serum albumin (HSA) has been investigated, using sensitive techniques like fluorescence spectroscopy and circular dichroism, to ascertain its binding mechanism and toxicological implications. Fluorescence studies revealed the quenching constant (Ksv) as 2.5 × 10⁴ M⁻¹ and binding affinity (Ka) as 2.96 × 10⁴ M⁻¹ (r² = 0.99), with a primary binding site of phorate at sub-domain IIA of HSA. Circular dichroism (CD) data demonstrated a noticeable reduction in secondary structure (α-helical content) of phorate treated HSA. Albumin treated with 200-1000 μM phorate released significant amounts of acid soluble amino and carbonyl groups, whereas higher concentrations resulted in protein fragmentation. It is postulated that the 1′-O and 3-O alkyl groups of phorate have a role in binding with electrophilic centers of Trp 214, and Arg 218/Lys 195, respectively. Moreover, the significant ultrastructural changes, reactive oxygen species (ROS) generation, mitochondrial damage and cell death in phorate treated cultured human amnion epithelial (WISH) cells, elucidated phorate induced cellular toxicity.     

Mechanism of action of AZD0865, a K+-competitive inhibitor of gastric H+,K+-ATPase

AZD0865 is a member of a drug class that inhibits gastric H⁺,K⁺-ATPase by K⁺-competitive binding. The objective of these experiments was to characterize the mechanism of action, selectivity and inhibitory potency of AZD0865 in vitro. In porcine ion-leaky vesicles at pH 7.4, AZD0865 concentration-dependently inhibited K⁺-stimulated H⁺,K⁺-ATPase activity (IC₅₀ 1.0 ± 0.2 μM) but was more potent at pH 6.4 (IC₅₀ 0.13 ± 0.01 μM). The IC₅₀ values for a permanent cation analogue, AR-H070091, were 11 ± 1.2 μM at pH 7.4 and 16 ± 1.8 μM at pH 6.4. These results suggest that the protonated form of AZD0865 inhibits H⁺,K⁺-ATPase. In ion-tight vesicles, AZD0865 inhibited H⁺,K⁺-ATPase more potently (IC₅₀ 6.9 ± 0.4 nM) than in ion-leaky vesicles, suggesting a luminal site of action. AZD0865 inhibited acid formation in histamine- or dibutyryl-cAMP-stimulated rabbit gastric glands (IC₅₀ 0.28 ± 0.01 and 0.26 ± 0.003 μM, respectively). In ion-leaky vesicles at pH 7.4, AZD0865 (3 μM) immediately inhibited H⁺,K⁺-ATPase activity by 88 ± 1%. Immediately after a 10-fold dilution H⁺,K⁺-ATPase inhibition was 41%, indicating reversible binding of AZD0865 to gastric H⁺,K⁺-ATPase. In contrast to omeprazole, AZD0865 inhibited H⁺,K⁺-ATPase activity in a K⁺-competitive manner (K_i 46 ± 3 nM). AZD0865 inhibited the process of cation occlusion concentration-dependently (IC₅₀ 1.7 ± 0.06 μM). At 100 μM, AZD0865 reduced porcine renal Na⁺,K⁺-ATPase activity by 9 ± 2%, demonstrating a high selectivity for H⁺,K⁺-ATPase. Thus, AZD0865 potently, K⁺-competitively, and selectively inhibits gastric H⁺,K⁺-ATPase activity and acid formation in vitro, with a fast onset of effect.     

Bivalent sequential binding of docetaxel to methyl-β-cyclodextrin

New docetaxel (Dtx) and cyclodextrin (CD) inclusion complexes having improved apparent water solubility (up to 9.98 mg mL⁻¹) were obtained from phase solubility diagrams. γ-CD and SBE-β-CD offered only poor solubility enhancements while considerable increases in apparent solubility were obtained with Me-β-CD (20%, w/w) and HP-β-CD (40%, w/w) (9.98 mg mL⁻¹ and 7.43 mg mL⁻¹, respectively). The complexation mechanism between Dtx and Me-β-CD was investigated by circular dichroism spectrometry, two-dimensional ¹H NMR (NOESY) in D₂O, isothermal titration calorimetry (ITC) and molecular docking calculations. Circular dichroism and NOESY confirmed the existence of non-covalent interactions between Dtx and Me-β-CD and suggested that the tert-butyl group (C₆-C₉) and two aromatic groups (C₂₄-C₂₉ and C₃₀-C₃₅) of Dtx interacted with the Me-β-CD molecules. The combination of ITC results to molecular docking calculations led to the identification of an unconventional sequential binding mechanism between Me-β-CD and Dtx. In this sequential binding, a Me-β-CD molecule first interacted with both tert-butyl and C₃₀-C₃₅ aromatic groups (K₁: 744 M⁻¹). Then a second Me-β-CD molecule interacted with the C₂₄-C₂₉ aromatic group (K₂: 202 M⁻¹). The entropy of the first interaction was positive, whereas a negative value of entropy was found for the second interaction. The opposite behavior observed for these two sites was explained by differences in the hydrophobic contact surface and functional group flexibility.     

Molecular spectroscopic studies on the interaction between Ractopamine and bovine serum albumin

To investigate the interaction between Ractopamine (RAC), an animal growth promoter, and bovine serum albumin (BSA), three spectroscopic approaches (fluorescence, UV–vis and FT-IR) and three different experiments (two mole-ratio and a Job's methods) were used to monitor the biological kinetic interaction procedure. The Stern–Volmer quenching constants K_SV, the binding constants K_a, and the number of binding sites n at 298, 301 and 304 K were evaluated by molecular spectroscopic approaches. The values of enthalpy (−13.47 kJ mol⁻¹) and entropy (78.39 J mol⁻¹ K⁻¹) in the reaction indicated that RAC bound to BSA mainly by electrostatic and hydrophobic interaction. The site markers competitive experiments indicated that the binding of RAC to BSA primarily took place in site I. The spectra data matrix was further investigated with multivariate curve resolution-alternating least squares (MCR-ALS), and the concentration profiles and the pure spectra for three species (BSA, RAC and RAC-BSA) existed in the kinetic interaction procedure, as well as the apparent equilibrium constants, were obtained.     

Dynamics of saxitoxin binding to saxiphilin c-lobe reveals conformational change

Thermodynamic parameters (ΔG, ΔH, ΔS, ΔC_p) have been determined to evaluate the dynamics of binding of saxitoxin to the c-lobe of saxiphilin. We have developed an improved method to rapidly express and purify recombinant saxiphilin c-lobe, and fully characterized it by mass spectrometry for the first time. Surface plasmon resonance (SPR) was used to characterize the interaction between saxitoxin and immobilized c-lobe. At 298 K, c-lobe binds saxitoxin with K_D=1.2 nM, ΔH°=−11.7±0.8 kcal/mol, and ΔS°=1.17±0.07 cal/mol K. Analysis of ΔC_p of toxin association at several temperatures suggests that hydrophobic forces contribute to the binding event. Additionally, changes in 8-anilino-1-naphthalene sulfonic acid (ANS) fluorescence upon binding to c-lobe in the presence and absence of saxitoxin support a conformational change in c-lobe upon saxitoxin binding.     

Potency, voltage-dependency, agonist concentration-dependency, blocking kinetics and partial untrapping of the uncompetitive N-methyl-d-aspartate (NMDA) channel blocker memantine at human NMDA (GluN1/GluN2A) receptors

Both the clinical tolerability and the symptomatic effects of memantine in the treatment of Alzheimer's disease have been attributed to its moderate affinity (IC₅₀ around 1 μM at −70 mV) for NMDA receptor channels and associated fast, double exponential blocking/unblocking kinetics and strong voltage-dependency. Most of these biophysical data have been obtained from rodent receptors. Some substances show large species-specific differences, so using human rather than rodent receptors and tissue may highlight important differences in the effects of drugs. In the present study we compared the potency of memantine, ketamine and (+)MK-801 in binding to NMDA receptors in post-mortem human cortical tissue and to antagonize intracellular Ca²⁺ responses of human GluN1/GluN2A receptors expressed in HEK-293 cells. In addition, the biophysical properties of memantine and ketamine were compared using patch clamp recordings from these cells.Memantine was confirmed to be a moderate affinity (IC₅₀ at −70 mV of 0.79 ± 0.02 μM, Hill = 0.92 ± 0.02), strongly voltage-dependent (δ = 0.90 ± 0.09) uncompetitive antagonist of human GluN1/GluN2A receptors. Moreover, the rapid double exponential blocking kinetics (e.g. at 10 μM - onset τ_fast = 273 ± 25 ms (weight 69%), onset τ_slow = 2756 ± 296 ms, offset τ_fast = 415 ± 82 ms (weight 38%) offset τ_slow = 5107 ± 1204 ms) and partial untrapping (around 20%) previously reported for memantine on rodent receptors were confirmed for human receptors. Ketamine showed similar potency (IC₅₀ at −70 mV of 0.71 ± 0.03 μM, Hill = 0.84 ± 0.02) but somewhat less pronounced voltage-dependency (δ = 0.79 ± 0.04), slower, single exponential kinetics (ketamine: k_on = 0.15 ± 0.05 × 10⁶ M⁻¹ s⁻¹, k_off = 0.22 ± 0.05 s⁻¹ c.f. memantine following normalization k_on = 0.32 ± 0.11 × 10⁶  M⁻¹ s⁻¹, k_off = 0.53 ± 0.10 s⁻¹) and was fully trapped.The present data closely match previously reported data from studies in rodent receptors and suggest that the proposed mechanism of action of memantine in Alzheimer's disease as a fast, voltage-dependent open-channel blocker of NMDA receptors can be confirmed for human NMDA receptors.     

Development of a selective competitive receptor binding assay for the determination of the affinity to NR2B containing NMDA receptors

A selective, rapid and efficient competitive binding assay for the determination of the affinity of compounds towards the ifenprodil binding site of NR2B subunit containing NMDA receptors has been developed. In the assay system, [³H]ifenprodil was used as radioligand and membrane homogenates from L(tk-) cells stably expressing recombinant human NR1a/NR2B receptors served as the receptor material. Sonication of the cells during work-up, performing all steps with 96-well multiplates and using a solid scintillator represent particular features of this assay. The binding kinetics was investigated by saturation and association/dissociation experiments. [³H]ifenprodil bound to a single, saturable site on human recombinant NR1a/NR2B receptors, resulting in a B_max-value of 25.8 pmol/μg protein and K_d-value of 7.6 ± 2.3 nM (SEM). The dissociation rate constant (k_off) was 0.03861 min⁻¹ and the association rate constant k_on resulted in 0.00625 nM⁻¹ min⁻¹. The specificity of the assay was proved with cells not treated with dexamethasone, which has to be added to induce NMDA receptor synthesis of the cells. Additionally, the absence of α₁, σ₁ and σ₂ receptors was shown. The K_i-values of the NR2B ligands ifenprodil and eliprodil determined with the new assay are in good accordance with reported data.     

Novel inclusion complex of ibuprofen tromethamine with cyclodextrins: Physico-chemical characterization

Guest–host interactions of ibuprofen tromethamine salt (Ibu.T) with native and modified cyclodextrins (CyDs) have been investigated using several techniques, namely phase solubility diagrams (PSDs), proton nuclear magnetic resonance (¹H NMR), differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), X-ray powder diffractometry (XRPD), scanning-electron microscopy (SEM) and molecular mechanics (MM). From the analysis of PSD data (A_L-type) it is concluded that the anionic tromethamine salt of ibuprofen (pK_a = 4.55) forms 1:1 soluble complexes with all CyDs investigated in buffered water at pH 7.0, while the neutral form of Ibu forms an insoluble complex with β-CyD (B_S-type) in buffered water at pH 2.0. Ibu.T has a lower tendency to complex with β-CyD (K₁₁ = 58 M⁻¹ at pH 7.0) compared with the neutral Ibu (K₁₁ = 4200 M⁻¹) in water. Complex formation of Ibu.T with β-CyD (ΔG° = −20.4 kJ/mol) is enthalpy driven (ΔH° = −22.9 kJ/mol) and is accompanied by a small unfavorable entropy (ΔS° = −8.4 J/mol K) change. ¹H NMR studies and MM computations revealed that, on complexation, the hydrophobic central benzene ring of Ibu.T and part of the isobutyl group reside within the β-CyD cavity leaving the peripheral groups (carboxylate, tromethamine and methyl groups) located near the hydroxyl group networks at either rim of β-CyD. PSD, ¹H NMR, DSC, FT-IR, XRPD, SEM and MM studies confirmed the formation of Ibu.T/β-CyD inclusion complex in solution and the solid state.     

Synthesis and pharmacological characterization of [I]MRS5127, a high affinity, selective agonist radioligand for the A3 adenosine receptor

A recently reported selective agonist of the human A₃ adenosine receptor (hA₃AR), MRS5127 (1′R,2′R,3′S,4′R,5′S)-4′-[2-chloro-6-(3-iodobenzylamino)-purine]-2′,3′-O-dihydroxy-bicyclo-[3.1.0]hexane, was radioiodinated and characterized pharmacologically. It contains a rigid bicyclic ring system in place of a 5′-truncated ribose moiety, and was selected for radiolabeling due to its nanomolar binding affinity at both human and rat A₃ARs. The radioiodination of the N⁶-3-iodobenzyl substituent by iododestannylation of a 3-(trimethylstannyl)benzyl precursor was achieved in 73% yield, measured after purification by HPLC. [¹²⁵I]MRS5127 bound to the human A₃AR expressed in membranes of stably transfected HEK 293 cells. Specific binding was saturable, competitive, and followed a one-site binding model, with a K_d value of 5.74 ± 0.97 nM. At a concentration equivalent to its K_d, non-specific binding comprised 27 ± 2% of total binding. In kinetic studies, [¹²⁵I]MRS5127 rapidly associated with the hA₃AR (t_1/2 = 0.514 ± 0.014 min), and the affinity calculated from association and dissociation rate constants was 3.50 ± 1.46 nM. The pharmacological profile of ligands in competition experiments with [¹²⁵I]MRS5127 was consistent with the known structure-activity-relationship profile of the hA₃AR. [¹²⁵I]MRS5127 bound with similar high affinity (K_d, nM) to recombinant A₃ARs from mouse (4.90 ± 0.77), rabbit (2.53 ± 0.11), and dog (3.35 ± 0.54). For all of the species tested, MRS5127 exhibited A₃AR agonist activity based on negative coupling to cAMP production. Thus, [¹²⁵I]MRS5127 represents a new species-independent agonist radioligand for the A₃AR. The major advantage of [¹²⁵I]MRS5127 compared with previously used A₃AR radioligands is its high affinity, low degree of non-specific binding, and improved A₃AR selectivity.     

Identification of permeability-related hurdles in oral delivery of curcumin using the Caco-2 cell model

Curcumin a poly-phenolic compound possesses diverse pharmacologic activities; however, its development as a drug has been severely impeded by extremely poor oral bioavailability. Poor aqueous solubility and extensive metabolism have been implicated for this but the role of membrane permeability has not been investigated. In the present study, permeability of curcumin was assessed using the Caco-2 cell line. Curcumin was poorly permeable with a P_app (A → B) value of 2.93 ± 0.94 × 10⁻⁶ cm/s. P_app value in (B → A) study was found out to be 2.55 ± 0.02 × 10⁻⁶ cm/s, thus ruling out the role of efflux pathways in poor oral bioavailability of curcumin. Studies using verapamil, a P-gp inhibitor, further confirmed this finding. Detailed mass balance studies showed loss of curcumin during transport. Further experiments using lysed cells revealed that 11.78% of curcumin was metabolized during transport. Studies using itraconazole, a CYP3A4 inhibitor, established its role in curcumin metabolism. Curcumin was also found to accumulate in cells as revealed by CLSM studies. Sorption and desorption kinetic studies further confirmed accumulation of curcumin inside the cells. Amount accumulated was quantitated by HPLC and found to be >20%. Thus, intestinal first-pass metabolism and intracellular accumulation played a role in poor permeability of curcumin. Based on its poor aqueous solubility and intestinal permeability, curcumin can be classified as a BCS Class IV molecule. This information can facilitate designing of drug delivery systems for enhancement of oral bioavailability of curcumin.     

Antioxidant activity of propolis extracts from Serbia: A polarographic approach

Antioxidant activity (AO) of commercial propolis extracts (PEs), available on Serbian market, was determined by direct current (DC) polarography. Polarographic anodic current of 5.0 mmol L⁻¹ alkaline solution of H₂O₂ was recorded at potentials of mercury dissolution. Decrease of the current was plotted against the volume of gradually added PEs. The volume of PE causing 20% current decrease was determined from the linear part of the plot. Antioxidant activity was expressed in H₂O₂ equivalent (HPEq), representing the volume of PE that corresponds to 1.0 mmol L⁻¹ H₂O₂ decrease. Resulting HPEq ranged between 1.71 ± 0.11 and 8.00 ± 0.18 μL. Range of 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical scavenging activity was from 0.093 ± 0.004% to 0.346 ± 0.006%. Total phenolic content (TCP) of PE with superior AO activity was 5.31 ± 0.05%g GAE, while the extract with the lowest activity contained 1.45 ± 0.02%g GAE. Antioxidant activity, determined by polarographic method, was correlated with DPPH scavenging activity (R² = 0.991) and TCP (R² = 0.985). Validity of obtained results was further confirmed using ANOVA and post hoc Tukey HSD test.     

Peptide aldehyde inhibitors challenge the substrate specificity of the SARS-coronavirus main protease

SARS coronavirus main protease (SARS-CoV M^pro) is essential for the replication of the virus and regarded as a major antiviral drug target. The enzyme is a cysteine protease, with a catalytic dyad (Cys-145/His-41) in the active site. Aldehyde inhibitors can bind reversibly to the active-site sulfhydryl of SARS-CoV M^pro. Previous studies using peptidic substrates and inhibitors showed that the substrate specificity of SARS-CoV M^pro requires glutamine in the P1 position and a large hydrophobic residue in the P2 position. We determined four crystal structures of SARS-CoV M^pro in complex with pentapeptide aldehydes (Ac-ESTLQ-H, Ac-NSFSQ-H, Ac-DSFDQ-H, and Ac-NSTSQ-H). Kinetic data showed that all of these aldehydes exhibit inhibitory activity towards SARS-CoV M^pro, with K_i values in the μM range. Surprisingly, the X-ray structures revealed that the hydrophobic S2 pocket of the enzyme can accommodate serine and even aspartic-acid side-chains in the P2 positions of the inhibitors. Consequently, we reassessed the substrate specificity of the enzyme by testing the cleavage of 20 different tetradecapeptide substrates with varying amino-acid residues in the P2 position. The cleavage efficiency for the substrate with serine in the P2 position was 160-times lower than that for the original substrate (P2 = Leu); furthermore, the substrate with aspartic acid in the P2 position was not cleaved at all. We also determined a crystal structure of SARS-CoV M^pro in complex with aldehyde Cm-FF-H, which has its P1-phenylalanine residue bound to the relatively hydrophilic S1 pocket of the enzyme and yet exhibits a high inhibitory activity against SARS-CoV M^pro, with K_i = 2.24 ± 0.58 μM. These results show that the stringent substrate specificity of the SARS-CoV M^pro with respect to the P1 and P2 positions can be overruled by the highly electrophilic character of the aldehyde warhead, thereby constituting a deviation from the dogma that peptidic inhibitors need to correspond to the observed cleavage specificity of the target protease.     

Post-exposure treatment of VX poisoned guinea pigs with the engineered phosphotriesterase mutant C23: A proof-of-concept study

The highly toxic organophosphorus (OP) nerve agent VX is characterized by a remarkable biological persistence which limits the effectiveness of standard treatment with atropine and oximes. Existing OP hydrolyzing enzymes show low activity against VX and hydrolyze preferentially the less toxic P(+)-VX enantiomer. Recently, a phosphotriesterase (PTE) mutant, C23, was engineered towards the hydrolysis of the toxic P(−) isomers of VX and other V-type agents with relatively high in vitro catalytic efficiency (k_cat/K_M = 5 × 10⁶ M⁻¹ min⁻¹). To investigate the suitability of the PTE mutant C23 as a catalytic scavenger, an in vivo guinea pig model was established to determine the efficacy of post-exposure treatment with C23 alone against VX intoxication. Injection of C23 (5 mg kg⁻¹ i.v.) 5 min after s.c. challenge with VX (∼2LD₅₀) prevented systemic toxicity. A lower C23 dose (2 mg kg⁻¹) reduced systemic toxicity and prevented mortality. Delayed treatment (i.e., 15 min post VX) with 5 mg kg⁻¹ C23 resulted in survival of all animals and only in moderate systemic toxicity. Although C23 did not prevent inhibition of erythrocyte acetylcholinesterase (AChE) activity, it partially preserved brain AChE activity. C23 therapy resulted in a rapid decrease of racemic VX blood concentration which was mainly due to the rate of degradation of the toxic P(−)-VX enantiomer that correlates with the C23 blood levels and its k_cat/K_M value. Although performed under anesthesia, this proof-of-concept study demonstrated for the first time the ability of a catalytic bioscavenger to prevent systemic VX toxicity when given alone as a single post-exposure treatment, and enables an initial assessment of a time window for this approach. In conclusion, the PTE mutant C23 may be considered as a promising starting point for the development of highly effective catalytic bioscavengers for post-exposure treatment of V-agents intoxication.     

Thermodynamics of the interaction between oxytocin and its myometrial receptor in sheep: A stepwise binding mechanism

Entropy (ΔS), enthalpy (ΔH) and heat capacity (ΔC_p) changes attending the oxytocin interaction with its two binding sites on myometrial cell membranes in sheep were derived from the temperature dependence of K_d values. The high affinity oxytocin site (K_d on the order of 10⁻⁹ mol l⁻¹, 25 °C), ascribed to the oxytocin receptor (OXTR), is entropy-driven in the temperature range 0–37 °C. Enthalpy component prevails as a driving force in the binding to the low affinity site (K_d ≈ 10⁻⁷) within the higher temperature range. ΔC_p values in both cases do not differ significantly from zero but become highly relevant in the presence of a GTP analog (10⁻⁴ M GTP-γS). Under these conditions, ΔC_p in the low site interaction becomes negative and ΔS is shifted toward negative values (enthalpy drift); ΔC_p of the high affinity site rises to a high positive value and the interaction is even more strongly entropy driven. Atosiban, a competitive antagonist of oxytocin at OXTR displays a single significant binding site on myometrial cells (K_d about 10⁻⁷ mol l⁻¹). Thermodynamic profiles of atosiban and the low affinity oxytocin site show conspicuous similarities, indicating that the inhibitor is bound to the low affinity site, and not, with a lower affinity, to the putative receptor protein. It is suggested that the interaction of oxytocin with its responding system on myometrial membranes follows in two distinct steps that are likely to be associated with several independent binding domains in the GPCR receptor.