Microcalorimetric method to determine competitive binding action of a psychotropic drug (dipotassium chlorazepate) on L-tryptophan · human serum albumin complex

A mathematical treatment and an original microcalorimetric method are developed to verify an eventual competitive binding between any two substances for the same macromolecule. To apply this method, a competitive binding of L-tryptophan and one benzodiazepin (dipotassium chlorazepate) for human serum albumin is perfectly demonstrated.The association constants and the enthalpy variations are equal to 14 000 ± 2000 M^?1 and ?6.6 ± 0.2 kcal/mol for human serum albumin · tryptophan complex and 13 000 ± 1000 M^?1 and ?10.0 ± 0.2 kcal/mol for human serum albumin · chlorazepate complex. In all cases the stoichiometry is equal to one.The binding of tryptophan to human serum albumin is partially stereospecific; the association constant and the enthalpy variation for D-tryptophan complex are equal, respectively, to 1000 ± 200 M^?1 and ?2.6 ± 0.3 kcal/mol.     

Presence of D2-dofamine Receptors in Human Term Placenta

Abstract

We studied the binding of [³H]-spiperone on human term placental membranes. This binding reached plateau level after 30 min incubation at 37°C and was reversed (t_1/2 ～ 5 min) by addition of an excess of unlabeled spiperone. Scatchard analysis of saturation experiments with increasing doses of [³H]-spiperone (0–25 nM) showed one class of high affinity binding sites with a dissociation constant (Kd) of 14 ± 2 nM and a maximal binding capacity (Bmax) of 222 ± 9 fmoles/mg protein. The affinity of 5 competitors was determined in competitive binding assays. The D₂-dopamine antagonists were the most potent inhibitors: Ki for spiperone and haloperidol were 8 ± 2 and 56 ± 22 nM respectively. Dopamine inhibited [³H]-spiperone binding with a Ki of 570 ± 50 μM whereas Schering 23390 (D₁ antagonist) and propranolol (β-adrenergic antagonist) were without effect. The binding was also inhibited by 100 μM GTPγS (38 ± 8% inhibition), indicating that the dopamine receptor is coupled with a GTP binding protein. These results demonstrate for the first time the presence of D₂-dopamine receptors in human placenta.     

[3H] verapamil binding sites in skeletal muscle tranverse tubule membranes

[³H]verapamil binding to muscle tubule membrane has the following properties. K_D = 27 ± 5 nM and maximum binding capacity B_max = 50 ± 5 pmol/mg of protein. A 1 = 1 stoichiometry of binding was found for the ratio of [³H]verapamil versus [³H] nitrendipine binding sites. The dissociation constant found at equilibrium is near that determined from the ratio of the rate constants for association (k₁) and dissociation (k_?1). Antiarrhythmic drugs like D600, diltiazem and bepridil are competitive inhibitors of [³H]verapamil binding with K_D values between 40 and 200 nM. Dihydropyridine analogs are apparent non competitive inhibitors of [³H]verapamil binding with half-maximum inhibition values (K_0.5) between 1 and 5 nM.     

Interaction of calcium ions with α-elastin: An equilibrium dialysis,circular dichroism,and microcalorimetric study

The interaction of calcium ions with α-elastin has been investigated by equilibrium dialysis, CD, and microcalorimetric techniques. Consistent with literature data, it was found that the interaction in water is very poor. In trifluoroethanol (TFE), equilibrium dialysis experiments showed that calcium ions bind to ～-elastin with an association constant of ～250 L × mol^?1. Such a figure is not consistent with highly specific, highly selective binding. It was also found that the CD response is not directly proportional to the amount of bound calcium but depends on the protein concentration. From microcalorimetric experiments it was found that the heat effect relative to the binding process is of the order of 1.9 kcal/g ion. From this figure and from the binding constant, a positive ΔS value of about 17 e.u. was evaluated, leading to the conclusion that the binding process is entropy driven. From microcalorimetric measurements a ΔH of 1.5 kcal/residue was found for the calcium-induced conformational transition of the protein.     

Microcalorimetric method to determine competitive binding. Action of a psychotropic drug (dipotassium chlorazepate) on L-tryptophan . human serum albumin complex.

A mathematical treatment and an original microcalorimetric method are developed to verify an eventual competitive binding between any two substances for the same macromolecule. To apply this method, a competitive binding of L-tryptophan and one benzodiazepin (dipotassium chlorazepate) for human serum albumin is perfectly demonstrated. The association constants and the enthalpy variations are equal to 14 000 +/- 2000 M-1 and --6.6 +/- 0.2 kcal/mol for human serum albumin . tryptophan complex and 13 000 +/- 1000 M-1 and --10.0 +/- 0.2 kcal/mol for human serum albumin . chlorazepate complex. In all cases the stoichiometry is equal to one. The binding of tryptophan to human serum albumin is partially stereospecific; the association constant and the enthalpy variation for D-tryptophan complex are equal, respectively, to 1000 +/- 200 M-1 and --2.6 +/- 0.3 kcal/mol.     

The effect of 7,8,4´‐trihydroxyflavone on tyrosinase activity and conformation: Spectroscopy and docking studies

Tyrosinase is a ubiquitous enzyme that plays an essential role in the production of melanin. Effective inhibitors of tyrosinase have extensive applications in the medical, cosmetic and food industries. In this study, a combination of enzyme kinetics, ultraviolet (UV)‐visible absorption, fluorescence spectroscopic techniques and a computational simulation method was used to characterize the inhibitory mechanism of 7,8,4´‐trihydroxyflavone on tyrosinase. 7,8,4´‐Trihydroxyflavone was found to strongly inhibit the oxidation of l ‐DOPA by tyrosinase with an IC₅₀ value of 10.31 ± 0.41 μM. The inhibitory mechanism was determined to be reversible and non‐competitive with a K_i of 9.50 ± 0.40 μM. The UV absorption spectra showed that 7,8,4´‐trihydroxyflavone could chelate with copper ions and form a complex with tyrosinase. The intrinsic fluorescence of tyrosinase was quenched by 7,8,4´‐trihydroxyflavone through a static quenching mechanism. 7,8,4´‐Trihydroxyflavone was found to occupy a single binding site with a binding constant of 7.50 ± 1.20 × 10⁴ M^?1 at 298 K. The conformation of tyrosinase changed, and the microenvironment became more hydrophilic after 7,8,4´‐trihydroxyflavone binding. Thermodynamics parameters indicated that the binding was a spontaneous process and involved hydrogen bonds and van der Waals forces. The binding distance was evaluated to be 4.54 ± 0.05 nm. Docking simulation analysis further authenticated that 7,8,4´‐trihydroxyflavone could form hydrogen bonds with the residues His244 and Met280 within the tyrosinase active site. Our results will contribute to further understanding of the inhibitory mechanisms of 7,8,4´‐trihydroxyflavone against tyrosinase and will facilitate future screening for tyrosinase inhibitors.     

Characteristics of an Adenylate Cyclase Coupled β-Adrenergic Receptor in a Smooth Muscle Tumor Cell Line

Abstract

We have shown that binding of ³H-dihydroalprenolol ([³H] DHA) to DDT₁ MF-2 cells and cell membranes was of high affinity, saturable, stereoselective and reversible. The [³H]DHA dissociation constants were 0.63 ± 0.15 nM (n=6) and 0.83 ± 0.04 nM (n=5) for intact cells and cell membranes, respectively, with a binding site concentration for cells of 27,300 ± 5,200 sites/ cell (n=6) and for membranes 468 ± 24 fmoles/mg protein (n=5). The order of agonist competition for the [³H]-DHA binding site of DDT₁ cell membranes was isoproterenol (K_i = 0.20 ± 0.07 μM) > epinephrine (K_i = 0.4 ± 0.2 μM) > norepinephrine (K_i = 66.5 ± 5.15 μM) consistent with a β₂-selective receptor interaction. Zinterol, a β₂-selective antagonist, (K_i = 0.05 ± 0.01 μM) was 18x more effective than metoprolol, a β₁-selective antagonist (K_i = 0.9 ± 0.1 μM), in competing for the DHA binding site. A nonlinear iterative curve fitting analysis of zinterol and metoprolol binding isotherms indicated that (p>0.05) DDT₁ cells possess a pure population of β₂-adrenergic receptors. Finally, we have shown that DDT₁ MF-2 cell β₂-adrenergic receptor is functionally coupled to adenylate cyclase via a G/F protein complex as demonstrated in part by a guanine nucleotide requirement for isoproterenol stimulation of adenylate cyclase activity. In addition, guanine nucleotide mediated a reduction in the affinities of isoproterenol and epinephrine for the [³H]DHA binding site.     

Cytosolic 25-hydroxycholesterol binding activity of Chinese hamster ovary cells

DEAE-Sephadex chromatography of cytosols of Chinese hamster ovary cells incubated with tritium-labeled 25-hydroxycholesterol shows a peak of specific binding activity. This binding activity can be assayed by determining the amount of labeled 25-hydroxycholesterol in cytosol which is refractory to adsorption to activated charcoal at high specific activity but can be made to adsorb to charcoal in the presence of a 50-fold excess of unlabeled 25-hydroxycholesterol. The binding activity shows positive cooperatively (Hill coefficient = 2.3 ± 0.3) and high affinity (dissociation constant = 1.4 × 10^?7m). Inactivation of binding by trypsin or boiling suggests that the binding activity is a protein. The sedimentation coefficient of the binding activity is 5 S. Binding of 25-hydroxycholesterol is competitive with several other sterols and correlates well with the concentrations of these compounds that inhibit cholesterol biosynthesis.     

A new 68Ga-labeled BBN peptide with a hydrophilic linker for GRPR-targeted tumor imaging

Bombesin (BBN) is a peptide exhibiting high affinity for the gastrin-releasing peptide receptor (GRPR), which is overexpressed on several types of cancers. Various GRPR antagonists and agonists have been labeled with radiometals for positron emission tomography (PET) imaging of GRPR-positive tumors. However, unfavorable hepatobiliary excretion such as high intestinal activity may prohibit their clinical utility for imaging abdominal cancer. In this study, the modified BBN peptide with a new hydrophilic linker was labeled with ⁶⁸Ga for PET imaging of GRPR-expressing PC-3 prostate cancer xenograft model. GRPR antagonists, MATBBN (Gly-Gly-Gly-Arg-Asp-Asn-d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHCH₂CH₃) and ATBBN (d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu-NHCH₂CH₃), were conjugated with 1,4,7-triazacyclononanetriacetic acid (NOTA) and labeled with ⁶⁸Ga. Partition coefficient and in vitro stability were also determined. GRPR binding affinity of both tracers was investigated by competitive radioligand binding assay. The in vivo receptor targeting potential and pharmacokinetic of ⁶⁸Ga-NOTA-MATBBN were also evaluated in PC-3 prostate tumor model and compared with those of ⁶⁸Ga-NOTA-ATBBN. NOTA-conjugated BBN analogs were labeled with ⁶⁸Ga within 20 min with a decay-corrected yield ranging from 90 to 95 % and a radiochemical purity of more than 98 %. The specific activity of ⁶⁸Ga-NOTA-MATBBN and ⁶⁸Ga-NOTA-ATBBN was at least 16.5 and 11.9 GBq/μmol, respectively. The radiotracers were stable in phosphate-buffered saline and human serum. ⁶⁸Ga-NOTA-MATBBN was more hydrophilic than ⁶⁸Ga-NOTA-ATBBN, as indicated by their log P values (?2.73 ± 0.02 vs. ?1.20 ± 0.03). The IC₅₀ values of NOTA-ATBBN and NOTA-MATBBN were similar (102.7 ± 1.18 and 124.6 ± 1.21 nM). The accumulation of ⁶⁸Ga-labeled GRPR antagonists in the subcutaneous PC-3 tumors could be visualized via small animal PET. The tumors were clearly visible, and the tumor uptakes of ⁶⁸Ga-NOTA-MATBBN and ⁶⁸Ga-NOTA-ATBBN were determined to be 4.19 ± 0.32, 4.00 ± 0.41, 2.93 ± 0.35 and 4.70 ± 0.40, 4.10 ± 0.30, 3.14 ± 0.30 %ID/g at 30, 60, and 120 min, respectively. There was considerable accumulation and retention of ⁶⁸Ga-NOTA-ATBBN in the liver and intestines. In contrast, the abdominal area does not have much retention of ⁶⁸Ga-NOTA-MATBBN. Biodistribution data were in accordance with the PET results, showing that ⁶⁸Ga-NOTA-MATBBN had more favorable pharmacokinetics and higher tumor to background ratios than those of ⁶⁸Ga-NOTA-ATBBN. At 1 h postinjection, the tumor to liver and intestine of ⁶⁸Ga-NOTA-MATBBN were 8.05 ± 0.56 and 21.72 ± 3.47 and the corresponding values of unmodified counterpart were 0.85 ± 0.23 and 3.45 ± 0.43, respectively. GRPR binding specificity was demonstrated by reduced tumor uptake of radiolabeled tracers after coinjection of an excess of unlabeled BBN peptides. ⁶⁸Ga-NOTA-MATBBN exhibited GRPR-targeting properties both in vitro and in vivo. The favorable characterizations of ⁶⁸Ga-NOTA-MATBBN such as convenient synthesis, specific GRPR targeting, high tumor uptake, and satisfactory pharmacokinetics warrant its further investigation for clinical cancer imaging.     

SMMC-7721肝癌细胞67kD层粘连蛋白受体的分离纯化

分离纯化肝癌细胞的 6 7kD层粘连蛋白受体 (6 7LR) ,以便进一步研究 6 7LR的结构、功能及其在肝癌浸润、转移过程中的作用 .以SMMC 772 1肝癌细胞和L 0 2正常肝细胞为材料 ,采用13 1I标记的层粘连蛋白测定其与细胞的结合能力 ;亲和层析法分离纯化层粘连蛋白受体 ,用SDS PAGE、放射自显影及体外竞争结合实验进行鉴定 .在相同条件下SMMC 772 1肝癌细胞与层粘连蛋白特异结合量为 17 5 4± 0 4 9ng 10 5细胞 ,而L 0 2正常肝细胞与层粘连蛋白的特异结合量为 8 36± 0 4 8ng 10 5细胞 .经过亲和层析 ,从SMMC 772 1肝癌细胞和L 0 2正常肝细胞均可获得纯化受体 ,SDS PAGE显示为单一条带 ,分子量为 6 7kD ,放射自显影及体外竞争结合实验表明其具有较强的与层粘连蛋白结合的活性 .体外竞争结合实验表明 ,SMMC 772 1肝癌细胞层粘连蛋白受体 (772 1LnR)的抑制率可达到 96 2 7± 2 2 9% ,而L 0 2正常肝细胞层粘连蛋白受体 (L 0 2LnR)的抑制率为 4 8 71± 3 79% ,这说明 772 1LnR与层粘连蛋白的亲和力明显高于L 0 2LnR(P <0 0 0 1) .结果表明 ,与L 0 2肝细胞比较 ,SMMC 772 1肝癌细胞具有与层粘连蛋白较强结合能力的特异受体 ,并从肝癌细胞膜上分离纯化到与层粘连蛋白有较强亲和力的 6 7LR     

A radioimmunoassay of androstenedione

A dextran-coated charcoal radioimmunoassay for androstenedione (4-androsten-3, 17-dione) is reported which uses an anti-testosterone antiserum raised in sheep, against a testosterone-17-hemisuccinate-Bovine Serum Albumin conjugate. It is more sensitive and rapid than previously published double dilution, gas chromatographic and competitive protein binding assays. Androstenedione is separated from cross-reacting Steroids by Celite column chromatography. The intra-assay and interassay coefficients of variation were 10.7 and 11.6 per cent, respectively. Using this method serum androstenedione in men was 1.15 ± 0.35 ng/ml; in women, 1.41 ± 0.30 ng/ml; in post-menopausal women, 0.88 ± 0.34 ng/ml; in ovariectomized women, 0.67 ± 0.17 ng/ml; and in ovariectomized-adrenalectomized women, 0.14 ± 0.05 ng/ml. The blank of the method was usually 4 to 5 pg, but ranged between 0 and 12 pg. The sensitivity of the standard curve was 8 pg.     

Oxytocin Receptors in Bovine Mammary Tissue

Abstract

Oxytocin receptors were identified and characterized in bovine mammary tissue. [³H]-oxytocin was specifically bound to the 105,000 × g particulate fractions from 5 lactating cows and 5 non-lactating cows. Binding reached equilibrium by 50 min at 20°C and by 8 hr at 4°C. The half-time of displacement at 20°C was approximately 1 hr. ACTH, TRH, angiotensin I, angiotensin II, pentagastrin, bradykinin, xenopsin and L-valyl-histidyl-L-leucyl-L-threonyl-L-prolyl-L-valyl-L-glutamyl-L-lysine were not competitive in the dose range tested at 20°C. The ability of other peptides to inhibit ³H-oxytocin binding was as follows: oxytocin > vasotocin > arginine - vasopressin >lysine - vaso-pressin > Pen¹ Phe² Thr⁴ - oxytocin. The Kd of the oxytocin receptor averaged 1.66 ± 1.19 nMol/L for lactating cows and 0.97± 0.49 nMol/L for non-lactating cows, respectively. The maximum number of binding sites was 0.14 ± 0.12 nM/mg protein and 0.15 ± 0.08 nM/mg protein for lactating cows and non-lactating cows, respectively. Identification and characterization of these receptors now makes it possible to study the dynamics of hormonal binding throughout various physiological states of the animal.     

Discrimination and Quantification of Glomerular Receptor Subtypes for Atrial Natriuretic Factor (Anf)

Abstract

Binding sites for atrial natriuretic factor (ANF) were determined on isolated rat glomeruli as well as on glomerular membranes. To define optimal conditions, binding of ANF was investigated varying incubation time, temperature and protein concentration. Binding conditions were found to be best at 4°C for 5 hours with 15 μg of glomerular protein. Saturation and affinity cross-linking experiments confirmed the presence of two distinct receptor subtypes – the B-receptor (130 kDa) and the C-receptor (65 kDa). Quantitative differentiation of both ANF binding sites was achieved by competitive displacement with two different unlabeled ANF ligands: a) rANF(99–126) (homologous displacement), b) des(18–22)rANF(4–23)NH₂(heterologous displacement). Intact glomeruli and glomerular membranes did not differ significantly in receptor density for the B-receptor (71 ± 37 vs. 94 ± 53 fmol/mg protein) or the C-receptor (976 ± 282 vs. 966 ± 167 fmol/mg protein) or in affinity constants for the B-receptor (43 ± 36 vs. 52 ± 44 pM) or the C-receptor (876 ± 377 vs. 307 ± 36 pM). Glomerular membranes compared to glomeruli showed less nonspecific binding and less intra-assay variation of measuring points done in triplicates. This method of selective displacement should allow to study the influence of various physiological and pathophysiological conditions on the binding properties of B-and C-receptors for ANF.     

Near native binding of a fluorescent serotonin conjugate to serotonin type 3 receptors

Described is the synthesis of 5-hydroxytryptamine-tetramethylrhodamine (5HT¹); an indole nitrogen linked fluorescent conjugate of serotonin. Through a series fluorescence quenching experiments and experiments in the presence of a known competitive antagonist (Granisetron), it was shown that 5HT¹ specifically binds to purified homo-pentameric type-3 human serotonin receptors (5HT_3A). The measured dissociation constant and Hill coefficient are K_d = 83 ± 3 nM and n = 3.1 ± 0.3, respectively which is indicative of multi-ligand binding and cooperativity similar to that of unconjugated serotonin.     

New Drugs for the Na+/H+ Exchanger. Influence of Na+ Concentration and Determination of Inhibition Constants with a Microphysiometer

The NHE-1 isoform of the Na⁺/H⁺ exchanger is excessively activated in cardiac cells during ischemia. Hence NHE-1 specific inhibitors are being developed since they could be of beneficial influence under conditions of cardiac ischemia and reperfusion. In this study, the Cytosensor™ microphysiometer was used to measure the potency of four new drug molecules, i.e., EMD 84021, EMD 94309, EMD 96785 and HOE 642 which are inhibitors of the isoform 1 of the Na⁺/H⁺ exchanger. The experiments were performed with Chinese hamster ovary cells (CHO K1) which are enriched in the NHE-1 isoform of the Na⁺/H⁺ antiporter. The Na⁺/H⁺ exchanger was stimulated with NaCl and the rate of extracellular acidification was quantified with the Cytosensor. The proton exchange rate was measured as a function of the NaCl concentration in the range of 10–138 mm NaCl stimulation. The proton exchange rate followed Michaelis-Menten kinetics with a K _M = 30 ± 4 mm for Na⁺. Addition of either one of the four inhibitors decreased the acidification rate. The IC₅₀ values of the four compounds could be determined as 23 ± 7 nm for EMD 84021, 5 ± 1 nm for EMD 94309, 9 ± 2 nm for EMD 96785 and 8 ± 2 nm for HOE 642 at 138 mm NaCl, in good agreement with more elaborate biological assays. The IC₅₀ values increased with the NaCl concentration indicating competitive binding of the inhibitor. The microphysiometer approach is a fast and simple method to measure the activity of the Na⁺/H⁺ antiporter and allows a quantitative kinetic analysis of the proton excretion rate. Received: 3 September 1998/Revised: 20 November 1998     

Inhibition of urease by bismuth(III): Implications for the mechanism of action of bismuth drugs

Bismuth compounds are widely used for the treatment of peptic ulcers and Helicobacter pylori infections. It has been suggested that enzyme inhibition plays an important role in the antibacterial activity of bismuth towards this bacterium. Urease, an enzyme that converts urea into ammonia and carbonic acid, is crucial for colonization of the acidic environment of the stomach by H. pylori. Here, we show that three bismuth complexes exhibit distinct mechanisms of urease inhibition, with some differences dependent on the source of the enzyme. Bi(EDTA) and Bi(Cys)₃ are competitive inhibitors of jack bean urease with K _i values of 1.74 ± 0.14 and 1.84 ± 0.15 mM, while the anti-ulcer drug, ranitidine bismuth citrate (RBC) is a non-competitive inhibitor with a K _i value of 1.17 ± 0.09 mM. A ¹³C NMR study showed that Bi(Cys)₃ reacts with jack bean urease during a 30 min incubation, releasing free cysteines from the metal complex. Upon incubation with Bi(EDTA) and RBC, the number of accessible cysteine residues in the homohexameric plant enzyme decreased by 5.80 ± 0.17 and 11.94 ± 0.13, respectively, after 3 h of reaction with dithiobis(2-nitrobenzoic acid). Kinetic analysis showed that Bi(EDTA) is both a competitive inhibitor and a time-dependent inactivator of the recombinant Klebsiella aerogenes urease. The active C319A mutant of the bacterial enzyme displays a significantly reduced sensitivity toward inactivation by Bi(EDTA) compared with the wild-type enzyme, consistent with binding of Bi³⁺ to the active site cysteine (Cys³¹⁹) as the mechanism of enzyme inactivation.     

Enhanced Expression of an Endothelin ETA Receptor in Capillaries from Human Glioblastoma: A Quantitative Receptor Autoradiographic Analysis Using a Radioluminographic Imaging Plate System

Abstract: We identified and characterized ¹²⁵I-endothelin-1 (¹²⁵I-ET-1) binding sites in tumor capillaries isolated from human glioblastomas, using the quantitative receptor autoradiographic technique with pellet sections. Quantification was done using the computerized radioluminographic imaging plate system. High-affinity ET receptors were localized in capillaries from glioblastomas and the surrounding brain tissues (K_D = 4.7 ± 1.0 × 10^?10 and 1.6 ± 0.3 × 10^?10M, respectively; B_max = 161 ± 38 and 140 ± 37 fmol/mg, respectively; mean ± SEM, n = 5). BQ-123, a selective antagonist for the ET_A receptor, potently competed for ¹²⁵I-ET-1 binding to sections of the microvessels with IC₅₀ values of 5.1 ± 0.3 and 5.1 ± 1.5 nM, and 10^?6M BQ-123 displaced 84 and 58% of ET binding to capillaries from tumors and brains, respectively. In addition, competition curves obtained in the presence of increasing concentrations of ET-3 showed two components (IC₅₀ = 5.7 ± 2.5 × 10^?10 and 1.4 ± 0.2 × 10^?6M for tumor microvessels, 1.8 ± 0.6 × 10^?10 and 1.1 ± 0.3 × 10^?6M for brain microvessels, respectively). Our results indicate that (a) the method we used is simple and highly sensitive for detecting and characterizing various receptors in tumor capillaries, especially in the case of a sparse specimen, and (b) capillaries in glioblastomas express specific high-affinity ET binding sites, candidates for biologically active ET receptors, which predominantly belong to the ET_A subtype.     

Histrionicotoxin and alkylguanidine interactions with the solubilized and membrane-bound muscarinic acetylcholine receptor from porcine atria

The interaction of alkylguanidines and decahydrohistrionicotoxin with the membrane-bound and solubilized muscarinic acetylcholine receptor (mAcChR) from porcine atria was described. Alkylguanidines with alkyl chain lengths from one to ten carbons displaced l-[³H]quinuclidinyl benzilate (l-[³H]QNB) competitively from a single class of sites for the membrane-bound mAcChR. From a plot of ?ln K_i versus alkyl carbon chain number, a value of ?(473 ± 30) cal/mol was estimated as the energetic contribution per methylene group to the total binding energy. The binding of alkylguanidines to the digitonin/cholate solubilized mAcChR was complex in nature resulting in titration curves that did not obey the law of mass action for simple competitive inhibition at higher alkyl carbon numbers and a sigmoidal plot of ?ln K_i versus carbon number. Decahydrohistrionicotoxin bound in a competitive manner versus l-[³H]QNB to both the membrane-bound (K_i = (6.9 ± 1.4) × 10^?6 M) and the solubilized (K_i = (1.5 ± 0.3) × 10^?5 M) preparations.     

Design,synthesis, and in vitro bioactivity evaluation of fluorine-containing analogues for sphingosine-1-phosphate 2 receptor

Twenty eight new aryloxybenzene analogues were synthesized and their in vitro binding potencies toward S1PR2 were determined using a [³²P]S1P competitive binding assay. Out of these new analogues, three compounds, 28c (IC₅₀ = 29.9 ± 3.9 nM), 28e (IC₅₀ = 14.6 ± 1.5 nM), and 28g (IC₅₀ = 38.5 ± 6.3 nM) exhibited high binding potency toward S1PR2 and high selectivity over the other four receptor subtypes (S1PR1, 3, 4, and 5; IC₅₀ > 1000 nM). Each of the three potent compounds 28c, 28e, and 28g contains a fluorine atom that will allow to develop F-18 labeled PET radiotracers for imaging S1PR2.     

Investigation of the binding characteristics between ligands and epidermal growth factor receptor by cell membrane chromatography

The binding property between a ligand and its receptor is very important for numerous biological processes. In this study, we developed a high epidermal growth factor receptor (EGFR)‐expression cell membrane chromatography (CMC) method to investigate the binding characteristics between EGFR and the ligands gefitinib, erlotinib, canertinib, afatinib, and vandetanib. Competitive binding analysis using gefitinib as the marker was used to investigate the interactions that occurred at specific binding sites on EGFR. The ability of displacement was measured from the HEK293‐EGFR/CMC column on the binding sites occupied by gefitinib for these ligands, which revealed the following order: gefitinib (K_D, 8.49 ± 0.11 × 10^?7 M) > erlotinib (K_D, 1.07 ± 0.02 × 10^?6 M) > canertinib (K_D, 1.41 ± 0.07 × 10^?6 M) > afatinib (K_D, 1.80 ± 0.12 × 10^?6 M) > vandetanib (K_D, 1.99 ± 0.03 × 10^?6 M). This order corresponded with the values estimated by frontal displacement analysis and the scores obtained with molecular docking. Furthermore, thermodynamic analysis indicated that the hydrogen bond or Van der Waals force was the main interaction force in the process of EGFR binding to all 5 ligands. Overall, these results demonstrate that a CMC method could be an effective tool to investigate the binding characteristics between ligands and receptors.