Binding kinetics differentiates functional antagonism of orexin-2 receptor ligands

Orexin receptor antagonism represents a novel approach for the treatment of insomnia that directly targets sleep/wake regulation. Several such compounds have entered into clinical development, including the dual orexin receptor antagonists, suvorexant and almorexant. In this study, we have used equilibrium and kinetic binding studies with the orexin-2 (OX₂) selective antagonist radioligand, [³H]-EMPA, to profile several orexin receptor antagonists. Furthermore, selected compounds were studied in cell-based assays of inositol phosphate accumulation and ERK-1/2 phosphorylation in CHO cells stably expressing the OX₂ receptor that employ different agonist incubation times (30 and 5 min, respectively). EMPA, suvorexant, almorexant and TCS-OX-29 all bind to the OX₂ receptor with moderate to high affinity (pK_I values ≥ 7.5), whereas the primarily OX₁ selective antagonists SB-334867 and SB-408124 displayed low affinity (pK_I values ca. 6). Competition kinetic analysis showed that the compounds displayed a range of dissociation rates from very fast (TCS-OX2-29, k_off = 0.22 min⁻¹) to very slow (almorexant, k_off = 0.005 min⁻¹). Notably, there was a clear correlation between association rate and affinity. In the cell-based assays, fast-offset antagonists EMPA and TCS-OX2-29 displayed surmountable antagonism of orexin-A agonist activity. However, both suvorexant and particularly almorexant cause concentration-dependent depression in the maximal orexin-A response, a profile that is more evident with a shorter agonist incubation time. Analysis according to a hemi-equilibrium model suggests that antagonist dissociation is slower in a cellular system than in membrane binding; under these conditions, almorexant effectively acts as a pseudo-irreversible antagonist.Linked ArticlesThis article is part of a themed section on Orexin Receptors. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2014.171.issue-2     

Interindividual Differences in the Protein Binding of Sulfonamides: The Effect of Disease and Drugs 1,2

Abstract: The variation in the plasma protein binding of sulfonamides in healthy adults was minimal. Additional evidence for consistency in binding came from a literature survey in which it was found that the plasma protein binding of several sulfonamides in man and laboratory animals was remarkably similar as reported by investigators from different countries. Some hospitalized patients, however, do show a binding deficiency which may be due to an interaction between drugs, disease and altered plasma proteins. Several drugs were found to compete with the sulfonamide for the same binding sites on the protein (albumin) molecule. The plasma protein binding of a sulfonamide in six anephric patients was studied before and after haemodialysis. As expected all showed a deficiency in binding. Unexpectedly, however, the binding was less in the post-dialysis plasma sample in three patients. In some patients there appeared to be a qualitative and a quantitative change in the binding sites on the protein. More definitive studies are needed with plasma protein fractions from healthy and ill subjects in the presence and absence of drugs. This may lead to a better understanding of the side effects to some drugs and could result in the development of useful displacing agents.     

Thermodynamics of Ligand Binding and Efficiency

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Revealing interaction mode between HIV-1 protease and mannitol analog inhibitor

HIV protease is a key enzyme to play a key role in the HIV-1 replication cycle and control the maturation from HIV viruses to an infectious virion. HIV-1 protease has become an important target for anti-HIV-1 drug development. Here, we used molecular dynamics simulation to study the binding mode between mannitol derivatives and HIV-1 protease. The results suggest that the most active compound (M35) has more stable hydrogen bonds and stable native contacts than the less active one (M17). These mannitol derivatives might have similar interaction mode with HIV-1 protease. Then, 3D-QSAR was used to construct quantitative structure-activity models. The cross-validated q(2) values are found as 0.728 and 0.611 for CoMFA and CoMSIA, respectively. And the non-cross-validated r(2) values are 0.973 and 0.950. Nine test set compounds validate the model. The results show that this model possesses better prediction ability than the previous work. This model can be used to design new chemical entities and make quantitative prediction of the bioactivities for HIV-1 protease inhibitors before resorting to in vitro and in vivo experiment.     

Design and structure of symmetry-based inhibitors of HIV-1 protease

Summary The concept of active-site symmetry along with structural and mechanistic considerations of aspartic proteases has been used to design novel,C ₂-symmetry-based inhibitors of HIV PR. A comparative molecular-modeling strategy was used in the design of a pseudo-C ₂-symmetric diaminoalcohol series and later a diastereomeric series ofC ₂-symmetric diaminodiols. The structure of the pseudosymmetric alcohol A-74704, complexed with HIV PR, confirmed the proposed symmetric mode of binding, and also proved useful in subsequent efforts to improve the solubility of the more potent diol series. The crystal-structure analysis of a pseudosymmetricR,S-diol provided insights into the relative contributions of the two hydroxyl groups to the stability of the complex. A comparison of the binding modes for symmetric and asymmetric, substrate-based inhibitors indicates that the influence of symmetry on binding is mainly expressed at the protein level, since the overall subsite symmetry is maintained even with highly asymmetric inhibitors. Thus, while the incorporation ofC ₂ symmetry does not necessarily lead to compounds with improved potency over asymmetric compounds, this strategy has led to the design of structurally novel and perhaps pharmacologically desirable inhibitors. The growing number of examples of symmetry-based inhibitors is evidence that symmetry has become a useful paradigm for HIV protease inhibitor design and that structure-based approaches to inhibitor design can contribute in unique and critical ways to the conceptualization of medicinal chemistry strategies that can lead to useful clinical candidates for AIDS, cancer and other diseases.     

Novel method for probing the specificity binding profile of ligands: applications to HIV protease

A detailed understanding of factors influencing the binding specificity of a ligand to a set of desirable targets and undesirable decoys is a key step in the design of potent and selective therapeutics. We have developed a general method for optimizing binding specificity in ligand-receptor complexes based on the theory of electrostatic charge optimization. This methodology can be used to tune the binding of a ligand to a panel of potential targets and decoys, along the continuum from narrow binding to only one partner to broad binding to the entire panel. Using HIV-1 protease as a model system, we probe specificity in three distinct ways. First, we probe interactions that could make the promiscuous protease inhibitor pepstatin more selective toward HIV-1 protease. Next, we study clinically approved HIV-1 protease inhibitors and probe ways to broaden the binding profiles toward both wild-type HIV-1 protease and drug-resistant mutants. Finally, we study a conformational ensemble of wild-type HIV-1 protease to 'design in' broad specificity to known drugs before resistance mutations arise. The results from this conformational ensemble were similar to those from the drug-resistant ensemble, suggesting the use of a conformational wild-type ensemble as a tool to develop escape-mutant-resistant inhibitors.     

Prediction of HIV-1 protease/inhibitor affinity using RosettaLigand

Predicting HIV-1 protease/inhibitor binding affinity as the difference between the free energy of the inhibitor bound and unbound state remains difficult as the unbound state exists as an ensemble of conformations with various degrees of flap opening. We improve computational prediction of protease/inhibitor affinity by invoking the hypothesis that the free energy of the unbound state while difficult to predict is less sensitive to mutation. Thereby the HIV-1 protease/inhibitor binding affinity can be approximated with the free energy of the bound state alone. Bound state free energy can be predicted from comparative models of HIV-1 protease mutant/inhibitor complexes. Absolute binding energies are predicted with R = 0.71 and SE = 5.91 kJ/mol. Changes in binding free energy upon mutation can be predicted with R = 0.85 and SE = 4.49 kJ/mol. Resistance mutations that lower inhibitor binding affinity can thereby be recognized early in HIV-1 protease inhibitor development.     

Saturable Binding of Cyclosporin A to Erythrocytes: Estimation of Binding Parameters in Renal Transplant Patients and Implications for Bioavailability Assessment

Cyclosporin (CyA) exhibits saturable binding to erythrocytes. A one-site binding model was fitted to data from renal transplant patients receiving CyA therapy. The average maximum binding capacity is 2560 µg CyA/liter of packed erythrocytes; the unbound CyA concentration associated with 50% saturation of the binding site is 67 µg/liter. Analysis indicates that whole-blood CyA measurement to monitor drug therapy should be viewed cautiously, particularly when the hematocrit varies considerably. The error in estimating absolute bioavailability at steady state from whole-blood measurements, resulting as a consequence of the saturable binding, has been explored. Although extrapolation to the therapeutic situation, which involves transient drug administration, is difficult, errors of up to 25% are anticipated. When an accurate estimate of bioavailability is required, analysis based on plasma data is proposed. For bioequivalence testing, both blood and plasma CyA data are equally acceptable.     

2,2-二甲基-1,3-二氧戊环-4,5-二羧酸衍生物的设计、合成及其神经营养活性

目的设计并合成具有神经营养活性的小分子化合物。方法根据FKBPs家族蛋白活性位点在序列和构象上高度保守的特点以及FKBP12配体的关键结构特征，设计并合成新结构的FKBPs配体。应用鸡胚背根神经节体外无血清培养模型和H202损伤NG108—15细胞模型对新结构的FKBPs配体的神经营养活性进行评价。结果与结论合成了27个2，2-二甲基-1,3-二氧戊环-4，5-二羧酸衍生物。初步药理实验结果显示化合物7d对H2O2损伤NG108细胞有明显的保护效果；化合物70具有一定的促神经生长作用。     

Binding of Basic Drugs to Rat Lung Mitochondria

The role of the mitochondria in the accumulation of basic amine drugs in the rat lung was studied. Drug binding to the mitochondria was rapid and reached maximum levels after 2.5 min of incubation. Lipophilic basic drugs accumulated in the mitochondria more than nonlipophilic basic drugs and non-basic drugs, and the accumulation was dose dependent. Schatchard plots revealed at least two independent sets of binding sites for basic drugs in the mitochondria. The binding was competitively inhibited by other basic drugs but not nonbasic drugs. The degree of inhibition by competing basic drugs was correlated with their lipid solubilities. These findings with isolated mitochondria agree with previous results obtained with the perfused lung preparation and indicate that the mitochondria play an important role in the accumulation of basic drugs in the lung.     

Binding of tricyclic antidepressants and perazine to human plasma

Summary The binding of amitriptyline (AT), nortriptyline (NT), imipramine (IP), desmethylimipramine (DMI) and perazine (PER) to plasma from healthy volunteers was measured by equilibrium dialysis at 37°C. Tritium-labeled drugs of high specific activity were prepared by methylation of the corresponding secondary and primary amines. The concentrations used in binding experiments were in the low range of those achieved in therapy. Under conditions of rigorous pH control unbound fractions were 7.8±1.0% for AT, 11.0±1.2% for NT, 11.5±1.4% for IP, 15.3±1.6% for DMI and 4.6±0.8% for PER in 43 subjects aged 16–58 years. Binding values did not depend on sex or age, and in females no difference was detected between users and non-users of sexual hormones. A significant negative correlation was established between plasma total cholesterol and the free fractions of AT, NT, DMI and PER. Corresponding alterations were observed intraindividually when the cholesterol levels fluctuated within several weeks or months. The phospholipid concentration in plasma did not correlate with drug binding. The concentration dependence of AT and NT binding pointed to the contribution of plasma constituents present in low concentrations and exhibiting high affinity towards the drugs. Part of these constituents may be lipoproteins.     

米卡芬净对厄他培南血浆蛋白结合率的影响

目的 建立HPLC测定人血浆中游离厄他培南浓度的方法,并研究米卡芬净对厄他培南血浆蛋白结合率的影响。方法 用超滤法进行样品处理,色谱柱为DiamonsiL C₁₈（4.6 mm×150 mm,5.0 μm）,流动相为乙腈-10 mmol·-L¹ 醋酸钠溶液（12.4∶87.6）,流速为1.0 mL·min^-1,检测波长为295 nm,柱温为35℃,进样量10 μL。采用超滤法结合HPLC,测定未加和加入米卡芬净后厄他培南的人血浆蛋白结合率。结果 游离厄他培南浓度在2～50 μg·mL^-1内线性关系良好（r=0.999）;低中高浓度厄他培南回收率分别为（102.3±2.5）%,（97.6±3.1）%和（98.6±2.2）%;日内、日间精密度均<5%。与米卡芬净合用后,厄他培南血浆蛋白结合率无显著变化。结论 该法可测定厄他培南游离浓度,方法准确快捷,灵敏度高。在人血浆中米卡芬净不影响厄他培南的体外蛋白结合率。     

pH Dependent Binding of Ligands to Serum Lipoproteins

Purpose. The binding interactions of binedaline, nicardipine and darodipine with lipoproteins (HDL, LDL, VLDL) were examined as a function of pH in order to evaluate the role of lipoprotein components and ligand protonation in the binding process. Methods. Binding studies were performed by equilibrium dialysis with radiolabeled ligands and differential UV-visible spectroscopy. Results. Deprotonated ligands had a markedly higher affinity for lipoproteins than the protonated forms, resulting in a concomitant decrease in the pK_a of bound ligands, i.e., a decrease in the basicity of the ligand in the bound state. The UV-visible difference spectra generated upon binding of auramine O to lipoproteins also showed that there was a contribution to the binding arising from the deprotonation of the ligand. Ligand binding was related to the phospholipid and cholesteryl ester content and to a lesser degree to the free cholesterol and protein content of lipoproteins, therefore to the surface monolayer components of lipoproteins. This relationship was even more accurate for the deprotonated, high-affinity, than for the protonated species. Conclusions. It is suggested that among other possible interactions, ligand binding to lipoproteins involves proton exchange between the reactants and that the high affinity ligand species interact more specifically with the phospholipids of the lipoprotein surface monolayer.     

Kinetics of the Binding of Salicylazosulfapyridine to Human Serum Albumin

Abstract In order to elucidate the possibility of the dissociation rate of drugs from plasma proteins presenting a rate limiting step in the elimination of drugs by secretion (renal or biliary) and metabolism, the kinetics of salicylazosulfa-pyridine (SASP) binding to human serum albumin (HSA) has been investigated by stopped-flow photometry. Equilibrium dialysis showed that HSA has three classes of binding sites for SASP with 0.93, 2.3 and 8.4 sites, respectively, and association constants of 2.1 · 10⁶, 1.4 · 10⁵ and 3.0 · 10³ M^-1, respectively. The association rate constants for the first and second classes are 4.4 · 10⁶ and 1.5 · 10⁷ M^-1 sec.^-1, and the dissociation rate constants are 2.1 and 109 sec.^-1. At SASP concentrations resulting from the usual therapeutic doses about 83 % will bind to the first class binding sites. The dissociation “half time” for this class being 0.34 sec., leads to the conclusion that dissociation rates of this order of magnitude are unlikely to reduce the rate of metabolism or biliary secretion whereas it may reduce renal tubular secretion. Whether this is the case depends on the intrinsic rate constant of secretion.     

Absorption of new HIV-1 protease inhibitor,KNI-272, after intraduodenal and intragastric administrations to rats: Effect of solvent

KNI-272 is a tripeptide drug that has a strong pharmacological potential for treating human immunodeficiency virus type 1 (HIV-1). We have already reported the pharmacokinetic characteristics of KNI-272 after intravenous and intraduodenal (ID) administrations to rats. In this study, KNI-272 was administered to rats as a solution and the effect of four kinds of solvent on the bioavailability (BA) of KNI-272 was determined using rats. The mixtures included propylene glycol (PG) and water (70% PG), a solution of PG (100% PG), a solution of Tween 80 (Tween 80), and a mixture of PG and HCO60, a polyoxyethylated, 60μmol, castor oil derivative (PG:HCO60=7:3). After ID administration to rats at a dose of 50.0 mg kg^?1, the mean peak plasma concentrations, C_max, were 2.58±0.53 (SE) (70% PG), 3.28±0.51 (100% PG), 3.15±0.51 (Tween 80), and 4.66±0.68 μg mL^?1 (PG:HCO60). The highest BA, 44.6%, was obtained after ID administration of KNI-272 dissolved in PG:HCO60. On the other hand, after intragastric (IG) administration of KNI-272 solution in which the drug was dissolved with PG:HCO60, the T_max, the C_max, and the BA were 1.25±0.60h, 2.33±0.65 μg mL^?1, and 24.2%, respectively. The C_max and BA values were equal to half of the values obtained after ID administration of KNI-272 dissolved in the same solution. In this study, as the PG concentration in the solution increased and the other additives (Tween 80 and HCO60) were coadministered, the BA of KNI-272 after ID administration increased. These results suggest that, for the development of an oral dosage form of KNI-272, a non-ionic surfactant that dissolves in the duodenum or small intestine and that enhances the absorption of this drug from the gastrointestinal tract into the enterocytes is needed.