The influence of drug distribution and drug-target binding on target occupancy: The rate-limiting step approximation.

The influence of drug-target binding kinetics on target occupancy can be influenced by drug distribution and diffusion around the target, often referred to as "rebinding" or "diffusion-limited binding". This gives rise to a decreased decline of the drug-target complex concentration as a result of a locally higher drug concentration that arises around the target, which leads to prolonged target exposure to the drug. This phenomenon has been approximated by the steady-state approximation, assuming a steady-state concentration around the target. Recently, a rate-limiting step approximation of drug distribution and drug-target binding has been published. However, a comparison between both approaches has not been made so far. In this study, the rate-limiting step approximation has been rewritten into the same mathematical format as the steady-state approximation in order to compare the performance of both approaches for the investigation of the influence of drug-target binding kinetics on target occupancy. While both approximations clearly indicated the importance of kon and high target concentrations, it was shown that the rate-limiting step approximation is more accurate than the steady-state approximation, especially when dissociation is fast compared to association and distribution out of the binding compartment. It is therefore concluded that the new rate-limiting step approximation is to be preferred for assessing the influence of binding kinetics on local target site concentrations and target occupancy.

Estimation of the dissociation rate of unlabelled ligand-receptor complexes by a 'two-step' competition binding approach.

BACKGROUND AND PURPOSE: Because the in vivo effectiveness of ligands may also be determined by the rate by which they dissociate from their target receptors, drug candidates are being increasingly screened for this kinetic property. The dissociation rate of unlabelled ligand-receptor complexes can be estimated indirectly from their ability to slow the association of subsequently added radioligand molecules. EXPERIMENTAL APPROACH: We used the 'two-step competition' binding approach consisting of pre-incubating the receptor preparation with a wide range of ligand concentrations, washing off free ligand molecules, adding radioligand and monitoring its receptor binding after a fixed time. Based on the rationale that binding of both ligands is mutually exclusive and that they bind according to the law of mass action to a single class of sites, the unlabelled ligand's dissociation rate can be estimated from the upward shift that the competition curve experiences after washing. KEY RESULTS: The relevance of the 'two-step competition' approach was explored by computer simulations and by comparing the dissociation behaviour of unlabelled D(2) dopamine and CB(1) cannabinoid receptor antagonists in this and alternative approaches. Besides providing satisfactory estimations of dissociation rates, the method also detects the ability of the unlabelled ligand molecules to be released from 'sinks' such as the cell membrane. CONCLUSIONS AND IMPLICATIONS: As the 'two-step competition' requires rapid intermediate washing steps and needs radioligand binding to be measured at only one time point, this approach is particularly suited for binding studies on intact plated cells. LINKED ARTICLES: This article is part of a themed section on Analytical Receptor Pharmacology in Drug Discovery. To view the other articles in this section visit http://dx.doi.org/10.1111/bph.2010.161.issue-6.

Ligands, their receptors and ... plasma membranes.

Ligand-receptor interactions are customarily described by equations that apply to solutes. Yet, most receptors are present in cell membranes so that sufficiently lipophilic ligands could reach the receptor by a two-dimensional approach within the membrane. As summarized in this review, this may affect the ligand-receptor interaction in many ways. Biophysicians calculated that, compared to a three-dimensional approach from the liquid phase, such approach could alter the time the ligands need to find a receptor. Biochemists found that ligand incorporation in lipid bilayers modifies their conformation. This, along with the depth at which the ligands reside in the bilayer, will affect the probability of successful receptor interaction. Novel mechanisms were also introduced, including "exosite" binding and ligand translocation between the receptor's alpha-helical transmembrane domains. Pharmacologists focused attention at ligand concentrations in membrane, their adsorption and release rates and the effects thereof on ligand potency and residence time at the receptor.

Molecular mechanisms for the persistent bronchodilatory effect of the beta 2-adrenoceptor agonist salmeterol.

BACKGROUND: Beta(2)-adrenoceptor agonists are effective bronchodilators. In vitro studies demonstrated long-lasting airway smooth muscle relaxation by salmeterol after washout, the quick disappearance of this effect in presence of antagonists and its recovery after antagonist removal. Current explanations invoke salmeterol accumulation in the membrane ('diffusion microkinetic' model) or the existence of salmeterol-binding 'exosites'. An alternative model based on 'rebinding' of a dissociated ligand to the receptor molecules also produces an apparent decrease in the ligand's dissociation rate in the absence of competing ligands. PURPOSE AND APPROACH: Computer-assisted simulations were performed to follow the receptor-occupation by a salmeterol-like ligand and a competing ligand as a function of time. The aptness of the models to describe the above in vitro findings was evaluated. KEY RESULTS: The 'diffusion microkinetic' model is sufficient to explain a long-lasting beta(2)-adrenoceptor stimulation and reassertion as long as the membrane harbors a high concentration of the agonist. At lower concentration, 'rebinding' and, in second place, 'exosite' binding are likely to become operational. CONCLUSIONS AND IMPLICATIONS: The 'rebinding' and 'exosite' binding mechanisms take place at a sub-cellular/molecular scale. Pending their demonstration by experiments on appropriate, simple models such as intact cells or membranes thereof, these mechanisms remain hypothetical in the case of salmeterol. Airway smooth muscle contraction could also be governed by additional mechanisms that are particular to this macroscopic approach.

Sartan-AT1 receptor interactions: in vitro evidence for insurmountable antagonism and inverse agonism.

Sartans are non-peptide AT(1) receptor antagonists used to treat hypertension and related pathologies. Their effects on the G protein-dependent responses of angiotensin II (Ang II) were the same in vascular tissues and in isolated cell systems. All are competitive but, when pre-incubated, they act surmountably (only rightward shift of the Ang II concentration-response curve) or insurmountably (also decreasing the maximal response). Insurmountable behaviour reflects the formation of tight sartan-receptor complexes; it is often partial due to the co-existence of tight and loose complexes. Their ratio positively correlates with the dissociation half-life of the tight complexes and depends on the sartan: i.e. candesartan>olmesartan>telmisartan approximately equal EXP3174>valsartan>irbesartan>>losartan. When AT(1) receptors display sufficient basal activity (in case of receptor over-expression, mutation and, especially, tissue stretching) sartans may also act as inverse agonists. This rather affects long-term, G protein-independent hypertrophic responses leading to cardiovascular remodelling.

Translocation of the insulin-regulated aminopeptidase to the cell surface: detection by radioligand binding.

BACKGROUND AND PURPOSE: Insulin-regulated aminopeptidase (IRAP) and the insulin-dependent glucose transporter GLUT4 colocalize in specific intracellular vesicles (that is, GLUT4 vesicles). These vesicles move slowly to the cell surface, but their translocation is markedly enhanced by insulin, resulting in higher glucose uptake. Previous studies of the insulin-mediated translocation of IRAP to the cell surface have been hampered by the laborious detection of IRAP at the cell surface. We aimed to develop a more direct and faster method to detect IRAP. To this end, we used model systems with well-characterized IRAP: CHO-K1 cells expressing endogenous IRAP and recombinant HEK293 cells expressing human IRAP. A more widespread application of the method was demonstrated by the use of 3T3-L1 adipocytes. EXPERIMENTAL APPROACH: After stimulation of the cells with insulin, internalization of IRAP was inhibited by the addition of phenyl arsine oxide (PAO). Then, cell-surface IRAP was detected by the high-affinity binding of radiolabelled angiotensin (Ang) IV (either 125I or 3H). KEY RESULTS: We monitored the time- and concentration dependence of insulin-mediated translocation of IRAP in both cell lines and 3T3-L1 adipocytes. A plateau was reached between 6 and 8 min, and 10(-7) M insulin led to the highest amount of IRAP at the cell surface. CONCLUSIONS AND IMPLICATIONS: Based on the capacity of the IRAP apoenzyme to display high affinity for radiolabelled Ang IV and on the ability of PAO to inhibit IRAP internalization, we developed a more direct and faster method to measure insulin-mediated translocation of IRAP to the cell surface.

Molecular characterisation of the interactions between olmesartan and telmisartan and the human angiotensin II AT1 receptor.

BACKGROUND AND PURPOSE: Whereas some angiotensin II (Ang II) type 1 receptor blockers (ARBs) produce surmountable antagonism of AT(1) receptors, others such as olmesartan and telmisartan display varying degrees of insurmountability. This study compared the molecular interactions of olmesartan and telmisartan with the human AT(1) receptor, using well characterised in vitro methods and model systems. EXPERIMENTAL APPROACH: CHO-K1 cells that stably express human AT(1) receptors (CHO-hAT(1) cells) were used in several pharmacological studies of olmesartan and telmisartan, including direct radioligand binding and inhibition of Ang II-induced inositol phosphate (IP) accumulation. KEY RESULTS: Both ARBs were found to be competitive antagonists that displayed high affinity, slow dissociation, and a high degree of insurmountability for the AT(1) receptor (the latter greater with olmesartan). Their receptor interactions could be described by a two-step process with the initial formation of a loose complex (IR) and subsequent transformation into a tight binding complex (IR*). In washout experiments, [(3)H] telmisartan dissociated from the receptor with a half-life of 29 min and the Ang II-mediated IP accumulation response was 50% maximally restored within 24 min, whereas values for [(3)H] olmesartan were 72 min and 76 min, respectively. CONCLUSIONS AND IMPLICATIONS: The high degree of insurmountability, slow dissociation, and high affinity of olmesartan for its receptor may relate to its ability to stabilise IR* via the carboxyl group of its imidazole core. In comparison, telmisartan displays a less potent interaction with the receptor.

G protein-coupled receptors: a count of 1001 conformations.

G protein-coupled receptors (GPCRs) were initially regarded to adopt an inactive and an active conformation and to activate a single type of G protein. Studies with recombinant cell systems have led to a more complex picture. First, GPCRs can activate distinct G protein species. Second, GPCR multistate models have been invoked to explain their complex behaviour in the presence of agonists, antagonists and other binding partners. The occurrence of intermediate receptor conformational states during GPCR activation and antagonist binding is suggested by fluorescence measurements and studies with constitutively active receptor mutants and insurmountable antagonists. Different agonists may trigger distinct effector pathways through a single receptor by dictating its preference for certain G proteins (i.e. 'agonist trafficking'). Structural modification and exogenous and endogenous (e.g. other cellular proteins, lipids) allosteric modulators also affect ligand-GPCR interaction and receptor activation. These new developments in GPCR research could lead to the development of more selective therapeutic drugs.

Evaluation of bioactive saponins and triterpenoidal aglycons for their binding properties on human endothelin ETA and angiotensin AT1 receptors.

Different types of triterpenes including saponins and aglycons were evaluated for their ability to inhibit [3H] BQ-123 and [3H] angiotensin II binding to the human endothelin 1 ETA and angiotensin II AT1 receptors, respectively. Selectivity for only one of the two receptors was exhibited by asiatic acid and its saponins (ETA) and oleanolic acid (AT1). To a lesser extent betulinic acid, beta-amyrin and friedelin also showed selectivity for the ETA receptor. To address the question whether the effect of saponins on cell membranes might interfere with the normal binding of specific radioligands to their receptors, the activity of saponins with different haemolytic properties were compared. Highly haemolytic saponins such as alpha-hederin and beta-escine showed partial (60%) inhibition of radioligand-binding to the ETA receptor and complete inhibition (100%) to the AT1 receptor. Moreover, the haemolytically inactive kryptoescine, at the same concentration, caused complete inhibiton of radioligand-binding to both receptors, indicating that inhibition of receptor binding was not related to the membrane-interacting properties of saponins.

AT1 receptor antagonists.

Type 1 receptors (AT1) for the peptide hormone angiotensin II play a crucial role in the cardiovascular homeostasis. In this regard, several selective, orally active non-peptide antagonists have been developed for the treatment of hypertension and congestive heart failure. Pre-clinically, they have been routinely tested for their ability to inhibit angiotensin II induced contraction of rabbit aorta strips. This led to the distinction between surmountable antagonists, which only produce a parallel rightward shift of the angiotensin II concentration- induced response curve, and insurmountable antagonists that also decrease the maximal response. The molecular mechanism that is responsible for insurmountable antagonism has been extensively investigated in Chinese Hamster Ovary cells transfected expressing the human AT1 receptor. These experiments revealed that all biphenyltetrazole-countering AT1 receptor antagonists are competitive with angiotensin II and that the insurmountable behaviour of some of them is related to the formation of a long lasting/tight binding state of the antagonist-receptor complex. This may contribute to their long lasting clinical effect. This paper also focuses on the influence of a number of methodological approaches used to study AT1 receptor antagonists on their observed in vitro receptor binding properties.

New insights in insurmountable antagonism.

Antagonists that produce parallel rightward shifts of agonist dose-response curves with no alteration of the maximal response are traditionally classified as surmountable, while insurmountable antagonists also depress the maximal response. Although the longevity of the antagonist-receptor complex is quoted in many studies to explain insurmountable antagonism, slowly interconverting receptor conformations, allosteric binding sites, and receptor internalization have been evoked as alternative explanations. To complicate matters even further, insurmountable antagonism is not only drug-related; it may also depend on the tissue, species and experimental design. For the sake of drug development, it is important to elucidate the molecular mechanisms of insurmountable antagonism. New experimental approaches, such as intact cell studies and the use of computer-assisted simulations based on dynamic receptor models, herald the advent of better insight in the future.

Effects of BIBP3226 and BIBP3435 on cytosolic calcium in neuropeptide Y Y1 receptor-transfected Chinese hamster ovary cells and wild type CHO-K1 cells.

The NPY Y1-receptor selective antagonist BIBP3226 exerts a dual control on the cytosolic free calcium concentration ([Ca2+]i) in NPY Y1 receptor-transfected Chinese Hamster Ovary Cells (CHO-Y1 cells). It is a potent inhibitor of the NPY-evoked increase in [Ca2+]i. This can be ascribed to its antagonistic properties for the NPY Y, receptor since its less active stereoisomer, BIBP3435, is much less potent. However, when its concentration exceeds 1 microM, BIBP3226 produces a large increase in [Ca2+]i on its own. This effect is mimicked by BIBP3435 and it also occurs in wild type CHO-K1 cells. These latter cells do not contain high affinity binding sites for [3H]NPY and [3H]BIBP3226 and, hence, no endogenous NPY Y1 receptors. It is concluded that, at moderately high concentrations, the NPY Y1 receptor antagonist BIBP3226 and its entantiomer BIBP3435 are able to increase the [Ca2+ ]i in CHO cells either by stimulating another receptor or by directly affecting cellular mechanisms that are involved in calcium homeostasis.

Tight binding of the angiotensin AT(1) receptor antagonist.

Angiotensin II induces angiotensin AT(1) receptor internalization via Clathrin coated pits formation. We investigated whether insurmountable inhibition by the non-peptide antagonist 2-ethoxy-1-[(2'-(1H-tetrazol-5-yl) biphenyl-4-yl) methyl]-1H-benzimidazoline-7-carboxylic acid (candesartan) was related to receptor internalization. Mild acid treatment can discriminate between internalized and cell surface bound [(3)H]angiotensin II. In contrast, it provides no information about the subcellular localization of bound [(3)H]candesartan since this binding is acid resistant. The internalization of [(3)H]angiotensin II is rapidly inhibited in the presence of 0.4 M sucrose. Yet, no such rapid effect was noticed for [(3)H]candesartan. [(3)H]candesartan displays insurmountable/long lasting binding to the vast majority of both wild type and L(314) truncated rat angiotensin AT(1A) receptors with impaired receptor internalization. In agreement with previously published AT(1) angiotensin receptor visualization experiments, the present data suggest that non-peptide antagonist-angiotensin AT(1) receptor complexes remain at the cell surface. Insurmountable antagonism of candesartan is therefore independent from receptor internalization via clathrin-coated pits.

Biological screening of selected medicinal Panamanian plants by radioligand-binding techniques.

Nineteen plants from the Republic of Panama were selected by their traditional uses in the treatment of hypertension, cardiovascular, mental and feeding disorders and 149 extracts were screened using radioligand-receptor-binding assays. The methanol:dicloromethane extracts of the bark and leaves of Anacardium occidentale L., the leaves of Begonia urophylla Hook., the roots of Bocconia frutescens L., the stems and leaves of Cecropia cf.obtusifolia Bertol., the branches of Clusia coclensis Standl., the bark of Cochlospermum vitifolium (Willd.)Spreng., the roots of Dimerocostus strobilaceus Kuntze, the bark of Guazuma ulmifolia Lam., the leaves of Persea americana Mill. and the branches of Witheringia solanaceae L'Her. inhibited the [3H]-AT II binding (angiotensin II AT1 receptor) more than 50%. Only extracts of the roots of Dimerocostus strobilaceus Kuntze and the stems of Psychotria elata (Sw.) Hammel were potent inhibitors of the [3H] NPY binding (neuropeptide Y Y1 receptor) more than 50% and the ethanolic extracts of the leaves of Cecropia cf. obtusifolia Bertol., the leaves of Hedyosmum bonplandianum H.B.K., the roots of Bocconia frutescens L., the stem of Cecropia cf. obtusifolia Bertol. and the branches of Psychotria elata (Sw.) Hammel showed high inhibition of the [3H] BQ-123 binding (endothelin-1 ET(A) receptor) in a preliminary screening. These results promote the further investigation of these plants using the same assays.

A two-state receptor model for the interaction between angiotensin II type 1 receptors and non-peptide antagonists.

The interaction between non-peptide antagonists and the human angiotensin II type 1 (AT1) receptor in CHO-K1 cells was investigated by incubating the cells with antagonist, followed by a brief exposure to angiotensin II and measurement of the resulting inositol phosphate accumulation. The experimental data, expressed either as angiotensin II concentration-response curves or as antagonist concentration-inhibition curves, were in good agreement with computer-generated data according to a single-state model for the surmountable antagonist losartan and according to a two-step, two-state receptor model for the insurmountable antagonists candesartan, EXP3174, and irbesartan. Experimental and computer-generated data concerning the simultaneous exposure of the receptors to EXP3174 and losartan indicated that losartan produced a concentration-dependent restoration of the maximal response (angiotensin II concentration-response curves) as well as a rightward shift of the insurmountable portion of the EXP3174 inhibition curves, thus counteracting the higher-affinity EXP3174 binding. In conclusion, these findings provide further support for the concept that insurmountable and surmountable AT1 antagonists are mutually competitive and that insurmountable antagonist-receptor complexes may adopt different states.

How can the differences among AT1-receptor antagonists be explained?

Over the last few years we have seen a new class of antihypertensive drug evolve, the angiotensin II subtype 1 receptor antagonists. Hypothetically, all substances in this class should have the same effect on blood pressure and on end-organ damage as they all block the AT1 receptor. However, there are distinctions between them that may explain the significant and clinically important differences that seem to exist within this class of drug. An explanation for the differences may be found in receptor-antagonist kinetics. The receptor-antagonist interaction may be fitted to a two-state, two-step model which determines how large a part of the binding that will be surmountable and how large a part that will be insurmountable. The proportion of surmountable/insurmountable binding fits nicely to the duration of binding of the antagonist to the receptor, which may be translated into efficacy for the antagonist as outlined in the following review.

Angiotensin II type 1 receptor antagonists. Why do some of them produce insurmountable inhibition?

Chinese hamster ovary (CHO) cells expressing human recombinant angiotensin II type 1 (AT(1)) receptors offer a useful experimental system in which antagonist binding and inhibition of AT-induced inositol mono-, bis-, and trisphosphate accumulation can be measured under identical experimental conditions. The major conclusions of the current work are: All investigated AT(1) antagonists are competitive with respect to AT. They bind to a common or overlapping binding site on the receptor in a mutually exclusive way. Reduction of the maximal angiotensin II response, i.e. insurmountable inhibition, is observed only when the cells are preincubated with candesartan, EXP3174, or irbesartan and is strictly related to the dissociation rate of the antagonist-receptor complex. On the other hand, inhibition by losartan is fully surmountable by AT, and its dissociation is very rapid. With respect to the binding kinetics, the antagonist-receptor complex can adopt a fast and a slow reversible state. The equilibrium between both states, which is dependent upon the nature of the antagonists, determines the extent of insurmountable inhibition. Consequently, the dissociation rate of the different antagonists correlates with the amount of insurmountable inhibition. In addition to the relatively slow dissociation of candesartan, reassociation to the receptor, which is measurable in CHO-AT(1) cells, likely contributes to its long-lasting blood pressure lowering effect in vivo.

Reversible and syntopic interaction between angiotensin receptor antagonists on Chinese hamster ovary cells expressing human angiotensin II type 1 receptors.

Evidence for a competitive type of interaction between angiotensin II type 1 (AT(1)) antagonists on Chinese hamster ovary cells expressing the human AT(1) receptor (CHO-AT(1)) was obtained by analyzing the binding of [(3)H]-2-ethoxy-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]-1H- ben zimidazoline-7-carboxylic acid ([(3)H]candesartan) and by measuring the AT-induced production of inositol phosphates. The AT(1) antagonists candesartan, 2-n-butyl-4-chloro-1-[(2'-(1H-tetrazol-5-yl)biphenyl-4-yl)methyl]+ ++imid azole-5-carboxylic acid (EXP3174), or 2-n-butyl-4-chloro-5-hydroxymethyl-1-[(2'-(1H-tetrazol-5-yl)bip hen yl- 4-yl)methyl]imidazole (losartan) produced a concentration-dependent increase in the apparent K(d) values of [(3)H]candesartan in saturation binding experiments, while the B(max) values were unchanged. Furthermore, the dissociation rate of the radioligand initiated by 1 microM unlabelled candesartan was not changed in the presence of 10 microM losartan, 10 microM EXP3174, or 10 microM irbesartan (2-n-butyl-4-spirocyclopentane-1-[(2'-(1H-tetrazol-5-yl)b iph enyl-4-yl) methyl]2-imidazolin-5-one)). Preincubation of the CHO-AT(1) cells with candesartan, EXP3174, and irbesartan caused a reduction in the maximal AT-induced inositol mono-, bis-, and trisphosphate production. This insurmountable effect was reversed in the presence of 1 microM losartan. In line with this finding, the insurmountable antagonist concentration-inhibition curves at 10 microM AT were shifted to the right in the presence of losartan. For candesartan this effect was concentration-dependent, yielding a pK(B) value for losartan of 7.7, which is similar to the pK(B) from previously obtained AT concentration-response curves. Finally, the dissociation rate of candesartan, EXP3174, irbesartan, and losartan was determined by measuring the recovery of AT responses after antagonist pretreatment and washing of the cells with medium containing 1 microM losartan to prevent re-association of the insurmountable antagonists. In addition, similar kinetic data were obtained from the slowing of the [(3)H]candesartan association rate to antagonist preincubated cells.

Interaction between the partially insurmountable antagonist valsartan and human recombinant angiotensin II type 1 receptors.

The interaction between the AT1 receptor-selective antagonist valsartan, and its human receptor, was investigated by direct radioligand binding as well as by its inhibition of angiotensin II induced inositol phosphate accumulation in CHO cells expressing human recombinant AT1 receptors. Specific binding of [3H]-valsartan rapidly reached equilibrium at 37 degrees C. It was saturable and occurred to a homogeneous class of sites with a K(D) of 0.88+/-0.076. It was inhibited by other AT1 receptor antagonists with the same potency order as previously described for the binding of [3H]-angiotensin II and [3H]-candesartan to human AT1 receptors (i.e. candesartan > or = EXP3174 > valsartan = irbesartan = angiotensin II > losartan). When valsartan and angiotensin II were applied simultaneously to the CHO-AT1 cells. the antagonist caused a rightward shift of the angiotensin II concentration response curve. Hence, valsartan interacts with the AT1 receptor in a manner that is competitive with angiotensin II. Pre-incubation of the cells with 0.5, 5 and 50 nM valsartan caused an additional, concentration-dependent, up to 55% decline of the maximal response. The partial nature of this insurmountable inhibition by valsartan was confirmed by biphasic antagonist concentration-inhibition curves. These data reflect the co-existence of a fast reversible/surmountable as well as a tight binding/insurmountable valsartan receptor complex. In agreement, pre-incubation of the CHO-AT1 cells with 5 and 50 nM valsartan produced a partial inhibition of the angiotensin II induced increase of the free intracellular calcium concentration. [3H]-Valsartan dissociated from its receptors with a half-life of 17 min. In functional recovery experiments with valsartan-pre-treated cells, the angiotensin II-mediated response was half-maximally restored within approximately 30 min. These kinetic data suggest that the insurmountable inhibition by valsartan is related to its relatively slow dissociation from the human AT1 receptors.