On the different experimental manifestations of two-state 'induced-fit' binding of drugs to their cellular targets.

'Induced-fit' binding of drugs to a target may lead to high affinity, selectivity and a long residence time, and this mechanism has been proposed to apply to many drugs with high clinical efficacy. It is a multistep process that initially involves the binding of a drug to its target to form a loose RL complex and a subsequent isomerization/conformational change to yield a tighter binding R'L state. Equations with the same mathematical form may also describe the binding of bivalent antibodies and related synthetic drugs. Based on a selected range of 'microscopic' rate constants and variables such as the ligand concentration and incubation time, we have simulated the experimental manifestations that may go along with induced-fit binding. Overall, they validate different experimental procedures that have been used over the years to identify such binding mechanisms. However, they also reveal that each of these manifestations only becomes perceptible at particular combinations of rate constants. The simulations also show that the durable nature of R'L and the propensity of R'L to be formed repeatedly before the ligand dissociates will increase the residence time. This review may help pharmacologists and medicinal chemists obtain preliminary indications for identifying an induced-fit mechanism.

The Role of Binding Kinetics in GPCR Drug Discovery.

Binding kinetics are the rates of association and dissociation of a drug-protein complex and are important molecular descriptors for the optimization of drug binding to G-protein coupled receptors (GPCRs). There are now many examples of binding kinetics in GPCR drug discovery. In this report, the first principles and examples of binding kinetics in GPCR drug discovery are reviewed. Addressed are the influence of binding kinetics on the translation of binding to the therapeutic window in the context of the equilibrium state of the system and molecular mechanisms of slow binding including induced fit, displacement of water, rebinding and heterovalency.

Radioligand binding to intact cells as a tool for extended drug screening in a representative physiological context.

Radioligand binding assays on intact cells offer distinct advantages to those on membrane suspensions. Major pharmacological properties like drug affinity and binding kinetics are more physiologically relevant. Complex mechanisms can be studied with a wider choice of experimental approaches and so provide insights into induced-fit type binding, receptor internalisation and even into pharmacomicrokinetic phenomena like drug rebinding and partitioning into the membrane. Hence, intact cell binding constitutes a valuable addition to the pharmacologist's toolbox.

Avidity and positive allosteric modulation/cooperativity act hand in hand to increase the residence time of bivalent receptor ligands.

Bivalent ligands bear two target-binding pharmacophores. Their simultaneous binding increases their affinity (avidity) and residence time. They become 'bitopic' when the binding sites at the target permit the pharmacophores the exert allosteric modulation of each other's affinity and/or activity. Present simulations reveal that positive cooperativity exacerbates these phenomena, whereas negative cooperativity curtails them, irrespective of whether the association or dissociation rates of the individual pharmacophores are affected. Positive cooperativity delays the attainment of equilibrium binding, yielding 'hemi-equilibrium' conditions and only apparent affinity constants under usual experimental conditions. Monovalent ligands that bind to one of the target sites decrease the bitopic ligand's residence time concentration-wise; their potency depends on their association rate and thereon acting cooperativity rather than on affinity. This stems from the repetitive, very fast reformation of fully bound bitopic ligand-target complexes by rebinding of freshly dissociated pharmacophores. These studies deal with kinetic binding properties (of increasing interest in pharmacology) of bitopic ligands (a promising avenue in medicinal chemistry).

Radioligand dissociation measurements: potential interference of rebinding and allosteric mechanisms and physiological relevance of the biological model systems.

INTRODUCTION: In many situations, optimal drug therapy requires continuing high levels of target occupancy and this notion has led pharmacologists to focus their attention on the rate by which drug candidates dissociate from their target. To this end, radioligand dissociation experiments are often carried out on in vitro models, such as intact cells and the membranes thereof, but the interpretation of the collected data is sometimes ambiguous. AREAS COVERED: Pharmacodynamics is concerned about what the drug does to the target and, in this respect, allosteric modulation constitutes a quite novel, very promising research topic. The ability of unlabeled drugs to accelerate radioligand dissociation is often advocated to be a hallmark of such mechanism. Yet, the present computerized simulations reveal that competitive drugs produce the same effect by preventing hindered diffusion- and "forced proximity"-related rebinding of the radioligand. Herein, the authors provide hints to discern among those mechanisms. EXPERT OPINION: A critical, but constructive appraisal of radioligand dissociation binding data leads to the viewpoint that, from a physiological perspective, dissociation from confluent target-expressing plated cells, when in a naïve medium, is likely to provide the most pertinent insight in that ligand's in vivo residence time.

Antagonist-radioligand binding to D2L-receptors in intact cells.

D(2)-dopamine receptors mediate most of the physiological actions of dopamine and are important recognition sites for antipsychotic drugs. Earlier binding studies were predominantly done with broken cell preparations with the tritiated D(2)-receptor antagonists [(3)H]-raclopride, a hydrophilic benzamide, and [(3)H]-spiperone, a highly hydrophobic butyrophenone. Here we compared [(3)H]-raclopride and [(3)H]-spiperone binding properties in intact Chinese Hamster Ovary cells stably expressing recombinant human D(2L)-receptors. Specific binding of both radioligands occurred to a comparable number of sites. In contrast to the rapid dissociation of [(3)H]-raclopride in both medium only and in the presence of an excess of unlabelled ligand [(3)H]-spiperone dissociation was only observed in the latter condition, and it was still slower than in broken cell preparations. However, this could not explain the pronounced difference in the potency of some unlabelled ligands to compete with both radioligands. To integrate these new findings, a model is proposed in which raclopride approaches the receptor from the aqueous phase, while spiperone approaches the receptor by lateral diffusion within the membrane.

Slow antagonist dissociation and long-lasting in vivo receptor protection.

The ability of antagonists to form slowly dissociating complexes with their cognate receptors has repeatedly been proposed to contribute to their long-lasting clinical actions. Yet specific conditions seem to be necessary for this to take place. The elimination rate of the free antagonist and the variation of agonist concentration with time have important roles. Slowly dissociating antagonists are likely to exert longer efficient receptor protection in vivo than are fast-dissociating antagonists when the half-life of the antagonist-receptor complex exceeds that of the free antagonist. However, when the half-life of the free antagonist prevails, longer effective protection by slowly dissociating antagonists occurs only if the receptor is exposed to rapid fluctuations in free agonist concentration.

Long-lasting angiotensin type 1 receptor binding and protection by candesartan: comparison with other biphenyl-tetrazole sartans.

BACKGROUND: The ability of biphenyl-tetrazole angiotensin type 1 (AT1) receptor antagonists (BTsartans) to block angiotensin II (Ang II)-mediated responses has been extensively investigated in vascular tissues and, more recently, in cell lines expressing the human AT1-receptor. When pre-incubated, BTsartans acted surmountably (shifting the Ang II concentration-response curve to the right) or insurmountably (also decreasing the maximal response). It was shown that their insurmountable behaviour is due to the formation of tight, long-lasting complexes with the receptor. Partial insurmountable antagonism is due to the co-existence of tight and loose complexes. The proportion of insurmountable antagonism, the potency and the dissociation rate of the BTsartans decreases in the order: candesartan > EXP3174 (losartan's active metabolite) > valsartan > irbesartan >> losartan. OBJECTIVE: It is of interest to explore how tight AT1-receptor binding of BTsartans such as candesartan might contribute to their long-lasting clinical effect. METHODS: Computer-assisted simulations (COPASI program) were performed to follow the receptor-occupation and protection by different antagonists as a function of time. Free antagonist concentrations were allowed to decrease exponentially with time. RESULTS: The simulations suggest that slow dissociation does not tangibly prolong receptor occupancy if the free antagonist is eliminated at a slower pace (as is the case for BTsartans). Yet when surmountable and insurmountable antagonists occupy the same amount of receptors, insurmountable antagonists offer appreciably better protection against fluctuations in natural messenger concentration. CONCLUSION: Slow receptor dissociation and slow antagonist elimination are likely to act in synergy to produce long-lasting receptor protection.

Models and methods for studying insurmountable antagonism.

Insurmountable antagonists depress the concentration-response curves of subsequently added agonists. The longevity of the antagonist-receptor complex and the existence of allosteric binding sites are the most frequent explanations for this phenomenon. Yet, observed antagonist behaviour often depends on the tissue, the animal species, the duration of the measured response and the study design. Intact cell studies allow greater flexibility and tighter control of the experimental conditions and therefore have the potential to offer a better insight into the molecular basis of insurmountable antagonism.