Competition in drug binding and … the race to equilibrium.

Binding kinetics has become a popular topic in pharmacology due to its potential contribution to the selectivity and duration of drug action. Yet, the overall kinetic aspects of complex binding mechanisms are still merely described in terms of elaborate algebraic equations. Interestingly, it has been recommended some 10 years ago to examine such mechanisms in terms of binding fluxes instead of the conventional rate constants.

Distinct In Vitro Binding Profile of the Somatostatin Receptor Subtype 2 Antagonist [Lu]Lu-OPS201 Compared to the Agonist [Lu]Lu-DOTA-TATE.

Treatment of neuroendocrine tumours with the radiolabelled somatostatin receptor subtype 2 (SST) peptide agonist [Lu]Lu-DOTA-TATE is effective and well-established. Recent studies suggest improved therapeutic efficacy using the SST peptide antagonist [Lu]Lu-OPS201. However, little is known about the cellular mechanisms that lead to the observed differences.

Induced fit versus conformational selection: From rate constants to fluxes… and back to rate constants.

Induced fit- (IF) and conformational selection (CS) binding mechanisms have long been regarded to be mutually exclusive. Yet, they are now increasingly considered to produce the final ligand-target complex alongside within a thermodynamic cycle. This viewpoint benefited from the introduction of binding fluxes as a tool for analyzing the overall behavior of such cycle.

Fluxes for Unraveling Complex Binding Mechanisms.

A decade ago, many high-affinity drugs were thought to bind to their target via an induced-fit pathway instead of conformational selection. Yet, both pathways make up part of a thermodynamic cycle, and, owing to binding flux-based approaches, it is now rather considered that they act in parallel and also that their relative contribution to the final ligand-target complex depends on the ligand concentration. Those approaches are of increasing interest, but published data still merely refer to the peculiar situation of equilibrium binding.

Mechanisms of PDZ domain scaffold assembly illuminated by use of supported cell membrane sheets.

PDZ domain scaffold proteins are molecular modules orchestrating cellular signalling in space and time. Here, we investigate assembly of PDZ scaffolds using supported cell membrane sheets, a unique experimental setup enabling direct access to the intracellular face of the cell membrane. Our data demonstrate how multivalent protein-protein and protein-lipid interactions provide critical avidity for the strong binding between the PDZ domain scaffold proteins, PICK1 and PSD-95, and their cognate transmembrane binding partners.

Link between a high for drug binding and a fast clinical action: to be or not to be?

Review articles on binding kinetics essentially focus on drugs that dissociate slowly from their target since this is required for the successful treatment of many pathophysiological conditions. Recently, the therapeutic benefit of a high ( the second order association rate constant) has also been linked to fast association and to a fast clinical action. Other studies, however, called this assertion into question since additional factors, like the dosing paradigm and the binding mechanism, are important as well.

Distinct in vivo target occupancy by bivalent- and induced-fit-like binding drugs.

Background And Purpose: Optimal drug therapy often requires long-lasting target occupancy While this attribute was usually linked to the drug's pharmacokinetic properties, the dissociation rate is now increasingly recognized to contribute as well. Nearly all the earlier pharmacokinetic-pharmacodynamic (PK-PD) simulations encompassed single-step binding drugs and focused on k . However, 'micro'-PK mechanisms and more complex binding mechanisms like bivalent- and induced-fit binding may contribute as well.

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.

Cell membranes… and how long drugs may exert beneficial pharmacological activity in vivo.

The time course of the beneficial pharmacological effect of a drug has long been considered to depend merely on the temporal fluctuation of its free concentration. Only in the last decade has it become widely accepted that target-binding kinetics can also affect in vivo pharmacological activity. Although current reviews still essentially focus on genuine dissociation rates, evidence is accumulating that additional micro-pharmacokinetic (PK) and -pharmacodynamic (PD) mechanisms, in which the cell membrane plays a central role, may also increase the residence time of a drug on its target.

Effects of target binding kinetics on in vivo drug efficacy: koff , kon and rebinding.

Background And Purpose: Optimal drug therapy often requires continuing high levels of target occupancy. Besides the traditional pharmacokinetic contribution, target binding kinetics is increasingly considered to play an important role as well. While most attention has been focused on the dissociation rate of the complex, recent reports expressed doubt about the unreserved translatability of this pharmacodynamic property into clinical efficacy.

Identification of Drug-Like Inhibitors of Insulin-Regulated Aminopeptidase Through Small-Molecule Screening.

Intracerebroventricular injection of angiotensin IV, a ligand of insulin-regulated aminopeptidase (IRAP), has been shown to improve cognitive functions in several animal models. Consequently, IRAP is considered a potential target for treatment of cognitive disorders. To identify nonpeptidic IRAP inhibitors, we adapted an established enzymatic assay based on membrane preparations from Chinese hamster ovary cells and a synthetic peptide-like substrate for high-throughput screening purposes.

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.

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.

On the 'micro'-pharmacodynamic and pharmacokinetic mechanisms that contribute to long-lasting drug action.

Introduction: Optimal drug therapy often requires continuing high levels of target occupancy. Besides the traditional pharmacokinetic (PK) contribution thereto, drug-target interactions that comprise successive 'microscopic' steps as well as the intervention of the cell membrane and other 'micro'-anatomical structures nearby may help attaining this objective.

Areas Covered: This article reviews the 'micro'-pharmacodynamic (PD) and PK mechanisms that may increase a drug's residence time.

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.

'Partial' competition of heterobivalent ligand binding may be mistaken for allosteric interactions: a comparison of different target interaction models.

Background And Purpose: Non-competitive drugs that confer allosteric modulation of orthosteric ligand binding are of increasing interest as therapeutic agents. Sought-after advantages include a ceiling level to drug effect and greater receptor-subtype selectivity. It is thus important to determine the mode of interaction of newly identified receptor ligands early in the drug discovery process and binding studies with labelled orthosteric ligands constitute a traditional approach for this.

Molecular mechanism of allosteric modulation at GPCRs: insight from a binding kinetics study at the human A1 adenosine receptor.

Background And Purpose: Many GPCRs can be allosterically modulated by small-molecule ligands. This modulation is best understood in terms of the kinetics of the ligand-receptor interaction. However, many current kinetic assays require at least the (radio)labelling of the orthosteric ligand, which is impractical for studying a range of ligands.

A study of the molecular mechanism of binding kinetics and long residence times of human CCR5 receptor small molecule allosteric ligands.

Background And Purpose: The human CCR5 receptor is a co-receptor for HIV-1 infection and a target for anti-viral therapy. A greater understanding of the binding kinetics of small molecule allosteric ligand interactions with CCR5 will lead to a better understanding of the binding process and may help discover new molecules that avoid resistance.

Experimental Approach: Using [(3) H] maraviroc as a radioligand, a number of different binding protocols were employed in conjunction with simulations to determine rate constants, kinetic mechanism and mutant kinetic fingerprints for wild-type and mutant human CCR5 with maraviroc, aplaviroc and vicriviroc.

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.

Simplified models for heterobivalent ligand binding: when are they applicable and which are the factors that affect their target residence time.

Bivalent ligands often display high affinity/avidity for and long residence time at their target. Thereto responsible is the synergy that emanates from the simultaneous binding of their two pharmacophores to their respective target sites. Thermodynamic cycle models permit the most complete description of the binding process, and thereto, corresponding differential equation-based simulations link the "microscopic" rate constants that govern the individual binding steps to the "macroscopic" bivalent ligand's binding properties.

[3H]IVDE77, a novel radioligand with high affinity and selectivity for the insulin-regulated aminopeptidase.

The hexapeptide angiotensin IV (Ang IV) induces diverse biological effects such as memory enhancement and protection against ischemic stroke. Studies on the mechanism of Ang IV however are hampered by its instability and its lack of selectivity. The high-affinity binding site for Ang IV is the insulin-regulated aminopeptidase (IRAP, EC 3.

Exploring avidity: understanding the potential gains in functional affinity and target residence time of bivalent and heterobivalent ligands.

Bivalent ligands are increasingly important therapeutic agents. Although the naturally occurring antibodies are predominant, it is becoming more common to combine different antibody fragments or even low molecular weight compounds to generate heterobivalent ligands. Such ligands exhibit markedly increased affinity (i.

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.

Clozapine, atypical antipsychotics, and the benefits of fast-off D2 dopamine receptor antagonism.

Drug-receptor interactions are traditionally quantified in terms of affinity and efficacy, but there is increasing awareness that the drug-on-receptor residence time also affects clinical performance. While most interest has hitherto been focused on slow-dissociating drugs, D(2) dopamine receptor antagonists show less extrapyramidal side effects but still have excellent antipsychotic activity when they dissociate swiftly. Fast dissociation of clozapine, the prototype of the "atypical antipsychotics", has been evidenced by distinct radioligand binding approaches both on cell membranes and intact cells.

Amino triazolo diazepines (Ata) as constrained histidine mimics.

Two synthetic routes for the synthesis of amino-triazolodiazepine (Ata) scaffolds are presented. The scope of both of these proceeding through key intra- and intermolecular Huisgen cycloaddition reactions is discussed. The replacement of the His-Pro dipeptide segment in angiotensin IV by the dipeptide mimetic Ata-Gly and subsequent biological evaluation in two inhibitory enzyme assays validated the use of the Ata moiety as a His mimic given the equipotency of both peptidic analogs.