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

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

Discovery of Dual-Action Membrane-Anchored Modulators of Incretin Receptors

The glucose-dependent insulinotropic polypeptide (GIP) and the glucagon-like peptide-1 (GLP-1) receptors are considered complementary therapeutic targets for type 2 diabetes. Using recombinant membrane-tethered ligand (MTL) technology, the present study focused on defining optimized modulators of these receptors, as well as exploring how local anchoring influences soluble peptide function.Serial substitution of residue 7 in membrane-tethered GIP (tGIP) led to a wide range of activities at the GIP receptor, with [G(7)]tGIP showing enhanced efficacy compared to the wild type construct. In contrast, introduction of G(7) into the related ligands, tGLP-1 and tethered exendin-4 (tEXE4), did not affect signaling at the cognate GLP-1 receptor. Both soluble and tethered GIP and GLP-1 were selective activators of their respective receptors. Although soluble EXE4 is highly selective for the GLP-1 receptor, unexpectedly, tethered EXE4 was found to be a potent activator of both the GLP-1 and GIP receptors. Diverging from the pharmacological properties of soluble and tethered GIP, the newly identified GIP-R agonists, (i.e. [G(7)]tGIP and tEXE4) failed to trigger cognate receptor endocytosis. In an attempt to recapitulate the dual agonism observed with tEXE4, we conjugated soluble EXE4 to a lipid moiety. Not only did this soluble peptide activate both the GLP-1 and GIP receptors but, when added to receptor expressing cells, the activity persists despite serial washes.These findings suggest that conversion of a recombinant MTL to a soluble membrane anchored equivalent offers a means to prolong ligand function, as well as to design agonists that can simultaneously act on more than one therapeutic target

Micro-pharmacokinetics: quantifying local drug concentration at live cell membranes

Fundamental equations for determining pharmacological parameters, such as the binding afnity of a ligand for its target receptor, assume a homogeneous distribution of ligand, with concentrations in the immediate vicinity of the receptor being the same as those in the bulk aqueous phase. It is, however, known that drugs are able to interact directly with the plasma membrane, potentially increasing local ligand concentrations around the receptor. We have previously reported an infuence of ligand-phospholipid interactions on ligand binding kinetics at the β2-adrenoceptor, which resulted in distinct “micro-pharmacokinetic” ligand profles. Here, we directly quantifed the local concentration of BODIPY630/650-PEG8-S-propranolol (BY-propranolol), a fuorescent derivative of the classical β-blocker propranolol, at various distances above membranes of single living cells using fuorescence correlation spectroscopy. We show for the frst time a signifcantly increased ligand concentration immediatel adjacent to the cell membrane compared to the bulk aqueous phase. We further show a clear role of both the cell membrane and the β2-adrenoceptor in determining high local BY-propranolol concentrations at the cell surface. These data suggest that the true binding afnity of BY-propranolol for the β2-adrenoceptor is likely far lower than previously reported and highlights the critical importance of understanding the “micro-pharmacokinetic” profles of ligands for membrane-associated proteins

Chemogenomic Analysis of G-Protein Coupled Receptors and Their Ligands Deciphers Locks and Keys Governing Diverse Aspects of Signalling

Understanding the molecular mechanism of signalling in the important super-family of G-protein-coupled receptors (GPCRs) is causally related to questions of how and where these receptors can be activated or inhibited. In this context, it is of great interest to unravel the common molecular features of GPCRs as well as those related to an active or inactive state or to subtype specific G-protein coupling. In our underlying chemogenomics study, we analyse for the first time the statistical link between the properties of G-protein-coupled receptors and GPCR ligands. The technique of mutual information (MI) is able to reveal statistical inter-dependence between variations in amino acid residues on the one hand and variations in ligand molecular descriptors on the other. Although this MI analysis uses novel information that differs from the results of known site-directed mutagenesis studies or published GPCR crystal structures, the method is capable of identifying the well-known common ligand binding region of GPCRs between the upper part of the seven transmembrane helices and the second extracellular loop. The analysis shows amino acid positions that are sensitive to either stimulating (agonistic) or inhibitory (antagonistic) ligand effects or both. It appears that amino acid positions for antagonistic and agonistic effects are both concentrated around the extracellular region, but selective agonistic effects are cumulated between transmembrane helices (TMHs) 2, 3, and ECL2, while selective residues for antagonistic effects are located at the top of helices 5 and 6. Above all, the MI analysis provides detailed indications about amino acids located in the transmembrane region of these receptors that determine G-protein signalling pathway preferences

The History, Relevance, and Applications of the Periodic System in Geochemistry

Geochemistry is a discipline in the earth sciences concerned with understanding the chemistry of the Earth and what that chemistry tells us about the processes that control the formation and evolution of Earth materials and the planet itself. The periodic table and the periodic system, as developed by Mendeleev and others in the nineteenth century, are as important in geochemistry as in other areas of chemistry. In fact, systemisation of the myriad of observations that geochemists make is perhaps even more important in this branch of chemistry, given the huge variability in the nature of Earth materials – from the Fe-rich core, through the silicate-dominated mantle and crust, to the volatile-rich ocean and atmosphere. This systemisation started in the eighteenth century, when geochemistry did not yet exist as a separate pursuit in itself. Mineralogy, one of the disciplines that eventually became geochemistry, was central to the discovery of the elements, and nineteenth-century mineralogists played a key role in this endeavour. Early “geochemists” continued this systemisation effort into the twentieth century, particularly highlighted in the career of V.M. Goldschmidt. The focus of the modern discipline of geochemistry has moved well beyond classification, in order to invert the information held in the properties of elements across the periodic table and their distribution across Earth and planetary materials, to learn about the physicochemical processes that shaped the Earth and other planets, on all scales. We illustrate this approach with key examples, those rooted in the patterns inherent in the periodic law as well as those that exploit concepts that only became familiar after Mendeleev, such as stable and radiogenic isotopes

An anti-TNF--α antibody mimetic to treat ocular inflammation

Infliximab is an antibody that neutralizes TNF-α and is used principally by systemic administration to treat many inflammatory disorders. We prepared the antibody mimetic Fab-PEG-Fab (FpFinfliximab) for direct intravitreal injection to assess whether such formulations have biological activity and potential utility for ocular use. FpFinfliximab was designed to address side effects caused by antibody degradation and the presence of the Fc region. Surface plasmon resonance analysis indicated that infliximab and FpFinfliximab maintained binding affinity for both human and murine recombinant TNF-α. No Fc mediated RPE cellular uptake was observed for FpFinfliximab. Both Infliximab and FpFinfliximab suppressed ocular inflammation by reducing the number of CD45+ infiltrate cells in the EAU mice model after a single intravitreal injection at the onset of peak disease. These results offer an opportunity to develop and formulate for ocular use, FpF molecules designed for single and potentially multiple targets using bi-specific FpFs

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&#8217;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&nbsp;receptor.</p