A Solid State \u3csup\u3e13\u3c/sup\u3eC-NMR Study of Diamonds and Graphites

The 13C-NMR spectra of gem quality and industrial diamonds show two resonances with the more intense resonance at high field. Two resonances are also shown in 13C-NMR spectra of various graphites; however, the low field resonance is of greater intensity than the high field resonance in the graphites. The resonances are very broad and they are assigned to graphite type (sp2) carbon and diamond type (sp3) carbon

Analysis of Ligand Bias in Functional Studies Involving the Allosteric Modulation of G Protein- Coupled Receptors

Introduction The affinity constants of a ligand for active and inactive states of a receptor ultimately determine its capacity to activate downstream signaling events. In this report, we describe a reverse-engineering strategy for estimating these microscopic constants. Methods Our approach involves analyzing responses measured downstream in the signaling pathway of a G protein-coupled receptor under conditions of allosteric modulation and reduced receptor expression or partial receptor inactivation. The analysis also yields estimates of the isomerization constant of the unoccupied receptor, the sensitivity constant of the signaling pathway, and the more empirical parameters of the receptor population including the observed affinities and efficacies of allosteric and orthosteric ligands – including inverse agonists – and the efficacy of the unoccupied receptor (i.e., constitutive activity). Results and discussion We validate our approach with an analytical proof and by analysis of simulated data. We also use our method to analyze data from the literature. We show that the values of the microscopic constants of orthosteric and allosteric ligands are constant regardless of the allosteric interaction and the nature of the receptor-signaling pathway as long as the same active state mediates the response. Our analysis is useful for quantifying probe-dependent allosteric interactions and the selectivity of agonists for different signaling pathways. Knowing the isomerization constant and sensitivity constant of a signaling pathway in a given cell line or tissue preparation enables future investigators to estimate the affinity constants of agonists for receptor states simply through analysis of their concentration–response curves. Our approach also provides a means of validating in silico estimates of ligand affinity for crystal structures of active and inactive states of the receptor

Analysis of Agonism and Inverse Agonism in Functional Assays with Constitutive Activity: Estimation of Orthosteric Ligand Affinity Constants for Active and Inactive Receptor States

We describe a modification of receptor theory for the estimation of observed affinities (Kobs) and relative efficacies of orthosteric ligands in functional assays that exhibit constitutive activity. Our theory includes parameters for the fractions of the occupied receptor population in the active (intrinsic efficacy, ε) and inactive (εi) states and analogous parameters for the fractions of the free receptor population in the active (εsys) and inactive (εi-sys) states. The total stimulus represents the summation of the active states of the free and occupied receptor populations. A modified operational model is developed that expresses the response as a logistic function of the total stimulus. This function includes the standard parameters related to affinity and efficacy (Kobs and τ) as well as a parameter proportional to the activity of the free receptor complex, τsys. Two related parameters are proportional to the fraction of the free (τi-sys) and occupied (τi) receptor populations in the inactive state. We show that the estimates of the affinity constants of orthosteric ligands for the active (Kb) and inactive (Ka) states of the receptor are equivalent to τKobs/τsys and τiKobs/τi-sys, respectively. We verify our method with computer simulation techniques and apply it to the analysis of M2 and M3 muscarinic receptors. Our method is applicable in the analysis of ligand bias in drug discovery programs

Analysis of Functional Responses at G Protein-Coupled Receptors: Estimation of Relative Affinity Constants for the Inactive Receptor Sstate

We describe a modification of receptor theory that enables the estimation of relative affinity constants for the inactive state of a G protein-coupled receptor. Our approach includes the traditional parameters of observed affinity (Kobs) and efficacy (fraction of ligand-receptor complex in the active state, ε) and introduces the concept of the fraction of the ligand-receptor complex in the inactive state (intrinsic inactivity, εi). The relationship between receptor activation and the ligand concentration is known as the stimulus, and the operational model expresses the response as a logistic function of the stimulus. The latter function includes Kobs and the parameter τ, which is proportional to ε. We introduce the parameter τi, which is proportional to εi. We have previously shown that the product, Kobsτ, of one agonist, expressed relative to that of another (intrinsic relative activity, RAi), is a relative measure of the affinity constant for the active state of the receptor. In this report, we show that the product, Kobsτi, of one agonist, expressed relative to that of another (intrinsic relative inactivity, RIi), is a relative measure of the affinity constant for the inactive state of the receptor. We use computer simulation techniques to verify our analysis and apply our method to the analysis of published data on agonist activity at the M3 muscarinic receptor. Our method should have widespread application in the analysis of agonist bias in drug discovery programs and in the estimation of a more fundamental relative measure of efficacy (RAi/RIi)

Parallelization of chip-based fluorescence immuno-assays with quantum-dot labelled beads

This paper presents an optical concept for the read-out of a parallel, bead-based fluorescence immunoassay conducted on a lab-on-a-disk platform. The reusable part of the modular setup comprises a detection unit featuring a single LED as light source, two emission-filters, and a color CCD-camera as standard components together with a spinning drive as actuation unit. The miniaturized lab-on-a-disk is devised as a disposable. In the read-out process of the parallel assay, beads are first identified by the color of incorporated quantum dots (QDs). Next, the reaction-specific fluorescence signal is quantified with FluoSpheres-labeled detection anti-bodies. To enable a fast and automated read-out, suitable algorithms have been implemented in this work. Based on this concept, we successfully demonstrated a Hepatitis-A assay on our disk-based lab-on-a-chip

A Simple Method for Estimation of Agonist Activity at Receptor Subtypes: Comparison of Native and Cloned M3 Muscarinic Receptors in Guinea Pig Ileum and Transfected Cells

We describe a simple method for calculating the pharmacological activity of an agonist (A) relative to a standard agonist (S) using only the concentration-response curves of the two agonists. In most situations, we show that the product of the ratios of maximal responses (E max − A/E max − S) and potencies (EC50 − S/EC50 − A) is equivalent to the product of the affinity and intrinsic efficacy of A expressed relative to that of S. We refer to this term as the IRA value of A. In a cooperative system where the concentration-response curve of the standard agonist is steep and that of the test agonist is flatter with a lower maximal response, the simple calculation of IRA described above underestimates agonist activity; however, we also describe a means of correcting the IRA in this situation. We have validated our analysis with modeling techniques and have shown experimentally that the IRA values of muscarinic agonists for stimulating contractions in the guinea pig ileum (M3 response) are in excellent agreement with those measured in the phosphoinositide assay on Chinese hamster ovary cells expressing the M3 muscarinic receptor

N-terminal myristoylation is required for membrane localization of cGMP-dependent protein kinase type II

The apical membrane of intestinal epithelial cells harbors a unique isozyme of cGMP-dependent protein kinase (cGK type II) which acts as a key regulator of ion transport systems, including the cystic fibrosis transmembrane conductance regulator (CFTR)-chloride channel. To explore the mechanism of cGK II membrane-anchoring, recombinant cGK II was expressed stably in HEK 293 cells or transiently in COS-1 cells. In both cell lines, cGK II was found predominantly in the particulate fraction. Immunoprecipitation of solubilized cGK II did not reveal any other tightly associated proteins, suggesting a membrane binding motif within cGK II itself. The primary structure of cGK II is devoid of hydrophobic transmembrane domains; cGK II does, however, contain a penultimate glycine, a potential acceptor for a myristoyl moiety. Metabolic labeling showed that cGK II was indeed able to incorporate [3H]myristate. Moreover, incubation of cGK II-expressing 293 cells with the myristoylation inhibitor 2-hydroxymyristic acid (1 mM) significantly increased the proportion of cGK II in the cytosol from 10 +/- 5 to 35 +/- 4%. Furthermore, a nonmyristoylated cGK II Gly2 --> Ala mutant was localized predominantly in the cytosol after transient expression in COS-1 cells. The absence of the myristoyl group did not affect the specific enzyme activity or the Ka for cGMP and only slightly enhanced the thermal stability of cGK II. These results indicate that N-terminal myristoylation fulfills a crucial role in directing cGK II to the membrane

The guinea pig ileum lacks the direct, high-potency, M2-muscarinic, contractile mechanism characteristic of the mouse ileum

We explored whether the M2 muscarinic receptor in the guinea pig ileum elicits a highly potent, direct-contractile response, like that from the M3 muscarinic receptor knockout mouse. First, we characterized the irreversible receptor-blocking activity of 4-DAMP mustard in ileum from muscarinic receptor knockout mice to verify its M3 selectivity. Then, we used 4-DAMP mustard to inactivate M3 responses in the guinea pig ileum to attempt to reveal direct, M2 receptor-mediated contractions. The muscarinic agonist, oxotremorine-M, elicited potent contractions in ileum from wild-type, M2 receptor knockout, and M3 receptor knockout mice characterized by negative log EC50 (pEC50) values ± SEM of 6.75 ± 0.03, 6.26 ± 0.05, and 6.99 ± 0.08, respectively. The corresponding Emax values in wild-type and M2 receptor knockout mice were approximately the same, but that in the M3 receptor knockout mouse was only 36% of wild type. Following 4-DAMP mustard treatment, the concentration–response curve of oxotremorine-M in wild-type ileum resembled that of the M3 knockout mouse in terms of its pEC50, Emax, and inhibition by selective muscarinic antagonists. Thus, 4-DAMP mustard treatment appears to inactivate M3 responses selectively and renders the muscarinic contractile behavior of the wild-type ileum similar to that of the M3 knockout mouse. Following 4-DAMP mustard treatment, the contractile response of the guinea pig ileum to oxotremorine-M exhibited low potency and a competitive-antagonism profile consistent with an M3 response. The guinea pig ileum, therefore, lacks a direct, highly potent, M2-contractile component but may have a direct, lower potency M2 component

Assessment of the molecular mechanisms of action of novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one allosteric modulators at the M1 muscarinic acetylcholine receptors

Positive allosteric modulators (PAMs) that target the M1 muscarinic acetylcholine (ACh) receptor (M1 mAChR) are potential treatments for cognitive deficits in conditions such as Alzheimer's disease and schizophrenia. We recently reported novel 4-phenylpyridine-2-one and 6-phenylpyrimidin-4-one M1 mAChR PAMs with the potential to display different modes of positive allosteric modulation and/or agonism (Mistry et al., 2016), but their molecular mechanisms of action remain undetermined. The current study compared the pharmacology of three such novel PAMs with the prototypical first-generation PAM, BQCA, in a recombinant Chinese hamster ovary (CHO) cell line stably expressing the human M1 mAChR. Interactions between the orthosteric agonists and the novel PAMs or BQCA suggested their allosteric effects were solely governed by modulation of agonist affinity. The greatest degree of positive co-operativity was observed with higher efficacy agonists, whereas minimal potentiation was observed when the modulators were tested against the lower efficacy agonist, xanomeline. Each PAM was investigated for its effects on the endogenous agonist, ACh, on three different signalling pathways, (ERK1/2 phosphorylation, IP1 accumulation and β-arrestin-2 recruitment), revealing that the allosteric potentiation generally tracked with the efficiency of stimulus-response coupling and that there was little pathway bias in the allosteric effects. Thus, despite the identification of novel allosteric scaffolds targeting the M1 mAChR, the molecular mechanism of action of these compounds is largely consistent with a model of allostery previously described for BQCA, suggesting that this may be a more generalized mechanism for M1 mAChR PAM effects than previously appreciated