Biomedical subjects

Mark R Dowling

Publications and source records attributed to Mark R Dowling.

8 recordsLinked to original sources

PubMed2006

Quantifying the association and dissociation rates of unlabelled antagonists at the muscarinic M3 receptor.

Slow receptor dissociation kinetics has been implicated in the long clinical duration of action of the muscarinic receptor antagonist tiotropium. However, despite the potential benefits of new drugs with slow dissociation kinetics, the rate parameters of new compounds are seldom measured due to technical difficulties and financial implications associated with radiolabeling multiple ligands. Here we describe the development and optimisation of a medium throughput assay which is capable of measuring the kinetic parameters of novel, unlabelled compounds. Radioligand binding studies were performed with membranes derived from CHO cells recombinantly expressing the human M(3) muscarinic receptor.Initial characterisation of the radioligand [(3)H]-NMS yielded on and off rates of 4.1+/-0.2 x 10(8) M(-1) min(-1) and 0.015+/-0.0005 min(-1), respectively. The specific binding of [(3)H]-NMS was measured over time in the presence and absence of several concentrations of unlabelled competitor compounds. These data were analysed using a competition kinetic model to provide on and off rates for the unlabelled competitor. Comparison of the kinetically derived Kd (k(off)/k(on)) with K(i) values generated at equilibrium showed an excellent correlation (r(2)=0.99), providing good validation of the method. The on and off rates were also used in theoretical computer simulations to successfully predict the effect of incubation time on apparent IC(50) values. This study demonstrates that a medium-throughput competition kinetic binding assay can be used to determine accurate on and off rates of unlabelled compounds, providing the opportunity to optimise for kinetic parameters early in the drug discovery process.

Algorithms↗

PubMed2006

Quantum computation as geometry.

Quantum computers hold great promise for solving interesting computational problems, but it remains a challenge to find efficient quantum circuits that can perform these complicated tasks. Here we show that finding optimal quantum circuits is essentially equivalent to finding the shortest path between two points in a certain curved geometry. By recasting the problem of finding quantum circuits as a geometric problem, we open up the possibility of using the mathematical techniques of Riemannian geometry to suggest new quantum algorithms or to prove limitations on the power of quantum computers.

Journal Article↗

PubMed2006

A single point mutation (N514Y) in the human M3 muscarinic acetylcholine receptor reveals differences in the properties of antagonists: evidence for differential inverse agonism.

A single asparagine-to-tyrosine point mutation in the human M muscarinic acetylcholine (mACh) receptor at residue 514 (N514Y) resulted in a marked increase (approximately 300%) in agonist-independent [3H]inositol phosphate ([3H]IPx) accumulation compared with the response observed for the wild-type (WT) receptor. All the antagonists tested were able to inhibit both the WT-M3 and (N514Y)M3 mACh receptor-mediated basal [3H]IPx accumulation in a concentration-dependent manner. However, significant differences in both potency and binding affinity were only seen for those antagonists that possess greater receptor affinity. Despite being transfected with equivalent amounts of cDNA, cells expressed the (N514Y)M3 mACh receptor at levels that were only 25 to 30% of those seen for the WT receptor. Differences in the ability of chronic antagonist exposure to up-regulate (N514Y)M3 mACh receptor expression levels were also seen, with 4-diphenylacetoxy-N-methylpiperidine (4-DAMP) producing only 50% of the receptor up-regulation produced by atropine or pirenzepine. Basal phosphorylation of the (N514Y)M3 mACh receptor was approximately 100% greater than that seen for the WT-M3 receptor. The ability of antagonists to decrease basal (N514Y)M3 mACh receptor phosphorylation revealed differences in inverse-agonist efficacy. Atropine, 4-DAMP, and pirenzepine all reduced basal phosphorylation to similar levels, whereas methoctramine, a full inverse agonist with respect to reducing agonist-independent [3H]IPx accumulation, produced no significant attenuation of basal receptor phosphorylation. This study shows that mACh receptor inverse agonists can exhibit differential signaling profiles, which are dependent on the specific pathway investigated, and therefore provides evidence that the molecular mechanism of inverse agonism is likely to be more complex than the stabilization of a single inactive receptor conformation.

Cell Line↗

PubMed2006

In vitro and in vivo pharmacological characterization of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel inhaled beta(2) adrenoceptor agonist with a 24-h duration of action.

Here, we describe the preclinical pharmacological profile of 5-[(R)-2-(5,6-diethyl-indan-2-ylamino)-1-hydroxy-ethyl]-8-hydroxy-1H-quinolin-2-one (indacaterol), a novel, chirally pure inhaled beta(2) adrenoceptor agonist, in comparison with marketed drugs. Indacaterol is close to a full agonist at the human beta(2) adrenoceptor (E(max) = 73 +/- 1% of the maximal effect of isoprenaline; pEC(50) = 8.06 +/- 0.02), whereas salmeterol displays only partial efficacy (38 +/- 1%). The functional selectivity profile of indacaterol over beta(1) human adrenoceptors is similar to that of formoterol, whereas its beta(3) adrenoceptor selectivity profile is similar to that of formoterol and salbutamol. In isolated superfused guinea pig trachea, indacaterol has a fast onset of action (30 +/- 4 min) similar to formoterol and salbutamol, and a long duration of action (529 +/- 99 min) comparable with salmeterol. In the conscious guinea pig, when given intratracheally as a dry powder, indacaterol inhibits 5-hydroxytryptamine-induced bronchoconstriction for at least 24 h, whereas salmeterol, formoterol, and salbutamol have durations of action of 12, 4, and 2 h, respectively. When given via nebulization to anesthetized rhesus monkeys, all of the compounds dose-dependently inhibit methacholine-induced bronchoconstriction, although indacaterol produces the most prolonged bronchoprotective effect and induces the lowest increase in heart rate for a similar degree of antibronchoconstrictor activity. In conclusion, the preclinical profile of indacaterol suggests that this compound has a superior duration of action compatible with once-daily dosing in human, together with a fast onset of action and an improved cardiovascular safety profile over marketed inhaled beta(2) adrenoceptor agonists.

Administration, Inhalation↗

PubMed2005

Modelling cell lifespan and proliferation: is likelihood to die or to divide independent of age?

In cell lifespan studies the exponential nature of cell survival curves is often interpreted as showing the rate of death is independent of the age of the cells within the population. Here we present an alternative model where cells that die are replaced and the age and lifespan of the population pool is monitored until a steady state is reached. In our model newly generated individual cells are given a determined lifespan drawn from a number of known distributions including the lognormal, which is frequently found in nature. For lognormal lifespans the analytic steady-state survival curve obtained can be well-fit by a single or double exponential, depending on the mean and standard deviation. Thus, experimental evidence for exponential lifespans of one and/or two populations cannot be taken as definitive evidence for time and age independence of cell survival. A related model for a dividing population in steady state is also developed. We propose that the common adoption of age-independent, constant rates of change in biological modelling may be responsible for significant errors, both of interpretation and of mathematical deduction. We suggest that additional mathematical and experimental methods must be used to resolve the relationship between time and behavioural changes by cells that are predominantly unsynchronized.

Animals↗

PubMed2005

Time-reversal test for stochastic quantum dynamics.

The calculation of quantum dynamics is currently a central issue in theoretical physics, with diverse applications ranging from ultracold atomic Bose-Einstein condensates to condensed matter, biology, and even astrophysics. Here we demonstrate a conceptually simple method of determining the regime of validity of stochastic simulations of unitary quantum dynamics by employing a time-reversal test. We apply this test to a simulation of the evolution of a quantum anharmonic oscillator with up to 6.022x10(23) (Avogadro's number) of particles. This system is realizable as a Bose-Einstein condensate in an optical lattice, for which the time-reversal procedure could be implemented experimentally.

Journal Article↗

PubMed2005

An investigation of whether agonist-selective receptor conformations occur with respect to M2 and M4 muscarinic acetylcholine receptor signalling via Gi/o and Gs proteins.

1. A range of muscarinic acetylcholine (mACh) receptor agonists (methacholine (MCh), oxotremorine-M (OXO-M), oxotremorine (OXO), arecoline (AREC), bethanechol (BETH), pilocarpine (PILO)) have been investigated with respect to their binding to, and activation of, M(2) and M(4) mACh receptors, recombinantly expressed in Chinese hamster ovary cells, to explore the possibility that these agonists may differentially affect mACh receptor-G(i/o) and -G(s) coupling. 2. M(2)/M(4) mACh receptor coupling to the adenylyl cyclase/cyclic AMP signalling pathway has been explored in intact cells. G(i/o)-mediated negative coupling to adenylyl cyclase was explored functionally by assessing the ability of the mACh receptor agonists to inhibit forskolin-stimulated enzymic activity. Following pertussis toxin treatment (100 ng ml(-1), 18-20 h) to inactivate G(i/o) proteins, each agonist caused a G(s)-mediated enhancement of forskolin-stimulated adenylyl cyclase activity. 3. At both M(2) and M(4) mACh receptors, all agonists tested were more potent in mediating G(i/o)- versus G(s)-coupled responses. This difference (determined as the pIC(50) (G(i/o) coupling) minus pEC(50) (G(s) coupling) value) was greatest for AREC (65-75-fold) and least for BETH and PILO (</=10-fold). 4. Using apparent binding affinities (pK(B)), and potency (EC(50)/IC(50)) and responsiveness (E(max)/I(max)) estimates, relative efficacy (e(rel)) values for each agonist with respect to M(2) and M(4) mACh receptor coupling to G(i/o)- and G(s)-mediated signalling were also calculated. While the e(rel) values obtained for MCh and OXO-M in CHO-m2 cells were similar, OXO-M behaved as a 'super-agonist' at the M(4) mACh receptor giving greater e(rel) values for both G(i/o) and G(s) coupling relative to MCh. 5. The experimental data indicate that while interesting differences between agonists with respect to M(2)/M(4) mACh receptor activation and receptor-G(i/o) and -G(s) coupling can be discerned, no clear examples of agonist trafficking of signal have emerged.

Animals↗

PubMed2004

Measurement of agonist-stimulated [35S]GTPgammaS binding to assess total G-protein and Galpha-subtype-specific activation by G-protein-coupled receptors.

On activation, G-protein-coupled receptors (GPCRs) exert many of their cellular actions through promotion of guanine nucleotide exchange on the Galpha-subunit of heterotrimeric G-proteins to release free Galpha-GTP and betagamma-subunits. In membrane preparations, GTP can be substituted by 35S-labeled guanosine 5'-O-(3-thio)triphosphate ([35S]GTPgammaS) and on agonist stimulation a stable [35S]GTPgammaS-Galpha complex will form and accumulate. Separation of 35S-bound GTPgammaS-Galpha complexes from free [35S]GTPgammaS allows differences between basal and agonist-stimulated rates of [35S]GTPgammaS-Galpha complex formation to be used to obtain pharmacological information on receptor-G-protein information transfer. Further, by releasing Galpha-subunits into solution following the [35S]GTPgammaS binding step, Galpha-subunit-specific antibodies can be used to investigate the Galpha-protein subpopulations activated by receptors by immunoprecipitation of [35S]GTPgammaS-Galpha complexes and quantification by scintillation counting. Here we describe a total [35S]GTPgammaS binding assay and a modification of this method that incorporates a Galpha-specific immunoprecipitation step.

Cell Membrane↗