10 research outputs found
Advances in GPCR modeling evaluated by the GPCR Dock 2013 assessment: Meeting new challenges
© 2014 Elsevier Ltd All rights reserved. Despite tremendous successes of GPCR crystallography, the receptors with available structures represent only a small fraction of human GPCRs. An important role of the modeling community is to maximize structural insights for the remaining receptors and complexes. The community-wide GPCR Dock assessment was established to stimulate and monitor the progress in molecular modeling and ligand docking for GPCRs. The four targets in the present third assessment round presented new and diverse challenges for modelers, including prediction of allosteric ligand interaction and activation states in 5-hydroxytryptamine receptors 1B and 2B, and modeling by extremely distant homology for smoothened receptor. Forty-four modeling groups participated in the assessment. State-of-the-art modeling approaches achieved close-to-experimental accuracy for small rigid orthosteric ligands and models built by close homology, and they correctly predicted protein fold for distant homology targets. Predictions of long loops and GPCR activation states remain unsolved problems
Atomistic studies of the dynamics of P-glycoprotein and its ligands
A signifficant obstacle facing the healthcare industry is the phenomenon of multidrug resistance
(MDR) in which a cell acquires simultaneous resistance to many unrelated drugs that it has
never been exposed to. At the molecular level, MDR can be characterised by a reduction of
intracellular drug levels due to their active efflux by multidrug transporters such as P-glycoprotein
(Pgp). Pgp is able to efflux a phenomenally wide variety of chemically unrelated drugs and
causal relationships have been established between its expression and the acquisition of MDR to
numerous anticancer and central nervous system (CNS) drugs. There has thus been much effort
to understand the molecular biology of Pgp and how it functions. However, many aspects of its
functioning remain unclear. From a drug discovery viewpoint, we have yet to fully understand
what features make some drugs susceptible to Pgp-mediated efflux (substrates) and what makes
others able to inhibit Pgp function (inhibitors). From a mechanistic viewpoint, it is still uncertain
what the exact nature of Pgp's binding site is, the role of ATP binding and hydrolysis in transport
and how both of these interplay with ligand binding.
The work presented in this thesis attempts to answer these questions from two perspectives.
Firstly the mouse Pgp crystal structure [PDB 3G60] was used as a unique starting point for
molecular dynamics (MD) simulations to characterise the dynamics and conformational
exibility of Pgp, properties believed to be integral to its function. The simulations revealed Pgp to be
a highly dynamic molecule at both its transmembrane (TM) and nucleotide binding domains
(NBDs). The latter exhibited a conformational asymmetry that supports the Constant Contact
model of ATPase activity. In the presence of the Pgp substrate, daunorubicin, the NBDs exhibited
tighter asymmetric dimerisation leading to increased affinity for ATP. In contrast, the presence
of the Pgp inhibitor, QZ59-RRR led to NBD conformational changes that reduced their affinity
for ATP. Thus providing an appealing mechanism for how QZ59-RRR inhibits Pgp ATPase
activity. MD simulation was also used to provide atomic-detail interpretations of multiple binding
stoichiometries of drug and lipid molecules observed by collaborator-led mass spectrometry
experiments. This also provided opportunity to validate the Pgp simulations against novel
experimental data.
The second strand of the thesis explored the membrane permeation dynamics of CNS therapeutics
in order to identify differences in protonation states, conformations, orientations and membrane
localisation that might distinguish those that are Pgp substrates and from those that are not. These
properties were studied using complementary MD simulation and nuclear magnetic resonance
(NMR) techniques. The simulations revealed a novel set of criteria that in
uence the likelihoodof a drug to 'flip-flop' across a membrane, a behaviour that may make drugs more susceptible to
Pgp efflux. These observations were broadly consistent with the NMR experiments. However,
the NMR data also highlighted limitations in the simulation approaches used in this thesis and
emphasised the need to also consider the kinetics of permeation in addition to its thermodynamics.This thesis is not currently available via ORA
Design of a ratiometric reporter to improve the dynamic range and sensitivity of a bacterial biosensor
For applications from biosensor generation to synthetic biology, the ability to accurately and quantitatively generate input-output data from biological systems over a wide dynamic range is becoming increasingly important. This study has demonstrated that a simple approach utilising multiple promoters, recognising the same transcriptional activator but with differing affinities, linked to compatible reporter genes can achieve this goal. This simple system highlights that even for complex promoters with multiple binding sites we can use the ratio of two reporters to obtain accurate and reproducible input-output data for reporters contained on a high copy number plasmid without the need for any background subtraction or accurate determination of cell number. It has also demonstrated that the precise promoter strengths are not too crucial to the design, provided they are significantly different, as taking the ratio of either the high affinity/low affinity or medium affinity/low affinity channels give similar improvements over any single channel alone.
The modularity of this system means that it should be possible to exchange the IPTG inducible promoter and hence place the ntrC* gene under the regulation of any environmentally important promoter.
It should also be possible to simply construct different strength promoters for the same transcriptional activator by standard DNA synthesis techniques, therefore allowing direct ratiometric detection of specific active transcription factors. Finally, there is considerable interest in the biosensor field in the use of immobilised cells for field based applications. In these cases, determining the exact number of viable cells present at the time of use may be problematic and obtaining high signal levels may also be essential. In this regard, the use of ratiometric reporters from constructs on high copy number plasmids could alleviate many of these potential problems and significantly simplify the required biosensor detection equipment.This thesis is not currently available in OR
New Paleointensities From the Skinner Cove Formation, Newfoundland, Suggest a Changing State of the Geomagnetic Field at the Ediacaran-Cambrian Transition
Extremely weak early Cambrian dipole moment similar to Ediacaran: Evidence for long-term trends in geomagnetic field behaviour?
Molecular Determinants for Competitive Inhibition of α4β2 Nicotinic Acetylcholine Receptors
The Erythrina alkaloids erysodine and dihydro-β-erythroidine (DHβE) are potent and selective competitive inhibitors of α4β2 nicotinic acetylcholine receptors (nAChRs), but little is known about the molecular determinants of the sensitivity of this receptor subtype to inhibition by this class of antagonists. We addressed this issue by examining the effects of DHβE and a range of aromatic Erythrina alkaloids on [3H]cytisine binding and receptor function in conjunction with homology models of the α4β2 nAChR, mutagenesis, and functional assays. The lactone group of DHβE and a hydroxyl group at position C-16 in aromatic Erythrina alkaloids were identified as major determinants of potency, which was decreased when the conserved residue Tyr126 in loop A of the α4 subunit was substituted by alanine. Sensitivity to inhibition was also decreased by substituting the conserved aromatic residues α4Trp182 (loop B), α4Tyr230 (loop C), and β2Trp82 (loop D) and the nonconserved β2Thr84; however, only α4Trp182 was predicted to contact bound antagonist, suggesting α4Tyr230, β2Trp82, and β2Thr84 contribute allosterically to the closed state elicited by bound antagonist. In addition, homology modeling predicted strong ionic interactions between the ammonium center of the Erythrina alkaloids and β2Asp196, leading to the uncapping of loop C. Consistent with this, β2D196A abolished sensitivity to inhibition by DHβE or erysodine but not by epierythratidine, which is not predicted to form ionic bonds with β2Asp196. This residue is not conserved in subunits that comprise nAChRs with low sensitivity to inhibition by DHβE or erysodine, which highlights β2Asp196 as a major determinant of the receptor selectivity of Erythrina alkaloids