-
[show abstract]
[hide abstract]
ABSTRACT: Their ubiquitous nature, wide cellular distribution and versatile molecular recognition and signalling help make G-protein binding receptors (GPCRs) the most important class of membrane proteins in clinical medicine, accounting for ∼40% of all current therapeutics. A large percentage of current drugs target the endogenous ligand binding (orthosteric) site, which are structurally and evolutionarily conserved, particularly among members of the same GPCR subfamily. With the recent advances in GPCR X-ray crystallography, new opportunities for developing novel subtype selective drugs have emerged. Given the increasing recognition that the extracellular surface conformation changes in response to ligand binding, it is likely that all GPCRs possess an allosteric site(s) capable of regulating GPCR signalling. Allosteric sites are less structurally conserved than their corresponding orthosteric site and thus provide new opportunities for the development of more selective drugs. Constitutive oligomerisation (dimerisation) identified in many of the GPCRs investigated, adds another dimension to the structural and functional complexity of GPCRs. In this review, we compare 60 crystal structures of nine GPCR subtypes (rhodopsin, ß(2)-AR, ß(1)-AR, A(2a)-AR, CXCR4, D(3)R, H(1)R, M(2)R, M(3)R) across four subfamilies of Class A GPCRs, and discuss mechanisms involved in receptor activation and potential allosteric binding sites across the highly variable extracellular surface of these GPCRs. This analysis has identified a new extracellular salt bridge (ESB-2) that might be exploited in the design of allosteric modulators.
Biochemical pharmacology 09/2012; · 4.25 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: A dual-pharmacophoric peptide was engineered by grafting the integrin binding RGD motif between the C- and N-termini of a disulfide-rich noradrenaline transporter inhibiting χ-conotoxin resulting in a stable backbone cyclized peptide. The construct maintained two independent biological activities and showed increased plasma stability with no adverse effects observed following administration to rats, highlighting the potential value of pharmacophore grafting into constrained peptide scaffolds.
Organic & Biomolecular Chemistry 05/2012; 10(30):5791-4. · 3.70 Impact Factor
-
Sulan Luo,
Kalyana Bharati Akondi,
Dongting Zhangsun,
Yong Wu,
Xiaopeng Zhu,
Yuanyan Hu,
Sean Christensen,
Cheryl Dowell,
Norelle L Daly,
David J Craik, Ching-I Anderson Wang,
Richard J Lewis,
Paul F Alewood,
J Michael McIntosh
[show abstract]
[hide abstract]
ABSTRACT: Different nicotinic acetylcholine receptor (nAChR) subtypes are implicated in learning, pain sensation, and disease states, including Parkinson disease and nicotine addiction. alpha-Conotoxins are among the most selective nAChR ligands. Mechanistic insights into the structure, function, and receptor interaction of alpha-conotoxins may serve as a platform for development of new therapies. Previously characterized alpha-conotoxins have a highly conserved Ser-Xaa-Pro motif that is crucial for potent nAChR interaction. This study characterized the novel alpha-conotoxin LtIA, which lacks this highly conserved motif but potently blocked alpha3beta2 nAChRs with a 9.8 nm IC(50) value. The off-rate of LtIA was rapid relative to Ser-Xaa-Pro-containing alpha-conotoxin MII. Nevertheless, pre-block of alpha3beta2 nAChRs with LtIA prevented the slowly reversible block associated with MII, suggesting overlap in their binding sites. nAChR beta subunit ligand-binding interface mutations were used to examine the >1000-fold selectivity difference of LtIA for alpha3beta2 versus alpha3beta4 nAChRs. Unlike MII, LtIA had a >900-fold increased IC(50) value on alpha3beta2(F119Q) versus wild type nAChRs, whereas T59K and V111I beta2 mutants had little effect. Molecular docking simulations suggested that LtIA had a surprisingly shallow binding site on the alpha3beta2 nAChR that includes beta2 Lys-79. The K79A mutant disrupted LtIA binding but was without effect on an LtIA analog where the Ser-Xaa-Pro motif is present, consistent with distinct binding modes.
Journal of Biological Chemistry 02/2010; 285(16):12355-66. · 4.77 Impact Factor
-
[show abstract]
[hide abstract]
ABSTRACT: Monoamine transporters are a group of transmembrane neurotransmitter sodium symporter (NSS) transporters that play a crucial role in regulating biogenic monoamine concentrations at peripheral and central synapses. Given the key role played by serotonin, dopamine and noradrenaline in addictive and disease states, structure-function studies have been conducted to help guide the development of improved central nervous system therapeutics. Extensive pharmacological, immunological and biochemical studies, in conjunction with three-dimensional homology modeling, have been performed to structurally and functionally characterise the monoamine transporter substrate permeation pathway, substrate selectivity, and binding sites for ions, substrates and inhibitors at the molecular level. However, only recently has it been possible to start to construct an accurate molecular interaction network for the monoamine transporters and their corresponding substrates and inhibitors. Crystal structures of Aquifex aeolicus leucine transporter (LeuT(Aa)), a homologous protein to monoamine transporters that has been experimentally demonstrated to share similar structural folds with monoamine transporters, have been determined in complex with amino acids and inhibitors. The molecular interactions of leucine and tricyclic antidepressants (TCA) has supported many of the predictions based on the mutational studies. Models constructed from LeuT(Aa) are now allowing a rational approach to further clarify the molecular determinants of NSS transporter-ligand complexes, and potentially the ability to better manipulate drug specificity and affinity. In this review, we compare the structure-function relationships of other SLC6 NSS family transporters with monoamine transporters, and discuss possible mechanisms involved in substrate binding and transport, and modes of inhibition by TCAs.
Biochemical pharmacology 11/2009; 79(8):1083-91. · 4.25 Impact Factor
-
Ching-I Anderson Wang
[show abstract]
[hide abstract]
ABSTRACT: Plants do not possess an adaptive immune system and are unable to circulate antibodies. Therefore plants have highly coordinated defense mechanisms, which efficiently prevent the spread of infections by foreign pathogens. Plants use preformed antimicrobials, basal defenses and gene-for-gene resistance. Gene-for-gene resistance is initiated upon the direct or indirect recognition of pathogen-derived avirulence (Avr) gene products by the plant disease resistance (R) gene products. Recent studies suggest that the indirect recognition (guard theory) is mediated by one or several host components. While the Avr-R interaction has been very well characterised genetically for a range of plant and pathogen species, surprisingly little is known, especially at the molecular level, regarding the recognition mechanism between Avr and R proteins. Because the recognition of Avr by R protein is the key to activate the disease resistance, the interaction between Avr and R and the mechanism of resistance initiation are of intrinsic interest and importance, and are investigated in this study. This thesis covers the expression, purification, characterisation and crystallisation of four flax rust (Melampsora lini) AvrL567 proteins, AvrL567-A, AvrL567-B, AvrL567-C and AvrL567-D, with distinctive recognition specificities, and of their corresponding flax (Linum usitatissimum) R protein L6 and the closely related protein M. Significantly, AvrL567-A and AvrL567-D protein structures were determined. This allowed the interaction mechanism between AvrL567 and L proteins to be modelled. By using site-directed mutagenesis on polymorphic amino residues of AvrL567 variants to confirm the role of each individual amino acid residue in conferring recognition specificity, it was determined that the interactions between AvrL567 and L proteins were the results of cumulative effects of multiple contact points. The structures provided some insights into possible pathogenicity-associated functions as nucleic acid binding activity was observed in vitro. Importantly, this demonstrates that AvrL567 proteins may play a role in influencing host gene expression during infection. Furthermore, binding of the large subunits of ribulose-1,5-bisphosphate carboxylase / oxygenase (RuBisCo), which plays a crucial role in energy metabolism in plants, was also detected by pull-down assays. This highlights the possible virulence effects of these Avr proteins. This study highlights several aspects of Avr-R interaction at the molecular level and possible virulence functions of Avr proteins. The results imply a significant step towards defining the resistance response at a molecular level, bringing us a step closer towards being able to engineer new plant disease resistance genes to control diseases for which naturally occurring resistances are not adequate.
