Christopher Higgs

Dimerization and Domain Swapping in G-Protein-Coupled Receptors: A Computational Study

In recent years there has been an increasing number of reports describing G protein-coupled receptor (GPCR) dimerization and heterodimerization. However, the evidence on the nature of the dimers and their role in GPCR activation is inconclusive. Consequently, we present here a review of our computational studies on G protein-coupled receptor dimerization and domain swapping. The studies described include molecular dynamics simulations on receptor monomers and dimers in the absence of ligand, in the presence of an agonist, and in the presence of an antagonist (or more precisely an inverse agonist). Two distinct sequence-based approaches to studying protein interfaces are also described, namely correlated mutation analysis and evolutionary trace analysis. All three approaches concur in supporting the proposal that the dimerization interface includes transmembrane helices 5 and 6. These studies cannot distinguish between domain swapped dimers and contact dimers as the models used were restricted to the helical part of the receptor. However, it is proposed that for the purpose of signalling, the domain swapped dimer and the corresponding contact dimer are equivalent. The evolutionary trace analysis suggests that every GPCR family and subfamily (for which sufficient sequence data is available) has the potential to dimerize through this common functional site on helices 5 and 6. The evolutionary trace results on the G protein are briefly described and these are consistent with GPCR dimerization. In addition to the functional site on helices 5 and 6, the evolutionary trace analysis identified a second functional site on helices 2 and 3. Possible roles for this site are suggested, including oligomerization.

Dimerization of G-protein-coupled receptors.

The evolutionary trace (ET) method, a data mining approach for determining significant levels of amino acid conservation, has been applied to over 700 aligned G-protein-coupled receptor (GPCR) sequences. The method predicted the occurrence of functionally important clusters of residues on the external faces of helices 5 and 6 for each family or subfamily of receptors; similar clusters were observed on helices 2 and 3. The probability that these clusters are not random was determined using Monte Carlo techniques. The cluster on helices 5 and 6 is consistent with both 5,6-contact and 5,6-domain swapped dimer formation; the possible equivalence of these two types of dimer is discussed because this relates to activation by homo- and heterodimers. The observation of a functionally important cluster of residues on helices 2 and 3 is novel, and some possible interpretations are given, including heterodimerization and oligomerization. The application of the evolutionary trace method to 113 aligned G-protein sequences resulted in the identification of two functional sites. One large, well-defined site is clearly identified with adenyl cyclase, beta/gamma and regulator of G-protein signaling (RGS) binding. The other G-protein functional site, which extends from the ras-like domain onto the helical domain, has the correct size and electrostatic properties for GPCR dimer binding. The implications of these results are discussed in terms of the conformational changes required in the G-protein for activation by a receptor dimer. Further, the implications of GPCR dimerization for medicinal chemistry are discussed in the context of these ET results.

Identification and hit-to-lead exploration of a novel series of histamine H4 receptor inverse agonists.

The identification and hit-to-lead exploration of a novel, potent and selective series of histamine H(4) receptor inverse agonists is described. The initial hit, 3A (IC(50) 19 nM) was identified by means of a ligand-based virtual screening approach. Subsequent medicinal chemistry exploration yielded 18I which possessed increased potency (R-enantiomer IC(50) 1 nM) as well as enhanced microsomal stability.

Evidence for dimerization in the beta(2)-adrenergic receptor from the evolutionary trace method

Gkoutos, G. V., Higgs, C., Bywater, R. P., Gouldson, P. R., Reynolds, C. A. (1999). Evidence for dimerization in the beta(2)-adrenergic receptor from the evolutionary trace method. International Journal of Quantum Chemistry, 74 (3), 371-379

From virtual to clinical: The discovery of PGN-1531, a novel antagonist of the prostanoid EP4 receptor.

In this Letter, we present the results of a hit-finding and lead optimization programme against the EP4 receptor (EP4R). In a short time period, we were able to discover five structurally diverse series of hit compounds using a combination of virtual screening methods. The most favoured hit, compound 6, was demonstrated to be a competitive antagonist of the EP4R. Compound 73 was identified following several rounds of optimization, which centred on improving both the primary EP4R affinity and selectivity against the related EP2R as well as the aqueous solubility. This work culminated in the preparation of PGN-1531, the sodium salt of 73, which showed a marked improvement in solubility (>10 mg/mL). PGN-1531 is a potent and selective antagonist at EP4Rs in vitro and in vivo, with the potential to alleviate the symptoms of migraine that result from cerebral vasodilatation.