Miles Congreve

Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol–Bound β1-Adrenergic Receptor

Comparisons between structures of the β1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser5.46, coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of β1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both β1AR and β2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey β1AR and an inverse agonist of human β2AR. The structure of 7-methylcyanopindolol–bound β1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound β1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol–bound β1AR and the hydroxyl group of Ser5.46 is positioned 0.8 Å further from the ligand, with respect to the position of the Ser5.46 side chain in cyanopindolol-bound β1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists.

Engineering G Protein-Coupled Receptors for Drug Design

G protein-coupled receptors (GPCRs) play a crucial role in many diseases and are the site of action of 25–30 % of current drugs (Overington et al., Nat Rev Drug Discov 5(12):993–996, 2006). As such GPCRs represent a major area of interest for the pharmaceutical industry. Despite the rich history of this target class there remain many opportunities for clinical intervention and there is a scarcity of high quality drug-like molecules for many receptors. High-throughput screening has often failed to unlock the potential of members of this superfamily and new, complementary approaches to GPCR drug discovery are required. However, the instability of GPCRs when removed from the cell membrane has severely limited the application of the techniques of structure-based and fragment-based drug discovery. The Heptares approach is successfully overcoming this fundamental challenge and facilitates both biophysical and biochemical fragment screening and also the generation of structural information. Heptares uses its StaR® technology to thermostabilise GPCRs using mutations in precisely defined biologically-relevant conformations (Robertson et al., Neuropharmacology 60(1):36–44, 2011). StaR proteins are amenable to techniques that cannot be readily used with wild-type GPCRs, including fragment screening, biophysical kinetic profiling and X-ray crystallography. Crystal structures of multiple GPCRs have been solved using this approach in the last 5 years (Dore et al., Structure 19(9):1283–1293, 2011; Dore et al., Nature 511:557–562, 2014; Hollenstein et al., Nature 499(7459):438–443, 2013).