Soluble adenylate cyclases catalyse the synthesis of the second messenger cAMP through the cyclisation of ATP and are the only known enzymes to be directly activated by bicarbonate. Here, we report the first crystal structure of the human enzyme that reveals a pseudosymmetrical arrangement of two catalytic domains to produce a single competent active site and a novel discrete bicarbonate binding pocket. Crystal structures of the apo protein, the protein in complex with α,β-methylene adenosine 5'-triphosphate (AMPCPP) and calcium, with the allosteric activator bicarbonate, and also with a number of inhibitors identified using fragment screening, all show a flexible active site that undergoes significant conformational changes on binding of ligands. The resulting nanomolar-potent inhibitors that were developed bind at both the substrate binding pocket and the allosteric site, and can be used as chemical probes to further elucidate the function of this protein.

A new approach to generating information on ligand receptor interactions within the binding pocket of G protein-coupled receptors has been developed, called Biophysical Mapping (BPM). Starting from a stabilized receptor (StaR), minimally engineered for thermostability, additional single mutations are then added at positions that could be involved in small molecule interactions. The StaR and a panel of binding site mutants are captured onto Biacore chips to enable characterization of the binding of small molecule ligands using surface plasmon resonance (SPR) measurement. A matrix of binding data for a set of ligands versus each active site mutation is then generated, providing specific affinity and kinetic information (K(D), k(on), and k(off)) of receptor-ligand interactions. This data set, in combination with molecular modeling and docking, is used to map the small molecule binding site for each class of compounds. Taken together, the many constraints provided by these data identify key protein-ligand interactions and allow the shape of the site to be refined to produce a high quality three-dimensional picture of ligand binding, thereby facilitating structure based drug design. Results of biophysical mapping of the adenosine A(2A) receptor are presented.

Virtual screening was performed against experimentally enabled homology models of the adenosine A(2A) receptor, identifying a diverse range of ligand efficient antagonists (hit rate 9%). By use of ligand docking and Biophysical Mapping (BPM), hits 1 and 5 were optimized to potent and selective lead molecules (11-13 from 5, pK(I) = 7.5-8.5, 13- to >100-fold selective versus adenosine A(1); 14-16 from 1, pK(I) = 7.9-9.0, 19- to 59-fold selective).

Accumulation of extracellular adenosine within the microenvironment is a strategy exploited by tumors to escape detection by the immune system. Adenosine signaling through the adenosine 2A receptor (A2AR) on immune cells elicits a range of immunosuppressive effects which promote tumor growth and limit the efficacy of immune checkpoint inhibitors. Preclinical data with A2AR inhibitors have demonstrated tumor regressions in mouse models by rescuing T cell function; however, the mechanism and role on other immune cells has not been fully elucidated.

The orexin system, which consists of the two G protein-coupled receptors OX1 and OX2, activated by the neuropeptides OX-A and OX-B, is firmly established as a key regulator of behavioral arousal, sleep, and wakefulness and has been an area of intense research effort over the past two decades. X-ray structures of the receptors in complex with 10 new antagonist ligands from diverse chemotypes are presented, which complement the existing structural information for the system and highlight the critical importance of lipophilic hotspots and water molecules for these peptidergic GPCR targets. Learnings from the structural information regarding the utility of pharmacophore models and how selectivity between OX1 and OX2 can be achieved are discussed.

Biophysical fragment screening of a thermostabilized β1-adrenergic receptor (β1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein-ligand crystal structures of the β1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized β1AR complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 Å, respectively.

HTL0018318 is a selective M1 receptor partial agonist currently under development for the symptomatic treatment of cognitive and behavioural symptoms in Alzheimer's disease and other dementias. We investigated safety, tolerability, pharmacokinetics and exploratory pharmacodynamics (PD) of HTL0018318 following single ascending doses.

The cholinergic system and M1 receptor remain an important target for symptomatic treatment of cognitive dysfunction. The selective M1 receptor partial agonist HTL0018318 is under development for the symptomatic treatment of Dementia's including Alzheimer's disease (AD) and dementia with Lewy bodies (DLB). We investigated the safety, tolerability, pharmacokinetics and exploratory pharmacodynamics of multiple doses of HTL0018318 in healthy younger adults and elderly subjects.