StaR-led drug discovery in action
Heptares’ most advanced programme targeting the A2A receptor as a potential new treatment for Parkinson’s disease and cognition has entered candidate selection phase. The A2A receptor is clinically validated in Parkinson’s disease as a target for monotherapy, and has potential as a target for molecules that modify or treat other CNS indications such as ADHD and neurodegeneration, and cognition. Using A2A StaRs, Heptares generated completely novel compound series and by employing drug discovery techniques not previously applicable to GPCRs, has rapidly advanced the programme to candidate identification; the properties of this candidate (completely novel chemical structure, low molecular weight and polar surface area, excellent oral bioavailability, low clearance combined with high potency and efficacy) clearly differentiate it from competitor compounds.
Heptares is currently in discussions to partner this candidate for further preclinical and clinical development.
Until recently the only GPCR that had been crystallised was rhodopsin – an unusual GPCR located in the retina and responsible for sensing light. This receptor has been used to construct models of the entire superfamily – however its homology is less than 20% to the Family A subgroup and significantly less to the other GPCR subgroups.
Very recently crystal structures of the human beta 2 adrenergic receptor have been obtained using either an antibody complex or enzyme fusion.
GPCRs are particularly difficult to crystallise due mainly to their instability in the detergents used for crystallography and their conformational flexibility and heterogeneity which inhibits the formation of crystals. StaRs however are stable in a wide range of detergents and are held in a single defined conformation – they are therefore more amenable to crystallisation than conventional GPCRs.
The first high resolution X-ray structure of a GPCR (the beta 1 adrenergic receptor) stabilised using the StaR process has now been solved by Heptares’ founding scientists at the MRC Laboratory of Molecular Biology (Warne et al (2008) Nature 454 (7203); 486-491).
All of the structures obtained so far represent inactive conformations of the receptors. No single GPCR structure, which necessarily depicts a given conformation, will capture all the information needed for structure-based drug design. What is needed are multiple structures bound with compounds of different chemical series and different pharmacological profiles.
In addition, structures of GPCRs from different families in both active and inactive conformations are required to understand receptor function and compound activity at the molecular level.
Once a GPCR structure has been obtained it can be used to:
Run in silico virtual screens of compound libraries to find starting points for a chemistry programme
Design chemical hits de novo
Understand structure activity relationships of compounds (including existing drugs and candidates) binding to their receptor and use these insights to direct lead generation and optimisation
Model the structure of other closely related GPCRs and use these for drug discovery or for improving compound selectivity (homology modelling).
Such approaches are likely to generate new chemical templates for GPCRs that may overcome issues such as low selectivity, poor pharmacokinetic profiles or toxicity, which may be present in existing chemotypes identified using high-throughput screening.
Screening and Molecular profiling
StaRs can be immobilised on chips or beads using a variety of coupling strategies. They retain normal GPCR function and expected pharmacology whilst immobilised in detergent. These properties of StaR™s enable them to be used in a variety of ultra high throughput affinity based screening platforms.
StaR™s can be immobilised on Biacore chips. Surface plasmon resonance (SPR) based technology using the Biacore system enables the generation of detailed mechanistic measurements including:
Kinetics (on- and off-rates) of compounds. These parameters have a major impact on the properties of drugs such as duration of action. Drugs with a slow off-rate are often more effective than those with a rapid off-rate, but depending on the target, can be more susceptible to side effects.
Specificity. Compounds identified in GPCR screens using whole cells often have effects downstream of the receptor. The Biacore method allows a direct measurement of activity on the purified protein.
Affinity. Affinities of compounds at GPCRs are usually measured by displacement of another labelled ligand. The Biacore can be used to obtain a ‘pure’ direct measurement of affinity of binding interaction. This is useful for screening small ‘fragment’ compounds that can bind with a low affinity.
Using StaRs for antibody generation
There are a significant number of opportunities to drug GPCRs with antibody rather than small molecule therapeutics. This is particularly the case for GPCRs involved in cancer and inflammation; however, historically it has proved difficult to obtain functional antibodies to GPCRs due to their low stability.
StaRs represent a novel approach to raising antibodies to GPCRs. They can either be used directly to immunize animals (such as mice) to make monoclonal antibodies or immobilised on plates and used for screening antibody libraries in vitro. The fact that StaRs retain a single conformation during these processes may increase the probability of identifying functional
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