Fragment screening of stabilized G-protein-coupled receptors using biophysical methods.

Biophysical studies with G-protein-coupled receptors (GPCRs) are typically very challenging due to the poor stability of these receptors when solubilized from the cell membrane into detergent solutions. However, the stability of a GPCR can be greatly improved by introducing a number of point mutations into the protein sequence to give a stabilized receptor or StaR®. Here, we present the utility of StaRs for biophysical studies and the screening of fragment libraries. Two case studies are used to illustrate the methods: first, the screening of a library of fragments by surface plasmon resonance against the adenosine A(2A) receptor StaR, demonstrating how very small and weakly active xanthine fragments can be detected binding to the protein on chips; second, the screening and detection of fragment hits of a larger fragment library in an NMR format called TINS (target-immobilized NMR screening) against the ß(1) adrenergic StaR.

Target immobilization as a strategy for NMR-based fragment screening: comparison of TINS, STD, and SPR for fragment hit identification.

Fragment-based drug discovery (FBDD) has become a widely accepted tool that is complementary to high-throughput screening (HTS) in developing small-molecule inhibitors of pharmaceutical targets. Because a fragment campaign can only be as successful as the hit matter found, it is critical that the first stage of the process be optimized. Here the authors compare the 3 most commonly used methods for hit discovery in FBDD: high concentration screening (HCS), solution ligand-observed nuclear magnetic resonance (NMR), and surface plasmon resonance (SPR). They selected the commonly used saturation transfer difference (STD) NMR spectroscopy and the proprietary target immobilized NMR screening (TINS) as representative of the array of possible NMR methods. Using a target typical of FBDD campaigns, the authors find that HCS and TINS are the most sensitive to weak interactions. They also find a good correlation between TINS and STD for tighter binding ligands, but the ability of STD to detect ligands with affinity weaker than 1 mM K(D) is limited. Similarly, they find that SPR detection is most suited to ligands that bind with K(D) better than 1 mM. However, the good correlation between SPR and potency in a bioassay makes this a good method for hit validation and characterization studies.

Target immobilization and NMR screening of fragments in early drug discovery.

Using localized NMR spectroscopy on immobilized targets provides us with a method to simultaneously assess binding of small molecules to two different samples. This Target Immobilized NMR Screening (TINS) has a number of advantages, not least is the requirement for minimal quantities of non-isotopically labeled protein and the applicability to insoluble or unstable targets. The technique is sensitive to binding with K(D) values in the range of 100 nM to 20 mM, while careful selection of the reference protein reduces the number of false positive hits. This combination ensures a maximal number of valid hits from which to select starting points for hit elaboration projects. Hits can be prioritized using biological assays when appropriate, as well as an array of biophysical techniques. So far a variety of soluble proteins, including kinases, GTPases, viral targets and proteases, as well as a membrane protein, have been successfully screened against our fragment library. Here we illustrate our experiences with a number of examples which emphasize the usefulness of the method in selecting and prioritizing fragment hits for elaboration towards leads.

Perspectives on NMR in drug discovery: a technique comes of age.

In the past decade, the potential of harnessing the ability of nuclear magnetic resonance (NMR) spectroscopy to monitor intermolecular interactions as a tool for drug discovery has been increasingly appreciated in academia and industry. In this Perspective, we highlight some of the major applications of NMR in drug discovery, focusing on hit and lead generation, and provide a critical analysis of its current and potential utility.

Integration of fragment screening and library design.

With more than 10 years of practical experience and theoretical analysis, fragment-based drug discovery (FBDD) has entered the mainstream of the pharmaceutical and biotech industries. An array of biophysical techniques has been used to detect the weak interaction between a fragment and the target. Each technique presents its own requirements regarding the fragment collection and the target; therefore, in order to optimize the potential of FBDD, the nature of the target should be a driving factor for simultaneous development of both the library and the screening technology. A roadmap is now available to guide fragment-to-lead evolution when structural information is available. The next challenge is to apply FBDD to targets for which high-resolution structural information is not available.