Drug Design for G‐Protein‐Coupled Receptors by a Ligand‐Based NMR Method

Abstract

More than 50 % of all drug targets are membrane proteins. [1] Recent progress in membrane protein crystallography has made a few of these targets amenable to established structurebased design methods.[2] However, crystallization of a membrane protein target remains a challenge, and medicinal chemists must rely on ligand-based design approaches for the targets that do not crystallize. These approaches can benefit from the knowledge of the bioactive conformation of the ligand conformation and the relative orientation of different chemotypes in the receptor binding site (cross-chemotype alignments). The generation of hypotheses for the bioactive conformation and cross-chemotype alignments depend on the availability of sufficient data on ligand structure-activity relationships (SARs). Herein, we demonstrate that these insights can be derived in the absence of protein-ligand crystal structures by straightforward ligand-based approaches relying on NMR spectroscopy. We show that a qualitative and fast analysis of INPHARMA NMR data [3,4] can be used within the timelines of the drug-discovery process without extended modeling and detailed data interpretation. We performed this study using intact biological membranes rather than reconstituted proteins [5,6] to eliminate solubilization-related artefacts, to expand the applicability to receptors that do not tolerate solubilization, and to significantly enhance the speed of the method. Here we demonstrate that a straightforward ligand-based NMR approach can be used to establish a nonradioactive binding assay for a Gprotein-coupled receptor (GPCR) and to give access to the relative orientation of multiple chemotypes supporting ligand-based drug design.

Complex Pharmacology of Free Fatty Acid Receptors

“…163 By the use of an NMR-based method for GPCR screening, Bartoschek and co-workers were able to identify novel submicromolar FFA1 agonists such as 9. 164 Using free fatty acids as the starting point and inspiration from the early thiazolidinediones in the design of small druglike free fatty acid-mimicking compounds, Christiansen et al identified a phenoxyacetic acid hit containing a central alkyne that was optimized to the potent and selective TUG-424 (10), which was confirmed to enhance insulin secretion at high but not low glucose levels in a FFA1-dependent manner. 165 This compound, however, displayed lower than ideal in vitro metabolic stability and short plasma half-life, suspected to reflect metabolism of the ortho-methyl group and/or betaoxidation.…”

Drug Discovery Opportunities and Challenges at G Protein Coupled Receptors for Long Chain Free Fatty Acids

“…Two other binding techniques have developed enhanced specificity for the ligand–receptor interaction under study. Bartoschek et al (2010) generated FFAR1 binding data from saturation transfer difference 1 H nuclear magnetic resonance (NMR) measurements, in which NMR spectra are obtained only when ligands bind a macromolecular complex, such as a receptor protein. They showed that this method allowed estimation of agonist IC 50 values from competition binding experiments, which displayed close correspondence with functional potencies.…”

Structural and Dynamic Information on Ligand Binding

“…The ligand-based methods, which typically involve comparison of the NMR parameters of a mixture of compounds in the presence and absence of the protein molecules, rely on the exchange-mediated transfer of bound state information to the free state. This biases ligand-based methods towards identification of weakly binding ligands (rapid exchange), but it does render the molecular weight of the protein molecule largely irrelevant, and indeed the approach has been applied to the ribosome, to receptors in membranes and to whole cells [64][65][66][67][68][69][70]. Competition experiments can be used to extend the range of the relaxation approach to tighter binding ligands, including those which are not in fast exchange.…”