Identifying Receptor-Ligand Binding Sites
Information on how and where a ligand binds provides guidance in optimizing compound activity. One of the strengths of NMR is the ability to provide details on molecular structure and interaction with the most recognized approach being to follow changes in NMR chemical shifts as a function of molecular interaction. However, these methods are not fundamentally fast or low-barrier in as much as they are a use of the detailed knowledge obtained from NMR structural work. Specifically, the time-consuming steps of cloning and expressing the receptor, collecting and assigning the multidimensional NMR data sets and computing a three dimensional structure are prerequisite. The constraints do not reduce the utility of these methods, they do however, have the combined effect of excluding their application on many targets likely to be encountered in a pharmaceutical setting.
We have explored a more rapid alternative to the chemical shift perturbation approach which, in principle, can be employed with less overhead. The method is based on using a NMR water exchange (WEX) experiment to selectively detect protons that have exchanged from water to a position on the molecule of interest. WEX spectra can be used follow site-specific differences in the molecular access of water (hydrogen bonding or other structural changes) that result from interactions. Essentially, if an interaction introduces a hydrogen bond (or protection) to a resonance exchanging with water, the signal from that proton disappears from the WEX spectrum. Conversely if an interaction breaks a hydrogen bond (or exposes) an exchangeable proton, a new peak appears in the WEX spectrum.
Figure 3
Figure 3 shows the WEX data of a Ribonuclease A the absence and presence of 2', 5' -CpA, an inhibitor that binds at the active site. The data clearly show a resonance at about 13 ppm (bottom spectrum in Figure 3) disappearing when 2', 5' -CpA is added (top spectrum in Figure 3). This resonance has been assigned to His 19 sidechain and has been demonstrated to be involved in a hydrogen bond between the He2 proton of His 19 and the O12-O2P oxygen atoms of 2', 5' -CpA. Furthermore there are indications that the hydrogen bond between Phe 120 backbone amide proton and O2P oxygen atom of 2', 5' -CpA can be observed as a result of a missing resonance also in Figure 3. These data suggest that 2', 5' -CpA stabilizes labile protons at the Ribonuclease A active site by forming hydrogen bonds manifesting as a change in the exchange features of protons at key contact points. Analyses such as these, while not providing the all of the detail, are a route to rapid assessment of receptor-ligand interaction.
 
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