Crystallography‐Independent Determination of Ligand Binding Modes

Abstract

Within the last few decades, structure-based drug design (SBDD) has evolved into a powerful tool for the optimization of many low-molecular-weight lead compounds into highly potent drugs.[1] The principle of SBDD lies in the combination of different chemical moieties with the aim of obtaining a molecule that, while possessing the pharmacological properties necessary for a drug, is complementary in shape to the receptor binding pocket. This process requires knowledge of the exact structure of the protein/ligand complex. At present, structural genomics initiatives provide protein structures of biomedically relevant targets at an increasing rate [2] and recent structures of ion channels [3] and G-protein-coupled receptors (GPCRs) [4][5][6] bring these protein classes within reach for SBDD. Despite these successes, the daily work of pharmaceutical discovery is often limited by the ability to obtain high-resolution crystal structures of the target proteins in complex with the lower affinity ligands (lead structures) that are commonly identified by high-throughput screening or by fragment-based lead discovery.[1] In view of this limitation, SBDD would benefit from methods providing the relative orientations of different chemical fragments binding competitively to a receptor site. Such an approach would provide protein/ligand structures of novel ligands or fragments in relation to the known cocrystal structure of a reference ligand.Recently, we reported the observation by NMR spectroscopy of interligand NOE peaks occurring between two small ligands binding weakly and competitively to the same binding pocket of a common macromolecular receptor (Figure 1). [7,8] The measured mixture in solution contained two ligands (L A and L B ) in a 10-to 50-fold excess relative to the target receptor (T). As the ligands were competitive binders, these NOEs did not originate from a direct transfer of magnetization between the two ligands, [9] but rather from a spindiffusion process mediated by the protons of the receptor binding pocket. We proposed that such interligand NOEs can be used to define the relative orientation of the two ligands in the receptor binding pocket (the relative binding mode) and we termed the novel effect INPHARMA (internuclear NOEs for pharmacophore mapping; [8] Figure 1). INPHARMA can be observed for complexes with a dissociation constant (K d ) in the low micromolar to millimolar range.The scope of this work is to demonstrate for the first time that the INPHARMA method allows the determination of the relative, and in favorable cases even the absolute, binding mode of two low-affinity ligands binding competitively to a common receptor site and that it can thus be applied in the context of SBDD. In accordance with existing SBDD workflows, the experimental information derived from INPHARMA was used to select the correct binding mode

An NMR-based scoring function improves the accuracy of binding pose predictions by docking by two orders of magnitude

“…Previously, we demonstrated that INPHARMA is able to determine the binding mode of the two ligands L 1 and L 2 to the catalytic subunit of PKA (Orts et al 2008). In this work we aim at establishing INPHARMA as an effective scoring function for binding modes in high-throughput docking campaigns.…”

“…The INPHARMA data were measured on a mixture of the Chinese hamster Ca catalytic subunit of cyclic adenosine monophosphate (cAMP) dependentent protein kinase A (PKA) (25–30 uM), L 1 (150 uM) and L 2 (450 uM), as described in (Orts et al 2008). The values of the measured INPHARMA NOEs used in this work are in Table S1.…”

“…These approaches have the advantage of observing only the resonances of the ligands and of being applicable to protein targets of any size. Recently, we have developed an NMR-based methodology, INPHARMA (Interligand NOEs for PHARmacophore MApping) (Sanchez-Pedregal et al 2005; Reese et al 2007; Orts et al 2008), which is able to reveal the relative, and in favourable cases even the absolute, binding mode of competitively binding, low-affinity ligands, with the sole requirement of a structural model of the apo-receptor. The relative binding mode of two ligands interacting competitively with a common receptor allows pharmacophore or ligand superimposition.…”

“…S1), for which the binding modes determined by INPHARMA could be compared to existing crystal structures (Orts et al 2008). This test established the value of INPHARMA, and confirmed that the combination of tr-NOEs and INPHARMA NOEs, in the presence of a structural model for the apo-potein, allows discriminating between a few, very diverse docking modes (Orts et al 2008). …”

The NMR2 Method to Determine Rapidly the Structure of the Binding Pocket of a Protein–Ligand Complex with High Accuracy

“…CORCEMA [46,68,69] and INPHARMA [48,67,72,73] are methods that back predict intra-ligand, intra-protein and protein-ligand NOEs or spin diffusion using the full relaxation matrix formalism. They are powerful tools that can also handle systems undergoing multistate conformational exchange and chemical exchange between the free and bound states.…”