Specific Methyl Group Protonation for the Measurement of Pharmacophore‐Specific Interligand NOE Interactions

Abstract

In the recent decades NMR spectroscopy has emerged as a powerful tool in the drug-discovery field. Several NMRbased methods have proved beneficial for the optimization of low-molecular-weight lead structures. Among those, techniques relying on the nuclear Overhauser effect (NOE) can provide information on pharmacophores at atomic resolution. The transferred-NOEs (tr-NOEs) [1] allow access to the bioactive conformation of ligands and can also be applied as a screening tool to identify the interaction of a small molecule with a macromolecular receptor. Recently, we have reported on the measurement of protein-mediated interligand NOEs (INPHARMA), which are observed for a mixture of two ligands binding competitively and weakly to the same binding pocket of a common target.[2-4] These interligand NOEs do not result from a direct magnetization transfer between the protons of the two ligands, since the small compounds, being competitive binders, never occupy the protein binding pocket at the same time. Rather, a spin diffusion process, mediated by the protons of the receptor-binding pocket, leads to cross-peaks between the two ligands. We proposed and demonstrated that these interligand NOEs can be used to determine the relative binding mode of two drug leads or, in favorable cases, even their absolute binding pose (INPHARMA approach).[3]The principle underlying the methodology relies on the dependence of the INPHARMA NOEs on the protein environment and more specifically on the distances between the protons of each ligand and the protons of the receptor. Consequently, the size of the INPHARMA NOEs depends on the binding mode of each ligand to the receptor and a quantitative interpretation of such effects can be used to derive the binding modes. However, extensive spin diffusion among the receptor protons reduces the specificity of the INPHARMA NOE signals, and the pharmacophore signature has to be retrieved by theoretically simulating the effect of spin diffusion. Thus, the determination of the ligands binding modes relies on computationally intensive calculations based on the full-relaxation matrix approach in presence of chemical exchange, [5] including the protons of the ligands and all protons of the receptor within a reasonable distance from the binding pocket typically 10 . [1,3] In the daily workflow of drug discovery, it is desirable to extract information from the INPHARMA NOEs without need for the demanding full-relaxation matrix calculations. We reasoned that a clearer fingerprint of the binding modes of the two ligands to the receptor would be obtained from the values of the INPHARMA NOEs if spin diffusion inside the protein could be either switched off or considerably reduced. An efficient way to attenuate intramolecular spin-diffusion is to reduce the proton density in the receptor, for example, by deuteration. In the past few years, a robust protocol has been developed for bacterial expression of proteins, in which methyl groups are selectively protonated in a highly deuterated background.[6] Here we empl...

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

“…In recent decades, various methods have been developed in order to derive protein-ligand complex structures faster than with the classical NMR structure calculation protocol, but these methods mostly rely on a preliminary docking step rather than on experimentally driven calculations. Moreover, sometimes partial resonance assignments of the receptor are required [39][40][41][42][43][44][45]47,49,72]. A complex structure calculation method that is based on defined and accurate NOEs [78][79][80] but also bypasses the long and tedious protein assignment step was missing.…”

“…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.…”

The INPHARMA technique for pharmacophore mapping: A theoretical guide to the method

“…However, we have recently demonstrated that the INPHARMA approach is much more powerful and allows in favorable cases the de novo description of the binding mode of both L 1 and L 2 [11,12]. In this work we have shown that the methodology is precise enough to unambiguously select one docking mode per ligand and accurate enough to select the correct docking mode, as compared to the crystal structures available for the investigated test cases [12].…”

“…To overcome this problem, we developed the INPHARMA methodology (Interligand Noes for PHARmacophore MApping) that allows mapping the structure of the binding pocket of a macromolecule on the NMR resonances of two competitively binding ligands [11][12][13][14]. The method is based on the observation of interligand, spin-diffusion mediated, transferred-NOE data, between two ligands L 1 and L 2 , binding competitively and weakly to a macromolecular receptor T (Fig.…”

Estudos estruturais e de interação de novos inibidores e ativadores da tirosinase

“…O INPHARMA permite observar a orientação de ligantes no sítio do receptor, e para isso, são utilizados dois ligantes (L 1 e L 2 ) competitivos pelo mesmo sítio e com baixa ou média afinidade pelo receptor (Orts, 2008;.…”