Sensitive NMR Approach for Determining the Binding Mode of Tightly Binding Ligand Molecules to Protein Targets

Abstract

Structure-guided drug design relies on detailed structural knowledge of protein-ligand complexes, but crystallization of cocomplexes is not always possible. Here we present a sensitive nuclear magnetic resonance (NMR) approach to determine the binding mode of tightly binding lead compounds in complex with difficult target proteins. In contrast to established NMR methods, it does not depend on rapid exchange between bound and free ligand or on stable isotope labeling, relying instead on a tert-butyl group as a chemical label. tert-Butyl groups are found in numerous protein ligands and deliver an exceptionally narrow and tall (1)H NMR signal. We show that a tert-butyl group also produces outstandingly intense intra- and intermolecular NOESY cross-peaks. These enable measurements of pseudocontact shifts generated by lanthanide tags attached to the protein, which in turn allows positioning of the ligand on the protein. Once the ligand has been located, assignments of intermolecular NOEs become possible even without prior resonance assignments of protein side chains. The approach is demonstrated with the dengue virus NS2B-NS3 protease in complex with a high-affinity ligand containing a tert-butyl group.

Trimethylsilyl tag for probing protein–ligand interactions by NMR

“…This often makes it possible to assign and track tBu signals in the presence of modest spectral overlap with protein signals. Recent examples have demonstrated this for ligands containing a tBu group and proteins containing a siteselectively incorporated tBu-tyrosine residue (Chen et al 2015;Chen et al 2016;Jabar et al 2017). When the spectral overlap is pronounced, however, the 1 H-NMR signal even of a tBu group may be difficult to identify and track.…”

Busca Virtual De Compostos Bioativos: Conceitos E Aplicações

“…27 Ao mesmo tempo, o planejamento de compostos bioativos, em especial de fármacos, se beneficiou grandemente do desenvolvimento da cristalografia de raios-X de proteínas e de técnicas multidimensionais em Ressonância Magnética Nuclear (RMN). 8,13,[28][29][30] Estas técnicas permitiram elucidar grande número de estruturas tridimensionais (3D) de potenciais alvos biológicos, sendo grande parte delas disponível no Protein Data Bank (PDB). 31 No entanto, embora a Química Combinatória e o HTS tenham gerado um vasto número de compostos com diversidade estrutural, possibilitando a seleção de ligantes que apresentassem um ótimo ajuste a um determinado alvo biológico, é consenso na literatura 10,29,32 que estas técnicas não levaram necessariamente à descoberta de novos fármacos.…”

Integrative protein modeling in RosettaNMR from sparse paramagnetic restraints

“…The components of the Δ χ -tensor(s) can be calculated from the proteins' PCS values prior to docking. We tested this method on three protein-ligand complexes (Tab 3): the SH2 domain of Grb2 bound to a low-affinity phosphorylated pYTN tripeptide ligand as well as a high-affinity macrocyclic inhibitor (45), and a complex of dengue virus NS2B-NS3 protease (DENpro) with a high-affinity ligand (46). PCS-assisted docking of the two SH2 ligands was converged (see score-vs-RMSD plots in Fig 6C and Fig S9).…”

“…We applied PCS-guided RosettaLigand to three systems for which experimental PCS data had been published: two complexes of the Src homology 2 (SH2) domain of the growth factor receptorbound protein 2 (Grb2) with a low-affinity pYTN tripeptide and a high-affinity inhibitor (4-[(10S,14S,18S)-18-(2-amino-2-oxoethyl)-14-(1-naphthylmethyl)-8,17,20-trioxo-7,16,19triazaspiro [5.14]icos-11-en-10-yl]benzylphosphonic acid) (45), and a complex between the dengue virus NS2B-NS3 protease with a high affinity ligand (46). 22 12 25 17 26 22 30 24 † Top 1% RMSD: average RMSD 100 of best 1% of models ranked by RMSD.…”