Fragment Growing Induces Conformational Changes in Acetylcholine-Binding Protein: A Structural and Thermodynamic Analysis

Abstract

Optimization of fragment hits toward high-affinity lead compounds is a crucial aspect of fragment-based drug discovery (FBDD). In the current study, we have successfully optimized a fragment by growing into a ligand-inducible subpocket of the binding site of acetylcholine-binding protein (AChBP). This protein is a soluble homologue of the ligand binding domain (LBD) of Cys-loop receptors. The fragment optimization was monitored with X-ray structures of ligand complexes and systematic thermodynamic analyses using surface plasmon resonance (SPR) biosensor analysis and isothermal titration calorimetry (ITC). Using site-directed mutagenesis and AChBP from different species, we find that specific changes in thermodynamic binding profiles, are indicative of interactions with the ligand-inducible subpocket of AChBP. This study illustrates that thermodynamic analysis provides valuable information on ligand binding modes and is complementary to affinity data when guiding rational structure- and fragment-based discovery approaches.

Molecular actions of smoking cessation drugs at α4β2 nicotinic receptors defined in crystal structures of a homologous binding protein

“…Similar to the complex in AcAChBP (23), lobeline exposes a subpocket in Ct-AChBP by changing the rotameric state of F102 (loop A) from the g-to tconformation (homologous to Y91 in Ac-AChBP; ref. 24). Lobeline forms four hydrogen bonds that converge upon residues of loop B (shown as dashed lines in Fig.…”

Exploration of the molecular architecture of the orthosteric binding site in the α4β2 nicotinic acetylcholine receptor with analogs of 3-(dimethylamino)butyl dimethylcarbamate (DMABC) and 1-(pyridin-3-yl)-1,4-diazepane

“…Two other aromatic box residues observed to adopt alternative conformation in AChBP structures are TyrA and TrpD (TyrD in Ac-AChBP). Alternative conformations of these residues have revealed two different extensions to the binding pocket that can accommodate non-classical ligands, including lobeline [17,20] (2, Figure 1 and Figure 2B) and the synthetic compound, 3α-(benzoyloxy)-8β-((R)-2-hydroxy-2-phenylethyl)-8α-methyl-8 azoniabicyclo[3.2.1]octane methiodide (3, Figure 1 and Figure 2C) [27]. From crystallographic studies of Ac-AChBP it is known that a sub-pocket is made accessible by rotameric shift of TyrA upon binding of 2 ( Figure 2B) [17].…”

“…From crystallographic studies of Ac-AChBP it is known that a sub-pocket is made accessible by rotameric shift of TyrA upon binding of 2 ( Figure 2B) [17]. However, mutational studies indicate that the flip is dependent on specific residues on Loop F on the complementary subunit interface to stabilize the tyrosine in its alternative conformation [27], and it remains unknown if the pocket exists in nAChRs. The second sub-pocket is an extension of the binding site along the dimer interface towards the ion channel and has been observed by co-crystallization of 3 with Ac-AChBP [27].…”

Synthesis of n-acetylglucosaminides dimers

“…7а). Сполука-лідер із Kd = 320 нM була спроектована шляхом накладання кристалічних структур природного алкалоїду ло-буліну та ідентифікованого бензоат-заміщеного фрагменту нортропіну [49].…”