Abstract
We present a robust protocol based on iterations of free energy perturbation (FEP) calculations, chemical synthesis, biophysical mapping and X‐ray crystallography to reveal the binding mode of an antagonist series to the A2A adenosine receptor (AR). Eight A2AAR binding site mutations from biophysical mapping experiments were initially analyzed with sidechain FEP simulations, performed on alternate binding modes. The results distinctively supported one binding mode, which was subsequently used to design new chromone derivatives. Their affinities for the A2AAR were experimentally determined and investigated through a cycle of ligand‐FEP calculations, validating the binding orientation of the different chemical substituents proposed. Subsequent X‐ray crystallography of the A2AAR with a low and a high affinity chromone derivative confirmed the predicted binding orientation. The new molecules and structures here reported were driven by free energy calculations, and provide new insights on antagonist binding to the A2AAR, an emerging target in immuno‐oncology.
Highlights
Computational estimation of shifts in binding free energy, associated with ligand modifications or point mutations in the receptor macromolecule, can provide the missing link between the structure of a protein-ligand complex and a panel of experimental binding affinities
These mutations involve residues in direct contact with the ligand, such as N2536.55, L853.33, M1775.38, N1815.42 and I662.64 (Ballesteros Weinstein numbering[31] in superscript), as well as residues not directly in contact with the ligand, namely S2777.42, Y2717.36 and L167EL2. This approach was extended for 1,2,4-triazines as A2AAR antagonists,[32] and the binding mode was later confirmed by X-ray crystallography.[29]
We initially examine the surface plasmon resonance (SPR) data available for these A2AAR antagonist families through a recently developed in silico mutagenesis tool based on free energy perturbation (FEP) simulations.[11,14,15]
Summary
Computational estimation of shifts in binding free energy, associated with ligand modifications or point mutations in the receptor macromolecule, can provide the missing link between the structure of a protein-ligand complex and a panel of experimental binding affinities. The first application of this technique was based around the co-crystallized A2AAR antagonist ZM241385 in combination with 8 receptor mutants (see Figure 1 A).[22] These mutations involve residues in direct contact with the ligand, such as N2536.55, L853.33, M1775.38, N1815.42 and I662.64 (Ballesteros Weinstein numbering[31] in superscript), as well as residues not directly in contact with the ligand, namely S2777.42, Y2717.36 and L167EL2 (see Figure 1) This approach was extended for 1,2,4-triazines as A2AAR antagonists,[32] and the binding mode was later confirmed by X-ray crystallography (see Figure 1 B).[29] In the same HTS campaign, a series of chromones were identified as a novel family of A2AAR antagonists,[32] and consecutively optimized to yield the potent and selective Chromone 14 (see Figure 2).[30] At that point, the leadoptimization program was successful in improving the affinity of the initial HTS hit, while not focusing on pharmacokinetic optimization (i.e., the most potent compound Chromone 14 contains a metabolically unstable ester group). Experimental structures of two chromone-A2AAR complexes were solved which confirmed the binding mode hypothesis from the computational studies
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