Abstract
A congeneric series of 21 phosphodiesterase 2 (PDE2) inhibitors are reported. Crystal structures show how the molecules can occupy a ‘top-pocket’ of the active site. Molecules with small substituents do not enter the pocket, a critical leucine (Leu770) is closed and water molecules are present. Large substituents enter the pocket, opening the Leu770 conformation and displacing the waters. We also report an X-ray structure revealing a new conformation of the PDE2 active site domain. The relative binding affinities of these compounds were studied with free energy perturbation (FEP) methods and it represents an attractive real-world test case. In general, the calculations could predict the energy of small-to-small, or large-to-large molecule perturbations. However, accurately capturing the transition from small-to-large proved challenging. Only when using alternative protein conformations did results improve. The new X-ray structure, along with a modelled dimer, conferred stability to the catalytic domain during the FEP molecular dynamics (MD) simulations, increasing the convergence and thereby improving the prediction of ΔΔG of binding for some small-to-large transitions. In summary, we found the most significant improvement in results when using different protein structures, and this data set is useful for future free energy validation studies.
Highlights
Drug discovery lead optimisation (LO) requires synthesising analogues of important compounds
The H-loop (702–728) covers the ligand in the binding site in contrast to entirely open H-loop conformations typically observed in published PDE2A catalytic domain crystal structures (Fig. 2 and S2)
We considered if the waters were left in the top-pocket, could better results be attained despite the clashes with the large ligands? A 5 ns per λ window free energy perturbation (FEP) protocol was performed but again with little impact on results, mean unsigned error (MUE) for the ΔG and ΔΔG was 1.18 ± 0.52 and 1.30 ± 0.56 kcal/mol respectively and no improvement for the small-to-large ΔΔG transitions, with the MUE still >3 kcal/mol
Summary
Drug discovery lead optimisation (LO) requires synthesising analogues of important compounds. As well as the small-scale binding site motions that open and close the top-pocket, we focus on another aspect of protein dynamics, namely large-scale conformational change. Via crystallography we disclose a previously unreported conformational state of the PDE2 catalytic domain We call this an intermediate H-loop conformation, and show how this large-scale change of H-loop from open to intermediate states affects the calculation of binding free energies. This is an attractive study: i) test conformational changes at different scales supported with X-ray structures, ii) perturbations can be relatively small amongst the 21 close analogues with incremental increases in substituent size, iii) a greater than 3 kcal/mol range of activity, iv) water structure adaptation. The overall difficulties to predict affinity for this real-life scenario demonstrate that effort is needed to improve and diagnose FE calculations
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