Inclusion of Enclosed Hydration Effects in the Binding Free Energy Estimation of Dopamine D3 Receptor Complexes

Confined hydration and conformational flexibility are some of the challenges encountered for the rational design of selective antagonists of G-protein coupled receptors. We present a set of C3-substituted (-)-stepholidine derivatives as potent binders of the dopamine D3 receptor. The compounds are characterized biochemically, as well as by computer modeling using a novel molecular dynamics-based alchemical binding free energy approach which incorporates the effect of the displacement of enclosed water molecules from the binding site. The free energy of displacement of specific hydration sites is obtained using the Hydration Site Analysis method with explicit solvation. This work underscores the critical role of confined hydration and conformational reorganization in the molecular recognition mechanism of dopamine receptors and illustrates the potential of binding free energy models to represent these key phenomena.Comment: This is the first report of using enclosed hydration in estimating binding free energies of protein-ligand complexes using implicit solvatio

Ligand Selectivity in the Recognition of Protoberberine Alkaloids by Hybrid-2 Human Telomeric G-Quadruplex: Binding Free Energy Calculation, Fluorescence Binding, and NMR Experiments

The human telomeric G-quadruplex (G4) is an attractive target for developing anticancer drugs. Natural products protoberberine alkaloids are known to bind human telomeric G4 and inhibit telomerase. Among several structurally similar protoberberine alkaloids, epiberberine (EPI) shows the greatest specificity in recognizing the human telomeric G4 over duplex DNA and other G4s. Recently, NMR study revealed that EPI recognizes specifically the hybrid-2 form human telomeric G4 by inducing large rearrangements in the 50-flanking segment and loop regions to form a highly extensive four-layered binding pocket. Using the NMR structure of the EPI-human telomeric G4 complex, here we perform molecular dynamics free energy calculations to elucidate the ligand selectivity in the recognition of protoberberines by the human telomeric G4. The MM-PB(GB)SA (molecular mechanics-Poisson Boltzmann/Generalized Born) Surface Area) binding free energies calculated using the Amber force fields bsc0 and OL15 correlate well with the NMR titration and binding affinity measurements, with both calculations correctly identifying the EPI as the strongest binder to the hybrid-2 telomeric G4 wtTel26. The results demonstrated that accounting for the conformational flexibility of the DNA-ligand complexes is crucially important for explaining the ligand selectivity of the human telomeric G4. While the MD-simulated (molecular dynamics) structures of the G-quadruplex-alkaloid complexes help rationalize why the EPI-G4 interactions are optimal compared with the other protoberberines, structural deviations from the NMR structure near the binding site are observed in the MD simulations. We have also performed binding free energy calculation using the more rigorous double decoupling method (DDM); however, the results correlate less well with the experimental trend, likely due to the difficulty of adequately sampling the very large conformational reorganization in the G4 induced by the protoberberine binding

Distinguishing Binders from False Positives by Free Energy Calculations: Fragment Screening Against the Flap Site of HIV Protease

Molecular docking is a powerful tool used in drug discovery and structural biology for predicting the structures of ligand–receptor complexes. However, the accuracy of docking calculations can be limited by factors such as the neglect of protein reorganization in the scoring function; as a result, ligand screening can produce a high rate of false positive hits. Although absolute binding free energy methods still have difficulty in accurately rank-ordering binders, we believe that they can be fruitfully employed to distinguish binders from nonbinders and reduce the false positive rate. Here we study a set of ligands that dock favorably to a newly discovered, potentially allosteric site on the flap of HIV-1 protease. Fragment binding to this site stabilizes a closed form of protease, which could be exploited for the design of allosteric inhibitors. Twenty-three top-ranked protein–ligand complexes from AutoDock were subject to the free energy screening using two methods, the recently developed binding energy analysis method (BEDAM) and the standard double decoupling method (DDM). Free energy calculations correctly identified most of the false positives (≥83%) and recovered all the confirmed binders. The results show a gap averaging ≥3.7 kcal/mol, separating the binders and the false positives. We present a formula that decomposes the binding free energy into contributions from the receptor conformational macrostates, which provides insights into the roles of different binding modes. Our binding free energy component analysis further suggests that improving the treatment for the desolvation penalty associated with the unfulfilled polar groups could reduce the rate of false positive hits in docking. The current study demonstrates that the combination of docking with free energy methods can be very useful for more accurate ligand screening against valuable drug targets

The Role of Displacing Confined Solvent in the Conformational Equilibrium of β-Cyclodextrin

This study investigates the role of hydration and its relationship to the conformational equilibrium of the host molecule β-cyclodextrin. Molecular dynamics simulations indicate that the unbound β-cyclodextrin exhibits two state behavior in explicit solvent due to the opening and closing of its cavity. In implicit solvent, these transitions are not observed and there is one dominant conformation of β-cyclodextrin with an open cavity. Based on these observations, we investigate the hypothesis that the expulsion of thermodynamically unfavorable water molecules into the bulk plays an important role in controlling the accessibility of the closed macrostate at room temperature. We compare the results of the molecular mechanics analytical generalized Born plus non-polar solvation approach to those obtained through Grid Inhomogeneous Solvation Theory analysis with explicit solvation to elucidate the thermodynamic forces at play. The calculations help to illustrate the deficiencies of continuum solvent models and demonstrate the key role of the thermodynamics of enclosed hydration in driving the conformational equilibrium of molecules in solution. </p

Large Scale Affinity Calculations of Cyclodextrin Host–Guest Complexes: Understanding the Role of Reorganization in the Molecular Recognition Process

Host–guest inclusion complexes are useful models for understanding the structural and energetic aspects of molecular recognition. Due to their small size relative to much larger protein–ligand complexes, converged results can be obtained rapidly for these systems thus offering the opportunity to more reliably study fundamental aspects of the thermodynamics of binding. In this work, we have performed a large scale binding affinity survey of 57 β-cyclodextrin (CD) host–guest systems using the binding energy distribution analysis method (BEDAM) with implicit solvation (OPLS-AA/AGBNP2). Converged estimates of the standard binding free energies are obtained for these systems by employing techniques such as parallel Hamiltonian replica exchange molecular dynamics, conformational reservoirs, and multistate free energy estimators. Good agreement with experimental measurements is obtained in terms of both numerical accuracy and affinity rankings. Overall, average effective binding energies reproduce affinity rank ordering better than the calculated binding affinities, even though calculated binding free energies, which account for effects such as conformational strain and entropy loss upon binding, provide lower root-mean-square errors when compared to measurements. Interestingly, we find that binding free energies are superior rank order predictors for a large subset containing the most flexible guests. The results indicate that, while challenging, accurate modeling of reorganization effects can lead to ligand design models of superior predictive power for rank ordering relative to models based only on ligand–receptor interaction energies

Thermodynamic Decomposition of Solvation Free Energies with Particle Mesh Ewald and Long-Range Lennard-Jones Interactions in Grid Inhomogeneous Solvation Theory

Grid Inhomogeneous Solvation Theory (GIST) maps out solvation thermodynamic properties on a fine meshed grid and provides a statistical mechanical formalism for thermodynamic end-state calculations. However, differences in how long-range non-bonded interactions are calculated in molecular dynamics engines and in the current implementation of GIST have prevented precise comparisons between free energies estimated using GIST and those from other free energy methods such as thermodynamic integration (TI). Here, we address this by presenting PME-GIST, a formalism by which particle mesh Ewald (PME) based electrostatic energies and long-range Lennard-Jones (LJ) energies are decomposed and assigned to individual atoms and the corresponding voxels they occupy in a manner consistent with the GIST approach. PME-GIST yields potential energy calculations that are precisely consistent with modern simulation engines and performs these calculations at a dramatically faster speed than prior implementations. Here, we apply PME-GIST end-states analyses to 32 small molecules whose solvation free energies are close to evenly distributed from 2 kcal/mol to -17 kcal/mol and obtain solvation energies consistent with TI calculations (R2 = 0.99, mean unsigned difference 0.8 kcal/mol). We also estimate the entropy contribution from the 2nd and higher order entropy terms that are truncated in GIST by the differences between entropies calculated in TI and GIST. With a simple correction for the high order entropy terms, PME-GIST obtains solvation free energies that are highly consistent with TI calculations (R2 = 0.99, mean unsigned difference = 0.4 kcal/mol) and experimental results (R2 = 0.88, mean unsigned difference = 1.4 kcal/mol). The precision of PME-GIST also enables us to show that the solvation free energy of small hydrophobic and hydrophilic molecules can be largely understood based on perturbations of the solvent in a region extending a few solvation shells from the solute. We have integrated PME-GIST into the open-source molecular dynamics analysis software CPPTRAJ

Ligand Selectivity in the Recognition of Protoberberine Alkaloids by Hybrid-2 Human Telomeric G-Quadruplex: Binding Free Energy Calculation, Fluorescence Binding, and NMR Experiments

The human telomeric G-quadruplex (G4) is an attractive target for developing anticancer drugs. Natural products protoberberine alkaloids are known to bind human telomeric G4 and inhibit telomerase. Among several structurally similar protoberberine alkaloids, epiberberine (EPI) shows the greatest specificity in recognizing the human telomeric G4 over duplex DNA and other G4s. Recently, NMR study revealed that EPI recognizes specifically the hybrid-2 form human telomeric G4 by inducing large rearrangements in the 5′-flanking segment and loop regions to form a highly extensive four-layered binding pocket. Using the NMR structure of the EPI-human telomeric G4 complex, here we perform molecular dynamics free energy calculations to elucidate the ligand selectivity in the recognition of protoberberines by the human telomeric G4. The MM-PB(GB)SA (molecular mechanics-Poisson Boltzmann/Generalized Born) Surface Area) binding free energies calculated using the Amber force fields bsc0 and OL15 correlate well with the NMR titration and binding affinity measurements, with both calculations correctly identifying the EPI as the strongest binder to the hybrid-2 telomeric G4 wtTel26. The results demonstrated that accounting for the conformational flexibility of the DNA-ligand complexes is crucially important for explaining the ligand selectivity of the human telomeric G4. While the MD-simulated (molecular dynamics) structures of the G-quadruplex-alkaloid complexes help rationalize why the EPI-G4 interactions are optimal compared with the other protoberberines, structural deviations from the NMR structure near the binding site are observed in the MD simulations. We have also performed binding free energy calculation using the more rigorous double decoupling method (DDM); however, the results correlate less well with the experimental trend, likely due to the difficulty of adequately sampling the very large conformational reorganization in the G4 induced by the protoberberine binding

Resolving the Ligand-Binding Specificity in c‑MYC G‑Quadruplex DNA: Absolute Binding Free Energy Calculations and SPR Experiment

We report the absolute binding free energy calculation and surface plasmon resonance (SPR) experiment for ligand binding with the c-MYC G-quadruplex DNA. The unimolecular parallel DNA G-quadruplex formed in nuclease hypersensitivity element III₁ of the c-MYC gene promoter regulates the c-MYC transcription and is recognized as an emerging drug target for cancer therapy. Quindoline derivatives have been shown to stabilize the G-quadruplex and inhibit the c-MYC expression in cancer cells. NMR revealed two binding sites located at the 5′ and 3′ termini of the G-quadruplex. Questions about which site is more favored and the basis for the ligand-induced binding site formation remain unresolved. Here, we employ two absolute binding free energy methods, the double decoupling and the potential of mean force methods, to dissect the ligand-binding specificity in the c-MYC G-quadruplex. The calculated absolute binding free energies are in general agreement with the SPR result and suggest that quindoline has a slight preference for the 5′ site. The flanking residues around the two sites undergo significant reorganization as the ligand unbinds, which provides evidence for ligand-induced binding pocket formation. The results help interpret experimental data and inform rational design of small molecules targeting the c-MYC G-quadruplex