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

The mechanism of H171T resistance reveals the importance of Nδ-protonated His171 for the binding of allosteric inhibitor BI-D to HIV-1 integrase

Background: Allosteric HIV-1 integrase (IN) inhibitors (ALLINIs) are an important new class of anti-HIV-1 agents. ALLINIs bind at the IN catalytic core domain (CCD) dimer interface occupying the principal binding pocket of its cellular cofactor LEDGF/p75. Consequently, ALLINIs inhibit HIV-1 IN interaction with LEDGF/p75 as well as promote aberrant IN multimerization. Selection of viral strains emerging under the inhibitor pressure has revealed mutations at the IN dimer interface near the inhibitor binding site. Results: We have investigated the effects of one of the most prevalent substitutions, H171T IN, selected under increasing pressure of ALLINI BI-D. Virus containing the H171T IN substitution exhibited an ~68-fold resistance to BI-D treatment in infected cells. These results correlated with ~84-fold reduced affinity for BI-D binding to recombinant H171T IN CCD protein compared to its wild type (WT) counterpart. However, the H171T IN substitution only modestly affected IN-LEDGF/p75 binding and allowed HIV-1 containing this substitution to replicate at near WT levels. The x-ray crystal structures of BI-D binding to WT and H171T IN CCD dimers coupled with binding free energy calculations revealed the importance of the Nδ- protonated imidazole group of His171 for hydrogen bonding to the BI-D tert-butoxy ether oxygen and establishing electrostatic interactions with the inhibitor carboxylic acid, whereas these interactions were compromised upon substitution to Thr171. Conclusions: Our findings reveal a distinct mechanism of resistance for the H171T IN mutation to ALLINI BI-D and indicate a previously undescribed role of the His171 side chain for binding the inhibitor. Electronic supplementary material The online version of this article (doi:10.1186/s12977-014-0100-1) contains supplementary material, which is available to authorized users

A New Class of Allosteric HIV-1 Integrase Inhibitors Identified by Crstallographic Fragment Screening of the Catalytic Core Domain

HIV-1 integrase (IN) is essential for virus replication and represents an important multifunctional therapeutic target. Recently discovered quinoline-based allosteric IN inhibitors (ALLINIs) potently impair HIV-1 replication and are currently in clinical trials. ALLINIs exhibit a multimodal mechanism of action by inducing aberrant IN multimerization during virion morphogenesis and by competing with IN for binding to its cognate cellular cofactor LEDGF/p75 during early steps of HIV-1 infection. However, quinoline-based ALLINIs impose a low genetic barrier for the evolution of resistant phenotypes, which highlights a need for discovery of second-generation inhibitors. Using crystallographic screening of a library of 971 fragments against the HIV-1 IN catalytic core domain (CCD) followed by a fragment expansion approach, we have identified thiophenecarboxylic acid derivatives that bind at the CCD-CCD dimer interface at the principal lens epithelium-derived growth factor (LEDGF)/p75 binding pocket. The most active derivative (5) inhibited LEDGF/p75-dependent HIV-1 IN activity in vitro with an IC50 of 72 μm and impaired HIV-1 infection of T cells at an EC50 of 36 μm. The identified lead compound, with a relatively small molecular weight (221 Da), provides an optimal building block for developing a new class of inhibitors. Furthermore, although structurally distinct thiophenecarboxylic acid derivatives target a similar pocket at the IN dimer interface as the quinoline-based ALLINIs, the lead compound, 5, inhibited IN mutants that confer resistance to quinoline-based compounds. Collectively, our findings provide a plausible path for structure-based development of second-generation ALLINIs

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

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

Molecular Dynamics Free Energy Simulations Reveal the Mechanism for the Antiviral Resistance of the M66I HIV-1 Capsid Mutation

While drug resistance mutations can often be attributed to the loss of direct or solvent-mediated protein−ligand interactions in the drug-mutant complex, in this study we show that a resistance mutation for the picomolar HIV-1 capsid (CA)-targeting antiviral (GS-6207) is mainly due to the free energy cost of the drug-induced protein side chain reorganization in the mutant protein. Among several mutations, M66I causes the most suppression of the GS-6207 antiviral activity (up to ~84,000-fold), and only 83- and 68-fold reductions for PF74 and ZW-1261, respectively. To understand the molecular basis of this drug resistance, we conducted molecular dynamics free energy simulations to study the structures, energetics, and conformational free energy landscapes involved in the inhibitors binding at the interface of two CA monomers. To minimize the protein−ligand steric clash, the I66 side chain in the M66I−GS-6207 complex switches to a higher free energy conformation from the one adopted in the apo M66I. In contrast, the binding of GS-6207 to the wild-type CA does not lead to any significant M66 conformational change. Based on an analysis that decomposes the absolute binding free energy into contributions from two receptor conformational states, it appears that it is the free energy cost of side chain reorganization rather than the reduced protein−ligand interaction that is largely responsible for the drug resistance against GS-6207

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

Stratified UWHAM and Its Stochastic Approximation for Multicanonical Simulations Which Are Far from Equilibrium

We describe a new analysis tool called Stratified unbinned Weighted Histogram Analysis Method (Stratified-UWHAM), which can be used to compute free energies and expectations from a multicanonical ensemble when a subset of the parallel simulations is far from being equilibrated because of barriers between free energy basins which are only rarely (or never) crossed at some states. The Stratified-UWHAM equations can be obtained in the form of UWHAM equations but with an expanded set of states. We also provide a stochastic solver, Stratified RE-SWHAM, for Stratified-UWHAM to remove its computational bottleneck. Stratified-UWHAM and Stratified RE-SWHAM are applied to study three test topics: the free energy landscape of alanine dipeptide, the binding affinity of a host–guest binding complex, and path sampling for a two-dimensional double well potential. The examples show that when some of the parallel simulations are only locally equilibrated, the estimates of free energies and equilibrium distributions provided by the conventional UWHAM (or MBAR) solutions exhibit considerable biases, but the estimates provided by Stratified-UWHAM and Stratified RE-SWHAM agree with the benchmark very well. Lastly, we discuss features of the Stratified-UWHAM approach which is based on coarse-graining in relation to two other maximum likelihood-based methods which were proposed recently, that also coarse-grain the multicanonical data