Elucidating the Molecular Determinants of the Binding Modes of a Third-Generation HIV-1 Integrase Strand Transfer Inhibitor: The Importance of Side Chain and Solvent Reorganization.

The first- and second-generation clinically used HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) are key components of antiretroviral therapy (ART), which work by blocking the integration step in the HIV-1 replication cycle that is catalyzed by a nucleoprotein assembly called an intasome. However, resistance to even the latest clinically used INSTIs is beginning to emerge. Developmental third-generation INSTIs, based on naphthyridine scaffolds, are promising candidates to combat drug-resistant viral variants. Among these novel INSTIs, compound 4f exhibits two distinct conformations when binding with intasomes from HIV-1 and the closely related prototype foamy virus (PFV) despite the high structural similarity of their INSTI binding pockets. The molecular mechanism and the key active site residues responsible for these differing binding modes in closely related intasomes remain elusive. To unravel the molecular determinants governing the two distinct binding modes, we applied a novel molecular dynamics-based free energy method that utilizes alchemical pathways to overcome the sampling challenges associated with transitioning between the two bound conformations of ligand 4f within the crowded environments of the INSTI binding pockets in these intasomes. The calculated conformational free energies successfully recapitulate the experimentally observed binding mode preferences in the two viral intasomes. Analysis of the simulated structures suggests that the observed binding mode preferences are caused by amino acid residue differences in both the front and the central catalytic sub-pocket of the INSTI binding site in HIV-1 and PFV. Additional free energy calculations on mutants of HIV-1 and PFV revealed that while both sub-pockets contribute to binding mode selection, the central sub-pocket plays a more important role. These results highlight the importance of both side chain and solvent reorganization, as well as the conformational entropy in determining the ligand binding mode, and will help inform the development of more effective INSTIs for combatting drug-resistant viral variants.

Elucidating the molecular determinants for binding modes of a third-generation HIV-1 integrase strand transfer inhibitor: Importance of side chain and solvent reorganization.

The first and second-generation clinically used HIV-1 integrase (IN) strand transfer inhibitors (INSTIs) are key components of antiretroviral therapy (ART), which work by blocking the integration step in the HIV-1 replication cycle that is catalyzed by a nucleoprotein assembly called an intasome. However, resistance to even the latest clinically used INSTIs is beginning to emerge. Developmental third-generation INSTIs, based on naphthyridine scaffold, are promising candidates to combat drug-resistant viral variants. Among these novel INSTIs, compound 4f exhibits two distinct conformations when binding to intasomes from HIV-1 and the closely related prototype foamy virus (PFV), despite the high structural similarity of their INSTI binding pockets. The molecular mechanism and the key active site residues responsible for these differing binding modes in closely related intasomes remain elusive. To unravel the molecular determinants governing the two distinct binding modes, we employ a novel molecular dynamics-based free energy approach that utilizes alchemical pathways to overcome the sampling challenges associated with transitioning between two ligand conformations within crowded environments along physical pathways. The calculated conformational free energies successfully recapitulate the experimentally observed binding mode preferences in the two viral intasomes. Analysis of the simulated structures suggests that the observed binding mode preferences are caused by amino acid residue differences in both the front and the central catalytic sub-pocket of the INSTI binding site in HIV-1 and PFV. Additional free energy calculations on mutants of HIV-1 and PFV revealed that while both sub-pockets contribute to the binding mode selection, the central sub-pocket plays a more important role. These results highlight the importance of both side chain and solvent reorganization, as well as the conformational entropy in determining the ligand binding mode and will help inform the development of more effective INSTIs for combatting drug-resistant viral variants.

Mechanisms of HIV-1 integrase resistance to dolutegravir and potent inhibition of drug-resistant variants.

HIV-1 infection depends on the integration of viral DNA into host chromatin. Integration is mediated by the viral enzyme integrase and is blocked by integrase strand transfer inhibitors (INSTIs), first-line antiretroviral therapeutics widely used in the clinic. Resistance to even the best INSTIs is a problem, and the mechanisms of resistance are poorly understood. Here, we analyze combinations of the mutations E138K, G140A/S, and Q148H/K/R, which confer resistance to INSTIs. The investigational drug 4d more effectively inhibited the mutants compared with the approved drug Dolutegravir (DTG). We present 11 new cryo-EM structures of drug-resistant HIV-1 intasomes bound to DTG or 4d, with better than 3-Å resolution. These structures, complemented with free energy simulations, virology, and enzymology, explain the mechanisms of DTG resistance involving E138K + G140A/S + Q148H/K/R and show why 4d maintains potency better than DTG. These data establish a foundation for further development of INSTIs that potently inhibit resistant forms in integrase.

Structure-based virtual screening workflow to identify antivirals targeting HIV-1 capsid.

We have identified novel HIV-1 capsid inhibitors targeting the PF74 binding site. Acting as the building block of the HIV-1 capsid core, the HIV-1 capsid protein plays an important role in the viral life cycle and is an attractive target for antiviral development. A structure-based virtual screening workflow for hit identification was employed, which includes docking 1.6 million commercially-available drug-like compounds from the ZINC database to the capsid dimer, followed by applying two absolute binding free energy (ABFE) filters on the 500 top-ranked molecules from docking. The first employs the Binding Energy Distribution Analysis Method (BEDAM) in implicit solvent. The top-ranked compounds are then refined using the Double Decoupling method in explicit solvent. Both docking and BEDAM refinement were carried out on the IBM World Community Grid as part of the FightAIDS@Home project. Using this virtual screening workflow, we identified 24 molecules with calculated binding free energies between - 6 and - 12 kcal/mol. We performed thermal shift assays on these molecules to examine their potential effects on the stability of HIV-1 capsid hexamer and found that two compounds, ZINC520357473 and ZINC4119064 increased the melting point of the latter by 14.8 °C and 33 °C, respectively. These results support the conclusion that the two ZINC compounds are primary hits targeting the capsid dimer interface. Our simulations also suggest that the two hit molecules may bind at the capsid dimer interface by occupying a new sub-pocket that has not been exploited by existing CA inhibitors. The possible causes for why other top-scored compounds suggested by ABFE filters failed to show measurable activity are discussed.

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.

Computational design of small molecular modulators of protein-protein interactions with a novel thermodynamic cycle: Allosteric inhibitors of HIV-1 integrase.

Targeting protein-protein interactions for therapeutic development involves designing small molecules to either disrupt or enhance a known PPI. For this purpose, it is necessary to compute reliably the effect of chemical modifications of small molecules on the protein-protein association free energy. Here we present results obtained using a novel thermodynamic free energy cycle, for the rational design of allosteric inhibitors of HIV-1 integrase (ALLINI) that act specifically in the early stage of the infection cycle. The new compounds can serve as molecular probes to dissect the multifunctional mechanisms of ALLINIs to inform the discovery of new allosteric inhibitors. The free energy protocol developed here can be more broadly applied to study quantitatively the effects of small molecules on modulating the strengths of protein-protein interactions.

Molecular Simulations of Hydrogen Bond Cluster Size and Reorientation Dynamics in Liquid and Glassy Azole Systems.

We simulated the dynamics of azole groups (pyrazole, imidazole, 1,2,3-triazole, 1,2,4-triazole, and tetrazole) as neat liquids and tethered via linkers to aliphatic backbones to determine how tethering and varying functional groups affect hydrogen bond networks and reorientation dynamics, both factors which are thought to influence proton conduction. We used the DL_Poly_2 molecular dynamics code with the GAFF force field to simulate tethered systems over the temperature range 200-900 K and the corresponding neat liquids under liquid state temperatures at standard pressure. We computed hydrogen bond cluster sizes; orientational order parameters; orientational correlation functions associated with functional groups, linkers, and backbones; time scales; and activation energies associated with orientational randomization. All tethered systems exhibit a liquid to glassy-solid transition upon cooling from 600 to 500 K, as evidenced by orientational order parameters and correlation functions. Tethering the azoles was generally found to produce hydrogen bond cluster sizes similar to those in untethered liquids and hydrogen bond lifetimes longer than those in liquids. The simulated rates of functional group reorientation decreased dramatically upon tethering. The activation energies associated with orientational randomization agree well with NMR data for tethered imidazole systems at lower temperatures and for tethered 1,2,3-triazole systems at both low- and high-temperature ranges. Overall, our simulations corroborate the notion that tethering functional groups dramatically slows the process of reorientation. We found a linear correlation between gas-phase hydrogen bond energies and tethered functional group reorientation barriers for all azoles except for imidazole, which acts as an outlier because of both atomic charges and molecular structure.

Phosphorus removal from aqueous solutions using a synthesized adsorbent prepared from mineralized refuse and sewage sludge.

Mineralized refuse and sewage sludge generated from solid waste from municipal landfills and sewage treatment plants were sintered as a cost-effective adsorbent for the removal of phosphorus. Compared with the Freundlich model, phosphorus adsorption on the synthesized adsorbent, zeolite and ironstone was best described by the Langmuir model. Based on the Langmuir model, the maximum adsorption capacity of the synthesized adsorbent (9718 mg kg(-1)) was 13.7 and 25.4 times greater than those of zeolite and ironstone, respectively. The desorbability of phosphorus from the synthesized adsorbent was significantly lower than that of zeolite. Moreover, phosphorus removal using the synthesized adsorbent was more tolerant to pH fluctuations than zeolite and ironstone for the removal of phosphorus from aqueous solutions. The immobilization of phosphorus onto the synthesized adsorbent was attributed to the formation of insoluble calcium, aluminium and iron phosphorus. The heavy metal ion concentrations of the leachate of the synthesized adsorbent were negligible. The synthesized adsorbent prepared from mineralized refuse and sewage sludge was cost-effective and possessed a high adsorptive capacity for phosphorus removal from aqueous solutions.

Effects of nitrogen conversion and environmental factors on landfill CH4 oxidation and N2O emissions in aged refuse.

We determined the effects of nitrification capacity and environmental factors on landfill methane oxidation potential (MOP) using an aged refuse in laboratory batch assays and compared it with two different types of soils. The nitrogen conversion in the three experimental materials after 120 h incubation yielded first-order reaction kinetics at an initial concentration of 200 mg kg(-1) NH4(+)-N. The net nitrification rate for the aged refuse was 1.50 (p < 0.05) and 2.08 (p < 0.05) times that of the clay soil and the sandy soil, respectively. The net NO3(-)-N generation rate by the aged refuse was 1.93 (p < 0.05) and 2.57 (p < 0.05) times that of the clay soil and the sandy soil, respectively. When facilitated by ammonia-oxidizing bacteria during CH4 co-oxidation, the average value of the MOP in the aged refuse at a temperature range of 4-45 °C was 2.34 (p < 0.01) and 4.71 (p < 0.05) times greater than that of the clay soil and the sandy soil, respectively. When the moisture content ranged from 8 to 32% by mass, the average values for the MOP in the aged refuse were 2.08 (p < 0.01) and 3.15 (p < 0.01) times greater than that of the clay soil and the sandy soil, respectively. The N2O fluxes in the aged refuse at 32% moisture content were 5.33 (p < 0.05) and 12.00 (p < 0.05) times more than in the clay and the sandy soil, respectively. The increase in N2O emissions from a municipal solid waste landfill can be neglected after applying an aged refuse bio-cover because of the much higher MOP in the aged refuse. The calculated maximum MOP value in the aged refuse was 12.45 µmol g(-1) d.w. h(-1), which was much higher than the documented data.

Structural heterogeneity in the collision complex between organic dyes and tryptophan in aqueous solution.

The heterogeneity on photoinduced electron transfer (PET) kinetics between a labeled fluorophore and an amino acid residue has been extensively studied in biopolymers. However in aqueous solutions, the heterogeneity on PET kinetics between a fluorophore and a quencher has rarely been reported. Herein, we selected four commonly used fluorophores, such as tetramethylrhodamine (TMR), Rhodamine B (RhB), Alexa fluor 546 (Alexa546), and Atto655, and studied their respective PET kinetics in 50 mM tryptophan solutions with femtosecond transient absorption spectroscopy to explore the structural heterogeneity in their corresponding collision complexes. We measured the decay of the first excited electronic state of respective fluorophore with and without 50 mM tryptophan in aqueous solutions, and derived the charge separation rate in their corresponding collision complexes. We found that the PET process of all selected fluorophores in 50 mM tryptophan solutions has two charge separation rates, which indicates that the relevant states in the collision complex between respective fluorophore and tryptophan have strong structural heterogeneity. These femtosecond PET measurements are in agreement with Vaiana's molecular dynamics simulation (J. Am. Chem. Soc.2003, 125, 14564). In addition, with the obtained PET kinetic parameters, we derived the relative brightness of the collision complex between respective fluorophore and tryptophan, which are important parameters for the PET based fluorescence correlation spectroscopy study involving these fluorophores in biopolymers.