We present the first receptor-based pharmacophore model for HIV-1 integrase. The development of "dynamic" pharmacophore models is a new method that accounts for the inherent flexibility of the active site and aims to reduce the entropic penalties associated with binding a ligand. Furthermore, this new drug discovery method overcomes the limitation of an incomplete crystal structure of the target protein. A molecular dynamics (MD) simulation describes the flexibility of the uncomplexed protein. Many conformational models of the protein are saved from the MD simulations and used in a series of multi-unit search for interacting conformers (MUSIC) simulations. MUSIC is a multiple-copy minimization method, available in the BOSS program; it is used to determine binding regions for probe molecules containing functional groups that complement the active site. All protein conformations from the MD are overlaid, and conserved binding regions for the probe molecules are identified. Those conserved binding regions define the dynamic pharmacophore model. Here, the dynamic model is compared to known inhibitors of the integrase as well as a three-point, ligand-based pharmacophore model from the literature. Also, a "static" pharmacophore model was determined in the standard fashion, using a single crystal structure. Inhibitors thought to bind in the active site of HIV-1 integrase fit the dynamic model but not the static model. Finally, we have identified a set of compounds from the Available Chemicals Directory that fit the dynamic pharmacophore model, and experimental testing of the compounds has confirmed several new inhibitors.

Development of the new generation of therapeutics against the influenza viral coat protein neuraminidase is a response to the continuing threat of influenza epidemics. A variety of structurally similar compounds have been reported that vary greatly in their ability to inhibit neuraminidase, a critical enzyme that cleaves sialic acid and promotes virion release. To determine how neuraminidase exhibits this wide range of affinities with structurally similar compounds, molecular dynamic simulations, coupled with free energy calculations, were used to determine the binding components of a series of neuraminidase inhibitors. Using four cocrystal structures of neuraminidase-inhibitor complexes, we examined the structural and energetic components of ligand potency and selectivity. An in-depth energetic analysis, including internal energy, entropy, and nonbonded interactions, reveals that potency of ligand binding is governed by nonpolar contacts. Electrostatic components generally oppose binding, although two of the best inhibitors use electrostatic interactions to orient the ligand. This investigation suggests that the enhanced selectivity and potency of the better ligands may arise from an improved positioning of their ligand atoms in the active site due to polar and hydrophobic functionalities. Simulations that included crystal water molecules in the active site indicate that the more potent ligands make less use of water-mediated interactions.

We have recently presented a new pharmacophore design method that allows for the incorporation of the inherent flexibility of a target active site. The flexibility of the enzymatic system is described by collecting many conformations of the uncomplexed protein; this ensemble of conformational states can come from a molecular dynamics (MD) simulation, multiple crystal structures, or many NMR structures. Binding sites for functional groups that complement the active site are determined through multiple-copy calculations. These calculations are conducted for each protein conformation, providing a large collection of potential binding sites. The Cartesian coordinates from each protein conformation are overlaid through RMS fitting of essential catalytic residues, and the pharmacophore model is described by binding regions that are conserved over many protein conformations. Previously, we developed a "dynamic" pharmacophore model for HIV-1 integrase using 11 conformations of the protein from an MD simulation; the MUSIC procedure was used to calculate binding positions for methanol molecules in each configuration of the active site. Here we present "static" pharmacophore models developed with a single conformation of the protein from two new crystal structures (standard protocol for multiple-copy methods). The static models do not perform as well as the previous dynamic model in fitting known inhibitors of HIV-1 integrase. To test the applicability of the dynamic pharmacophore method and the assumption that any reliable source of protein conformations is applicable, we have now developed a second dynamic pharmacophore model based on the two crystal structures also used for the development of the static models. Though the dynamic model based on the two crystal structures does not fit as many known inhibitors as the previous dynamic model, it is a significant improvement over the static models. Even better performance is expected with the addition of new crystal structures as they become available. However, it is notable that using only two structures leads to great improvement in the models.

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