Balancing target flexibility and target denaturation in computational fragment-based inhibitor discovery.

Accounting for target flexibility and selecting "hot spots" most likely to be able to bind an inhibitor continue to be challenges in the field of structure-based drug design, especially in the case of protein-protein interactions. Computational fragment-based approaches using molecular dynamics (MD) simulations are a promising emerging technology having the potential to address both of these challenges. However, the optimal MD conditions permitting sufficient target flexibility while also avoiding fragment-induced target denaturation remain ambiguous. Using one such technology (Site Identification by Ligand Competitive Saturation, SILCS), conditions were identified to either prevent denaturation or identify and exclude trajectories in which subtle but important denaturation was occurring. The target system used was the well-characterized protein cytokine IL-2, which is involved in a protein-protein interface and, in its unliganded crystallographic form, lacks surface pockets that can serve as small-molecule binding sites. Nonetheless, small-molecule inhibitors have previously been discovered that bind to two "cryptic" binding sites that emerge only in the presence of ligand binding, highlighting the important role of IL-2 flexibility. Using the above conditions, SILCS with hydrophobic fragments was able to identify both sites based on favorable fragment binding while avoiding IL-2 denaturation. An important additional finding was that acetonitrile, a water-miscible fragment, fails to identify either site yet can induce target denaturation, highlighting the importance of fragment choice.

CHARMM additive all-atom force field for carbohydrate derivatives and its utility in polysaccharide and carbohydrate-protein modeling.

Monosaccharide derivatives such as xylose, fucose, N-acetylglucosamine (GlcNAc), N-acetylgalactosamine (GlaNAc), glucuronic acid, iduronic acid, and N-acetylneuraminic acid (Neu5Ac) are important components of eukaryotic glycans. The present work details development of force-field parameters for these monosaccharides and their covalent connections to proteins via O-linkages to serine or threonine sidechains and via N-linkages to asparagine sidechains. The force field development protocol was designed to explicitly yield parameters that are compatible with the existing CHARMM additive force field for proteins, nucleic acids, lipids, carbohydrates, and small molecules. Therefore, when combined with previously developed parameters for pyranose and furanose monosaccharides, for glycosidic linkages between monosaccharides, and for proteins, the present set of parameters enables the molecular simulation of a wide variety of biologically-important molecules such as complex carbohydrates and glycoproteins. Parametrization included fitting to quantum mechanical (QM) geometries and conformational energies of model compounds, as well as to QM pair interaction energies and distances of model compounds with water. Parameters were validated in the context of crystals of relevant monosaccharides, as well NMR and/or x-ray crystallographic data on larger systems including oligomeric hyaluronan, sialyl Lewis X, O- and N-linked glycopeptides, and a lectin:sucrose complex. As the validated parameters are an extension of the CHARMM all-atom additive biomolecular force field, they further broaden the types of heterogeneous systems accessible with a consistently-developed force-field model.

Mechanism of binding site conformational switching in the CD44-hyaluronan protein-carbohydrate binding interaction.

The transmembrane protein CD44, which has been implicated in cancer biology and inflammation, mediates cell adhesion through multimeric interactions with the linear extracellular glycosaminoglycan hyaluronan (HA; in megadaltons). Affinity switching of CD44 from a low-affinity state to a high-affinity state is required for normal CD44 physiological function; crystal structures of the CD44 hyaluronan binding domain complexed with HA oligomers point to a conformational rearrangement at a binding site loop, leading to the formation of direct contact between the oligomer and an arginine side chain as a molecular basis for affinity switching. Here, all-atom explicit-solvent molecular dynamics simulations were used to characterize the dynamics and thermodynamics of oligomeric hyaluronan (oHA) and its two crystallographic complexes with the CD44 hyaluronan binding domain: the "A-form," which lacks arginine-HA close contact, and the "B-form," which has direct arginine side-chain-HA contact. From the simulations, the conformational properties of oHA are essentially unaltered in going from the unbound state to either the A-form or the B-form bound state, with the oligomer retaining its flexibility when bound and with only two of the eight monosaccharides in the oligomer maintaining uninterrupted contact with the protein. Biased simulations revealed that altering the backbone conformation of a tyrosine residue in the arginine loop can induce the A-form→B-form conformational transition and that a large free-energy barrier prevents ready interconversion between the two forms, thereby suggesting that the tyrosine backbone forms a molecular switch.