Synthesis and evaluation of antineurotoxicity properties of an amyloid-ß peptide targeting ligand containing a triamino acid.

Peptide-like compounds containing an arginine have been shown to bind and stabilize the central helix of the Alzheimer's disease related amyloid-ß peptide (Aß) in an α-helical conformation, thereby delaying its aggregation into cytotoxic species. Here we study a novel Aß targeting ligand AEDabDab containing the triamino acid, N(γ)-(2-aminoethyl)-2,4-diaminobutanoic (AEDab) acid. The new AEDab triamino acid carries an extra positive charge in the side chain and is designed to be incorporated into a ligand AEDabDab where the AEDab replaces an arginine moiety in a previously developed ligand Pep1b. This is done in order to increase the Aß-ligand interaction, and molecular dynamics (MD) simulation of the stability of the Aß central helix in the presence of the AEDabDab ligand shows further stabilization of the helical conformation of Aß compared to the previously reported Pep1b as well as compared to the AEOrnDab ligand containing an N(δ)-(2-aminoethyl)-2,5-diaminopentanoic acid unit which has an additional methylene group. To evaluate the effect of the AEDabDab ligand on the Aß neurotoxicity the AEDab triamino acid building block is synthesized by reductive alkylation of N-protected-glycinal with α-amino-protected diaminobutanoic acid, and the Aß targeting ligand AEDabDab is prepared by solid-phase synthesis starting with attachment of glutarate to the Wang support. Replacement of the arginine residue by the AEDab triamino acid resulted in an improved capability of the ligand to prevent the Aß1-42 induced reduction of gamma (γ) oscillations in hippocampal slice preparation.

Amyloid-ß Peptide Targeting Peptidomimetics for Prevention of Neurotoxicity.

A new generation of ligands designed to interact with the α-helix/ß-strand discordant region of the amyloid-ß peptide (Aß) and to counteract its oligomerization is presented. These ligands are designed to interact with and stabilize the Aß central helix (residues 13-26) in an α-helical conformation with increased interaction by combining properties of several first-generation ligands. The new peptide-like ligands aim at extended hydrophobic and polar contacts across the central part of the Aß, that is, "clamping" the target. Molecular dynamics (MD) simulations of the stability of the Aß central helix in the presence of a set of second-generation ligands were performed and revealed further stabilization of the Aß α-helical conformation, with larger number of polar and nonpolar contacts between ligand and Aß, compared to first-generation ligands. The synthesis of selected novel Aß-targeting ligands was performed in solution via an active ester coupling approach or on solid-phase using an Fmoc chemistry protocol. This included incorporation of aliphatic hydrocarbon moieties, a branched triamino acid with an aliphatic hydrocarbon tail, and an amino acid with a 4'- N, N-dimethylamino-1,8-naphthalimido group in the side chain. The ability of the ligands to reduce Aß1-42 neurotoxicity was evaluated by gamma oscillation experiments in hippocampal slice preparations. The "clamping" second-generation ligands were found to be effective antineurotoxicity agents and strongly prevented the degradation of gamma oscillations by physiological concentration of monomeric Aß1-42 at a stoichiometric ratio.

Effect of mutations on internal dynamics of an RNA hairpin from hepatitis B virus.

Conformational dynamics plays a key role in mediating specific interactions between RNAs and proteins. Flexible parts, such as the loop regions, are often involved in protein binding. Characterization of the factors that influence the flexibility of loop regions will improve our understanding of RNA-protein binding. Here we use molecular dynamics simulations to study the dynamical features of the apical stem-loop of hepatitis B virus and a mutant, with two consensus-based secondary structure mutations (A-U→C-G) in the stem region. The mutations reduce the dynamics of the system and influence the hairpin conformations. The simulations show that inducing rigidity in the stem affects the loop conformational flexibility: the loop residues become less mobile and less accessible to the solvent, and thus less accessible to a possible targeting protein.

Elucidating the Relation between Internal Motions and Dihedral Angles in an RNA Hairpin Using Molecular Dynamics.

Molecular dynamics simulations were performed to characterize the internal motions of the ribonucleic acid apical stem loop of human hepatitis B virus. The NMR relaxation rates calculated directly from the trajectory are in good agreement with the experiment. Calculated order parameters follow the experimental pattern. Order parameters lower than 0.8 are observed for nucleotides that are weakly hydrogen bonded to their base pair partner, unpaired, or part of the loop. These residues show slow decay of the internal correlation functions of their base and sugar C-H vectors. Concerted motions around backbone dihedral angles influence the amplitude of motion of the sugar and base C-H vectors. The order parameters for base C-H vectors are also affected by the fluctuation of the glycosidic dihedral angle.

Implicit Solvent Models and Stabilizing Effects of Mutations and Ligands on the Unfolding of the Amyloid ß-Peptide Central Helix.

We have systematically evaluated the ability of molecular dynamics simulation with implicit solvation models (EEF1.1, SASA, ASPENR, SCPISM, RUSH, ACE2, GBORN, GBSW, GBMV II, FACTS) to characterize the unfolding of the amyloid beta (Aß) peptide and the stabilizing effects of mutations and ligands. The 13-26 region of Aß (Aß13-26) unfolds and leads to the formation of amyloid fibrils, the causative agent of Alzheimer's disease. Stabilization of Aß13-26 decreases Aß polymerization as well as the formation of intermediate structures, which may also be toxic. The unfolding behavior of wild-type Aß13-26 with an increase in temperature led us to select GBORN, GBMV II, and SCPISM for further investigation considering their ability to reproduce the stabilizing effect on the Aß13-26 helical conformation due to mutations (V18A/F19A/F20A and V18L/F19L/F20L) and ligands (Dec-DETA and Pep1b) designed to stabilize Aß13-26. Structural parameters (RMSD, helicity) of the peptide were used to assess the performance of the implicit solvent models with reference to previous explicit solvent simulations.

Influence of spacer-receptor interactions on the stability of bivalent ligand-receptor complexes.

Experiments show that a ligand-receptor complex formed by binding a bivalent ligand (D) in which the two ligating units are joined covalently by a flexible polymeric spacer (S) can be orders of magnitude more stable than the corresponding complex formed with monomeric ligands. Although molecular models rationalizing this "enhancement effect" have been proffered, they ignore spacer-receptor (S-R) interactions, which can substantially influence the relative stability of complexes. Here, the results of a computational study designed to assess the impact of S-R interactions in the prototypic bivalent complex are presented and compared to results of experiments. The S-R interactions mimicking general features of biological systems are modeled by contoured R surfaces with hills (or depressions) at the binding sites. In the fictitious limit of vanishing S-R interactions, the enhancement is pronounced, as observed in experiments. For strictly repulsive S-R interactions (hard R surface), the enhancement vanishes, or even reverses. This is particularly the case if the R surface is convex (i.e., rising between the binding sites), while the enhancement is only moderately reduced if the R surface is concave. Alternatively, a weak S-R attraction close to the R surface can increase the enhancement. It is concluded that large enhancement should be observed only if both features are present: a concave R surface plus a weak S-R attraction. The latter occurs for spacer material such as polyethylene glycol (PEG), which is weakly hydrophobic and thus attracted by protein surfaces. It is shown that the enhancement of bivalent binding can be characterized by a single key parameter, which may also provide guidelines for the design of multivalent complexes with large enhancement effect.

Merging Implicit with Explicit Solvent Simulations: Polyethylene Glycol.

We constructed an accurate polyether force field for implicit solvent (IS) molecular dynamics (MD) simulations that matches local and global conformations of 1,2-dimethoxy-ethane (DME) and polyethylene glycol (PEG), respectively. To make appropriate force field adjustments for IS models of PEG, we used long-term MD simulation data of 1 µs in explicit solvent (ES) based on the most recent CHARMM35 ether force field that includes adjustments for PEG in explicit water. In IS models, competition of attractive van der Waals (vdW) interactions between solute-solute and solute-solvent atom pairs is often not considered explicitly. As a consequence, the attractive vdW interactions between solute atom pairs that remain in IS models explicitly can yield equilibrium structures that are too compact. This behavior was observed in the present study comparing MD simulation data of the DME and PEG ES model with corresponding IS models that use generalized Born (GB) electrostatics combined with positive surface energy terms favoring compact structures. To regain balance of attractive vdW interactions for IS models, we considered the IS generalized Born with simple switching (GBSW) model in detail, where we turned off surface energy terms and reduced attractive vdW interactions to 90%, or we used alternatively even slightly negative surface energies. However, to obtain quantitatively the same local and global distributions of PEG conformers as in ES, we needed additional force field adjustments involving torsion potentials and 1-4 and 1-5 atom pair Coulomb interactions. This CHARMM ether force field, specifically optimized for IS simulation conditions, is equally valid for dimeric and polymeric ethylene glycol. To explore the conformational space of PEG with MD simulations, an IS GBSW model requires 2 orders of magnitude less CPU time than the corresponding ES model. About a factor of 5 of this gain in efficiency is due to the lack of solvent viscosity in IS models.

Bound Ligand Conformer Revealed by Flexible Structure Alignment in Absence of Crystal Structures: Indirect Drug Design Probed for HIV-1 Protease Inhibitors.

In the absence of structural knowledge on the target protein, the bound ligand conformer (BLC) can be constructed approximately by an indirect drug-design approach that uses a set of ligands binding to the same target. Once the bound ligand conformer (BLC) is known, different strategies of drug design can be pursued. The indirect drug-design approach of the present study is based on the assumption that a set of ligands with chemically different architecture binding to the same target protein carry hidden information of their corresponding true BLCs. It is shown how this information can be extracted by pairwise flexible structure alignment (FSA) using molecular dynamics (MD) simulations with attractive intermolecular interactions that derive from the molecular similarity of the ligands and allow the ligands to adopt the same space. The FSA approach is performed with a newly designed module overlap in the experimental CHARMM-29a1, which soon will become publicly available. Combining the conformations obtained from FSA of different ligand pairs yields consensus ligand conformers (CLCs) that should be similar to the BLCs. This procedure was validated on HIV-1 protease (HIV-P), where at present 44 crystal structures with bound ligands of sufficiently diverse chemical composition are available. The FSA approach identifies four different clusters of HIV-P BLCs. These clusters are consistent with the H-bond patterns of the ligands bound to HIV-P in the crystal structures exhibiting four different binding modes. The cluster-specific CLCs are indeed very similar (rmsd ≈ 2 Å) to the corresponding BLCs from the crystal structure, demonstrating the feasibility of the present approach.