The amyloid-␤ peptide (A␤) can generate cytotoxic oligomers, and their accumulation is thought to underlie the neuropathologic changes found in Alzheimer's disease. Known inhibitors of A␤ polymerization bind to undefined structures and can work as nonspecific aggregators, and inhibitors that target conformations that also occur in larger A␤ assemblies may even increase oligomerderived toxicity. Here we report on an alternative approach whereby ligands are designed to bind and stabilize the 13-26 region of A␤ in an ␣-helical conformation, inspired by the postulated A␤ native structure. This is achieved with 2 different classes of compounds that also reduce A␤ toxicity to cells in culture and to hippocampal slice preparations, and that do not show any nonspecific aggregatory properties. In addition, when these inhibitors are administered to Drosophila melanogaster expressing human A␤ 1-42 in the central nervous system, a prolonged lifespan, increased locomotor activity, and reduced neurodegeneration is observed. We conclude that stabilization of the central A␤ ␣-helix counteracts polymerization into toxic assemblies and provides a strategy for development of specific inhibitors of A␤ polymerization.amyloid fibrils ͉ neurodegenerative disease ͉ protein misfolding ͉ Alzheimer's disease A lzheimer's disease is a progressive neurodegenerative disorder that is characterized by cerebral extracellular amyloid plaques and intracellular neurofibrillary tangles (1). Classically, the amyloid cascade hypothesis (2) states that Alzheimer's disease is caused by fibril and plaque formation of amyloid-␤ peptide (A␤) in the central nervous system. More recently, the hypothesis has been modified to include A␤ assemblies of sizes intermediate to monomeric and fibrillar forms, which today are considered to be the main source of cytotoxicity (3). Such A␤ assemblies include low-number oligomers and larger assemblies known as protofibrils, globulomers, Alzheimer's disease diffusible ligands, or A␤*56 (4-7). A␤ is cleaved from an integral membrane protein, the amyloid ␤ precursor protein (A␤PP), predominantly as a 40-residue peptide (A␤ 1-40 ). In addition, a C-terminally elongated 42-residue version can be excised (A␤ 1-42 ); it is this longer variant that is the main constituent of parenchymal amyloid deposits (8).The link between A␤ aggregation and Alzheimer's disease implies that inhibitors of this process should be able to slow down disease progression. A number of low-molecular-mass A␤ aggregation inhibitors have been identified by use of screens of compound libraries as well as rational design strategies. The resulting inhibitors include such chemically diverse compounds as curcumin, inositol, and nicotine (9, 10). The screens have identified inhibitors of fibril formation that similarly to the rationally designed inhibitors are predicted to bind to A␤ in an elongated, ␤-strand-like conformation and prevent its polymerization. A potential problem with this strategy is that blocking the later stages of fibril formation will favor t...

Analogues of alpha-factor, Saccharomyces cerevisiae tridecapeptide mating pheromone (H-Trp-His-Trp-Leu-Gln-Leu-Lys-Pro-Gly-Gln-Pro-Met-Tyr-OH), containing p-benzoylphenylalanine (Bpa), a photoactivatable group, and biotin as a tag, were synthesized using solid-phase methodologies on a p-benzyloxybenzyl alcohol polystyrene resin. Bpa was inserted at positions 1, 3, 5, 8, and 13 of alpha-factor to generate a set of cross-linkable analogues spanning the pheromone. The biological activity (growth arrest assay) and binding affinities of all analogues for the alpha-factor receptor (Ste2p) were determined. Two of the analogues that were tested, Bpa(1) and Bpa(5), showed 3-4-fold lower affinity than the alpha-factor, whereas Bpa(3) and Bpa(13) had 7-12-fold lower affinities. Bpa(8) competed poorly with [(3)H]-alpha-factor for Ste2p. All of the analogues tested except Bpa(8) had detectable halos in the growth arrest assay, indicating that these analogues are alpha-factor agonists. Cross-linking studies demonstrated that [Bpa(1)]-alpha-factor, [Bpa(3)]-alpha-factor, [Bpa(5)]-alpha-factor, and [Bpa(13)]-alpha-factor were cross-linked to Ste2p; the biotin tag on the pheromone was detected by a NeutrAvidin-HRP conjugate on Western blots. Digestion of Bpa(1), Bpa(3), and Bpa(13) cross-linked receptors with chemical and enzymatic reagents suggested that the N-terminus of the pheromone interacts with a binding domain consisting of residues from the extracellular ends of TM5-TM7 and portions of EL2 and EL3 close to these TMs and that there is a direct interaction between the position 13 side chain and a region of Ste2p (F55-R58) at the extracellular end of TM1. The results further define the sites of interaction between Ste2p and the alpha-factor, allowing refinement of a model for the pheromone bound to its receptor.

Interaction of CCR5 with the HIV-1 gp120-CD4 complex involves its amino-terminal domain (Nt-CCR5) and requires sulfation of 2-4 tyrosine residues in Nt-CCR5. The conformation of a 27-residue Nt-CCR5 peptide, sulfated at Y10 and Y14, was studied in both its free form and in a ternary complex with deglycosylated-gp120 and a CD4-mimic peptide. NMR experiments revealed a helical conformation at the center of Nt-CCR5(1-27) which is induced upon gp120 binding, as well as a helical propensity for the free peptide. A well-defined structure for the bound peptide was determined for residues 7-23, increasing by two-fold the length of Nt-CCR5's known structure. Two-dimensional saturation transfer experiments and measurement of relaxation-times highlighted Nt-CCR5 residues Y3, V5, P8-T16, E18, I23 and possibly D2 as the main binding determinant. A calculated docking model for Nt-CCR5(1-27) suggests that residues 2-22 of Nt-CCR5 interact with the bases of V3 and C4 while the C-terminal segment of Nt-CCR5(1-27) points towards the target cell membrane reflecting an Nt-CCR5 orientation that differs by 180° from a previous model. A gp120 site that could accommodate CCR5Y3 in a sulfated form has been identified. The present model attributes a structural basis for binding interactions to all gp120 residues previously implicated in Nt-CCR5 binding. Moreover, the strong interaction of sulfated CCR5Tyr14 with gp120Arg440 revealed by the model and the previously found correlation between E322 and R440 mutations shed light on the role of these residues in HIV-1 phenotype conversion furthering our understanding of CCR5 recognition by HIV-1.