The rational design of amyloid oligomer inhibitors is yet an unmet drug development need. Previous studies have identified the role of tryptophan in amyloid recognition, association and inhibition. Furthermore, tryptophan was ranked as the residue with highest amyloidogenic propensity. Other studies have demonstrated that quinones, specifically anthraquinones, can serve as aggregation inhibitors probably due to the dipole interaction of the quinonic ring with aromatic recognition sites within the amyloidogenic proteins. Here, using in vitro, in vivo and in silico tools we describe the synthesis and functional characterization of a rationally designed inhibitor of the Alzheimer's disease-associated β-amyloid. This compound, 1,4-naphthoquinon-2-yl-L-tryptophan (NQTrp), combines the recognition capacities of both quinone and tryptophan moieties and completely inhibited Aβ oligomerization and fibrillization, as well as the cytotoxic effect of Aβ oligomers towards cultured neuronal cell line. Furthermore, when fed to transgenic Alzheimer's disease Drosophila model it prolonged their life span and completely abolished their defective locomotion. Analysis of the brains of these flies showed a significant reduction in oligomeric species of Aβ while immuno-staining of the 3rd instar larval brains showed a significant reduction in Aβ accumulation. Computational studies, as well as NMR and CD spectroscopy provide mechanistic insight into the activity of the compound which is most likely mediated by clamping of the aromatic recognition interface in the central segment of Aβ. Our results demonstrate that interfering with the aromatic core of amyloidogenic peptides is a promising approach for inhibiting various pathogenic species associated with amyloidogenic diseases. The compound NQTrp can serve as a lead for developing a new class of disease modifying drugs for Alzheimer's disease.

Amyloid aggregation is linked to a number of neurodegenerative syndromes, the most prevalent one being Alzheimer's disease. In this pathology, the b-amyloid peptides (Ab) aggregate into oligomers, protofibrils, and fibrils and eventually into plaques, which constitute the characteristic hallmark of Alzheimer's disease. Several low-molecular-weight compounds able to impair the Ab aggregation process have been recently discovered; yet, a detailed description of their interactions with oligomers and fibrils is hitherto missing. Here, molecular dynamics simulations are used to investigate the influence of two relatively similar tricyclic, planar compounds, that is, 9, 10-anthraquinone (AQ) and anthracene (AC), on the early phase of the aggregation of the Ab heptapeptide segment H 14 QKLVFF 20 , the hydrophobic stretch that promotes the Ab self-assembly. The simulations show that AQ interferes with b-sheet formation more than AC. In particular, AQ intercalates into the b-sheet because polar interactions between the compound and the peptide backbone destabilize the interstrand hydrogen bonds, thereby favoring disorder. The thioflavin T-binding assay indicates that AQ, but not AC, sensibly reduces the amount of aggregated Ab 1-40 peptide. Taken together, the in silico and in vitro results provide evidence that structural perturbations by AQ can remarkably affect ordered oligomerization. Moreover, the simulations shed light at the atomic level on the interactions between AQ and Ab oligomers, providing useful insights for the design of small-molecule inhibitors of aggregation with therapeutic potential in Alzheimer's disease.

Aggregation of the amyloid-β peptide (Aβ) into fibrillar structures is a hallmark of Alzheimer's disease. Thus, preventing self-assembly of the Aβ peptide is an attractive therapeutic strategy. Here, we used experimental techniques and atomistic simulations to investigate the influence of carnosine, a dipeptide naturally occurring in the brain, on Aβ aggregation. Scanning force microscopy, circular dichroism and thioflavin T fluorescence experiments showed that carnosine does not modify the conformational features of Aβ42 but nonetheless inhibits amyloid growth. Molecular dynamics (MD) simulations indicated that carnosine interacts transiently with monomeric Aβ42 by salt bridges with charged side chains, and van der Waals contacts with residues in and around the central hydrophobic cluster ((17)LVFFA(21)). NMR experiments on the nonaggregative fragment Aβ12-28 did not evidence specific intermolecular interactions between the peptide and carnosine, in agreement with MD simulations. However, a close inspection of the spectra revealed that carnosine interferes with the local propensity of the peptide to form backbone hydrogen bonds close to the central hydrophobic cluster (residues E22, S26 and N27). Finally, MD simulations of aggregation-prone Aβ heptapeptide segments show that carnosine reduces the propensity to form intermolecular backbone hydrogen bonds in the region 18-24. Taken together, the experimental and simulation results (cumulative MD sampling of 0.2 ms) suggest that, despite the inability of carnosine to form stable contacts with Aβ, it might block the pathway toward toxic aggregates by perturbing the hydrogen bond network near residues with key roles in fibrillogenesis.

SUMMARY Increasing evidence suggests that extracellular miRNAs may serve as biomarkers of diseases, but the physiological relevance of extracellular miRNA is unclear. We find that intradermal cheek injection of miR-711 induces TRPA1-depedent itch (scratching) without pain (wiping) in naïve mice. Extracellular perfusion of miR-711 induces TRPA1 currents in both Trpa1-expressing heterologous cells and native sensory neurons through the core sequence GGGACCC. Computer simulations reveal that the core sequence binds several residues at the extracellular S5-S6 loop of TRPA1, which are critical for TRPA1 activation by miR-711 but not allyl isothiocyanate. Intradermal inoculation of human Myla cells induces lymphoma and chronic itch in immune- deficient mice, associated with increased serum levels of miR-711, secreted from cancer cells. Lymphoma-induced chronic itch is suppressed by miR-711 inhibitor and a blocking peptide that disrupts the miR-711/TRPA1 interaction. Our findings demonstrated an unconventional physiological role of extracellular naked miRNAs as itch mediators and ion channel modulators.

Background: Inhibition of pathogenic protein aggregation by small molecules is poorly understood. Results: Aggregation inhibitors alter the free energy landscape of a relevant fragment of Alzheimer amyloid-␤ peptide in subtle but complex fashion. Conclusion: Intrinsic disorder of disease proteins persists at the level of binding small molecules. Significance: Hallmark characteristics and efficacy of inhibitors can be reconciled with lack of specificity.

Errors in protein folding may result in premature clearance of structurally aberrant proteins, or in the accumulation of toxic misfolded species or protein aggregates. These pathological events lead to a large range of conditions known as conformational diseases. Several research groups have presented possible therapeutic solutions for their treatment by developing novel compounds, known as pharmacological chaperones. These cell-permeable molecules selectively provide a molecular scaffold around which misfolded proteins can recover their native folding and, thus, their biological activities. Here, we review therapeutic strategies, clinical potentials, and cost-benefit impacts of several classes of pharmacological chaperones for the treatment of a series of conformational diseases.

The primary molecular target for clinically used opioids is the μ-opioid receptor (MOR). Besides the major seven-transmembrane (7TM) receptors, the MOR gene codes for alternatively spliced six-transmembrane (6TM) isoforms, the biological and clinical significance of which remains unclear. Here, we show that the otherwise exclusively intracellular localized 6TM-MOR translocates to the plasma membrane upon coexpression with β2-adrenergic receptors (β2-ARs) through an interaction with the fifth and sixth helices of β2-AR. Coexpression of the two receptors in BE(2)-C neuroblastoma cells potentiates calcium responses to a 6TM-MOR ligand, and this calcium response is completely blocked by a selective β2-antagonist in BE(2)-C cells, and in trigeminal and dorsal root ganglia. Co-administration of 6TM-MOR and β2-AR ligands leads to substantial analgesic synergy and completely reverses opioid-induced hyperalgesia in rodent behavioral models. Together, our results provide evidence that the heterodimerization of 6TM-MOR with β2-AR underlies a molecular mechanism for 6TM cellular signaling, presenting a unique functional responses to opioids. This signaling pathway may contribute to the hyperalgesic effects of opioids that can be efficiently blocked by β2-AR antagonists, providing a new avenue for opioid therapy.

An intense effort is made by pharmaceutical and academic research laboratories to identify and develop selective antagonists for each adenosine receptor (AR) subtype as potential clinical candidates for "soft" treatment of various diseases. Crystal structures of subtypes A and AARs offer exciting opportunities for structure-based drug design. In the first part of the present work, Maybridge HitFinder library of 14400 compounds was utilized to apply a combination of structure-based against the crystal structure of AAR and ligand-based methodologies. The docking poses were rescored by CHARMM energy minimization and calculation of the desolvation energy using Poisson-Boltzmann equation electrostatics. Out of the eight selected and tested compounds, five were found positive hits (63% success). Although the project was initially focused on targeting AAR, the identified antagonists exhibited low micromolar or micromolar affinity against A/A, ARs, or AAR, respectively. Based on these results, 19 compounds characterized by novel chemotypes were purchased and tested. Sixteen of them were identified as AR antagonists with affinity toward combinations of the AR family isoforms (A/A, A/A, A/A/A, and A). The second part of this work involves the performance of hundreds of molecular dynamics (MD) simulations of complexes between the ARs and a total of 27 ligands to resolve the binding interactions of the active compounds, which were not achieved by docking calculations alone. This computational work allowed the prediction of stable and unstable complexes which agree with the experimental results of potent and inactive compounds, respectively. Of particular interest is that the 2-amino-thiophene-3-carboxamides, 3-acylamino-5-aryl-thiophene-2-carboxamides, and carbonyloxycarboximidamide derivatives were found to be selective and possess a micromolar to low micromolar affinity for the A receptor.