Mirror-Image Phage Display for the Selection of D-Amino Acid Peptide Ligands as Potential Therapeutics.

In neurodegenerative diseases like Alzheimer's disease (AD), endogenous proteins or peptides aggregate with themselves. These proteins may lose their function or aggregates and/or oligomers can obtain toxicity, causing injury or death to cells. Aggregation of two major proteins characterizes AD. Amyloid-ß peptide (Aß) is deposited in amyloid plaques within the extracellular space of the brain and Tau in so-called neurofibrillary tangles in neurons. Finding peptide ligands to halt protein aggregation is a promising therapeutical approach. Using mirror-image phage display with a commercially available, randomized 12-mer peptide library, we have selected D-amino acid peptides, which bind to the Tau protein and modulate its aggregation in vitro. Peptides can bind specifically and selectively to a target molecule, but natural L-amino acid peptides may have crucial disadvantages for in vivo applications, as they are sensitive to protease degradation and may elicit immune responses. One strategy to circumvent these disadvantages is the use of non-naturally occurring D-amino acid peptides as they exhibit increased protease resistance and generally do not activate the immune system. To perform mirror-image phage display, the target protein needs to be synthesized as D-amino acid version. If the target protein sequence is too long to be synthesized properly, smaller peptides derived from the full length protein can be used for the selection process. This also offers the possibility to influence the binding region of the selected D-peptides in the full-length target protein. Here we provide the protocols for mirror-image phage display selection on the PHF6* peptide of Tau, based on the commercially available Ph.D.™-12 Phage Display Peptide Library Kit, leading to D-peptides that also bind the full length Tau protein (Tau441), next to PHF6*. In addition, we provide protocols and data for the first characterization of those D-peptides that inhibit Tau aggregation in vitro. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Mirror image phage display selection against D-PHF6* fibrils Support Protocol 1: Single phage ELISA Basic Protocol 2: Sequencing and D-peptide generation Basic Protocol 3: Thioflavin-T (ThT) test to control inhibition of Tau aggregation Support Protocol 2: Purification of full-length Tau protein Basic Protocol 4: ELISA to demonstrate the binding of the generated D-peptides to PHF6* and full-length Tau fibrils.

Open-Label Placebo Administration Decreases Pain in Elderly Patients With Symptomatic Knee Osteoarthritis - A Randomized Controlled Trial.

Background: Recent studies indicate that the administration of open-label placebos (OLP) can improve symptoms in various medical conditions. The primary aim of this 3-week randomized controlled trial was to examine the effects of OLP treatments on pain, functional disability, and mobility in patients with arthritic knee pain. Methods: Sixty patients (55% females; mean age, 66.9 ± 9.7 SD years) were randomized to one of two OLP treatments (n = 41) or no treatment (NT; n = 19). OLP treatments were accompanied by the verbal suggestion "to decrease pain" (OLP-pain, n = 20) or "to improve mood" (OLP-mood, n = 21). Pain and mood levels were monitored on 11-point Numeric Rating Scales (NRSs) in a patient diary, and global clinical improvement (CGI-I) was assessed at the end of the study. At baseline and after 21 days, patients filled in validated questionnaires to assess symptoms and functional disability of the knee (WOMAC), mental and physical quality of life (SF-36), state anxiety (STAI-state), perceived stress (PSQ-20), and self-efficacy (GSE). In addition, knee mobility (neutral zero-method), heart rate variability (HRV), and diurnal cortisol levels were evaluated before and after treatment. Results: Evaluation of daily pain ratings indicated significant pain decrease in the OLP groups compared to NT (p = 0.013, d = 0.64), with no difference between the OLP-pain and the OLP-mood groups (p = 0.856, d = 0.05). OLP treatment also improved WOMAC pain (p = 0.036, d = 0.55), again with no difference between the two OLP groups (p = 0.65, d = 0.17). WOMAC function and stiffness, knee mobility, stress, state anxiety, quality of life, and self-efficacy did not change differently between groups. Conclusion: OLP treatment improved knee pain in elderly patients with symptomatic knee osteoarthritis (OA), while functional disability and mobility of the knee did not change. The content of the verbal suggestion was of minor importance. OLP administration may be considered as supportive analgesic treatment in elderly patients with symptomatic knee OA. Trial Registration: German Clinical Trials Register (https://www.drks.de/), DRKS00015191 (retrospectively registered).

Potent Tau Aggregation Inhibitor D-Peptides Selected against Tau-Repeat 2 Using Mirror Image Phage Display.

Alzheimer's disease and other Tauopathies are associated with neurofibrillary tangles composed of Tau protein, as well as toxic Tau oligomers. Therefore, inhibitors of pathological Tau aggregation are potentially useful candidates for future therapies targeting Tauopathies. Two hexapeptides within Tau, designated PHF6* (275-VQIINK-280) and PHF6 (306-VQIVYK-311), are known to promote Tau aggregation. Recently, the PHF6* segment has been described as the more potent driver of Tau aggregation. We therefore employed mirror-image phage display with a large peptide library to identify PHF6* fibril binding peptides consisting of D-enantiomeric amino acids. The suitability of D-enantiomeric peptides for in vivo applications, which are protease stable and less immunogenic than L-peptides, has already been demonstrated. The identified D-enantiomeric peptide MMD3 and its retro-inverso form, designated MMD3rev, inhibited in vitro fibrillization of the PHF6* peptide, the repeat domain of Tau as well as full-length Tau. Dynamic light scattering, pelleting assays and atomic force microscopy demonstrated that MMD3 prevents the formation of tau ß-sheet-rich fibrils by diverting Tau into large amorphous aggregates. NMR data suggest that the D-enantiomeric peptides bound to Tau monomers with rather low affinity, but ELISA (enzyme-linked immunosorbent assay) data demonstrated binding to PHF6* and full length Tau fibrils. In addition, molecular insight into the binding mode of MMD3 to PHF6* fibrils were gained by in silico modelling. The identified PHF6*-targeting peptides were able to penetrate cells. The study establishes PHF6* fibril binding peptides consisting of D-enantiomeric amino acids as potential molecules for therapeutic and diagnostic applications in AD research.

Selection and Characterization of Tau Binding á´ -Enantiomeric Peptides with Potential for Therapy of Alzheimer Disease.

A variety of neurodegenerative disorders, including Alzheimer disease (AD), are associated with neurofibrillary tangles composed of the tau protein, as well as toxic tau oligomers. Inhibitors of pathological tau aggregation, interrupting tau self-assembly, might be useful for the development of therapeutics. Employing mirror image phage display with a large peptide library (over 109 different peptides), we have identified tau fibril binding peptides consisting of d-enantiomeric amino acids. d-enantiomeric peptides are extremely protease stable and not or less immunogenic than l-peptides, and the suitability of d-peptides for in vivo applications have already been demonstrated. Phage display selections were performed using fibrils of the d-enantiomeric hexapeptide VQIVYK, representing residues 306 to 311 of the tau protein, as a target. VQIVYK has been demonstrated to be important for fibril formation of the full lengths protein and forms fibrils by itself. Here, we report on d-enantiomeric peptides, which bind to VQIVYK, tau isoforms like tau3RD (K19) as well as to full lengths tau fibrils, and modulate the aggregation of the respective tau form. The peptides are able to penetrate cells and might be interesting for therapeutic and diagnostic applications in AD research.