Humboldt-Universität zu Berlin, Department of Chemistry, Laboratory for Organic Synthesis of Functional Systems, Brook-Taylor-Str. 2, 12489 Berlin, Germany; Bundesanstalt für Materialforschung und -prüfung (BAM), Division 1.5 Protein Analysis, Richard-Willstätter-Str. 11, 12489 Berlin, Germany.
Deutsches Zentrum für Neurodegenerative Erkrankungen (DZNE), Sigmund-Freud-Straße 27, 53127 Bonn, Germany; CAESAR Research Institute, 53175 Bonn, Germany.
J Control Release. 2018 Sep 10;285:96-105. doi: 10.1016/j.jconrel.2018.06.032. Epub 2018 Jun 28.
A 2-dimensional high-throughput screening method is presented to select peptide sequences from large peptide libraries for precision formulation additives, having a high capacity to specifically host a drug of interest and provide tailored drug release properties. The identified sequences are conjugated with poly(ethylene glycol) (PEG) to obtain peptide-PEG conjugates that proved to be valuable as solubilizers for small organic molecule drugs to overcome limitations of poor water-solubility and low bio-availability. The 2D-screening method selects peptide sequences on both (i) high loading capacities and (ii) preferred drug-release capabilities as demonstrated on an experimental Tau-protein aggregation inhibitor/Tau- deaggregator with potentials for an anti-Alzheimer disease drug (BB17). To enable 2D-screening, a one-bead one-compound (OBOC) peptide library was immobilized on a glass slide, allocating individual beads to permanent positions. While the first screening step involved incubation of the supported OBOC library with BB17 to identify beads with high drug binding capacities by fluorescence scanner readouts, the second step reveals release properties of the high capacity binders by incubation with blood plasma protein model solutions. Efficiently peptides with high BB17 capacities and either keeper or medium or fast releaser properties can be identified by direct sequence readouts from the glass slide supported resin beads via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Four peptides are synthesized as peptide-PEG solubilizers representing strong, medium, weak releasers and non-binders. Loading capacities reached up to 1:3.4 (mol drug per mol carrier) and release kinetics (fast/medium/slow) are in agreement with the selection process as investigated by fluorescence anisotropy and fluorescence correlation spectroscopy. The ability of BB17/conjugate complexes to inhibit the aggregation of Tau (four repeat Tau ((M)Q244-E372 with deletion of K280), 129 residues) in N2a cells is studied by a Tau-pelleting assay showing the modulation of cellular Tau aggregation. Promising effects such as the reduction of 55% of total Tau load are observed for the strong releaser additive. Studies of in vitro Thioflavin S Tau-aggregation assays show half-maximal inhibitory activities (IC50 values) of BB17/conjugates in the low micro-molar range.
提出了一种二维高通量筛选方法，用于从大型肽文库中选择肽序列作为精密配方添加剂，这些添加剂具有高容量特异性地容纳目标药物，并提供定制的药物释放特性。鉴定出的序列与聚乙二醇（PEG）缀合，得到肽-PEG 缀合物，这些缀合物已被证明是小分子药物的有效增溶剂，可克服水溶性差和生物利用度低的限制。二维筛选方法选择具有（i）高载药量和（ii）优选药物释放能力的肽序列，这在实验性 Tau 蛋白聚集抑制剂/Tau 去聚集剂上得到了证明，该抑制剂具有治疗阿尔茨海默病药物的潜力（BB17）。为了实现二维筛选，将单珠单化合物（OBOC）肽文库固定在玻璃载玻片上，将单个珠子分配到永久位置。虽然第一步筛选涉及将支持 OBOC 文库与 BB17 孵育，以通过荧光扫描仪读数鉴定具有高药物结合能力的珠子，但第二步通过与血浆蛋白模型溶液孵育揭示高容量结合物的释放特性。通过从玻璃载玻片支持的树脂珠上的直接序列读出，可以有效地识别具有高 BB17 容量和保持剂或中速或快速释放剂特性的肽，方法是通过基质辅助激光解吸/电离飞行时间串联质谱法。合成了四种肽作为肽-PEG 增溶剂，代表强、中、弱释放剂和非结合剂。载药量高达 1:3.4（摩尔药物/摩尔载体），释放动力学（快/中/慢）与荧光各向异性和荧光相关光谱研究的选择过程一致。通过 Tau 沉淀测定法研究了 BB17/缀合物复合物抑制 Tau（四重复 Tau（(M)Q244-E372 缺失 K280），129 个残基）在 N2a 细胞中聚集的能力，结果表明细胞 Tau 聚集得到了调节。在 Tau 沉淀测定中观察到强释放剂添加剂的总 Tau 载量降低了 55%，这表明具有有希望的效果。在体外 Thioflavin S Tau 聚集测定中，BB17/缀合物的半数最大抑制活性（IC50 值）在低微摩尔范围内。
