1.5 Proteinanalytik
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Organisationseinheit der BAM
Spec2Seq
(2018)
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 Tau4RDΔK (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.
Small organic molecule drugs are one of the key classes, taking increasingly important roles in modern drug development strategies. With the focus on small molecule drugs, difficulties originate frequently from a pronounced lipophilic character, resulting in poor water solubility, low bioavailability and unfavored pharmacokinetics. Recently, peptide-poly(ethylene glycol) conjugates (peptide-PEG conjugates) were described as precisely tunable platforms to solubilize a broad scope of fluorescent or non-fluorescent small organic molecules [1-2]. Selection of drug hosting peptides was achieved by combinatorial means, which can further be extended by implementation of a drug release screening step.
One-bead-one-compound peptide libraries are powerful tools to select high affinity binders. However, the selection of positive hits from the peptide libraries remains tedious as it occurs by handpicking, strongly limiting the pool of investigated beads. Here we report our recent results on improving the analytical platform, using automated fluorescence scanning and MALDI-ToF-ToF MS/MS imaging to screen larger sets of beads, broadening the statistical base and unraveling more precisely suitable peptides. The screening puts special emphasis on loading capacities and drug-release of transporters by performing additional washing steps in different media (cf. Fig. 1). Peptides representing strong, medium, weak releaser were chosen for further analysis and synthesized as peptide-polymer transporters. Release was analyzed by fluorescence anisotropy and fluorescence correlation spectroscopy, due to the fluorescent characteristics of the drug.
Solubilization studies confirmed sufficient loading capacities for a potential anti-Alzheimer disease drug of three transporter molecules representing strong/weak drug releaser, reaching solubilization of up to 1:3.4 (µmol drug/µmol conjugate). Fluorescence anisotropy and fluorescence correlation spectroscopy of the drug-loaded transporter showed significant differences in drug releasing properties, confirming the screening process.