Screening on-bead

An alternative to microarrays, especially in the area of (cyclic) peptides, are one-bead-one-compound (OBOC) libraries. The technology for solid-phase peptide synthesis is well developed. Split-mix synthesis can be assisted by, for example, sorting equipment using radiolabels. All beads can then be screened at once, at least in theory, and active compounds can be detected by, for example, fluorescence [167]. There are three major challenges [168]  

1) Beads containing active compounds need to be sorted from the inactive ones. This can be accomplished with automated bead sorters or by using protein-coated magnetic nanoparticles. 

2) Active compounds need to be structurally identified. For peptides, Edman sequencing can be used. For nonpeptides, the analysis often remains an unsolved problem for large libraries. 

3) As every bead presents multiple copies of a compoimd, the fluorescence signal may not correlate to the actual potency of the structure due to multiple binding. However, this effect can also be utilized to identify low-affinity binders with a polyvalent assay system [169]. 

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On-Bead Screening of Encoded Peptidomimetic and Small Molecule Libraries... 

The second group can be represented by single bead methods, and relies on either bioanalytical methods to select the active compounds or on-bead screening to determine the beads carrying active compounds. It is limited to solid-phase chemistry and does not require chemical steps after library synthesis but does require sophisticated analytical methods to determine the structure of the active compounds. A recent hybrid deconvolution-single beaddecoding method named DRED (dual recursive deconvolution) requires both deconvolutive techniques and sophisticated analytical capacities. 

In the selected example by Lam et al. [101] many peptide libraries were prepared using the mix and split technique and tested in different on-bead screens. Incomplete libraries were tested (the population of most of them was more than a million compounds), and the positive structures were exploited through focused libraries. Some libraries were screened against an anti-insulin monoclonal antibody tagged with alkaline phosphatase, which allowed an enzyme-linked colorimetric detection. Only the beads bound to the murine MAb showed a tourquoise color, while the vast majority remained colorless (details of the technical realization of the assay can be found elsewhere [101, 102]). The chemical structure linked to the positive beads was then easily determined via Edman degradation of the peptide sequences. 

FIGURE 8.13 On-bead screened oligomeric peptide libraries. 

An on-bead screening often produces many active structures, sometimes significantly different, giving a preliminary SAR to orient further efforts. A panel of on-bead assays may allow a complete characterization on many different screens and a more detailed SAR for the molecules composing the library. Nevertheless, major concerns must be considered before thinking of... 

The encoding constructs used require many additional molecules, such as glycine alanine, lysine and so on, which may prove sensitive to some reaction conditions for library synthesis. The physical bulk of the encoding moieties may also hinder the reactivity of some library intermediates, or produce side reactions which decrease the library quality and cannot be detected by the encoding method, or disturb on-bead screening, preventing or hindering... 

An Example On-Bead Screening and Structure Determination of Positives from SH3 Domain-Directed Libraries... 

On-bead screening of L3 produced 32 positive beads, which were rapidly decoded (111). Thirty out of 32 beads contained a specific monomer in position Mi, while position M2 showed a higher tolerance for different monomers. The structures of resynthetized positives 7.18-7.25 with their binding constants are reported in Fig. 7.14. The larger hbrary L4 was tested and decoded using the very same protocol, and, while some sequences were discarded because they were unrelated to any other structure, a few related sequences (7.26-7.28, Fig. 7.15) were determined. The... 

Figure 9.49 On-bead screening and decoding protocol for the inverted SP peptide library of artificial receptors L35.

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