Combinatorial libraries – from solution to 2D microarrays

Abstract

Enzymatic modifications of split and mix libraries were followed by "pulling down" onto a 2-dimensional DNA microarray, via PNA tagging; this allowed complete library interrogation of all members of the split and mix library.

Probing Biology with Small Molecule Microarrays (SMM)

“…12 Profiling substrate selectivity of protease using fluorogenic substrates also sufficiently sensitive to measure differences in proteolytic activity between apoptotic and healthy cells as well as between sera from patient on anticoagulation therapy and healthy patients [55]. Conversely, it was also shown in a proof of principle that the activity of pepsin could be detected from a PNA-encoded library of substrates using dabcyl as an internal quencher and fluorescein [58]. More recently, PNA-encoded substrate containing fluorescent probes was used to perform β-secretase assay, a protease involved in Alzheimer's disease.…”

“…5). This PNA-encoded strategy has been validated with the discovery of inhibitors from libraries of > 1000 compounds and the profiles of substrate specificity and enzymatic activity from complex proteomic mixtures [54][55][56][57][58]. Starting from a bifunctional linker with orthogonal protecting groups, a library is prepared by split and mix combinatorial syntheses where a specific PNA codon is used to encode every building block.…”

“…As for kinases, there is widespread interest in methods that define the preferred substrate of a protease and in being able to correlate their activity to the cellular state. Three detection methods have been developed based on irreversible inhibitors that selectively label active proteases [52], fluorogenic substrates [55,83], and substrates flanked by two FRETing fluorophores [58].…”

“…Two different detection approaches have generally been used, the first makes use of [γ -32/33 P]-ATP to label the immobilized substrate with a radioactive phosphate, which can be detected by autoradiography, phosphor imager, or silver staining [9,13,[75][76][77][78][79]. The second detection method relies on phosphospecific antibodies, which are fluorescently labeled [13,51,58,76,80]. While the fluorescent detection may be preferable as it avoids working with radioactive ATP, it has been shown that only monoclonal anti-phosphotyrosine antibodies showed reliable results [76].…”

Discovery of Inhibitors of Protein-Protein Interactions from Combinatorial Libraries

“…These offer a strategy to isolate peptide ligands to target proteins and to define interaction sites between proteins [79]. In phage displayed libraries, instead of using inter-convertible building blocks as it is done in dynamic libraries, possible Both, solid-phase and solution-phase strategies can be easily automated, however, the major limitation to solution-phase is the isolation and purification of the library compounds [80,81]. To date, most of the isolation/purification steps are based on acidbase chemistry and sequestration-enabling reagents [82][83][84].…”