Direct screening of a dynamic combinatorial library using mass spectrometry

Abstract

A dynamic combinatorial library (DCL) screening approach is described that permits direct identification of the effective (from ineffective) combination of building blocks in the equilibrating DCL. The approach uses Fourier transform ion cyclotron resonance mass spectrometry (FTICR MS) together with sustained off-resonance irradiation collision activated dissociation (SORI-CAD) to detect noncovalent protein-DCL ligand complexes under native conditions. It was shown that in a single, rapid experiment one could concurrently identify all the ligands of interest from the DCL against a background of inactive DCL ligands while still in the presence of the target protein. This result has demonstrated that mass spectrometry may provide a fast preliminary screening approach to identify DCL candidates for later verification with more traditional but time-consuming analysis. The MS/MS enables DCL mixtures to be effectively deconvoluted without the need for either chromatography, synthesis of DCL sub-libraries, conversion of the DCL to a static library, or disruption of the protein-ligand complexes before analysis-all typically necessary for the current screening method for DCLs. ( -3]. The benefit of this additional dimension becomes readily evident when dynamic combinatorial libraries (DCLs) are prepared in the presence of a therapeutic target molecule. The reversible chemistry, together with the target acting as a template, permits self-screening owing to selection and amplification of the "best binders" by molecular recognition events between DCL constituents and the target. There are now multiple examples of ligand discovery from dynamic combinatorial libraries (DCLs) generated in the presence of protein targets [4 -20]. In principle, DCC has the potential to circumvent the need to individually synthesize, characterize, and screen each possible library constituent, however rapid detection of the enriched DCL product(s) still poses a serious analytical problem and is a critical limitation to DCC taking a prominent place in drug discovery [21]. The standard DCL screening approach must carry out the screening of identical DCLs twice-with and without the targetand then compare the equilibrium concentration profiles of all library ligands when generated in both the absence and presence (following disruption of the ligand-target complexes) of target protein [4, 7, 10, 14 -17]-the detection of ligand enrichment in the targeted DCL is the basis of identifying the "best binders". Unfortunately, this screening approach typically necessitates synthesis of individual library components to validate chromatographic (e.g., HPLC) or spectral (e.g., NMR) assignments; this is both labor-and time-intensive and has the effect of undermining the promoted advantages of DCC. The complexity of screening with this indirect method also increases with the size of the DCL owing to chromatographic and/or spectral overlap so that the preparation and screening of DCL sub-libraries becomes necessary for deconvolution of larger DCLs. For DCC to have ...

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