A matrix-assisted laser desorption/ionization tandem mass spectrometry method for direct screening of small molecule mixtures against an aminoglycoside kinase

•Developed a quantitative MALDI-MS/MS method for small molecule screening.•Demonstrated ability to screen mixtures by this method.•Identified several new kinase inhibitors and obtained IC50 data using MALDI.•Demonstrated advantages of MALDI over ESI for such assays.

Aminoglycoside phosphotransferase 3′IIIa (APH3′IIIa) is a bacterial enzyme involved in antibiotic resistance through phosphorylation of aminoglycosides, which can potentially be overcome by co-administration of an APH3′IIIa inhibitor with the antibiotic. Current assay methods for discovery of APH3′IIIa inhibitors suffer from low specificity and high false positive/negative hit rates. Here, we describe a method for screening APH3′IIIa inhibitors based on direct detection of kanamycin A phosphorylation using MALDI-MS/MS, which is more rapid than conventional assays and does not require secondary assays or sample cleanup. The MALDI-MS/MS assay operates at an ionic strength of 45 mM and co-factors can be utilized at near-physiological levels for optimal enzyme activity. Detection via MALDI-MS/MS allowed for improved reproducibility when compared to ESI-MS/MS. Furthermore, the use of MS/MS provided better signal-to-noise ratios relative to MS alone on the MALDI instrument. The assay was validated via generation of Z′-factors, with values of 0.78 and 0.56 in the absence and presence of 0.2% DMSO, respectively. The assay was used to screen a kinase directed library of >200 compounds, assayed as 21 mixtures of 10 compounds each. Five novel synthetic inhibitors were identified following mixture deconvolution. Inhibition constants were obtained for the aforementioned inhibitors using the MALDI-MS/MS assay, revealing several low to mid micromolar “hits”, and highlighting the quantitative nature of the assay.

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