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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