Simultaneous inhibition assay for human and microbial kinases via MALDI-MS/MS.
Selective inhibition of one kinase over another is a critical issue in drug development. For antimicrobial development, it is particularly important to selectively inhibit bacterial kinases, which can phosphorylate antimicrobial compounds such as aminoglycosides, without affecting human kinases. Previous work from our group showed the development of a MALDI-MS/MS assay for the detection of small molecule modulators of the bacterial aminoglycoside kinase APH3'IIIa. Herein, we demonstrate the development of an enhanced kinase MALDI-MS/MS assay involving simultaneous assaying of two kinase reactions, one for APH3'IIIa, and the other for human protein kinase A (PKA), which leads to an output that provides direct information on selectivity and mechanism of action. Specificity of the respective enzyme substrates were verified, and the assay was validated through generation of Z'-factors of 0.55 for APH3'IIIa with kanamycin and 0.60 for PKA with kemptide. The assay was used to simultaneously screen a kinase-directed library of mixtures of ten compounds each against both enzymes, leading to the identification of selective inhibitors for each enzyme as well as one non-selective inhibitor following mixture deconvolution.
Biointerfaces Institute and Department of Chemistry & Chemical Biology, McMaster University, Hamilton, Ontario, L8S 4L8 (Canada), Homepage: brennanlab.ca; biointerfaces.mcmaster.ca.
Anne Marie E
AM
Smith
John D
JD
Brennan
D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D006801:Humans; D007612:Kanamycin / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D047428:Protein Kinase Inhibitors / Q000737:chemistry / Q000378:metabolism*; D054852:Small Molecule Libraries / Q000737:chemistry / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity
D001426:Bacterial Proteins; D047428:Protein Kinase Inhibitors; D054852:Small Molecule Libraries; D007612:Kanamycin; D017868:Cyclic AMP-Dependent Protein Kinases
MALDI; antibiotic resistance; high throughput screening; kinases; small molecule inhibitors