Assay Platform
for Clinically Relevant Metallo-β-lactamases
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Abstract
Metallo-β-lactamases
(MBLs) are a growing threat to the use
of almost all clinically used β-lactam antibiotics. The identification
of broad-spectrum MBL inhibitors is hampered by the lack of a suitable
screening platform, consisting of appropriate substrates and a set
of clinically relevant MBLs. We report procedures for the preparation
of a set of clinically relevant metallo-β-lactamases (i.e.,
NDM-1 (New Delhi MBL), IMP-1 (Imipenemase), SPM-1 (São Paulo
MBL), and VIM-2 (Verona integron-encoded MBL)) and the identification
of suitable fluorogenic substrates (umbelliferone-derived cephalosporins).
The fluorogenic substrates were compared to chromogenic substrates
(CENTA, nitrocefin, and imipenem), showing improved sensitivity and
kinetic parameters. The efficiency of the fluorogenic substrates was
exemplified by inhibitor screening, identifying 4-chloroisoquinolinols
as potential pan MBL inhibitors
