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HTS Assay for Discovery of Novel Metallo-Beta-lactamase (MBL) Inhibitors.

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Probe Reports from the NIH Molecular Libraries Program [Internet]. Bethesda (MD): National Center for Biotechnology Information (US); 2010-.
2009 Nov 13 [updated 2011 May 5].

Excerpt

VIM-2 is an Ambler class B metallo-β-lactamase (MBL) capable of hydrolyzing a broad-spectrum of β-lactam antibiotics. Although the discovery and development of MBL inhibitors continue to be an area of active research, an array of potent, small molecule inhibitors is yet to be fully characterized for VIM-2. This report claims CID4870494/SID 24790728 as a potent, selective and non-competitive VIM-2 probe (ML121). This compound was discovered via screening of more than 290,000 MLSMR compounds in a nitrocefin-based enzyme activity assay. Various secondary assays were run on cherry-picked MLSMR samples to determine its potency (VIM-2 IC50= 54 nM) and selectivity (it was inactive in IMP-1 and TEM-1 beta-lactamase assays). Other tests were run to verify it was not an optical artifact of the detection formats used for screening; this testing included assays with two different VIM-2 substrates (nitrocefin and CCF2). Analogs of CID4870494 were less active against VIM-2. A powder sample of CID4870494 was obtained for more detailed studies. Although the potency of the powder sample was slightly less (IC50= 0.223 ± 0.003 μM) than that of the MLSMR cherry-picked liquid stock, CID4870494 remained inactive in IMP-1 and VIM-2 assays. Kinetic analyses demonstrated that it behaves as a non-competitive inhibitor, with a submicromolar Ki (Ki = 148 ± 14 nM). This represents a 10-fold improvement in potency over the prior art probe, Mitoxantrone. It is also important to note that Mitoxantrone’s intense color limits its use in some assay detection formats; as described above, CID4870494 does not appear as an artifact in fluorescence- (CCF2) and absorbance-based (nitrocefin) assays. Further studies employing the TFA salt form of the probe (SID111978109/CID50909789) demonstrate that probe ML121 can potentiate the antibiotic activity of imipenem in VIM-2 transformed E. coli. Thus, these assays reveal the probe ML121 is a novel, selective, and potent VIM-2 inhibitor. This probe will serve as a valuable tool to elucidate the role of VIM-2 in nosocomial beta-lactam antibiotic resistance.

PMID:
21961119
[PubMed]
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