TY  - JOUR
A1  - Scheuplein, Nicolas Julian
A1  - Lohr, Theresa
A1  - Vivoli Vega, Mirella
A1  - Ankrett, Dyan
A1  - Seufert, Florian
A1  - Kirchner, Lukas
A1  - Harmer, Nicholas J.
A1  - Holzgrabe, Ulrike
T1  - Fluorescent probe for the identification of potent inhibitors of the macrophage infectivity potentiator (Mip) protein of Burkholderia pseudomallei
T2  - SLAS Discovery
N2  - Highlights
• Synthesis of a new tracer molecule.
• Robust and easy screening method for a broad range of compound activities.
• FP assay validation considering limited use of starting material, DMSO tolerance, variation in incubation time and temperature.
• Possibility of extension to HTP assay.

Abstract
The macrophage infectivity potentiator (Mip) protein belongs to the immunophilin superfamily. This class of enzymes catalyzes the interconversion between the cis and trans configuration of proline-containing peptide bonds. Mip has been shown to be important for the virulence of a wide range of pathogenic microorganisms, including the Gram-negative bacterium Burkholderia pseudomallei. Small molecules derived from the natural product rapamycin, lacking its immunosuppression-inducing moiety, inhibit Mip's peptidyl-prolyl cis-trans isomerase (PPIase) activity and lead to a reduction in pathogen load in vitro. Here, a fluorescence polarization assay (FPA) to enable the screening and effective development of BpMip inhibitors was established. A fluorescent probe was prepared, derived from previous pipecolic scaffold Mip inhibitors labeled with fluorescein. This probe showed moderate affinity for BpMip and enabled a highly robust FPA suitable for screening large compound libraries with medium- to high-throughput (Z factor ∼ 0.89) to identify potent new inhibitors. The FPA results are consistent with data from the protease-coupled PPIase assay. Analysis of the temperature dependence of the probe's binding highlighted that BpMip's ligand binding is driven by enthalpic rather than entropic effects. This has considerable consequences for the use of low-temperature kinetic assays.
KW  - PPIase
KW  - fluorescence polarization
KW  - anisotropy
KW  - high throughput screening
KW  - Burkholderia pseudomallei Mip
KW  - Mip inhibitor
Y1  - 2023
UR  - https://opus.bibliothek.uni-wuerzburg.de/frontdoor/index/index/docId/34978
UR  - https://nbn-resolving.org/urn:nbn:de:bvb:20-opus-349784
VL  - 28
IS  - 5
ER  -