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Elucidating the Origin of Long Residence Time Binding for Inhibitors of the Metalloprotease Thermolysin

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Institute of Pharmaceutical Chemistry, University of Marburg, Marbacher Weg 6, 35032 Marburg, Germany
GE Healthcare Bio-Sciences AB, SE-751 84 Uppsala, Sweden
*Phone: +49 6421 28 21313. E-mail: [email protected]
Cite this: ACS Chem. Biol. 2017, 12, 1, 225–233
Publication Date (Web):November 29, 2016
Copyright © 2016 American Chemical Society

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    Kinetic parameters of protein–ligand interactions are progressively acknowledged as valuable information for rational drug discovery. However, a targeted optimization of binding kinetics is not easy to achieve, and further systematic studies are necessary to increase the understanding about molecular mechanisms involved. We determined association and dissociation rate constants for 17 inhibitors of the metalloprotease thermolysin by surface plasmon resonance spectroscopy and correlated kinetic data with high-resolution crystal structures in complex with the protein. From the structure–kinetics relationship, we conclude that the strength of interaction with Asn112 correlates with the rate-limiting step of dissociation. This residue is located at the beginning of a β-strand motif that lines the binding cleft and is commonly believed to align a substrate for catalysis. A reduced mobility of the Asn112 side chain owing to an enhanced engagement in charge-assisted hydrogen bonds prevents the conformational adjustment associated with ligand release and transformation of the enzyme to its open state. This hypothesis is supported by kinetic data of ZFPLA, a known pseudopeptidic inhibitor of thermolysin, which blocks the conformational transition of Asn112. Interference with this retrograde induced-fit mechanism results in variation of the residence time of thermolysin inhibitors by a factor of 74 000. The high conservation of this structural motif within the M4 and M13 metalloprotease families underpins the importance of this feature and has significant implications for drug discovery.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acschembio.6b00979.

    • SPR measurements, experimental data from SPR measurements, experimental data from SPR measurements at varying ionic strength conditions, sensograms from SPR measurements, comparison of kinetic data from SPR and photometric inhibition assay, crystallographic tables, ligand synthesis and purification, and sequence alignment of representative M4 and M13 proteases (PDF)

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    Atomic coordinates and experimental details for the crystal structures of 5 and 6 (PDB codes 5LIF and 5LWD) will be released upon publication.

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    Cited By

    This article is cited by 13 publications.

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