caa a22 4500 605546002 CHVBK 20210128100938.0 cr unu---uuuuu 210128e20150801xx s 000 0 eng 10.1007/s00723-015-0698-8 doi (NATIONALLICENCE)springer-10.1007/s00723-015-0698-8 ESR and X-ray Structure Investigations on the Binding and Mechanism of Inhibition of the Native State of Myeloperoxidase with Low Molecular Weight Fragments [Elektronische Daten] [Balagopalakrishna Chavali, Thierry Masquelin, Mark Nilges, David Timm, Stephanie Stout, William Matter, Najia Jin, Prabhakar Jadhav, Gary Deng] As an early visitor to the injured loci, neutrophil-derived human Myeloperoxidase (hMPO) offers an attractive protein target to modulate the inflammation of the host tissue through suitable inhibitors. We describe a novel methodology of using low temperature ESR spectroscopy (6K) and FAST™ technology to screen a diverse series of small molecules that inhibit the peroxidase function through reversible binding to the native state of MPO. Our initial efforts to profile molecules on the inhibition of MPO-initiated nitration of the Apo-A1 peptide (AEYHAKATEHL) assay showed several potent (with sub-micro molar IC50s) but spurious inhibitors that either do not bind to the heme pocket in the enzyme or retain high (>50%) anti oxidant potential. Such molecules when taken forward for X-ray did not yield inhibitor-bound co-crystals. We then used ESR to confirm direct binding to the native state enzyme, by measuring the binding-induced shift in the electronic parameter g to rank order the molecules. Molecules with a higher rank order—those with g-shift R relative≥15—yielded well-formed protein-bound crystals (n=33 structures). The co-crystal structure with the LSN217331 inhibitor reveals that the chlorophenyl group projects away from the heme along the edges of the Phe366 and Phe407 side chain phenyl rings thereby sterically restricting the access to the heme by the substrates like H2O2. Both ESR and antioxidant screens were used to derive the mechanism of action (reversibility, competitive substrate inhibition, and percent antioxidant potential). In conclusion, our results point to a viable path forward to target the native state of MPO to tame local inflammation. The Author(s), 2015 Chavali Balagopalakrishna Division of Tailored Therapeutics and Imaging, Lilly Corporate Center, Eli Lilly and Company, Bldg.87/C04, Column S17 DC 1940, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut Masquelin Thierry Discovery Chemistry Research and Technologies, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut Nilges Mark School of Molecular and Cellular Biology and Illinois EPR Research Center, Illinois EPR Research Center, 506 S. Mathews St., 61801, Urbana, IL, USA aut Timm David Discovery Chemistry Research and Technologies, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut Stout Stephanie Discovery Chemistry Research and Technologies, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut Matter William Division of Endocrine and Cardiovascular Research, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut Jin Najia Division of Endocrine and Cardiovascular Research, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut Jadhav Prabhakar Discovery Chemistry Research and Technologies, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut Deng Gary Division of Endocrine and Cardiovascular Research, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut Applied Magnetic Resonance Springer Vienna 46/8(2015-08-01), 853-873 0937-9347 46:8<853 2015 46 723 https://doi.org/10.1007/s00723-015-0698-8 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00723-015-0698-8 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Chavali Balagopalakrishna Division of Tailored Therapeutics and Imaging, Lilly Corporate Center, Eli Lilly and Company, Bldg.87/C04, Column S17 DC 1940, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut NATIONALLICENCE 700 1- Masquelin Thierry Discovery Chemistry Research and Technologies, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut NATIONALLICENCE 700 1- Nilges Mark School of Molecular and Cellular Biology and Illinois EPR Research Center, Illinois EPR Research Center, 506 S. Mathews St., 61801, Urbana, IL, USA aut NATIONALLICENCE 700 1- Timm David Discovery Chemistry Research and Technologies, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut NATIONALLICENCE 700 1- Stout Stephanie Discovery Chemistry Research and Technologies, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut NATIONALLICENCE 700 1- Matter William Division of Endocrine and Cardiovascular Research, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut NATIONALLICENCE 700 1- Jin Najia Division of Endocrine and Cardiovascular Research, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut NATIONALLICENCE 700 1- Jadhav Prabhakar Discovery Chemistry Research and Technologies, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut NATIONALLICENCE 700 1- Deng Gary Division of Endocrine and Cardiovascular Research, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, 46285, Indianapolis, IN, USA aut NATIONALLICENCE 773 0- Applied Magnetic Resonance Springer Vienna 46/8(2015-08-01), 853-873 0937-9347 46:8<853 2015 46 723