ESR and X-ray Structure Investigations on the Binding and Mechanism of Inhibition of the Native State of Myeloperoxidase with Low Molecular Weight Fragments

Journal Article · Tue May 19 00:00:00 EDT 2015 · Applied Magnetic Resonance

DOI:https://doi.org/10.1007/s00723-015-0698-8· OSTI ID:1343556

Chavali, Balagopalakrishna ^[1]; Masquelin, Thierry ^[2]; Nilges, Mark J. ^[3]; Timm, David E. ^[2]; Stout, Stephanie L. ^[2]; Matter, William F. ^[4]; Jin, Najia ^[5]; Jadhav, Prabhakar K. ^[2]; Deng, Gary G. ^[5]

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1. Eli Lilly and Company, Indianapolis, IN (United States). Division of Tailored Therapeutics and Imaging, Lilly Corporate Center
2. Eli Lilly and Company, Indianapolis, IN (United States). Discovery Chemistry Research and Technologies, Lilly Corporate Center
3. Illinois EPR Research Center, Urbana, IL (United States). School of Molecular and Cellular Biology and Illinois EPR Research Center
4. Eli Lilly and Company, Indianapolis, IN (United States). Division of Endocrine and Cardiovascular Research, Lilly Corporate Center
5. Division of Endocrine and Cardiovascular Research, Lilly Corporate Center

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 (6 K) 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. Also, 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.

• Research Organization: Eli Lilly and Company, Indianapolis, IN (United States)
• Sponsoring Organization: USDOE Office of Science (SC)
• Grant/Contract Number: AC02-06CH11357
• OSTI ID: 1343556
• Journal Information: Applied Magnetic Resonance, Vol. 46, Issue 8; ISSN 0937-9347
• Country of Publication: United States
• Language: English

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