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

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• 作者：Balagopalakrishna Chavali ; Thierry Masquelin ; Mark J. Nilges…
• 刊名：Applied Magnetic Resonance
• 出版年：2015
• 出版时间：August 2015
• 年：2015
• 卷：46
• 期：8
• 页码：853-873
• 全文大小：1,593 KB
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• 作者单位：Balagopalakrishna Chavali (1)
  Thierry Masquelin (2)
  Mark J. Nilges (4)
  David E. Timm (2)
  Stephanie L. Stout (2)
  William F. Matter (3)
  Najia Jin (3)
  Prabhakar K. Jadhav (2)
  Gary G. Deng (3)
  
  1. Division of Tailored Therapeutics and Imaging, Lilly Corporate Center, Eli Lilly and Company, Bldg.87/C04, Column S17 DC 1940, 893 S Delaware Street, Indianapolis, IN, 46285, USA
  2. Discovery Chemistry Research and Technologies, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, Indianapolis, IN, 46285, USA
  4. School of Molecular and Cellular Biology and Illinois EPR Research Center, Illinois EPR Research Center, 506 S. Mathews St., Urbana, IL, 61801, USA
  3. Division of Endocrine and Cardiovascular Research, Lilly Corporate Center, Eli Lilly and Company, 893 S Delaware Street, Indianapolis, IN, 46285, USA
  
• 刊物类别：Chemistry and Materials Science
• 刊物主题：Chemistry
  Analytical Chemistry
  
• 出版者：Springer Wien
• ISSN：1613-7507

文摘

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. 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??5—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.