Abstract
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 Rrelative ≥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.
Highlights
As an early visitor to the injured loci, neutrophil-derived human Myeloperoxidase 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 FASTTM technology to screen a diverse series of small molecules that inhibit the peroxidase function through reversible binding to the native state of MPO
Inflammation arising from the innate defenses is a pervasive clinical parameter of concern across atherosclerosis, diabetes, cancer, chronic kidney disease, and neuro degeneration [1–5]
Summary
Inflammation arising from the innate defenses is a pervasive clinical parameter of concern across atherosclerosis, diabetes, cancer, chronic kidney disease, and neuro degeneration [1–5]. We took an approach that focused on the inhibitor reversibly binding to the heme pocket in the native state of the enzyme (Fe3?) as against the catalytic forms Compound I/II in hMPO. ESR was complimented with FASTTM technology (a method of screening mixtures of small molecule fragments for binding to the protein molecules in the crystalline state) to initiate a fragment based drug design (FBDD) approach for the identification and confirmation of actives [14, 15] Both ESR and antioxidant screens were used to derive the mechanism of action (reversibility, competitive substrate inhibition, and percent antioxidant potential). The current study describes our successful efforts toward identifying molecular scaffolds that bind to the active site in the native state (confirmed with ESR and X-ray results), which do not act via the anti oxidant pathway, and are mechanistically competitive and reversible in binding. These compounds evolved from a SAR design of five structural scaffolds (Manuscript describing the SAR design, syntheses, X-ray structures, a MPO-specific chemiluminescence method to derive the enzyme-inhibition-constants, mouse inflammation model development, and in vivo results is under preparation)
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