Structural and mechanistic basis of differentiated inhibitors of the acute pancreatitis target kynurenine-3-monooxygenase

Abstract

Kynurenine-3-monooxygenase (KMO) is a key FAD-dependent enzyme of tryptophan metabolism. In animal models, KMO inhibition has shown benefit in neurodegenerative diseases such as Huntington's and Alzheimer's. Most recently it has been identified as a target for acute pancreatitis multiple organ dysfunction syndrome (AP-MODS); a devastating inflammatory condition with a mortality rate in excess of 20%. Here we report and dissect the molecular mechanism of action of three classes of KMO inhibitors with differentiated binding modes and kinetics. Two novel inhibitor classes trap the catalytic flavin in a previously unobserved tilting conformation. This correlates with picomolar affinities, increased residence times and an absence of the peroxide production seen with previous substrate site inhibitors. These structural and mechanistic insights culminated in GSK065(C1) and GSK366(C2), molecules suitable for preclinical evaluation. Moreover, revising the repertoire of flavin dynamics in this enzyme class offers exciting new opportunities for inhibitor design.

Biochemistry and structural studies of kynurenine 3‐monooxygenase reveal allosteric inhibition by Ro 61‐8048

“…The hKMO consists of 486 aa with hydrophobic regions near the C‐termini and was predicted as a membrane‐associated protein (http://www.uniprot.org). The crystal structure of an N‐terminal 396‐aa fragment of the yeast KMO and the crystal structure of a bacterial KMO homolog [Pseudomonas fluorescens KMO (pfKMO)] have been determined (20, 21, 23, 24). The yeast KMO N‐terminal fragment is a slightly elongated structure with a deep groove bound to the essential cofactor FAD.…”

“…3). The crystal structure of the full‐length pfKMO has been determined recently (21, 23, 24). The pfKMO has a similar structure to that of the yeast KMO, except for an all‐α‐C‐terminal domain that is missing in the yeast KMO structure.…”

“…Ro 61‐8048 binds in an allosteric tunnel that connecting the substrate‐binding lumen and the external environment, stabilizing the salt bridge formed between residues Glu372 and Arg84 (corresponding to hKMO residues Glu366 and Arg85, respectively), thus inhibiting KMOs by blocking substrate loading or product release and by preventing the necessary conformational changes needed to catalyze the reaction. Except for Ro 61‐8048, other KMO inhibitors, including the recently developed UPF648, GSK180, and GSK428 (21, 23, 24), all bind the substrate‐binding lumen with their binding sites completely or partially overlapped with that of the substrate l ‐KYN (Supplemental Fig. 5 A, B ).…”

“…Although new competitive KMO inhibitors have been developed recently (20–22), they have not shown promising neuroactive as does Ro 61‐8048. In spite of recent structural studies of different KMOs (20, 21, 23, 24), molecular mechanisms involved in the Ro 61‐8048 mediated KMO inhibition are not clearly understood, impeding its further optimization and clinic applications.…”

Kynurenine‐3‐monooxygenase: A new direction for the treatment in different diseases

“…Therefore, the activation of KMO leads to an increase in kynurenine levels, which are critical for the development of post‐traumatic sepsis, and an increase in kynurenine and 3‐HK levels is related to the development of organ failure in acute pancreatitis (Abdel‐Magid, ). Some KMO inhibitors which possess differentiated binding modes and kinetics had been reported recently (Hutchinson et al, ; Liddle et al, ; Walker et al, ). However, human pancreatitis is highly heterogeneous, so further studies are necessary to demonstrate whether KMO inhibitors are suitable for all pancreatitis (Ray, ).…”