Full-length in meso structure and mechanism of rat kynurenine 3-monooxygenase inhibition

Abstract

The structural mechanisms of single-pass transmembrane enzymes remain elusive. Kynurenine 3-monooxygenase (KMO) is a mitochondrial protein involved in the eukaryotic tryptophan catabolic pathway and is linked to various diseases. Here, we report the mammalian full-length structure of KMO in its membrane-embedded form, complexed with compound 3 (identified internally) and compound 4 (identified via DNA-encoded chemical library screening) at 3.0 Å resolution. Despite predictions suggesting that KMO has two transmembrane domains, we show that KMO is actually a single-pass transmembrane protein, with the other transmembrane domain lying laterally along the membrane, where it forms part of the ligand-binding pocket. Further exploration of compound 3 led to identification of the brain-penetrant compound, 5. We show that KMO is dimeric, and that mutations at the dimeric interface abolish its activity. These results will provide insight for the drug discovery of additional blood-brain-barrier molecules, and help illuminate the complex biology behind single-pass transmembrane enzymes.

Discovery of Novel, Potent Inhibitors of Hydroxy Acid Oxidase 1 (HAO1) Using DNA-Encoded Chemical Library Screening

“…Methylation of indazole (17) and phenol (18) also led to a decrease in potency by 113-and 57-fold, respectively. Removing phenolic OH (19) or replacing it with a chlorine (20) led to a 5-fold loss in potency in both instances. However, for compounds that retained potency the change in tPSA (from 131 to 111) and c log P (from 2.2 to 2.4 (19) or 3.1 (20)) did not lead to improved permeability compared to parent Compound 5.…”

“…The phenolic hydroxy forms an internal hydrogen bond with the nitrogen of the oxadiazole, stabilizing a planar conformation of this portion of the inhibitor. Removal of the hydroxyl group (19) results in a 5fold loss in potency (Table 2). Finally, the dimethylamine is situated in a pocket formed by residues Asp170, Asn173, Arg174, Ser22, and Ala206.…”

“…In our efforts to identify a small-molecule inhibitor of HAO1, we applied our DNA-Encoded Chemical Library (DECL) platform, which has been an attractive strategy to provide small molecule leads for a wide range of targets. − This platform allows the simultaneous screening of billions of diverse compounds. − Additionally, known binders can be incorporated into the screens to help understand the binding modes of identified hits. In this paper, we report the discovery of potent, novel, carboxylic acid-containing HAO1 inhibitors via a DECL screen, their absorption, distribution, metabolism, and excretion (ADME) optimization, and structural understanding of protein–ligand interactions.…”

Kynurenine‐3‐monooxygenase (KMO): From its biological functions to therapeutic effect in diseases progression

“…Without substrate, both domains lining the KMO active site reflect different flexibility as the observation of different conformations. The first Phe residue intimately interacts with aromatic residues such as Phe and Tyr by forms of π−π bonds which appear to contribute to the structural stabilization of KMO (Mimasu et al, 2021). Thus, with the further understanding of the structure, the functions of KMO in human body can be illuminated clearly and promote the advancement of therapeutic methods for related diseases.…”

“…In addition, Rat‐KMO shares 93% similarity with hKMO. Based on its monomer structure, the Rat‐KMO consists of three domains, including FAD‐binding domain I, FPMO enzyme‐conserved domain II, and C‐terminal domain III, containing only one TMD unexpectedly, and the other one partially embed in the membrane, a part of the ligand‐binding pocket (Mimasu et al, 2021). Discovery of Rat‐KMO structure in complex with a DNA‐encoded chemical library hit promotes the development of KMO inhibitors (Goodnow et al, 2017).…”