Dale E. Edmondson

The therapeutic potential of monoamine oxidase inhibitors.

Monoamine oxidase inhibitors were among the first antidepressants to be discovered and have long been used as such. It now seems that many of these agents might have therapeutic value in several common neurodegenerative conditions, independently of their inhibition of monoamine oxidase activity. However, many claims and some counter-claims have been made about the physiological importance of these enzymes and the potential of their inhibitors. We evaluate these arguments in the light of what we know, and still have to learn, of the structure, function and genetics of the monoamine oxidases and the disparate actions of their inhibitors.

Structure of human monoamine oxidase B, a drug target for the treatment of neurological disorders

Monoamine oxidase B (MAO B) is a mitochondrial outermembrane flavoenzyme that is a well-known target for antidepressant and neuroprotective drugs. We determined the structure of the human enzyme to 3 Å resolution. The enzyme binds to the membrane through a C-terminal transmembrane helix and apolar loops located at various positions in the sequence. The electron density shows that pargyline, an analog of the clinically used MAO B inhibitor, deprenyl, binds covalently to the flavin N5 atom. The active site of MAO B consists of a 420 Å3-hydrophobic substrate cavity interconnected to an entrance cavity of 290 Å3. The recognition site for the substrate amino group is an aromatic cage formed by Tyr 398 and Tyr 435. The structure provides a framework for probing the catalytic mechanism, understanding the differences between the B- and A-monoamine oxidase isoforms and designing specific inhibitors.

Insights Into the Mode of Inhibition of Human Mitochondrial Monoamine Oxidase B from High-Resolution Crystal Structures

Monoamine oxidase B (MAO-B) is an outer mitochondrial membrane-bound enzyme that catalyzes the oxidative deamination of arylalkylamine neurotransmitters and has been a target for a number of clinically used drug inhibitors. The 1.7-Å structure of the reversible isatin–MAO-B complex has been determined; it forms a basis for the interpretation of the enzymes structure when bound to either reversible or irreversible inhibitors. 1,4-Diphenyl-2-butene is found to be a reversible MAO-B inhibitor, which occupies both the entrance and substrate cavity space in the enzyme. Comparison of these two structures identifies Ile-199 as a “gate” between the two cavities. Rotation of the side chain allows for either separation or fusion of the two cavities. Inhibition of the enzyme with N-(2-aminoethyl)-p-chlorobenzamide results in the formation of a covalent N(5) flavin adduct with the phenyl ring of the inhibitor occupying a position in the catalytic site overlapping that of isatin. Inhibition of MAO-B with the clinically used trans-2-phenylcyclopropylamine results in the formation of a covalent C(4a) flavin adduct with an opened cyclopropyl ring and the phenyl ring in a parallel orientation to the flavin. The peptide bond between the flavin-substituted Cys-397 and Tyr-398 is in a cis conformation, which allows the proper orientation of the phenolic ring of Tyr-398 in the active site. The flavin ring exists in a twisted nonplanar conformation, which is observed in the oxidized form as well as in both the N(5) and the C(4a) adducts. An immobile water molecule is H-bonded to Lys-296 and to the N(5) of the flavin as observed in other flavin-dependent amine oxidases. The active site cavities are highly apolar; however, hydrophilic areas exist near the flavin and direct the amine moiety of the substrate for binding and catalysis. Small conformational changes are observed on comparison of the different inhibitor–enzyme complexes. Future MAO-B drug design will need to consider “induced fit” contributions as an element in ligand–enzyme interactions.

Biochemical, structural, and biological evaluation of tranylcypromine derivatives as inhibitors of histone demethylases LSD1 and LSD2

LSD1 and LSD2 histone demethylases are implicated in a number of physiological and pathological processes, ranging from tumorigenesis to herpes virus infection. A comprehensive structural, biochemical, and cellular study is presented here to probe the potential of these enzymes for epigenetic therapies. This approach employs tranylcypromine as a chemical scaffold for the design of novel demethylase inhibitors. This drug is a clinically validated antidepressant known to target monoamine oxidases A and B. These two flavoenzymes are structurally related to LSD1 and LSD2. Mechanistic and crystallographic studies of tranylcypromine inhibition reveal a lack of selectivity and differing covalent modifications of the FAD cofactor depending on the enantiomeric form. These findings are pharmacologically relevant, since tranylcypromine is currently administered as a racemic mixture. A large set of tranylcypromine analogues were synthesized and screened for inhibitory activities. We found that the common evolutionary origin of LSD and MAO enzymes, despite their unrelated functions and substrate specificities, is reflected in related ligand-binding properties. A few compounds with partial enzyme selectivity were identified. The biological activity of one of these new inhibitors was evaluated with a cellular model of acute promyelocytic leukemia chosen since its pathogenesis includes aberrant activities of several chromatin modifiers. Marked effects on cell differentiation and an unprecedented synergistic activity with antileukemia drugs were observed. These data demonstrate that these LSD1/2 inhibitors are of potential relevance for the treatment of promyelocytic leukemia and, more generally, as tools to alter chromatin state with promise of a block of tumor progression.

Molecular and mechanistic properties of the membrane-bound mitochondrial monoamine oxidases.

The past decade has brought major advances in our knowledge of the structures and mechanisms of MAO A and MAO B, which are pharmacological targets for specific inhibitors. In both enzymes, crystallographic and biochemical data show their respective C-terminal transmembrane helices anchor the enzymes to the outer mitochondrial membrane. Pulsed EPR data show both enzymes are dimeric in their membrane-bound forms with agreement between distances measured in their crystalline forms. Distances measured between active site-directed spin-labels in membrane preparations show excellent agreement with those estimated from crystallographic data. Our knowledge of requirements for development of specific reversible MAO B inhibitors is in a fairly mature status. Less is known regarding the structural requirements for highly specific reversible MAO A inhibitors. In spite of their 70% level of sequence identity and similarities of C(alpha) folds, the two enzymes exhibit significant functional and structural differences that can be exploited in the ultimate goal of the development of highly specific inhibitors. This review summarizes the current structural and mechanistic information available that can be utilized in the development of future highly specific neuroprotectants and cardioprotectants.

Demonstration of Isoleucine 199 as a Structural Determinant for the Selective Inhibition of Human Monoamine Oxidase B by Specific Reversible Inhibitors

Several reversible inhibitors selective for human monoamine oxidase B (MAO B) that do not inhibit MAO A have been described in the literature. The following compounds: 8-(3-chlorostyryl)caffeine, 1,4-diphenyl-2-butene, and trans,trans-farnesol are shown to inhibit competitively human, horse, rat, and mouse MAO B with Ki values in the low micromolar range but are without effect on either bovine or sheep MAO B or human MAO A. In contrast, the reversible competitive inhibitor isatin binds to all known MAO B and MAO A with similar affinities. Sequence alignments and the crystal structures of human MAO B in complex with 1,4-diphenyl-2-butene or with trans,trans-farnesol provide molecular insights into these specificities. These inhibitors span the substrate and entrance cavities with the side chain of Ile-199 rotated out of its normal conformation suggesting that Ile-199 is gating the substrate cavity. Ile-199 is conserved in all known MAO B sequences except bovine MAO B, which has Phe in this position (the sequence of sheep MAO B is unknown). Phe is conserved in the analogous position in MAO A sequences. The human MAO B I199F mutant protein of MAO B binds to isatin (Ki = 3 μm) but not to the three inhibitors listed above. The crystal structure of this mutant demonstrates that the side chain of Phe-199 interferes with the binding of those compounds. This suggests that the Ile-199 “gate” is a determinant for the specificity of these MAO B inhibitors and provides a molecular basis for the development of MAO B-specific reversible inhibitors without interference with MAO A function in neurotransmitter metabolism.

Bioactive Contaminants Leach from Disposable Laboratory Plasticware

Disposable plasticware such as test tubes, pipette tips, and multiwell assay or culture plates are used routinely in most biological research laboratories. Manufacturing of plastics requires the inclusion of numerous chemicals to enhance stability, durability, and performance. Some lubricating (slip) agents, exemplified by oleamide, also occur endogenously in humans and are biologically active, and cationic biocides are included to prevent bacterial colonization of the plastic surface. We demonstrate that these manufacturing agents leach from laboratory plasticware into a standard aqueous buffer, dimethyl sulfoxide, and methanol and can have profound effects on proteins and thus on results from bioassays of protein function. These findings have far-reaching implications for the use of disposable plasticware in biological research.

Structural Basis for Benzothiazinone-Mediated Killing of Mycobacterium tuberculosis

The crystal structure of the mycobacterial DprE1 reveals how the TB drug benzothiazinone BTZ043 blocks this microbial enzyme target. New TB Drug Snapped in Action Tuberculosis (TB) is a major global health problem that claimed 1.4 million lives in 2010. TB is becoming incurable with existing antibiotics, as infections with multidrug-resistant strains of the causative pathogen Mycobacterium tuberculosis continue to climb. To make matters worse, many patients with TB also suffer from HIV/AIDS, making both diseases even more difficult to treat. It has been more than 40 years since a new drug for TB was approved for clinical use. In 2009, a study published in Science described a promising new drug candidate, a synthetic organic molecule known as BTZ043, which is active in the low nanomolar range against mycobacteria. BTZ043 inhibits a bacterial epimerase enzyme that produces the sugar d-arabinose, the sole precursor for the synthesis of a polysaccharide that is an essential component of the bacterial cell wall. In a key follow-up study, Neres et al. use x-ray crystallography to obtain a picture of the epimerase at the atomic level. They demonstrate that the drug serves as a suicide substrate that is converted by the epimerase into a highly reactive species, and they present a snapshot that shows covalent binding of this species to the active site of the enzyme. Together with biochemical work, the three-dimensional structure explains why BTZ043 inactivates its target so effectively, thus killing the bacteria. By attaching a fluorescent probe to one side of the drug, the authors discovered that the epimerase enzyme becomes localized to the poles of live bacteria, thus pinpointing the site of action. The availability of the epimerase structure and a deeper understanding of its catalytic properties open a host of avenues for rational drug discovery that hopefully will result in new medicines for fighting TB. The benzothiazinone BTZ043 is a tuberculosis drug candidate with nanomolar whole-cell activity. BTZ043 targets the DprE1 catalytic component of the essential enzyme decaprenylphosphoryl-β-d-ribofuranose-2′-epimerase, thus blocking biosynthesis of arabinans, vital components of mycobacterial cell walls. Crystal structures of DprE1, in its native form and in a complex with BTZ043, reveal formation of a semimercaptal adduct between the drug and an active-site cysteine, as well as contacts to a neighboring catalytic lysine residue. Kinetic studies confirm that BTZ043 is a mechanism-based, covalent inhibitor. This explains the exquisite potency of BTZ043, which, when fluorescently labeled, localizes DprE1 at the poles of growing bacteria. Menaquinone can reoxidize the flavin adenine dinucleotide cofactor in DprE1 and may be the natural electron acceptor for this reaction in the mycobacterium. Our structural and kinetic analysis provides both insight into a critical epimerization reaction and a platform for structure-based design of improved inhibitors.

Crystal structure of human monoamine oxidase B, a drug target enzyme monotopically inserted into the mitochondrial outer membrane

Monoamine oxidase B (MAO B) is an outer mitochondrial membrane protein that oxidizes arylalkylamine neurotransmitters and has been a valuable drug target for many neurological disorders. The 1.7 Å resolution structure of human MAO B shows the enzyme is dimeric with a C‐terminal transmembrane helix protruding from each monomer and anchoring the protein to the membrane. This helix departs perpendicularly from the base of the structure in a different way with respect to other monotopic membrane proteins. Several apolar loops exposed on the protein surface are located in proximity of the C‐terminal helix, providing additional membrane‐binding interactions. One of these loops (residues 99–112) also functions in opening and closing the MAO B active site cavity, which suggests that the membrane may have a role in controlling substrate binding.

The FAD Binding Sites of Human Monoamine Oxidases A and B

The structural details of the interactions of the covalent 8alpha-S-cysteinyl-FAD with the protein moiety in monoamine oxidase B (MAO B) based on the MAO B crystal structure are described. The dinucleotide is bound to the protein in an extended conformation with the majority of the bonds to the protein identified as hydrogen bonds with amino acid side chains, amide bonds, and water molecules. Since those amino acids interacting with the FAD are conserved in monoamine oxidase A (MAO A), it is proposed that the FAD binding site in MAO A is quite similar to that in MAO B. The redox-active isoalloxazine ring is buried in the protein without direct access to bulk solvent. An electrostatic interaction is observed between the anionic pyrophosphate moiety and Arg42. The normally flat oxidized flavin ring is in a bent, puckered conformation in the MAO B binding site which is suggested to contribute to its reactivity in catalysis. This structural information is then used to explain previous studies on flavin analog incorporation into either MAO B or into MAO A.