A scaffold hopping approach to identify novel monoamine oxidase B inhibitors.

Monoamine oxidase B (MAO-B) inhibitors are used to treat Parkinson's disease. In this study, we searched for novel MAO-B inhibitors using a scaffold hopping approach based on our experience with the thiazolidinedione (TZD) class of compounds as MAO-B inhibitors. Several novel compounds were identified, with potencies in the low nanomolar and low micromolar range. We also found that derivatives of the natural product sulfuretin are potent MAO-A and MAO-B inhibitors.

Complexes of the outer mitochondrial membrane protein mitoNEET with resveratrol-3-sulfate.

Binding of the thiazolidinedione antidiabetic drug pioglitazone led to the discovery of a novel outer mitochondrial membrane protein of unknown function called mitoNEET. The protein is homodimeric and contains a uniquely ligated two iron-two sulfur cluster in each of its two cytosolic domains. Electrospray ionization mass spectrometry was employed to characterize solutions of the soluble cytosolic domain (amino acids 32--108) of the protein. Ions characteristic of dimers containing the cofactors were readily detected under native conditions. mitoNEET responded to exposure to solutions at low pH by dissociation to give monomers that retained the cofactor, followed by dissociation of the cofactor in a concerted fashion. mitoNEET formed complexes with resveratrol-3-sulfate, one of the primary metabolites of the natural product resveratrol. Resveratrol itself showed no tendency to interact with mitoNEET. The formation of complexes was evident in both electrospray ionization mass spectrometry and isothermal titration calorimetry measurements. Up to eight molecules of the compound associated with the dimeric form of the protein in a sequential fashion. Dissociation constants determined by micorcalorimetry were in the range 5-16 µM for the various binding sites. The only other known naturally occurring binding partner for mitoNEET at present is NADPH. It is very interesting that the iron-sulfur cluster containing protein interacts with two potentially redox active substances at the surface of mitochondria. These findings provide a new direction for research into two poorly understood, yet biomedically relevant, species.

A novel binding assay identifies high affinity ligands to the rosiglitazone binding site of mitoNEET.

A novel outer mitochondrial membrane protein containing [2Fe-2S] clusters, mitoNEET was first identified through its binding to the anti-diabetic drug pioglitazone. Pioglitazone belongs to a family of drugs that are peroxisome proliferator-activated receptor (PPAR) gamma agonists, collectively known as glitazones. With the lack of pharmacological tools available to fully elucidate mitoNEET's function, we developed a binding assay to probe the glitazone binding site with the aim of developing selective and high affinity compounds. We used multiple thiazolidine-2,4-dione (TZD), 2-thioxothiazolidin-4-one (TTD), and 2-iminothiazolidin-4-one (ITD) compounds to establish several trends to enhance ligand development for the purpose of elucidating mitoNEET function.

Structure-activity relationship and docking studies of thiazolidinedione-type compounds with monoamine oxidase B.

The neuroprotective activity of pioglitazone and rosiglitazone in the MPTP parkinsonian mouse prompted us to evaluate a set of thiazolidinedione (TZD) type compounds for monoamine oxidase A and B inhibition activity. These compounds were able to inhibit MAO-B over several log units of magnitude (82 nM to 600 µM). Initial structure-activity relationship studies identified key areas to modify the aromatic substituted TZD compounds. Primarily, substitutions on the aromatic group and the TZD nitrogen were key areas where activity was enhanced within this group of compounds.

Structure-based design of a thiazolidinedione which targets the mitochondrial protein mitoNEET.

Several PPAR-gamma agonists containing a thiazolidinedione moiety (referred to as glitazones) have been proposed to be neuroprotective and appear to alter mitochondrial function. Recently, a search for mitochondrial proteins that bind pioglitazone identified a novel protein, mitoNEET, which was later shown to regulate the oxidative capacity of the mitochondria. This identified an alternative target for the glitazones suggesting a possible new drug target for the treatment of neurodegenerative diseases. Molecular docking studies employing the reported crystal structure revealed five possible binding pockets on mitoNEET. We focused on two sites based on their physical characteristics. Using binding information gained from the analysis of two glitazones docked in these pockets, we designed and synthesized a ligand (NL-1) that would preferentially bind to site 1. Based on [(3)H]-binding data of the glitazones and comparisons to computer generated K(i)s, we were able to predict that site 1 was likely the target of the glitazones. NL-1 uncoupled isolated mitochondrial complex I respiration with an IC(50) of 2.4 microM and inhibited state III respiration up to 45%. To investigate the ability of NL-1 to block rotenone initiated free radicals from complex I, we found it was able to protect the human neuronal cell line SH-SY5Y against rotenone induced cell death. These data demonstrate that mitoNEET is a viable target for the design and synthesis of novel therapeutic agents aimed at altering mitochondrial function.

Identification of novel monoamine oxidase B inhibitors by structure-based virtual screening.

Parkinson's disease is a severe debilitating neurodegenerative disorder. Recently, it was shown that the peroxisome proliferating-activator receptor-gamma agonist pioglitazone protected mice from 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine neurotoxicity due to its ability to inhibit monoamine oxidase B (MAO-B). Docking studies were initiated to investigate pioglitazone's interactions within the substrate cavity of MAO-B. Modeling studies indicated that the thiazolidinedione (TZD) moiety was a likely candidate for its specificity to MAO-B. To explore this potential novel MAO-B scaffold, we performed a structure-based virtual screen to identify additional MAO-B inhibitors. Our search identified eight novel compounds containing the TZD-moiety that allowed for a limited study to identify structural requirements for binding to MAO-B. Inhibition assays identified two TZDs (A6355 and L136662) which were found to inhibit recombinant human MAO-B with IC(50) values of 82 and 195 nM, respectively.

EPR spectroscopy and electrospray ionization mass spectrometry reveal distinctive features of the iron site in leukocyte 12-lipoxygenase.

The procedure for the expression and purification of recombinant porcine leukocyte 12-lipoxygenase using Escherichia coli [K.M. Richards, L.J. Marnett, Biochemistry 36 (1997) 6692-6699] was updated to make it possible to produce enough protein for physical measurements. Electrospray ionization tandem mass spectrometry confirmed the amino acid sequence. The redox properties of the cofactor iron site were examined by EPR spectroscopy at 25K following treatment with a variety of fatty acid hydroperoxides. Combination of the enzyme in a stoichiometric ratio with the hydroperoxides led to a g4.3 signal in EPR spectra instead of the g6 signal characteristic of similarly treated soybean lipoxygenase-1. Native 12-lipoxygenase was also subjected to electrospray ionization mass spectrometry. There was evidence for loss of the mass of an iron atom from the protein as the pH was lowered from 5 to 4. Native ions in these samples indicated that iron was lost without the protein completely unfolding.

Electrospray ionization mass spectrometry of soybean lipoxygenases: N-terminal acetylation, chemical modification, and solution conformation.

Electrospray ionization mass spectrometry was used to examine both the covalent structure and solution conformation of the soybean lipoxygenases. The post-translational modifications of two lipoxgyenases were identified as N-terminal acetylations by tandem mass spectrometry of peptides generated by trypsin digestion. The N-terminal sequence suggests that the proteins were substrates for the plant homolog of the N-terminal acetyltransferase complex C in yeast. Analysis of samples of native lipoxygenase-3 produced ions corresponding within experimental error to the mass of the N-acetylated polypeptide and one iron atom. The precision of the measurements was within roughly 100 ppm for the 96,856 Da protein. This made it possible to detect the addition of a single oxygen atom to the enzyme in a chemical modification reaction with cumene hydroperoxide. The acid-induced denaturation of lipoxygenase-3, which was accompanied by nearly complete loss of catalytic activity, was observed below pH 3.5 with the appearance of ions in the mass spectrum derived from the apoprotein. There was no evidence for the loss of iron in the absence of unfolding. Solutions of lipoxygenase-3 incubated in 0.1M acetic acid produced ions with a novel charge state distribution suggesting a unique conformation. Circular dichroism measurements showed that the secondary structure features of the native protein were retained in the new conformation. Dynamic light scattering revealed that the new conformation was not a consequence of protein aggregation as the hydrodynamic radius of lipoxygenase-3 was significantly smaller in acetic acid solution than at pH 7.0. Remarkably, the enzyme incubated in acetic acid retained full catalytic activity.

Crystal structure of 12-lipoxygenase catalytic-domain-inhibitor complex identifies a substrate-binding channel for catalysis.

Lipoxygenases are critical enzymes in the biosynthesis of families of bioactive lipids including compounds with important roles in the initiation and resolution of inflammation and in associated diseases such as diabetes, cardiovascular disease, and cancer. Crystals diffracting to high resolution (1.9 Å) were obtained for a complex between the catalytic domain of leukocyte 12-lipoxygenase and the isoform-specific inhibitor, 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP). In the three-dimensional structure of the complex, the inhibitor occupied a new U-shaped channel open at one end to the surface of the protein and extending past the redox-active iron site that is essential for catalysis. In models, the channel accommodated arachidonic acid, defining the binding site for the substrate of the catalyzed reaction. There was a void adjacent to the OPP binding site connecting to the surface of the enzyme and providing a plausible access channel for the other substrate, oxygen.

Secondary alkyl hydroperoxides as inhibitors and alternate substrates for lipoxygenase.

Lipoxygenase plays a central role in polyunsaturated fatty acid metabolism, inaugurating the biosynthesis of eicosanoids in animals and phytooxylipins in plants. Redox cycling of the non-heme iron cofactor represents a critical element of the catalytic mechanism. Paradoxically, the isolated enzyme contains Fe(II), but the catalytically active form contains Fe(III), and the natural oxidant for the iron is the hydroperoxide product of the catalyzed reaction. Controlling the redox state of lipoxygenase iron with small molecules, inhibitors or activators, could be a means to modulate the activity of the enzyme. The effects of secondary alkyl hydroperoxides and the corresponding alcohols on soybean lipoxygenase-1 reaction rates were investigated and found to be very different. Secondary alcohols were noncompetitive or linear mixed inhibitors with inhibition constants in the millimolar concentration range, with more hydrophobic compounds producing lower values. Secondary alkyl hydroperoxides were inhibitors of lipoxygenase-1 primarily at high substrate concentration. They were more effective inhibitors than the alcohols, with dissociation constants in the micromolar concentration range. The hydroperoxides bearing longer alkyl substituents were the more effective inhibitors. Oxidation of the iron in lipoxygenase-1 by 2-hydroperoxyalkanes was evident in electron paramagnetic resonance (EPR) measurements, but the enzyme was neither activated nor was it inactivated. Instead there was evidence for an entirely different reaction catalyzed by the enzyme, a homolytic dehydration of the hydroperoxide to produce the corresponding carbonyl compound.

Interaction between non-heme iron of lipoxygenases and cumene hydroperoxide: basis for enzyme activation, inactivation, and inhibition.

Lipoxygenase catalysis depends in a critical fashion on the redox properties of a unique mononuclear non-heme iron cofactor. The isolated enzyme contains predominantly, if not exclusively, iron(II), but the catalytically active form of the enzyme has iron(III). The activating oxidation of the iron takes place in a reaction with the hydroperoxide product of the catalyzed reaction. In a second peroxide-dependent process, lipoxygenases are also inactivated. To examine the redox activation/inactivation dichotomy in lipoxygenase chemistry, the interaction between lipoxygenase-1 (and -3) and cumene hydroperoxide was investigated. Cumene hydroperoxide was a reversible inhibitor of the reaction catalyzed by lipoxygenase-1 under standard assay conditions at high substrate concentrations. Reconciliation of the data with the currently held kinetic mechanism requires simultaneous binding of substrate and peroxide. The enzyme also was both oxidized and largely inactivated in a reaction with the peroxide in the absence of substrate. The consequences of this reaction for the enzyme included the hydroxylation at C beta of two amino acid side chains in the vicinity of the cofactor, Trp and Leu. The modifications were identified by mass spectrometry and X-ray crystallography. The peroxide-induced oxidation of iron was also accompanied by a subtle rearrangement in the coordination sphere of the non-heme iron atom. Since the enzyme retains catalytic activity, albeit diminished, after treatment with cumene hydroperoxide, the structure of the iron site may reflect the catalytically relevant form of the cofactor.

Protein micelles from lipoxygenase 3.

Heat-induced conformational changes in lipoxygenase 3 were characterized by differential scanning calorimetry. The positions of the observed transitions were sensitive to the composition of the buffer. In particular, lipoxygenase 3 heated in carbonate buffer at pH 8.0 formed large soluble aggregates. Variable-temperature circular dichroism revealed that the formation of the aggregates was not accompanied by the unfolding of the C-terminal domain, which is composed primarily of alpha-helix. The aggregates were investigated using size exclusion chromatography, native polyacrylamide gel electrophoresis, dynamic light scattering, and electron microscopy. The data were consistent with the formation of roughly spherical particles with an average hydrodynamic radius of 26 nm and an approximate composite molecular weight of 10,000,000 Da. To account for the formation of soluble aggregates from lipoxygenase 3, we propose that hydrophobic amino acid residues are exposed by unfolding of the N-terminal beta-barrel domain of the protein resulting in the formation of protein micelles with a hydrophilic surface composed of the C-terminal domains.