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WO1996020946A1 - Analogues du flavine-adenine-dinucleotide, leurs compositions pharmaceutiques et leur activite en tant qu'inhibiteurs de la monoamine oxydase - Google Patents

Analogues du flavine-adenine-dinucleotide, leurs compositions pharmaceutiques et leur activite en tant qu'inhibiteurs de la monoamine oxydase Download PDF

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Publication number
WO1996020946A1
WO1996020946A1 PCT/US1995/017106 US9517106W WO9620946A1 WO 1996020946 A1 WO1996020946 A1 WO 1996020946A1 US 9517106 W US9517106 W US 9517106W WO 9620946 A1 WO9620946 A1 WO 9620946A1
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monoamine oxidase
mao
type
fad
inhibitor
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PCT/US1995/017106
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English (en)
Inventor
Creed W. Abell
Sau-Wah Kwan
Binhua Zhou
Blain M. Mamiya
Duane A. Lewis
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Research Development Foundation
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Priority claimed from US08/449,311 external-priority patent/US5756479A/en
Application filed by Research Development Foundation filed Critical Research Development Foundation
Priority to AU47454/96A priority Critical patent/AU4745496A/en
Publication of WO1996020946A1 publication Critical patent/WO1996020946A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7048—Compounds having saccharide radicals and heterocyclic rings having oxygen as a ring hetero atom, e.g. leucoglucosan, hesperidin, erythromycin, nystatin, digitoxin or digoxin

Definitions

  • the present invention relates generally to the fields of neuropharmacology and protein chemistry. More specifically, the present invention relates to novel flavin adenine dinucleotide analogues as inhibitors of monoamine oxidase. Description of the Related Art
  • concentration of neurotransmitters is controlled in part by enzymes that produce and metabolize the neurotransmitters.
  • Two such enzymes, monoamine oxidase A and B [MAO A and B; amine: oxygen, oxidoreductase (deaminating, flavin-containing) , EC 1.4.3.4] are the major intracellular enzymes in the central nervous system and peripheral tissues of mammals that catalyze the oxidative deamination of neuroactive and vasoactive amines.
  • These enzymes are integral proteins of the outer mitochondrial membrane and can be distinguished by differences in substrate preference, inhibitory specificity, tissue and cell distribution, and immunological properties.
  • nucleotide and deduced amino acid sequences definitively show that monoamine oxidase A and B are two distinct proteins. Comparative studies between the human and rat sequences show that the monoamine oxidase A and B proteins have approximately 70% identity and the respective A and B enzymes have very high sequence identity in both species, particularly in the two regions of the molecule that bind flavin adenine dinucleotide (FAD) covalently and non- covalently.
  • FAD flavin adenine dinucleotide
  • Monoamine oxidases A and B have been linked to various psychiatric and neurological disorders and each is a target for certain psychiatric and neurologic drugs.
  • the monoamine oxidase A inhibitor moclobemide and the monoamine oxidase B inhibitor deprenyl have been used for the treatment of unipolar depression and Parkinson's disease, respectively.
  • none of the available drugs is totally satisfactory.
  • some of these drugs, such as parnate do not distinguish between monoamine oxidase A and B, and some monoamine oxidase inhibitors produce life threatening side-effects such as hypertensive crisis.
  • monoamine oxidase A Current interest in monoamine oxidase A has been intensified by the discovery of an alteration in its gene. Defective monoamine oxidase A, due to a mutation of CAG (glutamine) to TAG (termination codon) , was identified in some members of a Dutch family who exhibit abnormal aggressive behavior. A role for monoamine oxidase B in idiopathic Parkinson's disease has received support through the identification of 1-methyl-4-pheny1-1, 2, 3, 6,- tetrahydropyridine (MPTP) , a powerful neurotoxin. This substance was found as a contaminant in a synthetic heroin and produced a Parkinsonian syndrome in some individuals who self-administered the synthetic heroin.
  • MPTP 1-methyl-4-pheny1-1, 2, 3, 6,- tetrahydropyridine
  • MPTP is oxidized by monoamine oxidase B to a reactive species (MPP*) .
  • MPP* a reactive species
  • Pretreatment of primates with deprenyl (a monoamine oxidase B selective inhibitor) , but not clorgyline (a monoamine oxidase A selective inhibitor) prevents the development of MPTP-induced Parkinsonism.
  • Treatment of hundreds of Parkinson's patients with deprenyl showed that this drug significantly slowed the rate of deterioration and the course of the disease.
  • an alternate allele of the monoamine oxidase B gene has been identified in patients with Parkinson's disease, a finding which suggests that an inherited variant form of monoamine oxidase B may be associated with a genetic predisposition for this neurological disorder.
  • Novel monoamine oxidase inhibitors that are designed to interact with functionally important regions of the enzymes may have high efficacy for treatment of depression and Parkinson's disease with minimal side effects.
  • Monoamine oxidase A and B are large proteins that have subunit molecular weights of approximately 60,000. Neurotransmitters and drugs that bind to these enzymes have small molecular weights in the range of hundreds, not thousands. Consequently, the drugs interact with only a small segment of the enzyme, such as the active site or the site to which an essential cofactor binds. Monoamine oxidase A and B use the cofactor, flavin adenine dinucleotide (FAD) . FAD binds to two different sites on both enzymes, one covalently and the other non-covalently, and these interactions are essential steps for monoamine oxidase A and B to function.
  • FAD flavin adenine dinucleotide
  • monoamine oxidase A and B would be of great value for the design of new drugs that have high efficacy for these diseases with minimal side effects. Since the monoamine oxidases are integral proteins of the outer mitochondrial membrane, however, it has been difficult to obtain crystals suitable for X-ray diffraction studies. Alternatively, a search for sequence similarities between monoamine oxidase A and B and other proteins of known structure may be useful for identifying common structural motifs.
  • a and B for the endpoint of therapeutically treating various psychiatric and neurological disorders without side effects.
  • the present invention fulfills this longstanding need and desire in the art.
  • an inhibitor of monoamine oxidase comprising a modified synthetic flavin adenine dinucleotide compound.
  • a pharmaceutical composition of the present invention and a pharmaceutically acceptable carrier.
  • a method of inhibiting monoamine oxidase enzymatic activity in a cell comprising the step of contacting said cell with a pharmacologically effective dose of a modified synthetic flavin adenine dinucleotide compound.
  • a method of treating a brain pathophysiological state in an individual comprising administering a therapeutically effective dose of a modified biosynthetic flavin adenine dinucleotide compound to said individual.
  • Figure 1 shows the human monoamine oxidase B mutants. Oligonucleotide primers of wild-type and mutants, and side chains corresponding to their amino acid substitutions are shown. Lower case letters indicate base substitutions. Bold letters indicate amino acid substitutions for the gluta ate residue in position 34 in mutants E34A, E34Q and E34D, and valine in position 10 in mutant V10I.
  • Figure 2 shows the monoamine oxidase B expression in COS-7 cells.
  • Figure 3 shows a western blot of wild type and mutant monoamine oxidase B in transfected COS-7 cells.
  • Expressed wild type and mutant monoamine oxidase B were immunoprecipitated using a polyclonal goat anti-monoamine oxidase B antibody and protein G beads as described below.
  • the immunoprecipitated enzymes were separated on 10% sodium dodecyl sulfate-polyacryla ide gel electrophoresis (SDS-PAGE) , transferred to a nitrocellulose membrane and analyzed by Western blotting using the monoamine oxidase B specific monoclonal antibody, monoamine oxidase B-1C2.
  • Lane 1 prestained MW marker
  • Lane 2 wild type monoamine oxidase B
  • Lane 3 E34A monoamine oxidase B
  • Lane 4 E34Q monoamine oxidase B
  • Lane 5 E34D monoamine oxidase B
  • Lane 6 V10I monoamine oxidase B
  • Lane 7 untransfected COS-7 cells
  • Lane 8 biotinylated MW markers.
  • Figure 4 shows a diagram of the putative dinucleotide- binding site DBS ( ⁇ -a- ⁇ 2 motif) and the covalent attachment site for FAD in human monoamine oxidase B. Thick and thin lines represent the peptide backbone and covalent bonds, respectively.
  • Amino acids 6 through 34 comprise the dinucleotide-binding site in monoamine oxidase B. The initial residue, is usually a hydrophilic amino acid. In monoamine oxidase B, this residue is at position 6 and is aspartate. Glutamate at position 34, hydrogen bonds with the 2'-hydroxy group of ribose in the adenine monophosphate moiety of FAD.
  • Figure 5 shows nucleotide sequences of mutagenic primers in site-directed mutagenesis studies. Lowercase letters indicate base substitutions. The codon for wild-type and mutants at positions 44 and 46 are indicated by a single line above the nucleotides. Base substitutions which do not alter the amino acid coding sequence were also included in each mutagenic primer to create a new restriction site (double underline) for the purpose of screening. Side chains corresponding to amino acid substitutions are also shown.
  • Figure 6 shows a western blot analysis of wild-type and mutant MAO B cDNAs were transfected in COS-7 cells. Expressed wild-type and variant MAO B enzymes were adjusted to equal concentrations based on ELISA results before immunoprecipitation. The immunoprecipitated enzymes were separated on 10% SDS-PAGE, transferred to a nitrocellulose membrane and analyzed by Western blotting using the MAO B specific monoclonal antibody, MAO B-1C2.
  • Lane 1 Prestained MW marker
  • Lane 2 wild-type MAO B
  • Lane 3 Y44F MAO B
  • Lane 4 L46V MAO B
  • Lane 5 Y44A MAO B
  • Lane 6 Y44S MAO B
  • Lane 7 untransfected COS-7 cells
  • Lane 8 Biotinylated MW marker.
  • Figure 7 shows a fluorogram of wild-type and mutant cDNAs were transfected in riboflavin-depleted COS-7 cells with the addition of exogenous [ 14 C] FAD during electroporation.
  • Expressed wild-type and variant MAO B enzymes were adjusted to equal concentrations based on ELISA before immunoprecipitation.
  • the immunoprecipitated enzymes were separated on 10% SDS-PAGE and analyzed by fluorography.
  • Lane 1 [ U C] methylated MW marker
  • Lane 2 wild-type MAO B
  • Lane 3 Y44F MAO B
  • Lane 4 L46V MAO B
  • Lane 5 Y44A MAO B
  • Lane 6 Y44S MAO B
  • Lane 7 untransfected riboflavin-depleted COS-7 cells
  • Lane 8 [ U C] methylated MW marker.
  • Figure 8 shows a model of FAD interacting with three distinct binding sites of MAO B.
  • Amino acids 6-34 comprise the dinucleotide-binding motif in MAO B.
  • the glutamine residue of MAO B at position 34 forms a hydrogen bond with the 2'-hydroxy group of ribose in the AMP moiety of FAD.
  • Cys-397 forms a covalent linkage with the flavin through the 8- ⁇ -methyl group of FAD.
  • a third FAD binding site is shown in which the aromatic moiety of Tyr-44 forms an essential contact with the isoalloxazine moiety of FAD. Variants that lack an aromatic ring at residue 44 show a dramatic decrease in their ability to incorporate FAD and in catalytic activity.
  • the present invention illustrates the structural requirements for binding of FAD to monoamine oxidase B.
  • Replacing glutamate in position 34 with aspartate, glutamine or alanine and replacing valine in position 10 with isoleucine by site-directed mutagenesis had dramatic and unexpected effects.
  • the effect of these amino acid substitutions on monoamine oxidase B activity was demonstrated in COS-7 cells transfected with wild-type and mutant cDNAs encoding the normal and variant enzymes.
  • Glu 34 plays a critical role in the catalytic integrity of monoamine oxidase B and provides increased insight into the structure and function of this ⁇ -a- ⁇ 2 motif.
  • the cDNA clones for monoamine oxidase A and B have been isolated and the nucleotide and deduced amino acid sequences determined.
  • Monoamine oxidase A and B consist of 527 and 520 amino acid residues with calculated subunit molecular weights of 59,700 and 58,800, respectively. These enzymes have a high sequence identity (approximately 70%) but those residues that differ occur at the same place throughout the polypeptide chains, indicating that monoamine oxidase A and B are derived from separate genes rather than by a splicing mechanism.
  • the genes for human monoamine oxidase A and B consist of 15 exons and 14 introns with identical exon-intron organization.
  • Monoamine oxidase A and B contain at least four functional regions, including a FAD-binding site at the amino terminal end that is found in the vast majority of flavin- requiring enzymes.
  • the other functional regions include a region of unknown function near the middle of the molecule, the FAD- covalent binding site toward the C-terminal end and the 28 amino acid residues on the carboxyl terminal end of the protein that anchors the enzymes to the outer mitochondrial membrane.
  • the present invention is directed to a composition of matter that is an inhibitor of monoamine oxidase comprising a modified synthetic flavin adenine dinucleotide compound.
  • the inhibitor of the present invention is effective against both monoamine oxidase A and monoamine oxidase B.
  • the flavin adenine dinucleotide compound disclosed by the instant application is one which interacts with glutamate-34 of monoamine oxidase to disrupt its enzymatic efficacy.
  • the flavin adenine dinucleotide compound is modified by removing a hydroxyl group from its adenosine onophosphate moiety.
  • the hydroxyl group removed is either the 2' hydroxyl group and/or the 3' hydroxyl group.
  • the inhibitor is selected from the group consisting of flavin deoxyadenosine dinucleotide and flavin dideoxyadenosine dinucleotide.
  • the present invention also provides a method of inhibiting monoamine oxidase enzymatic activity in a cell, comprising the step of contacting said cell with a pharmacologically effective dose of a modified synthetic flavin adenine dinucleotide compound.
  • the method of inhibiting monoamine oxidase enzymatic activity is effective against both monoamine oxidase A and B.
  • the present invention also provides a method of treating a brain pathophysiological state in an individual, comprising administering a therapeutically effective dose of a modified synthetic flavin adenine dinucleotide compound to said individual.
  • Representative examples of brain pathophysiological states include depression, Parkinson's disease, phobic-anxiety states, bulemia, post-traumatic reactions, obsessive-compulsive disorders and narcolepsy.
  • compositions may be prepared using the novel modified biosynthetic flavin adenine dinucleotide compound of the present invention.
  • the pharmaceutical composition comprises the novel modified synthetic flavin adenine dinucleotide compound of the present invention and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier A person having ordinary skill in this art would readily be able to determine, without undue experimentation, the appropriate dosages and routes of administration of the novel modified synthetic flavin adenine dinucleotide compound of the present invention.
  • the pharmaceutical composition of the present invention is administered in a dose of from about 0.1 mg/kg to about 20 mg/kg.
  • the following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion.
  • EXAMPLE 1 Site-directed Mutagenesis Mutagenesis was carried out with a "Transformer Site- directed Mutagenesis" kit (Clontech) according to the manufacturer's protocol.
  • a second oligonucleotide (Selection Primer) is used in addition to the mutagenic oligonucleotide (Mutagenic Primer) to introduce a restriction site mutation unique to the plasmid for the purpose of selection.
  • Selection Primer The human monoamine oxidase B cDNA (Bach et al . , 1988, Proc. Natl. Acad. Sci.. 85:4934-4938) was cloned into the EcoRI site of the pBluescript SK vector (Stratagene) .
  • a Hpal restriction site was introduced into the 30-mer "Selection Primer" to replace the Kpnl site in the plasmid vector (5'- CGAGGGGGGGCCCG GGTACCCAATTCGCCC-3 ' to 5 ' - CGAGGGGGGGCCCGGtTAaCCAATTCGCCC-3') .
  • All oligonucleotides were custom synthesized by National Biosciences ( Figure 1) .
  • the glutamate residue in position 34 was replaced with alanine in E34A, glutamine in E34Q, aspartate in E34D, and the valine residue in position 10 was replaced with isoleucine in V10I
  • mutant cDNAs were then subcloned into the pSVK3 expression vector (LKB- Pharmacia) at the EcoRI site and screened for clones with the mutant insert in the sense orientation.
  • This expression vector contains the colEl origin for efficient replication in E. coli, an SV40 origin and the SV40 early promoter. Both the wild-type and mutant plasmid DNAs were purified through CsCl-gradients for transfection studies.
  • EXAMPLE 2 Transfection and Transient Expression Wild-type and mutant monoamine oxidase B cDNAs were transiently transfected into COS-7 cells by electroporation using a method described by Zimmerman et al . , J. Membr. Biol.. 67:165- 182 (1982) . Cells were harvested during late log phase growth and resuspended to a concentration of 3.125 x 10 6 cells/ml in DMEM supplemented with 10% fetal bovine serum (FBS) . Fifteen micrograms of normal or mutant cDNA were added to 0.8 ml (2.5 x 10 6 cells) of cell suspension.
  • FBS fetal bovine serum
  • Electroporation was carried out in a Bio-Rad Gene Pulser with a setting of 0.25 kV and 500 uF. Cells were resuspended in 15 ml of DMEM/FBS and incubated at 37°C with 5% C0 2 for 48 hours.
  • Transfected COS-7 cells were harvested at 48 hours and homogenized in a lysis solution containing 20 mM Tris-HCl, 1.0 mM EDTA, and 0.5 mM phenylmethylsulfonyl fluoride (PMSF).
  • Sucrose was added to a final concentration of 0.25 M followed by centrifugation at 400 xg for 3 minutes.
  • the pellets were resuspended in 20 mM Tris-HCl, 0.5 mM EDTA, 0.5 mM PMSF, 0.25 M sucrose, pH 8.0, and centrifuged as above. The two supernatants were combined for each sample and centrifuged at 50,000 x g for 1 hour in a Beckman Airfuge.
  • Polyclonal antibodies to monoamine oxidase B were obtained from a goat immunized with purified bovine liver monoamine oxidase B.
  • the goat (Sunset Farm, Acadia TX) was given a subcutaneous injection of 0.5 mg of the antigen in Freund's complete adjuvant (Difco) followed by two subsequent injections in Freund's incomplete adjuvant at one week intervals.
  • Three weeks after immunization approximately 300 ml of blood was collected at two week intervals.
  • the blood was allowed to clot and serum subjected to a 50% ammonium sulphate precipitation, then dialyzed against PBS.
  • the antibody was further purified by passage through a CM Affi-Gel blue column (Bio-Rad) .
  • Protein concentrations of samples containing wild-type or mutant monoamine oxidase B were determined by a Micro-BCA kit
  • Monoamine oxidase B activity was assayed radiometrically by a modification of Wurtman and Axelrod, Biochem. Pharmacol. 12:1439-1440 (1963). Briefly, equal amounts of wild-type or mutant monoamine oxidase B (based on ELISA) from mitochondrial subcellular fractions were incubated in the assay mixture containing 0.05 M sodium phosphate buffer, pH 7.4, 3.6 ⁇ mole of 55 Ci/mmole [ U C] benzylamine hydrochloride (Amersham) , and 10 ⁇ moles of unlabeled benzylamine. Sample were run in triplicate, and one out of each set was denatured with 6 N HC1 prior to the addition of substrate to serve as an internal control.
  • the protein-G sepharose/goat antibody/monoamine oxidase B immunocomplex was collected by centrifugation at 10,000 xg for 20 seconds, and washed 6 times with 20 mM Tris buffer, pH 8.0. Bound proteins were eluted with SDS-PAGE sample buffer and subsequently analyzed.
  • the immunoprecipitated proteins (obtained as described above) were subjected to electrophoresis in a 10% SDS- polyacrylamide gel and electrotransferred to a nitrocellulose membrane (S&S) .
  • S&S nitrocellulose membrane
  • the membrane was first blocked with 5% nonfat dry milk, then incubated with mouse monoamine oxidase B-1C2 monoclonal antibody overnight. Secondary antibody (biotin- conjugated goat anti-mouse antibody) was added and the membrane further incubated for 3 hours with streptavidin and biotin- conjugated alkaline phosphatase complex (Bio-Rad) . Following extensive washing, the blot was developed by addition of nitro- blue-tetrazolium/5-bromo-4-chloro-3-indoyl phosphate (Bio-Rad) as substrate.
  • the parameters of wild-type monoamine oxidase B expression under varying conditions were characterized. Optimal electroporation was achieved when 3.1 x 10 6 COS-7 cells/ml were transfected with 15 ⁇ g of cDNA, resulting in the synthesis of approximately 1.3 ⁇ g of monoamine oxidase B per mg of total cellular protein. The use of larger cell numbers did not increase the total amount of monoamine oxidase B obtained and decreased the amount of monoamine oxidase B obtained per cell.
  • EXAMPLE 10 Activity of Wild-Type and Mutant monoamine oxidase B Monoamine oxidase B expressed in COS-7 cells was partially purified, and the amount of monoamine oxidase B for each sample was equalized prior to radiometric activity assays. As shown in TABLE I, the activity in mutants E34D, E34Q, and E34A was very low or undetectable. Of the three mutations at residue 34, only E34D, which is a highly conserved mutation from glutamate to aspartate, maintains any detectable monoamine oxidase B activity. Mutant V10I, is a highly conserved mutation in a region of the ⁇ -a- ⁇ z motif not thought to be critical for enzymatic activity.
  • This mutant was constructed as a control to ensure that the mutagenesis process was capable of producing active mutants, and any loss of catalytic activity in other mutants could be attributed to the identification of a critical residue required for enzymatic activity.
  • the V10I mutant maintained high activity, it was only -65% of that found for wild-type monoamine oxidase B (TABLE I) . Molecular activity measurements were obtained in duplicates for each of a series of three separate experiments.
  • Monoamine oxidase B samples obtained from immunoprecipitation were positively identified using the monoamine oxidase B-specific monoclonal antibody (monoamine oxidase B-1C2) .
  • monoamine oxidase B-1C2 monoamine oxidase B-specific monoclonal antibody
  • wild-type and monoamine oxidase B variants have a band corresponding to 59 kDa
  • untransfected COS-7 cells show no detectable monoamine oxidase B.
  • Wild-type, mutant, and untransfected COS-7 cells all have a faint band at a lower molecular weight (55 kDa) which is attributed to the heavy chain of the goat antibody. A faint band at about 80 kDa was consistently found but the origin of this is unknown.
  • EXAMPLE 12 The deduced amino acid sequences of human liver and placental monoamine oxidase A, bovine adrenal monoamine oxidase
  • Glu 34 plays a crucial role in the dinucleotide binding site of monoamine oxidase B. Based on the crystal structures of glutathione reductase, p-hydroxybenzoate hydroxylase, lipoamide dehydrogenase and other proteins, this site is known to consist of a ⁇ -a- ⁇ z motif that interacts with FAD, and it has a consensus sequence ofAsp-Val-Val-Val-Ile-Gly-x-Gly-x-x-x-Gly-Leu-x-x-Ala-x- x-Leu-x-x-x-x-x-x-x-Val-x-Val -Leu-Glu.
  • polypeptide Gly 11 -x- Gly 13 -x-x-Gly 16 which constitutes a turn between the first ⁇ - sheet and the beginning of the ⁇ -helix, is highly conserved in 28 flavoproteins. Also Ala 20 is highly conserved, and residues at positions 7-10, 12, 15, 17, 23, 30, 32, and 33 are usually hydrophobic. Furthermore, amino acids at position 6 are highly conserved hydrophilic residues, and the motif usually ends with glutamate residue 34.
  • the ⁇ -carboxylate group of glutamate is thought to bind to the 2'-hydroxy group of ribose of the AMP moiety of FAD to align this cofactor for participation in the oxidation-reduction cycle during catalysis of amines to their corresponding aldehydes.
  • the putative structure of the ⁇ - - ⁇ z motif in monoamine oxidase B is shown in Figure 4.
  • the E34A mutant was inactive since the alanine substitution has little structural similarity to the glutamate residue and does not carry a negative charge.
  • the negative charge is appears to be essential for interaction with the AMP moiety of FAD.
  • the glutamine residue in E34Q is closest to the wild-type glutamate in size, but it also lacks a negative charge and resulted in complete loss of activity.
  • the dramatic decrease in activity was unexpected since a negative charge was retained. This could be due to the shorter side chain of the aspartate residue, which results in a loss of contact between its 3-carboxylate group and the 2'- hydroxy group of the ribose in FAD. Assuming that the additional
  • C-C bond length of glutamate is 1.54 A and has a bond angle of
  • a fourth mutant (V10I) was also constructed to serve as a control.
  • This variant protein contained lie 10 in place of Val 10 , which is positioned at the end of the first /3,-sheet before the Gly ⁇ -x-Gly ⁇ -x-x-Gly 16 turn. Analysis of this variant protein showed that it had approximately 65% of the wild type activity.
  • Gin 34 , Ala 34 , or Asp 34 resulted in a dramatic reduction of enzymatic activity.
  • the present invention represents the first illustration of the critical role of selected amino acids in the dinucleotide binding site for generating enzymatic activity.
  • the present invention shows that Glu 34 is a crucial amino acid residue that participates in the function of monoamine oxidase B through non- covalent binding to the AMP moiety of FAD.
  • FAD is covalently bound to Cys 397 .
  • Cys 397 a crucial amino acid residue that participates in the function of monoamine oxidase B through non- covalent binding to the AMP moiety of FAD.
  • FAD is covalently bound to Cys 397 .
  • FAD is positioned in the macromolecule by dual binding to residues Glu 34 and Cys 397 . Because these two residues are located distally in the primary amino acid sequence of monoamine oxidase B, it is unknown whether FAD is first bound to the apoenzyme at the dinucleotide binding site and then delivered to Cys 397 or vice versa.
  • FAD functions as an electron acceptor in the oxidation of amines and is, therefore, likely to constitute part of or be adjacent to the catalytic site of monoamine oxidase. Furthermore, the dinucleotide binding site in the N-terminus of monoamine oxidase B and the site for covalent attachment of FAD towards the C-terminus of monoamine oxidase B are in close proximity through FAD linkage and consequently could form part of the active site for monoamine oxidase B.
  • the substrate binding site(s) in monoamine oxidase remains unknown, but it may be in close proximity to Glu 34 or Cys 397 .
  • site-directed mutagenesis was used to convert Glu 34 in monoamine oxidase B to Asp, Gin, and Ala.
  • the wild-type and mutant cDNAs were then transiently transfected into COS-7 cells. All three variants exhibited a dramatic decrease in enzymatic activity as compared to wild-type monoamine oxidase B, and only the Asp variant retained any detectable activity.
  • the present invention illustrates that the hydrogen bonding between the 7-carboxylate group of Glu 34 and the 2'-hydroxyl group of FAD is very critical for monoamine oxidase B enzymatic activity. Furthermore, FAD initially binds to the dinucleotide binding site and then is delivered through folding of the macromolecule to the covalent binding site.
  • EXAMPLE 13 Design of MAO inhibitors Based on the important role played by Glu 34 ' the present invention discloses novel FAD derivatives act as monoamine oxidase A and/or B inhibitors.
  • One of these FAD derivatives is flavin deoxyadenine dinucleotide (dFAD) , which lacks the critical 2'-hydroxyl group in the AMP moiety of FAD.
  • a second FAD derivative of the present invention is flavin dideoxyadenine dinucleotide (ddFAD) , which lacks both 2'- and 3'-hydroxyl group in the AMP moiety of FAD.
  • ddFAD flavin dideoxyadenine dinucleotide
  • the great advantage of the compounds of the present invention is that they mimic the native FAD structure but lack the critical 2'- and/or 3'-hydroxyl group.
  • the present invention discloses that these FAD derivatives compete with FAD for monoamine oxidase B binding during the synthesis of monoamine oxidase in vivo . If these compounds are coupled into monoamine oxidase B during synthesis of the nascent polypeptide, the newly synthesized monoamine oxidase B would not acquire its catalytic activity due to the loss of a critical hydrogen bonding between the ⁇ -carboxylate group of Glu 34 and the 2'-hydroxyl group of the AMP moiety of FAD. In other words, monoamine oxidase B enzymatic activity would be dramatically inhibited by these FAD analogs.
  • Synthesis of dFAD and ddFAD is carried out by a modified method of DeLuca et al., (1956) J Biol. Chem. 223, 569- 576, which has been used to prepare synthetic FAD for many years.
  • This method involves a direct chemical condensation to form a phosphate diester bond between riboflavin-5'-phosphate (pyridinium salt) and deoxy- (or dideoxy-) adenosine-5'- phosphoramidate under anaerobic conditions.
  • the yellow residue obtained from above is dissolved in distilled water.
  • the solution is adjusted to pH 7.0 with dilute ammonia and carefully applied to a DEAE-cellulose column
  • EXAMPLE IS dFAD or ddFAD as substituted cofactors and inhibitors of monoamine oxidase
  • Rib * and Rib " COS-7 cells which serve as positive and negative controls, respectively. Since dFAD and ddFAD cannot serve as substituted cofactors of monoamine oxidase B, monoamine oxidase
  • Flavin 2'-deoxyadenosine dinucleotide was synthesized as described above. Synthetic dFAD was purified by resolution on HPLC to yield a major sharp peak on the chromatogram. The authenticity of dFAD was confirmed by mass spectrometry and NMR analysis.
  • the present invention shows that Glu-34 is required for catalytic activity, presumably by forming a hydrogen bond between the carboxylate group of glutamate and the 2'-hydroxyl group of ribose in the AMP moiety of FAD.
  • a third FAD binding site in MAO B (residues 39-46) has been identified by sequence comparisons to other flavoenzymes.
  • the conserved sequence contains a tyrosine residue (Tyr-44) which, based on the X-ray crystal structure of ferredoxin-NADP * reductase, is postulated to participate in FAD binding through van der Waals contact with the isoalloxazine ring and a hydrogen bond to the 3'-hydroxy of the ribityl moiety.
  • the homologous sequence occurs in NADPH-sulfite reductase, NADH nitrate reductase, NADPH-cytochrome P-450 oxidoreductase, ferredoxin-NADP * reductase (FNR) , and NADH-cytochrome b5 reductase and is thought to be involved in noncovalent FAD binding in these enzymes.
  • the crystal structure of FNR has been determined, and the entire FAD binding domain was characterized. This domain was shown to consist of an antiparallel /3-barrel not previously observed in other flavoproteins. A short sequence containing a tyrosine residue was shown to reside within a ⁇ - sheet in close proximity to FAD.
  • the tyrosine residue is thought to make extensive van der Waals contact with the isoalloxazine moiety and to form a hydrogen bond with the 3'-hydroxy of the ribityl moiety of FAD. Since the similar sequence in MAO B is in close proximity to the known FAD dinucleotide binding region, it seems possible that the conserved tyrosine (Tyr-44) has a similar function in MAO B. To illustrate that this tyrosine interacts with FAD in MAO B, mutants that encode substitutions at Tyr-44 were prepared and expressed in mammalian COS-7 cells. Substitutions (Tyr to Phe, Ser or Ala) were selected to permit analysis of the aromatic and hydrogen bonding roles of the tyrosine residue with FAD.
  • the aromatic ring of the tyrosine residue is essential for FAD binding and catalytic activity in MAO B, but hydrogen bonding through the hydroxyl group is not critical. Based upon these findings, a model of how FAD interacts with three binding sites in MAO B was constructed, and the sequence of events that occur during the flavinylation process was examined.
  • Mutagenesis was performed by the method of Deng and Nickoloff (1992) using a "Transformer Site-directed Mutagenesis" kit (Clontech) as described above.
  • the mutagenic primers and the corresponding amino acid changes are shown in Figure 5. Tyr at position 44 was replaced with Phe in Y44F, Ser in Y44S and Ala in Y44A.
  • the Leu residue at position 46 was replaced with Val in L46V. All mutagenic primers were designed to create a new restriction site, without altering the coding sequence of any other amino acids, for the purpose of screening.
  • mutants Y44F and L46V were carried out in the pBluescript SK vector (Stratagene) with human MAO B cDNA inserted at the EcoRI site. A Hpal restriction site was introduced into a 30-mer selection primer to replace the only Kpnl site in the plasmid vector.
  • clones were picked from an NZCYM/ampicillin plate and inoculated into a microcentrifuge tube containing 0.5 ml NZCYM/ampicillin and incubated with shaking for 4 hours at 37 C C. An aliquot (50 ⁇ l) of the mini-culture was stored at 4°C for future propagation.
  • the remaining 450 ⁇ l was spun down and processed by the alkaline lysis method in a total volume of 45 ⁇ l.
  • the supernatant was ethanol precipitated and the DNA screened by restriction analysis for the presence of a new restriction site created by the mutagenic primer.
  • the mutant cDNAs were then subcloned into the pSVK3 expression vector (LKB- Pharmacia) at the .EcoRI site and screened for the sense orientation.
  • mutants Y44A and Y44S were carried out directly within the expression vector pSVK3 with human MAO B cDNA inserted into the .EcoRI site.
  • a Ifpal restriction site was introduced into a 29-mer selection primer to replace the only Jtpnl site in the pSVK3 vector.
  • the mutant clones were screened for the presence of the new restriction site created by the mutagenic primer as described above. The presence of the correct mutations in all mutant cDNAs were confirmed by double- stranded dideoxy DNA sequencing. Both wild-type and mutant plasmid DNAs were purified through CsCl-gradients prior to transfection studies.
  • Mammalian COS-7 cells used for MAO B expression were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum (FBS) and 5% C0 2 at 37°C. Riboflavin-depleted COS-7 cells were generated by maintaining these cells in riboflavin- free DMEM-FBS (Gibco) for greater than 100 days. Transient transfection by electroporation (Zimmerman et al., 1982) of wild- type or mutant MAO B cDNAs into COS-7 cells was carried out as described above. Briefly, cells were harvested during late log phase growth and resuspended in DMEM supplemented with 10% FBS.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • C0 2 fetal bovine serum
  • Riboflavin-depleted COS-7 cells were generated by maintaining these cells in riboflavin- free DMEM-FBS (Gibco)
  • Wild-type or mutant cDNA (15 ⁇ g) was then electroporated into the cells (2.5 x 10 6 cells/ 0.8 ml DMEM-FBS).
  • 20 ⁇ l of 0.8 mM [ 14 C] FAD and 15 ⁇ g of MAO B cDNA were simultaneously electroporated into riboflavin-depleted COS-7 cells in riboflavin-free medium (2.5 x 10° cells/0.8 ml).
  • Transfected cells were resuspended in 15 ml of DMEM/FBS (or riboflavin-free DMEM-FBS) and incubated at 37°C with 5% C0 2 .
  • Protein concentrations of samples containing wild-type or variant MAO B were determined by a Micro-BCA kit (Pierce) . All samples were then adjusted to equal protein concentration prior to quantitation of MAO B by ELISA using a modification of the method of Yeomanson and Billett (1992) as described above. Expression levels of wild-type or variant MAO B were determined in duplicate for three separate experiments. EXAMPLE 20
  • MAO B activity was measured by a modification of the method of Wurtman and Axelrod (1963) . This modification allows accurate activity measurements in small amounts of MAO B (as low as 5 ng) expressed in cultured cells.
  • all samples were adjusted to equal protein concentration and 10 ⁇ l of cell lysate (containing wild-type or variant MAO B) was incubated in an assay mixture (200 ⁇ l) containing 0.05 M sodium phosphate buffer, pH 7.4, 3.6 nmole of 55 mCi/mmole [ 14 C] benzylamine hydrochloride, and 10 nmoles of unlabeled benzylamine.
  • controls were assayed concurrently, including cell lysate-free assay buffer, and nontransfected COS-7 cell lysate. After incubation at 37°C for 9 minutes, all reactions were terminated with 6 N HCl (25 ⁇ l) and placed on ice for 2 minutes. The reaction product was extracted with toluene (500 ⁇ l) and centrifuged at 10,000 xg for 5 minutes. The organic phase was counted in liquid scintillation fluid (Bio-Safe) in a Beckman LB3801 model liquid scintillation counter. The activities of wild-type and variant MAO B enzymes were determined in duplicate in three separate experiments.
  • Transfected COS-7 cells were homogenized in 300 ⁇ l of 20 mM Tris HCl, 1 mM EDTA, 0.5 mM PMSF, pH 8.0 and MAO B was extracted with 0.25% Triton X-100 for 30 minutes at 4°C. After centrifugation at 1300 xg for 5 minutes, an aliquot of each supernatant was assayed by ELISA and all supernatants were adjusted to equal MAO B concentrations. The supernatants (300 ⁇ l) were then incubated with 10 ⁇ g of goat polyclonal anti-MAO B antibody overnight at 4 ⁇ C. Protein-G Sepharose beads were added (50 ⁇ l) and the samples were further incubated for 3 hours.
  • the protein-G sepharose/goat antibody/MAO B immunoco plex was collected by centrifugation at 10,000 xg for 20 seconds, and washed 6 times with 20 mM Tris buffer, pH 8.0. The immunocomplex was eluted with SDS-PAGE sample buffer and subsequently analyzed by Western blot or fluorography.
  • the immunoprecipitated proteins (obtained as described above) were subjected to electrophoresis in a 10% SDS- polyacrylamide gel and analyzed by Western blotting as described above.
  • Immunoprecipitated wild-type and variant MAO B were subjected to electrophoresis in a 10% SDS-polyacrylamide gel.
  • the gel was fixed in 7% acetic acid, 10% methanol and 83% H 2 0 for 1 hour and processed for fluorography as described by Bonner and Laskey (1974) .
  • the dried gel was exposed to Kodak X-OMAT AR film at -80°C.
  • MAO B was further identified by Western blotting. Wild-type and variant MAO B were adjusted to equal concentrations based on ELISA results, and immunoprecipitated with goat anti-MAO B polyclonal antibodies. The immunoprecipitated wild-type and variant MAO B enzymes were positively identified using a monoclonal antibody specific to MAO B (MAO B-1C2) . As shown in Figure 6, wild-type and variant MAO Bs have a band of equal intensity corresponding to 59 kDa, whereas the untransfected COS-7 cells show no MAO B band.
  • the activity of expressed wild-type and variant MAO B was determined in duplicate for three separate experiments by a radiometric assay using benzylamine as substrate. Substitution of Tyr-44 with phenylalanine in Y44F resulted in only a slight decrease (93% of wild-type) in enzymatic activity (Table II) . However, substitutions of tyrosine to serine and alanine in Y4 S and Y44A, respectively, resulted in a dramatic loss of MAO B activity. A fourth mutant, L46V, was constructed to serve as a control. This mutant cDNA encodes a variant protein containing Val-46 in place of Leu-46 at a site near the critical tyrosine residue.
  • Val-46 was not highly conserved among the other flavoenzymes and has not been postulated to play a role in FAD binding, a mutation at this site was not expected to have a dramatic effect on MAO B activity.
  • the L46V variant showed only a slight loss of enzymatic activity (83% of wild- type) .
  • the enzymatic activities (umol/min/mg MAO B) of all the variants closely correlated with their specific activities ( ⁇ mol/min/mg protein) .
  • MAO B variants retaining the aromatic moiety at position 44 retained enzymatic activity, whereas those without the aromatic moiety (Y44S and Y44A) were nearly devoid of enzymatic activity.
  • riboflavin-depleted COS-7 cells were produced by maintaining cells in riboflavin-free medium for greater than 100 days. Wild-type MAO B cDNA was transfected into these cells at 1 week intervals during the process of riboflavin depletion to monitor the effect on MAO B expression and activity. Expression levels of MAO B remained constant (approximately l ⁇ g/mg protein) throughout the process of riboflavin depletion. MAO B activity in sequential transfections, however, decreased rapidly as the endogenous riboflavin was depleted from the COS-7 cells.
  • transiently expressed apo-MAO B was completely devoid of activity.
  • Cells grown continuously in riboflavin-free medium for greater than five months showed no detectable change in morphology. Furthermore, trypan blue staining did not detect the presence of damaged cells.
  • MAO B is devoid of an FAD cofactor. Flavinylation of wild-type and variant MAO B enzymes was studied in these cells by simultaneous electroporation of [ 14 C] FAD with MAO B cDNA. The expressed enzymes were first adjusted to equal MAO B concentrations based on ELISA, followed by immunoprecipitation using goat anti-MAO B polyclonal antibodies. The immunoprecipitated MAO B was then subjected to SDS-PAGE and analyzed by fluorography. The amount of [ U C] FAD incorporated into wild-type or variant MAO B was determined by the intensity of banding on the fluorogram.
  • the wild-type and variants L46V and Y44F were capable of incorporating [ 14 C] FAD, as observed by dark bands of equal intensity at a molecular weight of about 59 kDa.
  • Y44S and Y44A which do not contain an aromatic moiety at position 44, showed only very faint bands.
  • Binding of Tyr-44 to the isoalloxazine ring requires an aromatic-aromatic interaction.
  • the analogous tyrosine in FNR appears on the si-face of the isoalloxazine ring tilted at an angle of approximately 53°.
  • Such a geometric arrangement allows the hydrogen atoms on the edge of the aromatic ring to approach the p-electron cloud of the isoalloxazine ring.
  • Burley and Petsko (1988) found that such edge-to-face interactions are approximately -1.5 Kcal/mol of stabilization energy, which is enthalpically favored over face-to-face aromatic stacking. Furthermore, they demonstrated that an interplanar angle of 55° is an enthalpically optimal geometric arrangement.

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Abstract

La présente invention concerne un inhibiteur de la monoamine oxydase comprenant un composé synthétique modifié du flavine-adénine-dinucléotide. L'invention concerne également un procédé pour inhiber l'activité enzymatique de la monoamine oxydase dans une cellule, lequel consiste entre autres à mettre en contact ladite cellule avec une dose pharmacologiquement efficace d'un composé synthétique modifié du flavine-adénine-dinucléotide. L'invention concerne encore un procédé de traitement d'un état cérébral physiopathologique chez un individu, consistant à administrer une dose thérapeutiquement efficace d'un composé synthétique modifié du flavine-adénine-dinucléotide audit individu.
PCT/US1995/017106 1994-12-29 1995-12-29 Analogues du flavine-adenine-dinucleotide, leurs compositions pharmaceutiques et leur activite en tant qu'inhibiteurs de la monoamine oxydase WO1996020946A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1997044483A1 (fr) * 1996-05-17 1997-11-27 University Of Leicester Ensemble electroaccepteur - enzyme
GB2330143A (en) * 1996-05-17 1999-04-14 Univ Leicester Enzyme-electron acceptor assembly
WO2008055945A1 (fr) 2006-11-09 2008-05-15 Probiodrug Ag Dérivés 3-hydr0xy-1,5-dihydr0-pyrr0l-2-one utiles en tant qu' inhibiteurs de la glutaminyl-cyclase dans le traitement des ulcères, du cancer et d'autres maladies
WO2008065141A1 (fr) 2006-11-30 2008-06-05 Probiodrug Ag Nouveaux inhibiteurs de glutaminylcyclase
WO2008104580A1 (fr) 2007-03-01 2008-09-04 Probiodrug Ag Nouvelle utilisation d'inhibiteurs de la glutaminyl cyclase
US7732162B2 (en) 2003-05-05 2010-06-08 Probiodrug Ag Inhibitors of glutaminyl cyclase for treating neurodegenerative diseases
WO2011029920A1 (fr) 2009-09-11 2011-03-17 Probiodrug Ag Dérivés hétérocycliques en tant qu'inhibiteurs de glutaminyle cyclase
WO2011107530A2 (fr) 2010-03-03 2011-09-09 Probiodrug Ag Nouveaux inhibiteurs
WO2011110613A1 (fr) 2010-03-10 2011-09-15 Probiodrug Ag Inhibiteurs hétérocycliques de la glutaminyl cyclase (qc, ec 2.3.2.5)
WO2011131748A2 (fr) 2010-04-21 2011-10-27 Probiodrug Ag Nouveaux inhibiteurs
WO2012123563A1 (fr) 2011-03-16 2012-09-20 Probiodrug Ag Dérivés de benzimidazole en tant qu'inhibiteurs de la glutaminyl cyclase
EP2865670A1 (fr) 2007-04-18 2015-04-29 Probiodrug AG Dérivés de thio-urée utilisés comme inhibiteurs de la glutaminyl cyclase
EP3461819A1 (fr) 2017-09-29 2019-04-03 Probiodrug AG Inhibiteurs de la glutaminyl-cyclase

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Title
BIOCHEM. BIOPHYS. RES. COMM., Volume 173, Number 3, issued 31 December 1990, WEYLER et al., "Catalytically Active Monoamine Oxidase Type A from Human Liver Expressed in Saccharomyces Cerevisiae Contains Covalent FAD", pages 1205-1211. *
BIOCHEMICAL JOURNAL, Volume 242, issued 1987, BARBER et al., "Anti-Flavin Antibodies", pages 89-95. *
BIOCHEMICAL PHARMACOLOGY, Volume 37, Number 18, issued 1988, GOMEZ et al., "A Comparative Study of Some Kinetic and Molecular Properties of Microsomal and Mitochondrial Monoamine Oxidase", pages 3407-3413. *
BIOCHIM. BIOPHYS. ACTA, Volume 89, issued 1964, McCORMICK et al., "Coenzyme Specificity of D-Amino Acid Oxidase for the Adenylate Moiety of FAD", pages 447-452. *
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J. CLIN. INVEST., Volume 67, Number 4, issued May 1981, PINTO et al., "Inhibition of Riboflavin Metabolism in Rat Tissues by Chlorpromazine, Imipramine and Amitriptyline", pages 1500-1506. *

Cited By (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB2330143A (en) * 1996-05-17 1999-04-14 Univ Leicester Enzyme-electron acceptor assembly
GB2330143B (en) * 1996-05-17 2000-04-05 Univ Leicester Enzyme-electron acceptor assembly
WO1997044483A1 (fr) * 1996-05-17 1997-11-27 University Of Leicester Ensemble electroaccepteur - enzyme
US7732162B2 (en) 2003-05-05 2010-06-08 Probiodrug Ag Inhibitors of glutaminyl cyclase for treating neurodegenerative diseases
US8809010B2 (en) 2003-05-05 2014-08-19 Probiodrug Ag Method for prophylactic treatment of alzheimer's disease using inhibitors of glutaminyl cyclase and glutamate cyclases
WO2008055945A1 (fr) 2006-11-09 2008-05-15 Probiodrug Ag Dérivés 3-hydr0xy-1,5-dihydr0-pyrr0l-2-one utiles en tant qu' inhibiteurs de la glutaminyl-cyclase dans le traitement des ulcères, du cancer et d'autres maladies
WO2008065141A1 (fr) 2006-11-30 2008-06-05 Probiodrug Ag Nouveaux inhibiteurs de glutaminylcyclase
EP2481408A2 (fr) 2007-03-01 2012-08-01 Probiodrug AG Nouvelle utilisation d'inhibiteurs glutaminyle cyclase
WO2008104580A1 (fr) 2007-03-01 2008-09-04 Probiodrug Ag Nouvelle utilisation d'inhibiteurs de la glutaminyl cyclase
EP2865670A1 (fr) 2007-04-18 2015-04-29 Probiodrug AG Dérivés de thio-urée utilisés comme inhibiteurs de la glutaminyl cyclase
WO2011029920A1 (fr) 2009-09-11 2011-03-17 Probiodrug Ag Dérivés hétérocycliques en tant qu'inhibiteurs de glutaminyle cyclase
WO2011107530A2 (fr) 2010-03-03 2011-09-09 Probiodrug Ag Nouveaux inhibiteurs
WO2011110613A1 (fr) 2010-03-10 2011-09-15 Probiodrug Ag Inhibiteurs hétérocycliques de la glutaminyl cyclase (qc, ec 2.3.2.5)
WO2011131748A2 (fr) 2010-04-21 2011-10-27 Probiodrug Ag Nouveaux inhibiteurs
WO2012123563A1 (fr) 2011-03-16 2012-09-20 Probiodrug Ag Dérivés de benzimidazole en tant qu'inhibiteurs de la glutaminyl cyclase
EP3461819A1 (fr) 2017-09-29 2019-04-03 Probiodrug AG Inhibiteurs de la glutaminyl-cyclase

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