EP1239863A2 - Nouveaux inhibiteurs de glycosidases et leurs applications pharmacologiques, notamment pour traiter le diabete - Google Patents

Nouveaux inhibiteurs de glycosidases et leurs applications pharmacologiques, notamment pour traiter le diabete

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Publication number
EP1239863A2
EP1239863A2 EP00990069A EP00990069A EP1239863A2 EP 1239863 A2 EP1239863 A2 EP 1239863A2 EP 00990069 A EP00990069 A EP 00990069A EP 00990069 A EP00990069 A EP 00990069A EP 1239863 A2 EP1239863 A2 EP 1239863A2
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Prior art keywords
polyamine
approximately
spermidine
advantageously
enzyme
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German (de)
English (en)
French (fr)
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Nushin Banu Hélène AGHAJARI
Xavier Guy Robert
Richard Michel Haser
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Centre National de la Recherche Scientifique CNRS
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Centre National de la Recherche Scientifique CNRS
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    • 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
    • A61K31/7008—Compounds having an amino group directly attached to a carbon atom of the saccharide radical, e.g. D-galactosamine, ranimustine
    • 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
    • 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
    • A61K31/13—Amines
    • 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
    • A61K31/13—Amines
    • A61K31/131—Amines acyclic
    • 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
    • A61K31/16—Amides, e.g. hydroxamic acids
    • 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
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/197—Carboxylic acids, e.g. valproic acid having an amino group the amino and the carboxyl groups being attached to the same acyclic carbon chain, e.g. gamma-aminobutyric acid [GABA], beta-alanine, epsilon-aminocaproic acid or pantothenic acid
    • A61K31/198—Alpha-amino acids, e.g. alanine or edetic acid [EDTA]
    • 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
    • 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
    • A61K31/7016—Disaccharides, e.g. lactose, lactulose
    • 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
    • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/46—Hydrolases (3)
    • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the subject of the invention is new glycosidase inhibitors and their pharmacological applications, in particular for treating diabetes.
  • glycosyl hydrolases some are responsible for the breakdown or digestion of sugars. Some of these enzymes, like ⁇ -amylases, are very important at the biotechnological level (bio-industries of detergents, derivatives and transformations of starch %), but also as targets of molecules of pharmacological interest, for example for the treatment of diabetes.
  • amylases are hydrolytic enzymes that are widely used in nature; they are found in particular in animals, microbes, plants and fungi. These enzymes are involved in the degradation of sugar oligosaccharides, such as starch and glycogen, by hydrolysis of the ⁇ -1,4 interglycosidic bonds (for ⁇ -amylases). Barley seeds contain two main isoenzymes of ⁇ -amylase, AMY1 and AMY2, which are both involved in the breakdown of starch to provide energy for the development of the plant embryo.
  • the function of these isoenzymes is to catalyze the transformation of polysaccharides (starch, various sugars %) at various stages of germination, with a view to the production of sugars assimilable by the plant for its physiological and energy needs.
  • the research made it possible to establish the detailed architecture of these proteins, as well as the precise topology of the active sites where the reactions catalyzed by these enzymes take place.
  • One of the major objectives is to better understand the remarkable differences in physicochemical properties of these two isoenzymes, and this despite their very strong sequence homology (almost 80% identity).
  • Inhibitors of the acarbose type (polysaccharide in nature) have proven themselves, especially in the treatment of non-insulin dependent diabetes. They are available on the market, now in many countries. Like any drug, these molecules are not devoid of any side effect, hence the interest of exploring other ways.
  • spermidine and other neighboring polyamines
  • polyamines interact with DNA.
  • One of the aspects of the invention is to propose a new class of glycosidase inhibitors, and in particular of ⁇ -amylases devoid of the side effects of the inhibitors known to date, and non-toxic.
  • Another aspect of the invention is to propose new glycosidase inhibitors whose production cost is lower than that required to prepare the inhibitors known to date.
  • the invention in general relates to the use of a polyamine-like molecule, a polyamine derivative or a polyamine to inhibit the active site of the glycosidases involved in the transformation of polysaccharides. in sugars, especially glucose, in a living organism.
  • polyamine-like molecule is meant any molecule belonging to the chemical superfamily of polyamines, which are molecules containing at least two amino functions.
  • polyamine derivative is meant any molecule belonging to the chemical superfamily of polyamines, but containing chemical modifications and / or grafted chemical functions not belonging to the chemical superfamily of polyamines.
  • the invention also relates to the use of a molecule of the polyamine type, of a polyamine derivative or of a polyamine for inhibiting in vitro the active site of the glycosidases involved in the transformation of polysaccharides into sugars, in particular into glucose, in a living organism.
  • the invention relates to the demonstration of a new class of ⁇ -glycosidase inhibitors, of polyamine type, therefore of chemical nature radically different from the inhibitors currently used in pharmacology for the treatment of diabetes and also of interest for the treatment of other metabolic disorders, such as obesity.
  • the representative of this new class of agents is a natural polyamine, spermidine: NH 2 - (CH 2 ) 4 -NH- (CH 2 ) 3 -NH 2 .
  • polyamines have an inhibitory activity on glycosidases, because of their affinity with respect to the enzymatic target constituted by glycosidases, in particular ⁇ -amylases.
  • a spermidine-type polyamine and a glycosidase, for example the isoenzyme 1 of barley ⁇ -amylase (AMY 1).
  • the polyamine inhibitor is preferentially recognized by the enzymatic target, demonstrating that it has a significantly higher affinity for the enzyme compared to acarbose-type inhibitors (of polysaccharide nature) currently on the market.
  • living organism denotes man as well as animals or plants.
  • the inhibition activity mentioned above is exerted both in vitro and in vivo, and involves an enzyme of the ⁇ -amylase type, therefore belonging to the family of ⁇ -glycosidases present and operational in all organisms. living.
  • the invention relates to the use of a polyamine derivative or of a polyamine for the preparation of a medicament intended for the diagnosis, prevention or treatment of pathologies involving metabolic disorders linked to glycosidases, and more particularly a deregulation of intestinal glucose absorption, such as non-insulin dependent diabetes, obesity, hyperglycemia, or hyperlipidemia.
  • a polyamine derivative or of a polyamine for the preparation of a medicament intended for the diagnosis, prevention or treatment of pathologies involving metabolic disorders linked to glycosidases, and more particularly a deregulation of intestinal glucose absorption, such as non-insulin dependent diabetes, obesity, hyperglycemia, or hyperlipidemia.
  • the invention relates to the use of polyamines, which comprise at least 2 positive charges, in particular at least 3 amino functions, and where appropriate at least 1 linear or branched osidic (or saccharide) function, said positive charges, in particular said amino functions being spaced apart by carbon chains the length of which is from approximately 2 carbon atoms to approximately 8 carbon atoms, in particular from approximately 3 carbon atoms to approximately 5 carbon atoms.
  • the distance between two adjacent positive charges, in particular between two amino functions is approximately 4 ⁇ to approximately 7 ⁇ .
  • the distance between the positive charges carried by two adjacent amino functions along the chain is approximately 5 ⁇ for N6-N10 and approximately 6.5 ⁇ for N1-N6.
  • the assembly advantageously forms a linear or branched chain of 7 to 19 carbon atoms, and preferably of 9 to 15 carbon atoms.
  • the invention also relates to a complex between a polyamine and a glycosidase enzyme, in particular glycosyl hydrolase and more particularly ⁇ -amylase, present in all living organisms and responsible for the reactions of transformation and hydrolysis of oligosaccharides and polysaccharides into osidic molecules.
  • a glycosidase enzyme in particular glycosyl hydrolase and more particularly ⁇ -amylase
  • simpler ones like maltose and glucose in which the polyamine is fixed at the level of the active site of the enzyme, in particular by hydrogen bonds bringing into play the positive charges of the polyamine, corresponding to its amino functions, and the carboxylic functions side chains of amino acids of the above-mentioned enzyme, the number of hydrogen bonds advantageously being at least 4.
  • the invention relates to a crystal complex between a polyamine and a glycosidase enzyme.
  • active site of the enzyme is meant the specific region of the enzyme involved in the binding of a glucose type unit belonging to the oligosaccharide or polysaccharide fixed by the enzyme. For example, a tetrasaccharide binding to the enzyme in the active site will occupy four subsites.
  • At least two of the sub-sites of the active site of the enzyme are involved in the binding with the above-mentioned polyamine.
  • enzyme active site sub-site is meant a division of the active site of the enzyme corresponding, under physiological conditions, to the attachment of a single osidic unit of a polysaccharide.
  • the glycosidase enzyme when in the complex of the invention, is ⁇ -amylase, in particular barley ⁇ -amylase (AMY 1), the following four amino acids of the enzyme : Glu (205), Trp (207), Asn (209), Asp (180) are involved in the binding with polyamine.
  • the complex of the invention in particular between ⁇ -amylase and spermidine, can be characterized by at least one of the following interactions and in particular by all of the following interactions, which are hydrogen type bonds and which can be defined as indicated below:
  • “Wat” is an abbreviation corresponding to a molecule of water, in the environment of the enzyme, and is referenced by an arbitrary number (see Figure 1).
  • the complex of the invention in particular between ⁇ -amylase and spermidine can be characterized by the following additional interactions: Additional interactions (second level): Distances ( ⁇ ):
  • the expression “second level interaction” denotes, for example, weaker interactions than the hydrogen bonds, for example the Van der Waals interactions as well as the hydrogen bonds which do not directly participate
  • the polyamine is a chain from approximately 6 to approximately 20 atoms, in particular comprising from approximately 6 to 15, and advantageously from approximately 6 to 10 nitrogen atoms , advantageously two primary amino functions, respectively at each of the ends of the polyamine, at least one of the amino functions being optionally substituted by a substituent chosen from, linear or cyclic polysaccharides having, from approximately 1 to approximately 6 osidic units advantageously glucose, maltose, or cyclodextrin, and at least one of the nitrogen atoms inside the chain being optionally substituted by a substituent chosen from oligosaccharides, linear or cyclic, from 1 to 6, in particular 1 to 3 sugar units, in particular glucose, maltose or cyclodextrin.
  • the enzyme is ⁇ -amylase and the polyamine is chosen from spermidine and its derivatives and corresponding to one of the following general formulas:
  • R ! H, (CHR 3 ) y - [NH- (CHR 3 ) z ] r NHR 4
  • R 2 (CHR 3 ) y - [NH- (CHR 3 ) J r NHR 4
  • R 3 H, alkyl, aryl, alkenyl, ⁇ -carboxyalkyl
  • Boc butyloxycarbonyl, the length of the alkyl chains between the nitrogen atoms being able to vary from approximately 2 to approximately 8 carbons, and in particular from approximately 3 to approximately 5 carbons, the alkyl chains possibly also being substituted by chemical groups preferentially comprising an amino or derivative function, the alkyl chains also being able to contain nitrogen atoms other than those represented on the formulas above.
  • the invention also relates to the new polyamines, capable of entering into the constitution of the complex defined above and in particular constituted by a chain of approximately 6 to approximately 20 atoms, in particular comprising from approximately 6 to approximately 15, and advantageously d '' about 6 to about 10 nitrogen atoms, advantageously two primary amino functions, respectively at each of the ends of the polyamine, at least one of the amino functions being optionally substituted by a substituent chosen from, linear or cyclic polysaccharides having d '' approximately 1 to approximately 6 sugar units advantageously glucose, maltose, or cyclodextrin and at least one of the nitrogen atoms inside the chain being optionally substituted by a substituent chosen from linear or cyclic oligosaccharides of 1 to 6 units, in particular from 1 to 3 sugar units, in particular glucose, maltose or cyclodextrin - provided that the polyamine is different from the following products: spermidine spermine
  • the invention therefore also relates to the new polyamines defined above, capable of interacting with the active site of the target enzyme, according to a mode of interaction similar to that observed experimentally in the crystalline state with spermidine in contact with the enzyme under consideration.
  • the invention relates in particular to the polyamine derivatives of the following general formula:
  • R ! H, (CHR 3 ) y -
  • R 2 (CHR 3 ) y - [NH- (CHR 3 ) J r NHR 4
  • R 3 H, alkyl, a ⁇ yl, alkenyl, ⁇ -carboxyalkyl
  • polyamines of the invention one of the functions of which is substituted by one or more osidic units, and designated by glycoconjugates can be synthesized by conventional methods in glycochemistry (oxidation, reductive amination, peptide coupling, etc.).
  • FIG. 1 represents on the one hand, the numbering of the atoms of the spermidine molecule, and on the other hand the diagram of the interactions between the AMY 1 residues and the spermidine molecule in the AMY 1 / spermidine complex of the invention .
  • Figure 2 shows the electron density function (determined at a resolution of 2.44 ⁇ ) of the crystal of the AMY1 / spermidine complex in the region of the active site.
  • the elongated volume of electronic density corresponds to the binding of a spermidine molecule (NH 2 - (CH 2 ) 3 -N ⁇ - (CH 2 ) 4 -NH 2 ,) in the active region of the enzyme.
  • Figure 3 shows the interactions of the spermidine molecule with the active site of ⁇ -amylase.
  • the affinity of the polyamine for the enzyme is largely due to the interactions by hydrogen bonds with the three nitrogen atoms.
  • Figure 4 represents the superimposition of the experimental complex: AMY1 / spermidine (clear “stick” model) and of the model generated by the molecular modeling and dynamic calculations for the pig pancreas ⁇ / amermase / spermidine (dark “stick” model). ). To simplify the diagram, the water molecules are not shown.
  • Figure 5 represents the superimposition of the experimental complex: AMY1 / spermidine (clear "stick” model) and the model generated by molecular modeling and dynamic calculations for human salivary ⁇ -amylase / spermidine (dark “stick” model).
  • the water molecules are not shown. Only residues interacting with spermidine are shown. The nitrogen of spermidine (linear molecule in the center) are shown by balls (NI, N6 and N10). The residues numbered in italics belong to AMYl.
  • Figure 6 represents the superimposition of the experimental complex: AMYl / spermidine (clear "stick” model) and the model generated by molecular modeling and dynamic calculations for human pancreas ⁇ / amermase / spermidine (dark "stick” model) .
  • the water molecules are not shown. Only residues interacting with spermidine are shown. The nitrogen of spermidine (linear molecule in the center) are shown by balls (NI, N6 and N10). The residues numbered in italics belong to AMYl.
  • Figure 7 represents the superposition of 3 cycles of the acarbose-inhibiting pseudo-tetrasaccharide (represented by the dark “stick” model) and a spermidine molecule (clear “ball and stick” model) in their respective configuration within the active site of a barley ⁇ -amylase.
  • FIG. 8 represents a planar formula of the 3 cycles mentioned above corresponding to a molecule of acarbose after cutting of the ⁇ -1,4-interglycosidic bond leading to the loss of the glucose unit at the reducing end.
  • the 9 C-terminal residues of the recombinant isoenzyme 1 of barley ⁇ -amylase (AMY1) were cut in order to obtain a C-terminal end of the same length as that of isoenzyme 2 (AMY2).
  • the protein was then overexpressed in Pischia pastoris, purified and concentrated.
  • the protein preparation used for crystallization has a concentration of 5.1 mg / ml and is found in a solution of 10 mM MES (2- [N] -Morpholino ethane sulfonic acid), 100 mM CaCl 2 , 0.02% NaN 3 , pH 6.7.
  • the crystals were obtained by co-crystallization using the vapor diffusion principle using the suspended drop technique. To do this, to 2 ⁇ l of the protein solution described above, 2.5 ⁇ l of 21% polyethylene glycol 8000 at 21% were added as precipitant and 0.5 ⁇ l of a 0.1M spermidine solution as crystallization additive. This drop was balanced at 19 ° C against a reservoir containing 500 ⁇ l of 21% polyethylene glycol 8000. The crystals were then soaked for 20 hours in a 21% polyethylene glycol 8000 solution containing 10 mM of acarbose (a pseudotetrasaccharide).
  • Diffraction data collection The diffraction data collection was carried out using an X-ray generator (CuK ⁇ radiation - wavelength 1.5418 ⁇ ) with a rotating anode (Nonius 581) operating at 40kV and 90 mA (i.e. 3.6 kW) with a graphite monochromator coupled to a two-dimensional Image Plate detector (MarResearch 345) with a diameter of 34.5 cm.
  • a set of 180 diffraction images (each corresponding to 1 ° of oscillation of the crystal) was collected at 15 ° C, the crystal being mounted in a capillary and positioned 120 mm from the detector, with a time of exposure of the crystal to 500s x-rays per shot. The highest resolution for this dataset is 2.44 ⁇ .
  • the molecular replacement method was used thanks to the software AmoRe (Navaza, 1994) with as a guide model the structure of the isoenzyme 2 of barley ⁇ -amylase (AMY2 - accession code PDB: 1AMY (Kadziola et al., 1994 and Kadziola et al., 1998) due to the significant sequence homology (80%) between this initial model and our protein of interest (AMYl).
  • the structure considered is characterized by an R factor of 17.2% and a free R factor of 20.3% (the latter being based on 10% of diffraction data randomly selected - Briinger, 1992). Additional statistics regarding this structure are presented in the table below.
  • glycopolyamine type molecules which integrate possibilities for various interactions with glycosidases, on the one hand thanks to the positive charges (NH groups) of the polyamine skeleton, on the other hand thanks also to the interactions between the grafted sugars with aromatic side chains of the active site.
  • the following molecules which are spermidine-maltooligosaccharide conjugates and whose synthesis protocols are detailed below, represent good candidates, inhibitors of ⁇ -glycosidases:
  • the 3 N ⁇ N'-diprotected spermidine is obtained in three stages from the monoprotected putrescine 1.
  • the first stage is a Michael-type addition of putrescine on acrylonitrile thus generating the corresponding nitrile.
  • the secondary amine function of derivative 2 is then protected by a tert-butyloxycarbonyl group.
  • Protected spermidine 4 N ⁇ di ⁇ -di is obtained in three stages from putrescine.
  • the first step is an alkylation with 4-chloro-butan-1-ol, followed by the protection of the two amino functions by tert-butyloxycarbonyl groups.
  • spermidine 5 N ⁇ ⁇ ⁇ -protected is directly obtained from spermidine by treatment with tert-butyloxycarbonyloxyimino-2-phenylacetonitrile (Boc-on) for 1 h at 0 ° C (Hesse et a, 1996).
  • the oligosaccharide 6 (35 mmol) is dissolved in a water-methanol mixture (36 mL, [1: 3]), then is then added a solution of iodine (17 g) in methanol (240 mL) and heated to 40 ° C. A solution of potassium hydroxide (16 g) in methanol (400 ml) is then added and the reaction mixture is vigorously stirred at 40 ° C for 35 minutes. At the end of this period, the disappearance, the coloring and the appearance of a white-yellow precipitate are observed. The reaction mixture is then cooled in an ice bath and the suspension is filtered through a buchner, then rinsed with cold methanol and then with ethyl ether.
  • the solid collected is taken up in a minimum of water and then precipitated again by adding methanol.
  • the solid is then dried, taken up in water and then lyophilized, to give the oxidized oligosaccharide in the form of potassium salt 7 (Kobayashi et al, 1985).
  • the acid salt in water solution 60 mL is treated with Amberlite IRN resin
  • the spermidine conjugate 8 (2 mmol) is treated with trifluoroacetic acid in water (30 mL, [9: 1]) for 30 n at room temperature. The reaction mixture is then concentrated in vacuo and the solid is taken up in water and washed 3 times with ethyl acetate. The aqueous phase is then lyophilized to give the spermidine conjugate
  • the 3 structures were first superimposed with that of the AMYl / spermidine complex. This superposition was carried out by superposing the ⁇ carbons of the 3 catalytic residues of each of these structures, so as to make the active sites of these 4 structures coincide as well as possible.
  • the spermidine molecule In the active site of each of the ⁇ -amylases considered, the spermidine molecule has been modeled in its original conformation, retaining the interactions observed for the AMYl / spermidine complex. All the manipulations of the structures (superposition and insertion of the spermidine into the active site) were carried out with the TURBO-FRODO software (Roussel et al, 1991).
  • This step was to minimize the energy of the system, firstly, by displacing the atoms which could establish bad contacts between them (in particular the water molecules which had not been displaced during the insertion of the spermidine. in the active site), but also to position the atoms so as to maximize the interactions by hydrogen bond between them and thus go towards a model of structure most stable energetically.
  • amylase-spermidine does not reveal bad contacts between the spermidine molecule and the respective enzymes.
  • each amino acid residue, each water molecule but also the spermidine inserted are positioned respecting an optimal stereochemistry to ensure adequate inhibition. of the system.
  • the number of direct hydrogen bonds between spermidine and a residue or a water molecule is given in the following table for the experimental complex AMYl / spermidine and for the 3 calculated models: pig pancreas ⁇ -amylase / spermidine, ⁇ - human salivary amylase / spermidine and human pancreatic ⁇ -amylase / spermidine.
  • spermidine is capable, according to these models, of carrying out 4 or 5 direct hydrogen bonds with a residue of the ⁇ -amylases studied and of making 1, 6 or 7 hydrogen bonds with water molecules present in the active site, themselves stabilized by other hydrogen bonds.
  • Acarbose a pseudo-tetrasaccharide (an anti-diabetes drug (GLUCOR ® from Bayer) on the market), binds to amylases to block the active site, as has been shown with different amylases of various origins, and in particular the porcine enzyme.
  • Figure 8 shows a truncated acarbose molecule at the reducing end (right of the figure), after switching off the terminal glucose unit.
  • PROCHECK a program to check the stereochemistry of protein structures. J. Appl. Cryst. 26, 283-291.

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EP00990069A 1999-12-23 2000-12-20 Nouveaux inhibiteurs de glycosidases et leurs applications pharmacologiques, notamment pour traiter le diabete Withdrawn EP1239863A2 (fr)

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FR9916409A FR2802817B1 (fr) 1999-12-23 1999-12-23 Nouveaux inhibiteurs de glycosidases et leurs applications pharmacologiques, notamment pour traiter le diabete
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FR2802817B1 (fr) 2002-10-11
AU2687301A (en) 2001-07-09
JP2003518501A (ja) 2003-06-10
US20030143713A1 (en) 2003-07-31
WO2001047528A3 (fr) 2002-06-20
FR2802817A1 (fr) 2001-06-29
CA2395305A1 (fr) 2001-07-05
WO2001047528A2 (fr) 2001-07-05

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