Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/0004—Oxidoreductases (1.)
  • C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
  • C12N9/0073—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14) with NADH or NADPH as one donor, and incorporation of one atom of oxygen 1.14.13
  • C12N9/0075—Nitric-oxide synthase (1.14.13.39)
• • 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
  • A61K31/33—Heterocyclic compounds
  • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
  • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
  • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
  • A61K31/513—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim having oxo groups directly attached to the heterocyclic ring, e.g. cytosine
• • 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
  • A61P3/08—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
  • A61P5/00—Drugs for disorders of the endocrine system
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
  • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
  • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2500/00—Screening for compounds of potential therapeutic value
  • G01N2500/02—Screening involving studying the effect of compounds C on the interaction between interacting molecules A and B (e.g. A = enzyme and B = substrate for A, or A = receptor and B = ligand for the receptor)

Definitions

• the invention relates to a novel method for screening inhibitors of the binding between the neuronal nitric oxide synthase protein (nNOS) and the neuronal nitric oxide synthase inhibiting protein (PIN).
• nNOS neuronal nitric oxide synthase protein
• PIN neuronal nitric oxide synthase inhibiting protein
• the invention also relates to the pancreatic form of the protein nNOS of rats and of any animal species including man, as well as the nucleic acid coding for said protein.
• the invention also relates to the use of proteins or peptides for the preparation of medicaments intended for the treatment of prediabetic or hyperinsulinic conditions.
• NOS nitric oxide synthase
• eNOS endothelial NOS
• nNOS neuronal NOS
• Neural NOS was first identified and cloned by DS Bredt in the rat brain in 1991 (Bredt et al, 1991). At the level of the central nervous system, it has a role in the phenomenon of long-term memorization (LTP), ⁇ l ; ' at the peripheral level it is present in the non-adrenergic and non-cholinergic (NANC) neurons of the vessels, the intestine, etc.
• LTP long-term memorization
• ⁇ l ⁇ l
• NANC non-adrenergic and non-cholinergic
• Current studies are more oriented towards the search for nNOS inhibitors capable of blocking the toxic effects of NO on neuronal survival.
• PIN neuronal nitric oxide synthase inhibitor protein
• Type 2 diabetes also previously called non-insulin-dependent diabetes (DNTD)
• DNTD non-insulin-dependent diabetes
• This disease linked to the socio-economic context of industrialized countries, is a major public health problem: an estimated 220 million people are affected by 2007.
• Type 2 diabetes is characterized by two major defects, the relative importance of which is variable: the first is linked to a decrease in the ability of insulin to increase glucose consumption in peripheral tissues, i.e. insulin resistance, and the second is a secretory dysfunction of the pancreatic ⁇ cell which makes the pancreas unable to secrete enough msulin to compensate for insulin resistance.
• These two anomalies are preceded by a silent prodromal period, called prediabetes, of variable length, which can range from a few years to a few decades.
• This pre-diabetic state is characterized not by an insulin deficit, but on the contrary, by the development of secretory hyperactivity resulting in hyperinsulinemia.
• Hyperinsulinemia is an abnormality very commonly associated with obesity, which is an important risk factor in the development of type 2 diabetes in genetically predisposed subjects, in particular in certain populations, such as for example the PIMA Indians, where the prevalence obesity and type 2 diabetes is very high. If hyperinsulinemia develops frequently to compensate for insulin resistance, states of secretory hyperactivity or hyperinsulinism are also manifested by elevated plasma levels of proinsulin and its conversion intermediates. This inappropriate secretion of insulin which is not or poorly processed has been demonstrated in the majority of studies on subjects with glucose intolerance, that is to say on prediabetic subjects. This secretion of insulin which is not or badly matured constitutes a very poor prognosis; indeed an elevation of the ratio proinsulin / insulin is associated with the development of type 2 diabetes and cardiovascular complications within 2 to 5 years.
• type 2 diabetes makes it essential to search for innovative drugs to offer patients a wider range of therapies in order to correct pancreatic dysfunctions at different stages of the disease, especially in the prediabetic stage and in the stage of type 2 diabetes.
• One of the aspects of the invention is to provide a new detection method allowing high-throughput screening of compounds which decrease the interaction between the PIN and nNOS proteins.
• One of the other aspects of the invention is to provide a new method for detecting inhibitors of the interaction between the PIN proteins and nNOS, without modifying the catalytic activity of neuronal NOS.
• One of the other aspects of the invention is to detect compounds making it possible to restore a normal insulin response in prediabetic, hyperinsulinic or type 2 diabetic patients.
• the invention relates to a method for detecting compounds modulating the complexation between the neuronal nitric oxide synthase protein (nNOS), represented by the sequence SEQ ID NO: 2 or one of its variants, and the nitric oxide synthase inhibiting protein.
• nNOS neuronal nitric oxide synthase protein
• PIN neuronal
• nNOS neuronal nitric oxide synthase protein
• PIN neuronal nitric oxide synthase inhibitor protein
• the nNOS protein or one of its variants is meant all the neuronal NOS of any species, in particular human, expressed in different tissues can present either point mutations, or a particular alternative splicing, or both at the same time.
• Modification of the insulin response means either a decrease in the insulin response under the effect of the compound modulating the complexation between the nNOS protein and the PIN protein, or an increase in the insulin response under the effect of the above-mentioned compound.
• the modification of the insulin response induced by the compound modulating the complexation between the nNOS protein and the PLN protein can be measured by cell tests, and in particular by using the LNS-1 cell line (Asfari et al., 1992) in two types. conditions:
• the cells are stimulated by increasing concentrations of glucose (0.5-1-1.5-2 g / 1) in the presence of the compound to be tested in buffer Krebs Ringer albumen (Asfari et al., 1992), - if the test compound is not liposoluble and therefore incapable of crossing cell membranes, the cells are permeabilized in the presence of ⁇ toxin from Staphylococcus aureus (Maechler et al, 1997) and stimulated with an insulin-secreting compound, active in these cells, in the presence or in the absence of the above-mentioned compound (Maechler et al., 1997).
• a detection method is used involving the molecular labeling of at least one of the partners, namely the PIN protein and / or the nNOS protein, by a substance , such as a radioactive element, a fluorescent element, a luminescent element, an enzyme or biotin.
• This marking allows a direct or indirect quantitative physical measurement, by emission (radioactive, luminescent or fluorescent radiation) or signal consumption (absorption of light or fluorescent signal), spontaneously or after addition of an enzymatic substrate or light excitation.
• emission radioactive, luminescent or fluorescent radiation
• signal consumption absorption of light or fluorescent signal
• Biacore AB surface plasmon resonance technique
• the amount of complex formed can be determined in solution if the PIN protein and the nNOS protein are labeled with substances capable of energy exchange with one another.
• the formation of the complex is accompanied by the bringing together of the two partners, which allows an energy transfer between the two markers and then causes an increase or a decrease in the intensity of the fluorescence signal emitted by one of the two markers.
• the determination of the amount of complex can be carried out in the solid phase if one of the two partners is immobilized on the solid support and if the binding of the another partner is detected by surface plasmon resonance or by labeling this partner by the revelation system as described above.
• the first partner namely the nNOS protein or the protein & PLN
• the first partner can be immobilized either covalently (chemical reaction), or non-covalently by physico-chemical interactions (adsorption on a hydrophobic plastic surface) or by biospecific interactions where the biological sensor is previously immobilized on the plate (antibody specific to the first partner or avidin if the first partner is coated with biotin).
• the second partner namely the PLN protein or the nNOS protein respectively, makes it possible to determine the amount of complex formed (or its variation) either directly by surface plasmon resonance, or indirectly by quantitative revelation of its marker.
• the marker can be either an element attached to the second partner by chemical bond (radioactive, fluorescent, luminescent element, enzyme, biotin) or another biospecific substance such as an antibody directed against the second partner (itself labeled directly or indirectly) or avidin (labeled directly or indirectly).
• chemical bond radioactive, fluorescent, luminescent element, enzyme, biotin
• biospecific substance such as an antibody directed against the second partner (itself labeled directly or indirectly) or avidin (labeled directly or indirectly).
• the reference inhibitor is for example chosen from the peptides represented by the sequences SEQ ID NO: 3 and SEQ LD NO: 4.
• the complex formed between PLN and nNOS can be detected according to the techniques described above.
• This test makes it possible to target the early stages of type 2 diabetes, hyperinsulinic states and patent type 2 diabetes, by using the insulin-modulating properties of the pancreatic form of NOS and its endogenous inhibitor PLN.
• the present invention also relates to a detection method as defined above, characterized in that the compound does not substantially modify the catalytic activity of the nNOS protein or of one of its variants.
• the expression "without substantially modifying the catalytic activity of the nNOS protein or of one of its variants" designates the capacity of the test compound to only slightly influence the NO-producing activity of nNOS.
• nNOS an absence of modification or an increase or decrease not exceeding 20 to 30% of the basal catalytic activity of nNOS, corresponding to the absence of said compound, if said compound binds to nNOS.
• the catalytic activity of nNOS can be estimated, for example, by its capacity to produce radiolabelled citrulline from its substrate: radiolabelled arginine and in the presence of cofactors such as BL * ., FAD, FMN, NADPH, Ca 2+ and calmodulin.
• the citrulline produced can be separated from arginine by ion exchange chromatography and quantified by counting radioactivity.
• the invention relates to a method for detecting compounds which decrease the complexation between the neuronal nitric oxide synthase protein (nNOS) or one of its variants and the neuronal nitric oxide synthase inhibitor protein (PIN), the reduction of this complexation resulting in a reduction of the insulin response regulated by the nNOS protein or one of its variants, in which: - a mixture comprising said compound, the PIN protein and the nNOS protein or one of its variants is incubated, step of incubation being carried out under conditions allowing:
• “Possible significant reduction in the amount of complex formed between the PIN protein and the nNOS protein or one of its variants” means a variation of at least about 20%, and preferably at least about 50% , the amount of complex formed in the presence of the test compound relative to a control value corresponding to the absence of test compound.
• the invention also relates to a method for detecting compounds which increase the complexation between the neuronal nitric oxide synthase protein (nNOS) or one of its variants and the neuronal nitric oxide synthase inhibitor protein (PIN), the increase in this complexation resulting in an amplification of the insulin response regulated by the nNOS protein or one of its variants, in which:
• PIN or the nNOS protein or one of its variants which leads to an increase in the complexation between the PIN protein and the nNOS protein or one of its variants.
• amplification of the insulin response is meant an increase of at least about 20% ", and preferably at least about 50%, of the secretion of insulin under the effect of the compound increasing the complexation between nNOS protein and PIN protein, compared to a control value corresponding to the absence of test compound.
• the invention relates to a detection method, as defined above, in which the nNOS protein used is either the pancreatic form of the nNOS protein or the form present in the brain.
• pancreatic nNOS The rat nNOS protein found in pancreatic cells is pancreatic nNOS: it is the mutated form of rat neuronal NOS.
• the rat pancreatic nNOS has three amino acid mutations compared to the rat brain nNOS; these mutations are localized neither in the functional domains of the enzyme (binding domains of the cofactors as mentioned above), nor in the interaction zone between PIN and nNOS, but they affect its three-dimensional conformation and thus confer it pancreatic specificity.
• the nNOS protein used can also be the rat nNOS protein, present in the rat brain.
• An advantageous detection method according to the invention is a detection method as defined above, in which the variation is detected, in particular the possible significant reduction in the amount of complex formed between the PIN protein and the nNOS protein compared to a first control value, to a second value of control and a third control value, one of these control values corresponding to the amount of complex formed between the PIN protein and the nNOS protein in the absence of the compound subjected to the detection process, the other of these values control corresponding to the absence of complex between the PIN protein and the nNOS protein, resulting either from the absence of the PIN protein or from the absence of the nNOS protein, and the other of these control values corresponding to the amount of complex formed between the PIN protein and the nNOS protein in the presence of a reference inhibitor.
• the amount of complex formed between the PIN protein and the nNOS protein or one of its variants can be detected according to one of the techniques described above.
• the first control value is obtained, for example, by performing the following experiment:
• a mixture comprising the PIN protein and the nNOS protein or one of its variants is incubated, under conditions allowing the formation of a complex between the PIN protein and the nNOS protein or one of its variants,
• the second control value is obtained, for example, by carrying out the following experiment corresponding: x - either to the incubation of the PIN protein alone, which results in an absence of complex formed between the nNOS protein or one of its variants and the PIN protein,
• the third control value is obtained, for example, by performing the following experiment:
• the third control value can also be obtained by performing the following experiment:
• the PIN protein, preincubated with the reference inhibitor, is added to the nNOS protein immobilized on a biosensor (for example, Biacore AB), - Detecting, in particular by surface plasmon resonance, the amount of complex formed between the PLN protein and the nNOS protein or one of its variants, this amount corresponding to said control value.
• a biosensor for example, Biacore AB
• - Detecting in particular by surface plasmon resonance, the amount of complex formed between the PLN protein and the nNOS protein or one of its variants, this amount corresponding to said control value.
• An advantageous detection method according to the invention is a detection method as defined above, in which the mixture comprising the PLN protein, the nNOS protein and the compound subjected to the detection method is prepared:
• the mixture comprising the PLN protein, the nNOS protein and the compound subjected to the detection process is prepared by simultaneously adding the PIN protein, the nNOS protein and the said compound, the detection of compounds which bind to the complex formed between the protein is promoted.
• the detection of compounds which bind the PIN protein is favored, but also promotes the search for very bpns ligands of the nNOS protein (ligands having a very strong affinity for the nNOS protein, of the order of ⁇ M, and preferably of nM), which are disadvantaged from the kinetic point of view.
• the detection of compounds which bind the nNOS protein is favored but the also the search for very good ligands of the PLN protein (ligands having a very strong affinity for the PIN protein, of the order of ⁇ M, and preferably of nM) which are disadvantaged from the kinetic point of view.
• the mixture comprising the PLN protein, the nNOS protein and the compound subjected to the detection process is prepared by adding said compound beforehand incubated with the PLN protein and the nNOS protein, the binding of said compound with the PLN protein is facilitated before the addition of the nNOS protein, which promotes the detection of compounds binding the PIN protein.
• the mixture comprising the PIN protein, the nNOS protein and the compound subjected to the detection process is prepared by adding said compound previously incubated with the nNOS protein, and the PIN protein, the binding of said compound with the nNOS protein is facilitated before the addition of the PIN protein, which promotes the detection of compounds binding the nNOS protein.
• the quantification of the complexes formed can be carried out in solution using fluorescent markers and by fluorescence polarization (or transfer). This process has the advantage of being quick (a single incubation step, no washing) and offers a direct detection system.
• the PIN protein, the above-mentioned compound and the nNOS protein are added successively, or after preincubation of the PLN or nNOS protein with the above-mentioned compound, one of the two partners must be immobilized beforehand on a solid support.
• the quantification of the complexes formed is carried out either by analysis by surface plasmon resonance, or by labeling the other partner. This process makes it possible to determine to which part the compound binds, that is to say the PIN protein, the "nNOS protein or the complex formed between the two proteins.
• An advantageous detection method is a detection method as defined above, in which the nNOS protein is previously fixed on a solid support.
• the expression "fixed on a solid support” designates a process in which the nNOS protein is immobilized covalently (chemical reaction) or non-covalent (non-specific adsorption on plastic, avidin-biotin system, antibody) on a solid support.
• the binding of the PLN protein is detected by surface plasmon resonance or by labeling with a detection system (fluorescent, luminescent, radioactive, enzyme, biotin marker) which makes it possible to measure the amount of complex formed.
• a detection system fluorescent, luminescent, radioactive, enzyme, biotin marker
• the simultaneous addition of the PIN protein and of the compound subjected to the detection process, not previously mixed, makes it possible to detect both ligands of the nNOS protein, of the PIN protein and also of the complex formed between the two proteins. This process therefore promotes the detection of molecules which inhibit the association between the PIN protein and the nNOS protein and thus allows the search for compounds which dissociate the complex between the two proteins.
• the successive addition of the compound subjected to the detection process and of the PLN protein makes it possible to detect compounds inhibiting only the nNOS protein. Indeed, if the compound tested does not bind to the nNOS protein, it is eliminated during the washing which takes place before the addition of the PIN protein.
• the addition of the compound subjected to the detection process previously incubated with the PIN protein makes it possible to detect both ligands of the PIN protein, of the nNOS protein and also of the complex formed between the PIN protein and the nNOS protein.
• This embodiment facilitates the binding of said compound with the PIN protein before incubation with the nNOS protein and makes it possible to search for ligands of the PIN protein.
• This embodiment also makes it possible to select very good ligands of the nNOS protein.
• the invention also relates to a detection method as defined above in which the PIN protein is previously fixed on a solid support.
• the expression “fixed on a solid support” designates a process in which the PIN protein is immobilized covalently (chemical reaction) or non-covalent (non-specific adsorption on plastic, avidin-biotin system, antibody) on a solid support.
• the binding of the nNOS protein is detected by surface plasmon resonance or by labeling with a detection system (fluorescent, luminescent, radioactive, enzyme, biotin marker) which makes it possible to measure the amount of complex formed.
• a detection system fluorescent, luminescent, radioactive, enzyme, biotin marker
• the simultaneous addition of the nNOS protein and of the compound subjected to the detection process, not previously mixed, makes it possible to detect both ligands of the nNOS protein, of the PLN protein and also of the complex formed between the two proteins.
• This process therefore promotes the detection of molecules which inhibit the association between the PIN protein and the nNOS protein and thus allows the search for compounds which dissociate the complex between the two proteins.
• the PIN protein is previously fixed on a solid support, the successive addition of the compound subjected to the detection process and of the nNOS protein makes it possible to detect compounds which only inhibit the PLN protein. Indeed, if the compound tested does not bind to the PIN protein, it is eliminated during the washing which takes place before the addition of the nNOS protein.
• An advantageous detection method according to the invention is a detection method as defined above, in which the PIN protein and the nNOS protein are in solution.
• the quantification of the complexes formed is carried out by labeling the two proteins with a fluorescent compound and by measuring the fluorescence polarization.
• the invention also relates to a protein characterized in that it comprises or consists of the sequence SEQ ÎD NO: 2 or element of said protein comprising at least 100 amino acids provided that said fragment contains the amino acid in position ( 269).
• the sequence SEQ LD NO: 2 is a new protein, isolated in rats corresponding to the pancreatic form of neuronal NOS.
• nNOS pancreatic form of nNOS. It has four nucleotide mutations in position (269), (953), (1008) and (1299). The last of the mutations is a silent mutation; it therefore does not cause an amino acid change.
• the invention relates to the peptides of sequence:
• peptides being compounds which can be detected by the method as defined above, and having a greater affinity for the PIN protein than that presented by one of the proteins as defined above, with respect to PIN protein.
• peptides which mimic nNOS are elements of the nNOS protein, selected by simple or combined mutational analysis, and are obtained by chemical synthesis.
• the two peptides represented by the sequences SEQ ID NO: 3 and SEQ LD
• sequences SEQ LD NO: 3 to SEQ LD NO: 106 correspond to fragments of the mutated nNOS protein.
• the invention also relates to the nucleic acids encoding one of the proteins, one of the protein fragments or one of the peptides as defined above. It relates in particular to the nucleotide sequence having the sequence SEQ ID NO: 1
• the sequence SEQ LD NO: 1 is a new nucleic acid sequence, identified in the rat, coding for the new protein corresponding to the pancreatic form of neuronal NOS, represented by the sequence SEQ LD NO: 2.
• the invention relates a pharmaceutical composition characterized in that it comprises a protein, a protein fragment or a peptide as defined above, in combination with a pharmaceutically acceptable vehicle.
• the doses used can vary from approximately 10 mg to 1 g per day for an adult of average weight equal to 60 kg.
• the invention also relates to a pharmaceutical composition characterized in that it comprises any non-peptide substance detected by the screening method as defined above, in association with a pharmaceutically acceptable vehicle.
• the invention also relates to a pharmaceutical composition characterized in that it comprises the molecule of the following formula:
• the invention also relates to the use of proteins, protein fragments or peptides as defined above, for the preparation of medicaments intended for the treatment of alterations of the insulin response in prediabetic, hyperinsulinic states or in diabetes mellitus. type 2 patent.
• the doses used can vary from approximately 10 mg to 1 g per day for an adult of average weight equal to 60 kg.
• the invention also relates to the use of any non-peptide substance detected by the screening method as defined above, for the preparation of medicaments intended for the treatment of alterations in the insulin response in prediabetic, hyperinsulinic states or in diabetes. type 2 patent.
• the invention also relates to the use of the molecule of the following formula:
• a prediabetic state is characterized by a slight basal hyperglycemia between 6 mM (108 mg / dl) and 7 mM (126 mg / dl).
• the prediabetic state is characterized by glucose intolerance, i.e. blood glucose levels between 7.8 mM (140 mg / dl) and 11 mM (200 mg / dl) two hours after an oral glucose test.
• Hyperinsulmism corresponds to an insulinemia representing 1.5 to 10 times the plasma levels given in the literature in normal humans (10 ⁇ U / ml ⁇ 20 pmole / 1).
• Type 2 diabetes is characterized by fasting blood sugar greater than or equal to 7 mM (126 mg / dl). Type 2 diabetes is also characterized by hyperglycemia greater than 11 mM (200 mg / dl), 2 hours after an oral glucose tolerance test.
• the drugs as mentioned above and obtained using proteins, protein fragments, peptides or non-peptide substances as defined above, are capable of restoring in prediabetic or hyperinsulinic patients a secretion of normal and biphasic insulin.
• biphasic and normal insulin response designates an insulin secretion having a first secretion phase of 5 to 10 minutes as well as a second longer phase of variable intensity depending on the glucose intake (approximately
• the drugs as mentioned above and obtained using proteins, protein fragments, peptides or " non-peptide substances as defined above, are capable of restoring in diabetic patients of the type
• the invention relates to a kit or a kit for detecting a modulating compound, in particular reducing the complexation between the PIN protein and the nNOS protein, comprising; - the nNOS protein, in particular the pancreatic form of the nNOS protein,
• the media or buffers necessary for dilution are for example
• - PBS added with 0.1% of T een 20 and of 1% of BSA (bovine serum albumin).
• BSA bovine serum albumin
• the suitable washing means are for example PBS supplemented with 0.1% of
• the media or buffers allowing the formation of a complex between the PIN protein and the nNOS protein and the formation of a complex between the PLN protein or the nNOS protein and the compound subjected to the detection process are for example PBS supplemented with 0.1% Tween 20 and 1% BSA.
• the means for detecting the variation in the amount of complex formed between the nNOS protein and between the PIN protein are for example:
• Figure 1 shows the RT-PCR analysis of PIN expression in rat pancreatic islets and in INS-1 cells (Asfari et al., 1992). The total RNAs are isolated, the complementary DNA is synthesized by reverse transcription then it is amplified by PCR with primers based on the PLN sequence and on that of ⁇ microglobulin ( ⁇ 2 Hi), which is used as positive control. '' A negative control is carried out in the absence of complementary DNA (C). DNA fragments of known size (2000, 1200, 800, 400, 200 and 100 base pairs) are used as molecular weight (PM) markers.
• Figure 2 shows the protein blot analysis (Western blot) of the presence of the PIN protein in LNS-1 cells. The proteins extracted from INS-1 cells and from the rat brain (Cerv.) Are separated on a 13.5% tricine gel, transferred to a nitrocellulose membrane and incubated with an anti-PIN monoclonal antibody.
• the signal is detected by an anti-mouse antibody coupled to peroxidase followed by a chemiluminescence reaction. 16 and 7 kDa indicate molecular weight markers.
• Figures 3A and 3B show the co-localization of the PIN protein and neuronal NO synthase in LNS-1 cells by immunofluorescence.
• INS-1 cells are doubly labeled with an anti-PIN monoclonal antibody ( Figure 3A) and with a rabbit neuronal anti-NO synthase antibody ( Figure 3B).
• the fluorescence is revealed by an anti-mouse antibody coupled to fluorescein and an anti-rabbit antibody coupled to rhodamine and then it is analyzed by a confocal dual channel microscope.
• the scale bar indicates 10 ⁇ m.
• Figures 4A and 4B show the effect of overexpression of PLN in INS-1 cells on glucose-induced insulin secretion.
• a X Figure 4A represents the RT-PCR analysis of PIN overexpression in LNS-1 cells.
• INS-1 cells are transfected with an empty expression vector (column C) or containing DNA complementary to PLN (PIN column).
• the total RNAs are isolated and the complementary DNA is amplified by RT-PCR with primers based on the PIN sequence.
• DNA fragments of known size 2000, 1200, 800, 400, 200 and 100 base pairs
• FIG. 4B represents the analysis of the insulin secretion of INS-1 cells overexpressing PLN (column PIN) compared to the control cells (column C). 48 hours after transfection, the cells are incubated in the presence of glucose at 1 g / l and the insulin secretion is measured by a radioimmunoassay.
• FIGS. 5A and 5B represent sensorgrams corresponding to the analysis by surface plasmon resonance of the interaction between PIN and a normal nNOS peptide (FIG. 5A) or a mutated nNOS peptide (FIG. 5B), represented by the sequence SEQ ID NO: 3.
• the peptides are immobilized on a channel of a CM5 biosensor (Biacore AB) and the PIN protein, resulting from a digestion of GST-PIN by thrombin, is injected at increasing concentrations: 5 ⁇ g / ml (curve 4 with regular dotted lines in Figure 5 A and curve d with regular dotted lines in Figure 5B), 10 ⁇ g / ml (curve 3 with alternated dotted lines in Figure 5A and curve c with alternate dotted lines in Figure 5B),
• FIGS. 6A and 6B represent the inhibition of the binding of the GST-PIN protein on the nNOS protein for different concentrations of normal peptide and mutated peptides, represented by the sequences SEQ ID NO: 3 and SEQ ID NO: 4.
• the nNOS protein is immobilized on an ELISA plate and brought into contact with the GST-PIN protein, previously incubated with increasing concentrations of peptide.
• the interaction between the two proteins is revealed by an anti-GST antibody coupled to peroxidase and then by measuring the absorbance at 490 n.
• Figure 6A shows the absorbance as a function of the concentration of peptide used (in ⁇ g / ml).
• the curve with black circles corresponds to the normal peptide, those with black squares to the peptide represented by the sequence SEQ ID NO: 3 and that with black diamonds to the peptide represented by the sequence SEQ LD NO: 4.
• Figure 6B represents the percentage inhibition of protein binding
• FIGS. 7 A, 7B, 7C and 7D represent sensorgrams corresponding to the analysis by surface plasmon resonance of the inhibition of the interaction between the PIN protein and the nNOS protein by a normal nNOS peptide (FIG. 7A) or mutated nNOS peptides represented by the sequences SEQ ID NO: 3 and SEQ LD NO: 4 ( Figures 7B and 7C).
• Figures 7A, 7B and 7C show the RU response, which corresponds to the amount of protein bound on the biosensor, as a function of time.
• FIG. 7A represents the inhibition of the binding of the PIN protein to the nNOS protein by the normal peptide (Lys Asp Thr Gly Ile Gin Nal Asp Arg Asp).
• Curve 1 corresponds to the absence of peptide;
• curves 2, 3 and 4 correspond respectively to a concentration of the normal peptide equal to 20 ⁇ g / ml, 50 ⁇ g / ml and
• FIG. 7B represents the inhibition of the binding of the PL ⁇ protein to the n ⁇ OS protein by the mutant peptide, represented by the sequence SEQ LD ⁇ O: 3.
• Curve a corresponds to the absence of said peptide;
• curves b, c, d, e and f correspond respectively to a concentration of said peptide equal to 5 ⁇ g / ml, 10 ⁇ g / ml, 20 ⁇ g / ml, 30 ⁇ g / ml and 40 ⁇ g / ml.
• FIG. 7C represents the inhibition of the binding of the PIN protein to the nNOS protein by the mutant peptide, represented by the sequence SEQ LD NO: 4.
• Curve 1 corresponds to the absence of said peptide;
• curves 2, 3, 4, 5 and 6 correspond respectively to a concentration of said peptide equal to 1 ⁇ g / ml, 2 ⁇ g / ml, 3 ⁇ g / ml, 5 ⁇ g / ml and 10 ⁇ g / ml.
• FIG. 7D represents the curve of inhibition of the binding of the PIN protein to the nNOS protein by the mutant peptide, represented by the sequence SEQ ID NO: 3
• Figure 8 represents the analysis of the insulin secretion of islets of Zucker fa / fa rats, in the presence of increasing concentrations of the molecule of formula C 24 H 18 N 4 ⁇ 5 S, compared to the secretion of insulin obtained in the absence of said molecule.
• the islets of rats are first stabilized in the presence of glucose at 0.75 g / l, then incubated in groups of three in the presence of glucose at the stimulating concentration of 2 g / l with or without said molecule. Insulin secretion is then measured by radioimmunoassay.
• insulin secretion in ng / ml
• the black column corresponds to the control measurement (without said molecule); the gray column corresponds to the measurement in the presence of said molecule at 20 ⁇ M; the white column corresponds to the measurement in the presence of said molecule at 50 ⁇ M and the white column with vertical stripes corresponds to the measurement in the presence of said molecule at 100 ⁇ M.
• Overlapping fragments of complementary DNA are obtained by RT-PCR from islets of rat Langerhans and from the insulin-secreting cell line INS-1 (Asfari et al., 1992) .
• the islets are isolated from the pancreas of a male Wistar rat by collagenase digestion and are separated from the exocrine tissue by a Ficoll gradient (Shibata et al., 1976).
• the INS-1 cells are derived from a rat insulinoma and are cultured in RPMI 1640 containing 10% fetal calf serum, 100 U / ml of penicillin, 100 ⁇ g / ml of streptomycin, 2 mM L-glutamine, 10 mM Hepes, 1 mM sodium pyruvate and 50 ⁇ M ⁇ -mercaptoethanol.
• the total RNAs of the isolated islets and of the LNS-1 cells are extracted with TRIzol (Life technologies).
• the first strand of complementary DNA is synthesized from 10 ⁇ g of total RNA in the presence of 3 ⁇ g of random primers (Life technologies), 1 ⁇ g of oligo primer (dT) (Life technologies) and of the transcriptase reverse Superscript LT RNAse H- (Life technologies).
• the PCR is then carried out in the presence of Taq Polymerase (Life technologies) with the pairs of primers listed in the following table: List of sense and antisense primers used for sequencing the pancreatic form of neuronal NO synthase
• the PCR will be carried out in 40 cycles comprising a denaturation step at 94 ° C for 1 minute, a hybridization step at 60 ° C for 1 minute, a step d elongation at 72 ° C for 1 minute then a final elongation of 10 minutes.
• the complementary DNA fragments are purified after migration on a 1.5% agarose gel using the QiaEx II extraction kit (Qiagen).
• the fragments are then sequenced twice manually using dCTP '[- ⁇ S 35 ] and the Thermosequenase Cycle Sequencing kit (Amersham) and once using an automatic sequencer (ABI PRISM 377, PE Applied Biosystems) and the dRhodamine Terminator Sequencing Ready Reaction kit (PE Applied Biosystems).
• Amersham Thermosequenase Cycle Sequencing kit
• ABS automatic sequencer
• PE Applied Biosystems dRhodamine Terminator Sequencing Ready Reaction kit
• a valine is mutated to isoleucine at position 269, an alanine to proline at position 953 and a serine to phenylalanine at position 1008.
• the neuronal NO synthase of the pancreas is therefore slightly different from NO neuronal synthase previously identified in the rat cerebellum (Bredt et al., 1991).
• PLN neuronal nitric oxide synthase inhibitor protein
• the first strand of complementary DNA is synthesized from 10 ⁇ g of total RNA in the presence of 3 ⁇ g of random primers (Life technologies), 1 ⁇ g of oligo primer (dT) (Life technologies) and of the transcriptase reverse Superscript II RNAse H- (Life technologies).
• the PCR is then carried out in the presence of Taq Polymerase (Life technologies) with the following pairs of primers:
• the PCR will be carried out in 40 cycles comprising a denaturation step at 94 ° C for 1 minute, a hybridization step at 60 ° C for 1 minute, a step d elongation at 72 ° C for 1 minute then a final elongation of 10 minutes.
• the PCR products are then separated on a 1.5% agarose gel and visualized by staining with ethidium bromide.
• a fragment of the expected size (443 base pairs) is obtained with the PIN primers both in the pancreatic islets and in the LNS-1 cells (see Figure 1).
• RT-PCR analysis therefore highlights the presence of PIN messenger RNA in rat pancreatic ⁇ cells.
• PIN messenger RNA in rat pancreatic ⁇ cells.
• the simultaneous expression of neuronal NO synthase and its natural inhibitor PIN has been demonstrated in the insulin-secreting cells of the endocrine pancreas.
• the PLN complementary DNA was sequenced and its total homology with the PIN sequence from the rat brain was noted (Jaffrey et al, 1996). Presence of the PIN protein in INS-1 cells.
• the LNS-1 cells and the rat brains are homogenized in a lysis buffer containing 50 mM Tris (pH 7.4), 150 mM NaCl, 2 mmol / 1 of EDTA, 1 mmol / 1 of phenylmethylsulfonyl fluoride, 10 ⁇ g / ml leupeptin and 10 ⁇ g / ml aprotinin. Insoluble material is removed by centrifugation. The protein concentration of the supernatant is determined using Coomassie blue (Coomassie Protein Assay Reagent, Pierce). 80 ⁇ g of proteins are separated by electrophoresis on a 13.5% tricine gel) and then transferred to a nitrocellulose membrane.
• the membranes are first saturated with skimmed milk powder at 5% in PBS added with 0.1% Tween 20 and they are then incubated overnight with an anti-PIN monoclonal antibody (diluted to l / 250 e , Transduction Laboratories). After 3 washes in PBS-Tween, the membrane is finally incubated with a anti-mouse antibody coupled to peroxidase (diluted l / e 5000, Sigma Aldrich). The immunoreactivity is revealed using a chemiluminescence reaction (ECL, Amersham Life Science). Analysis by
• INS-1 cells are seeded in Lab-Tek ® slide systems and cultured for 4 days before use. They are then fixed with parafo ⁇ naldehyde at 2% in PBS (tammon phosphate saline solution) for 20 minutes and permeabilized for 5 minutes with 0.1% Triton X-100. After saturation of the non-specific sites with 2% BSA (bovine serum albumin), the cells are incubated with a monoclonal anti-PIN antibody (diluted 1/100 th , Transduction Laboratories) and an anti-NO synthase neuronal antibody rabbit (diluted 1/100 th , Euro-Diagnostica) overnight.
• PBS tammon phosphate saline solution
• BSA bovine serum albumin
• an anti-mouse antibody coupled to fluorescein (diluted 1/100 th , Biosys) and an anti-rabbit antibody coupled to rhodamine (diluted 1/100 th , Biosys) are applied to the cells for one hour .
• the cells are mounted in Citifluor (Citiffuor Ltd) and observed with a confocal argon and krypton laser microscope (Biorad).
• the PLN protein is present in the cytoplasm of INS-1 cells ( Figure 3A).
• the signal fluorescent of PIN ( Figure 3A) and that of neuronal NO synthase (see Figure 3B) are highly superimposable, which indicates that the two proteins are strongly collocated in rat pancreatic ⁇ cells.
• the PIN protein has been shown to interact with neuronal NO synthase in vitro and in vivo (Jaffrey et al., 1996) at the level of its amino acids 163 to 245. It therefore seems that neuronal NO synthase and PIN interact inside pancreatic ⁇ cells.
• the PIN protein is overexpressed in LNS-1 cells and the insulin response to glucose is measured.
• the complementary DNA of PIN obtained after RT-PCR (see above) is cloned into a eukaryotic expression vector pCR3.1 (TA cloning Kit, Invitrogen). Then the LNS-1 cells (approximately 8.10 5 ) are transfected with 1.5 ⁇ g of plasmid (empty or containing PLN) using the reagent Lipofectamine plus Reagent
• the PLN overexpression is then verified by RT-PCR using 5 ⁇ g of total RNA and the primers mentioned above (see table 1). 48 hours after transfection, the cells are washed in Krebs Ringer bicarbonate buffer pH 7.4) (108 mM NaCl; 1.19 mM KH 2 PO 4 ; 4.74 mM KC1; 2.54 mM CaCl 2 ; 1, 19 M MgSO 4 , 7H 2 O; 18 mM NaHCO 3 ) without glucose, then preincubated in this same buffer for one hour at 37 ° C. After removing the medium, the cells are incubated in Krebs supplemented with 1 g / l of glucose for one hour at 37 ° C.
• the PN complementary DNA obtained after RT-PCR (see example 1 below) is cloned into the vector pET21b (containing the polyhistidine label in the C-terminal position, (HIS) 6 , supplied by example by
• the vector pGEX-2T containing the label glutathione S-transferase in the N-terminal position, GST, supplied for example by Pharmacia.
• the bacteria After transformation of the BL21 (DE3) bacteria (supplied for example by Novagen) by the recombinant plasmids, the bacteria are cultured at 37 ° C. in LB medium up to an OD of 0.6. The protein is then produced after induction with 1 mM of LPTG (isopropylthio- ⁇ -
• D-galactoside for 5 hours at 30 ° C.
• the bacteria are recovered by centrifugation and are lysed according to standard conditions (Short Protocols in Molecular Biology, 2 nd Edition, John Wiley and Son). Insoluble material is removed by centrifugation and the protein is purified on a nickel column for the polyhistidine label (for example Ni NTA agarose, supplied by Qiagen) or on a glutathione sepharose column for the label GST (for example supplied Pharmacia) according to the supplier's recommendations. The PIN protein is then stored at -80 ° C.
• pancreatic having a polyhistidine tag was cloned into the vector pll9L (Poul et al., 1995) under control of the viral promoter P10 (Poul et al., 1995).
• the recombinant virus is obtained by cotransfection of the loaded vector and of the baculovirus DNA in the Sf9 insect cells (ATCC CRL 1711) using the lipofection technique (DOTAP, Roche Diagnostics).
• DOTAP lipofection technique
• the virus clones are then isolated by lysis range and selected for their capacity to produce the nNOS protein by protein transfer (Western Blot) with an anti-nNOS antibody (Transduction
• the protein is finally purified on a nickel column (for example Ni NTA agarose, supplied by Qiagen) according to the supplier's recommendations.
• Ni NTA agarose supplied by Qiagen
• the pancreatic nNOS protein is then stored at -80 ° C.
• the pancreatic nNOS is immobilized at the bottom of a Maxisorp plastic plate (Nunc) at the concentration of 1 to 5 ⁇ g / ml in 200 ⁇ l of PBS overnight at 4 ° C. . After washing in PBS containing 0.1% Tween 20, the plate is saturated with 100 ⁇ l of PBS-1% BSA for one hour at 37 ° C.
• PIN protein comprising a GST label (GST-PIN) or polyhistidine (PLN- (HIS) 6 ) at a concentration of 0.1 to 10 ⁇ g / ml in 100 ⁇ l of PBS-0.1% Tween 20-1% BSA in the presence or absence of the test compound for two hours at 37 ° C.
• the plate is then washed and then incubated with 100 ⁇ l of anti-label, anti-GST or anti (HIS) 6 antibody coupled to peroxidase (diluted to l / 2000 e in PBS-0.1% Tween 20-1 % BSA, Sigma Aldrich) for one hour at 37 ° C.
• nNOS-PIN-antibody complex The formation of the nNOS-PIN-antibody complex is revealed by a colored reaction in the presence of the peroxidase substrate, O-phenylenediamine, for 30 minutes in the dark, and the intensity of the coloration is measured at 490 nm.
• the PLN protein in GST-PIN or PLN- (HIS) 6 form, is immobilized at the bottom of a plastic plate and then brought into contact with the pancreatic nNOS. Reactivity is detected by an anti-nOS antibody coupled to peroxidase.
• nNOS soluble peptides of nNOS are prepared by an AMS 422 robot (Abimed) by chemical synthesis Fmoc on solid phase (Gausephol, 1992). These nNOS peptides from AMS 422 robot (Abimed) by chemical synthesis Fmoc on solid phase (Gausephol, 1992). These nNOS peptides from AMS 422 robot (Abimed) by chemical synthesis Fmoc on solid phase (Gausephol, 1992). These nNOS peptides from AMS 422 robot (Abimed) by chemical synthesis Fmoc on solid phase (Gausephol, 1992). These nNOS peptides from AMS 422 robot (Abimed) by chemical synthesis Fmoc on solid phase (Gausephol, 1992). These nNOS peptides from AMS 422 robot (Abimed) by chemical synthesis Fmoc on solid phase (Gausephol, 1992). These nNOS
• 10 amino acids correspond to the zone of interaction with PIN (amino acids 229 to 238 of nNOS).
• the peptides are then deprotected and cleaved from the resin by a treatment with trifluoroacetic acid in the presence of the appropriate sensors.
• the peptides are lyophilized and their purity is checked by analytical HPLC. If necessary, the peptides are then purified to 90% by preparative HPLC, then analyzed by mass spectrometry.
• the binding of PIN on the immobilized peptides is analyzed by BIACORE 2000 (Biacore AB).
• the peptides at a concentration of 10 ⁇ g / ml in a 10 mM acetate buffer, pH 4, are coupled on a channel of a CM5 biosensor (Biacore AB) using the NHS-EDC protocol (N-hydroxysuccinimide (NHS), Biacore AB; N-ethyl-N'-
• association and dissociation constants are determined from the sensorgram using the BIAevaluation 3.0 software (Biacore AB) and a so-called global analysis method 5 (simultaneous analysis of the kinetic association and dissociation constants for the sensorgrams corresponding to all concentrations of PIN used).
• Biacore AB BIAevaluation 3.0 software
• global analysis method 5 Simultaneous analysis of the kinetic association and dissociation constants for the sensorgrams corresponding to all concentrations of PIN used.
• any peptide (Lys Ala Val Asp Leu Ser His Gin Pro Ser Ala Ser Lys Asp Gin Ser Leu), which is a fragment of nNOS (delimited from amino acid at position 131 to amino acid at position 147 of the protein nNOS)
• nNOS peptide SEQ LD NO: 3, arginine mutated into tryptophan at position 9 increases its affinity for the PIN protein.
• LD NO: 3 and SEQ LD NO: 4 having a better affinity for the PIN protein than the nNOS protein, are capable of reducing the interaction between the nNOS and PIN proteins by using the first embodiment of the invention, described above.
• nNOS is immobilized on an ELISA plate, Maxisorp (Nunc) at the
• nNOS-PLN complex The formation of the nNOS-PLN complex is revealed by a colored reaction in the presence of the peroxidase substrate, O-phenylenediamine, for 20 minutes in the dark, and the intensity of the coloration is measured at 490 nm.
• the control is carried out using any peptide as defined above.
• the binding of the PLN protein to the nNOS protein is inhibited by 14% for a concentration of said peptide equal to 50 ⁇ g / ml.
• the two mutant peptides represented by the sequences SEQ ID NO: 3 and SEQ LD NO: 4, cause an inhibition of the binding of the PIN protein on the nNOS protein reaching 71% (for the peptide represented by the sequence SEQ ID NO: 3
• the peptide represented by the sequence SEQ JJD NO: 3 has an inhibition constant (K *: IC 50 ) of 5 ⁇ M and the peptide represented by the sequence SEQ ID NO: 4 has an inhibition constant of 0.5 ⁇ M, while the normal peptide inhibits this interaction only with an inhibition constant of 300 ⁇ M.
• mutant peptides of the nNOS protein represented by the sequences SEQ ID NO: 3 or SEQ LD NO: 4, having a better affinity for the PIN protein than the nNOS protein, are capable of inhibiting the interaction between nNOS and PIN proteins using, surface plasmon resonance analysis.
• nNOS protein The PIN binding on the immobilized nNOS protein (Alexis) is analyzed by BIACORE 2000 (Biacore AB) in the presence or absence of the synthetic peptides.
• the nNOS protein at a concentration of 10 ⁇ g / ml in 10 mM acetate buffer pH 5.5, is coupled on a channel of a CM5 biosensor (Biacore AB, for example) using the NHS-EDC protocol, which leads to an immobilized protein density of approximately
• the PIN protein at 5 ⁇ g / ml is premcubed in the presence of increasing concentrations of peptides (1 to 100 ⁇ g / ml) for 30 minutes then is injected onto the chip with a flow of 30 ⁇ l / in.
• the sensorgrams corresponding to the binding of the peptides to the nNOS protein are recorded with an association time of 180 seconds and a dissociation time of 400 seconds.
• the association and dissociation constants are determined from the sensorgram using the BIAevaluation 3.0 software (Biacore AB) and a so-called global analysis method (simultaneous analysis of the kinetic association and dissociation constants for the sensorgrams corresponding to all the concentrations of peptides used).
• the controls are carried out by injecting the nNOS with any peptide (defined above), preincubated with PIN.
• the binding of PIN to the nNOS protein is inhibited at 19% for 20 ⁇ g / ml of peptide, inhibition capping at 45% for 50 and 100 ⁇ g / ml.
• the mutant peptide represented by the sequence
• SEQ LD NO: 3 results in inhibition of PIN binding to the nNOS protein by 59% at 5 ⁇ g / ml and almost completely blocks this interaction at 30 and 40 ⁇ g / ml (90 and 91% respectively) .
• the mutant peptide represented by the sequence SEQ LD NO: 4 (see FIG. 7C) which inhibits the binding of PIN to the nNOS protein by 75% from the concentration of 1 ⁇ g / ml and totally inhibits this interaction at 10 ⁇ g / ml (98%>).
• the inhibition curves for PIN binding to the nNOS protein as a function of the concentration of peptide used (FIG.
• the peptide represented by the sequence SEQ LD NO: 3 exhibits an inhibition constant ( Kj: IC50) of 4 ⁇ M and the peptide, represented by the sequence SEQ ID NO: 4, an inhibition constant of 0.4 ⁇ M.
• the two mutant peptides are therefore capable of inhibiting the interaction between the PIN and nNOS proteins.
• C 24 H 18 NO 5 S obtained by in vitro screening of a chemical bank of 3000 chemical compounds (Chembridge) according to the method of the invention described above, is capable of decreasing the secretion of insulin from obese animals hyperinsulinic and insulin resistant (Zucker fa / fa rats: line of rats presenting a mutation of the gene named fa (abbreviation of "fatty")).
• the chemical molecule used corresponds to the following formula:
• nNOS 100 ng
• nNOS 100 ng
• the nNOS is placed in the presence of 5 ⁇ l of molecules (at the final concentration of 10 ⁇ M) and of the GST-PIN protein at 0.5 ⁇ g / ml for 2 hours at 37 ° C.
• the formation of the PIN-nNOS complex is then detected by incubation with the anti-GST antibody (diluted to 1/2000) for one hour at 37 ° C., then revealed by incubation with O-phenylenediamine for 30 minutes and reading at 490 nm.
• the molecules considered positive are those resulting in an inhibition of approximately 30 to 50% of the interaction.
• Islets of rats' Zucker fa / fa are isolated after collagenase digestion according to the technique Lacy et al. (Diabetes, 1967). After isolation, the islets are stabilized in Krebs Ringer containing 0.75 g / l of glucose for 45 minutes at 37 ° C. Groups of 3 islets are then incubated in Krebs Ringer with 2 g / 1 of glucose containing increasing concentrations of the molecule of formula C24H18N4O5S (from 20 to 100 ⁇ M) for one hour at 37 ° C. »The supernatant is then collected and the insulin secretion is measured by radioimmunology (see
• the molecule of formula C24H ⁇ sN4 ⁇ sS decreasing the PIN-nNOS interaction, blocks in a dose-dependent manner (from the concentration of 50 ⁇ M) the secretion of insulin induced by 2 g / 1 of glucose in islets isolated from hyperinsulinic rats . Indeed, the insulin response is reduced by 36% and by 79% at the respective concentrations of

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