Patent Application
20200131495
The present invention relates to the treatment or prevention of inflammation, such as obesity, overweight and/or obesity-related diseases and disorders. The present invention relates to methods of inducing satiation, prolonging satiety, reducing food intake, controlling weight gain and stimulating weight loss.
Obesity is one of such poorly treatable chronic conditions accompanied by numerous comorbidities. Obesity and overweight are typically characterized by increased food intake and decreased energy expenditure suggesting altered role of peptidergic systems regulating energy balance. Indeed, the regulation of appetite and feeding behavior involves interaction between intestinal hunger and satiety peptide hormones with the brain neuronal circuitries containing orexigenic and anorexigenic neuropeptides (Schwartz et al., 2000. Nature. 404(6778):661-71).
The current model of food intake control implicates gut-derived hunger and satiety hormones signalling to several brain circuitries regulating homeostatic and hedonic aspects of feeding (Berthoud H.-R., 2011. Curr. Opin. Neurobiol. 21(6):888-896; Murphy et al., 2006. Nature. 444(7121):854-859). Prominent amongst these are the anorexigenic and orexigenic pathways originating from the hypothalamic arcuate nucleus (ARC) that include the proopiomelanocortin (POMC) and neuropeptide Y (NPY)/agouti-related protein (AgRP) neurons, respectively, relayed in the paraventricular nucleus (PVN) (Atasoy et al., 2012. Nature. 488(7410):172-177; Garfield et al., 2015. Nat. Neurosci. 18(6):863-71; Shi et al., 2013. Cell Metab. 17(2):236-248).
The central melanocortin (MC) system consisting of melanocortin peptides including α-melanocyte-stimulating hormone (α-MSH) derived from its precursor proopiomelanocortin (POMC) and acting on the MC type 4 receptors (MC4R) is critically involved in regulation of energy balance (Cone, 2006. Endocr. Rev. 27(7):736-49). In fact, deficit in both POMC expression and MC4R signalling leads to hyperphagia and obesity in both human and genetically modified rodents (Huszar et al., 1997. Cell. 88(1):131-41; Krude et al., 1998. Nat. Genet. 19(2):155-7; Farooqi et al., 2003. N. Engl. J. Med. 348(12):1085-95). Selective stimulation of the central MC4R appears hence as a very attractive target to treat hyperphagia and obesity and several α-MSH peptide analogues have been developed and clinically tested, such as for instance setmelanotide as a replacement therapy during POMC deficiency and for treatment of hyperphagia in Prader-Willi syndrome (Chen et al., 2015. J. Clin. Endocrinol. Metab. 100(4): 1639-45; Kuhnen et al., 2016. N. Engl. J. Med. 375(3):240-6). The penetration of high affinity α-MSH like drugs in the brain has however the risk of unwanted side-effects such as increasing blood pressure.
Recently, the gut commensal Escherichia coli (E. coli) caseinolytic protease B (ClpB) protein has been identified as a conformational mimetic of α-MSH, suggesting potential use of specific bacterial proteins as a new type of peptide-like drugs (Tennoune et al., 2014. Transl. Psychiatry. 4:e458). The ClpB protein has a molecular weight superior to 95 kDa, which raises some difficulties (such as for example production, stability, and the like).
It was shown that activation of the MC4R in the gut enteroendocrine cells stimulates release of satiety hormones glucagon-like peptide-1 (GLP-1) and peptide YY (PYY) (Panaro et al., 2014. Cell Metab. 20(6):1018-1029).
A recent study showed that a ClpB fragment corresponding to amino acid residues 534-548 of ClpB sequence from E. coli is a full micromolar MC1R agonist with partial activities on MC3R and MCSR but not on MC4R (Ericson et al., Bioorganic & Medicinal Chemistry Letters, 2015, 25:5306-5308). However, the activity of the full ClpB or larger fragments containing α-MSH-like epitope on MCR may differ from the modified short fragment studied by Ericson et al.
The hormone α-MSH is also known to have potent anti-inflammatory effects and protective effects on cells of the immune system and on peripheral nonimmune cell types expressing melanocortin receptors, such as MCIR and MC3R (Brzoska et al., 2008. Endrocr. Rev. 29(5):581-602). Moreover, recent studies show that α-MSH is an interesting target for treating psoriasis, allergic rhinitis, osteoarthritis and neuroinflammatory diseases (Auriemma et al., 2012. J. Invest. Dermatol. 132(7):1814-24; Kleiner et al. Clin. Exp. Allergy. 46(8):1066-74; BOhm et al., 2016. Biochem. Pharmacol. 116:89-99; Mykicki et al., 2016. Sci. Transl. Med. 8(362):362ra146).
Therefore, there is still a need to identify new polypeptides or proteins that retain the biological activity of α-MSH, in particular binding to melanocortin receptors.
The inventors surprisingly showed that ClpB fragments comprising a sequence homology with α-MSH epitope have a direct action on intestinal mucosal cells to stimulate secretion of PYY.
The present invention thus relates to polypeptides and proteins comprising a fragment of a ClpB protein or variant thereof, and uses thereof.
The present invention relates to a polypeptide or protein of at least 15 amino acids comprising a fragment of a ClpB protein or variant thereof, comprising a negatively charged residue and two consecutive Arg and Trp residues, wherein said polypeptide or protein is not a full-length ClpB protein.
In one embodiment, the polypeptide or protein of the invention comprises the amino acid sequence SEQ ID NO: 2. In one embodiment, the polypeptide or protein of the invention comprises the amino acid sequence SEQ ID NO: 3. In one embodiment, the polypeptide or protein of the invention comprises the amino acid sequence SEQ ID NO: 4.
In one embodiment, the polypeptide or protein of the invention further comprises a sequence having at least 75% sequence homology with the sequence GKPV. In one embodiment, the polypeptide or protein of the invention comprises the amino acid sequence SEQ ID NO: 6.
The present invention also relates to a composition comprising the polypeptide or protein as described herein.
The present invention further relates to a pharmaceutical composition comprising the polypeptide or protein as described herein and at least one pharmaceutically acceptable excipient.
Another object of the present invention is a medicament comprising the polypeptide or protein according to the invention.
The present invention also relates to a dietary supplement comprising the polypeptide or protein as described herein. The present invention further relates to a food composition comprising the polypeptide or protein as described herein.
The present invention further relates to the polypeptide or protein, the pharmaceutical composition, or the medicament according to the invention for use in the treatment or prevention of obesity, overweight and/or obesity-related diseases and disorders in a subject in need thereof.
Another object of the present invention is the use of the polypeptide or protein, the dietary supplement or the food composition according to the invention for reducing weight in a subject. In one embodiment, the subject is not obese.
The present invention further relates to a kit comprising a polypeptide or protein, a composition, a pharmaceutical composition, a medicament, a dietary supplement or a food composition according to the invention.
In the present invention, the following terms have the following meanings:
The present invention thus relates to a polypeptide or protein comprising or consisting of at least a fragment of a ClpB protein or variant thereof.
In one embodiment, the polypeptide or protein of the invention comprises or consists of at least 15 amino acids in length. In one embodiment, the polypeptide or protein of the invention comprises or consists of at least 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 75, 100 or more amino acids. In one embodiment, the polypeptide of the invention comprises or consists of less than 100, 75, 60, 50, 40, 35, 30, 25, 20, 19, 18, 17, or 16 amino acids in length.
In one embodiment, the polypeptide or protein of the invention comprises from 4 to 100 or more amino acids, from 10 to 80 amino acids, from 15 to 70 amino acids, from 20 to 60 amino acids, from 25 to 50 amino acids, from 30 to 45 amino acids, or from 35 to 40 amino acids.
In another embodiment, the polypeptide of the invention comprises from 10 to 100 amino acids, or from 10 to 80, 70, 60, 50, 45, 40, 35, 30, 25, 20 or 15 amino acids. In another embodiment, the polypeptide of the invention comprises from 15 to 100 amino acids, or from 15 to 80, 70, 60, 50, 45, 40, 35, 30, 25, or 20 amino acids. In another embodiment, the polypeptide of the invention comprises from 20 to 100 amino acids, or from 20 to 80, 70, 60, 50, 45, 40, 35, 30, or 25 amino acids. In another embodiment, the polypeptide of the invention comprises from 25 to 100 amino acids, or from 25 to 80, 70, 60, 50, 45, 40, 35, or 30 amino acids.
In one embodiment, the polypeptide of the invention comprises or consists of 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 amino acids. In another embodiment, the polypeptide of the invention comprises or consists of 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 amino acids.
In one embodiment, the polypeptide of the invention does not comprise less than 15 amino acids. In one embodiment, the polypeptide of the invention comprises at least 15 amino acids. In one embodiment, the polypeptide of the invention does not comprise less than 20 amino acids. In one embodiment, the polypeptide of the invention comprises at least 20 amino acids.
In another embodiment, the protein of the invention comprises or consists of at least 100, 125, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600 or more amino acids in length.
In one embodiment, the protein of the invention comprises from 100 to 1500 or more amino acids, from 100 to 1000 amino acids, from 100 to 900 amino acids, from 100 to 850 amino acids, from 100 to 800 amino acids, from 100 to 750 amino acids, or from 100 to 700 amino acids. In another embodiment, the protein of the invention comprises from 150 to 1500, 1000, 900, 850, 800, 750 or 700 amino acids. In another embodiment, the protein of the invention comprises from 200 to 1500, 1000, 900, 850, 800, 750 or 700 amino acids. In another embodiment, the protein of the invention comprises from 300 to 1500, 1000, 900, 850, 800, 750 or 700 amino acids.
In one embodiment, the polypeptide or protein of the invention comprises from 15 to 1500 or more amino acids, from 15 to 1000 amino acids, from 15 to 900 amino acids, from 15 to 800 amino acids, from 15 to 700 amino acids, from 15 to 600 amino acids, or from 15 to 500 amino acids. In another embodiment, the polypeptide or protein of the invention comprises from 20 to 1500, 1000, 900, 800, 700, 600 or 500 amino acids. In another embodiment, the polypeptide or protein of the invention comprises from 25 to 1500, 1000, 900, 800, 700, 600 or 500 amino acids. In another embodiment, the polypeptide or protein of the invention comprises from 30 to 1500, 1000, 900, 800, 700, 600 or 500 amino acids.
A ClpB protein according to the present invention refers to a caseinolytic protease B protein, which is a member of the Hsp100/ClpB family of hexameric AAA+-ATPases.
In one embodiment, the ClpB protein according to the present invention is a bacterial ClpB protein. In one embodiment, the ClpB protein according to the present invention is a Enterobacteriaceae protein. Enterobacteriaceae include, but are not limited to, Arsenophonus, Brenneria, Buchnera, Budvicia, Buttiauxella, Cedecea, Citrobacter, Cosenzaea, Cronobacter, Dickeya, Edwardsiella, Enterobacillus, Enterobacter, Erwinia, Escherichia, Ewingella, Franconibacter, Gibbsiella, Hafnia, Izhakiella, Kosakonia, Klebsiella, Kluyvera, Leclercia, Lelliottia, Leminorella, Levinea, Lonsdalea, Mangrovibacter, Moellerella, Morganella, Obesumbacterium, Pantoea, Pectobacterium, Phaseolibacter, Photorhabdus, Plesiomonas, Pluralibacter, Pragia, Proteus, Providencia, Pseudocitrobacter, Rahnella, Raoultella, Rosenbergiella, Rouxiella, Saccharobacter, Salmonella, Samsonia, Serratia, Shigella, Shimwellia, Siccibacter, Sodalis, Tatumella, Thorsellia, Trabulsiella, Wigglesworthia, Xenorhabdus, Yersinia and Yokenella.
In one embodiment, the ClpB protein according to the present invention is a ClpB from Hafnia, preferably from Hafnia alvei (SEQ ID NO: 1). In one embodiment, the ClpB protein according to the present invention is a ClpB from E. Coli, preferably from E. Coli K12 (SEQ ID NO: 21).
In Hafnia alvei, the chaperone protein ClpB is a protein of 857 amino acids. In one embodiment, the ClpB protein has the amino acid sequence of the chaperone protein ClpB from Hafnia alvei at set forth in SEQ ID NO: 1 (GenBank Reference Number: KIC99545.2). In one embodiment, the amino acid sequence of ClpB comprises or consists of an amino acid sequence at least about 95 to about 100% identical to the amino acid sequence of SEQ ID NO: 1. In the context of the present application, the percentage of identity is calculated using a global alignment (i.e. the two sequences are compared over their entire length).
In E. Coli K12, the chaperone protein ClpB is a protein of 857 amino acids. In one embodiment, the ClpB protein has the amino acid sequence of the chaperone protein ClpB from E. Coli K12 as set forth in SEQ ID NO: 21 (GenBank Reference Number: BAA16476.1). In one embodiment, the amino acid sequence of ClpB comprises or consists of an amino acid sequence at least about 95 to about 100% identical to the amino acid sequence of SEQ ID NO: 21. In the context of the present application, the percentage of identity is calculated using a global alignment (i.e. the two sequences are compared over their entire length).
In one embodiment, the ClpB protein according to the present invention is a ClpB variant.
In one embodiment, a ClpB variant as used herein comprises or consists of a sequence presenting a sequence identity of at least about 70% with amino acid sequence SEQ ID NO: 1, preferably of at least about 80%, more preferably of at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.
A ClpB variant may also, or alternatively, contain non-conservative changes. In one embodiment, variant polypeptides differ from a native sequence by substitution, deletion or addition of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids. Variants may also (or alternatively) be modified by, for example, the deletion or addition of amino acids that have minimal influence on the immunogenicity, secondary structure and hydropathic nature of the polypeptide.
In one embodiment, a variant of SEQ ID NO: 1 is capable of inducing satiation, prolonging satiety, reducing food intake, controlling weight gain, stimulating weight loss and/or reducing fat mass on lean mass ratio in a subject in need thereof with an efficiency at least equivalent to the one of SEQ ID NO: 1.
In one embodiment, a variant of SEQ ID NO: 1 comprises conservative amino acid substitutions as compared to the sequence of SEQ ID NO: 1, such as, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more conservative amino acid substitutions.
In another embodiment, a variant of SEQ ID NO: 1 is a polypeptide wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids from the sequence of SEQ ID NO: 1 is/are absent, or substituted by any amino acid, or wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids (either contiguous or not) is/are added.
In one embodiment, the polypeptide or protein of the invention comprises or consists of at least a fragment of ClpB, preferably at least a fragment of the ClpB having the amino acid sequence SEQ ID NO: 1.
In one embodiment, a ClpB fragment comprises or consists of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 75, 100 or more amino acids of a ClpB protein, preferably of a ClpB having the amino acid sequence SEQ ID NO: 1.
In one embodiment, a ClpB fragment comprises or consists of less than 100, 75, 60, 50, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10 or less amino acids in length.
In one embodiment, a ClpB fragment comprises from 4 to 100 or more amino acids, from 10 to 80 amino acids, from 15 to 70 amino acids, from 20 to 60 amino acids, from 25 to 50 amino acids, from 30 to 45 amino acids, or from 35 to 40 amino acids.
In another embodiment, a ClpB fragment comprises from 4 to 80 amino acids, or from 4 to 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 or 10 amino acids. In another embodiment, a ClpB fragment comprises from 6 to 100 amino acids, or from 6 to 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 or 10 amino acids. In another embodiment, a ClpB fragment comprises from 8 to 100 amino acids, or from 8 to 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 15 or 10 amino acids. In another embodiment, a ClpB fragment comprises from 10 to 100 amino acids, or from 10 to 80, 70, 60, 50, 45, 40, 35, 30, 25, 20 or 15 amino acids.
In one embodiment, a ClpB fragment comprises or consists of 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 amino acids. In another embodiment, a ClpB fragment comprises or consists of 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 80, 90 or 100 amino acids.
In another embodiment, a ClpB fragment comprises from 100 to 500 or more amino acids, from 100 to 400 amino acids, from 100 to 300 amino acids, from 100 to 200 amino acids, or from 100 to 150 amino acids. In another embodiment, a ClpB fragment comprises from 150 to 500 or more amino acids, from 150 to 400 amino acids, from 150 to 300 amino acids, or from 150 to 200 amino acids. In another embodiment, a ClpB fragment comprises from 100 to 350 amino acids, from 150 to 300 amino acids, or from 200 to 250 amino acids.
In one embodiment, a ClpB fragment comprises or consists of 100, 125, 150, 175, 200, 250, or 300 amino acids. In another embodiment, a ClpB fragment comprises or consists of 220 amino acids. In one embodiment, a ClpB fragment comprises or consists of 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, or 230 amino acids.
In one embodiment, a ClpB fragment comprises or consists of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 75, 100 or more amino acid residues of SEQ ID NO: 1 or variant thereof.
In one embodiment, a ClpB fragment comprises or consists of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 75, 100 or more contiguous amino acid residues of SEQ ID NO: 1. In one embodiment, a ClpB fragment comprises or consists of at least 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 50, 60, 75, 100 or more discontiguous amino acid residues of SEQ ID NO: 1 or variant thereof. In one embodiment, a ClpB fragment comprises or consists of at least 5 discontiguous amino acid residues of SEQ ID NO: 1 or variant thereof. In one embodiment, a ClpB fragment comprises or consists of at least 6 discontiguous amino acid residues of SEQ ID NO: 1 or variant thereof.
In one embodiment, a ClpB fragment of the invention comprises a negatively charged residue and two consecutive Arg and Trp residues. In one embodiment, a ClpB fragment of the invention comprises consecutive Arg and Trp residues and a negatively charged residue upstream of the consecutive Arg and Trp residues.
In one embodiment, a ClpB fragment of the invention comprises at least a negatively charged residue upstream of consecutive Arg and Trp residues. As used herein, the term “upstream of consecutive Arg and Trp residues” means at position −1, −2, −3, −4 or −5 of the Arg residue. In others words, in one embodiment, the Arg residue of the consecutive Arg and Trp sequence is at position +1, +2, +3, +4 or +5 of the negatively charged residue.
In one embodiment, the negatively charged residue is a Glu or Asp residue. In a particular embodiment, the negatively charged residue is a Glu residue.
In one embodiment, a ClpB fragment comprises at least amino acid residue E538 of SEQ ID NO: 1 or variant thereof.
In one embodiment, a ClpB fragment comprises or consists of at least amino acid residues R542 and/or W543 of SEQ ID NO: 1 or variant thereof. In one embodiment, a ClpB fragment comprises or consists of at least amino acid residues R542 and W543 of SEQ ID NO: 1 or variant thereof.
In one embodiment, a ClpB fragment comprises or consists of at least amino acid residue E538, R542 and W543 of SEQ ID NO: 1.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 2.
wherein X is any amino acid and X2-4 is 2, 3 or 4 of any amino acid, preferably wherein E/D is E.
In another particular embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 3.
wherein X is any amino acid, preferably wherein E/D is E.
In another particular embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 4.
In a preferred embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 5.
In a particular embodiment, the polypeptide or protein of the invention comprises the amino acid sequence SEQ ID NO: 2, 3, 4 or 5 and has a length of at least 15 amino acids, preferably at least 20 amino acids.
In one embodiment, a ClpB fragment of the invention further comprises a sequence having at least 75% sequence homology with the sequence GKPV.
In one embodiment, a ClpB fragment further comprises at least amino acid residues G545, P547 and/or V548 of SEQ ID NO: 1 or variant thereof. In a particular embodiment, a ClpB fragment comprises at least amino acid residues G545, 1546, P547 and/or V548 of SEQ ID NO: 1 or variant thereof. In a preferred embodiment, a ClpB fragment comprises at least amino acid residues G545, 1546, P547 and V548 of SEQ ID NO: 1 or variant thereof.
In one embodiment, a ClpB fragment according to the invention comprises or consists of at least amino acid residues E538, R542, W543, G545, P547 and V548 of SEQ ID NO: 1 or variant thereof. In one embodiment, a ClpB fragment according to the invention comprises or consists of at least amino acid residues E538, R542, W543, G545, 1546, P547 and V548 of SEQ ID NO: 1 or variant thereof.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 6.
wherein X is any amino acid and X2-4 is 2, 3 or 4 of any amino acid, preferably wherein E/D is E.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence 10 SEQ ID NO: 7.
wherein X is any amino acid and X2-4 is 2, 3 or 4 of any amino acid.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 8.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 9.
wherein X is any amino acid and X2-4 is 2, 3 or 4 of any amino acid.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 10.
wherein X2-4 is 2, 3 or 4 of any amino acid.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 11.
wherein X is any amino acid.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 12.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 13.
wherein X is any amino acid and X2-4 is 2, 3 or 4 of any amino acid.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 14.
wherein X2-4 is 2, 3 or 4 of any amino acid.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 15.
wherein X is any amino acid.
In one embodiment, a ClpB fragment comprises or consists of amino acid sequence SEQ ID NO: 16.
In a particular embodiment, the polypeptide or protein of the invention comprises the amino acid sequence SEQ ID NO: 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 and has a length of at least 15 amino acids, preferably at least 20 amino acids.
In one embodiment, a ClpB fragment comprises or consists of an amino acid sequence presenting a sequence identity of at least 70% with amino acid sequence 538-548 of SEQ ID NO: 1 (EVLARWTGIPV, SEQ ID NO: 12), preferably of at least 80%, more preferably of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In one embodiment, a ClpB fragment comprises or consists of the amino acid sequence SEQ ID NO: 12.
In one embodiment, a ClpB fragment comprises or consists of an amino acid sequence presenting a sequence identity of at least 70% with amino acid sequence 536-548 of SEQ ID NO: 1 (IAEVLARWTGIPV, SEQ ID NO: 18), preferably of at least 80%, more preferably of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In one embodiment, a ClpB fragment comprises or consists of the amino acid sequence SEQ ID NO: 18.
In one embodiment, a ClpB fragment comprises or consists of an amino acid sequence presenting a sequence identity of at least 70% with amino acid sequence 534-548 of SEQ ID NO: 21 (AEIAEVLARWTGIPV, SEQ ID NO: 19), preferably of at least 80%, more preferably of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In one embodiment, a ClpB fragment comprises or consists of the amino acid sequence SEQ ID NO: 19.
In one embodiment, a ClpB fragment comprises or consists of an amino acid sequence presenting a sequence identity of at least 70% with amino acid sequence 534-548 of SEQ ID NO: 1 (VEIAEVLARWTGIPV, SEQ ID NO: 17), preferably of at least 80%, more preferably of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In one embodiment, a ClpB fragment comprises or consists of the amino acid sequence SEQ ID NO: 17.
In one embodiment, a ClpB fragment comprises or consists of an amino acid sequence presenting a sequence identity of at least 70% with amino acid sequence 537-756 of SEQ ID NO: 21 (SEQ ID NO: 22), preferably of at least 80%, more preferably of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more. In one embodiment, a ClpB fragment comprises or consists of the amino acid sequence SEQ ID NO: 22.
In one embodiment, a polypeptide or protein comprising or consisting of at least a fragment of a ClpB protein or variant thereof does not consist of the amino acid sequence of a ClpB protein. In other words, in one embodiment, the polypeptide or protein of the invention is not a full-length ClpB protein. Accordingly, in one embodiment, the amino acid sequence of the polypeptide or protein of the invention is not 100% identical to a ClpB sequence. In a particular embodiment, the amino acid sequence of the polypeptide or protein of the invention is not 100% identical to SEQ ID NO:1 and/or is not 100% identical to SEQ ID NO:21.
In one embodiment, a polypeptide or protein comprising or consisting of at least a fragment of a ClpB protein or variant thereof does not consist of the amino acid sequence of α-MSH. In a particular embodiment, a polypeptide or protein comprising or consisting of at least a fragment of a ClpB protein or variant thereof does not consist of the amino acid sequence as set forth in SEQ ID NO:20.
In one embodiment, the polypeptide or protein of the invention or variant thereof has homology with α-MSH (SEQ ID NO: 20). In one embodiment, the polypeptide or protein of the invention or variant thereof has conformational homology with α-MSH. In another embodiment, the polypeptide or protein of the invention or variant thereof has sequence homology with α-MSH. In another embodiment, the polypeptide or protein of the invention or variant thereof has conformational and sequence homology with α-MSH.
In one embodiment, the polypeptide or protein of the invention or variant thereof is recognized by an anti-α-MSH antibody. In other words, in one embodiment, the polypeptide or protein of the invention or variant thereof serves as an epitope for an anti-α-MSH antibody. In one embodiment, the polypeptide or protein of the invention or variant thereof serves as a conformational epitope for an anti-α-MSH antibody.
In one embodiment, a polypeptide or protein comprising or consisting of at least a fragment of a ClpB protein or variant thereof is a polypeptide derivative.
In one embodiment, the polypeptide or protein of the invention or variant thereof is modified by means well-known in the art, e.g., by the addition of one or more functional group such as a phosphate, acetate, lipid or carbohydrate group, and/or by the addition of one or more protecting group. For example, the polypeptide or protein of the invention or variant thereof can be modified by the addition of one or more functional groups such as phosphate, acetate, or various lipids and carbohydrates.
The polypeptide or protein of the invention or variant thereof described herein can be produced synthetically by chemical synthesis or enzymatic synthesis as it is well known in the art. Alternatively, nucleotide sequences encoding the polypeptide or protein of the invention or variant thereof of the invention can be introduced into a protein expression vector and produced in a suitable host organism (e.g., bacteria, insect cells, etc.), then purified. In one embodiment, the polypeptide or protein of the invention or variant thereof is obtained by a cloning method, such as, for example, using any production system known in the art, such as, for example, E. coli, yeast, baculovirus-insect cell, or mammalian cells such as HEK or CHO expression system.
An additional polypeptide (“tag”) can be added on for the purpose of purifying or identifying or purifying the polypeptides. Protein tags make it possible, for example, for the polypeptides to be adsorbed, with high affinity, to a matrix, and for the matrix then to be washed stringently with suitable buffers without the complex being eluted to any significant extent, and for the adsorbed complex subsequently to be eluted selectively.
Examples of protein tags which are known to the skilled person are a (His)6 tag, a Myc tag, a FLAG tag, a hemagglutinin tag, a glutathione transferase (GST) tag, intein having an affinity chitin-binding tag or maltose-binding protein (MBP) tag. These protein tags can be located N-terminally, C-terminally and/or internally.
The present invention further relates to a polynucleotide or nucleic acid encoding a polypeptide or protein comprising or consisting of at least a fragment of the ClpB protein or variant thereof as described herein.
In one embodiment, the polynucleotide or nucleic acid of the invention is DNA. In another embodiment, the polynucleotide or nucleic acid of the invention is RNA, for example, in the form of messenger RNA (mRNA).
In one embodiment, the polynucleotide or nucleic acid of the present invention is single stranded. In another embodiment, the polynucleotide or nucleic acid of the present invention is double stranded.
Another object of the present invention is a vector encoding a polypeptide or protein comprising or consisting of at least a fragment of the ClpB protein or variant thereof as described herein, or comprising a polynucleotide or nucleic acid encoding a polypeptide or protein comprising or consisting of at least a fragment of the ClpB protein or variant thereof as described herein.
Examples of vector include, but are not limited to, a plasmid, a bacteriophage, a virus, a cationic vesicle or any other type of vector.
In one embodiment, the vector of the invention is an expression vector.
Another object of the present invention is a composition comprising a polypeptide or protein or variant thereof as described herein, or a polynucleotide or nucleic acid as described herein, or a vector as described herein.
Another object of the present invention is a pharmaceutical composition comprising a polypeptide or protein or variant thereof as described herein, or a polynucleotide or nucleic acid as described herein, or a vector as described herein, and at least one pharmaceutically acceptable excipient.
Another object of the present invention is a medicament comprising a polypeptide or protein or variant thereof as described herein, or a polynucleotide or nucleic acid as described herein, or a vector as described herein.
In one embodiment, the pharmaceutical composition or the medicament of the invention comprises a therapeutically effective amount of the polypeptide, protein, polynucleotide or nucleic acid, or vector of the invention.
Another object of the present invention is a vaccine comprising a polypeptide or protein or variant thereof as described herein, or a polynucleotide or nucleic acid as described herein, or a vector as described herein.
In one embodiment, the vaccine according to the present invention is for use in the prevention of overweight and/or obesity-related diseases and disorders.
Another object of the present invention is a dietary supplement or protein dietary supplement comprising a polypeptide comprising a fragment of a ClpB protein as described herein, preferably a polypeptide comprising a ClpB fragment comprising the amino acid sequence SEQ ID NO:2.
Another object of the present invention is a food composition comprising a polypeptide comprising a fragment of a ClpB protein as described herein, preferably a polypeptide comprising a ClpB fragment comprising the amino acid sequence SEQ ID NO:2.
In one embodiment, the dietary supplement or food composition comprises or consists of about 1% to about 80% by weight of the polypeptide or protein comprising a fragment of a ClpB protein according to the invention. In one embodiment, the dietary supplement or food composition comprises or consists of about 1% to about 50% by weight, preferably from about 2% to about 25% by weight of the polypeptide or protein comprising a fragment of a ClpB protein according to the invention.
In one embodiment, the dietary supplement or food composition comprises or consists of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80% by weight or more of the polypeptide comprising a fragment of a ClpB protein according to the invention.
In one embodiment, the dietary supplement or food composition of the present invention comprises at least one additional ingredient selected from among simple and/or complex carbohydrates, lipids, fibers, or minerals.
In one embodiment, the dietary supplement or food composition of the present invention comprises at least one essential amino acid. Preferably, said at least one essential amino acid is isolated or free, i.e., not bonded in a protein chain.
In one embodiment, the dietary supplement or food composition according to the present invention is in the form of anhydrous powder or powder containing water or moisture.
In one embodiment, the dietary supplement or food composition further comprises carriers or vehicles. “Carriers” or “vehicles” mean materials suitable for administration and include any such material known in the art such as, for example, any liquid, gel, solvent, liquid diluent, solubilizer, or the like, which is non-toxic and which does not interact with any components, in particular with the bacterial strain, of the composition in a deleterious manner. Examples of nutritionally acceptable carriers include, for example, water, salt solutions, alcohol, silicone, waxes, petroleum jelly, vegetable oils, polyethylene glycols, propylene glycol, liposomes, sugars, gelatin, lactose, amylose, magnesium stearate, talc, surfactants, silicic acid, viscous paraffin, perfume oil, fatty acid monoglycerides and diglycerides, petroethral fatty acid esters, hydroxymethyl-cellulose, polyvinylpyrrolidone, and the like.
In one embodiment, the dietary supplement or food composition of the present invention comprises an amount of dietary fibers. Dietary fibers pass through the small intestine undigested by enzymes and functions as a natural bulking agent and laxative. Dietary fibers may be soluble or insoluble and in general a blend of the two types is preferred. Suitable sources of dietary fibers include soy, pea, oat, pectin, guar gum, gum Arabic, fructooligosaccharides, galacto-oligosaccharides, sialyl-lactose and oligosaccharides derived from animal milks. In some embodiments, the dietary fiber is selected among mannans. Mannans (such as glucomannans and galactomannans), such as guar gum, locust bean gum, konjac, and xanthan gum, are present in some plant cell walls. The glucomannans are generally comprised of (1-4)-β-linked glucose and mannose units, while the galactomannans are generally comprised of a (1-4)-β-mannan backbone substituted with single units of (1-6)-α-galactose. Many endospermic legumes, such as guar and locust bean, contain galactomannans in the endosperm during seed development. Glucomannans have also been found as a minor component of cereal grains.
In one embodiment, the dietary supplement or food composition of the present invention contains minerals and micronutrients such as trace elements and vitamins in accordance with the recommendations of Government bodies such as the USRDA. For example, the composition may contain per daily dose one or more of the following micronutrients in the ranges given: 300 to 500 mg calcium, 50 to 100 mg magnesium, 150 to 250 mg phosphorus, 5 to 20 mg iron, 1 to 7 mg zinc, 0.1 to 0.3 mg copper, 50 to 200 μg iodine, 5 to 15 μg selenium, 1000 to 3000 μg beta carotene, 10 to 80 mg Vitamin C, 1 to 2 mg Vitamin B1, 0.5 to 1.5 mg Vitamin B6, 0.5 to 2 mg Vitamin B2, 5 to 18 mg niacin, 0.5 to 2.0 μg Vitamin B12, 100 to 800 μg folic acid, 30 to 70 μg biotin, 1 to 5 μg Vitamin D, 3 to 10 μg Vitamin E.
In one embodiment, the dietary supplement or food composition of the present invention further comprises emulsifiers. Examples of food grade emulsifiers typically include diacetyl tartaric acid esters of mono- and di-glycerides, lecithin and mono- and di-glycerides. Similarly, suitable salts and stabilizers may be included.
In one embodiment, the dietary supplement or food composition according to the present invention can be used for the preparation of dietary food.
In one embodiment, the dietary supplement or food composition according to the present invention can be used for introduction into daily meals.
The present invention also relates to a polypeptide or protein comprising or consisting of at least a fragment of the ClpB protein or variant thereof, a polynucleotide or nucleic acid, a vaccine, a pharmaceutical composition or a medicament as described hereinabove, for use for treating inflammation in a subject in need thereof.
Another object of the invention relates to a method for treating inflammation in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide or protein comprising or consisting of at least a fragment of the ClpB protein or variant thereof, polynucleotide or nucleic acid, vaccine, pharmaceutical composition or medicament as described hereinabove.
Within the meaning of the invention, by “inflammation”, it is meant, as defined in Dorland's Medical Dictionary, “a localized protective response, elicited by injury or destruction of tissues, which serves to destroy, dilute or wall off both the injurious agent and the injured tissue”. It is characterized by fenestration of the microvasculature, leakage of the elements of blood into the interstitial spaces, and migration of leukocytes into the inflamed tissue. On a macroscopic level, this is usually accompanied by the familiar clinical signs of erythema, edema, hyperalgesia (tenderness), and pain.
In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vaccine, pharmaceutical composition or medicament according to the invention is for use in the treatment of inflammation, wherein said inflammation is selected from the group comprising obesity, obesity-related diseases or disorders, neuroinflammation, multiple sclerosis, atherosclerosis, allergies, ankylosing spondylitis, arthritis (osteoarthritis, rheumatoid arthritis, or psoriatic arthritis), asthma, graft versus host disease, Parkinson's disease, Alzheimer's disease, Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome, systemic lupus erythematous, nephritis, and ulcerative colitis.
In one embodiment, the inflammation of the invention is an acute inflammation. In another embodiment, the inflammation of the invention is a chronic inflammation.
In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vaccine, pharmaceutical composition or medicament according to the invention is for use in the treatment of inflammation, wherein said inflammation is selected from the group comprising or consisting of obesity, obesity-related diseases or disorders, neuroinflammation, multiple sclerosis, psoriasis, allergic rhinitis, osteoarthritis and neuroinflammatory diseases. In a particular embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vaccine, pharmaceutical composition or medicament according to the invention is for use in the treatment of inflammation, wherein said inflammation is selected from the group comprising or consisting of obesity, neuroinflammation, multiple sclerosis, psoriasis, allergic rhinitis, osteoarthritis and neuroinflammatory diseases.
An object of the present invention relates to a polypeptide or protein comprising or consisting of at least a fragment of the ClpB protein or variant thereof, polynucleotide or nucleic acid, vaccine, pharmaceutical composition or medicament according to the invention for use in the treatment or prevention of obesity in a subject in need thereof.
Another object of the present invention relates to a method for treating or preventing obesity in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide or protein comprising or consisting of at least a fragment of the ClpB protein or variant thereof, polynucleotide or nucleic acid, vaccine, pharmaceutical composition or medicament as described hereinabove.
One aspect of the present invention relates to a polypeptide or protein comprising or consisting of at least a fragment of the ClpB protein or variant thereof, polynucleotide or nucleic acid, vaccine, pharmaceutical composition or medicament as described hereinabove, for use in the treatment or prevention of overweight and/or obesity-related diseases and disorders.
Another aspect of the present invention relates to a method for treating or preventing overweight and/or obesity-related diseases and disorders in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide or protein comprising or consisting of at least a fragment of the ClpB protein or variant thereof, polynucleotide or nucleic acid, vaccine, pharmaceutical composition or medicament as described hereinabove.
Overweight and/or obesity-related diseases and disorders include, but are not limited to, high blood pressure, diabetes (in particular, type 2 diabetes), glucose intolerance, insulin resistance, cardiovascular disease (such as atherosclerosis, coronary artery disease, narrowed arteries, angina, heart attack, blood clots), high cholesterol, fatty liver disease, hepatic steatosis, cholelithiasis, joint problems, osteoarthritis, orthopedic problems, impaired balance, skin conditions, sleep apnea, respiratory problems, asthma, heavy snoring, cancer (including breast, colon, gallbladder, uterus, colon and prostate cancers), metabolic syndrome, menstrual abnormalities and psychosocial effects.
One aspect of the present invention relates to a method of inducing satiation in a subject in need thereof comprising administering to the subject an effective amount of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention.
One aspect of the present invention relates to a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention for use for inducing satiation in a subject in need thereof.
One aspect of the present invention concerns the non-therapeutic use of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, dietary supplement or food composition according to the invention for inducing satiation in a subject.
One aspect of the present invention relates to a method of prolonging satiety in a subject in need thereof comprising administering to the subject an effective amount of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention relates to a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention for use for prolonging satiety in a subject in need thereof.
One aspect of the present invention concerns the non-therapeutic use of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, dietary supplement or food composition according to the invention for prolonging satiety in a subject.
In one embodiment, the prolongation of satiety is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In a particular embodiment, the prolongation of satiety is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the time elapsed between meals, preferably of the time elapsed between meals prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention. In one embodiment, the prolongation of satiety is of at least 15%, 20% or 25%. In a particular embodiment, the prolongation of satiety is of at least 15%, 20% or 25% of the time elapsed between meals, preferably of the time elapsed between meals prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention relates to a method of reducing meal size in a subject in need thereof comprising administering to the subject an effective amount of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention relates to a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention for use for reducing meal size in a subject in need thereof.
One aspect of the present invention concerns the non-therapeutic use of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, dietary supplement or food composition according to the invention for reducing meal size in a subject.
In one embodiment, the reduction of meal size is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In a particular embodiment, the reduction of meal size is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the meal size prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention. In one embodiment, the reduction of meal size is of at least 15%, 20% or 25%. In a particular embodiment, the reduction of meal size is of at least 15%, 20% or 25% of the meal size prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention relates to a method of reducing food intake in a subject in need thereof comprising administering to the subject an effective amount of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention relates to a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention for use for reducing food intake in a subject in need thereof.
One aspect of the present invention concerns the non-therapeutic use of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, dietary supplement or food composition according to the invention for reducing food intake in a subject.
In one embodiment, the reduction of food intake is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In a particular embodiment, the reduction of food intake is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the food intake prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention. In one embodiment, the reduction of food intake is of at least 15%, 20% or 25%. In a particular embodiment, the reduction of food intake is of at least 15%, 20% or 25% of the food intake prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention also relates to a method of controlling, in particular reducing, weight gain in a subject in need thereof comprising administering to the subject an effective amount of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention relates to a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention for use for controlling, in particular reducing, weight gain in a subject in need thereof.
One aspect of the present invention concerns the non-therapeutic use of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, dietary supplement or food composition according to the invention for controlling, in particular reducing, weight gain in a subject.
One further aspect of the present invention also relates to a method of stimulating weight loss in a subject in need thereof comprising administering to the subject an effective amount of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention relates to a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention for use for stimulating weight loss in a subject in need thereof.
One aspect of the present invention concerns the non-therapeutic use of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, dietary supplement or food composition according to the invention for stimulating weight loss in a subject.
In one embodiment, the stimulation of weight loss is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In a particular embodiment, the stimulation of weight loss is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the weight loss of the subject prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention. In one embodiment, the stimulation of weight loss is of at least 15%, 20% or 25%. In a particular embodiment, the stimulation of weight loss is of at least 15%, 20% or 25% of the weight loss of the subject prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One further aspect of the present invention also relates to a method of reducing weight in a subject in need thereof comprising administering to the subject an effective amount of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention relates to a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention for use for s reducing weight in a subject in need thereof.
One aspect of the present invention concerns the non-therapeutic use of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, dietary supplement or food composition according to the invention for reducing weight in a subject.
In one embodiment, the reduction of weight is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In a particular embodiment, the reduction of weight is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the body weight of the subject prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention. In one embodiment, the reduction of weight is of at least 15%, 20% or 25%. In a particular embodiment, the reduction of weight is of at least 15%, 20% or 25% of the body weight of the subject prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention relates to a method of reducing fat mass on lean mass ratio in a subject in need thereof, comprising administering to the subject an effective amount of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
One aspect of the present invention relates to a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention for use for reducing fat mass on lean mass ratio in a subject in need thereof, in particular in an obese subject.
One aspect of the present invention concerns the non-therapeutic use of a polypeptide or protein, polynucleotide, vector, composition, dietary supplement or food composition according to the invention for reducing fat mass on lean mass ratio in a subject, in particular in a subject having normal weight or uncomplicated overweight.
In one embodiment, the reduction of fat mass on lean mass ratio is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10%. In a particular embodiment, the reduction of fat mass on lean mass ratio is of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% of the fat mass on lean mass ratio of the subject prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention. In one embodiment, the s reduction of fat mass on lean mass ratio is of at least 15%, 20% or 25%. In a particular embodiment, the reduction of fat mass on lean mass ratio is of at least 15%, 20% or 25% of the fat mass on lean mass ratio of the subject prior to administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
In one embodiment, the use of a polypeptide or protein, polynucleotide or nucleic acid, vector, composition, dietary supplement or food composition according to the invention is a cosmetic use.
In one embodiment, the subject is a female. In some embodiments, the subject is a male.
In one embodiment, the subject is a child, such as an individual aged less than 18 (in human years). In another embodiment, the subject is an adult, such as an individual aged 18 or more (in human years).
In one embodiment, the subject is obese. In one embodiment, the subject has a body mass index (BMI) above 30.
In one embodiment, the subject is moderately obese. In one embodiment, the subject has a BMI ranging from about 30 to about 35. In one embodiment, the subject is severely obese. In one embodiment, the subject has a BMI ranging from about 35 to about 40. In one embodiment, the subject is morbidly obese. In one embodiment, the subject has a BMI ranging from about 40 to about 50 or more.
In one embodiment, the subject is not obese. In one embodiment, the subject has a BMI below 30. In one embodiment, the subject is overweight. Accordingly, in one embodiment, the subject has a BMI ranging from about 25 to about 30.
In one embodiment, the subject is a healthy overweight subject. In one embodiment, the subject is a non-healthy overweight subject.
In one embodiment, the subject has a normal body weight. In one embodiment, the subject has a BMI ranging from about 18.5 and 25.
In one embodiment, the subject is under a slimming diet and/or wants to lose weight. In another embodiment, the subject is not under a slimming diet and/or does not want to lose weight.
In one embodiment, the subject is at risk of gaining weight. In some embodiments, the subject is at risk of accumulating excessive fat.
In one embodiment, the subject is at risk of developing overweight and/or obesity. In one embodiment, the subject is at risk of developing overweight and/or obesity-related diseases and disorders.
In one embodiment, the subject is a binge-eater. In one embodiment, the subject suffers from binge- or compulsive eating disorder.
In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, or vector according to the invention is administered to the subject in the form of a composition, pharmaceutical composition, medicament or vaccine. In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, or vector according to the invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.
In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention is to be administered orally, by injection, topically, nasally, buccally, rectally, vaginally, intratracheally, by endoscopy, transmucosally, or by percutaneous administration.
The disclosed polypeptides or proteins, polynucleotide or nucleic acid, vectors, compositions, pharmaceutical compositions, medicaments or vaccines according to the invention can be delivered to the target cells in a variety of ways. The delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro. The skilled artisan will be able to adapt the delivery of the polypeptides or nucleic acid sequences of the invention.
In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention is to be orally administered. Examples of formulations adapted to oral administration include, but are not limited to: solid forms, liquid forms and gels. Examples of solid forms adapted to oral administration include, but are not limited to, pill, tablet, capsule, soft gelatin capsule, hard gelatin capsule, caplet, compressed tablet, cachet, wafer, sugar-coated pill, sugar coated tablet, or dispersing/or disintegrating tablet, powder, solid forms suitable for solution in, or suspension in, liquid prior to oral administration and effervescent tablet. Examples of liquid form adapted to oral administration include, but are not limited to, solutions, suspensions, drinkable solutions, elixirs, sealed phial, potion, drench, syrup and liquor.
In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention is to be administered by injection, preferably systemically injected. Examples of formulations adapted to systemic injections include, but are not limited to: liquid solutions or suspensions, solid forms suitable for solution in, or suspension in, liquid prior to injection. Examples of systemic injections include, but are not limited to, intravenous, subcutaneous, intramuscular, intradermal, intravitreal, and intraperitoneal injection, or perfusion. In another embodiment, when injected, the composition, the pharmaceutical composition or the medicament of the invention is sterile. Methods for obtaining a sterile pharmaceutical composition include, but are not limited to, GMP synthesis (GMP stands for “Good manufacturing practice”).
In another embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention is to be topically administered. Examples of formulations adapted to topical administration include, but are not limited to, sticks, waxes, creams, lotions, ointments, balms, gels, masks, leave-on washes and/or the like.
In one embodiment of the invention, the ointment is an oleaginous ointment; an emulsified ointment such as, for example, oil-in-water or a water-in-oil ointment; or a water-soluble ointment, preferably is an oleaginous ointment.
In one embodiment of the invention, the oleaginous ointment uses bases such as, for example, plant and animal oils; plant and animal fats; waxes; vaseline, such as, for example, white vaseline or vaseline oil; and paraffin such as, for example, liquid paraffin or paraffin oil.
In another embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention can also be applied topically using a transdermal system, such as one of an acrylic-based polymer adhesive with a resinous crosslinking agent impregnated with the composition and laminated to an impermeable backing. Examples of formulations adapted to transdermal administration include, but are not limited to, ointment, paste, cream, film, balm, patch, such as, for example, transdermal patch, gel, liposomal forms and the like.
In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention can be administered topically as a transdermal patch, more particularly as a sustained-release transdermal patch. The transdermal patches can include any conventional form such as, for example, adhesive matrix, polymeric matrix, reservoir patch, matrix or monolithic-type laminated structure, and are generally comprised of one or more backing layers, adhesives, penetration enhancers, an optional rate controlling membrane and a release liner which is removed to expose the adhesives prior to application. Polymeric matrix patches also comprise a polymeric-matrix forming material. Suitable transdermal patches are well described in the art [see e.g., U.S. Pat. Nos. 5,262,165, 5,948,433, 6,010,715 and 6,071,531].
In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention is administered in a controlled-release, delayed-release, extended-release, long-acting-release, modified-release, sustained-release or timed-release form. Therefore, in one embodiment, the composition, pharmaceutical composition, medicament or vaccine according to the invention further comprises sustained-release matrices, such as biodegradable polymers.
In one embodiment, the dietary supplement or food composition according to the invention is to be orally administered. Examples of formulations adapted to oral administration include, but are not limited to: solid forms, liquid forms and gels. Examples of solid forms adapted to oral administration include, but are not limited to, pill, tablet, capsule, soft gelatin capsule, hard gelatin capsule, caplet, compressed tablet, cachet, wafer, sugar-coated pill, sugar coated tablet, or dispersing/or disintegrating tablet, powder, solid forms suitable for solution in, or suspension in, liquid prior to oral administration and effervescent tablet. Examples of liquid form adapted to oral administration include, but are not limited to, solutions, suspensions, drinkable solutions, elixirs, sealed phial, potion, drench, syrup and liquor.
In one embodiment, the dietary supplement or food composition according to the invention is formulated as powders, for example, for mixing with consumable liquids such as milk, juice, water or consumable gels or syrups for mixing into other dietary liquids or foods. In one embodiment, the dietary supplement or food composition according to the invention is formulated with other foods or liquids to provide premeasured supplemental foods, such as single serving bars, for example. Flavorings, binders, protein, complex carbohydrates, and the like may be added as needed.
In one embodiment, the dietary supplement or food composition according to the invention is formulated using any pharmaceutically acceptable forms of the vitamins, minerals and other nutrients discussed above, including their salts. Preferred forms are calcium carbonate, magnesium hydroxide or magnesium sulfate, sodium tetraborate, cupric oxide, manganese sulfate, zinc sulfate, cholecalciferol, ferrous fumarate, pyridoxine hydrochloride, chromium picolinate, d-alphatocopherol acetate, and ascorbic acid.
In one embodiment, a therapeutically effective amount of the polypeptide or protein, polynucleotide, vector, composition, pharmaceutical composition, medicament, or vaccine according to the invention is administered at least once a day, twice a day, or at least three times a day. In another embodiment, a therapeutically effective amount of the polypeptide or protein, polynucleotide, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention is administered every day, every two, three, four, five, six or seven days.
In another embodiment, a therapeutically effective amount of the polypeptide or protein, polynucleotide, vector, composition, pharmaceutical composition, medicament, or vaccine according to the invention is administered every week, twice a week, every two weeks, or once a month. In another embodiment, a therapeutically effective amount of the polypeptide or protein, polynucleotide, vector, composition, pharmaceutical composition, medicament or vaccine according to the invention is administered every month for a period at least 2, 3, 4, 5 or 6 months.
In another embodiment, a therapeutically effective amount of the polypeptide or protein, polynucleotide or vector according to the present invention ranges from about 1 μg to 5 g. In another embodiment, a therapeutically effective amount of the polypeptide or protein, polynucleotide or vector according to the present invention to be administered ranges from about 0.1 μg/kg to 1 g/kg, i.e., from about 0.1 μg per kilo body weight to 1 g per kilo body weight.
In one embodiment, the method of the invention is for a chronic treatment, i.e., the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or vaccine according to the invention, is administered for a prolonged period of time, such as, for example, for at least about 1 week, 1 month, 1 year or more.
In another embodiment, the method of the invention is for an acute treatment, such as, for example, a treatment with only 1, 2 or 3 administrations of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, or vaccine according to the invention.
In one embodiment, the administration of the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention is repeated, for example, 2 to 3 times a day, for one day or more and generally for a sustained period of at least 4 days, or even 4 to 15 weeks, with, where appropriate, one or more periods of interruption. In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention is administered simultaneously or sequentially with a meal of the subject. In one embodiment, the polypeptide or protein, polynucleotide or nucleic acid, vector, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention is administered prior to the meal of the subject.
The present invention also relates to a kit comprising a polypeptide or protein, polynucleotide or nucleic acid, composition, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition according to the invention.
In one embodiment, the kit of the invention further comprises means to administer the polypeptide or protein, polynucleotide or nucleic acid, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition to a subject in need thereof.
Means to administer the polypeptide or protein, polynucleotide or nucleic acid, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition include, but are not limited to, syringes, needles, and other materials. In some embodiments, a means to administer the polypeptide or protein, polynucleotide or nucleic acid, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition include syringes pre-filled with the polypeptide or protein, polynucleotide or nucleic acid, pharmaceutical composition, medicament or vaccine of the invention.
In one embodiment, the kit of the invention further comprises instructions for the administration of the polypeptide or protein, polynucleotide or nucleic acid, pharmaceutical composition, medicament, vaccine, dietary supplement or food composition to said subject.
In one embodiment, the kit of the invention is for use for treating or preventing obesity in a subject in need thereof. In one embodiment, the kit of the invention is for use for treating or preventing overweight and/or obesity-related diseases or disorders in a subject in need thereof.
In one embodiment, the kit of the invention is used for inducing satiation, prolonging satiety, reducing meal size, reducing food intake, controlling weight gain, reducing weight gain, stimulating weight loss, reducing weight and/or reducing fat mass on lean mass ratio in a subject in need thereof.
The invention will be further illustrated by the examples. However, these examples should not be interpreted in any way as limiting the scope of the present invention.
The present invention is further illustrated by the following examples.
Previous in silico analysis showed that several proteins of E. coli and some other microorganisms contain amino acid sequence homology with α-MSH, suggesting that they may be responsible for the production of α-MSH-reactive autoantibodies (Fetissov, in: Neuropsychiatric Disorders and Infection, Fatemi, S. H. (ed.), Taylor & Francis Books Ldt, 2004, pp 253-262; Fetissov et al., 2008. Nutrition. 24:348-359).
To find out which bacterial proteins may not be only theoretical but also functional α-MSH antigen-mimetics, a novel proteomic approach has been undertaken using E. coli strain K12. The novelty of this approach consisted in exploiting the well-known phenomenon of a so-called non-specific staining by primary antibodies in immunodetection protocols such as Western blot. In this approach, the “non-specific staining” of other proteins than the antigenic peptide used for antibody production was researched as a basis of a structural conformational homology between the α-MSH peptide and bacterial proteins.
Materiel and Methods
The proteomic approach reviewed here was used in the original study (Tennoune et al., 2014, Transl Psychiatry, 4:e458), and consisted in two-dimensional gel electrophoresis to separate total proteins of E. coli K12.
Two-Dimensional Polyacrylamide Gel Electrophoresis
For two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE), 400 μg of E. coli K12 protein extract were added to iso-electro focusing buffer (7 M urea, 2 M thiourea and 0.5% ampholytes, pH 4-7, 20 mM dithiothreitol, 2 mM tributyl phosphine, 2% CHAPS and 0.005% bromophenol blue) and solubilized for 60 min at room temperature with slight shaking. The first-dimensional gel separation was carried out using Ready Strip IPG Strip (18 cm, pH 4-7 NL, Bio-Rad, Mames-la-Coquette, France). After 24 h of passive rehydration of the strip with iso-electro focusing buffer, the protein sample was added to the strips through a loading cup placed at 1.5 cm from the cathode. Iso-electro focusing was performed with the Ettan IPGphor 3 System (GE Healthcare) in four steps (31 500 Vh): 500 V for 1 h, 1000 V gradient, 10 000 V gradient and 10 000 V for 2 h. After two equilibration steps with 2% dithiothreitol and 2.5% iodoacetamide, respectively, the second dimension, that is, a SDS-PAGE, (10% polyacrylamide gel, 20 cm×18 cm×1 mm) was performed on an Ettan Daltsix vertical electrophoresis system (GE Healthcare) with 12 mA per gel. After SDS-PAGE, the 2D gel was fixed for 2 h in 2% (vol:vol) orthophosphoric acid and in 50% (vol:vol) methanol at room temperature.
Gels were then rinsed with water, and the protein spots were visualized by CBB G-250 (Bio-Rad Laboratories, Hercules, Calif., USA) staining (34% (vol:vol) methanol, 17% (wt:vol) ammonium sulfate, 2% (vol:vol) orthophosphoric acid and 0.66 g CBB G-250 per liter).
Immunoblotting
Following 2D-PAGE, E. coli cytoplasmic proteins were transferred onto Hybond-ECL polyvinylidene difluoride membrane (GE Healthcare) via a dry transfer method (Trans Blot Cell, Bio-Rad) and a constant current of 0.8 mA·cm−2 of the membrane size for 2 h. After transfer, membranes were blocked with 5% (wt:vol) milk (Regilait, Macon, France) in phosphatebuffered saline (PBS; 10 mmol·L−1 Tris, pH 8, and 150 mmol·L−1 NaCl) plus 0.05% (vol:vol) Tween 20. After washes, membranes were incubated overnight at 4° C. with polyclonal rabbit anti-α-MSH IgG (1:1000, Peninsula Laboratories, San Carlos, Calif., USA), followed by washes and incubation with polyclonal swine anti-rabbit horseradish peroxidase-conjugated Igs (1:3000; Dako, Trappes, France). Immunoblots were revealed by the ECL detection system (GE Healthcare) and were scanned with ImageScanner II (GE Healthcare) and digitalized with Labscan 6.00 software (GE Healthcare).
The same procedure was performed after adsorption of rabbit anti-α-MSH IgG with 10−6 M of α-MSH peptide (Bachem AG, Bubendorf, Switzerland) overnight at 4° C.
Protein Identification
The protein spots of interest were excised from CBB G-250-stained 2D gels using the Ettan Spot Picker (GE Healthcare), and automated in-gel digestion of proteins was performed on the Ettan Digester (GE Healthcare). Protein extracts were then resuspended in 10 μl of 5% (vol:vol) acetonitrile/0.1% (vol:vol) formic acid and then analyzed with a nano-LC1200 system coupled to a 6340 Ion Trap mass spectrometer equipped with a nanospray source and an HPLC-chip cube interface (Agilent Technologies, Courtaboeuf, France). In brief, peptides were enriched and desalted on a 40-nl RPC 18 trap column and separated on a Zorbax (30-nm pore size, 5-μm particle size) C18 column (43 mm long×75 μm inner diameter; Agilent Technologies). A 9-min linear gradient (3-80% acetonitrile in 0.1% formic acid) at a flow rate of 400 nl·min−1 was used, and the eluent was analysed with an Ion Trap mass spectrometer. For protein identification, MS/MS peak lists were extracted and compared with the protein databases by using the MASCOT Daemon version 2.2.2 (Matrix Science, Boston, Mass., USA) search engine. The searches were performed with the following specific parameters: enzyme specificity, trypsin; one missed cleavage permitted; no fixed modifications; variable modifications, methionine oxidation, cysteine carbamidomethylation, serine, tyrosine and threonine phosphorylation; monoisotopic; peptide charge, 2+ and 3+; mass tolerance for precursor ions, 1.5 Da; mass tolerance for fragmentations, 0.6 Da; electrospray ionization-Trap as instrument; taxonomy, E. coli; National Center for Biotechnology Information (NCBI) database (NCBI Nr 20120531 (18280215 sequences, 6265275233 residues); Bethesda, Md., USA). Protein hits were automatically validated if they satisfied one of the following criteria: identification with at least two top-ranking peptides (bold and red) each with a MASCOT score of 454 (Po0.01), or at least two top-ranking peptides each with a MASCOT score of 447 (Po0.05). To evaluate false positive rates, all the initial database searches were performed using the ‘decoy’ option of MASCOT. Results were considered relevant if the false positive rate never exceeded 1%.
Results
Results of two-dimensional gel electrophoresis to separate total proteins of E. coli K12 are presented in
The ClpB protein of Enterobacteria has been shown to stimulate production of α-MSH-cross-reactive antibodies. The inventors therefore hypothesized the presence of an α-MSH-like epitope in the ClpB protein of Enterobacteria.
Sequence alignment between ClpB and α-MSH revealed a discontinuous 6-amino acid sequence homology within the central part (amino acid residues 534-548 of SEQ ID NO: 1) of E. coli and H. alvei ClpB (
ClpB sequence of Enterobacteria shares the following properties of the α-MSH peptide:
It is likely that combination of the 2 first properties is necessary for the unique functionality of the α-MSH-like epitope of ClpB (amino acid residues 534-548 of SEQ ID NO: 1), including both antigenic and α-MSH-like ligand interactions.
All commensal and pathogenic Enterobacteriaceae contained the identical α-MSH-like epitope of ClpB, suggesting the ability of bacteria belonging to this family to interfere with α-MSH signaling. Such bacteria include but are not limited to the genus of Escherichia. Salmonella. Shigella. Klebsiella. Enterobacter. Citrobacter. Cronobacter and Hafnia.
Material & Methods
ClpB Fragmentation
First the fragmentation was performed on different conditions: ClpB diluted at 1/10 (from the stock at 5 mg/mL) and ClpB 1/10 with 0.0025% trypsin. Those different conditions were placed during 20 minutes at 37° C. After incubation, the fragmentation was immediately stopped by placing tubes on ice and western blot was performed.
Western Blot
After fragmentation, Leamli solution without J-mercaptoethanol was added into tubes to perform western on 20% acrylamide SDS gel in Tris-Glycine buffer (BioRad, Hercules, USA) and transferred to a nitrocellulose membrane (GE Healthcare, Orsay, France). Then, the membrane was blocked for at least 1 h at room temperature with 5% (w/v) non-fat dry milk in TBS (10 mmol/L Tris, pH 8; 150 mmol/L NaCl) plus 0.05% (w/v) Tween 20. After blocking, the membrane was incubated overnight at 4° C. with anti-α-MSH antibody. The membrane was incubated overnight at 4° C. with rabbit polyclonal anti-α-MSH antibodies (1:1000, Phoenix Pharmaceuticals). After three washes in a blocking solution of 5% (w/v) non-fat dry milk in TBS/0.05% Tween 20, membranes were incubated for 1 h with peroxidase-conjugated anti rabbit IgG (1:1000, SantaCruz Biotechnology). After three washes, the peroxidase reaction was revealed using the ECL detection kit (GE Healthcare). Protein bands were compared with the molecular weight standard (Precision Plus, BioRad) and films were scanned using ImageScanner III (GE Healthcare).
Western Blot on Plasma, Hypothalamus and Colic Mucosa
All animal care and experimentation were in accordance with guidelines established by the National of Health, USA and complied with both French and European Community regulations (Official Journal of the European Community L358, 18, 18/12/1986). Male Sprague-Dawley Rats or Mice C57B16 (Janvier Labs, Genest-Saint-Isle, France) were kept in holding cages and controlled environmental conditions (22° C.+/−P° C. on a 12 hours light-dark cycle with lights on at 7:30 a.m.). During the acclimatization period, animals were given ad libitum access to water and RM1 standard chow (RM1 diet; Special Diet Service Ltd, Witham, Essex, UK).
Mucosa of colon were taken from these rats and mice and washed with PBS. Plasma and hypothalamus of these same animals were also taken and stored at −80° C.
Tissues were incubated with extracted buffer (PBS+protease inhibitor (1%)). The volume added depends of sample quantity. Samples were homogenized using potter. Then, samples were centrifuged (12000 g, 20 min, 4° C.). The supernatant was stored at −80° C. if not analysed immediately.
The protocol used is the same as previously described but f3-mercaptoethanol 10% (w/v) was added at Laemli solution. Then, samples were heated at 85° C. during 2 min before migration on gel. Two primary antibodies were tested: rabbit polyclonal anti-E. Coli ClpB antibodies (1:5000, Delphi Genetics) and anti-alpha-MSH antibodies (1:3000, Phoenix Pharmaceuticals). The secondary antibody used for the both was peroxidase-conjugated anti-rabbit IgG (1:5000, Dako).
Results
The western blot performed on ClpB protein pre-incubated with trypsin showed several bands when revealed with anti-α-MSH antibodies (
Moreover, the western blot showed that no full-length ClpB (˜96 kDa) is present in the solution after trypsinisation.
In addition, the results of
After sequencing of this ˜25 kDa fragment, it appears that it corresponds to the sequence of ClpB from amino acid 537 to amino acid 756 (SEQ ID NO: 22).
PYY Secretion Experiment in Rat Colons
Cell Culture
After euthanasia, rat colon was sampled and washed with fresh phosphate buffer saline (PBS). Intestinal tissue was then washed with L-15 medium (Leibovitz-15 medium; Sigma-Aldrich, Mo., US) maintaining a physiologic pH. Colic mucosa was scraped and digested with 0.4 mg/mL of collagenase IX (Psichas et al., 2015. Int J Obes (Lond). 39(3):424-9) in high glucose DMEM (Dulbecco's Modified Eagle Medium; Dominique Dutscher, France—supplemented with 5.5 mmol/L of L-glutamine, 100 U/mL of penicillin, 0.1 mg/mL of streptomycin and non-essential amino acids) at 37° C. during 5-10 minutes. Cell suspensions were centrifuged at 750 rpm during 8 minutes and intestinal cells were suspended in the same supplemented DMEM medium in which 10% of fetal bovine serum was added. Cell suspensions were filtered at 100 pm (Merck Millipore, Mass., USA) and cultured into 24 wells plate coated with 1% Matrigel (Corning, N.Y., US). Finally, plates were incubated overnight at 37° C. in a 95% O2/5% CO2 atmosphere.
Cell Lysate Preparation
Previously, bacteria culture of wild type (WT) and ClpB depleted (ΔClpB) E. coli K12 were performed to proceed to total protein extraction. The protein extraction was performed in PBS containing 1% protease inhibitor (Sigma-Aldrich, Mo., US) by sonication during 30 seconds. Then, a centrifugation at 10000 rpm during 30 minutes at 4° C. was realized. The supernatant containing proteins was then incubated with 0.0025% trypsin during 20 minutes at 37° C. After incubation, the fragmentation was immediately stopped by placing tubes on ice. The solution containing proteins was sampled and dosed to determine total protein concentration in each condition.
Concomitantly, intestinal cells were incubated in secretion buffer pH 7.4 (4.5 mM of KCl, 138 mM of NaCl, 4.2 mM of NaHCO3, 1.2 mM de NaH2PO4, 2.6 mM de CaCl2), 1.2 Mm de MgCl2 and 10 mM of HEPES). Then cells were incubated during 20 minutes at 37° C. in secretion buffer containing trypsinized fragments of proteins from E. coli K12 WT and E. coli K12 ΔClpB at 0.015 μg/μL (Breton et al., 2015. International Journal of Eating Disorders. 49:805-808; n=4 for each conditions). As a control, cells were also incubated in PBS.
After incubation, supernatants were sampled, centrifuged (10000 rpm during 3 minutes) and immediately stored at −80° C. Then cells were treated with a lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% IGEPAL-CA 630, 0.5% desoxycholic acid and protease inhibitor cocktail without EDTA) to extract intracellular peptides. Cell lysates were immediately frozen at −80° C. towards PYY measurements.
PYY Dosage
PYY dosage was performed on cell medium and cell lysates to measure PYY liberation (in the medium), production (within the lysates) and the total PYY relative production (medium and lysates). This dosage was realized using Fluorescent Immunoassay Kit® (Phoenix Pharmaceuticals, Inc.) according to the manufacturer instructions.
Briefly, after all reagent reconstitution, 50 μl of 1× assay buffer into 2 wells as total binding, 50 μl of prepared peptide standards (in duplicate), 50 l of rehydrated positive control (in duplicate) and 50 μl of prepared samples (in duplicate) were charged onto the immunoplate. Then, 25 μl of rehydrated primary antibody and 25 μl of rehydrated biotinylated peptide were added into each well except the blank well. The microplate was incubated for 2 hours at room temperature (20-23° C.) under orbital shaking at 300-400 rpm. After incubation, each well was washed four times with 350 μl of 1× assay buffer and 100 μl of SA-HRP antibody solution previously prepared by diluting 12 μl of SA-HRP into 12 ml of 1× assay buffer was added. The immunoplate was incubated again for 1 hour at room temperature (20-23° C.) under orbital shaking at 300-400 rpm. After incubation, each well was washed in the same way as before and 100 μl of TMB substrate solution were added. The plate was incubated and protected from the light for 1 hour at room temperature (20-23° C.) under orbital shaking at 300-400 rpm.
At the end, 100 μl 2N HCl were added into each well to stop the reaction (the colour in the well changed from blue to yellow) and the immunoplate was read onto a microtiter plate reader. Absorbance in optical density was read at 450 nm.
PYY Secretion Experiment in Rat Colons and Ileums
Colons and ileums were removed from male rats (Janvier Labs, Genest-Saint-Isle, France). They were washed with ice-cold PBS, then with ice-cold L-15 (Leibowitz) medium (PAA, Yeovil, UK). Then they were digested with 0.4 mg/mL collagenase XI (Sigma, Poole, UK) in High-Glucose DMEM (+1% L-Glutamine+1% Penicillin+1% Streptomycin+1% and non-essential amino acids) at 37° C. during 10 min. Cells suspensions were centrifuged (10 min at 400 g) and the pellets were resuspended in High-Glucose DMEM (the same that previously but with 10% Fetal Bovine Serum (FBS). Cell suspensions were filtered through a nylon mesh (pore size 100 Jpm) (Merck Millipore, Mass., USA) and plated onto 24 well, 1% Matrigel-coated plates (Corning, N.Y., US). The plates were incubated overnight at 37° C. in an atmosphere of 95% O2 and 5% CO2.
After 24 hours of culture, cells were incubated for 20 min in a water bath with secretion buffer (4 mM KCl, 138 mM NaCl, 1.2 mM NaHCO3, 1.2 mM NaH2PO4, 2.6 mM CaCl2), 1.2 mM MgCl2 and 10 mM HEPES) adjusted at pH 7.4) and full-length ClpB or the ˜25 kDa fragment at a concentration of 10 nM. As a control, cells were incubated with secretion buffer and PBS.
After incubation, the cells were treated with lysis buffer (50 mmol/L Tris-HCl, 150 mmol/L NaCl, 1% IGEPAL CA-630, 0.5% deoxycholic acid+cocktail of protease inhibitor tablets complete without EDTA) to extract intracellular peptides. Cells were collected with a cell scraper and the lysates were centrifuged (20 min at 12000 g). Lysates were stored at −80° C. if not analyzed immediately.
Secretion of intestinal hormones was assayed using PYY Fluorescent Immunoassay Kit® (Phoenix Pharmaceuticals, Inc.) as described above.
Results
PYY Release in Rat Colons
The same E. coli proteins pre-incubated with trypsin producing ClpB fragments as above were used for experiments on primary cultured intestinal cell from rat colon. Results show an increase of PYY release compared to control (
These results demonstrate that fragments of ClpB lead to a more efficient release of PYY, thereby demonstrating an activation of the MC4R (see Panaro et al., 2014. Cell Metab. 20(6): 1018-1029).
PYY Release in Rat Colons and Ileums
Results show that incubation of intestinal tissues from rats with full-length ClpB and with the fragment of ˜25 kDa both increase PYY secretion compared to the control (
Material & Methods
Male C57/B16 mice were placed in the BioDAQ mouse cages (Research Diets, Inc., New Brunswick, N.J.) alone to measure the food intake. Each mouse was administered either physiological serum (n=6), full-length ClpB, or fragment of ˜25 kDa.
Two modes of administration were tested: intraperitoneal (i.p.) injection at a concentration of 2 pmol in a volume of 200 μL (n=8), and oral administration at a concentration of 60 pmol in a volume of 200 μL (n=10).
Results
The results presented in
In particular, the oral administration of the fragment of ˜25 kDa decreases food intake while the oral administration of the full-length ClpB protein has no such effect (
| Number | Date | Country | Kind |
|---|---|---|---|
| 17305400.8 | Apr 2017 | EP | regional |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2018/058451 | 4/3/2018 | WO | 00 |
