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WO2013017830A1 - Structure des récepteurs de leptine - Google Patents

Structure des récepteurs de leptine Download PDF

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WO2013017830A1
WO2013017830A1 PCT/GB2012/051722 GB2012051722W WO2013017830A1 WO 2013017830 A1 WO2013017830 A1 WO 2013017830A1 GB 2012051722 W GB2012051722 W GB 2012051722W WO 2013017830 A1 WO2013017830 A1 WO 2013017830A1
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leptin
amino acid
seq
lbd
composition according
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Peter Artymiuk
Byron CARPENTER
Richard Ross
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University of Sheffield
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2869—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against hormone receptors
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/72—Receptors; Cell surface antigens; Cell surface determinants for hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2299/00—Coordinates from 3D structures of peptides, e.g. proteins or enzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

  • This disclosure relates to the characterisation of a binding site in leptin receptor for an antagonistic antibody and the use of this information in the design of leptin receptor antagonists.
  • Human leptin is a 16kD protein hormone encoded by the lep gene in humans and the ob gene in mice. Leptin acts through the leptin receptor which is a single transmembrane receptor of the cytokine family. There is a single gene that encodes leptin in humans which includes three exons and two introns and spans about 18kb of genomic DNA. Leptin links nutritional status and the immune system to control, inter alia, appetite and the immune function. The existence of mutations in either leptin or leptin receptor can result in an obese phenotype with attendant secondary symptoms associated with obesity (e.g. heart disease, diabetes type II).
  • obesity e.g. heart disease, diabetes type II
  • Leptin is mainly produced by adipose tissue in proportion to the body mass index (BMI) and, at lower levels, by organs such as the stomach and placenta. Leptin regulates body weight through inhibition of food intake and stimulation of energy expenditure. Moreover, leptin affects both the innate and adaptive immunity. On innate immunity, leptin modulates the activity of neutrophils, increases the phagocytosis of monocytes/macrophages and enhances the secretion of inflammatory mediators of the acute-phase response.
  • BMI body mass index
  • leptin affects both the innate and adaptive immunity. On innate immunity, leptin modulates the activity of neutrophils, increases the phagocytosis of monocytes/macrophages and enhances the secretion of inflammatory mediators of the acute-phase response.
  • leptin On adaptive immunity, leptin promotes proliferation and interleukin 2 (IL-2) secretion by na ' ive T cells whereas on memory T cells, it promotes the switch towards T helper 1 (Th1 ) immune response by increasing interferon- ⁇ (INF- ⁇ ) and tumor necrosis factor- a (TNF- a secretion). If leptin expression and/or production is perturbed then the pathological manifestation of disease is complicated with effects on energy metabolism and immune status. Apart from the established linkage to obesity, reduction in leptin is associated with infertility, osteoporosis and immune suppression.
  • INF- ⁇ interferon- ⁇
  • TNF- a secretion tumor necrosis factor- a
  • Leptin-antagonist therapy may have a role in the treatment of some immune-mediated disorders.
  • Leptin is permissive to a Th1 mediated immune response (Lord et al., 1998), and blockade of leptin, in animal models, imparts resistance to: antigen-induced arthritis (Busso et al., 2002), multiple sclerosis (Matarese et al., 2001 a; Matarese et al., 2001 b), atherosclerosis (Schafer et al., 2004), and certain types of breast cancer (Cleary et al., 2004).
  • leptin therapeutics both agonists and antagonists, with optimized pharmacological properties.
  • ObR obesity receptor
  • the extracellular domain of ObR is composed of: an N-terminal cytokine receptor homology domain (CRH-1 ); an immunoglobulin-like (Ig) domain; a second CRH domain (CRH-2), also referred to as the leptin-binding domain (LBD); and two Fibronectin type III ( N III) domains (Haniu et al., 1998).
  • ObR is known to dimerize in a ligand-independent fashion both on the cell surface and in solution (Couturier and Jockers, 2003; Devos et al., 1997; Nakashima et al., 1997; White et al., 1997).
  • This disclosure relates to the crystal structure of the leptin-binding domain [LBD] of the human obesity receptor complexed with an antagonizing Fab fragment derived from a monoclonal antibody.
  • LBD leptin-binding domain
  • 9F8 a monoclonal antibody
  • its mechanism of action was unknown. Therefore, we crystallized LBD in complex with 9F8 Fab and solved the structure at 1 .95 A resolution.
  • Using the LBD structure we were able to construct a molecular docking model of the leptin-LBD complex and propose that leptin binding involves an induced fit mechanism.
  • leptin-LBD model We also used the leptin-LBD model to propose a mechanism by which 9F8 Fab antagonizes leptin binding to LBD.
  • This disclosure identifies the epitope within LBD that bind 9F8 and thereby provides means to produce antagonistic antibodies which have use in the treatment of conditions which would benefit from the inhibition of leptin activity.
  • composition comprising a polypeptide, or antigenic fragment thereof, consisting essentially of amino acid residues from about 438 to 525 of SEQ ID NO: 1 .
  • said polypeptide, or antigenic fragment thereof consists essentially of amino acid residues from about 463 to 504 of SEQ ID NO: 1 .
  • said polypeptide includes the amino acid residues 463 to 473 of SEQ ID NO: 1 .
  • said polypeptide includes the amino acid residues 477 to 487 of SEQ ID NO: 1 .
  • said polypeptide includes the amino acid residues 500-504 of SEQ ID NO: 1 .
  • said composition is an immunogenic composition optionally comprising an adjuvant and/or carrier.
  • said composition comprises one or more peptide antigens comprising the amino acid sequences QLRYHRSSLYC, PSIHPISEPKD or FQPIF wherein said peptide antigen is less than 50 amino acids in length.
  • said peptide antigen is between 10-30 amino acids in length; preferably said peptide antigen is 8-18 amino acids in length.
  • composition comprises 2 or 3 peptide antigens.
  • composition comprises or consists essentially of peptide antigens consisting of the amino acid sequences QLRYHRSSLYC, PSIHPISEPKD and FQPIF.
  • said peptides are linked in a polytope antigen wherein said peptide antigens are linked either directly or indirectly together.
  • said composition includes an adjuvant and/or carrier.
  • An adjuvant is a substance or procedure which augments specific immune responses to antigens by modulating the activity of immune cells.
  • adjuvants include, by example only, Freunds adjuvant, muramyl dipeptides, liposomes.
  • An adjuvant is therefore an immunomodulator.
  • a carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter.
  • the term carrier is construed in the following manner.
  • a carrier is an immunogenic molecule which, when bound to a second molecule augments immune responses to the latter.
  • Such antigens contain B-cell epitopes but no T cell epitopes.
  • the protein moiety of such a conjugate provides T-cell epitopes which stimulate helper T-cells that in turn stimulate antigen-specific B-cells to differentiate into plasma cells and produce antibody against the antigen.
  • Helper T-cells can also stimulate other immune cells such as cytotoxic T-cells, and a carrier can fulfil an analogous role in generating cell-mediated immunity as well as antibodies.
  • Certain antigens which lack T- cell epitopes such as polymers with a repeating B-cell epitope (e.g. bacterial polysaccharides), are intrinsically immunogenic to a limited extent. These are known as T-independent antigens.
  • Such antigens benefit from association with a carrier such as tetanus toxoid, under which circumstance they elicit much stronger antibody responses.
  • composition according to the invention for use in the production of antagonistic antibodies that inhibit leptin binding to the leptin receptor.
  • said antagonistic antibodies are monoclonal antibodies.
  • the said immunocompetent mammal is a mouse.
  • said immunocompetent mammal is a rat.
  • composition according to the invention for use in the screening of antibody phage display libraries for the identification of antagonistic leptin receptor antibodies.
  • a method to screen a combinatorial antibody phage display library wherein said library comprises antibody variable heavy and variable light chain nucleic acids comprising the steps:
  • a modelling method to determine the association of an agent with leptin receptor comprising the steps of:
  • the Molecular Similarity application permits comparisons between different structures, different conformations of the same structure, and different parts of the same structure.
  • Each structure is identified by a name.
  • One structure is identified as the target (i.e., the fixed structure); all remaining structures are working structures (i.e. moving structures).
  • the working structure is translated and rotated to obtain an optimum fit with the target structure.
  • the person skilled in the art may use one of several methods to screen chemical entities or fragments for their ability to associate with a target.
  • the screening process may begin by visual inspection of the target on the computer screen, generated from a machine-readable storage medium. Selected fragments or chemical entities may then be positioned in a variety of orientations, or docked, within the binding pocket.
  • CAVEAT P. A. Bartlett et al, "CAVEAT: A Program to Facilitate the Structure- Derived Design of Biologically Active Molecules". In Molecular Recognition in Chemical and Biological Problems", Special Pub., Royal Chem. Soc, 78, pp. 182-196 (1989)).
  • CAVEAT is available from the University of California, Berkeley, California.
  • 3D Database systems such as MACCS-3D (MDL Information Systems, San Leandro, California). This is reviewed in Y. C. Martin, "3D Database Searching in Drug Design", J. Med. Chem., 35, pp.
  • substitutions may then be made in some of its atoms or side groups in order to improve or modify its binding properties.
  • initial substitutions are conservative, i.e., the replacement group will have approximately the same size, shape, hydrophobicity and charge as the original group.
  • the computational analysis and design of molecules, as well as software and computer systems are described in US Patent No 5,978,740 which is included herein by reference.
  • said fitting operation includes at least Phe-563 of SEQ ID NO: 1 .
  • a screening method for the identification of an agent which inhibits the interaction of leptin with leptin receptor comprising the steps of:
  • Figure 1 Analysis of leptin and 9F8 binding to ObR.
  • A Competition binding data demonstrating the ability of 9F8 mAb to displace leptin from the full-length extracellular domain of ObR. The binding of biotinylated leptin was measured in the presence of increasing concentrations of either 9F8 mAb or unlabelled leptin. The IC50 of leptin and 9F8 mAb binding to ObR is 0.76 nM and 1 .0 nM respectively. Data shown is from a single experiment, and error bars represent the mean of duplicate samples.
  • a 1 :1 molar ratio mixture of LBD and leptin resolves as a predominant peak with a retention volume of 15.32 ml.
  • a 1 :1 molar ratio mixture of LBD and 9F8 Fab resolves as a predominant peak with a retention volume of 13.99 ml.
  • a 1 :1 :1 molar ratio mixture of all three proteins resolves as a predominant peak with a retention volume of 14.04 ml, which relates well to the LBD-9F8 Fab complex.
  • the main peak contains a shoulder with a retention volume of approximately 15.35 ml, which is likely to contain both free 9F8 Fab (14.95 ml) and the LBD-leptin complex (15.32 ml).
  • FIG. 1 Crystal structure of the LBD-9F8 Fab complex.
  • A Ribbon representation of the two copies of the LBD-9F8 Fab complex in the asymmetric unit. The two LBD molecules are coloured blue and magenta, the 9F8 Fab molecules are coloured yellow (heavy chain) and green (light chain).
  • B A single copy of the LBD-9F8 Fab complex showing that the Fab binds to the N-terminal sub-domain of LBD.
  • C Secondary structural elements of LBD coloured by rainbow: N-terminus, blue; C-terminus, red. Key loops, which are discussed in the text, are labelled; unmodelled loops are indicated by dashed lines and marked with an asterisk. See also Figure S1 . Figures made using Pymol (DeLano, 2002);
  • Figure 3 Interaction of 9F8 Fab with LBD.
  • A Polar interactions between the 9F8 Fab heavy chain (yellow) and LBD (light blue).
  • B Positioning of LBD residue lle-482 (light blue) in a deep cavity within the solvent accessible surface of 9F8 Fab (white), at the interface between the Fab heavy (yellow) and light (green) chains, lle-482 makes van der Waals contacts with the hydrophobic pocket defined by six Fab residues: Trp-54 (heavy chain); His-103 (heavy chain); His-53 (light chain); Trp-94 (light chain); Tyr-96 (light chain); Leu-98 (light chain).
  • FIG. 4 A model of leptin binding to LBD.
  • LBD residues implicated in leptin binding by mutagenesis studies are shown as sticks. Important loops which are discussed in the text are coloured: C-D loop, yellow; E-F loop, cyan; J-K loop, magenta; L-M loop, green. The SSLY motif, which is located within the C-D loop, is coloured red.
  • B Differences in the flexible J-K loop between LBD molecules. Phe-563 appears to adopt a solvent exposed position in chain A, but is partially buried within the core of LBD in chain B.
  • FIG. S2 Flexible loop in LBD: related to figure 5. Comparison of the generally good electron density observed for LBD with the fragmented density of the flexible J-K loop. The potentially different positioning of this between LBD molecules A and B is displayed, and the resulting difference in surface contour is highlighted; Electron density map of LBD, related to Figure 5.
  • A A typical example of the good electron density observed for LBD. The Fobs-Fcalc electron density is shown as grey mesh (contoured at 1 .5 ⁇ level) around the SSLY motif and E-F loop of both LBD molecules.
  • Model S1 LBD-leptin docking models: related to figure 5.
  • the molecular-docking models of leptin binding to both LBD chains A and B are provided in a single supplemental Protein Data Bank (.pdb) file
  • Figure 5 shows SEQ ID NO: 1 which is the amino acid sequence of the leptin receptor;
  • Figure 6 shows the amino acid sequences of heavy and light Fab chains of antagonistic monoclonal antibody 9F8.
  • LBD (residues 428-635 of human ObR) was expressed, refolded and purified essentially as described previously (Sandowski et al., 2002). Briefly: LBD was cloned into the expression vector pET21 a(+) and expressed as insoluble inclusion bodies in E. coli strain BL21 (DE3)-RIPL. Cell pellets from 5 liters of culture were lysed by sonication in buffer A (10 mM TRIS, 1 mM EDTA, 10 mM DTT, 1 mM PMSF, 0.5 mg/ml lysozyme, protease inhibitor tablets (Roche), pH 8.0).
  • Inclusion bodies were harvested by centrifugation at 40,000g and purified by three washes in buffer B (50 mM TRIS, 1 mM EDTA, 10 mM DTT, 2% sodium deoxycholate, pH 8.0); a final wash was performed with deionised water.
  • buffer B 50 mM TRIS, 1 mM EDTA, 10 mM DTT, 2% sodium deoxycholate, pH 8.0
  • Inclusion bodies were solubilised in 1 liter of buffer C (10 mM TRIS, 4.5 M urea, pH 8.0) and the pH adjusted to 1 1 .3. Cysteine was added to a concentration of 10 mM and the solution was mixed at 4°C in the dark for 1 hour. The solution was added to 2 liters of 750 mM arginine (unbuffered) and mixed at 4°C for 15 minutes. The solution was dialyzed against 20 liters of pre-cooled buffer D (10mM TRIS, pH 9.0) for 72 hours at 4°C, with nine external buffer changes. Refolded LBD was loaded onto a 25 ml Q-Sepharose fast flow column (GE Healthcare), pre-equilibrated with buffer D.
  • buffer C 10 mM TRIS, 4.5 M urea, pH 8.0
  • Cysteine was added to a concentration of 10 mM and the solution was mixed at 4°C in the dark for 1 hour.
  • the solution was added to 2 liters of
  • the column was washed with 500 ml of buffer D and eluted stepwise with: 50 mM, 100 mM, 150 mM, 200 mM, 1 M NaCI (in buffer D).
  • the eluate was analyzed by SDS- and native-PAGE and fractions containing mainly monomeric LBD were dialyzed against buffer E (25 mM TRIS, 150 mM NaCI, pH 8.0) at 4°C.
  • LBD was concentrated to 10 mg/ml using a Vivacell-250 concentrator (Sartorius) and loaded on a Sephacryl S-200 HR 16/60 gel filtration column (GE Healthcare). The final yield of pure LBD was 0.6 mg per liter of E. coli culture.
  • a micro-titer plate was coated with the capture antibody 2H6 (500 ng/100 ⁇ /well). Plates were washed three times with wash buffer (PBS, 0.05% Tween-20). Soluble human leptin receptor was captured from normal human serum (100 ⁇ /well) diluted 1 :4 in assay buffer (50 mM TRIS, 154 mM NaCI, 20 ⁇ diethylenetriaminepenta-acetic acid, 0.01 % Tween 40, 0.5% BSA, 0.05% bovine gamma-globulin, 0.05% NaN 3 , pH 7.75), incubation was performed for 2 hours at room temperature, shaking at 500 rpm. Plates were washed three times with wash buffer.
  • assay buffer 50 mM TRIS, 154 mM NaCI, 20 ⁇ diethylenetriaminepenta-acetic acid, 0.01 % Tween 40, 0.5% BSA, 0.05% bovine gamma-globulin, 0.05% NaN 3
  • a 96-well micro-titer plate was coated with the capture antibody 6C5 (500 ng/100 ⁇ /well). Plates were washed three times with wash buffer (PBS, 0.05% Tween-20). LBD (2.5 ng), biotinylated-leptin (1 .6 ng) and unlabelled-leptin (varying concentrations), diluted in assay buffer (10 mM HEPES, 150 mM NaCI, 0.5 % BSA, 0.005% Tween-20, 0.02% NaN 3 , pH 7.4), were added to each well. Plates were incubated for 2 hours at room temperature, shaking at 500 rpm. Plates were washed three times with wash buffer.
  • Streptavidin horseradish peroxidase (GE Healthcare) was added to each well (10 ng/100 ⁇ /well) and plates were incubated for 30 minutes at room temperature, shaking at 500 rpm. Plates were washed six times with wash buffer. TMB substrate (Sigma-Aldrich) was added (100 ⁇ /well) and plates were incubated in the dark at room temperature for 15 minutes. Stop solution (5% sulphuric acid) was added (100 ⁇ /well) and the absorbance was measured at 450 nm.
  • 9F8 mAb was expressed in the monoclonal hybridoma cell line and was purified by protein A affinity chromatography (Pierce). Fab fragments were prepared by digestion of the purified mAb with immobilized papain (Pierce) at 37°C for 5 hours. Fab fragments were purified by reverse-affinity chromatography on a protein A column (Pierce). The resulting Fab fragments were further purified by gel filtration on a Sephacryl S-200 HR 16/60 column.
  • LBD and 9F8 Fab were mixed in a 1 :1 stochiometric ratio and incubated overnight at 4°C; uncomplexed material was removed by gel filtration on a Sephacryl S-200 16/60 column.
  • the best crystals were grown using the hanging-drop vapor diffusion technique against crystallization buffer (100 mM sodium acetate pH 4.6, 150 mM unbuffered sodium acetate, 5% PEG-4000), at a protein concentration of 7.5 mg/ml. Crystals grew to maximum size in 2-3 weeks at 7°C. Crystals were soaked in cryoprotectant (reservoir solution containing 30% ethylene glycol) for 1 minute, before freezing in a dry nitrogen stream at 100 K.
  • cryoprotectant containing 30% ethylene glycol
  • the amino acid sequence of LBD was automatically fitted using ARP-WARP (Cohen et al., 2008). Refinement of the structure was performed by the maximum likelihood method using REFMAC (Murshudov et al., 1997), with manual rebuilding using COOT (Emsley and Cowtan, 2004). The final model was assessed to be of good quality with a crystallographic R-factor and free R-factor of 17% and 21 % respectively, full refinement statistics are given in table 2. The model was validated using the MOLPROBITY web server (Davis et al., 2007), scoring in the 96 th percentile. Stereochemistry analysis showed that 97.2% of residues fall within the most favored regions, with residues occupying disallowed regions.
  • the approximately modeled J-K and L-M loops of LBD were included in the search model in order to maintain integrity and completeness of the molecular surface.
  • the final models were subjected to 20 cycles of energy minimization, using the GROMOS96 implementation of the Swiss-pdb viewer (Guex and Peitsch, 1997), to resolve a number of small stereo-chemical clashes.
  • the final docking models for both LBD chains are available as a supplemental data file online (Model S1 ).
  • the asymmetric unit of the crystal contains two copies of the LBD-9F8 Fab complex.
  • the overall electron density for the LBD and the Fab molecules is very good, but several loop regions of LBD, 39 residues in total, could not be satisfactorily modeled due to poor electron density (Figure S1 A).
  • One of the complexes (LBD, chain B; 9F8 Fab heavy chain, chain D; and 9F8 Light chain, chain F) had fewest unmodelled residues, and subsequent analysis refers to this complex.
  • the two copies of the LBD-9F8 Fab complex in the asymmetric unit interact through a major interface between the LBD molecules.
  • the LBD molecules are arranged in a cross-shaped complex, which displays almost perfect two-fold symmetry ( Figures 1 A and S1 B).
  • LBD is a ⁇ -sheet rich protein composed of two sub-domains that each adopts a fibronectin type III fold (Figure 2C). Three disulphide bonds are observed within the N-terminal sub-domain, between residues: 436-447; 473-528; and 488-498 ( Figure S1 C).
  • the two cysteine residues located within the C-terminal sub-domain (Cys-604 and Cys-613) are separated by 28 A and do not interact with one another. Cys-604 is exposed on the surface of the protein and is cysteinylated ( Figure S1 C); this modification is almost certainly a consequence of using cysteine as a reducing agent during the refolding of LBD.
  • cysteinylation is heavily involved in a major crystal lattice contact between LBD and the Fab heavy chain from a neighboring complex.
  • a cysteine mutant (C604A / C613A) improved the yield of purified LBD six-fold with no loss in affinity for leptin (data not shown).
  • the mutant failed to crystallize in complex with 9F8 Fab, demonstrating that the cysteinylation was integral for crystallization of LBD in this particular crystal form.
  • the C-terminal domain of LBD also contains the WSXWS motif, which is an important structural feature common to all cytokine receptors.
  • the WSNWS sequence of LBD forms the basis of a ⁇ -cation stack, which involves Arg-573, Trp-583, Arg-612 and Lys-614 ( Figure S1 C).
  • the structure of LBD was compared with all coordinates deposited in the Protein Data Bank using the program DALI (Holm et al., 2008).
  • 9F8 Fab is typical of that of most antibody Fab fragments, except that it is glycosylated at Asn-22 of the light chain, close to the complementarity determining regions (CDRs).
  • CDRs complementarity determining regions
  • LBD-9F8 Fab interaction 9F8 Fab binds within the N-terminal sub-domain of LBD ( Figure 2B).
  • the total buried surface area of the interface is 1500 A 2 , which compares well with other antibody-antigen interfaces reported in the literature (Braden and Poljak, 1995). All of the direct polar contacts, a total of eight hydrogen bonds and three salt-bridges (Table 1 ), are formed between LBD and the Fab heavy chain (CDRs: H1 , H2 and H3) ( Figure 3A).
  • the interface also involves an extensive network of van der Waals interactions involving both the heavy chain (CDRs: H1 , H2 and H3) and light chain (CDRs: L2 and L3) of 9F8 Fab (Table 1 ).
  • a network of water molecules mediates additional indirect contacts between LBD and both Fab chains.
  • the interacting surfaces of LBD and 9F8 Fab display a high degree of complementarity in both surface contour and electrostatic charge.
  • a protrusion caused by lle-482 of LBD fits tightly into a deep cavity on the Fab surface, forming extensive van der Waals interactions with residues from both Fab chains ( Figure 3B).
  • the highest scoring output models were analyzed to determine how well they aligned with other cytokine complexes, and how well they explained the published mutagenesis data for both LBD (Iserentant et al., 2005; Niv-Spector et al., 2005b) ( Figure 4B) and leptin (Peelman et al., 2004).
  • the third highest scoring model (Figure 4C) showed remarkable similarity to both the GCSF / GCSF receptor (PDB: 2D9Q) (Tamada et al., 2006) and IL-6 / gp130 (PDB: 1 P9M) (Boulanger et al., 2003) complexes.
  • the alpha carbon RMSD between the complexes was approximately 3 A in both cases, which is similar to the RMSD when the individual proteins are superimposed.
  • This model was replicated in docking simulations using both LBD chains: in both cases interface contacts were identical, except in the region immediately surrounding the J-K loop. This model explains the importance of the majority of residues identified by mutagenesis studies and is described herein.
  • the hydrophobic E-F loop of LBD contains six residues (Phe-500, lle-503, Phe-504, Leu-505, Leu-506, Ser-507) ( Figure 4B) which, when mutated to alanine, dramatically reduce leptin-binding affinity and signal transduction (Iserentant et al., 2005; Niv-Spector et al., 2005b). Interestingly, two of these residues (Phe-500 and lle-503) have their side chains almost totally buried in the core of the protein, and do not interact with leptin.
  • Leu-505 and Leu-506 align with the hydrophobic cavity between helices 1 and 3 of leptin, forming van der Waals interactions with Leu-13 and Leu-86 (Figure 4D).
  • This network of van der Waals interactions also involves part of the SSLY motif from LBD (Ser-469, Ser-470, Leu-471 and Tyr-472) ( Figure 4B) (Haniu et al., 1998).
  • Leu-471 and Tyr-472 form van der Waals contacts with Val-6, Leu-86 and Val-89 of leptin ( Figure 4D).
  • Phe-504 does not align with the hydrophobic cavity, instead it is surrounded by polar residues from leptin (Asn-78, Glu-81 and Asn-82). Although this positioning of Phe-504 seems energetically unfavorable, it may play a significant role in coordinating Arg-468 through a ⁇ -cation stacking interaction (Figure 4B).
  • Arg-468 has been shown to be very important for leptin binding (Niv-Spector et al., 2005b): mutation to alanine causes a 37-fold decrease in leptin binding affinity. In the model Arg-468 is positioned approximately 5 A from Asn-82 and Asp-85 of leptin.
  • Tyr-441 of LBD is another interesting residue, whose side-chain has been implicated in direct interactions with leptin ( Figure 4B) (Niv-Spector et al., 2005b; Sandowski et al., 2002).
  • the Tyr-441 side-chain does not directly interact with leptin, but instead it interacts with Leu-538 from the C-terminal sub-domain of LBD. Therefore Tyr-441 may be important for stabilization of the sub-domain interface of LBD, alternatively a rotamer shift upon complexation may allow it to form direct interactions with Arg-20, Arg-71 or Gln-75 of leptin.
  • Phe-563 might occupy two distinct positions in the two LBD molecules ( Figure 4A and S2).
  • Phe-563 forms extensive contacts with leptin (Thr-16, Thr-19, Arg-20 and Asp-23), but it lacks a specific hydrophobic or aromatic binding partner.
  • Phe-563 does not interact with leptin, instead adjacent residues on the J-K loop (Pro-564 and Glu-565) become more heavily involved in binding.
  • the buried conformation of Phe-563 would be energetically favorable for complex formation.
  • a deep cavity is observed beneath the J-K loop when Phe-563 is orientated in its solvent exposed position. This cavity may represent a novel target for small-molecule drugs aimed at disrupting the leptin-LBD interface.
  • the leptin and 9F8 Fab epitopes are predicted to partially overlap
  • Hydrogen bonds and salt bridges were calculated using PISA (Krissinel and Henrick, 2007) . Hydrogen bonds are defined as interactions exhibiting the necessary geometry with contact distances of 3.3 A or less. Salt bridges are defined as interactions exhibiting the necessary geometry, electrostatic charge and protonation state with contact distances of 4.0 A or less. Van der Waals interactions were calculated using the CONTACT function of CCP4i (Potterton et al., 2003) and are defined as: interactions with contact distances of 4.0 A or less, excluding hydrogen bonds and salt bridges. Only residues that form three or more van der Waals contacts are shown.
  • bR work ⁇
  • Leptin receptor oligomerizes with itself but not with its closely related cytokine signal transducer gp130. FEBS letters 403, 79-82.
  • GCSF granulocyte colony- stimulating factor

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Abstract

Cette invention concerne la caractérisation d'un site de liaison dans le récepteur de leptine pour un anticorps antagoniste et l'utilisation de ces informations dans la conception d'anticorps de récepteurs de leptine.
PCT/GB2012/051722 2011-08-03 2012-07-19 Structure des récepteurs de leptine Ceased WO2013017830A1 (fr)

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WO2017013270A1 (fr) 2015-07-23 2017-01-26 Universite De Strasbourg Utilisation d'un inhibiteur de la signalisation de la leptine pour la protection des reins de patients atteints de ciliopathie

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Publication number Priority date Publication date Assignee Title
WO2017013270A1 (fr) 2015-07-23 2017-01-26 Universite De Strasbourg Utilisation d'un inhibiteur de la signalisation de la leptine pour la protection des reins de patients atteints de ciliopathie

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