WO2017175022A1 - Methods and pharmaceutical compositions for inhibiting mast cell degranulation - Google Patents

Abstract

The present invention relates to methods and pharmaceutical compositions for inhibiting mast cell degranulation. In particular, the present invention relates to a method of inhibiting mast cell degranulation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent capable of inhibiting the formation the Kinesin-1/Slp3/Rab27b complex.

Description

METHODS AND PHARMACEUTICAL COMPOSITIONS FOR INHIBITING MAST

CELL DEGRANULATION

FIELD OF THE INVENTION:

The present invention relates to methods and pharmaceutical compositions for inhibiting mast cell degranulation.

BACKGROUND OF THE INVENTION:

Mast cells (MC) are granulated cells of hematopoietic origin homing to most tissues of the body. They are especially enriched under surfaces exposed to the external environment such as the skin, the airways and the intestine. MC are key effectors in innate immunity but are also notoriously known for their deleterious role in allergies, with the most serious manifestation being anaphylaxis (Galli et al., 2005a; Galli et al., 2005b).

MC express on their surface several receptors including the high-affinity IgE receptor (FceRI) responsible for allergic triggering. Crosslinking receptor-bound IgE by specific multivalent antigen or allergen launches within minutes the release of numerous inflammatory mediators from SGs including proteases, proteoglycans, lysosomal enzymes such as β- hexosaminidase and biogenic amines such as histamine and serotonin. This is followed within 15 to 30 min by the synthesis of lipid mediators such as leukotrienes and prostaglandins and after several hours by de novo synthesis and secretion of cytokines and chemokines that mediate the inflammatory response (Blank et al., 2014; Blank and Rivera, 2004; Wernersson and Pejler, 2014).

Degranulation is accompanied by an extensive reorganization of the cytoskeleton associated with membrane ruffling and spreading (Draber and Draber, 2015). It also involves the anterograde movement of SGs towards the plasma membrane where granules fuse to release their content. The FceRI-mediated anterograde movement of SGs has been shown to depend on the dynamics of microtubules (Nishida et al., 2005). This involved the activation of a Fyn/Gab2/RhoA signaling pathway, but was independent on calcium influx (Nishida et al., 2011; Nishida et al., 2005). Further studies showed a role of ARF1 activated via Fyn and PI3K, the latter being recruited via Gab2(Nishida et al., 2011). More recently, DOCK5 in association with Nck2 and Akt a downstream effector of phosphatidylinositol 3-kinase (PI3K) have been shown to regulate microtubules dynamics in mast cells (Ogawa et al., 2014). This involved Akt-mediated inactivation of glycogen synthase kinase 3 beta (GSK3 ), thereby promoting microtubule assembly. However, the molecular machinery that links the trafficking of SGs to microtubule dynamics in MC are largely unknown.

The mechanism that controls vesicular fusion between SG-SG and SG-plasma membrane in MC begins to be characterized and include the N-ethylmaleimide- sensitive factor attachment protein receptors (SNAREs) such as Syntaxin 3 (STX3), Syntaxin 4, SNAP- 23 and VAMP8 and an accessory molecule such as Muncl8-2 (Brochetta et al., 2014; Lorentz et al., 2012; Tiwari et al., 2008). The small GTPases Rab27, Rab27a and Rab27b, have been involved in MC degranulation, with a main role for Rab27b in de granulation (Mizuno et al., 2007). Their GTP-bound forms have been shown to recruit effectors of the synaptotagmin- like protein family (Slpl/JFCl, Slp2a, Slp3, Slp4/granuphilin and Slp5) that are involved in the trafficking and docking of secretory vesicles in different cell types (Fukuda et al., 2002; Kuroda et al., 2002; Menasche et al., 2008). Members of Sip family have a common N- terminal Rab27-binding Slp-homology domain (SHD) and a C-terminal tandem C2 domains (putative phospholipid-binding sites).

In cytotoxic T lymphocytes (CTLs) and in neuron, we and others reported that the plus-end movement of cytotoxic granules and synaptic vesicles, respectively is mediated by the microtubule-dependent motor protein Kinesin-l(Arimura et al., 2009; Kurowska et al., 2012). In CTLs, a Rab27a/Slp3/Kinesin-1 complex was shown to regulate cytotoxic granule transport, while in neuron a Rab27b/Slpl/CRMP-2/Kinesin-l molecular complex is involved in the anterograde transport of synaptic vesicle (Arimura et al., 2009; Kurowska et al., 2012). The conventional member of the kinesin superfamily, Kinesin-1, is a tetrameric protein constituted by 2 heavy chains (KIF5A, KIF5B or KIF5C) and 2 light chains (KLCl, KLC2 or KLC3)(Hirokawa, 1998). KIF5B and KLCl are ubiquitously distributed and mediate the plus- end-directed microtubule-dependent transport of cargos. The targeting invalidation of Kif5b in pancreatic β-cells has revealed a key role of Kinesin-1 in insulin secretion(Cui et al., 2011). Another mouse model targeting Kif5b specifically in myogenic cells has shown that Kinesin- 1 plays an important role in the anterograde transport of key elements that are required for myofibril assembly, as cc-sarcomeric actin, myosin IIB, nestin and desmin(Wang et al., 2013).

SUMMARY OF THE INVENTION:

The present invention relates to Methods and pharmaceutical compositions for inhibiting mast cell degranulation. In particular, the present invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:

Cross-linking of mast cell (MC) IgE receptors (FceRI) triggers degranulation and release of numerous allergic and inflammatory mediators stored in secretory granules (SGs) by a process involving microtubule-dependent anterograde transport. However, the molecular machinery coupling SGs for transport along microtubules towards the secretion site remains poorly characterized. Here the inventors report that Kinesin-1, a conventional member of the kinesin superfamily, regulates FceRI- stimulated translocation of SGs to the plasma membrane. Conditional mice lacking Kif5b (the heavy chain of Kinesin-1) in hematopoietic cells displayed a decreased sensitivity to IgE-mediated passive systemic anaphylaxis and MC derived from their bone marrow showed a severe reduction in IgE-induced degranulation. By contrast, deficiency in Kif5b did not affect cytokine secretion and early FceRI-initiated signaling pathways. The inventors identify Synaptotagmin-like protein 3 (Slp3) as the critical effector linking the Kinesin-1 motor protein to Rab27b-associated SGs. The recruitment of Kinesin-1 to the Slp3/Rab27b effector complex required stimulation and was dependent on phosphatidylinositol 3-kinase. Disruption of this association severely impaired the degranulation response. Thus, the data demonstrate that PI3K-dependent formation of a Kinesin- 1/Slp3/Rab27b complex is critical for the control of microtubule-dependent SG movement in the FceRI-mediated degranulation response but not for cytokine release.

Accordingly a first object of the present invention relates to a method of inhibiting mast cell degranulation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent capable of inhibiting the formation the Kinesin- 1/Slp3/Rab27b complex.

As used herein, the term "mast cell" refers to a bone marrow derived cell that mediates hypersensitivity reactions. Mast cells are characterized by the presence of cytoplasmic granules (histamine, chondroitin sulfate, proteases) that mediate hypersensitivity reactions, high levels of the receptor for IgE (FceRI), and require stem cell factor and IL3 (cytokines) for development. Mature mast cells are not found in the circulation, but reside in a variety of tissues throughout the body. In some embodiments, the method of the present invention is particularly suitable for the treatment of IgE-mediated disorders. As used herein, the term "IgE-mediated disorders" refers to disorders mediated by binding of an IgE antibody to its receptor on mast cells, resulting in mast cell degranulation. IgE mediated disorders include atopic disorders, which are characterized by an inherited propensity to respond immunologically to many common naturally occurring inhaled and ingested antigens and the continual production of IgE antibodies. Specific atopic disorders includes allergic asthma, allergic rhinitis, atopic dermatitis and allergic gastroenteropathy. However, disorders associated with elevated IgE levels are not limited to those with an inherited (atopic) etiology. Other disorders associated with elevated IgE levels, that appear to be IgE-mediated and are treatable with the method of the present invention include hypersensitivity (e. g., anaphylactic hypersensitivity), eczema, urticaria, allergic bronchopulmonary aspergillosis, parasitic diseases, hyper-lgE syndrome, ataxia- telangiectasia, Wiskott-Aldrich syndrome, thymic alymphoplasia, IgE myeloma and graft- versus-host reaction.

In some embodiments, the method of the present invention is particularly suitable for the treatment of allergic disorder. As used herein, "allergic disorder" refers to any disorder resulting from antigen activation of mast cells that results in an "allergic reaction" or state of hypersensitivity and influx of inflammatory and immune cells. Those disorders include without limitation:

systemic allergic reactions, systemic anaphylaxis or hypersensitivity responses, anaphylactic shock, drug allergies, and insect sting allergies;

respiratory allergic diseases, such asthma, hypersensitivity lung diseases, hypersensitivity pneumonitis and interstitial lung diseases (ILD) (e.g. idiopathic pulmonary fibrosis, ILD associated with rheumatoid arthritis, or other autoimmune conditions);

rhinitis, hay fever, conjunctivitis, allergic rhinoconjunctivitis and vaginitis;

skin and dermatological disorders, including psoriasis and inflammatory dermatoses, such as dermatitis, eczema, atopic dermatitis, allergic contact dermatitis, dermatitis herpetiforms, linear IgA disease, acute and chronic urticaria and scleroderma;

vasculitis (e.g. necrotizing, cutaneous, and hypersensitivity vasculitis); spondyloarthropathies; and intestinal reactions of the gastrointestinal system (e.g., inflammatory bowel diseases such as Crohn's disease, ulcerative colitis, ileitis, enteritis, nontropical sprue and celiac disease).

In some embodiments, the subject suffers from allergic rhinitis. As used herein, the term "allergic rhinitis" refers to allergic inflammation of the nasal airways resulting in excess nasal secretion, itching and nasal obstruction. This condition is frequently mediated by IgE antibodies to pollen which subsequently activate mast cells.

In some embodiments, the subject suffers from asthma. As used herein, the term "asthma" refers to an inflammatory disease of the respiratory airways that is characterized by airway obstruction, wheezing, and shortness of breath.

In some embodiments, the subject suffers from anaphylaxis. As used herein, the term "anaphylaxis" refers to a life threatening allergic reaction characterized by decreased blood pressure, respiratory failure with bronchoconstriction, and skin rash due to release of mediators from cells such as mast cells.

As used herein, the term "Kinesin-1/Slp3/Rab27b complex" refers to the complex as described in the EXAMPLE which results from the association of Kinesin-1/Slp3 and Rab27b".

As used herein, the term "kinesin-1" has its general meaning in the art and refers to the tetrameric protein constituted by 2 heavy chains (KIF5A, KIF5B or KIF5C) and 2 light chains (KLC1, KLC2 or KLC3) (Hirokawa, 1998). Human exemplary nucleic and amino acid sequences of KIF5A are represented by the NCBI reference sequences NM_004984.2 and NP_004975.2 respectively. Human exemplary nucleic and amino acid sequences of KIF5B are represented by the NCBI reference sequences NM_004521.2 and NP_004512.1 respectively. Human exemplary nucleic and amino acid sequences of KIF5C are represented by the NCBI reference sequences NM_004522.2 and NP_004513.1 respectively. Human exemplary nucleic and amino acid sequences of KLC1 are represented by the NCBI reference sequences NM_001130107.1 and NP_001123579.1 respectively. Human exemplary nucleic and amino acid sequences of KLC2 are represented by the NCBI reference sequences NM_001134774.1 and NP_001128246.1 respectively. Human exemplary nucleic and amino acid sequences of KLC3 are represented by the NCBI reference sequences XM_005258536.3 and XP_005258593.1 2 respectively.

As used herein, the term "slp3" has its general meaning in the art and refers to the synaptotagmin like 3 protein. Human exemplary nucleic and amino acid sequences of slp3 are represented by the NCBI reference sequences NM_001009991.3 and NP_001009991.2 respectively.

As used herein, the term "rab27b" has its general meaning in the art and refers to a member RAS oncogene family. Human exemplary nucleic and amino acid sequences of slp3 are represented by the NCBI reference sequences NM_004163.4and NP_004154.2 respectively.

As used herein, the term "agent" refers to, for example, nucleic acids (e.g., DNA and RNA), carbohydrates, lipids, proteins, peptides, peptidomimetics, small molecules and other drugs.

In some embodiments, the agent is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide. In particular, the agent is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide directed to KIF5A, KIF5B, KIF5C, KLC1, KLC2, KLC3, slp3 or Rab27b. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti- sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

In some embodiments, the agent is capable of inhibiting the interaction between Rab27b and Slp3. In some embodiments, the agent is capable of inhibiting the interaction between Slp3 and KLC1. In some embodiments, the agent is capable of inhibiting the interaction between Kif5b and Slp3.

In some embodiments, the agent is an intrabody having specificity for Rab27b, Slp3 and KLC1. In some embodiments, the intrabody inhibits the interaction between Rab27b and Slp3. In some embodiments, the intrabody inhibits the interaction between Slp3 and KLC1. In some embodiments, the intrabody inhibits the interaction between Kif5b and Slp3. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In another embodiment, the antibody according to the invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody®". According to the invention, sdAb can particularly be llama sdAb.

In some embodiments, the agent of the present invention is a PI3K inhibitor. As used, herein, the term "PI3K" inhibitor" refers to any agent which binds to and inhibits the PI3 Kinase. In addition, the inhibitors with a specific activity on PI3K may be preferred. Inhibitors of PI3K are, in most cases, compounds that interfere with the binding of ATP in the binding site of PI3K ATP, thus preventing a more or less specific activity of these kinases. In some cases, inhibitors of PI3K are allosteric inhibitors. Non-limiting examples of PI3K inhibitors include: LY294002; NVP-BEZ235 (BEZ235) (Novartis); GDC-0941 (Genentech/Roche); GDC-0980 (Genentech); PI- 103 (Piramed); XL147 (Exilixis/Sanofi- Aventis); XL418 (Exilixis); XL665 (Exelixis); LY29002 (Eli Lilly); ZSTK474 (Zenyaku Kogyo); BGT226 (Novartis); wortmannin; quercetin; tetrodotoxin citrate (Wex Pharmaceuticals); thioperamide maleate; IC87114; PIK93; TGX-115; deguelin; NU 7026; OSU03012; tandutinib (Millennium Pharmaceuticals); MK-2206 (Merck); OSU-03012; triciribine (M.D. Anderson Cancer Center); PIK75; TGX-221; NU 7441; PI 828; WHI-P 154; AS-604850; AS-041164 (Merck Serono); AS-252424; AS-605240; AS-604850; compound 15e;17-P-hydroxywortmannin; PP121; WAY-266176; WAY-266175; BKM120 (Novartis); PKI-587 (Pfizer); BYL719 (Novartis) ; XL765 (Sanofi-Aventis); GSK1059615 or GSK615 (GlaxoSmithKline); IC486068; SF1126 (Semafore Pharmaceuticals); CAL-101 (Gilead Sciences); LME00084; PX-478 (Oncothyreon); PX-866 (Oncothyreon); PX-867 (Oncothyreon), BAY 80-6946 (Bayer), GSK2126458 (GlaxoSmithKline), INK1117 (Intellikine), IPI-145 (Infinity Pharmaceuticals) Palomid 529 (Paloma Pharmaceuticals); ZSTK474 (Zenyaku Kogyo); PWT33597 (Pathway Therapeutics); TG100-115 (TargeGen); CAL263 (Gilead Sciences); SAR245408 (Sanofi-Aventis); SAR245409 (Sanofi-Aventis); GNE-477; CUDC-907; and BMK120 (Novartis). In some embodiments, the PI3K inhibitor is LY294002 (a morpholine derivative of quercetin) or 2-(4-Morpholinyl)-8-phenyl-4H-l- benzopyran-4-one. LY294002 may be obtained commercially or synthesized as described in U.S. Patent No. 5, 703, 075, the content of which is incorporated herein by reference. In some embodiments, the PI3K inhibitor is a prodrug of LY294002 comprising a reversibly quaternized nitrogen as described in international patent application WO2004/089925. On example of such prodrug is SF1226 (Semafore Pharmaceuticals) which is composed of the PI3K inhibitor LY294002 conjugated to an RGD targeting peptide. For example, the endoderm induction medium may contain 1 to 100 μΜ PI3K inhibitor, such as LY294002, preferably about 10 μΜ.

As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of patient at risk of contracting the disease or suspected to have contracted the disease as well as patients who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a patient during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a patient during treatment of an illness, e.g., to keep the patient in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., disease manifestation, etc.]). By a "therapeutically effective amount" of the agent as above described is meant a sufficient amount to provide a therapeutic effect. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

According to the invention, the agent is administered to the subject in the form of a pharmaceutical composition. Typically, the agent may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. In the pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The agent can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the typical methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The preparation of more, or highly concentrated solutions for direct injection is also contemplated, where the use of DMSO as solvent is envisioned to result in extremely rapid penetration, delivering high concentrations of the active agents. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

A further object of the present invention relates to a method of screening a drug suitable for inhibiting mast cell degranulation comprising i) providing a test compound and ii) determining the ability of said test compound to disrupt the formation of the Kinesin- 1/Slp3/Rab27b complex. Any biological assay well known in the art could be suitable for determining the ability of the test compound to disrupt the formation of the Kinesin-1/Slp3/Rab27b complex. In some embodiments, the assay fist comprises determining the ability of the test compound to bind to a KIF5A, KIF5B, KIF5C, KLC1, KLC2, KLC3, slp3 or Rab27b polypeptide. In some embodiments, when it is determined that the test compound binds to a KIF5A, KIF5B, KIF5C, KLC1, KLC2, KLC3, slp3 or Rab27b polypeptide, a population of mast cells is then contacted and activated so as to determine the ability of the test compound to inhibit the degranulation of said cells. In particular, the effect triggered by the test compound is determined relative to that of a population of mast cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity. With respect to the present invention, such control substances are inert with respect to an ability to modulate the formation of the Kinesin- 1/Slp3/Rab27b complex. Exemplary controls include, but are not limited to, solutions comprising physiological salt concentrations. Assays for determining the mast cell degranulation are well known in the art and are typically described in the EXAMPLE. It is to be understood that test compounds capable of inhibiting mast cell degranulation, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, antibodies (e.g. intraantibodies), aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules. As used herein, the term "small organic molecule" refers to a molecule of size comparable to those organic molecules generally sued in pharmaceuticals. The term excludes biological macromolecules (e.g.; proteins, nucleic acids, etc.); preferred small organic molecules range in size up to 2000da, and most preferably up to about 1000 Da.

The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention. FIGURES:

Figure 1: Absence of Kif5b impairs mast cell degranulation in vitro and in vivo.

A) Release of β-hexosaminidase from IgE-sensitized WT and cKO^Kif5b BMMC induced by 20ng/ml DNP-HSA determined at the indicated times. Results are mean ± SD of 10 experiments. Statistical analysis was performed using the unpaired t-test (** p<0.005; *** p<0.0001). B) WT (n=5) and ΰΚΟ^Εί¾ (n=5) mice were passively sensitized with anti-DNP IgE antibody (^g/g) and 24h hours later mice were challenged with DNP-HSA (500 μg). Systemic anaphylaxis was determined by the change in body temperature. C) Serum levels of MCPT-1 in WT (n=5) and cKO^Kif5b (n=5) mice measured at 60 min after DNP-HSA challenge. Statistical analysis was performed using the unpaired t-test (** p<0.005). D) IgE-sensitized WT and cKO^Kif5b BMMC were plated on fibronectin-coated glass coverslips and then stimulated by the addition of 20ng/ml DNP-HSA for the indicated time (NS: non stimulated). Cells were then fixed, permeabilized and stained with anti-STX3. Scale bar 2μιη. Statistical analysis was performed using the unpaired t-test (* p<0.05; ** p<0.005; *** p<0.0001). More than 100 cells were counted per setting.

Figure 2: Characterization of the Kinesin-1 -dependent transport machinery in

MC. A) Relative quantification of Rab27a, Rab27b, Slpl, Slp2, Slp3, Slp4 and Slp5 transcripts by real-time PCR with BMMC mRNA. Transcripts levels for each sample were expressed as a proportion of the mean value for Rab27b. B) BMMC were lysed and analyzed by western blot using anti-Kif5b, anti-Slp2, anti-Slp3 and anti-Rab27 antibodies. C) IgE- sensitized WT BMMC were left unstimulated (NS) or were stimulated with 20ng/ml DNP- HSA for 10 min and lysed. Cell lysates were immunoprecipitated with a polyclonal anti- Rab27 antibody or with isotope control antibody. The immunoblots were analyzed using anti- Kif5b, anti-Slp3 and anti-Rab27 antibodies. Data are representative of three independent experiments. D) Flag-Rab27b and Gfp-Slp3 were coexpressed in HEK 293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody (M2 beads) and then separated by SDS-PAGE. Coprecipitated Rab27b and Slp3 were immunoblotted with anti-Flag and anti- Gfp. The blots represent 3 independent experiments. E) Flag-Rab27b, -Slp3 and Gfp-KLCl were coexpressed in HEK 293T cells. Cells were left unstimulated or were stimulated with PMA/ionomycin and then cells lysates were immunoprecipitated with anti-Flag antibody (M2 beads) and then separated by SDS-PAGE. Coprecipitated Rab27b or Slp3 and KLCl were immunoblotted with anti-Flag and anti-Gfp. The blots are representative of 3 independent experiments. F) Flag-Rab27b, -Slp3 and Gfp-Kif5b were coexpressed into HEK 293T cells. Cells were left unstimulated or were stimulated with PMA/ionomycin and then cell lysates were immunoprecipitated with anti-Flag antibody (M2 beads) and then separated by SDS- PAGE. Coprecipitated Rab27b or Slp3 and Kif5b were immunoblotted with anti-Flag and anti-Gfp. The blots represent 3 independent experiments.

Figure 3: Role of Slp3 in SGs degranulation. A) BMMC were electroporated with siRNA targeting Slp3 or control siRNA. Transfected BMMC were lysed and analyzed by western blot using anti-Slp3 and anti-tubulin as a loading control. B) IgE- sensitized WT and cKO^Kif5b BMMC were left unstimulated or were stimulated with 20ng/ml DNP-HSA for lOmin. Cells were fixed and cell surface expression of CD63 as a surrogate degranulation marker was determinded by flow cytometry (left panel) and quantitated (delta MFI CD63+). Statistical analysis was performed using the unpaired t-test (*** p<0.0001). C) Control or Slp3 siRNA were co-transfected with GFP alone. GFP+ BMMC were then assessed for CD63 expression by flow cytometry (left panel) and expression was quantified (delta MFI CD63+). Statistical analysis was performed using the unpaired t-test (*** p<0.0001). D) BMMC were electroporated with siRNA targeting Slp3 or control siRNA along with Gfp. IgE-sensitized electroporated BMMC were left unstimulated or were stimulated with 20ng/ml DNP-HSA for lOmin. Cells were fixed permeabilized and stained with anti-STX3 antibody. Scale bar 2μιη. Statistical analysis was performed using the unpaired t-test (*** p<0.0001). More than 30 cells were counted per setting.

Figure 4: Kinesin-1 -dependent transport machinery formation is dependent on the activation of PI3K. A. IgE-sensitized BMMC were preincubated in normal medium containing DMSO, or medium containing LY-294002 (100μΜ)(ΡΙ3Κ inhibitor), or medium containing BAPTA (20mM) (intracellular calcium chelator). BMMC were stimulated with 20ng/ml DNP-HSA for lOmin. Cells were fixed and incubated with anti-CD63 antibody before flow cytometry analysis (left panel) and expression was quantified (delta MFI CD63+). Statistical analysis was performed using the unpaired t-test (*** p<0.0001). B) IgE-sensitized BMMC were preincubated in normal medium containing DMSO, or medium containing LY- 294002, or medium containing BAPTA. BMMC were stimulated with 20ng/ml DNP-HSA for lOmin then cells were lysed. Cell lysates were immunoprecipitated with a polyclonal anti- Rab27 antibody or with isotope control antibody. The immunoblots were analyzed using anti- Kif5b, anti-Slp3 and anti-Rab27 antibodies. Data are representative of three independent experiments.

EXAMPLE:

Material & Methods

Reagents and Abs

Rabbit Abs to STX3 and mouse monoclonal anti-DNP-IgE have been described(Brochetta et al., 2014). Anti-c-Kit-APC and anti-FceRI-FITC were purchased from eBioscience. AKT rabbit antibody (Cell Signaling) P-AKT rabbit antibody (Cell Signaling), ERK1/2 rabbit antibody (Cell Signaling), P-ERK1/2 rabbit antibody (Cell Signaling), monoclonal Kif5b antibody (Biolegend), polyclonal Kif5b (proteintech), polyclonal rabbit Rab27 antibody (SY synaptic systems), mouse monoclonal serotonin antibody (Novusbio), rabbit polyclonal Slp3 antibody (Santa Cruz biotechnology), mouse monoclonal a-tubulin (Abeam), mouse GADPH antibody (Millipore), purified rabbit anti-cc-Tubulin (Rockland), AlexaFluor-555-conjugated goat anti-mouse IgG F(ab')2 secondary antibodies (Invitrogen) and AlexaFluor-555-conjugated donkey anti-rabit IgG (H+L) secondary antibodies (Invitrogen) were used. The polyclonal rabbit antibody anti-Slp2 was previously described(Menasche et al., 2008). Alexa Fluor 488 wheat germ agglutinin (WGA) was purchased from Invitrogen. Ly-294002 and BAPTA were purchase from Sigma.

Mice

VAV-Cre transgenic mice from Jackson Laboratory were first crossed with Kif5b^+I~ to generate Kif5b^+I~; VAV-Cre mice. Then Kif5b^+I~; VAV-Cre mice were bred with Κί 5^^α mice to generate Kif5b^a~ ; VA V- Cre (cKO^) mutant mice and the control littermates Kif5b^a+ (WT) mice. Genotyping was performed by PCR using primers described in Cui et al(Cui et al., 2011).

Cell culture and transfection Bone marrow was isolated from femur and tibias of 8-12 week-old mice. Cells were then cultured in IMDM medium (In vitro gen) supplemented with 15% of FCS, 1% nonessential amino acids, ImM sodium pyruvate, 50μΜ 2-ME, lOOU/ml penicillin, lOOU/ml streptomycin, lOng/ml IL-3 and lOng/ml SCF (Miltenyi Biotec). After 4 weeks of culture, maturation of bone marrow-derived MC (BMMC) was verified by flow cytometry for c-KIT and FceRI expression. BMMC were transfected using the NEPA21 (Nepagen) according to the manufacturer's protocols.

Histology

Sections from back skin were subject to PFA fixation and paraffin embedding in a routine histology laboratory. Sections were then stained with 0.1% toluidine blue solution.

Quantitative PCR

cDNA was prepared from BMMC mRNA using Superscript II and Random primers (Invitrogen). Levels of KIF5A, KIF5B, KIF5C, KLC1, KLC2, KLC3, RAB27A, RAB27B, SYTL1, SYTL2, SYTL3, SYTL4 and SYTL5 transcripts were determined by quantitative PCR (qPCR) using: TaqMan Gene expression Master Mix (Applied Biosystems), primer (KIF5A: Mm00515265_ml, KIF5B: Mm00515276_ml, KIF5C: Mm00500464_ml, RAB27A: Mm00469997_ml, KLCh Mm00492936_ml, KLC2: Mm00492945_ml, KLC3: Mm00461422_ml, RAB27B: Mm00472653_ml, SYTL1: Mm00473300_ml, SYTL2 Mm01317927_ml, SYTL3: Mm00473333_ml, SYTL4: Mm00489110_ml, SYTL5: Mm00624760_ml and 18S: Mm03928990_gl Applied Biosystems) and cDNA. Each sample was amplified in triplicate on a real-time PCR instrument (an ABI 7900 cycler) and analyzed using Sequence Detection Systems software (version 2.2.2, Applied Biosystems). The relative mRNA levels were quantified via the comparative Cx method and normalized against the average values of 18S as endogenous control. KIF5A, KIF5B and KIF5C transcript levels were then expressed as a proportion of the mean value of KIF5B. RAB27A, RAB27B, SYTL1, SYTL2, SYTL3, SYTL4 and SYTL5 values were expressed as a proportion of the mean value of the gene showing the highest level of expression arbitrary put at 1 unit.

Plasmid constructs Mouse Rab27b was cloned into pCRII-TOPO. These constructs were then subcloned into pEGFP-Cl (Clontech). Cloning of Slp3 and Kif5b has been previously reported(Kurowska et al., 2012; Menasche et al., 2008).

Degranulation assays

Release of granule content was determined by β-hexosaminidase assay. Briefly, BMMC were sensitized with anti-DNP- specific IgE overnight. IgE-sensitized BMMC were stimulated with 20ng/ml DNP-HSA for the indicated time at 37°C. Cell were centrifugated at 4°C and supernatants were incubated in citrate buffer with p-nitrophenyl-N-acetyl- -D- glucosaminide for 60 min at 37°C. Reaction was terminated in carbonate buffer for 15 min and OD at 405nm was determined. Specific release was calculated as the percentage of total β-hexosaminidase content from a Triton X-100 cell lysate after subtraction of baseline degranulation in untriggered cells. Degranulation of BMMC was also evaluated by surface expression CD63 using flow cytometry. IgE-sensitized BMMC were either left unstimulated or were stimulated with 20ng/ml DNP-HSA for 10 min. Cells were fixed in 3.7% PFA and incubated with anti-CD63 antibody (MLB international) at 4°C, followed by incubation with Alexa Fluor 647-conjugated anti-rat IgG before analysis by flow cytometry using a Fortessa apparatus (BD Biosciences).

Elisa

BMMC were sensitized with DNP- specific IgE overnight. Then cells were washed and incubated for 3h in culture medium containing 20ng/ml DNP-HSA. Elisa was performed using the commercial kits for IL-6, TNFa and MCP-1 (eBioscience) according to the manufacturer's protocols.

Immunofluorescence

BMMC were sensitized with DNP- specific IgE overnight. BMMCs were plated onto glass coverslips coated with fibronectin (10 g/ml, Sigma) for 45 min at 37°C with ImM MnCl₂. BMMC were activated with 20ng/ml DNP-HSA for the indicated times. BMMC were fixed by incubation for 15 minutes on ice in 3.7% w/v paraformaldehyde and then for a further 10 minutes in NH₄C1 (50 mM in phosphate-buffered saline (PBS)). The cells were then incubated for 1 h with specific primary antibodies in permeabilization buffer (PBS, 1 mg/mL bovine serum albumin and 0.05% w/v saponin (Sigma)), washed twice and incubated for another hour with fluorescent-conjugated secondary antibody in permeabilization buffer. Lastly, the cells were mounted on slides in Prolong Gold antifade reagent in the presence or absence of DAPI (Invitrogen Carlsbad, CA). Confocal microscopy was performed with a Zeiss LSM 700 (Carl Zeiss, Oberkochen, Germany) at 63x, steps of 0.38μιη microscope. The images were processed with the Zeiss LSM Image Browser (Carl Zeiss) and ImageJ software (version 1.43, Wayne Rasband, NIH, Bethesda MD)

TIRF

The TIRF assay was performed using WGA-488-loaded BMMC sensitized with DNP- specific IgE overnight. Cells were allowed to attach to glass coverslips coated with fibronectin (10 g/ml, sigma) before stimulation with 20ng/ml DNP-HSA. TIRF images were then acquired for 15 min (exposure time: 200 ms). During the observation, the cells were kept at 37°C and 5% C0₂. Fluorescence data were acquired with an Eclipse Ti-E TIRF imaging system (Nikon). Images were acquired with a Ropper scientific QuanTEM 512 SC camera (Nikon) and NIS-Elements AR software (version 3.1). Image sets were processed with Image J software.

Calcium Flux

5xl0⁶ IgE-sensitized BMMC were loaded with 5μg Indo-1 (life technologies) in RPMI medium supplemented with lOmM Hepes pH 7 for 30 min at 37°C. RPMI medium supplemented with lOmM Hepes pH 7 and 5% FCS was then added for 30 min at 37°C. Cells were washed and resuspended in RPMI medium supplemented with 5% FCS before addition of 20ng/ml DNP-HSA to induce calcium flux. Calcium flux was measured using the FACSAria flow cytometer (BD Biosciences) to monitor the ratio of the fluorescent emission 405nm/475nm. Kinetic analyses were obtained using FlowJo software package (Tree Star).

Passive systemic Anaphylaxis

A temperature probe (Bio Medic Data Systems) was implanted subcutaneously into the back skin of 8 week-old- WT and cKO mice. The next day, mice were passively sensitized i.v. using ^g/g anti-DNP mouse IgE antibody (Η1-ε-26). 24 h later mice were challenged by intravenous administration of 500 μg DNP-HSA (Sigma). Temperatures were recorded every 5 min for 60 min using a DAS-7007S wireless reader (Bio Medic Data Systems). At the end blood samples were collected to measure serum concentration of MCPT- 1 with an Elisa kit (eBioscience).

I mm u n o blotting and immunoprecipitation

Proximal signaling: 10⁷ IgE-sensitized WT and cKO^ BMMC stimulated with 20ng/ml DNP-HSA for indicated times were lysed in lysis buffer (50mM Tris HC1 pH7.6, 150mM NaCl, 5mM EDTA, 0.5% Triton X-100) supplemented with 1 EDTA-free protease inhibitor cocktail tablet (Roche Diagnostics). For the immunoprecipitation, 40xl0⁶ IgE- sensitized WT were either left unstimulated or were stimulated with 20ng/ml DNP-HSA for 10 min. Cells were lysed in lysis buffer (50mM Hepes pH7,4 ; 150mM Nacl ; MgC12 ImM ; 1% Triton X-100; 10% Glycerol) supplemented with 1 EDTA-free protease inhibitor cocktail tablet and phosphatase inhibitors (sigma-Aldrich). Immunoprecipitation was performed with polyclonal rabbit Rab27 antibody (Synaptic Systems). Anti-Slp3, anti-Rab27 and anti-Kif5b antibodies were used to develop the western blots. siRNA

Specific duplex siRNA targeting Slp3 was purchased from quiagen (N°SI02899750). Statistical analysis

Statistical analysis was performed with Prism Version 6 software using an unpaired two-tailed t-test which determine the p values: * p<0.05; ** p<0.005; *** p<0.0001.

Results

Kif5b is expressed in MC and is not required for MC differentiation

To explore the molecular machinery involved in the anterograde transport of SGs during MC degranulation we focused on Kif5b, the major isoform of Kinesin-1 expressed in murine MC. We generated a conditional mouse model invalided for the heavy chain (Kif5b) of Kinesin-1 in all hematopoietic cell lineages (cKO^Kif5b). In the absence of Kif5b, progenitors of the bone marrow normally differentiated into MC in the presence of IL3 and SCF. The expression levels of FceRI and c-Kit were comparable in WT and cKO^⁵¹³ bone marrow-derived MC (BMMC). We confirmed that in ΰΚΟ^Εί¾ BMMC Kif5b was absent and that no compensatory upregulation of the other isoforms became apparent. As Kinesin-1 is a tetrameric protein constituted of 2 heavy chains and 2 light chains, we also analyzed which light chains are expressed in BMMC. Quantitative RT-PCR for the 3 KLC (KLC1, KLC2 and

KLC3) identified KLC1 as the main isoform present in BMMC. The absence of Kif5b in _cKQKif5b _BMMC was confirmed at the protein level. In vivo, the number of MC in back skin detected by toluidine blue staining and in the peritoneal cavity detected by flow cytometry did not significantly differ between WT and cKO^⁵¹³ indicating that Kinesin-1 is not essential for MC survival or tissue targeting. Taken together, these results show that Kinesin-1 -deficiency does not affect MC development in vitro and in vivo.

Kinesin-1 regulates SGs degranulation in MC in vitro and in vivo

To determine the role of Kinesin-1 in stimulus-secretion coupling in MC, WT and _cKQKif5b _BMMC were sensitized with anti-DNP-IgE antibody before stimulation with DNP- human serum albumin (HSA) in kinetic experiments. FceRI-mediated BMMC activation led to a rapid degranulation response and release of β-hexosaminidase enzyme contained in SGs. Kif5b-deficient BMMC exhibited a markedly impaired capacity to degranulate upon FceRI activation (Figure 1A).

To assess the role of Kinesin-1 in MC degranulation in vivo, we performed passive systemic anaphylaxis experiments. WT and cKO^Klf5b mice were sensitized with anti-DNP IgE antibody and 24h later mice were challenged with DNP-HSA. Body temperature was recorded, and release of the MC specific protease- 1 (MCPT-1) in the serum was quantified to monitor the severity of the anaphylactic response. Figure IB shows that in cKO^Kif5b mice the temperature drop notably during the later phases was reduced as compared to WT mice. Likewise, cKO^Klf5b mice exhibited a marked reduction in serum concentration of MCPT-1 indicating that Kif5b-deficient MC were impaired in their capacity to degranulate in vivo (Figure 1C).

To explore whether absence of Kinesin-1 in BMMC affected SG translocation, we compared the distribution of SGs in resting (NS) and stimulated cells (10 and 30 min) using an antibody against STX3. In resting BMMC, no difference in SG distribution was observed between WT and cKO^⁵¹³ BMMC (Figure ID). In contrast, 10 min after stimulation through FceRI, while at least 80% of STX3 located at the plasma membrane in WT BMMC indicating significant SG translocation and fusion with the plasma membrane less then 15% of STX3 labeled the cell periphery in cKO¹^⁵¹³ BMMC (Figure ID). After 30 min of activation, the proportion of cKO^⁵¹³ BMMC, which had reached the plasma membrane increased to 50% but remains at a lower level in comparison to WT BMMC (Figure ID). These results indicate that Kinesin-1 is the essential molecular motor to allow SGs to be translocated to the plasma membrane and secrete their content.

To further confirm and extend these findings, we used live cell imaging by TIRF microscopy to analyze the dynamic recruitment of SGs at the plasma membrane upon FceRI activation. We found that wheat germ agglutinin (WGA), previously used to label cytotoxic granule in CTLs(Sepulveda et al., 2015) following a brief endocytosis period constitutes a valuable live marker of SGs in BMMC. BMMC were pulsed 20 min with WGA conjugated to Alexa-fluor-488 (green) and chased for 18h. WGA uptake was similar in WT and cKO^Kif5b BMMC with WGA being present in the lumen of STX3 positive SGs likely labeling intraluminal vesicles as it was shown for cytotoxic granules(Sepulveda et al., 2015). Upon WGA loading, BMMC were sensitized with anti-DNP-IgE antibody and placed on fibronectin-coated glass coverslips. After addition of DNP-HSA, TIRF acquisition was initiated and analyzed for a period of 15 min. In WT BMMC, docking events of SGs became detectable around 8 min after stimulation and until the end of the acquisition period. In contrast, very rare docking events of SGs were detectable in cKO¹^ BMMC until 13 min of acquisition where a small rise of docking events could be observed, however, the number of docking events was still drastically impaired compared to WT BMMC. These results indicate that Kinesin-1 controls SGs translocation towards their secretion site.

Kinesin-1 is dispensable for cytokine secretion and activation upon FceRI stimulation in MC

As FceRI receptor aggregation also leads to de novo synthesis and secretion of cytokines, we assessed whether Kif5b-deficient BMMC could produce and secrete proinflammatory cytokines such as TNFa, IL6 and CCL2 (also known as MCPl). After 3h of stimulation through FceRI, no significant differences in the amounts of secreted cytokines were found between WT and Kif5b-deficient BMMC. These results indicate that Kinesin-1 is dispensable for cytokine secretion upon FceRI activation.

We also assessed whether early signaling pathways were dependent on Kinesin-1 function. Phosphorylation of Erk and Akt as well as calcium flux entrance were unchanged between WT and Kif5b-deficient BMMC. These results indicate that the defect in degranulation observed in the Kif5b-deficient BMMC is not caused by a defect in early signaling events induced by FceRI activation. Characterization of Kinesin-1 -dependent transport machinery in MC

As Kinesin-1 has been shown to regulate the transport of Rab27/Slp-associated vesicles on microtubules and as Rab27b was implicated MC degranulation(Arimura et al., 2009; Kurowska et al., 2012; Mizuno et al., 2007), we explored further the molecular complex of the Kinesin-1 granular transport machinery in MC. In agreement with data showing a MC secretion defect in Rab27b but not in Rab27a KO mice(Mizuno et al., 2007), we found that Rab27b represents the main Rab27 family member expressed in BMMC, while Rab27a was only marginally expressed (Figure 2A). When the expression of the 5 members of the Sip family was analyzed, Slp3 was found highly expressed in BMMC, while Slp2 was expressed to a lesser extent (Figure 5A). No Slp4 and 5 and only minute amounts of Slpl were expressed in BMMC. In the absence of any detectable Slp2 protein in BMMC lysate (Figure 2B), we focused on the role of Slp3 adaptor in the Kinesin-1 mediated transport mechanism. Figure 2C shows that Slp3 co-immunoprecipitated with Rab27 in unstimulated and FceRI stimulated cells. Interestingly, while Kif5b did not co-immunoprecipitate in unstimulated cells substantial amounts of Kif5b became associated with Rab27b in stimulated cells (Figure 2C).

To understand which proteins of the complex were able to interact which each other, we overexpressed in HEK 293T cells different combination of tagged proteins. We confirm the strong interaction between Rab27b and Slp3, and Slp3 and KLC1 (Figure 2D and 2E). In contrast, no interaction between Rab27b and the light chain or the heavy chain of Kinesin-1 could be observed (Figure 2E and 2F). In Figure 2F, an interaction between Kif5b and Slp3 was observed, albeit it is not clear to know whether this interaction is direct or required the endogenous KLC1 expressed in HEK 293T. Contrary to what was observed in MC, the activation by PMA/ionomycin of HEK 293T system does not seem to modulate interaction between Slp3 and Kinesin-1 (Figure 2E and 2F). These data suggest that the interaction between Slp3 and Kinesin-1 does not require a post-translational modification but rather seems to be actively suppressed in MC. As mast cell activation promotes Slp3/Kif5b complex formation, this suggests that activation will modulate the accessibility of Kinesin-1 for Slp3.

We next investigated the subcellular localization of the Kinesin-1 -dependent transport machinery. We engineered BMMC to coexpress Gfp-Rab27b and Dsred-Slp3. A strong overlap of the 2 fluorescence signals (Dsred-Slp3/Gfp-Rab27b 73.90% ± 2.54% overlap) was observed on the secretory granules labeled by STX3 (STX3/(Gfp-Rab27b/Gfp-Slp3), 73.22% ± 5.56% overlap). Upon FceRI activation, the Rab27b/Slp3 complexes (DRed-Slp3/Gfp- Rab27b 80.92% ± 2.6% overlap) were recruited to the plasma membrane largely colocalizing with STX3 (STX3/(Gfp-Rab27b/Gfp-Slp3), 70.9% ± 4.7% overlap). In Kif5b-deficient BMMC, like for STX3 the Rab27b/Slp3 complex translocation to the plasma membrane was dramatically impaired upon activation further supporting the critical role of the Kinesin-1 in the transport of Rab27b/Slp3 associated SG to the plasma membrane.

The endogenous Kif5b subcellular localization showed a distribution along the microtubules with a strong label found beneath the plasma membrane and to a lesser extent in the cytosol and around the microtubule- organizing center (MTOC). The Kif5b-deficient MC revealed a total absence of Kif5b labeling confirming the specificity of the antibody. Upon FceRI activation in WT cells, Kif5b seems predominantly recruited at the peripheral ring of tubulin beneath the plasma, a cellular area rich in microtubule formation. The microtubule organization does not seem to be affected in the absence of Kif5b in unstimulated or stimulated condition and the microtubule formation occurs normally upon FceRI activation. As our results indicate that Slp3 links Rab27b-associated SGs to Kinesin-1 upon FceRI activation, we further analyze the implication of Slp3 in SG secretion using siRNA to specifically knockdown Slp3 in BMMC. The efficacy of siRNA targeting Slp3 was verified by western blot analysis (Figure 3A). Following Slp3 knock-down BMMC were assessed for their ability to de granulate by evaluating CD63 expression at the plasma membrane as reported previously(Brochetta et al., 2014). We confirmed that Kif5b-deficient BMMC upon IgE/Ag stimulation had a markedly diminished capacity to expose CD63 at the cell surface (Figure 3B). Likewise, Slp3 silencing substantially impaired SGs secretion (Figure 3C). We next assessed whether the secretion defect-mediated by Slp3 silencing is also associated with a defect in SGs translocation. The localization of STX3 in BMMC expressing siRNA targeting Slp3 in unstimulated condition was not affected compared to BMMC expressing control siRNA (Figure 3D). Upon FceRI activation BMMC expressing Slp3 siRNA were unable to translocate their STX3-containing SGs to the plasma membrane unlike BMMC expressed control siRNA (Figure 3D). These data demonstrate that Slp3 is part of the molecular machinery coupling microtubule dynamics and SGs translocation to the plasma membrane upon FceRI activation.

Kinesin-l-dependent transport machinery formation is regulated by PI3K

Results above supported an active mechanism allowing recruitment of the Kinesin-l- dependent transport machinery to SG. To explore possible signaling pathways we employed drugs specifically targeting PI3K and intracellular calcium flux, notably LY-294002 and BAPTA. We confirmed that both drugs lead to a severe degranulation defect (Nishida et al., 2011; Nishida et al., 2005) (Figure 4A). We next assessed the effect on Kif5b recruitment to the granular Slp3/Rab27b effector complex upon FceRI activation. Interestingly, we observed that while LY-294002 completely disturbs the Kif5b/Slp3 complex formation BAPTA has no effect (Figure 4B). To further verify whether blockade of PI3K affected coupling of SG granules to microtubule dependent translocation we analyzed the effect of drugs on SG- associated STX3 relocation to the plasma membrane upon FceRI activation. In agreement with the observed uncoupling, LY-294002 dramatically impairs SGs translocation to the plasma membrane whereas BAPTA does not affect SGs translocation(Nishida et al., 2005). Our results support that activation of PI3K enables the stimulation dependent coupling of the Kinesin-1 motor protein to the molecular machinery that links SG trafficking to microtubule dynamics.

Discussion:

Activation of MC through IgE and antigens triggers the release of SG containing the preformed mediators responsible for anaphylactic responses. Although microtubule dynamics is known to play a critical role in SG movement(Nishida et al., 2011; Nishida et al., 2005; Ogawa et al., 2014), little is yet known about the molecular machinery that couples SG granules to the microtubule cytoskeleton to drive their movement towards their secretion site upon MC activation. Here we provide evidences that the Kif5b heavy chain isoform of Kinesin-1 is the critical motor protein that regulates the translocation of SGs to the plasma membrane upon FceRI activation enabling anaphylactic degranulation in vitro and in vivo. By contrast Kif5b is dispensable for the activation of the proximal signaling pathways or the secretion of cytokines upon FceRI stimulation. We further demonstrate that the Kinesin-1 motor protein links microtubules to SG through a newly characterized molecular machinery composed of granule associated Rab27b and the Slp3 adaptor protein. Interestingly, although the association of this complex can be achieved in the absence of any posttranslational modifications, we found that in MC complex formation requires stimulation through a PI3K- dependent mechanism.

Several evidences support that Kif5b is critical for SG secretion upon FceRI activation. While calcium flux entrance and proximal signaling pathways as well as cytokine secretion were normal, Kif5b deficiency dramatically impaired (1) the release of β-hexosaminidase enzyme stored in SG, (2) the subcellular translocation of granule-associated STX3 to the plasma membrane, (3) the dynamic recruitment of SG at the plasma membrane (evidenced by TIRF) and (4) the degranulation ability of BMMC as quantified by cell surface CD63 expression as well as (5) the anaphylactic response in in vivo PSA experiments.

Of note, we observed that in the absence of Kif5b, the release of granule content is only partially impaired, both in vitro and in vivo. The particular type of secretion that occurs in MC may explain this partial impairment. Indeed, MC use a degranulation process, called compound or multivesicular exocytosis, during which granules undergo fusion with each other before reaching the plasma membrane(Rohlich et al., 1971). Such sequential fusion events may facilitate the release of the content of several SGs by limiting vesicular trafficking before fusion with the plasma membrane. In addition, degranulation in MC also occurs through SG already closely docked at the PM and hence may not require active SG movement. In addition, we cannot exclude that beside Kinesin-1 other Kinesin family members may also regulate SGs transport or compensate for the loss of Kinesin-1 in MC.

MC activation also leads cytokine/chemokine synthesis and secretion. Our data show that secretion of TNFcc, IL6 and MCP1 in Kif5b-deficient BMMC are not affected. Together with additional evidences obtained with SNARE and SNARE accessory proteins such as VAMP8 and Muncl8-2 (Brochetta et al., 2014; Tiwari et al., 2008), these data highlight that cytokine secretion and SG secretion do not use the same vesicular trafficking pathways in MC.

To more precisely characterize the SGs transport machinery, we focused on effectors of granule-localized Rab27b, which has been previously reported to affect MC degranulation and was also shown to represent a link to proteins interacting with Kinesins notably through Sip adaptors (Arimura et al., 2009; Kurowska et al., 2012; Mizuno et al., 2007). When assessing transcript expression levels of the various Sip members in MC we found Slp3 to be endogenously expressed and to be coprecipitated with Rab27b. Both proteins were localized on SG in MC and upon stimulation readily translocated to the PM upon FceRTmediated stimulation. To determine whether Slp3 participates in SGs secretion we used siRNA to knockdown Slp3 in BMMC. SGs secretion was drastically impaired in Slp3 silenced cells. This was clearly attributable to a defect in SGs translocation closely mimicking the Kif5b defect. These data strongly support the critical function of Slp3 in the translocation of SG during MC degranulation. Interestingly, the Kif5b deficiency does not affect the microtubule organization and microtubule formation upon FceRI-mediated stimulation highlighting that the SG translocation defect is rigorously caused by a mobility defect.

An interesting observation was that, in resting MC, the Rab27b/Slp3 complexes localize to SGs labeled by STX3 but does not interact with Kinesin-1. In contrast, upon mast cell activation, Kinesin-1 is recruited on SG along with the Rab27b/Slp3 complexes. This indicate that cell activation modulates the interaction of Kinesin-1 with the cargo receptor molecule, Slp3. Previous studies have shown an autoinhibitory mechanism used by different kinesin motors including Kinesin-1 that consists in the adoption of a folded conformation(Verhey and Hammond, 2009). Non-motor domains come into the contact with the motor domain and block the binding and the mobility on microtubules (Verhey and Hammond, 2009). The release of autoinhibition has been shown to involve cargo binding and cellular activation changing the phosphorylation status of the molecular motor(Donelan et al., 2002; Verhey and Hammond, 2009). Previous studies have shown that FEZ1 (fasciculation and elongation protein-ζΐ) and JIPl (Jun N-terminal kinase-interacting protein 1) regulate Kinesin-1 microtubule mobility through the binding with inhibitory regions of Kinesin- l(Blasius et al., 2007). For Kinesin-5 family and Kinesin-7 family, phosphorylation has been shown to remove the autoinhibition conformation(Cahu et al., 2008; Espeut et al., 2008). In pancreatic β-cells elevation of intracellular calcium has shown to induce the dephosphorylation of Kif5b by calcineurin leading to the insulin secretion(Donelan et al., 2002). As the overexpression of Slp3 and KLC1 or Kif5b in HEK 293T cells has shown that Slp3 interacts with Kinesin-1 through an activation-independent mechanism, this suggests that Kinesin-1 could adopt an autoinhibitory conformation that is not accessible for Slp3 in resting MC.

To identify possible signaling effectors that trigger the formation of the Kinesin-1- dependent transport machinery, drugs were used to disrupt the PI3K or the calcium flux in response to the MC activation. Interestingly, our results show that in absence of PI3K activity no Slp3/Kif5b complexes could occur. In contrast, formation of Slp3/Kif5b complex seems to be independent on calcium. Our results are in agreement with a previous study that shows that the translocation of the SG to the plasma membrane is calcium independent but dependent on microtubule dynamic reorganisation(Nishida et al., 2005). However, calcium is required for the release of SG and controls the fusion between SG and the plasma membrane. Previous studies have already shown that PI3K through the recruitment to the Gab2 adaptor protein plays an important role in the regulation of microtubule dynamics and SG translocation in IgE stimulated MC(Nishida et al., 2011; Ogawa et al., 2014). This involved the regulation of microtubule dynamics through phosphorylation and inactivation of GSK3 via Akt, a downstream effector of PI3K as well as ARFl(Ogawa et al., 2014). Interestingly, a previous study has shown that active GSK3 inhibited anterograde transport in neurons through the phosphorylation of KLC2(Morfini et al., 2002). However, further studies are necessary to understand what signaling molecules downstream of PI3K are able to regulate Kinesin-1 accessibility for cargo receptors such as Slp3. This may include investigation of the phosphorylation status of Kinesin-1 in unstimulated and stimulated condition to demonstrate whether this post-translational modification participates in the modulation of the accessibility of Kinesin-1 for it cargo receptor has it was shown for Kinesin-5 family and Kinesin-7 family(Cahu et al., 2008; Espeut et al., 2008).

In conclusion, using a conditional mouse model invalidated for Kif5b in all the hematopoietic cell populations, we have demonstrated that Kinesin-1 regulates SGs transport upon FceRI activation through the recruitment of Rab27b/Slp3-containing SGs a process regulated by PI3K following IgE-mediated stimulation. As cKO^Kif5b mice exhibited a marked reduction in passive systemic anaphylaxis, this raises the possibility that Kinesin-1 could be a valuable target for new therapeutic approaches controlling IgE-mediated type I immediate hypersensitivity reaction.

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Claims

CLAIMS:

1. A method of inhibiting mast cell degranulation in a subject in need thereof comprising administering to the subject a therapeutically effective amount of an agent capable of inhibiting the formation the Kinesin-1/Slp3/Rab27b complex.

2. The method of claim 1 wherein the subject suffers from an IgE-mediated disorder selected from the group consisting of allergic asthma, allergic rhinitis, atopic dermatitis and allergic gastroenteropathy.

3. The method of claim 1 wherein the subject suffers from an IgE-mediated disorder selected from the group consisting of hypersensitivity (e. g., anaphylactic hypersensitivity), eczema, urticaria, allergic bronchopulmonary aspergillosis, parasitic diseases, hyper-lgE syndrome, ataxia- telangiectasia, Wiskott-Aldrich syndrome, thymic alymphoplasia, IgE myeloma and graft- versus-host reaction.

4. The method of claim 1 wherein the agent is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide directed to KIF5A, KIF5B, KIF5C, KLC1, KLC2, KLC3, slp3 or Rab27b.

5. The method of claim 1 wherein the agent is capable of inhibiting the interaction between Rab27b and Slp3.

6. The method of claim 1 wherein the agent is capable of inhibiting the interaction between Slp3 and KLC1.

7. The method of claim 1 wherein the agent is capable of inhibiting the interaction between Kif5b and Slp3.

8. The method of claim 1 wherein the agent is an intrabody having specificity for Rab27b, Slp3 and KLCl.

9. The method of claim 1 wherein the agent of the present invention is a PI3K inhibitor.

10. A method of screening a drug suitable for inhibiting mast cell degranulation comprising i) providing a test compound and ii) determining the ability of said test compound to disrupt the formation of the Kinesin-1/Slp3/Rab27b complex.