WO2001021173A1 - Novel uses of 2-bromopalmitate - Google Patents

Abstract

The present invention provides a method of inhibiting Fyn/Lck fatty acylation and protein palmitoylation in a cell in an individual in need of such treatment comprising the step of administering to said individual a pharmacologically effective dose of 2-bromopalmitate. Also provided is a method of treating an individual having a pathophysiological state comprising the step of administering to said individual a pharmacologically effective dose of 2-bromopalmitate.

Description

NOVEL USES OF 2-BROMOPALMITATE

BACKGROUND OF THE INVENTION

Federal Funding Legend

This invention was produced in part using funds obtained

through grants GM57966 and CA29502 from the National Institute o f

Health. Consequently, the federal government has certain rights in

this invention.

Field of the Invention

The present invention relates generally to the fields of th e

molecular biology of T cell signaling and fatty acid biochemistry and

pharmacology. More specifically, the present invention relates t o

novel uses of 2-bromopalmitate. Description of the Related Art

Many viral and cellular proteins are modified by fatty acid

acylation with myristate or palmitate (1,2). For example, all members

of the Src family of tyrosine protein kinases are covalently modified

by the 14 carbon fatty acid myristate. Myristate is co-translationally

attached to a glycine at position 2 of the protein through an amide

linkage, in a process catalyzed by N-myristoyl transferase (NMT) ( 3 -

5). Myristoylation has been shown to be necessary (6,7) but no t

sufficient (8) for membrane binding. In addition, all Src proteins u s e

a second membrane targeting signal. For seven out of the nine Src

family members, this second signal involves modification with the 1 6

carbon fatty acid palmitate. Palmitate is post-translationally attached

to a cysteine residue within an N-terminal myr-gly-cys consensus

motif (9).

Attachment of myristate and palmitate to Src family

kinases enhances the localization of these proteins to the plasma

membrane, where they must be present in order to function properly.

In addition, protein palmitoylation has been shown to be critical for

localization of proteins to specialized subdomains of the plasma

membrane that are resistant to detergent extraction ( 10-15). These

detergent resistant microdomains (detergent resistant microdomains) ,

also known as rafts, are enriched in cholesterol, glycosphingolipids,

and GPI-anchored proteins (16-18). Localization to detergent resistant microdomains influences the ability of key signaling

molecules to interact with each other and to participate in signaling

from the cell surface to the interior of the cell (11,19-21).

The importance of protein fatty acylation is best illustrated

in T cell receptor (TCR) mediated signal transduction. The Src related

kinases Fyn and Lck are highly expressed in cells of hematopoietic

origin, particularly lymphocytes (22), and are required for signaling

through the T cell receptor. Protein tyrosine phosphorylation is o ne

of the first events that occurs after binding of antigens to surface

receptors in T lymphocytes. Upon receptor engagement, Fyn and Lck

phosphorylate tyrosine residues found within multiple

immunoreceptor tyrosine-based activation motifs (ITAMS) located o n

the cytosolic portions of the TCRζ and CD3 chains. Immunoreceptor

tyrosine-based activation motifs phosphorylation recruits key

molecules that mediate downstream signaling, including the tyrosine

kinase ZAP-70 (19). One of the targets for activated ZAP-70 is LAT, a

palmitoylated transmembrane protein (10). Several recent studies

have established that the ability of Lck, Fyn and LAT to function in T

cell receptor-mediated signaling depends on their fatty acylation and

localization to detergent resistant microdomains. Palmitoylation o f

Lck was shown to be essential for its signaling function in T

lymphocytes (11). Fyn must be palmitoylated and localized t o

detergent resistant microdomains in order to interact with the ζ chain of the T cell receptor (23). Moreover, LAT must be palmitoylated an d

in detergent resistant microdomains in order to become tyrosine

phosphorylated and participate in downstream signaling (20).

To date, studies of the role of protein palmitoylation i n

various cellular pathways have suffered from two major drawbacks .

First, in contrast to N-myristoylation, very little is known about th e

enzymology and biochemistry of protein palmitoylation. Two

thioesterases, PPT1 and APT1, have been identified that deacylate

palmitoylated Ras and Gα proteins in vitro (24,25). However, th e

enzyme(s) that catalyze(s) attachment of palmitate to proteins have

not been definitively identified. Several recent studies have described

purification of palmitoyl acyl transf erase (PAT) activities (26-28 ) ,

while other reports have documented that non-enzymatic

palmitoylation can occur under certain conditions in vitro (29 , 30) .

Second, nearly all studies reported to date on the role o f

palmitoylation in cellular functions have been limited to expressing

non-acylated mutant forms of proteins in various systems ( 1 1 , 20) .

While this approach does provide useful information, it is limited b y

the need to overexpress the mutant proteins. Furthermore, the loss o f

a cysteine residue, and not the loss of palmitate per se, may impair

the ability of the protein to function properly. For example, Hepler e t

al showed that cysteine residues at the amino terminus of the Gq alpha subunit is important for its interaction with effector an d

receptor molecules, regardless of their state of palmitoylation (31).

Polyunsaturated fatty acids (PUFAs), particularly the n- 3

series, are used clinically as immunosuppressive agents (32) and in

the treatment of various inflammatory diseases (33-36). Recently, it

was reported that polyunsaturated fatty acids inhibit T cell signal

transduction by displacing Fyn and Lck from the detergent resistant

microdomains (37). The inhibitory effects of polyunsaturated fatty

acids were hypothesized to be mediated by modification of DRM

structure and composition.

The prior art is deficient in the lack of specific inhibitors

of Fyn and Lck fatty acylation and protein palmitoylation. The present

invention fulfills this longstanding need and desire in the art.

SUMMARY OF THE INVENTION

The present invention documents the discovery of 2 -

bromopalmitate as an inhibitor of Fyn/Lck fatty acylation in general,

and palmitoylation in particular. 2-bromopalmitate preferentially

blocks palmitoylation of N-terminally palmitoylated proteins, and

inhibits membrane binding and localization of Fyn to detergent

resistant microdomains in COS-1 cells. Moreover, treatment of Jurkat

T cells with 2-bromopalmitate partially blocks localization o f endogenous Fyn, Lck and LAT to rafts, and inhibits T cell receptor-

mediated signaling events including enhanced tyrosine

phosphorylation, calcium flux and activation of MAP kinase. The

identification of 2-bromopalmitate as an inhibitor of fatty acylation o f

Src family kinases serves to provide insight into the role of protein

palmitoylation in Src mediated signal tranduction pathways.

The present invention also demonstrates that

polyunsaturated fatty acids are inhibitors of Fyn palmitoylation, and

discloses a novel mechanism of action by which these agents exert

their immunosuppressive effects.

In one embodiment of the present invention, there is

provided a method of inhibiting Fyn/Lck fatty acylation and protein

palmitoylation in a cell in an individual in need of such treatment

comprising the step of administering to said individual a

pharmacologically effective dose of 2-bromopalmitate.

In another embodiment of the present invention, there is

provided a method of treating an individual having a

pathophysiological state comprising the step of administering to said

individual a pharmacologically effective dose of 2-bromopalmitate.

In yet another embodiment of the present invention, there

is provided a pharmaceutical composition comprising 2 -

bromopalmitate and a pharmaceutically acceptable carrier. Other and further aspects, features, and advantages of th e

present invention will be apparent from the following description o f

the presently preferred embodiments of the invention given for th e

purpose of disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features ,

advantages and objects of the invention, as well as others which will

become clear, are attained and can be understood in detail, more

particular descriptions of the invention briefly summarized above may

be had by reference to certain embodiments thereof which are

illustrated in the appended drawings. These drawings form a part o f

the specification. It is to be noted, however, that the appended

drawings illustrate preferred embodiments of the invention a nd

therefore are not to be considered limiting in their scope.

Figure 1 shows the effect of 2-bromopalmitate on Fyn

fatty acylation and subcellular localization in COS-1 cells. Transfected

cells were preincubated overnight with 100 μM 2-bromopalmitate, a s

described below. Figure 1A : cells were radiolabeled for 4 hours in

the absence (C) or presence (2BP) of 2-bromopalmitate with 1251-

IC13 or 125I-IC16 (top panel), or with Tran35S-label (bottom panel) , lysed and subjected to immunoprecipitation with anti-Fyn antibody.

Lysates were subjected to SDS-PAGE followed by phosphorimaging .

Figure IB : cells were radiolabeled with Tran³⁵S-label for 5 minutes ,

fractionated into particulate P100 (P) fractions and soluble S 100 (S )

fractions by centrifugation at 100,000 x g, and subjected t o

immunoprecipitation, SDS-PAGE and phosphorimaging. Figure 1C:

cells were lysed in buffer containing 1% Triton X-100. Detergent

soluble (S) and resistant (R) fractions were clarified at 100,000 x g

and subjected to immunoprecipitation and SDS-PAGE followed by

immunoblotting with anti Fyn antibodies.

Figure 2 shows the effect of 2-bromopalmitate o n

localization of Fyn(16)-eGFP in COS-1 cells. Figure 2A : cells

transiently expressing Fyn(16)-eGFP were preincubated overnight in

the absence (top) or presence of 100 μM 2-bromopalmitate (middle)

or 100 μM 2-hydroxymyristate (bottom) and examined live by

fluorescence microscopy. Figure 2B: duplicates of the above samples

were subjected to subcellular fractionation into P100 and S I 00

fractions and subjected to immunoblotting with anti-GFP antibody.

2BP: 2-bromopalmitate. 2OH myr: 2 hydroxymyristate.

Figure 3 shows the effect of 2-bromopalmitate o n

subcellular localization of palmitoylated proteins. COS-1 cells were

transiently transfected and treated as follows: Figure 3 A : GαO( l O)-

Fyn. Cells were treated overnight without (C) or with (2BP) 2 - bromopalmitate, labeled for 5 minutes with Tran35S-label and

subjected to cellular fractionation into P100 (P) and S 100 (S )

fractions, followed by immunoprecipitation with anti-Fyn antibody,

SDS-PAGE and phosphorimaging. Figure 3B : GAP43(10)-Fyn. Cells

were treated with or without 2-bromopalmitate as in (Figure 3A) with

the exception of labeling for 2 hours, to allow newly synthesized

protein to reach the plasma membrane. Figure 3C : G2A,C3SFyn-

HRas. Cells were treated with 2-bromopalmitate as in (Figure 3A) .

After fractionation, lysates were subjected to immunoprecipitation

followed by immunoblotting with anti-Fyn antibodies. Figure 3D :

G12V-Hras. Cells were treated with or without 2-bromopalmitate as in

(Figure 3 A) and fractionated, following by immunoblotting with anti

Ras antibody. The faster migrating band represents processed Ras.

The slower migrating band (() represents unprocessed, cytosolic Ras).

Figure 4 shows the effect of 2-bromopalmitate on Fyn

fatty acylation and DRM localization of palmitoylated proteins in

Jurkat T cells. Cells were transfected by electroporation and

preincubated for 3 hours with 100 μM 2-bromopalmitate, a s

described below. Figure 4A : cells were radiolabeled for 4 hours in

the absence (C) or presence (2BP) of 2-bromopalmitate with ^{1 5}I-IC 13

or ¹²⁵I-IC16 (top panel) . Lysates were immunoprecipitated with anti-

Fyn antibody. Bottom panel: to monitor total protein levels, aliquots

from each sample were subjected to immunobloting with anti-Fyn antibody. Figure 4B : Localization to detergent resistant

microdomains. Cells were cultured with 2-bromopalmitate as in

(Figure 4A ), lysed with buffer containing 0.5% Triton X- 100, and

layered on the bottom of a sucrose gradient as detailed below. After

overnight centrifugation, 1 ml fractions were collected and the DRM

localization of endogenous palmitoylated proteins was analyzed b y

SDS-PAGE and immunoblotting with anti-Lck (top), anti-Fyn (middle)

or anti-LAT (bottom) antibodies.

Figure 5 shows tyrosine phosphorylation of signaling

proteins in Jurkat T cells. Figure 5A : Jurkat Cells were treated

without or with 2-bromopalmitate for 3 hours, then either were left

unactivated (-) or activated (+) with OKT3 mAb (0.3 mg/ml) for 3

minutes and lysed. Lysates were subjected to SDS-PAGE followed by

immunoblotting with anti phosphotyrosine antibody (PY99). Figure

5B : cells were treated and lysed as described in (Figure 5 A ). Lysates

were immunoprecipitated with agarose-conjugated phophotyrosine

antibody (PY99) and immunblotted for specific proteins .

Alternatively, lysates were immunoprecipitated for specific proteins

and immunoblotted with antiphosphotyrosine antibody, as depicted in

the figure.

Figure 6 shows calcium mobilization in Jurkat T cells.

Figure 6A: untreated cells were preincubated with Fluo 3 as indicated

below and calcium release was measured by flow cytometry. Top: After obtaining a background fluorescence (Baseline), cells were

activated with OKT3 mAb, and fluorescence was measured for th e

indicated time (post OKT3). Bottom: Quantations of fluorescence

before (left - baseline) and after (right - post OKT3) CD3 stimulation.

Figure 6B: cells were pretreated with 2-bromopalmitate and analyzed

as described in (Figure 6A) .

Figure 7 shows the activation of MAP Kinase in Jurkat

Cells. Cells were treated with 2-bromopalmitate, activated with OKT3

mAb and lysed. Lysates were subjected to SDS-PAGE and immublotted

for active (Top - pERK), or for total (bottom -MAPK) MAP Kinase.

Figure 8 shows the effect of polyunsaturated fatty acids

on Fyn fatty acylation and localization to detergent resistant

microdomains in COS-1 cells. Cells expressing Fyn were preincubated

overnight with 50 μM arachidonic acid (20:4) or eicosapentaenoic

acid (20:5) or left untreated (C), as described below. Figure 8 A

shows the cells were radiolabeled for 4 hours in the absence (C) o r

presence of 20:4 or 20:5 with ¹²⁵I-IC13 or ¹²⁵I-IC16, lysed and

duplicate samples were subjected to immunoprecipitation with anti-

Fyn antibody. Lysates were subjected to SDS-PAGE and

phosphorimaging (top). Bottom: to monitor total protein levels,

aliquots from each sample were subjected to SDS-PAGE followed b y

immunoblotting with anti-Fyn antibody. Figure 8B and Figure 8C :

Quantitation of (Figure 8A) . Effect of polyunsaturated fatty acids on IC13 (Figure 8B) or IC16 (Figure 8C) incorporation into Fyn.

Bars represent the average of 3 sets of duplicate experiments. Figure

8D: DRM localization. Cells were lysed with buffer containing 0.5 %

Triton X-100, and layered on the bottom of a sucrose gradient a s

detailed below. After overnight centrifugations, 1 ml fractions were

collected and the DRM localization of Fyn was analyzed by SDS-PAGE

and immunoblotting with anti-Fyn antibody.

DETAILED DESCRIPTION OF THE INVENTION

This invention describes a palmitate analog, 2 -

bromopalmitate, that effectively blocks Fyn fatty acylation in general,

and palmitoylation in particular. Treatment of COS-1 cells with 2 -

bromopalmitate blocked myristoylation and palmitoylation of Fyn,

and inhibited membrane binding and localization of Fyn to detergent

resistant membranes (DRMs)¹. In Jurkat T cells, 2-bromopalmitate

¹ Abbreviations: DRMs, detergent resistant microdomains, ITAM,

immune-receptor tyrosine-based activation motif; PUFAs,

polyunsaturated fatty acids; GFP, Green Fluorescent Protein; IC13, 13-

iodotridecanoic acid; IC16, 16-iodohexadecanoic acid; NMT, N- blocked localization of the endogenous palmitoylated proteins Fyn,

Lck and LAT to detergent resistant microdomains. This resulted in

impaired signaling through the T cell receptor as evidenced by

reductions in tyrosine phosphorylation, calcium release and activation

of MAP kinase. The polyunsaturated fatty acids arachidonic acid and

eicosapentaenoic acid inhibit Fyn palmitoylation and consequently

block Fyn localization to detergent resistant microdomains.

In accordance with the present invention there may b e

employed conventional molecular biology, microbiology, and

recombinant DNA techniques within the skill of the art. Such

techniques are explained fully in the literature. See, e.g., Maniatis,

Fritsch & Sambrook, "Molecular Cloning: A Laboratory Manual ( 1982) ;

"DNA Cloning: A Practical Approach," Volumes I and II (D.N. Glover ed .

1985); "Oligonucleotide Synthesis" (M.J. Gait ed. 1984); "Nucleic Acid

Hybridization" [B.D. Hames & S.J. Higgins eds. (1985)] ; "Transcription

and Translation" [B.D. Hames & S.J. Higgins eds. (1984)] ; "Animal Cell

Culture" [R.I. Freshney, ed. (1986)] ; "Immobilized Cells And Enzymes"

[IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning"

( 1984) .

myristoyl transferase; PAT, palmitoyl acyl transferase; TCR, T cell

receptor. The present invention is directed to a method o f

inhibiting Fyn/Lck fatty acylation and protein palmitoylation in a cell

in an individual in need of such treatment comprising the step o f

administering to said individual a pharmacologically effective dose o f

2-bromopalmitate. Preferably, the 2-bromopalmitate is administered

in a dose of from about 0.1 mg/kg to about 100 mg/kg of total body

weight of said individual. Administration of 2-bromopalmitate

inhibits N-terminally palmitoylated proteins, myristoylation o f

proteins and T cell signalling events. In one aspect, the individual has

a autoimmune disease. Representative examples of autoimmune

disease include rheumatoid arthritis, Crohn's disease, diabetes ,

multiple sclerosis and systemic lupus erythematosus.

The present invention is directed to a method of treating

an individual having a pathophysiological state comprising the step o f

administering to said individual a pharmacologically effective dose o f

2-bromopalmitate. Preferably, the individual has an autoimmune

disease or abnormal T cell signalling.

The present invention is also directed to a pharmaceutical

compositions containing 2-bromopalmitate. In such a case, the

pharmaceutical composition comprises 2-bromopalmitate and a

pharmaceutically acceptable carrier. A person having ordinary skill in

this art would readily be able to determine, without undue experimentation, the appropriate dosages and routes o f

administration of 2-bromopalmitate.

Compounds of the present invention, pharmaceutically

acceptable salt thereof and pharmaceutical compositions

incorporating such, may be conveniently administered by any of the

routes conventionally used for drug administration, e.g., orally,

topically, parenterally, or by inhalation. 2-bromopalmitate may b e

administered in conventional dosage forms prepared by combining

the compound with standard pharmaceutical carriers according t o

conventional procedures. 2-bromopalmitate may also b e

administered in conventional dosages in combination with a known,

second therapeutically active compound. These procedures may

involve mixing, granulating and compressing or dissolving the

ingredients as appropriate to the desired preparation. It will b e

appreciated that the form and character of the pharmaceutically

acceptable carrier or diluent is dictated by the amount of active

ingredient with which it is to be combined, the route of administration

and other well known variable. The carrier(s) must be "acceptable" in

the sense of being compatible with the other ingredients of th e

formulation and not deleterious to the recipient thereof.

The pharaceutical carrier employed may be, for example,

either a solid or a liquid. Representative solid carriers are lactose,

terra alba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium sterate, stearic acid and the like. Representative liquid carriers

include syrup, peanut oil, olive oil, water and the like. Similarly, th e

carrier may include time delay material well known in the art such a s

glyceryl monosterate or glyceryl disterarate alone or with a wax.

A wide variety of pharmaceutical forms can be employed .

Thus, if a solid carrier is used, the preparation can be tableted, placed

in a hard gelatin capsule in powder or pellet form or in the form of a

troche or lozenge. The amount of solid carrier will vary widely b u t

preferably will be from about 25 mg to about 1 gram. When a liquid

carrier is used, the preparation will be in the form of a syrup,

emulsion, soft gelatin capsule, sterile injectable liquid such as a n

ampule or nonaqueous liquid suspension.

2-bromopalmitate may be administered topically (non-

systemically). This includes the application of 2-bromopalmitate

externally to the epidermis or the buccal cavity and the instillation o f

such a compound into the ear, eye and nose, such that the compound

does not significantly enter the bloodstream. Formulation suitable for

topical administration include liquid or semi-liquid preparations

suitable for penetration through the skin to the site of inflammation

such as liniments, lotions, creams, ointments, pastes and drop s

suitable for administration to the ear, eye and nose. The active

ingredient may comprise, for topical administration from 0.001 % t o

10% w/w, for instance from 1% to 2% by weight of the Formulation. It may however, comprise as much as 10% w/w but preferably will

comprise less than 5% w/w, more preferably from 0.1 % to 1 % w/w o f

the Formulation.

Lotions according to the present invention include those

suitable for application to the skin and eye. An eye lotion may

comprise a sterile aqueous solution optionally containing a

bactericide and may be prepared by methods similar to those for th e

preparation of drops. Lotions or liniments for application to the skin

may include an agent to hasten drying and to cool the skin, such as a n

alcohol or acetone, and/or a moisterizer such as glycerol or an oil

such as castor oil or arachis oil.

Creams, ointments or pastes according to the pre sent

invention are semi-solid formulations of the active ingredient for

external application. They may be made by mixing the active

ingredient in finely divided or powdered form, alone or in solution o r

suspension in an aqueous or non-aqueous fluid, with the aid o f

suitable machinery, with a greasy or non-greasy base. The base may

comprise hydrocarbons such as hard, soft or liquid paraffin, glycerol,

beeswax, a metallic soap, a mucilage, an oil of natural origin such a s

almond, corn, archis, castor, or olive oil; wool fat or its derivatives o r

a fatty acid such as steric or oleic acid together with an alcohol such

as propylene glycol or a macrogel. The formulation may incorporate

any suitable surface active agent such as an anionic, cationic or non- ionic surfactant such as a sorbitan ester or a polyoxyethylene

derivative thereof. Suspending agents such as natural gums, cellulose

derivatives or inorganic materials such as silicaceous silicas, and other

ingredients such as lanolin may also be included.

Drops according to the present invention may comprise

sterile aqueous or oily solutions or suspensions and may be prepared

by dissolving the active ingredient in a suitable aqueous solution of a

bactericidal and/or fungicidal agent and/or any other suitable

preservative, and preferably including a surface active agent. The

resulting solution may then be clarified by filtration, transferred to a

suitable container which is then sealed and sterilized by autoclaving .

Alternatively, the solution may be sterilized by filtration and

transferred to the container by an aseptic technique. Examples o f

bactericidal and fungicidal agents suitable for inclusion in the drop s

are phenymercuric nitrate or acetate (-0.002%), benzalkonium

chloride (-0.01 %) and chlorhexidine acetate (-0.01 %). Suitable

solvents for the preparation of an oily solution include glycerol,

diluted alcohol and propylene glycol.

2-bromopalmitate may be administered parenterally, i.e. ,

by intravenous, intramuscular, subcutaneous, intranasal, intrarectal,

intravaginal or intraperitoneal administration. The subcutaneous and

intramuscular forms of parenteral administration are generally

preferred. Appropriate dosage forms for such administration may b e prepared by conventional techniques. Compounds may also b e

administered by inhalation, e.g., intranasal and oral inhalation

administration. Appropriate dosage forms for such administration,

such as aerosol formulation or a metered dose inhaler may b e

prepared by conventional techniques well known to those having

ordinary skill in this art.

For all methods of use disclosed herein for 2 -

bromopalmitate, the daily oral dosage regiment will preferably b e

from about 0.1 to about 100 mg/kg of total body weight. The daily

parenteral dosage regimen will preferably be from about 0.1 to about

100 mg/kg of total body weight. The daily topical dosage regimen will

preferably be from about 0.01 to about 1 g, administered one to four,

preferably two to three times daily. It will also be recognized by o ne

of skill in this art that the optimal quantity and spacing of individual

dosages of 2-bromopalmitate, or a pharmaceutically acceptable salt

thereof, will be determined by the nature and extent of the condition

being treated and that such optimums can be determined b y

conventional techniques.

Suitable pharmaceutically acceptable salts are well known

to those skilled in the art and include basic salts of inorganic and

organic acids, such as hydrochloric acid, hydrobromic acid, sulphuric

acid, phophoric acid, methane sulphonic acid, ethane sulphonic acid,

acetic acid, malic acid, tartaric acid, citric acid, lactic acid, oxalic acid, succinic acid, fumaric acid, maleic acid, benzoic acid, salicylic

acid, phenylacetic acid and mandelic acid. In addition,

pharmaceutically acceptable salts of 2-bromopalmitate may also b e

formed with a pharmaceutically acceptable cation, for instance, if a

substituent group comprises a carboxy moiety. Suitable

pharmaceutically acceptable cations are well known in the art a nd

include alkaline, alkaline earth ammonium and quaternary ammonium

cations .

Autoimmune diseases are characterized by immune cell

destruction of self cells, tissues and organs. Representative examples

of such autoimmune diaseases are rheumatoid arthritis diabetes ,

multiple sclerosis, Crohn's disease and systemic lupus erythematosus.

2-bromopalmitate has potential uses in other immune cell

functions. For example, the IgE receptor uses palmitoylated Lyn

(another Src kinase family member) to signal for the inflammatory

response and Lyn must be palmitoylated and in membrane rafts in

order to function. Thus, 2-bromopalmitate could be used as an anti-

inflammatory agent.

The following examples are given for the purpose o f

illustrating various embodiments of the invention and are not me ant

to limit the present invention in any fashion. EXAMPLE 1

Cell Culture and Transfections

COS-1 cells were maintained and transfected as previously

described (9). Transfection with FUGENE™ 6 Transfection Reagent

(Boehringer Mannheim) was carried out according to th e

manufacturer' s instructions. Jurkat T cells were maintained in RPMI

1640 supplemented with 10% FBS, 100 μg of penicillin and

streptomycin per ml and 100 μg of sodium pyruvate and glutamine

per ml. Cells were transfected by electroporation as previously

described (38).

EXAMPLE 2

Antibodi es

Monoclonal anti-Fyn and anti-Lck antibodies used for

Western blotting were purchased from Transduction Laboratories

(Lexington, KY). The rabbit polyclonal antiserum to Fyn used for

immunoprecipitation was described previously (13). Monoclonal

anti-PLCγ-1 and rabbit polyclonals anti-LAT, anti PI3 kinase, anti-Vav

and anti-ZAP-70 were purchased from Upstate Biotechnology (Lake

Placid, NY). Monoclonals anti-Hras, anti-p-ERK anti-P-Tyr (PY99) an d agarose-conjugated PY99 were purchased from Santa Cruz

Biotechnology (Santa Cruz, CA). Anti MAPK antibody was purchased

from New England Biolabs (Beverly, MA). Fluorescein (FITC)-

conjugated Goat Anti-Mouse secondary antibody was purchased from

Jackson ImmunoResearch Laboratories (West Grove, PA). Anti GFP

antibody was purchased from CLONTECH Laboratories (Palo Alto, CA).

EXAMPLE 3

Fyn Chimeras

The Fyn Chimeras Gα₀(10)Fyn and GAP43(10)-Fyn have

been described previously (12). G2A, C3S Fyn-HRas was constructed

as follows. An antisense oligonucleotide primer was designed th at

corresponded to the last 6 amino acids of Fyn fused in frame to the C-

terminal 12 amino acids of H-Ras, followed by a stop codon, a Sail site

and a G/C clamp. A sense primer that began 57 bases upstream of a

unique Bglll site in Fyn was constructed. These two primers were u s ed

in a PCR reaction to amplify a fragment containing the C-terminal

region of Fyn fused to the H-Ras tail. The PCR reaction product was

cut with Bglll and Sail and used to replace the corresponding region o f

Fyn in G2AFyn/pSP65. G2A Fyn HRas/pSP65 was then digested with

Ncol and Bglll to remove the 5' coding region of Fyn, and ligated to a 1.7 kb Ncol/Bglll fragment from another Fyn clone containing th e

G2A, C3S mutation. G2A, C3S Fyn-HRas/pSP65 was digested with EcoRI

and Sail and ligated into EcoRI and Sail cut pCMV5. The construct was

verified by DNA sequencing prior to use in transfections.

EXAMPLE 4

Cell Labeling

The syntheses of 13- [¹²⁵I]-iodotridecanoic acid (IC 13)or

16- [ ¹²⁵I]-iodohexanoic acid (IC16) were carried out as described

previously (39). Cell labeling was carried out as described ( 1 2 , 1 3 )

with modifications. Briefly, each 60 mm plate of Fyn transfected COS-

1 cells was incubated O/N in DMEM containing 2.5% FBS, 0.5 %

defatted BSA (Sigma) with or without 100 μM 2-bromopalmitate or 5 0

μM polyunsaturated fatty acid. Prior to labeling, the cells were

incubated for lhr with 1 ml of DMEM containing 2% dialyzed FBS,

then labeled for 4 hours with 25-50 μCi IC13 or IC16 in DMEM

containing 2% dialyzed FBS, 0.5% defatted BSA with or without 2 -

bromopalmitate or polyunsaturated fatty acid. Labeled cells were

washed three times with cold STE (100 mM NaCl, 10 mM Tris pH 7.4, 1

mM EDTA) and lysed in 0.6 ml of cold RIPA buffer containing protease

inhibitors (10 μg/ml each of benzamidine, AEBSF, TPCK andTLCK, 1 .5 μg/ml each of Leupeptin, Pepstatin A and Aprotinin). Lysates were

clarified at 100,000 x g for 15 min at 4°C in a Beckman TL- 100

ultracentrifuge. Lysates were immunoprecipitated with rabbit anti-Fyn

antibody and protein A-agarose. Immunoprecipitates were washed

three times with cold RIPA buffer and suspended in IX sample buffer

containing 100 mM dithiothreitol and subjected to SDS-PAGE. Gels

were dried between cellophane and analyzed by phosphorimaging

after 12-36 hours exposure. Experiments in Jurkat T cells were

carried out according to the above protocol using 2x l 0⁷ cells p e r

experiment, and reducing the incubation time with 2-bromopalmitate

to 3-4 hours.

EXAMPLE 5

Sπhcellnlar Cell Fractionati n

Each 60 mm plate of Fyn transfected COS-1 cells was

starved for 1 hour in DMEM containing 2.5% FBS and 0.5 % defatted

BSA with or without 100 μM 2-bromopalmitate. After overnight

culture at 37°C, the cells were fractionated into P100 and S I 00

fractions, immunoprecipitated with anti-Fyn antibody, subjected t o

SDS-PAGE, and immunoblotted with anti-Fyn antibody as previously

described (9, 12, 13). Analysis of the G2A,C3S Fyn-HRas chimera was performed

according to the procedure described above. Analysis of the G12V

HRas construct was according to the procedure described above,

except that the samples were not immunoprecipiated, and were

subjected to immunoblotting with anti-HRas antibody.

For analysis of newly synthesized Fyn, cells were cultured

overnight as descibed above, then starved for 1 hr in DMEM minus

methionine and cysteine containing 2% dialyzed FBS, 0.5% defatted

BSA with or without 100 μM 2-bromopalmitate. Cells were labeled for

5 minutes with Trans³⁵S-Label (ICN, Irvine, CA), then fractionated a s

described above. Gels were treated for 20 minutes with 1M salicylic

acid prior to drying. The Gαo(10)-Fyn chimera was labeled for 5 min

and GAP-43 Fyn chimera was labeled for 2 hours and fractionated

according to the above conditions.

EXAMPLE 6

Tmmunofluorescence microscopy

COS-1 cells were transfected with a Fyn(16)-eGFP construct

(12) and seeded onto 25 -mm glass coverslips 2 days prior to th e

experiment. Cells were treated overnight with or without 2 -

bromopalmitate as described above. Plates were washed with PBS and coverslips were mounted onto glass slides in PBS and observed with a

40X and 100X oil immersion lens on a Zeiss Axiophot 2 microscope

and photographed with Kodak TMAX 400.

EXAMPLE 7

T cell activation and phosphotyrosine immunohlots

Jurkat T cells (2x l 0⁶- l x l 0⁷) were centrifuged at 1 ,000 x g

for 5 minutes, rinsed with RPMI, resuspended in RPMI supplemented

with 2% dialyzed FBS, 0.5% defatted BSA, and incubated with o r

without 100 μM 2-bromopalmitate at 8x l 0⁵ cells/ml for 3 hours. The

cells were centrifuged, washed with RPMI and resuspended in RPMI a t

I x l 0⁷- l x l 0⁸ cells/ml. The cells were then activated with anti-CD3

OKT3 mAb (0.3 mg/ml) for 3 min at 37°C, quickly spun down, washed

once with cold RPMI and once with cold STE, and lysed in RIPA.

Samples were solubilized in IX sample buffer containing 5% β-

mercaptoethanol and subjected to SDS-PAGE, followed by

immunoblotting with anti-phosphotyrosine antibody (PY99). For

analysis of specific proteins, RIPA lysates were immunoprecipitated

with the specific antibodies O/N, and immunoblotted for

phosphotyrosine. Alternatively, proteins were immunoprecipitated with agarose-conjugated anti phosphotyrosine antibody and blotted

for the specific proteins.

EXAMPLE S

Isolation of DRMs

Isolation of Triton X-100 resistant and soluble fractions

was carried out as described previously (12). Isolation of detergent

resistant microdomains by sucrose gradients were carried out a s

follows (19): Jurkat cells (5 x l 0⁷) were treated with 2 -

bromopalmitate, activated with OKT3 mAb as described above, an d

lysed in 1 ml lysis buffer (25 mM MES pH 6.5, 150 mM NaCl, 0.5 %

Triton X-100, 1 mM Na₃V0₄) supplemented with protease inhibitors

for 30 minutes at 0°C. After homogenizing 10 times with a loose fit

Dounce homogenizer, lysates were mixed with 1 ml 85% sucrose in

MBS (25 mM MES pH 6.5, 150 mM NaCl), and overlayered with 6 m l

30% sucrose in MBS, then with 4 ml 5% sucrose in MBS. Following

centrifugation for 16 hours at 145,000 g in an SW40 rotor, 1 m l

fractions were collected and analyzed by PAGE and immunoblotting

with anti -Fyn, anti-LAT, or anti-Lck antibodies.

For isolation of detergent resistant microdomains in PUFA-

treated COS-1 cells, a confluent 100 mm plate of COS-1 cells transiently transfected with Fyn cDNA was washed with STE an d

subjected to the same procedure described above. Fractions were

analyzed for the presence of Fyn by immunoblotting.

EXAMPLE 9

Calcium Mobilization Assay

Jurkat cells were treated with 2-bromopalmitate a s

described above. The cells (2x l 0⁵) were collected by centrifugation

and resuspended at 2xl0⁶ cells/ml in RPMI containing in 50 μM fluo-3

with or without 100 μM 2-bromopalmitate for 30 minutes at ro o m

temperature. Cells loaded with fluo-3 were then collected b y

centrigation, washed with Hank's Buffered Saline Solution (5.4 m M

KC1, 0.3 mM Na₂HP0₄, 0.4 mM KH₂P0₄, 4 mM NaHCO,, 1.3 mM CaCl₂,

0.5 mM MgCl₂, 0.6 mM MgS0₄, 137 mM NaCl, 5.6 mM glucose, 20 m M

Hepes pH 7.4) and resuspended in the same buffer at 5x l 0⁵ cells/ml

at 37°C. To initiate calcium flux, the cells were activated with OKT3

antibody as described above, and analyzed for free calcium ion by

measurement of fluo-3 fluoresence emission by flow cytometry.

For analysis of CD3 positive cells, l x l O⁶ cells were

centrifuged, washed and resuspended in 100 μl ice cold phosphate

buffered saline (PBS - 136 mM NaCl, 2.6 mM KC1, 4.3 mM Na₂HP0₄, 1 .5 mM KH₂P0₄) containing 1% FBS. OKT3 antibody was added to a final

concentration of 0.3 mg/ml. Following 30 minutes on ice, the cells

were washed twice with PBS/1% FBS and incubated at 0°C for a n

additional 30 minutes with an FITC-conjugated Goat Anti-Mouse

secondary antibody (1 :20 dilution). The cells were washed twice,

resuspended in PBS/1 % FBS, and subjected to FACS analysis.

EXAMPLE 10

Activation of MAP kinase

Jurkat cells were treated with 2-bromopalmitate and

activated as described above. Lysates were analyzed for the presence

of active MAP kinase by immunoblotting with a pERK antibody, and for

total MAP kinase by immunoblotting with an anti MAPK antibody.

EXAMPLE 11

Identificati n ol 2-bromopalmitate as an inhibitor of F_yn Fatty

Acylation

A number of palmitic acid analogs were screened for their

ability to inhibit Fyn palmitoylation. COS-1 cells were transfected with cDNA encoding Fyn. Three days after transfection, the cells were

labeled with either [³⁵S]-methionine, 13- [¹²⁵I]-iodotridecanoic acid

(IC13), an iodinated myristate analog, or 16- [^{l 5}I]-iodohexanoic acid

(IC16), an iodinated palmitic acid analog (39), in the presence o r

absence of nonradioactive palmitate analogs. Cells were lysed,

immunoprecipitated with anti-Fyn antibody, and analyzed by SDS-PAGE

and phosphorimaging. 2-Bromo-palmitate efficiently inhibited Fyn

fatty acylation (Figure 1A). When normalized for total protein levels,

70% of Fyn myristoylation and over 90% of Fyn palmitoylation was

inhibited in the presence of 2-bromopalmitate. Treatment of cells

with other analogs, including 2-hydroxypalmitate, palmitoleic acid and

16-hydroxypalmitate had no effect (data not shown).

EXAMPLE 12

Effect of 2-hromo-palmitate on subcellular localization of Fyn

Newly synthesized Fyn becomes plasma membrane b ou nd

within 5 minutes after biosynthesis (12). The rapid membrane

targeting is dependent on dual fatty acylation of Fyn with myristate

and palmitate. The effect of 2-bromopalmitate on the ability of newly

synthesized Fyn to localize to membranes was next examined.

Transfected COS-1 cells were incubated for 12- 16 hours with o r without 100 μM 2-bromopalmitate. Cells were then metabolically

labeled with [³⁵S]-methionine for 5 minutes followed by fractionation

into cytosolic (S 100) or membrane (P100) fractions.

As depicted in Figure IB, in untreated cells, 90% of th e

labeled Fyn was membrane bound. In cells treated with 2-bromo¬

palmitate, 50% of Fyn remained cytosolic, demonstrating the ability o f

the reagent to partially block membrane association of newly

synthesized Fyn. The effect of 2-bromo-palmitate on membrane

localization of steady-state Fyn was also examined. Transfected cells

were treated with 2-bromo-palmitate as described above, then

fractionated, immunoprecipitated with anti-Fyn followed by Western

blotting with anti-Fyn antibody. The effect of 2-bromo-palmitate o n

membrane localization of steady-state Fyn was identical to the effect

on newly synthesized Fyn, with 50% of the Fyn protein fractionating in

the cytosol (data not shown). These results mimic the fractionation

pattern of a non-palmitoylated Fyn mutant (C3,6SFyn), and of a non-

myristoylated Fyn mutant (G2AFyn), and strongly suggest that th e

redistribution of Fyn observed in 2-bromopalmitate treated cells is

due to inhibition of Fyn fatty acylation (13).

Previous experiments have shown that following rapid

membrane binding of newly synthesized Fyn, there is a slower

partitioning of Fyn (10-20 minutes) to regions of the plasma

membrane that are resistant to Triton X-100 extraction at 4°C ( 1 2 ) . Therefore the effect of 2-bromopalmitate on the localization of Fyn t o

Triton X-100 insoluble fractions was examined. Transfected COS-1

cells were left untreated or treated with 2-bromopalmitate a s

described above. Cells were extracted with buffer containing 1 %

Triton X-100, and samples were subjected to immunoprecipitation and

Western blotting with anti-Fyn antibodies.

As depicted in Figure 1C, in untreated cells the majority o f

Fyn was associated with detergent resistant fractions (R), in agreement

with previous experiments (12). In comparison, Fyn in 2-bromo-

palmitate treated cells was mostly soluble, demonstrating the ability

of 2-bromopalmitate to partially block association of Fyn with

detergent resistant membrane subdomains.

To investigate the effect of 2-bromopalmitate on the

intracellular localization of Fyn more precisely, COS-1 cells expressing

a Fyn(16)-eGFP construct were examined by immunofluorescence .

This construct contains the first 16 amino acids of Fyn fused in frame

to eGFP; the chimera is targeted to the plasma membrane and

detergent resistant microdomains (12). Cells were cultured with n o

treatment, with 2-bromopalmitate or with 2-hydroxymyristate, a

known myristoylation inhibitor (40,41), as described above, and were

examined live by fluorescence microscopy.

Figure 2A shows that Fyn(16)-eGFP is primarily distributed

in the plasma membrane (Top). In contrast, cells treated with 2 - bromopalmitate (middle) and 2-hydroxymyristate (bottom) showed

reduced plasma membrane staining, and instead exhibited a distinct

perinuclear staining, presumably representing cytosolic and

intracellular membrane distribution. Analysis of the subcellular

localization of Fyn(16)-eGFP in the presence of 2-bromopalmitate and

2-hydroxymyristate was performed as described above for steady-

state Fyn. As depicted in Fig 2B, in the absence of treatment, 80% o f

Fyn(16)-eGFP Fyn was membrane bound, whereas in cells treated with

2-bromopalmitate and 2-hydroxymyristate, 65% and 78% of Fyn( 16)-

eGFP were cytosolic, respectively. These results strengthen the

hypothesis that fatty acylation of Fyn is important for the proper

localization of the protein within the cell.

EXAMPLE 1

Effect of 2-bromo-palmitate on membrane localization of o th er

palmitoylated proteins

Palmitoylation has been shown to occur on a wide variety

of cellular proteins and the sites of palmitoylation can be quite

diverse. Whether 2-bromopalmitate can inhibit palmitoylation and

membrane binding of other palmitoylated proteins was next

examined. Three representative palmitoylated protein sequences were chosen as model systems. The Gαo subunit of the heterotrimeric Go

protein is myristoylated and palmitoylated on an N-terminal Gly-Cys

motif, similar to Fyn and Lck (42,43). The neuronal protein GAP43

(neuromodulin) is palmitoylated near the N-terminus at cysteines 3

and 4, but is not myristoylated (44). Finally, the oncogenic H-Ras

protein is palmitoylated just upstream of the C-terminal CAAX box

(45 ) .

Each of these three sequences was appended onto the Fyn

protein. Gαo(10)-Fyn and GAP43(10)-Fyn are chimeric constructs

with the first 10 amino acids of Gαo or GAP-43, respectively, in place

of the first 10 amino acids of wt Fyn. These constructs have been

previously described (12, 13). In addition, the H-Ras tail was fused t o

the C-terminus of a non-acylated Fyn mutant (G2A,C3SFyn-HRas). This

construct contains full length Fyn with mutations in the N-terminal

myristoylation and palmitoylation sites, but with the C-terminus of H-

Ras available for prenylation and palmitoylation.

Finally, an oncogenic G12V full length H-Ras construct was

tested. As depicted in Figures 3A and B, 2-bromopalmitate inhibited

membrane binding of the two N-terminal palmitoylated proteins ,

GαO(10)-Fyn and GAP43(10)-Fyn, to the same extent as wt Fyn. In

contrast, 2-bromopalmitate had only a minimal effect on th e

membrane localization of the two Ras constructs, inducing a 10-20%

shift from membrane to cytosol (Figure 3C and D). The G12V H-Ras construct migrates as a doublet on a gel. The slower migrating form

represents the non-processed cytosolic form of H-Ras, and the faster

migrating form represents the processed Ras, which is membrane

bound. Thus 2-bromopalmitate appears to possess some specificity

towards inhibiting membrane localization of N-terminal palmitoylated

proteins, in comparison to a protein palmitoylated near the C-

terminus. The absolute sequence surrounding the palmitoylated

cysteine residue did not seem to be important, as the GAP43(10)-Fyn

construct was affected to the same extent as wt Fyn and Gαo( 10)-Fyn.

EXAMPLE 14

2-bromopalmitate inhibits fatty acylation and localization of

palmitoylated proteins to DRMs in T cells

The Src family kinases Fyn and Lck play critical roles in T

cell receptor (TCR) mediated signaling. Palmitoylation of Fyn and Lck

has been shown to be essential for localization to detergent resistant

microdomains in T cells, and localization to detergent resistant

microdomains is required for efficient signaling by the activated TCR

(11). The ability of 2-bromopalmitate to inhibit Fyn fatty acylation

was tested in Jurkat T cells. Cells were transfected by electroporation

with cDNA encoding Fyn. Two days after transfection, cells were labeled with IC13 or IC16 as described above for COS cells. Total

protein levels were monitored by immunoprecipitation followed by

immunoblotting with anti-Fyn antibody.

As depicted in Figure 4A, myristoylation a n d

palmitoylation were inhibited by 75% and 90% respectively in 2 -

bromopalmitate treated cells, relative to untreated controls ,

demonstrating the ability of the reagent to inhibit Fyn fatty acylation

in Jurkat T-cells.

The ability of 2-bromopalmitate to inhibit localization o f

palmitoylated proteins to detergent resistant microdomains in

activated Jurkat cells was examined next. Cells were either left

untreated or treated with 100 μM 2-bromopalmitate for 3 hours ,

washed, resuspended in serum-free media, and the TCR was activated

with OKT3 mAb. Activated cells were extracted with Triton X- 100

containing buffer. Lysates were layered on the bottom of a sucro se

gradient as described above, and subjected to overnight

ultracentrifugation. Rafts, which contain detergent resistant

microdomains, were collected at the 35%/5% sucrose interface

(Figure 4B, fractions 8-11), whereas fractions 1-4 represented the

Triton soluble fractions (Fig 4B). Each fraction was analyzed b y

immunoblotting with specific antibodies. 59% of Lck was found in th e

rafts in control cells, compared with 19% in cells treated with 2 - bromopalmitate (Top). Likewise, the amount of Fyn found in the rafts

was 88% in control cells, but only 59% in treated cells (middle).

The effect of 2-bromopalmitate on localization of LAT,

another palmitoylated protein in T cells that has been shown to b e

localized to plasma membrane rafts ( 10,20) was also examined. The

majority of LAT (71 %) was found in the detergent resistant

microdomains in control cells, whereas in cells treated with 2 -

bromopalmitate only 41 % of the protein remained associated with this

fraction (bottom). These data indicate that in Jurkat T-cells, 2 -

bromopalmitate is able to partially block association of endogenous

Fyn, Lck and LAT with rafts.

EXAMPLE 15

Effect of 2-bromopalmitate on tyrosine phosphorylation in T cells

One of the earliest signaling events after T cell receptor

activation is the tyrosine phosphorylation of multiple intracellular

proteins. The initial phosphorylation events are mediated by

activation of Src family kinases. Whether 2-bromopalmitate c an

interfere with signaling through the T cell receptor was examined b y

analyzing the ability of the compound to block tyrosine

phosphorylation in activated T cells. Jurkat cells were incubated with 2-bromopalmitate an d

activated with OKT3 anti-CD3 antibody. Cell lysates were analyzed by

immunoblotting with anti phosphotyrosine antibodies. In control

cells, stimulation with OKT3 antibodies induced tyrosine

phosphorylation of multiple proteins (Fig 5 A). In cells treated with 2 -

bromopalmitate, the phosphorylation of several proteins was

significantly inhibited. The most dramatic effect was on a 36 kD a

protein, which represents LAT (see below).

In order to identify the individual proteins whose tyrosine

phosphorylation is affected by 2-bromopalmitate, lysates were

immunoprecipitated with a panel of specific antibodies, an d

immunoblotted for phosphotyrosine. Alternatively, lysates were

immunoprecipitated with an antiphosphotyrosine antibody, and

blotted with antibodies to specific proteins.

Figure 5B shows that in 2-bromopalmitate treated cells,

CD3-mediated tyrosine phosphorylation of LAT was inhibited

completely. PLC-γl phosphorylation was inhibited by 70%, Vav

phosphorylation was inhibited by 40%, ZAP-70 phosphorylation was

inhibited by 50%, and PI3K phosphorylation was inhibited by 50%. Low

to moderate increases in tyrosine phosphorylation were observed in

the presence of 2-bromopalmitate alone for some of the proteins .

The reason for this basal activation is unknown. To verify that the observed inhibition of T cell receptor-

mediated tyrosine phosphorylation was not a result of toxicity effects

of 2-bromopalmitate, aliquots of each sample were analyzed by

immunoblotting with anti-LAT and anti-actin antibodies. The levels o f

LAT and actin were not affected by 2-bromopalmitate (data no t

shown). Thus 2-bromopalmitate was able to inhibit signaling through

the T cell receptor, as assayed by its ability to inhibit tyrosine

phosphorylation of key substrate proteins.

EXAMPLE 16

2-bromopalmitate inhibits calcium mobilization in T cells

T cell receptor activation results in increased Ca⁺⁺

mobilization in stimulated T cells. The increase in Ca⁺⁺ flux is

mediated by tyrosine phosphorylation and activation of PLC-γl. PLC-γl

hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol

1 ,4,5-triphosphate (IP3), which promotes calcium release from the IR

(46). The ability of 2-bromopalmitate to interfere with calcium

release was assayed next by flow cytometry.

Jurkat cells were incubated with or without 2 -

bromopalmitate, washed and stained with the fluorescent dye fluo-3

(50 μM). Cells were activated with OKT3 antibody and analyzed b y flow cytometry. Figure 5 shows that in response to T cell receptor

activation, T cells treated with 2-bromopalmitate were severely

impaired in their ability to release calcium compared with control

cells (Figures 6A and 6B). Quantitation of the data revealed th at

calcium flux shut down almost completely in the presence of 2 -

bromopalmitate. No effect of 2-bromopalmitate on cells incubated in

the absence of OKT3 was noted.

To ensure that the observed inhibition of calcium flux was

not due to a decreased expression of CD3 in 2bromo-palmitate treated

cells, Jurkat cells were incubated with OKT3 antibody at 0°C, followed

by incubation with a Fluorescein (FITC) conjugated Goat Anti-Mouse

secondary antibody. The percentage of CD3 positive cells was

analyzed by FACS analysis. Over 95% of the cells were found to b e

positive for CD3 in control and 2-bromopalmitate treated cells (data

not shown).

EXAMPLE 17

2-bromopalmitate inhibits MAP Kinase Activation

One of the proximal events following T cell receptor

engagement is activation of the MAP Kinase pathway. The ability of 2 -

bromopalmitate to inhibit activation of MAP kinase was examined in Jurkat cells. Cells were cultured with or without 2bromo-palmitate

and activated as described above. Cell lysates were subjected to SDS-

PAGE and immunoblotted with anti-active MAPK kinase (pERKl). As

depicted in Figure 7, 2-bromopalmitate inhibited the activation o f

MAPK kinase by 70%. The levels of total MAPK kinase remained

unchanged (Fig 7).

EXAMPLE 18

PTTFAs inhibit Fyn palmitoylation and localization to DRMs in COS-1

cells

The data reported above identify 2-bromopalmitate as a n

inhibitor of protein fatty acylation and T cell receptor-mediated

signaling. Whether other fatty acids, particularly long chain

unsaturated compounds, might also interfere with protein fatty

acylation was examined next. It has recently been reported that

polyunsaturated fatty acids inhibit T cell signal transduction b y

displacing Src kinases Fyn and Lck from the detergent resistant

microdomains (37). This inhibition was speculated to be due t o

polyunsaturated fatty acid-induced disruption of DRM structure an d

composition. Based on these results with 2-bromopalmitate,

polyunsaturated fatty acid-induced displacement of Fyn/Lck from th e detergent resistant microdomains may actually be due to alterations

of S-acylation.

To test this hypothesis, Fyn transfected COS-1 cells were

incubated O/N with or without 50 μM arachidonic acid (20:4) o r

eicosapentaenoic acid (20:5), then labeled with IC13 or IC16. Total

protein levels were monitored by immunoblotting aliquots of each

sample with anti-Fyn antibody (Fig 8A, lower panel).

As depicted in Fig 8A, Fyn myristoylation was not affected

by polyunsaturated fatty acid treatment to a significant effect (Fig 8B) .

On the other hand, Fyn palmitoylation was affected quite dramatically.

Arachidonic acid inhibited incorporation of IC16 into Fyn by 55 % ,

and eicosapentaenoic acid by 65% (Fig 8C). These reductions in

palmitate incorporation correlate well with the previously reported

observation that 20:5 is slightly more potent than 20:4 in inhibiting T

cell signaling and in displacing Fyn and Lck from detergent resistant

microdomains (37). In the same report, the polyunsaturated fatty

acid docosahexaenoic acid (22:6) was less active than 20:4 and 20 : 5 ,

and only moderately inhibited Fyn/Lck displacement from detergent

resistant microdomains and TCR signaling. In agreement with these

findings, 22:6 was 10-20% less potent than 20:4 and 20:5 at inhibiting

Fyn palmitoylation (data not shown).

Whether 20:4 and 20:5 inhibited localization of Fyn t o

detergent resistant microdomains in COS-1 cells was examined next. Cells were treated with or without polyunsaturated fatty acids a s

described above and layered on the bottom of a sucrose gradient a s

described above. Fractions were collected and analyzed by

immunoblotting with anti-Fyn antibody.

As depicted in Figure 8D, in untreated cells, 30% of Fyn

localized to detergent resistant microdomains, in agreement with

previous findings (47). Treatment with polyunsaturated fatty acids

markedly reduced the ability of Fyn to localize to detergent resistant

microdomains, with only 16% of Fyn found in detergent resistant

microdomains in 20:4 treated cells, and 5.3% in 20:5 treated cells .

These finding clearly demonstrate that the displacement of Fyn from

detergent resistant microdomains is likely due to a polyunsaturated

fatty acid-induced reduction in Fyn palmitoylation.

Di scussion

2-bromopalmitate inhibits Fyn fatty acylation in COS-1 cells

The ability of Src family members Fyn and Lck t o

participate in signaling through the T-cell receptor is critically

dependent on their fatty acylation with myristate and palmitate. In

this study, 2-bromopalmitate was identified as an inhibitor of Fyn fatty

acylation and membrane targeting. This was accomplished by screening palmitate analogs for their ability to inhibit incorporation o f

myristate and palmitate into Fyn in transiently transfected COS-1 cells.

This inhibition results in decreased membrane binding and

localization to detergent resistant microdomains. Moreover, 2 -

bromopalmitate inhibits fatty acylation and localization of Fyn, Lck

and LAT to detergent resistant microdomains in Jurkat T cells .

Consequently, this results in impaired signaling through the T-cell

receptor, as shown by a reduction in tyrosine phosphorylation,

calcium flux and activation of the MAP kinase pathway. Furthermore,

polyunsaturated fatty acids arachidonic acid (20:4) an d

eicosapentaenoic acid (20:5) are specific inhibitors of Fyn

palmitoylation and localization to detergent resistant microdomains

in COS-1 cells. This may account for the ability of these compounds

to inhibit T cell signaling as reported previously (37), and may be a

mechanism by which these agents exert their immunosuppressive an d

anti-inflammatory effects.

Protein palmitoylation occurs within an N-terminal myr-

gly-cys motif, and that this event is dependent on myristoylation

(12, 13). The ability of 2-bromopalmitate to partially inhibit

myristoylation likely accounts for some of the reduction i n

palmitoylation. However, the extent of inhibition by 2 -

bromopalmitate on Fyn palmitoylation is always greater than that o n

myristoylation, implying that 2-bromopalmitate has additional, direct effects on palmitoylation (Fig 1A, 4A). A direct effect o n

palmitoylation is also supported by the observation that 2 -

bromopalmitate inhibits membrane localization of a GAP43(10)-Fyn

construct, which is palmitoylated but not myristoylated (Fig 3B) .

Furthermore, 2-hydroxymyristate, a known inhibitor of myristoylation

(40,41 ), inhibits membrane localization of Fyn and Fyn(16)-eGFP to a

greater extent than 2-bromopalmitate (Fig 2B). This implies that 2 -

bromopalmitate treated cells contain a population of myristoylated,

non-palmitoylated Fyn that has a greater affinity for membranes th an

non-acylated Fyn. Finally, the membrane localization of Fyn in th e

presence of 2-bromopalmitate resembles that of the myristoylated,

non-palmitoylated C3,6S Fyn mutant previously studied (13). Thus,

2-bromopalmitate is an inhibitor of protein fatty acylation with s ome

specificity for palmitoylation.

Two possible mechanisms may account for the inhibitory

effect of 2-bromopalmitate on palmitoylation. One possibility is that

2-bromopalmitate binds to PAT, but because of the steric bulk of the

bromine, it cannot be transferred to the acceptor protein.

Alternatively, 2-bromopalmitate may serve as a substrate for PAT. In

this case, Fyn would be S-acylated with 2-bromopalmitate, b u t

hydrophilic and steric effects of the bromine atom would reduce th e

protein's affinity for membranes. In the absence of a radiolabeled form of 2-bromopalmitate, it is not possible at this point t o

distinguish between these two possibilities.

2-bromopalmitate inhibits Fyn fatty acylation and signaling in Jurkat T

cells

The experiments depicted in Figure 4 indicate that 2 -

bromopalmitate inhibits Fyn fatty acylation and localization t o

detergent resistant microdomains in Jurkat T cells. As a result, there

is a marked reduction in tyrosine phosphorylation of key signaling

molecules in CD3 stimulated cells (Fig 5), suggesting that signaling via

the TCR is impaired. Interestingly, some proteins show an increase i n

the level of phosphorylation in 2-bromopalmitate treated cells a s

compared to control cells, even in the absence of CD3 stimulation.

While the basis for this basal activation is unknown, it does not seem

to be related to TCR activation, since there is no effect on Ca⁺² flux o r

activation of MAP kinase pathway in unstimulated 2-bromopalmitate

treated cells. If this basal activation is taken into account, then th e

reduction of tyrosine phosphorylation on the signaling molecules

examined ranges from 70-100% (Fig 5B). PUFAs inhibit Fyn palmitoylation and localization to DRMs in COS-1

cell s

Polyunsaturated fatty acids modulate immune responses by

affecting T cell function (48). Therefore these agents (particularly th e

n-3 series) have found clinical applications in the treatment of various

inflammatory diseases such as rheumatoid arthritis and Crohn' s

disease relapses (33,35,36) and as immunosuppressive agents ( 32) .

Despite the broad clinical use of polyunsaturated fatty acids, the

mechanism of polyunsaturated fatty acid-induced T cell inhibition h ad

not been elucidated. Recently, it was reported that th e

polyunsaturated fatty acid-induced inhibition of T cell activation is

due to displacement of Src family kinases from the cytoplasmic layer

of the detergent resistant microdomains (37). This displacement was

hypothesized to be mediated by modification of the DRM structure

and composition. Here it was shown that the exclusion of Src family

kinase Fyn from detergent resistant microdomains in polyunsaturated

fatty acid-treated cells is due to inhibition of palmitoylation.

In contrast to the saturated inhibitor 2-bromopalmitate,

polyunsaturated arachidonic acid (20:4) and eicosapentaenoic acid

(20:5) have almost no effect on Fyn myristoylation, and are rather

specific for palmitoylation. Several lines of evidence suggest that the

mechanism of inhibition of palmitoylation involves the use o f

polyunsaturated fatty acids as alternative substrates for S-acylation t o Fyn. First, studies of partially purified preparations of PAT reveal that

longer chain fatty acyl CoAs, including stearate (18:0) a nd

arachidonate (20:4) can compete with palmitate for incorporation

into Fyn and Gαo (26,27). Secondly, Gα subunits, P-selectin,

asialoglycoprotein receptor and several platelet proteins have been

shown to be S-acylated with stearate, arachidonate and

eicosapentaenoate, in addition to palmitate (49,50). These results

indicate that the fatty acid specificity of PAT and S-acylation is loose

in vivo and in vitro. Thirdly, Fyn localization to the plasma

membrane fraction is not affected by polyunsaturated fatty acids

(data not shown), even though incorporation of ¹²⁵I-IC16 is markedly

reduced. Since myristoylation alone is not sufficient for stable

membrane binding, it is likely that Fyn becomes dually fatty acylated

by N-myristoylation and S-acylation with a polyunsaturated fatty acid.

The presence of myristate and polyunsaturated fatty acid at the N-

terminus of Fyn would provide strong affinity for binding to a

membrane bilayer. However, the presence of a polyunsaturated,

bulky acyl chain in the polyunsaturated fatty acid would preclude

specific localization to DRMS which, due to their liquid ordered

domain structure, provide a local environment conducive to insertion

of saturated, but not unsaturated fatty acid chains (18).

In conclusion, specific fatty acids and fatty acid analogs

function as inhibitors of protein fatty acylation and TCR mediated signaling. The advantage of using inhibitors that interfere with

subcellular localization of a key protein is that it allows one to study

signaling by endogenous cellular proteins and eliminates the need t o

overexpress mutant proteins. Thus, 2-bromopalmitate can be used a s

a powerful tool to study the role of Src kinases in the endogenous T

cell signaling system, and may provide insight into the role o f

signaling in the onset of disease. PUFA-induced inhibition of T cells is

likely due to the inhibition of Src kinase palmitoylation. Though these

agents are currently used in the clinic, their mechanism of action is

still largely unknown. A novel mechanism, inhibition of protein

palmitoylation, may account for the abilities of polyunsaturated fatty

acids to treat or prevent a broad range of immune-based diseases.

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Any patents or publications mentioned in this specification

are indicative of the levels of those skilled in the art to which the

invention pertains. These patents and publications are herein incorporated by reference to the same extent as if each individual

publication was specifically and individually indicated to b e

incorporated by reference.

One skilled in the art will readily appreciate that th e

present invention is well adapted to carry out the objects and obtain

the ends and advantages mentioned, as well as those inherent therein .

The present examples along with the methods, procedures ,

treatments, molecules, and specific compounds described herein are

presently representative of preferred embodiments, are exemplary,

and are not intended as limitations on the scope of the invention.

Changes therein and other uses will occur to those skilled in the art

which are encompassed within the spirit of the invention as defined by

the scope of the claims.

Claims

WHAT IS CLAIMED IS:

1 . A method of inhibiting Fyn/Lck fatty acylation an d

protein palmitoylation in a cell in an individual in need of such

treatment comprising the step of administering to said individual a

pharmacologically effective dose of 2-bromopalmitate.

2. The method of claim 1, where said 2 -

bromopalmitate is administered in a dose of from about 0.1 mg/kg t o

about 100 mg/kg of total body weight of said individual.

3 . The method of claim 1, where said 2 -

bromopalmitate inhibits N-terminally palmitoylated proteins.

4. The method of claim 1, where said 2 -

bromopalmitate inhibits myristoylation of proteins.

5 . The method of claim 1, where said 2

bromopalmitate inhibits T cell signalling events.

6 . The method of claim 1, where said individual has a

autoimmune disease.

7 . The method of claim 1, where said autoimmune

disease is selected from the group consisting of rheumatoid arthritis,

Crohn's disease, diabetes, multiple sclerosis and systemic lupus

erythematosus .

8 . A method of treating an individual having a

pathophysiological state comprising the step of administering to said

individual a pharmacologically effective dose of 2-bromopalmitate.

9 . The method of claim 8, where said 2 -

bromopalmitate is administered in a dose of from about 0.1 to about

100 mg/kg of total body weight of said individual.

1 0. The method of claim 8, where said

bromopalmitate inhibits N-terminally palmitoylated proteins.

1 1 . The method of claim 8, where said 2 •

bromopalmitate inhibits myristoylation of proteins.

1 2. The method of claim 8, where said 2 -

bromopalmitate inhibits T cell signalling events.

1 3 . The method of claim 8, where said individual has a

inflammatory disease.

1 4. The method of claim 8, where said disease is a n

autoimmune disease.

1 5 . The method of claim 14, where said autoimmune

disease is selected from the group consisting of rheumatoid arthritis,

diabetes, Crohn's disease, multiple sclerosis and systemic lupus

erythematosus .

1 6. A pharmaceutical composition comprising 2

bromopalmitate and a pharmaceutically acceptable carrier.