WO2008112320A1 - Inhibition of ion channel function - Google Patents

Abstract

Sphingomyelinase (SMase), an ion channel modulator, suppresses immune host response. Smase is used in the treatment of cystic fibrosis, and other therapies. SMase may modulate a potassium or chlorine channel in a subject; a composition comprising a therapeutically effective amount of a bacterial SMase is administered to a subject, thereby cleaving sphingomyelin. SMase may inhibit or suppress bacterial immunosuppression of a host immune system; the host is contacted with an inhibitor of the bacteria's SMase, thereby inhibiting or suppressing modulation of the host's immune cell potassium channel. An inhibitor of the bacteria's SMase may be used to treat a bacterial infection.

Description

INHIBITION OF ION CHANNEL FUNCTION

FIELD OF INVENTION

[0001] This invention is directed to sphingomyelinase as an ion channel modulator. Specifically, the invention is directed to the role of Smase as an ion channel modulator in suppressing immune host response, the treatment of cystic fibrosis and other therapeutic uses thereof.

BACKGROUND OF THE INVENTION

[0002] Current therapeutic strategies are targeting the basic defects associated with ion channels with the goal of correcting these defects in vivo or alternatively, modulating these channels. Consequently, the characterization of the processes by which ion channels are regulated in normal and disease states will provide critical insights into normal ion channel regulation as well as aid in the development of new treatment approaches.

[0003] Likewise, application of medicines against various bacterial virulence factors including phospholipases and perhaps proteases, in conjunction with other effective measures, might be a viable near-term approach to combating chronic or acute bacterial infection.

[0004] Cystic fibrosis (CF) is an autosomal-recessive genetic disease caused by mutations in the single gene encoding the cystic fibrosis transmembrane conductance regulator (CFTR). CFTR is normally expressed on the apical plasma membrane of epithelial cells, where it functions as a cAMP-regulated chloride channel. CF is characterized by an ion and solute transport defect that, in the lungs, culminates in accumulation of dehydrated mucus, impaired mucociliary clearance, and chronic bacterial infection. Lung disease, the major cause of morbidity in CF patients, remains a significant detriment to a healthy life for CF patients. The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-dependent Cl^" channel that controls transepithelial electrolyte transport, fluid flow, and ion concentrations in the intestine, lungs, pancreas, and sweat glands. [0005] Current therapeutic strategies are targeting the basic defects associated with mutant CFTR proteins with the goal of correcting these defects in vivo. Consequently, the characterization of the processes by which CFTR is regulated in normal and disease states will provide critical insights into normal CFTR regulation as well as aid in the development of new treatment approaches.

[0006] Likewise, application of medicines against various bacterial virulence factors including phospholipases and perhaps proteases, in conjunction with other effective measures, might be a viable near-term approach to improving length and quality of life for CF patients. The same approach might also benefit patients with other types of chronic bacterial infection.

SUMMARY OF THE INVENTION

[0007] In one embodiment, the invention provides a sphingomyelinase as an ion channel modulator. In another embodiment, the invention provides therapeutic methods utilizing the effect of sphingomyelinase as an ion channel modulator.

[0008] In one embodiment, the invention provides a method of modulating a potassium channel, a chlorine channel or their combination in a subject, comprising the step administering to said subject a composition comprising a therapeutically effective amount of a bacterial sphingomyelinase (SMase), thereby cleaving sphingomyelin.

[0009] In another embodiment, the invention provides a method of inhibiting or suppressing bacterial immunosuppression of a host immune system, comprising the step of contacting the host with a composition comprising an inhibitor of the bacteria's sphingomyelinase (SMase), thereby inhibiting or suppressing modulation of the host's immune cell potassium channel.

[00010] In one embodiment, provided herein is a method of treating a bacterial infection of a host, comprising the step of comprising the step of contacting the host with a composition comprising an inhibitor of the bacteria's sphingomyelinase (SMase), thereby inhibiting or suppressing modulation of the host's immune cell potassium channel. [00011] In another embodiment, provided herein is a method of suppressing the immune system of a host, comprising the step of contacting the host with a bacterial sphingomyelinase, thereby modulating a potassium channel on the host's immune cells..

[00012] In one embodiment, the invention provides a method of treating cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

[00013] In another embodiment, the invention provides a method of inhibiting or suppressing cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

[00014] In one embodiment, the invention provides a method of reducing symptoms of cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

[00015] In another embodiment, the inventon provides a composition for treating cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising a bacterial sphingomyelinase inhibitor, wherein the bacterial sphingomyelinase inhibitor inhibits sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

[00016] In one embodiment, the invention provides a method for screening for an agent useful in the treatment of cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising the step of contacting a bacteria associated with cystic fibrosis infection with a candidate compound; and analyzing the bacteria for expression or function of sphingomyelinase, whereby an agent capable of reducing the expression of sphingomyelinase, inhibiting the function of sphingomyelinase, or reacting with a lipid product of sphingomyelinase -catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR) is useful in the treatment of cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection.

[00017] In another embodiment, the invention provides a method of treating pulmonary disorder in a subject having an acute or a chronic bacterial infection, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

[00018] In one embodiment, provided herein is a method of screening for compounds capable of inhibiting bacterial sphyngomyelinase activity comprising: contacting in each of a plurality of reaction vessels in a high throughput screening array, an epithelial cell and a bacterial sphyngomyelinase with a test compound and an indicator compound and measuring the effect of the test compound on the viability of the epithelial cell to thereby identify compounds cpabale inhibiting the activity of bacterial sphyngomyelinase.

[00019] Other features and advantages of the present invention will become apparent from the following detailed description examples and figures. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[00020] The invention will be better understood from a reading of the following detailed description taken in conjunction with the drawings in which like reference designators are used to designate like elements, and in which:

Figure 1 shows the reaction schemes of lipid hydrolysis and the effect of SMase D on Kv2.1 channels, a, b, Hydrolysis of sphingomyelin by SMases C or D (a) and of phosphatidylcholine by PC-PLC (b). R and R' represent acyl chains, c, Amplitude of Kv2.1 current, repeatedly elicited by stepping membrane voltage from -80 to -40 mV, where the arrow indicates the addition of recombinant SMase D to the bath, d, G-V relations obtained before treatment with SMase D (squares), and 30 min (circles) or 24 h (triangles) after exposure of oocytes to SMase D for 10 min. e, Oocytes were exposed to SMase D for 30 min and then washed with enzyme-free solution before being injected with Kv2.1 cRNA. Data were collected from each oocyte 18 h after the injection (circles), and again after an additional 15 min of treatment with SMase D (triangles). The control G-V relation was obtained without SMase D treatment (squares). All data in d and e are presented as means ±s.e.m. (n=5), and the controls were pooled;

Figure 2 shows shows and the effect of SMase C on Kv2.1, Kirl.l and a KcsA-Kir2.1 chimaera. a, Kv2.1 currents elicited at 5-s intervals with the voltage protocol shown, gradually declining after the addition of recombinant ^SαSMase C to the bath. The dotted line indicates zero current level, b, Time course of current, where the arrow indicates the addition of 14 ng μl^"1 (open circles, left) or 1.4 ng μl^"1 (open triangles, middle) ^5αSMase C, or 40 ng μl^{"1 δα}SMase C (filled squares, right), c, G-V curves of Kv2.1 before (squares) and after (circles) treatment with SMase C. d, Fraction of current remaining at various times after the addition of ^5αSMase C, plotted against voltage: the top three data sets correspond to different levels of partial enzymatic treatment and the bottom set to complete treatment. The currents were normalized to those before the addition of SMase C at the corresponding voltages, e, G-V curves without (open symbols) or with (filled symbols) incubation for 2 h in a solution containing 5mM MβCD, and before (squares) or after (circles) treatment with ^SαSMase C. f, Fraction of current remaining after the addition of 3 μM hanatoxin (squares, top) and subsequent treatment with ^SaSMase C (circles, middle), or after treatment with ^5αSMase C alone (triangles, bottom), g-j, Kv2.1 currents plotted against time, where the arrows labelled 1 indicate the addition of 1OmM phosphocholine (g), 0.25 mg ml^"1 ceramide (Cg) pre- dissolved in ethanol (h), 1OmM choline (i) or 0.25 mg ml^"1 ceramide- 1 -phosphate (Cn) pre- dissolved in dodecane/ methanol (1 :49 by vol.) (j). SMase C (g, h) or SMase D (i, j) were used as positive controls (arrows labelled T). Similar results were observed in at least three experiments for each case, k, I-V curves of Kirl.l before (squares) and after (circles) treatment with SMase C, and after subsequent application of 0.5 μM pore-blocking tertiapin- Q (triangles). 1, I-V curves of a KcsA-Kir2.1 chimaera before (squares) and after (circles) treatment with SMase C. All data in c-f, k and 1 are presented as means ± s.e.m. (n=5-8); Figure 3 shows the effect of SMase C and PC-PLC on ionic and gating currents of Shaker channels, a, b, Ionic currents of Shaker(-IR) (a) and gating currents of the V478WShaker mutant (b) elicited with the protocols shown, gradually decreasing on addition of recombinant ^βoSMase C. c, d, G-V (c) and Q_0n-V (d) relations before (squares) or after

₅ (circles) treatment with SMase C. e, Trace of ionic current after treatment with SMase C, normalized in amplitude to that before treatment in a. f, Normalized Q-V curves before (squares) and after (circles) treatment with SMase C in d. g, h, Integrals of on (g) and off (h) gating charges (before or after treatment with SMase C) against time; all traces are normalized in amplitude, i-k, On-gating currents of Shaker's V478Wmutant versus timeo (similar resultswere observedinat least three experiments for each case). Gating currents were collected by stepping the voltage from -100 mV to 0 mV (i, k) or -60 mV (j). The arrows indicate the addition and washout of SMase C (i), the addition and washout of SMase D and subsequent addition of SMase C (j), and the addition and washout of purified native PC-PLC (k). The current was on average decreased by 11.2+ 2.8% (means± s.e.m., n=3) after thes addition of PC-PLC. 1, Q-V curves of control (filled squares) or after treatment with SMase D (open triangles) and subsequent treatment with SMase C (open circles). All data in c, d, f and 1 are presented as means± s.e.m. (n=5-l l); the error bars in c, d and f are generally smaller than the symbols; o Figure 4 shows the inhibition of KvI .3 by SMase C. a, b, Ionic currents of wild-type KvI .3 (a) and gating currents of the W384F mutant (b) elicited at 5-s intervals with the voltage protocol shown, gradually declining after the addition of recombinant SaSMase C (the off- gating current is largely immobilized), c, d, G-V (c) and Q_0n-V (d) relations before (squares) and after (circles) treatment with ^5αSMase C. The data are presented as means + s.e.m. (n=9-5 1 1 ).

Figure 5 shows identification of CFTR-inhibitory activity. (A and B) 0.I mM IBMX-activated CFTR (A) and constitutively active CFTR-ΔR (B) currents elicited at 3-s intervals (only every fifth trace shown) with the voltage protocol shown, and gradually disappearingo following venom addition (1 :3,000 dilution). The dotted line indicates zero current. All currents throughout the study are uncorrected for background "leak". (C) Size exclusion chromatography of 10 μl venom; the bar identifies active fractions. (D) The asterisk-marked peak in panel C was further purified on a reverse phase column, which yielded a major peak containing the activity;

Figure 6 shows inhibition of CFTR and CFTR-ΔR currents by recombinant spider and bacterial SMase D. (A and B) plots of currents elicited by repeated (0.33 Hz) pulses to 50 mV (see Fig. 5A) against time. Top traces are from oocytes injected with cRNA of CFTR (A) or CFTR-ΔR (B); lower traces (A and E) were obtained from uninjected oocytes. In all four cases, recombinant ^²SMaSe D was added at the time indicated (8 ng/:l was used throughout the study). I-V relations were collected at the beginning (control) and end (treated) of six similar recordings for each case. (C-F) Normalized I-V relations of CFTR (C and E) and CFTR-ΔR (D and F) before and after treatment with recombinant ^²SMaSe D (C and D) or ^SMase (8 ng/:μl; E and F). Data shown are presented as mean ± sem (n = 6); error bars generally smaller than the symbols;

Figure 7 shows the effects of histidine mutations and pH, and Mg²⁺on SMase D activity. (A) Arrows indicate successive addition of SMase D with or without the HI lA and H47A mutations. (B) successive addition of SMase D that was or was not pre-treated with 0.1% trifluoroacetic acid (pH < 3) forl4 h. (C) No Mg²⁺ was present in the bath during the first ~3 minutes. Arrows indicate successive additions of SMase D and 1 mM Mg²⁺. For each of the three cases (A-C), similar results were observed in 3 - 4 experiments. (D) Reaction scheme of SMase D-catalyzed sphingomyelin hydrolysis. R and R' represent acyl chains;

Figure 8 whows the effects of sphingomyelin and its hydrolysis products on CFTR-ΔR currents. (A) Arrows indicate addition and washout of SMaseD (after 500 s, the data were collected at 30-s intervals instead of 3-s). (B) Arrows indicate successive additions of choline (10 mM) and ^Lr2SMase D. (C) Arrows indicate successive additions of ceramide-1 -phosphate (C-I-P; 0.25 mg/ml; the stock was in 49: 1 methanol/dodecane) and ^²SMaSe D [the bath solution contained 2%of methanol/dodecane (49:1)]. (D) Arrows indicate successive addition and washout of ^²SMaSe D and addition of sphingomyelin (C 16-SM; 0.5 mg/ml; stock was in methanol), where the bath solution contained 2% methanol. (E) Arrows indicate successive additions of phosphocholine (1 mM) and ^Lr2SMase D. (F) Arrows indicate successive additions of ceramide-Cg (0.25 mg/ml; the stock was in ethanol) and ^²SMaSe D, where the bath solution contained 2% ethanol. Similar results were observed in at least three experiments for each case. ^SMase D was used as a positive control for CFTR inhibition in B, C, E, and F. Concentrations of all chemical and lipid stocks are 50 times the final.;

Figure 9 shows inhibition of CFTR currents by SMase C and PC-PLC. (A) Reaction scheme of SMase C-catalyzed sphingomyelin hydrolysis. (B-D) CFTR currents at 50 mV plotted against time, before and after addition (arrows) of recombinant ^βαSMase C (B) or ^5oSMase C

(O (40 ng/ μl was used throughout the study) or "purified" native ^βcPC-PLC bound with

Zm₊ (50 ng/:l) (D). I-V relations for control (1), ^βflSMase C (2) or ^βcPC-PLC (3) were collected from six similar recordings for each case. (E-G) Normalized I-V relations before and after ^βαSMase C, ^5flSMase C or ^βcPC-PLC. ^βαSMase C was used as a positive control for

CFTR inhibition in D and G. Data shown are mean ± sem (n = 6); error bars generally smaller than the symbols;

Figure 10 shows inhibition of CFTR-)R by recombinant SMases C and Mg²⁺ dependence. (A-D) CFTR-)R currents at 50 mV were plotted against time. (A, B) Arrows indicate addition of ^BαSMase C or ⁵⁰SMaSe C in the presence of 1 mM Mg²⁺. (C and D) No Mg²⁺ was present in the bath solution during the first 4.5 and 6 minutes, respectively; arrows indicate successive additions of ^βαSMase C and 1 mM Mg²⁺. Similar results were observed in 3 - 4 individual oocytes.;

Figure 11 shows inhibition of disease-causing CFTR mutants by bacterial SMases C and D. Normalized I-V relations of ΔF508 (A and C) or Rl 17H (B and D) mutants before and after exposure to ^βαSMase C (A and B) or ^CpSMase D (C and D). Data shown are mean ± sem (n = 6); error bars generally smaller than the symbols.;

Figure 12 shows Dependence of SMase inhibition of CFTR and CFTR-)R currents on PKA activity. (A and B) Percent inhibition by ^βαSMase C and ^SMase D of CFTR current (at 50 mV; mean ± sem, n = 4 - 12) activated by 0.1 mM IBMX, 1 mM IBMX or 1 mM IBMX plus 50 :M forskolin. (C and D) Percent inhibition of CFTR current by ^βαSMase C and ^CpSMase D, plotted against the amount of PKA-C cRNA injected per oocyte (mean V sem, n = 3 - 6). (E and F) Percent inhibition of CFTR-)R current by ^BaSMase C (E) or ^CpSMase D (F) (at 50 mV; mean ± sem, n = 4 - 5) with and without co-injection of PKA-C cRNA (16 ng per oocyte). (G) Record of currents elicited by pulses to 50 mV in an oocyte injected with CFTR cRNA, following applications (horizontal bars) of 0.1 mM IBMX, 8 ng/:l ^cpSMase D, and 1 mM IBMX plus 50 :M forskolin. Similar results were observed in five individual oocytes;

Figure 13 shows Biochemical analyses of commercial SMase C samples. (A) Coomassie blue staining of SDS PAGE of native S. aureus (Sa; lot number 075K4003) and B. cereus (Bc; lot number 063K4079) SMase C samples purchased from Sigma Chemical Corp. (St. Louis, MO), with standard protein molecular weight markers on left. The presence of more than one band indicates impurity. Based on molecular weight, the band of B. cereus SMase C sample that is most likely to represent B. cereus SMase C is indicated by an arrow. (B) MS analysis of the B. cereus SMase C sample reveals an even greater degree of impurity than suggested by the two bands on the SDS gel. The arrow indicates the peak that probably corresponds to B. cereus SMase C, based on molecular weight;

Figure 14 shows Currents from oocytes uninjected (A) or injected with PKA-C cRNA (16 ng; B) or with PKA-C and CFTR cRNAs (16 ng each; C), recorded by stepping voltage from -80 mV to 50 mV in 10 mV increments from the -20 mV holding potential. Similar-sized currents were observed with at least five individual oocytes for each case; and

Figure 15 shows results when Calu-3 cells were cultured in a 48-well dish containing MEM Alpha medium supplemented with 10% fetal bovine serum (FBS) and maintained in a 5% CO2 incubator at 37°C. Upon reaching confluence, cells were washed with and cultured in defined MEM Alpha media that contained recombinant sphingomyelinase C (SMase C) of Staphylococcus aureus (9 ng/μl). An equal volume of the enzyme buffer solution was added to the control group (i.e., 1 μl buffer to 100 μl culture medium). Triplets were carried out for both the SMase C and the control groups in each trail. After the indicated period of SMase C treatment, cells were taken out the incubator, trypsinized, washed with and then resuspend in. PBS. To determine viability, cells were incubated in a 1 :1 (V: V) mixture of PBS and 0.4% Trypan blue solution (Sigma) at room temperature for 15 minutes. Blue-stained and non-stained cells were counted with a hemacytometer chamber. Percentage of non-viable (stained) cells was plotted as the mean (± s.e.m.) of six independent trails for each time point. DETAILED DESCRIPTION OF THE INVENTION

[00021] This invention relates in one embodiment to the role of sphingomyelinase as an ion channel modulator. In another embodiment, the invention provides the role of Smase as an ion channel modulator in suppressing immune host response, the treatment of cystic fibrosis and other therapeutic uses thereof.

[00022] This invention relates in one embodiment to Smase as an ion channel modulator. In another embodiment, the invention relates to the role of Smase as an ion channel modulator in suppressing immune host response and therapeutic uses thereof. In one embodiment, the term "modulator" refers to any compound or agent that can alter the activity of an ion channel, i. e., alter the control mechanism of the gates of ion conduction pores, or alter the movement or transport of ions through an ion channel. The modulator can be an organic molecule or chemical compound (naturally occurring or non-naturally occurring), such as a biological macromolecule (e.g., nucleic acid, protein, non-peptide, or organic molecule), or an extract made from biological materials such as bacteria, plants, fungi, or animal (particularly mammalian) cells or tissues, protein or protein fragment. Modulators are evaluated for the potential to act as inhibitors or activators of a biological process or processes, e.g., to act as agonist, antagonist, partial agonist, partial antagonist, antineoplastic agents, cytotoxic agents, inhibitors of neoplastic transformation or cell proliferation, and cell proliferation-promoting agents. The activity of a modulator may be known, unknown or partially known

[00023] Accordingly and in one embodiment, provided herein is a method of modulating a potassium channel, or a chlorine (Cl^") channel in another embodiment; in a subject, comprising the step administering to said subject a composition comprising a therapeutically effective amount of a bacterial sphingomyelinase (SMase), thereby cleaving sphingomyelin.

[00024] In one embodiment, voltage gated potassium channels interact with sphingomyelin present mainly in the outer leaf of plasma membranes

[00025] In one embodiment, voltage-gated potassium (Kv) channels refer to the members of the voltage-gated ion channel superfamily, which is important for initiation and propagation of action potentials in excitable cells. In another embodiment, voltage-gated potassium (Kv) channels are composed of four identical or homologous subunits, each containing six transmembrane segments: S1-S6. Segments S1-S4 form the voltage-sensing domain (VSD), and segments S5 and S6 connected by the P loop, which is involved in ion selectivity, comprise the pore-forming domain (PD). S4 has four gating-charge-carrying arginines (Rl- R4) spaced at intervals of three amino acid residues, which are highly conserved and are thought to play a key role in coupling changes in membrane voltage to opening and closing of the pore. In the Kv channels =13 electronic charges cross the membrane electrical field per channel between the closed and open states.

[00026] In one embodiment, Kv channels control ion conduction by adopting different molecular states in response to variations of the transmembrane (TM) voltage. In another embodiment, there are three main states, resting, activation, and inactivation. In contrast with the resting state that occurs at hyperpolarized potentials of the membrane, activation and inactivation are induced by membrane depolarization. Whereas the resting and the inactivated states present low or no level of ion conductance (closed channel), the activated state is related to a high ion conductance (open channel). Transitions between these states are referred to in one embodiment, as a general gating mechanism. In one embodiment, the term "activation" refers to the transition from a resting (non-conducting) state of an ion channel to the activated (conducting) state.

[00027] In one embodiment, the voltage sensor of Kv channels interacts strongly with multiple molecules of several kinds of phospholipid. In another embodiment, sphingomyelin is preferred over PC wherein these lipids differ in hydrogen-bonding characteristics. The apparent preference reflects in one embodiment, both inherent channel-lipid affinity or in yet another embodiment, relative abundance in the channels' lipid microenvironments. In one embodiment, interactions between voltage sensors and phospholipids are important for the early gating transitions, and others are important for late transitions. In another embodiment, the phospho-head groups of membrane lipids, together with certain acidic channel residues, provide the necessary counter-charges for the positively charged voltage-sensing residues during individual steps of the voltage-sensor movement. In one embodiment, this charge- neutralizing action lowers the free energy of the overall gating process as well as the energy barrier for individual transitions in another embodiment, so that a modest voltage can drive the charged sensors through a series of conformational steps in a low-dielectric cell membrane to open the channel gate. In one embodiment, potassium channels, expressed in hosts' cells interact with sphingomyelin present mainly in the outer leaf of plasma membranes. SMase inhibition of potassium channels current results in one embodiment from enzymatic hydrolysis of sphingomyelin, which makes it more difficult to activate the potassium channels. In one embodiment, SMase cleaves sphingomyelin into a small piece (choline or phosphocholine) and a large one (ceramide-1 -phosphate or ceramide). The large piece has two long hydrophobic chains, and, like sphingomyelin, has a very strong preference to remain within the membrane. The highly water-soluble hydrolysis products phosphocholine (of SMase C) or choline (of SMase D) are easily diluted out in the bathing fluid following hydrolysis, and in another embodiment, are responsible for transient inhibition.

[00028] In one embodiment, bacterial SMase D shifts the conductance -voltage (G-V) relation of Kv2.1 (See e.g.Fig. Id), allowing channels to be activated at a negative voltage at which otherwise, they remain largely deactivated (Fig. Ic). In one embodiment, the shift in conductance does not require direct exposure of channels to SMase D. In one embodiment, SMase D acts through its lipase activity rather than by direct binding to the channel. In another embodiment, an electrostatic mechanism by which removal of the positively charged choline favours the activated state of the positively charged voltage sensors. In one embodiment, the SMase used or inhibited using the compositionas and methods provided herein, is bacterial SMase D.

[00029] In one embodiment, the SMase used or inhibited using the compositions and methods provided herein, is bacterial SMase D. In another embodiment, SMase C removes the negatively charged phosphodiester group as well as choline. In another embodiment, bacterial SMase C decreases potassium channel current ( See e.g.Fig. 2a-c) and the decrease is independent of voltage for both partial and complete enzymatic treatment (Fig. 2d). In one embodiment, SMase C of different species of bacteria, causes a comparable level of inhibition but with different kinetics, and only the kinetics, not the extent of inhibition, are dependent in one embodiment, on enzyme concentration (Fig. 2b). In one embodiment, SMase C inhibits the potassium channels by means of its enzymatic activity rather than by direct binding. [00030] In one embodiment, the methods and compositions provided herein, compose an agent capable of inhibiting the function or expression of bacterial SMase, whereby the agent is an antibody, or a fraction thereof, an antagonist, or a combination thereof.

[00031] In one embodiment, the potassium channel modulated in one embodiment, or inhibited in another embodiment, or in which current is decreased in opne embodiment or enhanced in another embodiment, is Kv2.1, or KvI.3 or their combination in another discrete embodiments.

[00032] In one embodiment, SMase C administration decreases both ionic and gating currents of KvI.3 channels. In one embodiment, the voltage-gated (Kv) KvI.3 channel is involved in the maintenance of the resting membrane potential in immune system cells. Kv 1.3 is abundantly expressed on "Effector Memory Cells" (TEM), which in another embodiment are key mediators in autoimmune inflammatory diseases. In another embodiment, KvI .3 blockers show more specificity for autoreactive TEM cells than any molecular target expressed on all T cells. In one embodiment, KvI.3 inhibitors ameliorate the symptoms of several T-cell mediated diseases. In addition to the level of Kv expression, the proper plasma membrane localization and protein partnerships are critical for the regulation of channel steady-state properties.

[00033] In another embodiment, the voltage-gated KvI .3 channel belonging to the Shaker family of channels dominates the K⁺ conductance in human T and B cells, with quiescent human T lymphocytes expressing Kv 1.3 channels (~300 per cell). In one embodiment, the major physiological function of this channel is the maintenance of a negative membrane potential, (Nernst potential for K⁺ is about -85 mV) and a large driving force for Ca²⁺ entry through Ca²⁺-release-activated Ca²⁺ channels ( which facilitates sustained Ca²⁺ signaling during T-cell activation by providing the electrical driving force for Ca²⁺ entry through voltage-independent Ca²⁺ channels). Peptide and small-molecule inhibitors of the channels depolarize the membrane, resulting in the inhibition of Ca²⁺ signaling and lymphocyte proliferation. The difference in the K⁺-channel dominance in T-cell subsets allows in certain embodiments, specific interference with their activation using selective blockers of KvI .3. In one embodiment specific SMase C action against KvI .3 helps S. aureus to neutralize host defences, by supparessing the host's immune system. [00034] In another embodiment, provided herein is a method of treating a bacterial infection of a host, comprising the step of contacting the host with a composition comprising an agent capable of inhibiting the function or expression of the bacteria's sphingomyelinase (SMase), thereby inhibiting or suppressing modulation of the host's immune-cell potassium channel. In one embodiment, the potassium channel modulated by the bacterial SMase sought to be inhibited ore suppressed using the compositions described herein, is KvI .3.

[00035] In one embodiment, sphingomyelinase C activates Kirl .l channels. In another embodiment, Kirl.l (ROMK) is a member of the inwardly rectifying (Kir) family of potassium channels, characterized by its sensitivity to intracellular pH (pFL) in the physiological range. Kirl .l channels are found in the apical membrane of epithelial cells lining the distal nephron of the kidney, where they secrete excess K⁺ into the urine and are therefore the principal regulators of K⁺ homeostasis in the body. In one embodiment, inhibition by H⁺ links K⁺ transport and electrical activity to cellular H⁺ homeostasis.

[00036] In another embodiment, CFTR is a regulator of the renal secretory renal outer medullar potassium (ROMK) channel. In another embodiment, ROMK mediates apical K recycling in the thick ascending limb (TAL) and net K secretion by aldosterone-sensitive distal nephron cells in the connecting segment and cortical collecting duct (CCD).

[00037] In one embodiment, inherited mutations in Kirl .l that disrupt pH-sensing mechanism, hypersensitizes the channel to H⁺ and result in type II Bartter syndrome, a hypokalaemic disorder. In another embodiment, the term Banter's syndrome, refers to a hypokalemic alkalosis with dehydration, hypotension, and severe polyuria which develops before birth or during infancy. In another embodiment, the disorder is caused by null mutations in any of four genes encoding proteins involved in NaCl absorption in the renal thick ascending limb of Henle (TALH) such as the ROMK potassium channel. In one embodiment provided herein is a method of treating hydronephrosis, polydipsia, polyuria, dehydration, or their combination in a subject, resulting from ROMK disfunction, comprising the step of administering to the subject a composition comprising a therapeutically effective amount of sphingomyelinase C, thereby producing diureasis.

[00038] The CFTR Cl^" channel is activated in one embodiment when its R (regulatory) domain is phosphorylated by cyclic AMP-dependent protein kinase A (PKA). Activation is assayed in another embodiment, from the Cl^" current that flows through activated channels in voltage-clamp experiments. The R domain, in the middle of the CFTR peptide chain, contains many phosphorylation sites. In one embodiment, activation increases with phosphorylation up to a point, but phosphorylation of all the sites is not necessary for full activation of current. If the N- and C-terminal peptides formed from genetic excision of the R domain are covalently linked together, the resulting single peptide forms a Cl^" channel that in another embodiment, is constitutively active but with very low open probability. If, in another embodiment, the N- and C-terminal peptides are expressed as separate transcripts (in Xenopus oocytes), the two unlinked peptides form a channel that is constitutively active with much higher open probability, but not as high as the fully activated wild type channel. The channel, formed by the unlinked N- and C-terminal peptides, will be referred to in one embodiment, as CFTR-ΔR.

[00039] In another embodiment, sphingomyelinase D (SMase D) from the Brown spider venom suppresses the Cl^" current that flows through the activated CFTR. In another embodiment, SMases C and D from respiratory tract bacteria found in cystic fibrosis patients in another embodiment, profoundly suppress CFTR current.

[00040] Accordingly and in one embodiment, provided herein is a method of treating, or in another embodiment inhibiting or suppressing, or in another embodiment, reducing the symptoms associated with cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

[00041] SMase inhibition of CFTR current results in one embodiment from enzymatic hydrolysis of sphingomyelin, which makes it more difficult to activate CFTR via phosphorylation of the R domain. In another embodiment, higher levels of phosphorylation by PKA can overcome the inhibitory effect when the R domain is present, but not in its absence (Fig. 12). In one embodiment, SMase cleaves sphingomyelin into a small piece (choline or phosphocholine) and a large one (ceramide- 1 -phosphate or ceramide). The large piece has two long hydrophobic chains, and, like sphingomyelin, has a very strong preference to remain within the membrane. In one embodiment, the sphingomyelin hydrolysis products might be the active inhibitors. The highly water-soluble hydrolysis products phosphocholine (of SMase C) or choline (of SMase D) would be easily diluted out in the bathing fluid following hydrolysis, and in another embodiment, are responsible for transient inhibition. In one embodiment, as shown in Fig. 8 there is no significant inhibition of CFTR-ΔR current following acute addition of choline or phosphocholine. In one embodiment the poorly water soluble products ceramide (of SMase C) and ceramide-1- phosphate (of SMase D) are unlikely to leave the membrane. In one embodiment either ceramide (from SMase C treatment), or ceramide-1- phosphate (from SMase D treatment), or both in other embodiments, remain attached to CFTR, thus accounting in one embodiment for the difference in percentage inhibition. In one embodiment sphingomyelin complexed to CFTR facilitates opening of the channel in a way that cannot be duplicated by ceramide or ceramide-1 -phosphate. In another embodiment ceramide and ceramide- 1-phospahte differ in their ability to influence activation gating.

[00042] In one embodiment, the methods for treating CF in an infected subject as provided herein, further comprise the step of removing a lipid product of sphingomyelinase -catalyzed sphingomyelin hydrolysis, thereby removing compounds aggrevating inflammation, whereby the sphingomyelinase is SMase-D, SMase-C or a combination thereof. In one embodiment, the lipid product of sphingomyelinase -catalyzed sphingomyelin hydrolysis removed in accordance with the methods provided herein is ceramide. In another embodiment, the lipid product of sphingomyelinase -catalyzed sphingomyelin hydrolysis is ceramide-1 -phosphate. In another embodiment, the lipid product of sphingomyelinase -catalyzed sphingomyelin hydrolysis is choline. In another embodiment, the lipid product of sphingomyelinase - catalyzed sphingomyelin hydrolysis is phosphocholine or in another embodiment, the lipid product of sphingomyelinase -catalyzed sphingomyelin hydrolysis is a combination thereof.

[00043] Many respiratory pathogens have sphingomyelinase activity. In one embodiment, S. aureus is a major respiratory threat for CF patients (>50% infection rate), causing recurring infection and inflammation that in another embodiment, significantly impair lung function. In another embodiment 5. aureus causes severe pneumonia and other types of infection in non- CF patients in another embodiment. In another embodiment S. aureus produces SMase-C, referred to in another embodiment as β-hemolysin. In one embodiment, Pseudomonas aeruginosa is another major offender in CF. In one embodiment, Pseudomonas aeruginosa permanently colonizes the airways of virtually all late-stage CF patients either in planktonic form in one embodiment, or as a biofilm following quorum sensing in another embodiment, and produces a phospholipase (termed in one embodiment PLC-H) that in one embodiment, can hydrolyze sphingomyelin.

[00044] In one embodiment, the chronic or acute bacterial infection sought to be treated using the compositions and methods provided herein is caused by Helicobacter Pilory, Arcanobacterium haemolyticum, S. aureus, B. cereus, P. aeruginosa , Corynebacterium pseudotuberculosis, M. tuberculosis, M. marinum, M. bovis or a combination thereof.

[00045] Although in one embodiment, CF disease originates from a genetic defect in CFTR, the severity of the pulmonary disease does not correlate well with genotype. The lung damage in CF patients is not specific, i.e., its histopathology is not fundamentally different from that seen in some other types of chronic pulmonary infection/inflammation. The fibrosis seen in CF, although very severe, is not so different from that following severe tissue damage from a variety of other causes. In one embodiment, aggressive antibiotic treatments and supportive measures increase the median life span of CF patients from five to thirty-seven years. In another embodiment CFTR defects predispose patients to bacterial infection that in another embodiment play a pivotal role in pathogenesis of CF.

[00046] In one embodiment, bacterial SMases C and D inhibit CFTR current, an action leading to the production of thick mucus that clogs the airways and thereby fosters further bacterial growth. In another embodiment, SMase D causes severe tissue necrosis. In one embodiment, ceramide and ceramide-1 -phosphate induce inflammation and in another embodiment trigger cell death.

[00047] In one embodiment, a reduction in CFTR activity in CF patients leads to thick mucus that clogs airways and thus fosters infection. Infection with bacteria that produce SMase activity would further suppress CFTR activity. In another embodiment, inhibition of CFTR activity arises since ceramide and ceramide-1- phosphate cannot substitute for sphingomyelin in some action that facilitates CFTR gating, which in another embodiment is initiated primarily by phosphorylating the R domain. In one embodiment, the lipid products of SMase-catalyzed sphingomyelin hydrolysis, ceramide and ceramide-1 -phosphate, further aggravate the disease by triggering inflammation in one embodiment or cell death or both in other embodiments. In one embodiment, these adverse effects associated with SMase activity are important pathogenic factors in bacterial infection in both CF and non-CF patients.

[00048] In one embodiment, CFTR plays a critical role in cholera toxin (CTX)-induced intestinal fluid secretion (secretory diarrhea)through activation of chloride channels. In one embodiment provided herein is a method of treating V. cholera induced secretory diarrhea, comprising the step of administerin to a subject in need thereof a composition comprising a bacterial SMase-C, thereby inhibiting CFTR current.

[00049] In one embodiment, the methods and compositions provided herein, comprise an agent capable of inhibiting the function or expression of bacterial SMase, whereby the agent is an antibody, or a fraction thereof, an antagonist, or a combination thereof.

[00050] In another embodiment, the antibody used to inhibit the function of the bacterial SMase used in the methods and compositions provided herein, or fraction thereof is specific against an amino acid having no less than 30% homology to the amino acid sequence represented by SEQ ID. No.'s 3,4, and 10-19.

[00051] In one embodiment, the term "antibody" includes complete antibodies (e.g., bivalent IgG, pentavalent IgM) or fragments of antibodies which contain an antigen binding site in other embodiments. Such fragments include in one embodiment Fab, F(ab')₂, Fv and single chain Fv (scFv) fragments. In one embodiment, such fragments may or may not include antibody constant domains. In another embodiment, Fab's lack constant domains which are required for Complement fixation. ScFvs are composed of an antibody variable light chain (V_L) linked to a variable heavy chain (V_H) by a flexible hinge. ScFvs are able to bind antigen and can be rapidly produced in bacteria or other systems. The invention includes antibodies and antibody fragments which are produced in bacteria and in mammalian cell culture. An antibody obtained from a bacteriophage library can be a complete antibody or an antibody fragment. In one embodiment, the domains present in such a library are heavy chain variable domains (V_H) and light chain variable domains (V_L) which together comprise Fv or scFv, with the addition, in another embodiment, of a heavy chain constant domain (C_HI) and a light chain constant domain (C_L)- The four domains (i.e., V_H - C_HI and V_L - C_L) comprise an Fab. Complete antibodies are obtained in one embodiment, from such a library by replacing missing constant domains once a desired V_H - V_L combination has been identified. [00052] Antibodies of the invention can be monoclonal antibodies (mAb) in one embodiment, or polyclonal antibodies in another embodiment. Antibodies of the invention which are useful in the compositions, methods and kits of the invention can be from any source, and in addition may be chimeric. In one embodiment, sources of antibodies can be from a mouse, or a rat, a plant, or a human in other embodiments. Antibodies of the invention which are useful for the compositions, and methods of the invention have reduced antigenicity in humans (to reduce or eliminate the risk of formation of anti-human andtibodies), and in another embodiment, are not antigenic in humans. Chimeric antibodies for use the invention contain in one embodiment, human amino acid sequences and include humanized antibodies which are non-human antibodies substituted with sequences of human origin to reduce or eliminate immunogenicity, but which retain the antigen binding characteristics of the non-human antibody.

[00053] In one embodiment, the terms "homology", "homologue" or "homologous", indicate that the sequence referred to, whether an amino acid sequence, or a nucleic acid sequence, exhibits, in one embodiment at least 30 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 72 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 75 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 80 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 82 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 85 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 87 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 90 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 92 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 95 % or more correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 97% correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits at least 99 % correspondence with the indicated sequence. In another embodiment, the amino acid sequence or nucleic acid sequence exhibits 95 % - 100 % correspondence with the indicated sequence. Similarly, as used herein, the reference to a correspondence to a particular sequence includes both direct correspondence, as well as homology to that sequence as herein defined.

[00054] In another embodiment, homology refers to sequence identity, or in yet another embodiment, may refer to structural identity, or functional identity. By using the term "homology" and other like forms, it is to be understood that any molecule, whether nucleic acid or peptide, that functions similarly, and/or contains sequence identity, and/or is conserved structurally so that it approximates the reference sequence, is to be considered as part of this invention.

[00055] Protein and/or peptide homology for any peptide sequence listed herein may be determined by immunoblot analysis, or via computer algorithm analysis of amino acid sequences, utilizing any of a number of software packages available, via methods well known to one skilled in the art. Some of these packages may include the FASTA, BLAST, MPsrch or Scanps packages, and may employ the use of the Smith and Waterman algorithms, and/or global/local or BLOCKS alignments for analysis, for example.

[00056] In one embodiment, the compositions provided herein are used in the methods provided hereinabove. In one embodiment, provided herein is a composition for treating cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising a bacterial sphingomyelinase inhibitor, wherein the bacterial sphingomyelinase inhibitor inhibits sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

[00057] In one embodiment, application of medicines against various bacterial virulence factors including phospholipases in one embodiment, or proteases, in conjunction with other effective measures, in another embodiment, is a viable near-term approach to improving length and quality of life for CF patients. In another embodiment, the same approach also benefits patients with other types of chronic bacterial infection. [00058] In one embodiment, the compositions used in the methods provided herein further comprises a carrier, excipient, emulsifier, stabilizer, sweetener, flavoring agent, diluent, coloring agent, solubilizing agent or a combination thereof.

5 [00059] In one embodiment, the compsitions described herein, used in the methods described herein is administered intravenously, intracavitarily, subcutaneously, intratumoraly, or a combination thereof.

[00060] "Intracavitary administration", as used herein, refers to administering a substanceo directly into a body cavity of a mammal. Such body cavities include the peritoneal cavity, the pleural cavity and cavities within the central nervous system, including the orbit of the eye.

[00061] In another embodiment of this invention, the small molecule agent described herein, or antibodies and their fragment is administered via the subcutaneous route. According to the5 present invention the antibodies described herein may be administered as a pharmaceutical composition containing a pharmaceutically acceptable carrier. The carrier must be physiologically tolerable and must be compatible with the active ingredient. Suitable carriers include, sterile water, saline, dextrose, glycerol and the like. In addition, the compositions may contain minor amounts of stabilizing or pH buffering agents and the like. Theo compositions are conventionally administered through parenteral routes, with intravenous, intracavitary or subcutaneous injection being preferred.

[00062] The preparation comprising the small molecule agent, polyclonal antibodies described herein, or their fragments; are administered in another embodiment, in a therapeutically5 effective amount. The actual amount administered, and the rate and time-course of administration, will depend in one embodiment, on the nature and severity of the condition being treated. Prescription of treatment, e.g. decisions on dosage, timing, etc., is within the responsibility of general practitioners or specialists, and typically takes account of the disorder to be treated, the condition of the individual subject, the site of delivery, the methodo of administration and other factors known to practitioners. Examples of techniques and protocols can be found in Remington's Pharmaceutical Sciences. [00063] Biologically active derivatives or analogs of the proteins described herein include in one embodiment peptide mimetics. Peptide mimetics can be designed and produced by techniques known to those of skill in the art. (see e.g., U.S. Pat. Nos. 4,612,132; 5,643,873 and 5,654,276, the teachings of which are incorporated herein by reference). These mimetics can be based, for example, on the protein's specific amino acid sequence and maintain the relative position in space of the corresponding amino acid sequence. These peptide mimetics possess biological activity similar to the biological activity of the corresponding peptide compound, but possess a "biological advantage" over the corresponding amino acid sequence with respect to, in one embodiment, the following properties: solubility, stability and susceptibility to hydrolysis and proteolysis.

[00064] Methods for preparing peptide mimetics include modifying the N-terminal amino group, the C-terminal carboxyl group, and/or changing one or more of the amino linkages in the peptide to a non-amino linkage. Two or more such modifications can be coupled in one peptide mimetic molecule. Other forms of the proteins and polypeptides described herein and encompassed by the claimed invention, include in another embodiment, those which are "functionally equivalent." In one embodiment, this term, refers to any nucleic acid sequence and its encoded amino acid which mimics the biological activity of the protein, or polypeptide or functional domains thereof in other embodiments.

[00065] In one embodiment, the composition further comprises a carrier, excipient, lubricant, flow aid, processing aid or diluent, wherein said carrier, excipient, lubricant, flow aid, processing aid or diluent is a gum, starch, a sugar, a cellulosic material, an acrylate, calcium carbonate, magnesium oxide, talc, lactose monohydrate, magnesium stearate, colloidal silicone dioxide or mixtures thereof.

[00066] In another embodiment, the composition further comprises a binder, a disintegrant, a buffer, a protease inhibitor, a surfactant, a solubilizing agent, a plasticizer, an emulsifier, a stabilizing agent, a viscosity increasing agent, a sweetner, a film forming agent, or any combination thereof. [00067] In one embodiment, the composition is a particulate composition coated with a polymer (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms protective coatings, protease inhibitors or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal and 5 oral. In one embodiment the pharmaceutical composition is administered parenterally, paracancerally, transmucosally, transdermally, intramuscularly, intravenously, intradermal Iy, subcutaneously, intraperitonealy, intraventricularly, or intracranially.

[00068] In one embodiment, the compositions of this invention may be in the form of ao pellet, a tablet, a capsule, a solution, a suspension, a dispersion, an emulsion, an elixir, a gel, an ointment, a cream, or a suppository.

[00069] In another embodiment, the composition is in a form suitable for oral, intravenous, intraarterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, or topicals administration. In one embodiment the composition is a controlled release composition. In another embodiment, the composition is an immediate release composition. In one embodiment, the composition is a liquid dosage form. In another embodiment, the composition is a solid dosage form.. o [00070] The compounds utilized in the methods and compositions of the present invention may be present in the form of free bases in one embodiment or pharmaceutically acceptable acid addition salts thereof in another embodiment. In one embodiment, the term "pharmaceutically-acceptable salts" embraces salts commonly used to form alkali metal salts and to form addition salts of free acids or free bases. The nature of the salt is not critical,₅ provided that it is pharmaceutically-acceptable. Suitable pharmaceutically-acceptable acid addition salts of compounds of Formula I are prepared in another embodiment, from an inorganic acid or from an organic acid. Examples of such inorganic acids are hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid. Appropriate organic acids may be selected from aliphatic, cycloaliphatic, aromatic, araliphatic, heterocyclic,o carboxylic and sulfonic classes of organic acids, example of which are formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, 4-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, 2-hydroxyethanesulfonic, toluenesulfonic, sulfanilic, cyclohexylaminosulfonic, stearic, algenic, b-hydroxybutyric, salicylic, galactaric and galacturonic acid. Suitable pharmaceutically-acceptable base addition salts include metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N-methylglucamine) and procaine. All of these salts may be prepared by conventional means from the corresponding compound by reacting, in another embodiment, the appropriate acid or base with the compound.

[00071] In one embodiment, the term "pharmaceutically acceptable carriers" includes, but is not limited to, may refer to 0.01-O.lM and preferably 0.05M phosphate buffer, or in another embodiment 0.8% saline. Additionally, such pharmaceutically acceptable carriers may be in another embodiment aqueous or non-aqueous solutions, suspensions, and emulsions, Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.

[00072] In one embodiment, the compounds of this invention may include compounds modified by the covalent attachment of water-soluble polymers such as polyethylene glycol, copolymers of polyethylene glycol and polypropylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone or polyproline are known to exhibit substantially longer half-lives in blood following intravenous injection than do the corresponding unmodified compounds (Abuchowski et al., 1981 ; Newmark et al., 1982; and Katre et al., 1987). Such modifications may also increase the compound's solubility in aqueous solution, eliminate aggregation, enhance the physical and chemical stability of the compound, and greatly reduce the immunogenicity and reactivity of the compound. As a result, the desired in vivo biological activity may be achieved by the administration of such polymer-compound abducts less frequently or in lower doses than with the unmodified compound. [00073] The pharmaceutical preparations of the invention can be prepared by known dissolving, mixing, granulating, or tablet-forming processes. For oral administration, the active ingredients, or their physiologically tolerated derivatives in another embodiment, such as salts, esters, N-oxides, and the like are mixed with additives customary for this purpose, such as vehicles, stabilizers, or inert diluents, and converted by customary methods into suitable forms for administration, such as tablets, coated tablets, hard or soft gelatin capsules, aqueous, alcoholic or oily solutions. Examples of suitable inert vehicles are conventional tablet bases such as lactose, sucrose, or cornstarch in combination with binders such as acacia, cornstarch, gelatin, with disintegrating agents such as cornstarch, potato starch, alginic acid, or with a lubricant such as stearic acid or magnesium stearate.

[00074] Examples of suitable oily vehicles or solvents are vegetable or animal oils such as sunflower oil or fish-liver oil. Preparations can be effected both as dry and as wet granules. For parenteral administration (subcutaneous, intravenous, intraarterial, or intramuscular injection), the active ingredients or their physiologically tolerated derivatives such as salts, esters, N-oxides, and the like are converted into a solution, suspension, or emulsion, if desired with the substances customary and suitable for this purpose, for example, solubilizers or other auxiliaries. Examples are sterile liquids such as water and oils, with or without the addition of a surfactant and other pharmaceutically acceptable adjuvants. Illustrative oils are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, or mineral oil. In general, water, saline, aqueous dextrose and related sugar solutions, and glycols such as propylene glycols or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions.

[00075] In addition, the composition can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents which enhance the effectiveness of the active ingredient. An active component can be formulated into the composition as neutralized pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the polypeptide or antibody molecule), 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 from 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, 2-ethylamino ethanol, histidine, procaine, and the like.

[00076] In one embodiment, the term "administering" or "contacting" refers to bringing a subject in contact with the compositions provided herein. For example, in one embodiment, the compositions provided herein are suitable for oral administration, whereby bringing the subject in contact with the composition comprises ingesting the compositions. A person skilled in the art would readily recognize that the methods of bringing the subject in contact with the compositions provided herein, will depend on many variables such as, without any intention to limit the modes of administration; the cardiovascular disorder treated, age, preexisting conditions, other agents administered to the subject, the severity of symptoms, location of the affected area and the like. In one embodiment, provided herein are embodiments of methods for administering the compounds of the present invention to a subject, through any appropriate route, as will be appreciated by one skilled in the art.

[00077] Alternatively, targeting therapies may be used in another embodiment, to deliver the active agent more specifically to certain types of cell, by the use of targeting systems such as antibodies or cell specific ligands. Targeting may be desirable in one embodiment, for a variety of reasons, e.g. if the agent is unacceptably toxic, or if it would otherwise require too high a dosage, or if it would not otherwise be able to enter the target cells.

[00078] The compositions of the present invention are formulated in one embodiment for oral delivery, wherein the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The tablets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added or a flavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar, or both. Syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor. In addition, the active compounds may be incorporated into sustained-release, pulsed release, controlled release or postponed release preparations and formulations.

[00079] Controlled or sustained release compositions include formulation in lipophilic depots (e.g. fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g. poloxamers or poloxamines) and the compound coupled to antibodies directed against tissue-specific receptors, ligands or antigens or coupled to ligands of tissue-specific receptors.

[00080] In one embodiment, the composition can be delivered in a controlled release system. For example, the agent may be administered using intravenous infusion, an implantable osmotic pump, a transdermal patch, liposomes, or other modes of administration. In one embodiment, a pump may be used (see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med. 321:574 (1989). In another embodiment, polymeric materials can be used. In another embodiment, a controlled release system can be placed in proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984). Other controlled release systems are discussed in the review by Langer (Science 249:1527-1533 (1990).

[00081] Such compositions are in one embodiment liquids or lyophilized or otherwise dried formulations and include diluents of various buffer content (e.g., Tris-HCl., acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e.g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g., ascorbic acid, sodium metabi sulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the protein, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). Also comprehended by the invention are particulate compositions coated with polymers (e.g., poloxamers or poloxamines). Other embodiments of the compositions of the invention incorporate particulate forms, protective coatings, protease inhibitors, or permeation enhancers for various routes of administration, including parenteral, pulmonary, nasal, and oral.

[00082] In another embodiment, the compositions of this invention comprise one or more, pharmaceutically acceptable carrier materials. In one embodiment, the carriers for use within such compositions are biocompatible, and in another embodiment, biodegradable. In other embodiments, the formulation may provide a relatively constant level of release of one active component. In other embodiments, however, a more rapid rate of release immediately upon administration may be desired. In other embodiments, release of active compounds may be event-triggered. The events triggering the release of the active compounds may be the same in one embodiment, or different in another embodiment. Events triggering the release of the active components may be exposure to moisture in one embodiment, lower pH in another embodiment, or temperature threshold in another embodiment. The formulation of such compositions is well within the level of ordinary skill in the art using known techniques. Illustrative carriers useful in this regard include microparticles of poly(lactide-co-glycolide), polyacrylate, latex, starch, cellulose, dextran and the like. Other illustrative postponed- release carriers include supramolecular biovectors, which comprise a non-liquid hydrophilic core (e.g., a cross-linked polysaccharide or oligosaccharide) and, optionally, an external layer comprising an amphiphilic compound, such as phospholipids. The amount of active compound contained in one embodiment, within a sustained release formulation depends upon the site of administration, the rate and expected duration of release and the nature of the condition to be treated suppressed or inhibited.

[00083] In one embodiment, the compositions described hereinabove are used in the methods described herein. Accordingly and in one embodiment, provided herein is a method of inhibiting or suppressing bacterial immunosuppression of a host immune system, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing current flowing through host's T-cell potassium channel.

[00084] Accordingly and in one embodiment, provided herein is a method for screening for an agent useful in the treatment of cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising the step of contacting a bacteria associated with cystic fibrosis infection with a candidate compound; and analyzing the bacteria for expression or function of sphingomyelinase, whereby an agent capable of reducing the expression of sphingomyelinase, inhibiting the function of sphingomyelinase, or reacting with a lipid product of sphingomyelinase -catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR) is useful in the treatment of cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection. In another embodiment, the methods and compositions provided hereinabove comprise the compounds identified by the methods provided herein.

[00085] In one embodiment, provided herein are methods and compositions for treating, or inhibiting or suppressing, or reducing the symptoms associated with cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising a compound identified by contacting a bacteria associated with cystic fibrosis infection with a candidate compound; and analyzing the bacteria for expression or function of sphingomyelinase, whereby an agent capable of reducing the expression of sphingomyelinase, inhibiting the function of sphingomyelinase, or reacting with a lipid product of sphingomyelinase -catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^* current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

[00086] In one embodiment, provided herein is a method of treating pulmonary disorder in a subject having an acute or a chronic bacterial infection, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR). In another embodiment, the compositions and methods provided hereinabove, are effective in treatment of pulmonary disorder in a subject having an acute or a chronic bacterial infection, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR). [00087] In another embodiment, the pulmonary disorder associated with chronic or acute bacterial infection is CF, infective asthma caused by bacterial, fungal, protozoal, or viral infection, non-allergic asthma, incipient asthma, wheezy infant syndrome or bronchiolytis, chronic eosinophilic pneumonia, chronic obstructive pulmonary disease (COPD), COPD that includes chronic bronchitis, pulmonary emphysema or dyspnea associated or not associated with COPD, COPD that is characterized by irreversible, progressive airways obstruction, adult respiratory distress syndrome (ARDS).

[00088] "Treating" or "treatment" embraces in another embodiment, the amelioration of an existing infection. The skilled artisan would understand that treatment does not necessarily result in the complete absence or removal of symptoms. Treatment also embraces palliative effects: that is, those that reduce the likelihood of a subsequent medical condition. The alleviation of a condition that results in a more serious condition is encompassed by this term.

[00089] In another embodiment, the term "treating" in its various grammatical forms refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of a disease state, disease progression, disease causitive agent or other abnormal condition.

[00090] In another embodiment, the term "contacting" refers to a union or junction, or apparent touching or mutual tangency of a any of the compositions described herein with a bacteria or SMase sought to be contacted. Under no circumstances does the term imply any further limitations to the process, such as by mechanism of inhibition. The methods provided herein, such as the method for inhibiting or suppressing a bacterial infection in one embodiment, are defined to encompass the inhibition of bacterial activity by the action of the compounds and their capabilities of inhibiting or suppressing the expression or function of SMase.

[00091] In another embodiment, the term "contacting" refers to providing conditions to bring the compound into proximity to a sphingomyelinase, such as SMase C in one embodiment, to allow for inhibition of activity of the sphingomyelinase. For example, contacting a compound of the present invention with an sphingomyelinase can be accomplished by administering the compound to a subject or a host, or by isolating cells, e.g., cells in lymph nodes, and admixing the cells with the compound under conditions sufficient for the compound to diffuse into or be actively taken up by the cells, in vitro or ex vivo, into the cell interior. When ex vivo administration of the compound is used, for example, in treating a bacterial s infection, the treated cells can then be reinfused into the host from which they were taken.

[00092] The term "about" as used herein means in quantitative terms plus or minus 5%, or in another embodiment plus or minus 10%, or in another embodiment plus or minus 15%, or in another embodiment plus or minus 20%.

I₀

[00093] The term "subject" refers in one embodiment to a mammal including a human in need of therapy for, or susceptible to, a condition or its sequelae. The subject may include dogs, cats, pigs, cows, sheep, goats, horses, rats, and mice and humans. The term "subject" does not exclude an individual that is normal in all respects, is

[00094] The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.

EXAMPLES

20

Materials and Methods:

Summary

2₅ [00095] Channel currents were recorded under two-electrode voltage clamping from oocytes injected with cRNA encoding relevant channels. The bath solution contained (in mM): 95/5 NaCl/KCl (or 80/20 for tail current measurements), 0.3 CaCl₂, 1 MgCl₂ and 10 HEPES, pH 7.6. Chemical reagent and lipase stock solutions (2 μl) were added manually to a 100-μl recording chamber. Unless specified otherwise, the final concentrations of recombinant

30 ^BaSMase C, ^SaSMase C, SMase D and native PC-PLC were 40, 14, 4 and 50 ng ml^"1, respectively.

Molecular bioloev and electrophysiological recordines [00096] The CFTR, CTFR-ΔR, and PKA-C were subcloned in the pGEMHE plasmid (46) (see acknowledgment for the source of all cDNA constructs). The ΔF508 and Rl 17H mutant CFTR cDNAs were obtained through PCR-based mutagenesis and confirmed by DNA sequencing. The cRNAs were synthesized with T3 or T7 polymerase using the corresponding linearized cDNAs as templates. Channel currents were recorded from whole oocytes previously injected with the corresponding cRNAs and stored at 18 ⁰C, using a two-electrode voltage clamp amplifier (Warner OC-725C). Background leak currents were not corrected. The resistance of electrodes filled with 3 M KCl was 0.2 - 0.3 MΩ. Unless specified otherwise, the bath solution contained (in mM): 95 Na₊ (Cl- + OH-), 5 KCl, 0.3 CaCl₂, 1 MgCl₂ and 10 HEPES; pH was adjusted to 7.6 with NaOH. SMases (2 - 5 μl) were manually added to the recording chamber (100 :1). B. cereus PC-PLC was purchased from Sigma.

Identification, purification and production of recombinant SMase D

[00097] For purification, Loxosceles reclusa venom samples purchased from Spider Pharm (Yarnell AZ) were loaded onto a size-exclusion FPLC column (Superdex G200, Pharmacia) where the running buffer contained 50 mM NaCl and 5 mM Tris chloride (pH 6.4). The most abundant active fraction was subsequently loaded onto a reverse-phase HPLC column (C 18, Beckman) and eluted with a water- acetonitrile gradient (1% per minute). Aqueous and organic mobile phases contained 0.1% and 0.07% trifluoroacetic acid, respectively. The SMase D activity was drastically reduced after HPLC purification because of exposure to low pH. The sample corresponding to the large peak on the HPLC chromatogram contained the activity, and was subsequently analyzed with MALDI-TOF mass spectrometry (MS) for mass identification and also run on SDS-PAGE for further purification. The single visible band on SDS-PAGE was excised and digested with trypsin. The digestion products were then analyzed with LCMS/MS, yielding 9 and 12 partial peptide sequences corresponding to LrI and LrI isoforms of SMase D (see sequence listing in the table below)

A B

"WENFNDFlK" "WEYFSDFtK"

"ATTPGDSKYHEK^M "LVLWFDLK"

"LVLWFDLK" "TGSLVDNQAYDAGKK¹*

"TGSLYDNQAYDAGKKT¹⁴ "¹NlXKHYWNNGNNGGFi*¹* '"NILQHYWNNGNNGGR"" "¹HYWNNGNNGGR⁰*

•"AYIVLSIPNIAHYK""¹ "VITSFKETLK¹¹'

"'ETLTSEGMPELMEK™ "'EUKSEGHPELMOK¹** "VGYOFSGNDDIDK¹"³ "¹SEGHPeLMDK «* ""IATYDDNPWETPKN'⁷' ^M1VGHDFSGNDAIGDvGNAYK'" "¹VTTWIVDK *»

"VYΥWTVOKR∞ '"IATYDDNPWETFKN¹"

Molecular cloning ofSMase C

[00098] Full length and "truncated" cDNAs of SMase C were produced with PCR, primed with a pair of oligonucleotides corresponding to the 5' or 3' translated regions against the genomic DNA isolated from B. anthracis STI and 5. aureus ATCC 29213, respectively. The truncated cDNA of S. aureus was further extended to full length with PCR.

Production of recombinant SMases [00099] To produce the mature recombinant wild-type and mutant SMases, E. coli BL21 (DE3) cells were transformed with the respective cDNAs cloned into pET30 vector (Novagen, San Diego, CA), grown in LB medium to -0.6 OD at 600 nm, and induced with 1 mM IPTG for 2 hours. The bacteria were harvested, resuspended, and sonicated. The resulting samples were loaded onto a cobalt affinity column and eluted by stepping the imidazole concentration from 50 mM to 500 mM (all SMase proteins contain N- and C- terminal His tags). The imidazole was later removed by dialysis.

Example 1: The Phosphodiester Group is Significant in Voltage Gating of Ion Channel

[000100] Kv2.1 channels, expressed in Xenopus oocytes, interact with sphingomyelin present mainly in the outer leaf of plasma membranes. To investigate the importance of phospho-head groups of membrane lipids in Kv-channel gating bacterial sphingomyelinases C and D (SMases C and D) was used. Both enzymes specifically hydrolyse sphingomyelin, but in different ways (Fig. Ia): SMase D removes only choline and leaves the lipid ceramide- 1 -phosphate behind in the membrane, whereas SMase C removes phosphocholine, leaving ceramide behind. A comparison of the effects of these two enzymes on the channels will help to elucidate the functional significance of the phosphodiester group in voltage gating.

[000101] SMase D of Corynebacterium pseudotuberculosis (^CpSMase D) shifts the conductance -voltage (G-V) relation of Kv2.1 by about -3OmV (ref. 8) (Fig. Id), allowing channels to be activated at a negative voltage at which they otherwise remain largely deactivated (Fig. Ic). The effect is maximal within 2 min and persists for at least 24 h (Fig.

Ic, d; cells cannot regenerate sphingomyelin from ceramide- 1 -phosphate). It does not require direct exposure of channels to SMase D, because it also occurred in Kv2.1 -expressing oocytes that had been treated with SMase D and then thoroughly washed before injection with Kv2.1 complementary RNA (cRNA; Fig. Ie). Additional exposure of such oocytes to

SMase D, as expected, caused no further shift. SMase D therefore acts through its lipase activity rather than by direct binding to the channel. Certain studies by the present applicants detailed in US Provisional Application Serial No. 60/918,069, incoporated herein by reference in its entirety, shows an electrostatic mechanism by which removal of the positively charged choline favours the activated state of the positively charged voltage sensors.

[000102] Unlike SMase D, SMase C removes the negatively charged phosphodiester group as well as choline. SMase C of Bacillus anthracis (^fiαSMase C) decreases Kv2.1 current by about 90% (Fig. 2a-c) and the decrease is independent of voltage for both partial and complete enzymatic treatment (Fig. 2d). SMase C of Staphylococcus aureus (^5αSMase C) caused a comparable level of inhibition but with faster kinetics, and only the kinetics, not the extent of inhibition, are dependent on enzyme concentration (Fig. 2b). SMase C therefore inhibits the channels by means of its enzymatic activity rather than by direct binding.

Example 2: The Effect of SMase C Disruption of Membrane Rafts on Ion Channel

Gating is Negligible

[000103] Sphingomyelin and cholesterol may form membrane domains called 'lipid rafts'. A cholesterol-extracting agent methyl-β-cyclodextrin (MβCD) was used to test whether the disruption of sphingomyelin-cholesterol interactions underlies the SMase C effect shown inExample 1. Exposure of oocytes to 5mM MβCD for 2 h (a common regimen) had little effect on the G-V curves obtained before and after SMase C treatment (Fig. 2e). The channel-inhibitory eftect ot SMase C therefore does not seem simply to reflect the disruption of sphingomyelin- cholesterol interactions.

Example 3: SMase C Inhibits Ion Channel by Removing Phospho-Heads Around Voltage Sensors

[000104] Looking for evidence that the observed inhibition of Kv2.1 by SMase C results from the removal of sphingomyelin phospho-heads around voltage sensors, it was tested whether hanatoxin, which is known to bind to voltage sensors, prevents SMase C from reaching the lipids and thus mitigates its effect (kinetics of hanatoxin inhibition and recovery are slow compared with the rate of inhibition caused by SMase C). Exposure to 3 μM hanatoxin inhibits Kv2.1 current by about 70% at 30 mV and by about 30% at 60 mV(Fig. 2f). (The extent of current reduction does not reflect the extent of hanatoxin binding; this is because a channel bound with hanatoxin can still be activated by a depolarization that is stronger than usual) In the absence of hanatoxin, SMase C decreases current by 90% at all voltages, but in the presence of hanatoxin the observed decrease in current is much smaller (Fig. 2f). Hanatoxin binding therefore protects a large fraction of channels against SMase C, which supports the notion that SMase C inhibits the channels by removing sphingomyelin phosphoheads around voltage sensors.

Example 4: Sphingomyelin Phospho-Heads are Essential in Allowing Voltage Sensors to Undergo Early Transitions Involving the Bulk of Gating-Charge Movement

[000105] The issue of whether SMase C affects gating current, a capacitive current arising from voltage sensor movement was examined. Unfortunately, measuring the gating current of Kv2.1 is technically challenging as a result of the lack of high-expression mutants that produce large gating currents but little ionic current. To circumvent this limitation, the well-studied Shaker Kv channel for ionic current, and its non-conducting V478W mutant was used for gating current measurements. SMase C treatment decreases both ionic and gating currents by about half (Fig. 3a-d). Such a proportional decrease could be a reflection of the fact that about half of the channels interact with sphingomyelin in such a manner that removal of the negatively charged phosphate groups creates an insuperable energy barrier for the positive gating charges to move during early gating transitions, effectively precluding activation by experimentally accessible depolarizations. Alternatively, the proportional decrease might be a coincidence peculiar to this particular Kv subtype, in which half of the gating charges in individual channels remain functional and these channels are therefore susceptible to partial activation. However, an approximately proportional decrease in ionic and gating currents was observed in Kv 1.3 channels. Additionally, SMase C does not decrease the slope of the Q-V curve (Fig. 3f), a parameter related to the effective number of gating charges. It is thus evident that in about half of the Shaker channels (more than half for Kv 1.3 and Kv2.1), sphingomyelin phospho-heads are essential in allowing voltage sensors to undergo early transitions involving the bulk of gating-charge movement. Phospholipids other than sphingomyelin presumably fulfill that role in the remaining channels. The two channel populations may exist in membrane domains that differ in their sphingomyelin content.

[000106] The above inference that the head groups of other phospholipids besides sphingomyelin enable early gating transitions in about half of Shaker channels expressed in Xenopus oocytes does not exclude the possibility that voltage sensors in these channels still interact with sphingomyelin, but in a manner that is important for late (downstream) transitions only. Rather, the very existence of channels whose early transitions do not require sphingomyelin permits investigation of the impact of sphingomyelin phospho-head groups on late transitions. It was found that the kinetics of ionic currents that remain after SMase C treatment is markedly slowed (Fig. 3e), whereas that of the remaining gating currents is barely affected (Fig. 3g, h). Removal of phospho-head groups of sphingomyelin must therefore also increase (but not insuperably) the energy barrier for one or more gating transitions downstream of the bulk of the gating charge movement. This modest increase in the transition energy for one or more late rate-limiting steps is consistent with the occurrence of only 5-10% of total gating charge movement late in the gating sequence. The resulting small change in the shallow part of the normalized Q-V curve at more depolarized potentials (Fig. 3f) is reminiscent of an S4 mutant (L370V) whose late transition is altered.

Example 5: SMase C Acts on Kv Channels by Means of its Lipase Activity

[000107] Consistent with the conclusion that SMase C acts by means of its lipase activity (See e.g. Example 1), was the observation that gating current did not recover after washout of SMase C (Fig. 3i). For further confirmation, the fact that SMase C acts only on sphingomyelin, not on ceramide- 1 -phosphate was exploited. Conversion of sphingomyelin to ceramide-1 -phosphate with SMase D was found to shift the activation curve to the left (Fig. 31); that is, it allows the mobilization of gating charges at more negative voltages (Fig. 3j). Indeed, pretreatment with SMase D prevents subsequently added SMase C from causing current inhibition (Fig. 3j, 1). The differing effects of SMases C and D indicate that the lipid product of one or both of the enzymes continues to interact with the channel protein for the duration of the experiment. The persistence probably reflects strong channel-lipid interactions and/or the confinement of relevant lipid molecules to microdomains. Consistent with this view was the observation that channel current was not significantly affected by the acute addition of products of sphingomyelin hydrolysis catalysed by either enzyme (Fig. 2g- j). In one embodiment, the term "channel protein" refers to voltage-gated ion channels, including the pore-forming α-subunit proteins ("α-subunits") and the cytoplasmic.beta.subunit proteins (also known in the art as "auxiliary subunits'Or " β- subunits").

[000108] To rule out the possibility that the observed effect of SMase C results from non-specific hydrolysis of the predominant outer-leaf phospholipid phosphatidylcholine (PC), a PC-specific phospholipase (PC-PLC) (Fig. Ib) from Bacillus cereus was tested directly against the channels, and was found to be only modest (11.2+2.8%) gating current depression (Fig. 3k), in contrast with about 50% for SMase C. SMase C therefore does not cause channel inhibition by hydrolysing PC. The small inhibition caused by PC-PLC is, again, consistent with the interaction of some channels with phospholipids other than sphingomyelin.

Example 6: SMase C Acts as an Immunosuppressant by Interaction with Kyl.3

SMase C was originally termed β-haemolysin after its haemolytic effect in vitro, yet its role in pathogenesis remains largely unknown. The fact that inhibition of KvI .3 in lymphocytes is immunosuppressive motivated the test ^5αSMase C interaction with human KvI .3. It was found that ^SαSMase C eliminates well over half of the ionic current of KvI .3 and of the gating current of its non-conducting W384F mutant (Fig. 4), in an approximately proportional manner as with Shaker channels. This finding raises the intriguing possibility that the SMase C action against KvI .3 helps S. aureus to neutralize host defences.

Example 7: Discovery and identification of CFTR-inhibiting activity in Brown spider venom [000109] Venoms from over 100 invertebrates were screened and those from

Loxosceles reclusa (Brown spider) and Loxosceles arizonica were found to have CFTR- inhibiting activity. An example with Brown spider venom is shown in Fig. 5A. Current (assayed during voltage steps) was activated by elevating intracellular cAMP with 0.1 mM EBMX (isobutylmethylxanthine, a phosphodiesterase inhibitor) and was dramatically reduced on addition of the venom. The venom also inhibited current through the constitutively active mutant CFTR-ΔR channel, as shown in Fig. 5B.

[000110] To isolate the CFTR-inhibiting activity, 10 μl of Brown spider venom were subjected to size-exclusion FPLC (fast protein liquid chromotography) which yielded several active fractions (Fig. 5C). The most abundant fraction was subjected to reverse-phase HPLC (high performance liquid chromatography; Fig. 5D), and the large peak obtained was run on SDS PAGE (sodium dodecyl sulphate polyacrylamide gel electrophoresis). The single band from SDS PAGE was digested into fragments with trypsin. Of these fragments, nine were identified by LCMS/MS (HPLC coupled with tandem mass spectrometry) as emanating from the LrI isoform of sphingomyelinase D (SMase D), and twelve from the LrI isoform (see SEQ. Listing). The observed masses of the active materials matched the theoretical masses of the two isoforms (for LrI , 31.2 kD theoretical and 31.2 kD observed; and for Lr2, 31.4 kD theoretical and 31.3 kD observed).

[000111] To confirm that the CFTR-inhibiting activity of the venom was from SMase

D, a recombinant version of Lr2SMase D using the E. coli expression system (see Methods). The recombinant "SMase D inhibited currents through both wild-type CFTR and CFTR-ΔR (Fig. 6A-D). To be certain that "SMase D rather than extraneous material in the bacterial product was responsible, two histidine residues that are known to be essential to the enzymatic activity of SMase D were replaced by alanine. This mutant of "²SMase D had no effect on CFTR current (Fig. IA), confirming that the observed CFTR inhibition by the venom was caused by Lr2 and/or other isoforms of SMase D. A further test, consistent with this conclusion, is that exposure of "SMase D to low pH, a procedure known to inactivate the enzyme eliminated its ability to inhibit CFTR current (Fig. IB).

[0001 12] Whether SMase D inhibit CFTR current by virtue of its lipase activity as suggested by the above, or by directly binding to CFTR was investigated. Irreversible enzymatic hydrolysis is suggested by the fact that no recovery occurred after washout of "SMase D for 30 minutes (Fig. 8/4). Also, the active site of SMase D contains a Mg²* ion, and a bound Mg2+ is essential for lipase activity. The CFTR-inhibiting effect of "SMase D, like its lipase activity, requires Mg²* (Fig. 1C). Altogether, these observations strongly suggest that the lipase activity of SMase D is important in suppressing CFTR current.

[000113] Whether the products of sphingomyelin hydrolysis is responsible for the inhibition was investigated. The enzyme catalyzes removal of the choline group of sphingomyelin (which is naturally present mainly in the outer leaf of the membrane bilayer). The hydrolysis products are choline and ceramide-1 -phosphate (Fig. ID). Acute addition of either to the bath solution (with ceramide-1 -phosphate pre-dissolved in methanol/dodecane) caused no significant inhibition of CFTR-.R current (Fig. SB and C). Acute addition of sphingomyelin (pre-dissolved in methanol) failed to restore the current after SMase D treatment (Fig. 8D).

Example 8: Inhibition of CFTR currents by bacterial SMase D

[000114] The bacterial pathogen Corynebacterium pseudotuberculosis produces

^SMase D that has -30% amino-acid identity to SMase D from spider venom. Recombinant ^c'SMase D, like "SMase D (Fig. 6C and D), inhibited currents through both CFTR and CFTR-.R (Fig. 6E and F). The respiratory pathogen Arcanobacterium haemolyticum also produces SMase D. Significantly, infection with this bacterium is invariably associated with respiratory symptoms.

Example 9: Inhibition of CFTR current by bacterial SMase C

[000115] The genome of many clinically important pathogenic respiratory tract bacteria, including B. anthracis and S. aureus, encodes SMase C instead of SMase D. SMase C hydrolyzes sphingomyelin to phosphocholine and ceramide, rather than to choline and ceramide-l - phosphate (Fig. 9A). The role of SMase C in infection remains largely unknown, in part because studies with "purified" native bacterial SMase C have been notoriously difficult to interpret due to the presence of additional pathogenic factors, including other phospholipases. For example, when native ^SaSMase C (from S. aureus) and ^BcSMase C (from B. cereus) that were obtained from a common commercial source were analyzed, the former showed numerous bands on SDS PAGE, and the latter showed two bands on SDS PAGE and many peaks in mass spectrum (supporting Fig. 6, where the molecular weight of SMase C is 30 - 35kDa).

[000116] To circumvent the problem of impurity, recombinant SMase C with cDNAs that were cloned from B. anthracis and S. aureus were produced. Recombinant SMase C from either bacterium inhibited currents through both CFTR (Fig. 9B, C, E, and F) and

CFTR-ΔR (Fig. 1OA and B). The inhibitory effect required the presence of Mg²* (Fig. 1OC and D), as did inhibition with ¹^²SMaSe D (Fig. 7C). Acute addition of the SMase C hydrolysis products phosphocholine or ceramide caused no significant current inhibition (Fig. 8E and F).

[000117] The specificity of SMases for sphingomyelin over phosphatidylcholine is almost certainly not absolute, which raises the question of whether the observed effect of SMases on CFTR might result from enzymatic hydrolysis of phosphatidylcholine, which is usually the predominant type of phospholipid in the outer leaf of the membrane . This possibility was tested with a phospholipase C (^BcPC-PLC) from Bacillus cereus, which (relatively) specifically removes phosphocholine from phosphatidylcholine. No significant inhibition of CFTR-ΔR current following addition of the enzyme (Fig. 9D and G) were found.

Example 10: Inhibition of natural CFTR mutants by sphingomyelinases

[0001 18] About 70% of CF cases are caused by deletion of residue F508 (CFTR-

ΔF508). The mutation impairs folding and trafficking of the protein but, once inserted into the plasmalemma, the ΔF508 mutant channel exhibits robust activity comparable to that of the wild type. It is unclear whether a small but functionally important amount of CFTR- ΔF508 is present in the airway epithelial cells of these CF patients. In contrast, the product of CFTR-Rl 17H, another relatively common mutant, folds properly, is well transported to the cytoplasmic membrane in affected patients, but is only partially functional. SMases C and D inhibited currents through both ΔF508 and Rl 17H mutant CFTR channels (Fig. 11).

Example 11: Dependence of SMase-induced inhibition on CFTR phosphorylation level [000119] 0.1 mM IBMX were initially used to elevate intracellular cAMP of oocytes.

This treatment activated CFTR current to 30 - 40% of maximum, and SMases typically inhibited 80 - 90% of the activated current (Fig. 124 and B). ImM IBMX activated more CFTR current (40 - 90% of maximum), but the extent of inhibition by SMase D became highly variable (Fig. 124 and B). To test the likely possibility that this variability reflects different cAMP levels in individual oocytes, the cAMP level were further boosted with the combination of 1 mM IBMX and 50 μM forskolin (an adenylate cyclase stimulator) to maximally activate the CFTR channel. Under this condition, SMase C (which removes both choline and phosphoryl groups) inhibited 75% of the current (Fig. 124), while SMase D (which removes only the choline group) inhibited only 20% (Fig. \2B), as investigated further below. SMase D inhibition is reversible, and can be overcome by high cAMP concentration. In Fig. 12G, CTFR current activated by 0.1 mM IBMX was almost completely inhibited by addition of SMase D; raising the cAMP level with 1 mM IBMX plus 50 μM forskolin not only restored the current, but boosted it to about twice the original level. Such reactivation of SMase D-suppressed current demonstrates that the enzymatic treatment does not render the CFTR channel irreversibly inactive.

[000120] To check whether the antagonistic effect of cAMP on SMase inhibition results from PKA mediated phosphorylation, CFTR was co-expressed with the catalytic subunit of PKA (PKA-C), which has constitutive kinase activity independent of cAMP. Co-injection of PKA-C and CFTR cRNAs induced robust CFTR current, while the background non-CFTR- current following PKAC cRNA injection alone remained minimal (Fig. 14). A 0.32 ng dose of PKA-C cRNA produced practically maximal current: the current only increased 5 "1 % (mean " sem; n = 3) on addition of 1 mM IBMX plus 50 μM forskolin. As expected, the ability of SMase to inhibit CFTR current decreased with increasing amounts of co-injected PKA-C cRNA (Fig. 12C and D). A relatively high dose of PKA-C cRNA was needed to overcome the inhibition caused by ^δαSMase C, a result which parallels the finding above that SMase C inhibition was more difficult to overcome with a boost of cAMP than was SMase D inhibition (Fig. 124 and B).

[000121] These findings strongly suggest that overcoming SMase inhibition requires a higher level of R domain phosphorylation. This idea is supported by findings that SMase inhibition of CFTR-ΔR (which has no phosphorylatable R domain) was not significantly affected by coexpression of PKA-C (Fig. 12£ and F). This finding is consistent with the idea that the open state of CFTR-ΔR expressed in an oocyte is not as stable as that of the wild- type channel "fully" activated by PKA.

Example 12: SMase induces cell death in epithelial cell cultures

[000122] As shown in Fig. 15, Calu-3 cells were cultured in a 48-well dish containing

MEM Alpha medium supplemented with 10% fetal bovine serum (FBS) and maintained in a 5% CO₂ incubator at 37°C. Upon reaching confluence, cells were washed with and cultured in defined MEM Alpha media that contained recombinant sphingomyelinase C (SMase C) of Staphylococcus aureus (9 ng/μl). An equal volume of the enzyme buffer solution was added to the control group (i.e., 1 μl buffer to 100 μl culture medium). Triplets were carried out for both the SMase C and the control groups in each trail. After the indicated period of SMase C treatment, cells were taken out the incubator, trypsinized, washed with and then resuspend in. PBS. To determine viability, cells were incubated in a 1 :1 (VTV) mixture of PBS and 0.4% Trypan blue solution (Sigma) at room temperature for 15 minutes. Blue-stained and non-stained cells were counted with a hemacytometer chamber. Percentage of non-viable (stained) cells was plotted as the mean (± s.e.m.) of six independent trails for each time point.

[000123] The demonstrates that SMase can induce cell death in epithelial cell cultures, thereby providing a new cell based assay/viability readout for identifying SMase inhibitors.

[000124] Having described preferred embodiments of the invention with reference to the accompanying drawings, it is to be understood that the invention is not limited to the precise embodiments, and that various changes and modifications may be effected therein by those skilled in the art without departing from the scope or spirit of the invention as defined in the appended claims.

Claims

What is claimed is:

1. A method of modulating a potassium channel (K⁺), a chlorine channel (Cl^") or their combination in a subject, comprising the step administering to said subject a composition comprising a therapeutically effective amount of a bacterial sphingomyelinase (SMase), thereby cleaving sphingomyelin.

2. The method of claim 1, whereby the potassium channel is Kv2.1, Kvl.3 or their combination.

3. The method of claim 1, whereby the bacterial sphingomyelinase is SMase C, SMase D or their combination.

4. The method of claim 1, whereby the SMase is P. aeruginosa, B. anthracis, S. aureus, C. pseudotuberculosis, or their combination.

5. The method of claim 1 , whereby the composition further comprises a carrier, excipient, emulsifϊer, stabilizer, sweetener, flavoring agent, diluent, coloring agent, solubilizing agent or a combination thereof.

6. The method of claim 1, whereby said composition is in the form of a pellet, a tablet, a capsule, a solution, a suspension, a dispersion, an emulsion, an elixir, a gel, an ointment, a cream, or a suppository.

7. The method of claim 1, whereby said composition is in a form suitable for oral, intravenous, intraaorterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, intracavital, intracranial, or topical administration.

8. The method of claim 1, whereby said composition is a controlled release composition.

9. The method of claim 1, whereby said composition is an immediate release composition

10. The method of claim 1, whereby said composition is a liquid dosage form.

1 1. The method of claim 1 , whereby said composition is a solid dosage form.

12. The method of claim 3, whereby the SMase is SMase D.

13. The method of claim 12, whereby the step of administering results in a shift of the conductance -voltage (G-V) relation of the potassium channel.

14. The method of claim 3, whereby the SMase is SMase C

15. The method of claim 14, whereby the step of administering results in decreasing the current of the potassium channel.

16. A method of inhibiting or suppressing bacterial immunosuppression of a host immune system, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing current flowing through host's immune- cell potassium channel.

17. The method of claim 16, whereby the host's immune cell potassium channel is KvI .3

18. The method of claim 16, whereby the bacteria source of the SMase is P. aeruginosa, B. 5 anthracis, S. aureus, C. pseudotuberculosis, or their combination.

19. The method of claim 16, whereby contacting is via oral, intravenous, intraaorterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, intracranial, intracavital, or topical administration.

20. The method of claim 16, whereby the baterial SMase is SMase C. 0

21. The method of claim 16, whereby inhibiting or suppressing the bacterial immunosuppression of a host immune system, comprises contacting the subject with an agent capable of inhibiting the function or expression of bacterial sphingomyelinase.

22. The method of claim 16, whereby the agent is an antibody, or a fraction thereof, an antagonist, or a combination thereof. 5

23. The method of claim 16, whereby the host's immune cell is a T-cell

24. A method of treating a bacterial infection of a host, comprising the step of contacting the host with a composition comprising an agent capable of inhibiting the function or expression of the bacteria's sphingomyelinase (SMase), thereby inhibiting or suppressing modulation of the host's T-cell potassium channel. 0

25. The method of claim 24, whereby the host's T-CeIl potassium channel is KvI .3

26. The method of claim 24, whereby the bacteria is P. aeruginosa, B. anthracis, S. aureus, C. pseudotuberculosis, or their combination.

27. The method of claim 24, whereby contacting is via oral, intravenous, intraaorterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, intracranial,5 intracavital, or topical administration.

28. The method of claim 24, whereby the baterial SMase is SMase C.

29. The method of claim 24, whereby the agent is an antibody, or a fraction thereof, an antagonist, or a combination thereof.

30. A method of suppressing the immune system of a host, comprising the step ofo contacting the host with a bacterial sphingomyelinase, thereby modulating a potassium channel on the host's immune-cell.

31. The method of claim 30, whereby the host's immune cell potassium channel is KvI .3

32. The method of claim 30, whereby the bacteria source of the SMase is P. aeruginosa, B. anthracis, S. aureus, C. pseudotuberculosis, or their combination.

33. The method of claim 30, whereby contacting is via oral, intravenous, intraaorterial, intramuscular, subcutaneous, parenteral, transmucosal, transdermal, intracranial, intracavital, or topical administration.

34. The method of claim 30, whereby the baterial SMase is SMase C.

5 35. The method of claim 30, whereby the host's immune cell is a T-cell.

36. The method of claim 24, whereby treating comprises inhibiting, suppressing, reducing incidence, alleviating symptoms, eliminating recurrence, preventing recurrence, delaying incidence, preventing incidence, improving symptoms, improving prognosis, curing or combination thereof. o

37. A method of treating cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising the step of inhibiting a bacterial sphingomyelinase- catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

38. A method of inhibiting or suppressing cystic fibrosis (CF) in a subject having a chronics or an acute bacterial infection, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

39. A method of reducing symptoms of cystic fibrosis (CF) in a subject having a chronic0 or an acute bacterial infection, comprising the step of inhibiting a bacterial sphingomyelinase-catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

40. The method of any one of claims 37-39, further comprising the step of removing a lipidS product of sphingomyelinase -catalyzed sphingomyelin hydrolysis, thereby removing compounds aggrevating inflammation.

41. The method of any one of claims 37-39, whereby the sphingomyelinase is SMase-D, SMase-C or a combination thereof.

42. The method of any one of claims 37-39, whereby the chronic or acute bacterial0 infection is caused by 5. aureus, B. cereus, P. aeruginosa , Corynebacterium pseudotuberculosis or a combination thereof.

43. The method of claim 40, whereby the lipid product of sphingomyelinase-catalyzed sphingomyelin hydrolysis is ceramide, ceramide- 1 -phosphate, choline, phosphocholine or a combination thereof.

44. The method of any one of claims 37-39, whereby the step of inhibiting, comprises contacting the subject with an agent capable of inhibiting the function or expression of bacterial sphingomyelinase.

45. The method of claim 44, whereby the agent is an antibody, or a fraction thereof, an s antagonist, or a combination thereof.

46. The method of claim 45, whereby the antibody or fraction thereof is specific against an amino acid having no less than 30% homology to the amino acid sequence represented by SEQ ID. No.'s 3,4, and 10-19.

47. A composition for treating cystic fibrosis (CF) in a subject having a chronic or ano acute bacterial infection, comprising a bacterial sphingomyelinase inhibitor, wherein the bacterial sphingomyelinase inhibitor inhibits sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR).

48. The composition of claim 47, wherein the sphingomyelinase is SMase-D, SMase-C ors a combination thereof.

49. The composition of claim 47, wherein the chronic or acute bacterial infection is caused by S. aureus, B. cereus, P. aeruginosa , Corynebacterium pseudotuberculosis or a combination thereof.

50. The composition of claim 47, wherein the bacterial sphingomyelinase inhibitor is an0 antibody, or a fraction thereof, an antagonist, or a combination thereof.

51. The composition of claim 47, wherein the antibody or fraction thereof is specific against an amino acid having no less than 30% homology to the amino acid sequence represented by SEQ ID. No.'s 3,4, and 10-19.

52. A method for screening for an agent useful in the treatment of cystic fibrosis (CF) in a5 subject having a chronic or an acute bacterial infection, comprising the step of contacting a bacteria associated with cystic fibrosis infection with a candidate compound; and analyzing the bacteria for expression or function of sphingomyelinase, whereby an agent capable of reducing the expression of sphingomyelinase, inhibiting the function of sphingomyelinase, or reacting with a lipid product of sphingomyelinaseo -catalyzed sphingomyelin hydrolysis, thereby enhancing Cl^" current flowing through an activated cystic fibrosis transmembrane conductance regulator (CFTR) is useful in the treatment of cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection.

53. The method of claim 52, whereby the lipid product of sphingomyelinase-catalyzed sphingomyelin hydrolysis is ceramide, ceramide-1 -phosphate, choline, phosphocholine or a combination thereof.

54. The method of claim 52, whereby the sphingomyelinase is SMase-D, SMase-C or a combination thereof.

55. The method of claim 52, whereby the bacteria is 5. aureus, B. cereus, P. aeruginosa , Corynebacterium pseudotuberculosis or a combination thereof.

56. The method of claim 52, whereby the agent capable of reducing the expression of sphygomyelinase is specific against a nucleic acid having no less tha 30% homology with the nucleic acid encoding SEQ K). No.'s 3,4 and 10-19.

57. A compound identified by the method of claim 52.

58. A method of treating cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising the step of contacting the subject with the compound of claim 57.

59. A method of inhibiting or suppressing cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising the step of contacting the subject with the compound of claim 57.

60. A method of reducing symptoms associated with cystic fibrosis (CF) in a subject having a chronic or an acute bacterial infection, comprising the step of contacting the subject with the compound of claim 57.

61. A method of screening for compounds capable of inhibiting bacterial sphyngomyelinase activity comprising: contacting in each of a plurality of reaction vessels in a high throughput screening array, an epithelial cell and a bacterial sphyngomyelinase with a test compound and an indicator compound and measuring the effect of the test compound on the viability of the epithelial cell to thereby identify compounds cpabale inhibiting the activity of bacterial sphyngomyelinase.

62. The method of claim 61 , whereby the sphingomyelinase is SMase-D, SMase-C or a combination thereof.

63. The method of claim 61 , whereby the indicator is trypan blue solution.