WO2006075172A2

WO2006075172A2 - Methods for identifying compounds capable of modulating cftr/ck2 interaction and/or cftr phosphorylation by ck2

Info

Publication number: WO2006075172A2
Application number: PCT/GB2006/000122
Authority: WO
Inventors: Anil Mehta; Russell Crawford; Kathryn Treharne
Original assignee: University Of Dundee
Priority date: 2005-01-13
Filing date: 2006-01-13
Publication date: 2006-07-20
Also published as: WO2006075172A3; GB2437681B; GB0715741D0; GB2437681A

Abstract

A method of identifying a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 comprising exposing CFTR and/or CK2 to a test compound and assessing the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. A method of screening for compounds of use in preventing or treating a secretory state or disorder and/or a cancer and/or cystic fibrosis wherein CFTR and/or CK2 is exposed to a test compound and the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 is assessed.

Description

Methods

The present invention relates to preventing or treating a secretory state or disorder, cancer or cystic fibrosis.

The listing or discussion of a prior-published document in this specification should not necessarily be taken as an acknowledgement that the document is part of the state of the art or is common general knowledge.

Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in a nucleotide-gated, small conductance epithelial chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan et al (1989) 245(4922): 1066-1073).

The CFTR polypeptide is a membrane protein and contains 1480 amino acids. The commonest mutant form of CFTR that causes most CF occurs when one phenylalanine (F) is missing at position 508 in CFTR (ΔF508, also named F508 del): depending on the geography 70-90% of patients with cystic fibrosis (CF) are either homozygous for the common mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) or compound heterozygous with another cftr mutation.

The consequences of the loss of a single amino acid from a total of 1480 residues that comprise wild type CFTR are difficult to reconcile with the wide spectrum of CF pathology if ion channel dysfunction is used as the sole pathogenic mechanism. It is presently unknown how mutation of CFTR induces its pleiotropic effects that range from defective embryogenesis in the foetal gut to enhanced salt absorption in the adult lung and an excess of white cell migration into the lung in the absence of infection. It is as if the CF state over primes the epithelia of the body against attack and in the process this destroys the epithelial lining of the lung (Ratgen et al (2003) Lancet 22:361 (9358): 681-689). Principally CF is a disease with thick secretions and too much inflammation. There are suggestions that mutation of CFTR is protective against human pathogens as the mutant CFTR gene is so prevalent, occurring in around 2.4 million UK citizens alone: protection against cholera has been proposed as one example; other examples may include salmonella infection and typhoid.

Current thinking suggests that the ΔF508 mutant CFTR polypeptide fails to fold properly, fails to exit the endoplasmic reticulum and is destroyed in the proteosome. Others find that this mutant fails to reside in the apical epithelium for long enough and the problem lies in the recycling of ΔF508 CFTR (Bradbury et al (1999) Am J Physiol 276 (4 Pt l):L659-68).

Because mutant CFTR (ΔF508) fails to leave the ER, many different compounds are in clinical trial with the specific aim of increasing the trafficking of the ΔF508 mutant from the endoplasmic reticulum to the plasma membrane. The hypothesis that underpins this approach is that the delivery of the mutant form of CFTR to the apical membrane will correct the defect. Whilst it may be true that increased presence of the mutant form of the protein at the apical membrane might restore channel activity, this has yet to be proven to correct all of the symptoms of the disease. Given that there is no correlation between chloride secretion and disease activity, this is not all together surprising (Davis et al (2004) Ped PuIm 38: 204- 209).

The ΔF508 CFTR mutation induces unexplained defects in multiple cellular pathways. CFTR interacts with multiple proteins, but the relatively low expression of CFTR and its restricted distribution to certain epithelial tissues make it difficult to understand how a mutation in CFTR can have such a broad effect on the body. We reasoned that: firstly, CFTR may interact with a regulatory protein that has multiple roles in cell biology; secondly we speculated that the site of the commonest mutation in CFTR normally binds this putative regulator; finally, we considered the notion that loss of F508 disrupted this putative interaction. We have found that CFTR contains a previously unrecognised target site for the pleiotropic serine/threonine protein kinase CK2 (formally casein kinase 2). This protein kinase targets acidic sequences within proteins as opposed to protein kinases such as protein kinase A and PKC that targets basic sequences. Moreover, CFTR and CK2 interact at the apical membrane of the cell and, importantly, this interaction acts to localise the CK2 holoenzyme to the apical membrane. Using selective pharmacological CK2 inhibition, we find that CK2-mediated CFTR phosphorylation maintains wild type Cl^" conductance through CFTR. This was confirmed by site directed mutagenesis of the proposed phosphorylation target residue serine 511 (i.e. downstream of F508) We have also found that CK2 binds normal, but not ΛF508 CFTR, and that F508 is critical for CK2 binding and channel function. Hence F508 is part of a binding site for CK2. The binding site is disrupted in the absence of the ΔF508 amino acid and CK2α subunit (CK2 is a heterotetramer) can no longer bind.

CK2 is a pleiotropic serine/threonine protein kinase with more than 300 potential targets identified. This number rises over the years and the final number of CK2 targets may be in the thousands. It has been proposed that CK2 provides a steady state background level of phosphorylation, and CK2 is therefore proposed as the 'eminence gi'ise of cellular regulation' by L. A. Pinna (Pinna, (1990) Biochim Biophys Acta. 1990 Sep 24;1054(3):267-84). The constitutive activity of CK2 inhibited to its discovery, could explain why CK2 has a role in a wide range of cellular functions such as RNA synthesis, apoptosis and cell cycle progression. It is known that CK2 malfunction causes cancer (Wang et al (2001) MoI Cell Biochem 227(l-2):167-74; Channavajhala et al (2002) Oncogene 21(34):5280-8).

In addition, since CK2 is spontaneously active in DNA/RNA/protein synthesis, CK2 activity can be used by pathogens to gain an advantage during infection by diverting CK2 function to enhance pathogen replication (Barroso et al (2002) Biochem Biophys Res Commun. 296(5): 1334-9; Shimada et al (2004) Microbiol Immunol. 48(3):205-10). In this model the spatial localisation of CK2 to particular parts of the cell may be important to restrict its activity. The apical membrane of epithelia is one such ke}' entry point for cell invasion from outside the body.

Interestingly, carriers of the ΔF508 CFTR allele (i.e. heterozygous for the mutant CFTR gene) appear to have a reduced likelihood to develop cancer (Padua et al (1997) Hum Mutat 10, 45-48), as well as a reduced tendency to develop secretory states or diseases such as cholera (Gabriel et al (1994) Science 266(5182): 107-9).

A first aspect of the invention provides a method of identifying a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 comprising exposing CFTR and/or CK2 to a test compound and assessing the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

A further aspect of the invention provides a method of screening for compounds of use in preventing or treating a secretory state or disorder and/or a cancer and/or cystic fibrosis wherein CFTR and/or CK2 is exposed to a test compound and the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 is assessed.

The methods of the invention relate to screening methods for drugs or lead compounds.

An embodiment of screening methods of the invention is wherein the methods comprise exposing a cell comprising CFTR polypeptide and CK2 polypeptide and assessing the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

An alternative embodiment of screening methods of the invention is wherein the methods comprise exposing a non-human animal comprising CFTR polypeptide and CK2 polypeptide and assessing the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. The screening methods of the invention may comprise native and/or recombinant CFTR and CK2 polypeptides. Methods for producing recombinant polypeptides are well known in the art. Polynucleotides encoding CFTR or CK2 polypeptides are provided below. Preferably, the non-human animal comprises cells comprising native CFTR and CK2 polypeptides.

CFTR interacts with and is phosphorylated by CK2, and this interaction acts to localise CK2 polypeptide to the apical membrane. In the absence of functional CFTR, CK2 is no longer concentrated at the apical membrane. Hence, the screening methods of the invention may also include assessing the effect of a test compound on the location of CK2 in a cell.

A number of secretory states or disorders would benefit from a reduction of CFTR function, for example pathogens such as cholera subverts the normal function of CFTR which leads to the gut secreting litres of fluid every hour. Therefore, compounds that reduce CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 may be of use in repressing CFTR function and so alleviate the effects of these disorders.

By "secretory states or disorders" we include those disorders that caused by bacteria such as Vibrio cholerae and enteropathogenic E. coli (EPEC) (Barroso et al (2002) Biochem Biophys Res Commun 295(5): 1334-1339; Shimada et al

(2004) Microbiol Immunol 48(3):205-210), and Typhoid (van de Vosse et al

(2005) Human Genetics 6, 123-142).

We also include both autosomal dominant and autosomal recessive polycystic kidney disorders. The formation of cysts in polycystic kidney disease is dependent on CFTR activity. Therefore, a compound that can mediate CFTR activity may be used to shrink cyst formation and hence preserve kidney function (Li et al (2004) Kidney Int. 66, 1926-1938; Li et al (2005). As discussed above, CO is a spontaneously active pleiotropic serine/threonine protein kinase. CK2 activity can be used by pathogens to gain an advantage during infection. In cells having normal CFTR, CK2 is localised to the apical cell membrane, while in cells having the ΔF508 form of CFTR, CK2 is no longer apical and remains distributed throughout the cell. Therefore, compounds that reduce CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 may be of use in modifying the activity of CK2 either by altering the location of the protein kinase in the cell or changing the capacity of CK2 to interact with and/or phosphorylate further polypeptides. Such a change in CK2 activity may be of use in the prevention or treatment of pathogen infection. Moreover, compounds that interfere with CK2/CFTR interaction may also be used to prevent viral entry to cells of, for example, the lung.

As discussed above carriers of the ΔF508 form of CFTR have a reduced likelihood of developing cancer (Padua et al, supra). Since the ΔF508 form of CFTR does not interact with CK2, compounds that mimic the activity of the ΔF508 form of CFTR by reducing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 may be of use in the treatment of cancer. By "cancer" we include malignant melanomas, colon cancer, breast cancer.

Since cystic fibrosis symptoms are caused at least to some extent by a reduced amount of functional CFTR, compounds that enhance CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 may be of benefit in activating CFTR function, including ΔF508 CFTR function, and so alleviate the effects of cystic fibrosis.

By "CFTR" we include any cystic fibrosis transmembrane conductance regulator polypeptide, fragments or variants of such polypeptide as well as further homologues, orthologues orparalogues of CFTR. We also include the ΔF508 form of CFTR. Examples of CFTR polypeptides and polynucleotides encoding CFTR polypeptides are known in the art. Examples of such polypeptides and polynucleotides are given in the following GenBank Accessions: P26361 (mouse); Q00555 (Sheep); P13569 (Human); P35071 (cow); AAK07685 (Bufό); AAL40378 (Takifugu rubripes); AAD38404 (Salmo) AAR16315 (Rat).

By "CK2" we include any serine/threonine protein kinase CK2 polypeptide, fragments or variants of such polypeptides as well as further homologues, orthologues or paralogues of CK2.

Examples of CK2 polypeptides and polynucleotides encoding CK2 polypeptides are known in the art. Examples of such polypeptides and polynucleotides are given in the following GenBank Accessions: NP_808227 CK2 αl subunit isoform a Homo sapiens; NP_808228 CK2 αl subunit isoform b Homo sapiens; NP_001311 CK2 β subunit Homo sapiens; NPJ)Ol 887 CK2 α¹ Homo sapiens; NP_031814.2 CK2 αl Mus musculus; P19139 CK2 αl Rattus norvegicus; P33674 CK2 αl Oryctolagus cuniculus; P21868 CK2 α Gallus gallus; P28020 CK2 α Xenopus sp; P08181 CK2 α Drosophϊla melanogaster al l.

Methods by which homologues, orthologues or paralogues of polypeptides can be identified are well known to those skilled in the art: for example, in silico screening or database mining. Preferably, such polypeptides have at least 40% sequence identity, preferably at least 60%, at least 70%, at least 80%, at least 90% or at least 95% sequence identity to the polypeptide sequence of CFTR or CK2 polypeptide.

Methods of determining the percent sequence identity between two polypeptides are well known in the art. For example, the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequence has been aligned optimally. The alignment may alternatively be carried out using the Clustal W program (Thompson et al., (1994) Nucleic Acids Res 22, 4673-80). The parameters used may be as follows:

Fast pairwise alignment parameters: K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05. Scoring matrix: BLOSUM.

As would be understood by the skilled person, in the methods of the invention the terms "CFTR" and "CK2" also includes fragments or variants and fusions of fragments or variants of these polypeptides.

A "fragment" or "variant" of the CFTR or CK2 polypeptide is one that may be usable in the screening methods of the invention. Such a variant may be encoded by a gene in which different codons can be substituted which code for the same amino acid(s) as the original codons. Alternatively, the substitute codons may code for a different amino acid that will not affect the function or immunogenicity of the protein or which may improve its function or immunogenicity. For example, site- directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis," Science, 229: 193-1210 (1985), which is incorporated herein by reference.

By "fusion of fragments or variants" we include said polypeptide fused to any other polypeptide or lipids such as myristic acid. For example, the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art. Similarly, the said polypeptide may be fused to an oligo-histidine tag such as His6 or to an epitope recognised by an antibody such as the well known Myc tag epitope. Fusions to any variant, fragment or derivative of said polypeptide are also included in the scope of the invention. It will be appreciated that fusions (or variants, fragments, derivatives or fusions thereof) which retain desirable properties, such as binding properties (for example, the ability to interact with a known interacting polypeptide) or phosphorylation properties or other biological functions, of the said polypeptide It is also particularly preferred if the fusions are one which are suitable for use in the screening assays described earlier.

In particular, a peptide comprising the amino acid sequence from 500 to 518 of CFTR and having a maximum of 50 amino acids, or a fragment or a variant or fusion of said peptide may be used in the screening methods of the invention. As is demonstrated in the accompanying examples, a peptide comprising of this amino acid region of CFTR retains the potential to interact with and be phosphorylated by CK2.

Furthermore, a peptide comprising an amino acid region of CFTR which retains the potential to interact with and be phosphorylated by CK2 also modulates CFTR/CK2 interaction and/or CFTR phosphorylation by CK2, i.e. it is a competitive inhibitor of CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. Therefore, such a peptide can be considered to be a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

By "500 to 518 of CFTR" we include the peptide sequence GTIKENIIFGVSYDEYRYR.

Preferably the peptide has 50 amino acids, 45, 40, 35, 30, 25, 24, 23, 22, 21, 20 or 19 amino acids and comprising the amino acid sequence from 500 to 518 of CFTR. The peptide may also have less than 19 amino acids, for example, 18, 17, 16, 15, 14, 13, 12, 11 or 10 amino acids, and comprise some of the amino. acid sequence from 500 to 518 of CFTR. A "fragment" of such a peptide will preferably comprise at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 of the amino acids of the amino acid sequence from 500 to 518 of CFTR. Preferably, the fragment comprises amino acid sequence from 500 to 518 of CFTR.

A "variant" of the peptide will have a region which has at least 10, 11, 12, 13, 14, 15, 16, 17, 18, or 19 amino acids of the amino acid sequence from 500 to 518 of CFTR. Methods of determining peptide or polypeptide identity are provided above. Preferably, the variant comprises amino acid sequence from 500 to 518 of CFTR.

A "variant" also refers to a peptide wherein at one or more positions there have been amino acid insertions, deletions, or substitutions, either conservative or non- conservative, provided that such changes result in a protein whose basic properties, for example protein interaction, thermostability, activity in a certain pH-range (pH- stability) have not significantly been changed. "Significantly" in this context means that one skilled in the art would say that the properties of the variant may still be different but would not be unobvious over the ones of the original protein.

By "conservative substitutions" is intended combinations such as GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.

Such variants may be made using the methods of protein engineering and site- directed mutagenesis as would be well known to those skilled in the art.

More preferably the peptide has 20 amino acids and comprises the amino acid sequence from 500 to 518 of CFTR. Most preferably the peptide comprises the amino acid sequence GTIKENFfFGVSYDEYRYR.

We also include a fusion of the said peptide. By "fusion" we include where the said peptide fused to any other polypeptide. As discussed above, CK2 does not interact with or phosphorylate the ΔF508 form of CFTR. However, a fragment of the ΔF508 form of CFTR can be used in the screening methods of the invention to identify compounds that modulate CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. For example, a compound may increase CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 where the CFTR polypeptide is derived from the ΔF508 form of CFTR. Hence the ΔF508 CFTR polypeptide can be used in the screening methods of the invention.

It is preferred that the CFTR polypeptide used in the screening methods of the invention comprises at least one CK2 binding site.

Although CK2-dependent phosphorylation of serine 511 is likely, this is not the only possibility because the protein sequence of CFTR reveals a number of potential CK2 consensus sequences in a cluster close to the R domain. In addition, there is a very acidic CK2 consensus sequence near the C terminus of CFTR and truncation of the region of CFTR incorporating this acidic stretch of residues has been reported by others to make CFTR less active (Effects of C-terminal deletions on cystic fibrosis transmembrane conductance regulator function in cystic fibrosis airway epithelia. Ostedgaard LS, Randak C, Rokhlina T, Karp P, Vermeer D, Ashbourne Excoffon KJ, Welsh MJ. Proc Natl Acad Sci U S A. 2003 Feb 18;100(4):1937-42).

In addition, there are a number of tyrosine residues near the serine 511 that, if phosphorylated, could increase the acidity of the region. CK2-induced tyrosine phosphorylation can occur in yeast but has not been shown to occur in mammals to date (Marchetta et al (2004) Cell MoI Life Sci 61(10): 1176-84). Therefore, the CFTR polypeptide used in the screening methods of the invention could contain tyrosine phosphorylation sites in addition to the S511 residue.

It is further preferred that the CFTR polypeptide used in the screening methods of the invention contains the CK2 binding site that promotes phosphorylation of the serine amino acid at position 511 of CFTR; this site includes the phenylalanine amino acid at position 508 of CFTR. The CK2 binding site may also include ΔI506 and ΔI507 since these ΔI mutants are very rare compared to ΔF508, and so these residues are required for the binding.

Fragments of the CK2 polypeptide which may be used in the screening methods of the invention include the alpha subunit of CK2, which is critical for the binding and phosphorylation of CFTR.

By "CFTR/CK2 interaction" we include that a CK2 polypeptide may bind to a CFTR polypeptide, as well as where a CFTR polypeptide may bind to a CK2 polypeptide. Moreover, CFTR binding to CK2 may act to reduce the activity of CK2. This may be important since, as described above, changing the capacity of CK2 to interact with and/or phosphorylate further polypeptides may be of use in the prevention or treatment of pathogen infection, particularly but not exclusively in secretory states.

By "CFTR phosphorylation by CK2" we include that CK2 catalyses the transfer of a phosphate group from a substrate molecule, for example ATP or GTP₅ to an amino acid present in the CFTR polypeptide. For example, as discussed herein, CK2 can phosphorylate the serine residue at position 511 of the CFTR polypeptide.

In addition to CFTR, the in-vivo recognised substrates of CK2 include the regulatory subunit II of protein kinase A. This polypeptide is present in the CFTR complex. Further in-vivo recognised substrates of CK2 include those polypeptides referred to in Litchfield (2003) Biochem J 369 1-15; and Meggio and Pinna (2003) FASEB J 17, 349-368.

By "exposing CFTR and/or CK2 to a test compound" we include where isolated CFTR and/or CK2 polypeptide(s) is incubated with a quantity of the test compound, or where a cell comprising CFTR and/or CK2 polypeptide(s) is incubated with a quantity of the test compound, or where a non-human animal comprising CFTR and/or CK2 polypeptide(s) is incubated with a quantity of the test compound. Hence this aspect of the invention includes both in vitro and in vivo testing of the test compound.

By "compounds" we include drug-like or lead compounds, as discussed below; peptides or polypeptides including antibodies and antibody fragments; nucleic acids such as catalytic RNA molecules.

As mentioned above, it will be appreciated that in the methods of the invention, which may be drug screening methods, a term well known to those skilled in the art, the compound may be a drug-like compound or lead compound for the development of a drug-like compound.

The term "drug-like compound" is well known to those skilled in the art, and may include the meaning of a compound that has characteristics that may make it suitable for use in medicine, for example as the active ingredient in a medicament. Thus, for example, a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 Daltons and which may be water-soluble. A drug-like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate target cellular membranes, but it will be appreciated that these features are not essential.

The term "lead compound" is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drug (for example because it is only weakly potent against its intended target, nonselective in its action, unstable, poorly soluble, difficult to synthesise or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics. Examples of compounds that may be capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 include 4,5,6,7- tetrabromobenzotriazole (TBB), tetrahydrobenzothiophene, benzofuran, pyramidinetrone, dihydropyridine, and anthraquinone and related structures. These compounds can be obtained from: 4,5,6,7-tetrabromobenzotriazole (Calbiochem), tetrahydrobenzothiophene (Fluorochem), benzofuran (Fluorochem), pyramidinetrione (Fisher), dihydropyridine (Fisher), and anthraquinone (Fisher).

A further compound that may be capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 is 2-Dimethylamino-4,5,6,7-tetrabromo- lff-benzimidazole, as described in Pagano et al (2004) Biochem Biophys Res Comm 321, 1040-1044.

Still further compounds are described in Meggio et al (2004) Biochemistry 43, 12931-12936.

As can be seen in the accompanying examples, the previously reported CFTR inhibitors DPC, niflumic acid and DIDS are capable of inhibiting CK2. Hence these CFTR blocking compounds seem to act via CK2 and not directly on CFTR. Therefore, such compounds may be capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

The compounds mentioned above can also be used to develop further compounds which can subsequently be assayed for activity using the screening methods of the invention, e.g. TBB can be used as a lead compound from which to develop further compounds.

Also, Sarno et al (2002) Pharamcol Ther 93, 159-168 describe a process for the rational design of protein kinase CK2 inhibitors which may be of use in identifying further compounds that may be capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. Further compounds that may be capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 include: antibodies or antibody fragments that bind to and mask CFTR such that CK2 can not interact with CFTR, or antibodies that bind to and mask CK2 such that CFTR can not interact with CK2; further peptides or polypeptides that bind to CFTR and/or CK2 to modulate CFTR/CK2 interaction and/or CFTR phosphorylation.

Further compounds that may be capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 may include, for example, divalent antibodies that can bind both CK2 and CFTR, which may be of use in elevating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. Such a compound may be of use in the prevention or treatment of cystic fibrosis, as discussed above.

Furthermore, as can be seen in the accompanying examples, a peptide comprising the amino acid sequence from 500 to 518 of CFTR and having a maximum of 50 amino acids, or a fragment or a variant or fusion of said peptide, as described above in relation to the screening methods of the invention, is capable of interacting with CK2 and can modulate CFTR/CK2 interaction and/or CFTR phosphorylation by CK2, i.e.- it is a competitive inhibitor of CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. Therefore, such a peptide can be considered to be a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. In particular, the S511D and ΔF508- S511D peptides described in the accompanying examples are capable of interacting with CK2 and can therefore be considered to be a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

In particular, we include where the CFTR polypeptide is a peptide comprising the amino acid sequence from 500 to 518 of CFTR and having a maximum of 50 amino acids, or a fragment or a variant or fusion of said peptide, as described above in relation to the screening methods of the invention. As is demonstrated in the accompanying examples, a peptide comprising of this amino acid region of CFTR retains the potential to interact with and be phosphorylated by CK2. It is not necessary that the peptide is capable of being phosphorylated by CK2. Hence such a peptide can have the CK2 interaction domain, but may lack an amino acid residue which may be phosphorylated by CK2, for example the serine residue at position 511 of CFTR. Known polymorphisms of the CFTR in this , region that do not induce clinical disease could be used to generate a correcting peptide.

Furthermore, the peptide may be phosphorylated or unphosphorylated. For example, the peptide may comprise a phosphorylated form of the serine residue at position 511 of CFTR, or an unphosphorylated form of this residue. Where phosphorylated, the phosphorylation may be catalysed by CK2.

Alternatively, such a compound may comprise a complementary peptide to the CFTR binding site of CK2 (or just a poly-lysine tail). Such a compound can act as a 'molecular tether', and may be of use since such a compound can be smaller and may be easier to administer than antibody.

Further compounds that may be capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 may include, for example, a compound capable of binding to a CK2 polypeptide, for example an antibody or antibody fragment as set out above, wherein the compound is further linked to an entity capable of localising the CK2 polypeptide to a particular region of the cell. For example, an antibody that recognises and binds to CK2 may be linked to a polypeptide that is capable of anchoring the CK2 polypeptide to a plasma membrane, e.g. a transmembrane polypeptide. In this way, a compound can bind to CK2 and localise the polypeptide to a plasma membrane of the cell.

Alternatively, such a compound may comprise a peptide that is capable of binding to the CFTR binding site of CK2 as well as a further entity capable of localising the CK2 polypeptide to a particular region of the cell. As used herein, the term "antibody" (Ab) or "monoclonal antibody" (Mab) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab')2 fragments) which are capable of specifically binding to CFTR polypeptide and/or a CK2 polypeptide. Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (WaH et al, J. Nucl. Med. 24:316-325 (1983)).

The screening methods of the invention include assessing the effect of a test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 can be studied using a number of different methods.

Firstly, CFTR/CK2 interaction can be measured using co-immunoprecipitation of CFTR with CK2. This method is described in more detail in the accompanying examples. Briefly, a test reaction or a sample of protein of an animal or cell exposed to a test compound is incubated with an antibody that binds to CFTR or CK2, the unbound protein removed, and the protein purified by the antibody examined. If an antibody that binds to CFTR has co-purified CK2, or if an antibody that binds to CK2 has co-purified CFTR, then the CFTR and CK2 proteins are considered to be interacting. The affinity of this interaction is likely to be high because these interactions survive washing by salt in the IM range.

Alternatively, CFTR phosphorylation by CK2 can be measured using a phosphorylation assay. This method is described in more detail in the accompanying examples. Briefly, CFTR polypeptide is immobilised on a solid support. CK2 is incubated with the immobilised CFTR polypeptide in the presence of a labelled ATP or GTP molecule. As discussed above, CK2 transfers a phosphate group, usually from ATP or GTP, to CFTR. The assay is terminated and the amount of CFTR polypeptide having a labelled phosphate group is assessed. Alternatively, CFTR/CK2 interaction can be measured by a protein-protein interaction assay. This method is described in more detail in the accompanying examples. Briefly, CFTR polypeptide is immobilised on a solid support. CK2 is incubated with the CFTR polypeptide. Subsequent binding of CK2 to CFTR is measured using ELISA, as would be appreciated by a person skilled in the art.

Alternatively, CFTR/CK2 interaction can be measured by a yeast two-hybrid based protein-protein interaction assay. For example, polynucleotides encoding CFTR or CK2 polypeptide are cloned into a yeast expression vector in-frame with components of a yeast two-hybrid interaction assay, for example the GAL4 DNA Binding Domain and GAL4 Transactivation Domain. CFTR and CK2 polypeptide interaction can subsequently be assayed by assessing the function of the GAL4 transcription factor, as would be appreciated by a person skilled in the art.

Alternatively, the amount of CFTR polypeptide phosphorylation by CK2 can be assessed using an antibody that recognises phosphorylated amino acid residues. For example, a test reaction or a sample of protein of an animal or cell exposed to a test compound is incubated with an antibody that recognises and binds to the CFTR polypeptide and the unbound protein removed. The isolated CFTR polypeptide is then incubated with an antibody that recognises phosphorylated S511 serine residues of the CFTR. The degree of antibody cross-reaction to the CFTR polypeptide gives a measure of CFTR phosphorylation by CK2.

Since there are a number of further serine residues in CFTR that can be phosphorylated by any other kinases (e.g. Protein Kinase A) it is preferred that such an antibody specifically binds to phosphorylated S511 serine residues of the CFTR.

The screening methods of the invention include assessing the effect of a test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. By "assessing" we include where the screening methods of the invention determine whether the test compound has any effect on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

In assessing the effect of a test compound it is useful to have a "reference sample", i.e. a sample of polypeptide taken from test reaction, animal or cell which has not been exposed to the test compound. By comparing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in a sample taken from an animal or a cell or a test reaction which has not been exposed to the test compound, to CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in a sample taken from an animal or a cell or a test reaction which has been exposed to the test compound, it is possible to determine the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation.

By "modulating" we include that the test compound induces an elevation or reduction of CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

For example, the test compound may elevate CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 by 10%, 20%, 30%, 50%, 70%, 100%, 150%, 200%, 500%, 1000% in comparison to a reference sample.

Also, the test compound may reduce CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 by 10%, 20%, 30%, 50%, 70%, 100%, 150%, 200%, 500%, 1000% in comparison to a reference sample.

The test compound may also completely abolish CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

A further embodiment of the screening methods of the invention is wherein the method comprises the step of selecting a compound that modulates CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. In one embodiment, the selected compound reduces CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. In an alternative embodiment the selected compound elevates CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

The screening methods of the invention can be used in "library screening" methods, a term well known to those skilled in the art. Thus, for example, the methods of the invention may be used to detect (and optionally identify) a test compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. Aliquots of a library may be tested for the ability to give the required result.

An embodiment of any of the screening methods of the invention is wherein the method comprises assessing the specificity of the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

Test compounds that have been identified from the screening methods of the invention as capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 can subsequently be further studied to determine the specificity of their effect. As mentioned above, CK2 is a pleiotropic serine/threonine kinase and has more than 300 potential targets. Therefore it may be desirable to assess whether a test compound is able to modify the ability of CK2 to interact with or phosphorylate any polypeptide, or if the test compound is able to specifically modify the ability of CK2 to interact with or phosphorylate CFTR. Therefore, selected test compounds may be further assessed by determining whether further polypeptides which are known to be in vivo phosphorylation targets for CK2 are unaffected, i.e. the test compound does not affect their phosphorylation status by CK2. This may be important in ensuring that a test compound has no undesirable side effects.

Similarly,. a test compound may be able to modify the ability of CFTR to interact with any protein, or the test compound may be able to specifically modify the ability of CFTR to interact with CK2. Methods of assessing the specificity of the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 include those mentioned above in relation to the screening methods of the invention, but wherein a different polypeptide is substituted into the assay as appropriate. For example, the method may comprise incubating CK2 with a further substrate protein and assessing the effect of the test compound on the interaction with and/or phosphorylation of that further substrate.

A further embodiment of the screening methods of the invention is wherein the method comprises the step of selecting a compound that specifically modulates CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

By "non-human animal" we include non-human primates such as baboons, chimpanzees and gorillas, new and old world monkeys as well as other mammals such as cats, dogs, rodents, pigs or sheep, or other animals such as poultry, for example chickens, fish such as zebrafish, or amphibians such as frogs. A preferred embodiment of this aspect of the invention is wherein the animal is a rodent such as a mouse, rat, hamster, guinea pig or squirrel.

A further embodiment of the screening methods of the invention is wherein a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 is formulated into a pharmaceutically acceptable composition.

A further aspect of the invention provides a method of making a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 comprising synthesising a compound identified by any one of the previous embodiments.

A further aspect of the invention provides a method of making a pharmaceutical composition comprising the method of any one of the previous claims and the step of mixing the identified compound with a pharmaceutically acceptable carrier. In human therapy, the compounds can be administered alone but will generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice. The carrier(s) must be "acceptable" in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof. Typically, the carriers will be water or saline which will be sterile and pyrogen free.

A further aspect of the invention provides the use of CFTR and/or CK2 in a method of identifying a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

A further aspect of the invention provides the use of CFTR and/or CK2 in a method of identifying a compound of use in preventing or treating a secretory state or disorder and/or a cancer and/or cystic fibrosis.

By "CFTR" and "CK2" we include those polypeptides mentioned above in relation to the screening methods of the invention. In particular, we include where the CFTR polypeptide is a peptide comprising the amino acid sequence from 500 to 518 of CFTR and having a maximum of 50 amino acids, or a fragment or a variant or fusion of said peptide, as described above hi relation to the screening methods of the invention. As is demonstrated in the accompanying examples, a peptide comprising of this amino acid region of CFTR retains the potential to interact with and be phosphorylated by CK2.

Examples of methods hi which CFTR and/or CK2 can be used include those mentioned above in relation to the screening methods of the invention.

A further aspect of the invention provides a method of assessing a patient's risk of developing cystic fibrosis or progression of cystic fibrosis comprising: i) obtaining a sample containing protein from the patient; and,

ii) determining CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in the patient's sample.

This method may be useful in the diagnosis of cystic fibrosis or as a basis of genetic counselling.

Methods of determining CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in the patient's sample are mentioned above in relation to the screening methods of the invention.

An embodiment of this aspect of the invention is wherein if the sample has a reduced amount of CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in comparison to a reference sample then the patient is considered to be at risk of developing cystic fibrosis.

For example, as mentioned above, a mutation in the CFTR polypeptide may cause the polypeptide to be retained within the endoplasmic reticulum or the CFTR polypeptide may fail to reside in the epithelium for long enough. By determining CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in a patient's sample, and comparing the data to a reference sample, the method of this aspect of the invention provides an assay for CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in a patient. This may provide a measure of the patient's risk of developing cystic fibrosis or progression of cystic fibrosis.

The ability to be able to better determine the risk of and individual developing cystic fibrosis or the progression of cystic fibrosis very important for several reasons. Firstly, if an individual is incorrectly diagnosed as not having CF when the individual does, in fact, have CF, he or she may not be given appropriate treatment. Since it is particularly important that treatment is initiated at an early age in order to give the maximum chance of increasing life expectancy, a proper diagnosis is very desirable. Similarly if an individual is incorrectly diagnosed as having CF when the individual does not, in fact, have CF, he or she may be treated unnecessarily. Again this is very important since treatment is often in the form of intensive physiotherapy given many times a day by the parents or other carers of the child over many years; typically, the treatment takes two hours per day including medication. Thus, the ability to eliminate or reduce the chances of a child unnecessarily being treated in this way would reduce emotional and physical stress in the child and his or her carers, and would eliminate or reduce the chances of health service resources being incorrectly used.

hi some cases, an individual may have been classified genetically as having CF (because they have a mutation in the CFTR gene which, ostensibly, is one which may be associated with CF) but they only have very mild symptoms. Nevertheless, because they have been classified genetically as having CF they may be treated (inappropriately) in the same way as, for example, an individual who has much more severe CF (eg an individual who is homozygous for ΔF508 in CFTR).

Thus, in some cases, it would be useful to classify an individual as one who although genetically has CF, is not one who physiologically has CF to the extent that the individual requires treatment, or at least the same intensive treatment as an individual with a severe form of CF.

Similarly, it is useful to determine whether a CF patient is responding to a particular treatment.

Suitable samples that may be used in the methods of this aspect of the invention include those which contain representative samples of the patient's polypeptide. A preferred embodiment of this aspect of the invention is wherein the sample contains extracts of airway epithelial cells. The sample of epithelial cell may be prepared into any fractions and fractions may be used in which any one or more of the parameters described, such as CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 activity may be measured.

The epithelial cell sample tested may be any suitable cell sample in which dysfunction may be found, particularly dysfunction due to CF. Typically, the epithelial cell sample is a lung cell sample, or a nasal epithelial cell sample, or a gut epithelial cell sample or liver biopsy. Preferably, because of the ease with which it can be obtained, the epithelial cell sample is a nasal epithelial cell sample. Suitable nasal epithelial cell samples may be obtained by scraping or brushing the lining of the nose. A typical assay requires two brush strokes per nostril. Methods for separating appropriate membrane fractions from post nuclear supernatants using 2 ml sucrose gradients and a TST 55.5 rotor have been described previously (Treharne et al (1994) Am. J. Physiol. (Lung Cell. MoI. Physiol.) 267, L592-L601; incorporated herein by reference). Brushings may be stored, if necessary.

A control epithelial cell sample may also be taken. The control epithelial cell sample may be any suitable control sample. Suitable control samples include, for example, a known normal (ie non-CF) cell sample, or a known CF cell sample. Both types of control samples may advantageously be used. It will be appreciated that the control sample and the test sample may usefully be matched such that, for example, both the test and control cell samples are both from the same tissue type (eg both nasal samples), or both samples are otherwise genetically similar (eg from siblings), or age matched.

Because CF patients are susceptible to infections, particularly bacterial infections of the lungs it is particularly preferred if the patient is treated with an appropriate course of antibiotics before the sample of epithelial cells (particularly nasal or lung epithelial cells) is isolated for measurement of the given parameters (and indeed the control samples are preferably obtained from an individual who has been treated with antibiotics such that the epithelial cells are free of a bacterial infection, or are otherwise free of such an infection).

The comparison of the test sample with that of a control sample may be measured directly, for example by carrying out the determination of the test parameters in parallel in test samples (derived from the patient) and in control samples (derived from suitable controls as discussed above). However, comparison of the test sample with control sample may be carried out indirectly in the sense that parameters to be measured for the control sample may have been measured historically and tabulated in a look-up table or the like so that it is only the parameters of the test sample that are measured when undertaking the determination. Control samples are suitably obtained from an individual who has been certified as having normal noses by a specialist ear, nose and throat (ENT) surgeon. The control samples may also be genotyped.

A further aspect of the invention provides a method of determining the restitution of CK2 and/or CFTR function comprising:

i) obtaining a sample containing protein from the patient; and,

ii) determining CFTR and/or CK2 location in a cell.

As mentioned above, CFTR interacts with and is phosphorylated by CK2, and this interaction acts to localise CK2 polypeptide to the apical membrane. In the absence of functional CFTR, CK2 is no longer concentrated at the apical membrane, Therefore, "restitution of CK2 and/or CFTR function" includes where these polypeptides are predominately located to the apical membrane.

For example, a airway nasal brush biopsy or a rectal biopsy from a patient could be analysed using antibodies which bind to CK2 or CFTR, and the position of the antibodies, and so the bound protein, in a cell determined using immunofluorescence confocal microscopy, as would be appreciated by the skilled person.

Antibodies which recognise CK2 or CFTR include: a monoclonal antibody to the β subunit from Santa Cruz; a Neomarkers anti-CFTR antibody.

Further details as to the use of such antibodies for determining the location of CK2 or CFTR in cells are provided in the accompanying example.

Such a method may be of use in determining whether, for example, a compound identified from the screening methods of the invention may be of use in preventing or treating a specified disorder. This is because the method of this aspect of the invention can be used to determine whether the compound has reconstituted CK2 and/or CFTR function.

Suitable samples that may be used in the methods of these diagnostic aspects of the invention include those which contain representative samples of the patient's polypeptide. Preferred embodiments of this aspect of the invention include where the sample contains extracts of cells such as airway epithelial cells, skin cells, gut cells, and further samples as would be appreciated by the skilled person.

A further aspect of the invention provides a polypeptide comprising all or a fragment or a variant or a fusion of a pseudo-CK2 phosphorylated CFTR polypeptide.

By "CFTR" we include those CFTR polypeptides discussed above in relation to the screening methods of the invention.

By "pseudo-CK2 phosphorylated" we include that the CFTR polypeptide includes a negatively charged amino acid which replaces an amino acid phosphorylated by CK2. For example, the CFTR polypeptide of this aspect of the invention may have a Glutamic acid or Aspartic acid residue at position 511 in replacement of the native serine residue at this position. An example of such a "pseudo-CK2 phosphorylated" polypeptide is provided in the accompanying examples (the S51 ID peptide).

Such a polypeptide also includes a polypeptide having charged structures that mimic or enhance the binding of CK2 to the polypeptide, so as to increase the affinity of CK2 binding. Moreover, a polypeptide having such charged structures may also be used to gain entry to a cell and can further include poly-lysine moieties that are known to bind to regulatory elements in CK2.

A pseudo-CK2 phosphorylated CFTR polypeptide may still be able to interact with CK2, but CK2 will not be able to phosphorylate such a polypeptide.

The ΔF508 CFTR polypeptide present in cystic fibrosis sufferers cannot interact with CK2 (as discussed above) and thus does not act as a channel allowing Cl^" conductance.

Hence the pseudo-CK2 phosphorylated CFTR polypeptide may be of use in such patients to allow Cl^" conductance and thus alleviate the symptoms of cystic fibrosis. The pseudo-CK2 phosphorylated CFTR polypeptide may show reduced conductance. Such a polypeptide may have to be further modified so as to permit native CK2 to bind to that channel. However, a pseudo-phosphorylated CTFR polypeptide may still bind to CK2, as shown in the Examples.

An example of a polypeptide according to this aspect of the invention is a CFTR polypeptide having a Glutamic acid or Aspartic acid residue at position 511 in replacement of the native serine residue at this position of the polypeptide. We provide details of a CFTR polypeptide having a Aspartic acid residue at position 511 in the accompanying examples (the S51 ID polypeptide). A "fragment" consists of any polypeptide sequence of the polypeptide of this aspect of the invention capable of functioning as a pseudo-CK2 phosphorylated CFTR polypeptide.

A "variant" of the polypeptide of the invention is one that is capable of functioning as a pseudo-CK2 phosphorylated CFTR polypeptide. Such a variant may be encoded by a gene in which different codons can be substituted which code for the same amino acid(s) as the original codons. Alternatively, the substitute codons may code for a different amino acid that will not affect the function or immunogenicity of the protein or which may improve its function or immunogenicity. For example, site-directed mutagenesis or other techniques can be employed to create single or multiple mutations, such as replacements, insertions, deletions, and transpositions, as described in Botstein and Shortle, "Strategies and Applications of In Vitro Mutagenesis," Science, 229: 193-1210 (1985), which is incorporated herein by reference.

A "fusion" includes where the said polypeptide fused to any other polypeptide, as described above.

An embodiment of this aspect of the invention is wherein the pseudo-CK2 phosphorylated CFTR polypeptide comprises a pseudo-phosphorylated region of amino acids from 500 to 518 of CFTR. An example of a polypeptide according to this embodiment of the invention is a CFTR polypeptide having a Glutamic acid or Aspartic acid residue at position 511 in replacement of the native serine residue at this position of the polypeptide, ie having the amino acid sequence GTIKENIIFGVEYDEYRYR or GTIKENIIFGVDYDEYRYR.

Peptides or polypeptides of the invention may be synthesised by the Fmoc- polyamide mode of solid-phase peptide synthesis as disclosed by Lu et al (1981) J Org. Chem. 46, 3433 and references therein. Temporary N-amino group protection is afforded by the 9-fluorenylmethyloxycarbonyl (Fmoc) group. Repetitive cleavage of this highly base-labile protecting group is effected using 20% piperidine in N₅N- dimethylformamide. Side-chain functionalities may be protected as their butyl ethers (in the case of serine threonine and tyrosine), butyl esters (in the case of glutamic acid and aspartic acid), butyloxycarbonyl derivative (in the case of lysine and histidine), trityl derivative (in the case of cysteine) and 4-methoxy-2,3,6- trimethylbenzenesulphonyl derivative (in the case of arginine). Where glutamine or asparagine are C-terminal residues, use is made of the 4,4'-dimethoxybenzhydryl group for protection of the side chain amido functionalities. The solid-phase support is based on- a polydimethyl-acrylamide polymer constituted from the three monomers dimethylacrylamide (backbone-monomer), bisacryloylethylene diamine (cross linker) and acryloylsarcosine methyl ester (functionalising agent). The peptide-to- resin cleavable linked agent used is the acid-labile 4-hydroxymethyl-phenoxyacetic acid derivative. All amino acid derivatives are added as their preformed symmetrical anhydride derivatives with the exception of asparagine and glutamine, which are added using a reversed N_jN-dicyclohexyl-carbodiimide/l-hydroxybenzotriazole mediated coupling procedure. All coupling and deprotection reactions are monitored using ninhydrin, trinitrobenzene sulphonic acid or isotin test procedures. Upon completion of synthesis, peptides are cleaved from the resin support with concomitant removal of side-chain protecting groups by treatment with 95% trifluoroacetic acid containing a 50% scavenger mix. Scavengers commonly used are ethanedithiol, phenol, anisole and water, the exact choice, depending on the constituent amino acids of the peptide being synthesised. Trifluoroacetic acid is removed by evaporation in vacuo, with subsequent trituration with diethyl ether affording the crude peptide. Any scavengers present are removed by a simple extraction procedure which, on lyophilisation of the aqueous phase, affords the crude peptide free of scavengers. Reagents for peptide synthesis are generally available from Calbiochem-Novabiochem (UK) Ltd, Nottingham NG7 2QJ, UK. Purification may be effected by any one, or a combination of, techniques such as size exclusion chromatography, ion-exchange chromatography and (principally) reverse-phase high performance liquid chromatography. Analysis of peptides may be carried out using thin layer chromatography, reverse-phase high performance liquid chromatography, amino-acid analysis after acid hydrolysis and by fast atom bombardment (FAB) mass spectrometric analysis. A further aspect of the invention provides a pol^ynucleotide encoding a pseudo- CK2 phosphorylated CFTR polypeptide according to the invention.

An example of a polynucleotide that encodes a pseudo-CK2 phosphorylated CFTR polypeptide is given the figures and repeated below:

aat ate ate ttt ggt gtt gag/gaa/gau/gac tat gat gaa tat aga

where the 7' denotes alternative trinucleotide sequences within the given polynucleotide sequence.

As would be appreciated by a person skilled in the art, the polynucleotide sequence of this aspect of the invention can vary from that provided herein and still encode a polypeptide according to the invention.

Methods of preparing a polynucleotide according to this aspect of the invention are well known and readily performed using standard molecular biology techniques as is well known to those skilled in the art and described further in Sambrook et ah, Molecular Cloning. A laboratory manual. 1989. Cold Spring Harbour publications.

A further aspect of the invention provides a therapeutic entity comprising a polypeptide according to the invention.

By "therapeutic entity" we include that the polypeptide of the invention is fused to another moiety or moieties, e.g., targeting moiety or another therapeutic agent. Such therapeutic entities may exhibit improved properties such as activity and/or stability and thus impart an improved therapeutic profile on the polypeptide of this invention. Examples of such entities which may be fused to the polypeptide of the invention include, for example, targeting moieties which provide for the delivery of the polypeptide to epithelial cells, e.g., antibodies to epithelial cells etc., as well as receptor and ligands expressed on epithelial cells. Other moieties which may be fused to the polypeptide include therapeutic agents which are used for treatment. Further therapeutic entities include fatty acids, cholesterol and polylysine.

For example, the pseudo-CK2 phosphorylated CFTR polypeptide as discussed herein may be fused to an antibody to target the peptide to an epithelial cell.

A further aspect of the invention is a peptidomimetic of any one of the polypeptides of the invention.

The term "peptidomimetic" refers to a compound that mimics the conformation and desirable features of a particular polypeptide as a therapeutic agent, but that avoids the undesirable features. For example, morphine is a compound which can be orally administered, and which is a peptidomimetic of the peptide endorphin.

Therapeutic applications involving peptides are limited, due to lack of oral bioavailability and to proteolytic degradation. Typically, for example, peptides are rapidly degraded in vivo by exo- and endopeptidases, resulting in generally very short biological half-lives. Another deficiency of peptides as potential therapeutic agents is their lack of bioavailability via oral administration. Degradation of the peptides by proteolytic enzymes in the gastrointestinal tract is likely to be an . important contributing factor. The problem is, however, more complicated because it has been recognised that even small, cyclic peptides which are not subject to rapid metabolite inactivation nevertheless exhibit poor oral bioavailability. This is likely to be due to poor transport across the intestinal membrane and rapid clearance from the blood by hepatic extraction and subsequent excretion into the intestine. These observations suggest that multiple amide bonds may interfere with oral bioavailability. It is thought that the peptide . bonds linking the amino acid residues in the peptide chain may break apart when the peptide drug is orally admini stered. There are a number of different approaches to the design and synthesis of peptidomimetics. In one approach, such as disclosed by Sherman and Spatola, J. Am. Chem. Soc, 112: 433 (1990), one or more amide bonds have been replaced in an essentially isoteric manner by. a variety of chemical functional groups. This stepwise approach has. met with some success in that active analogues have been obtained. In some instances, these analogues have been shown to possess longer biological half-lives than their naturally-occuring counterparts. Nevertheless, this approach has limitations. Successful replacement of more than one amide bond has been rare. Consequently, the resulting analogues have remained susceptible to enzymatic inactivation elsewhere in the molecule. When replacing the peptide bond it is preferred that the new linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond.

Retro-inverso peptidomimetics, in which the peptide bonds are reversed, can be synthesised by methods known in the art, for example such as those described in Meziere et al (1997) J Immunol. 159 3230-3237. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains. Retro-inverse peptides, which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.

In another approach, a variety of uncoded or modified amino acids such as D- amino acids and N-methyl amino acids have been used to modify mammalian peptides. Alternatively, a presumed bioactive conformation has been stabilised by a covalent modification, such as cyclisation or by incorporation of γ-lactam or other types of bridges. See, eg. Veber et al, Proc. Natl Acad. ScL USA, 75:2636 (1978) and Thursell et al, Biochem. Biophys. Res. Comm., 111:166 (1983).

A common theme among many of the synthetic strategies has been the introduction of some cyclic moiety into a peptide-based framework. The cyclic moiety restricts the conformational space of the peptide structure and this frequently results in an increased affinity of the peptide for a particular biological receptor. An added advantage of this strategy is that the introduction of a cyclic moiety into a peptide may also result in the peptide having a diminished sensitivity to cellular peptidases.

One approach to the synthesis of cyclic stabilised peptidomimetics is ring-closing metathesis (RCM). This method involves steps of synthesising a peptide precursor and contacting it with a RCM catalyst to yield a conformationally restricted peptide. Suitable peptide precursors may contain two or more unsaturated C-C bonds. The method may be carried out using solid-phase-peptide-synthesis techniques. In this embodiment, the precursor, which is anchored to a solid support, is contacted with a RCM catalyst and the product is then cleaved from the solid support to yield a conformationally restricted peptide.

Another approach, disclosed by D. H. Rich in Protease Inhibitors, Barrett and Selveson, eds., Elsevier (1986), has been to design peptide mimics through the application of the transition state analogue concept in enzyme inhibitor design. For example, it is known that the secondary alcohol of staline mimics the tetrahedral transition state of the scissile amide bond of the pepsin substrate. However, the transition state analogue concept has no apparent relevance to hormone agonist/antagonist design.

A further aspect of the invention provides a gene therapy vector comprising a polynucleotide according to the invention.

Gene therapy vectors typically allow for the expression of a desired polypeptide(s) in a human cell. The vector may be one that allows for selective expression in the target cell by using promoter sequences which work selectively in the target cell type, for example kidney cell-specific expression.

Gene therapy vectors of the invention may be made by any suitable method. For example, a variety of methods have been developed to operably link DNA to vectors via complementary cohesive termini. For instance, complementary homopolymer tracts can be added to the DNA segment to be inserted to the vector DNA. The vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.

Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors. The DNA segment, generated by endonuclease restriction digestion as described earlier, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerizing activities.

The combination of these activities therefore generates blunt-ended DNA segments. The blunt-ended segments are then incubated with a large molar excess of linker molecules in the presence of an enzyme that is able to catalyze the ligation of blunt- ended DNA molecules, such as bacteriophage T4 DNA ligase. Thus, the products of the reaction are DNA segments carrying polymeric linker sequences at their ends. These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.

Synthetic linkers containing a variety of restriction endonuclease sites are commercially available from a number of sources including International Biotechnologies Inc, New Haven, CN, USA.

A desirable way to modify the DNA encoding the polypeptide of the invention is to use the polymerase chain reaction as disclosed by Saiki et al (1988) Science 239, 487-491.

In this method the DNA to be enzymatically amplified is flanked by two specific oligonucleotide primers which themselves become incorporated into the amplified DNA. The said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art. It will be appreciated that the gene expression vector of the invention may readily be made using molecular biological techniques which are well known in the art, such as those described in Sambrook et άl (1989). Molecular cloning, a laboratory manual, 2ⁿ edition, Cold Spring Harbor Press, Cold Spring Harbor, New York.

An embodiment of this aspect of the invention is wherein the vector comprises a Moloney Leukaemia Virus (MLV) based retroviral vector or a lentiviral vector or adeno-associated vector (AAV). Such vectors are well known to those skilled in the art and may be constructed using the methods disclosed above in relation to this aspect of the invention.

A further aspect of the invention is a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 and identified by the screening methods of the invention and/or a polypeptide according to the invention and/or a polynucleotide according to the invention and/or a therapeutic entity according to according to the invention and/or a gene therapy vector according to according to the invention for use in medicine.

A further aspect of the invention is a pharmaceutical composition comprising a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 and identified by the screening methods of the invention and/or a polypeptide according to the invention and/or a polynucleotide according to the invention and/or a therapeutic entity according to according to the invention and/or a gene therapy vector according to according to the invention and a pharmaceutically acceptable carrier.

Example of suitable pharmaceutically acceptable carriers are discussed herein in relation to the screening methods of the invention.

A further aspect of the invention is the use of a compound capable of reducing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in the manufacture of a medicament for preventing or treating a secretory state or disorder and/or a cancer.

A further aspect of the invention is a method of treating a patient with or at risk of developing a secretory state or disorder and/or a cancer comprising administering to the patient an effective amount of a compound capable of reducing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

By "secretory states or disorders" we include those disorders that caused by bacteria such as Vibrio cholerae and Enteropathogenic E. coli (EPEC), Typhoid, Shigella. We also include both autosomal dominant and autosomal recessive polycystic lddney disorders.

By "cancer" we include malignant melanomas, colon cancer, breast cancer. Preferably these use or method aspects of the invention relate to the treatment of cancer.

An embodiment of these use or method aspects of the invention is wherein the compound is a compound identified by the screening methods of the invention and capable of reducing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

A further embodiment of the use or method aspects of the invention is wherein the compound is a peptide comprising the amino acid sequence from 500 to 518 of CFTR and having a maximum of 50 amino acids, or a fragment or a variant or fusion of said peptide, which is capable of interacting with CK2.

As has been discussed herein, such a peptide is a competitive inhibitor of CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 and may be of use in reducing CFTR function so as to alleviate the effects of these disorders. Such a peptide is further discussed above in relation to the screening methods of the invention. A further embodiment of the use or method aspects of the invention is wherein the compound is a polynucleotide encoding a peptide capable of reducing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. An example of a polynucleotide that encodes such a peptide of is given in the methods and shown below:

aat ate ate ttt ggt gtt tec tat gat gaa tat aga

A further embodiment of the use or method aspects of the invention is wherein the compound is a therapeutic entity comprising a peptide capable of reducing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. The term "therapeutic entity" has been discussed above in relation to a further aspect of the invention.

A further embodiment of the use or method aspects of the invention is wherein the compound is a peptidomimetic of a peptide capable of reducing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. The term "peptidomimetic" has been discussed above in relation to a further aspect of the invention.

A further embodiment of the use or method aspects of the invention is wherein the compound is a gene therapy vector according to the invention comprising a polynucleotide capable of reducing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. The term "gene therapy vector" has been discussed above in relation to a further aspect of the invention.

A further embodiment of the use or method aspects of the invention is wherein the compound is a polypeptide comprising all or a fragment or a variant of an amino acid sequence of a CFTR polypeptide and which is capable of binding to CK2.

Any fragment or variant of the CFTR polypeptide that is capable of binding to CK2 can be used as a compound in these use or method aspects of the invention. For example, such polypeptides may function as competitive inhibitors of CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

Examples of CFTR polypeptides and fragments or variants of CFTR are provided above in relation to the screening methods of the invention.

A further embodiment of these use or method aspects of the invention is wherein the compound is the previously reported CFTR inhibitors DPC, niflumic acid or, less preferably, DIDS. As mentioned above and shown in the accompanying examples, each of these compounds are capable of inhibiting CK2. Preferably these use or method aspects of the invention relate to the treatment of cancer.

A further aspect of the invention is the use of a compound capable of elevating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in the manufacture of a medicament for preventing or treating cystic fibrosis. As discussed above, by "CFTR" we include any cystic fibrosis transmembrane conductance regulator polypeptide, fragments or variants of such polypeptide as well as further homologues, orthologues or paralogues of CFTR. We also include the ΔF508 form of CFTR.

A further aspect of the invention is a method of treating a patient with or at risk of developing a cystic fibrosis comprising administering to the patient an effective amount of a compound capable of elevating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. As discussed above, by "CFTR" we include any cystic fibrosis transmembrane conductance regulator polypeptide, fragments or variants of such polypeptide as well as further homologues, orthologues or paralogues of CFTR. We also include the ΔF508 form of CFTR.

As discussed above, elevating CFTR phosphorylation by CK2- increases Cl^" conductance by CFTR. Cystic fibrosis causes a range of symptoms associated with the poor conductance of Cl^" by CFTR. Therefore, compounds that elevate CFTR/CK2 interaction and/or CFTR phosphorylation by CIC2 may be of use in repressing CFTR function so as to alleviate the effects of this disorder.

A further embodiment of these use or method aspects of the invention is wherein the compound is a compound capable of elevating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 and identified by the screening methods of the invention.

A further aspect of the invention is the use of a pseudo-CK2 phosphorylated CFTR polypeptide as discussed herein and/or a polynucleotide encoding such a polypeptide as discussed herein and/or a therapeutic entity comprising such a polypeptide as discussed herein and/or a peptidomemetic of such a polypeptide as discussed herein and/or a gene therapy comprising a polynucleotide encoding such a polypeptide as discussed, herein in the manufacture of a medicament for preventing or treating cystic fibrosis.

A further aspect of the invention is method of treating a patient with or at risk of developing cystic fibrosis comprising administering to the patient an effective amount of a pseudo-CK2 phosphorylated CFTR polypeptide as discussed herein and/or a polynucleotide encoding such a polypeptide as discussed herein and/or a therapeutic entity comprising such a polypeptide as discussed herein and/or a peptidomemetic of such a polypeptide as discussed herein and/or a gene therapy comprising a polynucleotide encoding such a polypeptide as discussed herein in the manufacture of a medicament for preventing or treating cystic fibrosis.

Preferably, the formulation is a unit dosage containing a daily dose or unit, daily sub- dose or an appropriate fraction thereof, of the active ingredient.

The compounds will normally be administered orally or by any parenteral route, in the form of a pharmaceutical formulation comprising the active ingredient; optionally in the form of a non-toxic organic, or inorganic, acid, or base, addition salt, in a pharmaceutically acceptable dosage form. Depending upon the disorder and patient to. be treated, as well as the route of administration, the compositions may be administered at varying doses.

For example, the compounds can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications. The compounds may also be administered via intracavernosal injection.

The compounds can also be administered parenterally, for example, intravenously, intra-arterially, intraperitoneally, intrathecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

It will be appreciated that it is particularly useful to administer the peptide to the site where the symptoms of CF are manifested. In particular, it is preferred if the peptides are administered to the lungs, for example using a nebuliser, or if they are administered to the gut by any convenient method. Similarly, in relation to treating chronic sputum producing disorders, the peptides are advantageously delivered to the lungs.

Means and methods of introducing a genetic construct into a cell in an animal body are known in the art. For example, the polynucleotide of use in the uses and methods aspects of the invention may be introduced into the target cells by any convenient method, for example methods involving adenoviruses.

Targeted retroviruses are also available for use in the invention; for example, sequences conferring specific binding affinities may be engineered into preexisting viral env genes (see Miller & Vile (1995) Faseb J. 9, 190-199 for a review of this and other targeted vectors for gene therapy). The tropism of a retroviral vector can be altered by the incorporation of foreign or hybrid envelope proteins (Battini JL, et al J. Virol. 66: 1468-1475; 1992). This can be achieved by insertion of monoclonal antibodies to mouse ecotropic retroviral particles. Alternatively, any chemical modification such as lactose binding to virus particles can increase the range possible target cells for transduction or confer a.predictably altered recognition specificity. Retrovirus particles displaying non-viral polypeptides may be used for specific target cells through the non-viral moiety.

Other methods involve simple delivery of the genetic construct into the cell for expression therein either for a limited time or, following integration into the genome, for a longer time. An example of the latter approach includes (preferably kidney-cell-targeted) liposomes (Nassander et al (1992) Cancer Res. 52, 646-653).

For the preparation of immuno-liposomes MPB-PE (N-[4-(p- maleimidophenyl)butyryl] -phosphatidyl ethanolamine) is synthesised according to the method of Martin & Papahadjopoulos (1982) J. Biol. Chem. 257, 286-288. MPB-PE is incorporated into the liposomal bilayers to allow a covalent coupling of the antibody, or fragment thereof, to the liposomal surface. The liposome is conveniently loaded with DNA or other genetic construct for delivery to the target cells, for example, by forming the said liposomes in a solution of the DNA or other genetic construct, followed by sequential extrusion through polycarbonate membrane filters with 0.6 μm and 0.2 μm pore size under nitrogen pressures up to 0.8 MPa. After extrusion, entrapped DNA construct is separated from free DNA construct by ultracentrifugation at 80 000 x g for 45 min. Freshly prepared MPB- PE-liposomes in deoxygenated buffer are mixed with freshly prepared antibody (or fragment thereof) and the coupling reactions are carried out in a nitrogen atmosphere at 4°C under constant end over end rotation overnight. The immunoliposomes are separated from unconjugated antibodies by ultracentrifugation at 80 000 x g for 45 min. Immunoliposomes may be injected intraperitoneally or directly into the target tissue. Examples of polynucleotides that may be used in the uses and methods of the invention are discussed herein.

Polypeptide may be delivered^' using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections. An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.

The polypeptide can be , administered by a surgically implanted device that releases the drug directly to the required site. For example, Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis. The direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.

Electroporation therapy (EPT) systems can also be employed for the administration of polypeptide. A device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.

Polypeptide can be delivered by electroincorporation (EI). EI occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In EI, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.

An alternative method of polypeptide delivery is the ReGeI injectable system that is thermo-sensitive. Below body temperature, ReGeI is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.

Polypeptide pharmaceuticals can also be delivered orally. The process employs a natural process for oral uptake of vitamin B₁₂ in the body to co-deliver proteins and peptides. By riding the vitamin B₁₂ uptake system, the protein or peptide can move through the intestinal wall. Complexes are synthesised between vitamin B₁₂ analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B₁₂ portion of the complex and significant bioactivity of the drug portion of the complex.

Polypeptides can be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins, which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targetting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient. See Derossi et al (1998), Trends Cell Biol 8, U-Sl.

Examples of peptides that may be used in the uses and methods of the invention are discussed herein.

A further aspect of the invention is a kit of parts useful in diagnosing cystic fibrosis comprising an agent capable of use in determining CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in a patient's sample.

Agents that can he used in this aspect of the invention to determine the CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 include those discussed above in relation to the method of determining a patient's risk of developing cystic fibrosis. An example of such an agent is an antibody capable of determining the phosphorylation status of CFTR, as would be appreciated by a person skilled in the art. A further aspect of the invention is. a kit of parts of use in determining the restitution of CK2 and/or CFTR function comprising one or more antibodies capable of binding to CK2 and/or CFTR in a patient's sample.

Further details of antibodies capable of binding to CK2 and/or CFTR are described above in relation to the method of determining the restitution of CO and/or CFTR function. Such a kit of parts may be of use for determining whether a compound as determined from the screening methods of the invention, or a gene therapy vector according to a previous aspect of the invention, has been successful in restoring the function of the CFTR and/or CK2 polypeptide in a patient.

A further embodiment of this aspect of the invention is a kit of parts further comprising a negative control and/or a positive control.

A further embodiment of this aspect of the invention is a kit of parts further comprising means for separating extracts of airway epithelial cells from a sample.

The invention will now be described in more detail, for the purposes of illustration only, in the following Examples and Figures.

Figure 1: CK2 regulates CFTR transport and colocalises with CFTR in wild type but not ΔF508 cells.

A. Preincubation of Calu-3 epithelia with TBB (1-100 μM, 30 min) prior to stimulation with forskolin (10 μM) attenuates cAMP-stimulated apical membrane Cl^" currents (I^cl(apical)). After mounting Calu-3 epithelia (R_t > 200 Ω) in Ussing chambers, the basolateral membranes were permeabilised with amphotericin B (10 μM) and an outwardly directed Cl^" gradient imposed ¹⁴'¹⁵. Representative traces show the effect of forskolin and the Cl^" channel blocker diphenylamine-2- carboxylate (DPC; 1 mM) on I^ci(apical). TBB had no effect on basal I^CI(apical) and DPC (1 mM) inhibited the current. The histogram quantifies the change in forskolin-stimulated I^cl(apical) evoked by TBB. Data show means ± SEM (0 μM TBB, n = 40; 1-100 μM TBB, n = 7); *, p < 0.01; **, p < 0.0001. B, left panel, time course of CFTR Cl current (I) in an excised inside-out multi-channel membrane patch from a Cl 27 cell expressing wild-type human CFTR. ATP (0.3 and 1.0 mM), PKA (75 nM) and TBB (40 μM) were present in the intracellular solution as indicated. Voltage was -50 mV; internal [Cl^"] = 147 mM; external [Cl^" ] = 10 mM. Upward deflections represent inward currents. TBB (40 μM) inhibited CFTR current by 76 ± 5% (n = 3) and inhibition was poorly reversed upon drug washout. .

B. Right panel, representative recordings show the effect of TBB (40 μM) on the single-channel activity of CFTR. Dashed lines indicate the closed state. Downward deflections correspond to channel openings. TBB (10 - 40 μM) decreased the single-channel current amplitude (i) and open probability (P₀) of CFTR (control for TBB (10 μM), i = -0.79±0.04 pA, P₀ = 0.47±0.02; TBB (10 μM), i = -0.64±0.05 pA, P₀ = 0.40±0.03; p < 0.05 : control for TBB (40 μM), i = - 0.76±0.03 pA; TBB (40 μM), i = -0.31±0.04 pA; p < 0.05; P₀ in presence of TBB (40 μM) could not be determined accurately; n = 6 for all values).

C. 4% paraformaldehyde-fixed human nasal ciliated airway cells from wild type (left panel) and Pseudomonas-fvQe homozygous ΔF5O8 CF (right panel) subjects (approved by local ethical committee) were stained with anti-CFTR rhodamine and anti-CK2 FITC. Images were acquired with a Zeiss LSM510 microscope using identical laser outputs and gain and are typical of samples from three separate individuals for each. Scale bar is 5 μm. Controls omitting primary antibodies were blank at the same settings.

Figure 2: CK2 interacts with wild type but not ΔF508 CFTR.

A. 1% octyl-glucoside extracted human bronchial epithelial (HBE) membranes overlaid onto dot blots of wild-type / ΔF508 peptide (GTIKENIIFGVSYDEYRYR / GTIKENΠGVSYDEYRYR; labelled as KENIIF / KENII respectively), and probed for CK2α or PKC (both Santa Cruz). Bottom panels omit primary antibody and fourth PKC panel is positive control; unrelated peptide is fromNBD2 (QRVGLLGRTGSGKSTLL). B. reciprocal co-immunoprecipitation (IP) of CK2 with CFTR from ovine tracheal (P4) ²⁰ and HBE membranes.

C. Western blots from HBE and CFBE membranes probed for CFTR showing that the level of CFTR in CFBE membranes is -10% of that in HBE membranes (left panel). Increasing CFBE protein tenfold gives similar CFTR levels to HBE; CK2α is detected in HBE, but not 1 Ox CFBE membranes (right panel).

D. co-immunoprecipitation of PKA and PKC, but not CK2 with CFTR from HBE and 1Ox CFBE membranes. All IPs were washed with IM NaCl.

Figure 3: CK2-dependent phosphorylation only occurs on wild-type (KENIIF) CFTR peptide; CK2 targets native CFTR.

A. quantification of recombinant CK2 (NEB) -dependent KENIIF / KENII / NBD2 CFTR peptide phosphorylation ± polylysine (CK2 activator) ¹² with γ[³²P]- ATP. n=3 ±range; inset, autoradiograph showing selective, polylysine-stimulated phosphorylation of immobilised KENIIF peptide.

B. as in A, but comparing PKA- and CK2-dependent phosphorylation of KENIIF / KENII. n=3 ±range; inset, KENIIF-specific inhibition of phosphorylation of immobilised peptides by competing free peptide (10 μM).

C. γ[³²P]-ATP and γ[³²P]-GTP CK2-dependent, TBB-inhibitable phosphorylation of 180 kDa proteins only in CFTR-expressing cell membranes (T84 and Calu-3 but not skeletal muscle).

D. CK2 activity is absent from CFBE membranes: Assay for CK2 activity in HBE and CFBE membranes and cytosol. ^'5 μg protein (or 50 μg in 10x samples) from the indicated sources were assayed for CK2 activity ⁹. Either γ[³²P]-ATP (black bars) or γ[³²P]-GTP (white bars) was used as phosphate donor to detect CK2 phosphorylation directed towards the specific target peptide RRRADDSDDDDD (n=3 ±range) (gift of F. Meggio, S. Sarno and L. Pinna). 10 μM TBB was used to ensure that activity was specific. CK2 activity is absent even from 10x CFBE membranes, but is equivalent to wild type levels in CFBE cytosol. Figure 4: CK2 phosphorylates wild type but not ΔF508 recombinant CFTR NBDl on S511.

A. Differential phosphor^ylation of wild type (NBDl) and ΔF508 NBDl protein by CK2 (NEB) from γ[³²P]-ATP/GTP; stimulation with poly-lysine (PoIyK) and inhibition with 10 μM TBB. n=3 ±range. Phosphorylation only occurs on wild- type NBDl.

B. Equal phosphorylation of wild-type and ΔF508 NBDl by PKA. n=3 ±range. PKAi is a pseudo-substrate inhibitor of PKA (100 nM)

C. CK2 and PKA phosphorylate wild-type NBDl additively at distinct sites (their respective inhibitors are specific). n=3 ±range.

D. CK2-, but not PKA-dependent phosphorylation is abolished in S511A NBDl protein. n=3 ±range. All C and D data are in the presence of poly-lysine. All constructs were sequence verified. Recombinant poly-His-tagged proteins were purified to homogeneity as described in supplementary material.

Figure 5: Amino acid sequence of human CFTR and CK2 αl subunit polypeptide

Figure 6: A. Using data to show that TBB inhibits Cl^" current when applied after a forskolin stimulus: Calu-3 monolayers were treated as described in Collett et al. 2002 and Cl^" current stimulated with 100 μM forskolin. TBB or DPC were added as indicated (left panel). TBB and DPC both inhibit I^cl(apical) to a similar extent (right panel, n=5, mean ±SEM).

B. Using data showing that TBB inhibits Cl^" current in HBE monolayers. n=5, mean ±SEM. *, pO.001. ^•

Figure 7: Characterisation of Neomarkers anti-NBDl of CFTR antibody used in the study: Specificity of Neomarkers antibody:

A Western blotting on 20μg of membrane extract from CFBE and HBE cells probed with a 1/1000 dilution of the Neomarkers NBDl -directed CFTR antibody. -180 kDa polypeptide corresponding to CFTR was detected. B The presence of CFTR was confirmed in the Calu-3 cells used in the experiments and the specificity of the antibody is indicated by the presence of a band in Calu-3 and HBE membranes and was absent in skeletal muscle. CFBE membranes showed faint staining after prolonged exposure (not shown). C Western blot of 4 - 12% SDS-PAGE gradient gel containing 20μg of CHO cell protein extract (membrane fraction) with and without methotrexate controlled CFTR expression. The nitrocellulose membrane was probed with a 1/1000 dilution of Neomarkers anti-NBDl CFTR antibody. A -180 kDa polypeptide corresponding to the predicted molecular mass of the CFTR protein was detected in CHO cell extracts to which methotrexate had been added and not in those CHO cell extracts without methotrexate added. Application of antibody in immunoprecipitations:

D Neomarkers anti-NBDl CFTR antibody or anti-CFTR gifted by J. Riordan were used to immunoprecipitate from 20μg of ovine tracheal membrane protein. The immunoprecipitates were blotted and probed with a 1/1000 dilution of Neomarkers anti-NBDl CFTR antibody. A -180 kDa polypeptide was detected in both samples.

Figure 8: CK2-dependent, heparin-inhibitable phosphorylation of 180 kDa proteins in human ciliated airway (AM), Calu-3 and HBE but not CFBE or CFTR- negative liver membranes. Heparin is a specific inhibitor of CK2 [Hathaway, 1980 #136]. Exogenous CK2 was added to lOμg membrane protein from the indicated sources and incubated with γ[ P]-ATP (100 mM) in a CK2 phosphorylation buffer

Figure 9: CKlα does not associate with CFTR:

DMP-linked NDPK (Santa Cruz), CFTR (Neomarkers), CKlα (gift of David Meek, Dundee) and CK2α (Santa Cruz)antibodies were used to immunoprecipitate from extracted P4 membranes (Muimo et άl. 1998, Am J Respir Cell MoI Biol 18(2): 270-8) (ovine tracheal membranes, 15μg each); Western blots were probed for either CKlα or CFTR as indicated. CKlα positive control was 5μg of rat liver whole extract and CKlα negative control was a pure cytosolic supernatant from a Calu-3 cell extract. No association between CKlα and CFTR was observed.

Figure 10: Phosphatases PP2Bα and PP2C associate with ΔF508 CFTR in the same quantities as with wild type CFTR:

15μg HBE and 150μg (1Ox) CFBE membrane were immunoprecipitated with anti- CFTR antibody. Western blots incorporating a positive control lane were probed for PP2Bα and PP2C as known CFTR-associated phosphatases. Quantities detected were proportional to the amount of CFTR in each sample. Thus two different phosphatases show no loss of binding to ΔF508 CFTR.

Figure 11: Coomassie blue stain (A), Western blot (Neomarkers, B) and silver stain (C) evidence to show purity of recombinant NBDl protein.

Figure 12: CK2 inhibitors - Dose-response

Using NBDl as substrate, active CK2 was incubated in the presence of 0, 50 nM, 500 nM, 1 mM, 10 mM and 100 mM of DIDS₅ TBB, Genistein, Nifiumic acid and DPC, phosphorylation, using P-ATP as donor.

Control was the substitution of PKA for CK2, and revealed over the entire range of compounds, that none of these compounds inhibited PKA phosphorylation of NBDl and that this phosphorylation is an ATP process (No effect using GTP as donor, not shown).

Figure 13: Titrated TBB or Genistein vs ATP

A. NBDl is NOT using TBB as an ATP analogue but does recognise Genistein as such. P-ATP using 100 mM final ATP concentration blocked using 2% BSA in TBS-tween washed 4 x 10 minutes and counted. Used 100 mg of NBDl / CK2α recombinant, protein per spot.

B. Comparison of genistein-evoked Cl^" current in Calu-3 monolayers in the presence of the indicated concentrations of TBB. Figure 14: CFTR inhibitors screened for ability to inhibit CK2

32

Using P-GTP, recombinant CK2 enzyme was used to phosphorylate CK2 peptide under normal conditions in the presence of a range of known CFTR inhibitors with various mechanisms of action.

As can been seen, DIDS, DPC, TBB and Nifiumic acid all inhibited CK2 activity at their reportedly usable concentrations, whilst the remaining compounds had no effect on CK2 activity.

Figure 15: Full length CFTR S511A / S511D / DF508 - S511D mutant binding to CK2α:

Recombinant CK2α protein was overlaid onto Nitrocellulose spotted with either full length wild type CFTR, full length S511A mutant, full length S511D mutant, NBDl alone, full length ΔF508 CFTR or full length ΔF508 - S511D mutant CFTR and probed with an antibody specifically directed against CK2α.

Figure 16: Full length CFTR mutant screen:

Neomarkers precipitation from 200 mg of total bacterial extract of wild-type (+ control)

S51 IA 1,2 or 3 mutants

S51 ID 1, 2, 3 or X mutants

Incubated with either CK2 + PoIyK or PKA as indicated on figure.

Anti-MEK antibody control was included for non-specific interactions.

Figure 17: DF508 - S511D mutant

Figure 18: DF508 - S511D Transfections into CFBE:

We used 30 ml of Lipofectamine and transfecting with 1 mg of DF508 - S511D cDNA, leaving to take up for 3 hours in 2.5 ml of media, washed in PBS and incubated in media for 22 hours. Membranes were harvested as normal and 100 mg of membrane fraction was loaded onto a 4 - 12% SDS-PAGE and probed for either CFTR or CK2 as above. Positive control was FfBE membranes and for CFTR, we included mock and DF transfection control lanes to eliminate the possibility that transfection was constituting a stress or that overexpression was having a non-specific effect and increasing thereby CFTR expression in the membrane. In both cases, the mutant increased CFTR expression whilst the mock and DF did not.

Figure 19: Further validation of the CFTR antibody

Membranes extracted from indicated cell lines; the CHO cell line contains a methotrexate-inducible CFTR. The protocols used are as described herein.

Figure 20: CK2α biots

A: HBE / CFBE membrane fractions (100 mg) were used either for CFTR, Kir6.2 or CK2α immunoprecipitation or Input as indicated in the figure labelling. A 1/1500 dilution on CK2α antibody was used to probe the 4-12% SDS-PAGE according to established laboratory protocols. Precipitating antibodies were DMP linked according to the methods previously detailed, eliminating the antibody heavy and light chains.

B: CFTR and CK2α immunoprecipitations from lOOmg of HBE/CFBE membranes probed with a 1/1000 dilution of CFTR antibody.

C: Comparison of CFTR content of 100 mg of HBE / CFBE membrane fractions. Membrane proteins were run on SDS-PAGE as normal and probed with a 1/1000 dilution of CFTR antibody.

Figure 21: CK2 / CFTR / ENaC co-precipitations:

20 mg of human nasal brushings loaded alone or precipitated as detailed in the figure.

A: A polypeptide of ~80 kDa was detected using a 1/3000 dilution of an anti-

ENaC antibody and this polypeptide was also present in a CFTR precipitation from an equivalent protein amount. B: Using the Kir6.2 antibody as a control for non-specific precipitation, we observe that both an ENaC and CK2a precipitation are able to specifically pull down CFTR.

C: CK2a visa versa preciptiations using ENaC, CFTR and NDPK antibodies. We observe that the CFTR protein complex is specifically associated with the ENaC protein in human nasal brushings.

Figure 22: CK2α vs CFTR

DMP -linked CK2α and CFTR antibodies were used to precipitate from ~100mg of P4 membrane extracts. These precipitates were run on a 4-12% gradient SDS- PAGE gel, transferred as normal and probed with a 1/1000 dilution of the alternate protein antibody as indicated in the figure.

Figure 23 : CK2β vs CFTR

DMP -linked CK2β and CFTR antibodies were used to precipitate from ~100mg of P4 membrane extracts. These precipitates were run on a 4-12% gradient SDS- PAGE gel, transferred as normal and probed with a 1/1000 dilution of the alternate protein antibody as indicated in the figure. + control for CK2β was pure recombinant NEB holoenzyme and - control for specificity was a CK2β precipitation from 20 mg of skeletal muscle probed for CFTR.

Figure 24: CK2α Validation from HBE/CFBE:

An increasing titration of HBE (5, 15, 30 and 50 mg respectively) and a bulked-up amount (200 mg) of CFBE membrane fractions were run on 4-12 % SDS-PAGE and probed first with a 1/1500 dilution of anti-mouse CFTR antibody, the nitrocellulose membrane was then stripped and re-probed with a 1/1000 dilution of anti-goat CK2α antibody. The resultant data indicated that: A: At a greatly diluted CFTR detection amount in HBE membranes (roughly comparable to the CFTR signal observed in CFBE membranes), a CK2α signal is still clearly detectable. B: At a greatly bulked-up level of CFBE membranes (where the CFTR signal is enhanced to approximately the same detection levels as in wild-type) no CK2α signal is detectable.

Figure 25: HBE / CFBE membranes vs CK2 beta

Used 20 mg of HBE or CFBE membrane fraction, run on a 4 - 12% SDS-PAGE gel and probed with a 1/2000 dilution of CK2 beta antibody. We observe surprisingly, that CFBE membranes contain CK2β protein and that it is roughly 8 - 10 times less abundant that in HBE, in keeping with the approximate 10:1 ration of CFTR, AMPK, NDPK and now, CK2β between HBE:CFBE membranes.

Figure 26: ATP-bound sites for NBDl and CK2

Figure 27: CK2 is localised to the apical membrane

Figure 28: Inhibition of Cl^" current with myr-KENIIF applied externally

Myristoylated KENIIF peptides applied externally shows inhibition of CY current. Myr-KENIIF inhibits CFTR at 5nM.

Figure 29:

A: The Gruenert (HBE/CFBE) and Riordan (CHO) cell lines; CHO contain methotrexate-inducible CFTR.

B: CK2-conditional mutant S51 IA-CFTR showing western blot, overlay and phosphorylation.

C: Membranes from nasal brushings (leftmost panel) or Gruenert cell lines. Note

CFBE membranes contain 10% of the CFTR of HBEs. ENaC and CK2 co-IP with

CFTR (leftmost panel). PP2B and PP2C co-IP with CFTR from both wild type and homozygous ΔF508 membranes (middle 2 panels). NDPK co-IPs with CFTR

(rightmost panel) Figure 30: Confirmation of the importance of phosphorylation of S511 and the dependence of CK2 binding on F508

A. Single-channel analysis of wild-type and S51 ID-CFTR. Left, representative single-channel recordings show the gating behaviour of wild-type (WT-) and S51 ID-CFTR in the presence of ATP (ImM) and PKA (75nM). The lower trace of each pair shows an expanded timescale. Right, quantification of i, Po and TBB ^• (40μM) blockade for both channels (n=6). Because of poor cell-surface expression, we did not study the effects of the non-phosphorylatable S511A mutation of CFTR function.

B. TBB inhibits wild-type, but not ΔF508-CFTR in Xenopus oocytes. Oocytes were injected with cRNA (1-lOng) encoding either wild-type or ΔF508-CFTR and macroscopic CFTR Cl^" currents were measured after 2-3 days using the two- electrode voltage-clamp technique. Conductances were calculated from clamp voltages and macroscopic currents. Following the activation of macroscopic currents with 3-isobutyl-l-methylxanthine (IBMX, 1 rnM) and forskolin (2 μM), TBB (1 μM) was added in the continuous presence of IBMX and forskolin. Histograms show the macroscopic conductance of wild-type CFTR (top panel, right) and ΔF508-CFTR (bottom panel, right) for the indicated conditions (FK=forskolin and IBMX)(n=7±SEM).

Example 1: Evidence to suggest that CK2 is a F508-dependent, CFTR-bound protein kinase.

Summary

The deletion of F508 (ΔF508) from the first nucleotide binding domain/fold (NBDl) of the cystic fibrosis transmembrane conductance regulator (CFTR) is the most frequent cause of cystic fibrosis (CF) ^1>2. This mutation disrupts CFTR's function in multiple ways perturbing CFTR biosynthesis ³, cell surface expression ⁴, channel gating ⁵ and regulation of epithelial ion channels ⁶. Structural studies indicate that F508 is located on the surface of NBDl in a position where it might participate in protein-protein interactions ⁷. However, to date, the molecular role of F508 in wild type CFTR remains uncertain. Here, we show that the pleiotropic protein kinase CK2 (casein kinase 2) ⁸'⁹, which is involved in gene transcription, protein trafficking, cell proliferation and development ¹⁰'^π ₅ binds and phosphorylates wild-type CFTR at S511. Moreover, we show that 4,5,6,7- tetrabromobenzotriazole, a specific inhibitor of CK2, inhibits the CFTR Cl^" channel. Importantly, the interaction of CK2 with NBDl of CFTR is abrogated by the ΔF508 mutation. We propose that ΔF508-dependent disruption of the CK2- CFTR interaction provides novel paradigms for both CK2 and CF research.

Results and Discussion

The amino acid sequence of CFTR immediately distal to F508 contains a consensus sequence (KENIIFsnsGVSYDEYR) for phosphorylation by protein kinase CK2 ⁿ with a potential target serine located at S511. CK2 is a pleiotropic kinase, with a wide spectrum of targets linking its activity to such diverse cellular functions as RNA synthesis, apoptosis and cell cycle progression ⁸'⁹. We investigated whether CK2 interacts functionally with this region of CFTR and, further, whether deletion of F508 disrupts binding of this signalling molecule to CFTR.

CFTR plays a fundamental role in fluid and electrolyte transport across epithelia ². To specifically examine CK2-dependent regulation of CFTR function in epithelia, we tested the effects of 4,5,6,7-tetrabromobenzotriazole (TBB), a selective inhibitor of CK2 ¹³ on cAMP-stimulated apical membrane Cl^" currents (I^α(apical)) using the Ussing chamber technique * ' ⁵. Figure IA demonstrates that preincubation of Calu-3 epithelial monolayers (a robust model for CFTR transport ¹⁶) with TBB caused a concentration-dependent decrease in I^cl(apical) with half- maximal inhibition occurring at a concentration (~15μM) that causes potent inhibition of CK2 ¹³. TBB also abolished the stimulated Cl^" current when applied after forskolin (Fig. 6A). Forskolin-stimulated I^cl(apical) was similarly abrogated in the 16HBE14o- bronchial epithelial cell line ¹⁶ (HBE - Fig. 6B). To test directly the effects of TBB on the CFTR Cl^" channel, we used excised inside-out membrane patches from C 127 (mouse mammary epithelial) cells expressing recombinant wild-type human CFTR ^π. Figure IB demonstrates that TBB (40 μM) caused a marked reduction in CFTR CF current, with inhibition being incompletely reversed upon washout of the drug (Fig. IB, left panel). At the single-channel level, TBB (10 and 40 μM) caused a fast flickery block of CFTR decreasing both single-channel current amplitude (i) and open probability (P₀; Fig. IB, right panel). These effects of TBB are similar to that produced by a variety of CFTR modulators (e.g. niflumic acid ¹⁷ and IBMX ¹⁸). They demonstrate that TBB, a selective inhibitor of CK2, inhibits the CFTR Cl^" channel.

To determine specifically whether CK2 binds and phosphorylates CFTR near F508, we generated wild-type and ΔF508-CFTR peptides, which both contain the CK2 consensus sequence. ^' Detergent extracts of airway epithelial membranes overlaid onto CFTR-derived peptide dot blots followed by detection with anti- CK2α antibody demonstrated that CK2 only binds the peptide sequence corresponding to wild-type CFTR (KENIIF; Fig. 2A). No interaction was detected with either the ΔF508-CFTR peptide (KENII; Fig. 2A) or a control peptide from another region of CFTR (unrelated peptide; Fig. 2A) and no peptide bound PKC as a control CFTR-directed kinase ¹⁹ (Fig. 2A). Next, we used immunofluorescence and confocal microscopy to test whether the subcellular localisation of CK2 coincides with that of CFTR in native cells with differentiated morphology (fresh biopsies of normal and homozygous ΔF5O8 CF ciliated human nasal epithelial cells). Figure 1C demonstrates that CK2α colocalises with CFTR at the apical membrane of normal but not ΔF508 CF epithelial cells.

To test CK2's association with CFTR in airway epithelial cell membranes, we isolated membranes from HBE ¹⁶ cells and sheep tracheal epithelium ²⁰. Figure 2B demonstrates that CFTR and CK2 co-immunoprecipitate under stringent conditions from both epithelial membranes. We tested the ΔF508 -dependence of the CFTR-CK2 interaction using a CF bronchial cell line homozygous for the ΔF508 mutation (CFBE41o-; CFBE) ¹⁶. Because low levels of ΔF508 CFTR protein are present in membranes from CFBE cells (~10% of wild-type CFTR levels; Fig. 2C, left panel), we used a ten-fold excess of membrane protein (1Ox CFBE). CK2 was not detected in 1Ox CFBE membranes (Fig. 2C, right panel). However, both PKA and PKC co-immunoprecipitate with CFTR from 1 Ox CFBE in a quantity comparable to that of HBE membranes (Fig 2D). Similarly, equivalent amounts of PP2Boc and PP2C ¹⁹ ₅ two protein phosphatases that dephosphorylate CFTR, were detected in both HBE and 10x CFBE (supplementary data, Fig. 10). Finally, CKl, another protein kinase requiring E/D amino acid residues near its target serine, was not detected in either cell membrane preparation (Fig. 9). Thus, the data demonstrate the F508-dependence of the CK2-CFTR interaction.

Next, we examined the potential signalling consequences. CK2 can use either ATP or GTP to phosphorylate its targets ⁹. Overlaying recombinant CK2 holoenzyme (2α-2β) onto peptide dot blots in the presence of γ[³²P]-ATP (or γ[³²P]-GTP, not shown) phosphorylated only the wild type peptide, despite the CK2 consensus sequence remaining intact in the ΔF508-CFTR peptide (Fig. 3A; phosphorylation enhanced eight-fold by the CK2 activator, polylysine ²¹). Consistent with these data, the phosphorylation of immobilised wild-type CFTR peptide was abolished by the presence of free wild-type CFTR peptide, but not ΔF508 peptide (10 μM) in the solution (Fig. 3B, inset). PKA failed to phosphorylate wild-type CFTR peptide (Fig. 3B), indicating that the phosphorylation of S511 is specific to CK2. The simplest explanation of these data is that F508 is critical for CK2 to bind CFTR and phosphorylate the only serine in either peptide, S511.

To determine whether CK2 can phosphorylate native CFTR₅ we isolated membranes from four different cell types that endogenously express CFTR. Addition of CK2 holoenzyme to membranes isolated from two different CFTR- expressing cell lines, T84 and Calu-3, in the presence of γ[³²P]-ATP or γ[³²P]-GTP caused the phosphorylation of a 180 kDa band consistent with the expected size of the mature form of CFTR; this phosphorylation was abolished by TBB (10 μM; Fig. 3C, upper panel) and the 180 kDa band was absent in CFTR-negative skeletal muscle. A similar 180 kDa band, inhibitable with another CK2 inhibitor, was phosphorylated in HBE (but not CFBE or liver) membranes, apically enriched human nasal epithelial membranes ²⁰ and Calu-3 membranes (supplementary data Fig. 8). The absence of CK2 from CFBE membranes observed in Fig. 2C was confirmed by testing for CK2 activity ⁹. CK2 activity was detected in HBE but not 1Ox CFBE membranes, whereas cytosolic activity was similar in both cell lines (Fig. 3D).

The ΔF508 dependence of CK2 phosphorylation was further demonstrated by the failure of CK2 to phosphorylate purified recombinant ΔF508-CFTR NBDl protein, whereas wild-type CFTR NBDl was phosphorylated as expected (Fig. 4A). Both proteins contain a dibasic PKA target at S660 ²². Consistent with these data, PKA. phosphorylated the wild type and ΔF508 proteins equivalently (Fig. 4B). Importantly, TBB (10 μM) abolished the CK2-dependent phosphorylation of wild-type CFTR NBDl whereas the PKA-dependent phosphorylation was unaffected, confirming the specificity of the inhibitor. Conversely, a specific PKA inhibitor was without effect on CK2-dependent phosphorylation, but abolished PKA-dependent phosphorylation (Fig. 4C). The incorporation of ³²PO₄ into the wild type NBDl was similar with both PKA and CK2, consistent with the NBDl containing one putative target serine for each kinase; furthermore; the phosphorylation of wild-type NBDl with both kinases was additive, indicating that CK2 and PKA target distinct sites (Fig. 4C, final column). Thus, wild type and ΔF508 NBDl domains exhibit differential phosphorylation only with respect to CK2. Finally, to verify that S511 alone is phosphorylated by CK2 in wild-type CFTR, we generated a mutant recombinant NBDl protein. Phosphorylation assays using NBDl and NBD1-S511 A protein as substrates for CK2 demonstrated J:hat PKA-dependent phosphorylation remains intact, whereas CK2 fails to phosphorylate the S 511 A mutant (Fig. 4D) .

Here, we demonstrate that CK2 is a novel NBDl -directed CFTR kinase and report that deletion of F508 selectively disrupts CK2 binding. Further, we suggest that wild-type CFTR is an anchoring protein for CK2. The absence of CK2 in ΔF508 apical membranes could thus not only close the channel but also disrupt apical- membrane-associated phosphorylation pathways dependent on this protein kinase. We believe that this will ultimately explain the plethora of cell functions that is abnormal in CF since the reported targets of CK2 ⁹'²³ overlap significantly with several unexplained aspects of CF disease ^24"26. Our studies on one such target ²⁴ support this hypothesis. The consistency of our results in cultured epithelial cells, CFTR-transfected cells and native tissue suggests that the CK2-CFTR interaction is a general feature of CFTR-expressing cells.

There is currently no cure for cystic fibrosis; one therapeutic approach proposes the stimulation of trafficking of ΔF508 CFTR to the apical membrane ²⁷'²⁸. Our data suggest that CK2-dependent regulation of ΔF508 CFTR would still be defective under such conditions, consistent with published data showing a failure to fully restore Cl^" current ^5>27-²⁹. Therefore, therapeutic strategies may be better directed towards the combined restoration of CK2 localisation and CFTR phosphorylation in the ΔF508 CF cell. Conversely, our data identifies an intensely-researched ion channel as an acutely-regulated CK2 target that might generate novel drugs for the treatment of secretory diarrhoeas (inhibitors of CFTR have been proposed as treatments for cholera ³⁰). We conclude that active, bound CK2 is essential for normal CFTR function and present a simple model of the commonest CF defect that directly links the deletion of F508 with defective binding of a pleiotropic kinase.

Supplementary Data

Fig 6A shows that, after CFTR stimulation with forskolin, TBB can inhibit the CFTR current despite the presence of both the chloride gradient across^' Calu-3 monolayers and forskolin. Right panel shows the similarity of the inhibition of CFTR current using DPC and TBB. Fig 6B. Since we used HBE cells to undertake the co-immunoprecipitation results with CK2, Fig 6B shows that TBB inhibits HBE CFTR currents in a similar manner. CFBE cells which bear the ΔF508 mutation showed no CFTR current (not shown n>5) in any experiment. Fig 7. The primary purpose of this work was to validate the antibody used in the experiments against a well characterised CFTR antibody supplied by J Riordan.

Blotted membranes were blocked for 30 minutes in TBS-T ween with 5% milk powder followed by 4 x 15 minute washes, anti-NBDl antibody (Neomarkers) was applied for 1.5 hours followed by 4 x 15 minute washes, then HRP-labelled anti-mouse secondary antibody was applied at a 1/5000 dilution for 45 minutes followed by 4 x 15 minute washes. Bound HRP was visualised using a chemiluminescence system and exposure to X-ray film.

Fig 8. Here we show that a phosphorylated band of the size expected for CFTR was present in membranes made from either native airway or the Calu-3 cell line used in this work. That phosphorylation of this band disappeared with another CK2 inhibitor (heparin) is consistent with our model.

Fig 9. CKl is another serine/threonine protein kinase that targets acid residue clusters. This protein kinase does not associate with CFTR in irnmunoprecipitation experiments using the antibody shown in the earlier figure.

Fig 10. Having shown that ΔF508 CFTR does not associate with CK2 (but does associate with PBCA and PKC) we now show that two protein phosphatases are unaffected by the loss of F508. Thus the loss of CK2 is selective.

Fig 11. Cloning of NBDl from CFTR: In order to clone an NBDl construct corresponding to amino acids 351 - 665 of human CFTR, we used full length cftr cDNA (gift; C. Boyd, Edinburgh) as template and designed the following primers (Fwd: 5'-ctcgagatgtggtatgactctcttgga-3 '; Rev: 5'-tcagttagccatcagtttacacgccggcg- 3') incorporating Xhol and Notl restriction sites 5" and 3" respectively. PCR was performed with these primers and template using a standard PCR protocol (92 ⁰C for 1 minute, 38 ⁰C for 2.5 minutes and 46 °C for 3.5 minutes, for 35 cycles) the resultant product (-950 base fragment) was gel purified and placed into a standard ligation reaction with XhoI/NotI digested pcDNA3.1-His⁺ (His -tagging plasmid vector (Invitrogen)). A small amount of this ligation reaction was transformed into JMl 09 competent E. coli bacteria. The transformation mixture was shake- incubated for 4 hours then 20 μl plated onto lOOμg/ml ampicillin-LB plates and incubated overnight.

Ampicillin-resistant bacterial colonies were used to inoculate 50 ml of ampicillin- LB broth and shake-incubated overnight. pcDNA3.1-His⁺ plasmid cDNAs encoding NBDl of CFTR were isolated from the bacterial culture using alkaline lysis (Qiagen) and verified by DNA sequencing using the ABI Prism DNA sequencer.

Purification of NBDl from bacteria: One litre of LB broth, inoculated with E. coli containing sequence-verified pcDNA3.1-His⁺ plasmid cDNA encoding NBDl of CFTR was grown for 10-12h in the presence of lOOμg/ml ampicillin. The bacteria were pelleted for 10 min at 4000rpm. The pellet was resuspended in 10 ml of lysis buffer (5OmM NaPO₄, 0.3M NaCl, 8M urea, 1OmM imidazole, 2% Tween- 20, pH 8 with protease inhibitors), then sonicated for 3 x 15s bursts. Sonicated bacteria were further lysed using a syringe and DNA/insoluble proteins pelleted by centrifugation at 13,000rpm for 5min. The supernatant was added to 5ml of start buffer (0.2M NaPO₄, 0.5M NaCl and 1OmM imidazole, pH 7.4) and applied to a Ni²⁺-Sepharose affinity column (BioRad) which was pre-equilibrated in start buffer. Sample was ran through the column three times and then washed with a further 10 column volumes of start buffer; His-tagged NBDl was eluted with 7 ml of elution buffer (0.2M NaPO₄, 0.5M NaCl, 50OmM imidazole, pH 7.4) 0.5ml fractions were collected by gravity flow. Column fractions were screened for NBDl protein by Bradford assay and Western blotting using the NBDl -directed anti-CFTR antibody. Purity of NBDl was verified by Coomassie blue and silver stain.

Generation of the S511A mutant in the NBDl region of CFTR: The cftr gene- specific primers; fwd: 5Mcatctttggtgttgcctatgatgaatat-3" and rev: 5"- atattcatcataggcaacaccaaagatga-3\ were used to introduce a S to A point mutation at the 511 site in the CFTR NBDl region using PfuTurbo DNA polymerase in a PCR-based method (Quickchange SDM kit, Invitrogen). Once the mutation had been generated and verified by DNA sequencing, the mutant was digested and subsequently ligated into our expression vector. The mutant was then transformed into a competent JMl 09 E. coli cell line for expression. Amplification and protein purification as described for the NBDl wild-type fragment.

Supplementary experimental details: Patch-clamp experimental methods:

CFTR Cl^" currents were recorded in excised inside-out membrane patches as described previously ¹⁶. The pipette (extracellular) solution contained (mM): 140 N-methyl-D-glucamine (NMDG)₅ 140 aspartic acid, 5 CaCl₂, 2 MgSO₄ and 10 TES, pH 7.3 with Tris ([Cl^'], 10 mM). The bath (intracellular) solution contained (mM): 140 NMDG, 3 MgCl₂, 1 CsEGTA, and 10 TES, pH 7.3 with HCl, ([Cr], 147 mM; [Ca²⁺]_free, <10^"8 M) and was maintained at 37 ⁰C. CFTR Cl^" currents were filtered at 500 Hz and digitised at 2.5 kHz (time-course studies) or 5 kHz (single-channel studies). In time-course studies, each data point is the average current for a 4 s period with data points collected continuously; no data were collected while solutions were changed. Average current (I) for a specific intervention is the average of all the data points collected during the intervention. To measure i, Gaussian distributions were fit to current amplitude histograms. To measure P₀₅ we created lists of open- and closed-times and calculated P₀ as described previously ¹ . The number of channels in a membrane patch was determined from the maximum number of simultaneous channel openings observed during the course of an experiment ¹⁶.

Summary of the known effects of CK2 and how this impacts on CF pathophysiology

CK2 phosphorylates targets in: (from Meggio and Pinna FASEB J. 2003 Mar;17(3):349-68; Table T). Cell signalling pathways (RII cAMP binding protein for PKA)₅

Endocytosis (caveolin),

Transcription (CREB/iκBβ),

DNA-RNA-protein- synthesis, (many including TOPO II)

Fatty acid metabolism (AMPK target Acetyl CoA carboxylase)

Bone turnover (osteopontin and dentine formation).

Table 1

References

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2. Welsh, M. J., Ramsey, B. W., Accurso, F. & Cutting, G. R. in The Metabolic and Molecular Basis of Inherited Disease (eds. Scriver, C. R., Beaudet, A. L., SIy₃ W. S. & Valle, D.) 5121-5188 (McGraw-Hill Inc., New York, 2001).

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6. Stutts, M. J. et al. Science 269, 847-50 (1995).

7. Lewis, H. A. et al. J Biol Chem (2004).

8. Litchfield, D. W. Biochem J 369 ', 1-15 (2003).

9. Meggio, F. & Pinna, L. A. Faseb J 17, 349-68 (2003).

10. Guerra, B . & Issinger, O. G. Electrophoresis 20, 391-408 (1999).

11. Wang, H., Davis, A., Yu, S. & Ahmed, K. MoI Cell Biochem 227, 167-74 (2001).

12. Meggio, F., Marin, O. & Pinna, L. A. Cell MoI Biol Res 40, 401-9 (1994).

13. Sarno, S. et al. FEBS Lett 496, 44-8 (2001).

14. Quinton, P. M. & Reddy, M. M. Nature 360, 79-81 (1992).

15. Collett, A., Ramminger, S. J., Olver, R. E. & Wilson, S. M. Am J Physiol Lung Cell MoI Physiol 282, L621-30 (2002).

16. Gruenert, D. C, Willems, M., Cassiman, J. J. & Frizzell, R. A. J Cyst Fibros 3 Suppl 2, 191-6 (2004).

17. Scott-Ward, T. S., Li, H., Schmidt, A., Cai, Z. & Sheppard, D. N. MoI Membr Biol 21, 27-38 (2004).

18. Schultz, B. D., Frizzell, R. A. & Bridges, R. J. J Membr Biol 170, 51-66 (1999).

19. Dahan, D. et al. Pflugers Arch 443 Suppl 1, S92-6 (2001).

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23. Ishihara, K., Yamagishi, N. & Hatayama, T. Biochem J 371, 917-25 (2003).

24. Treharne, K. J., Mehta, A. & Muimo, R. Pediatric Pulmonology Suppl 22, 195 (2001).

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29. Denning, G. M. et al. Nature 358, 761-4 (1992).

30. Thiagarajah, J. R. & Verkman, A. S. Curr Opin Pharmacol 3, 594-9 (2003).

Example 2: Application of the methods and kits of the invention

A patient is presented to a physician with symptoms of cystic fibrosis.

An airway nasal brush biopsy is performed on the patient to obtain a sample of polypeptides. The tissue contained in the biopsy is then analysed for CFTR/CK2 polypeptide location using a kit of parts of the invention; the kit of parts having an antibody capable of recognising CK2 and an antibody capable of recognising CFTR. The location of the CFTR/CK2 polypeptides in cells of the patient's sample is determined using confocal microscopy.

If the CFTR/CK2 polypeptides do not interact at the apical membrane of the cell then the patient can be considered to have or be at risk of developing cystic fibrosis.

Once so diagnosed, a patient can be treated with an appropriate quantity of a compound identified from the screening methods of the invention which is capable of elevating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

Restitution of CK2 and/or CFTR function can subsequently be measured to determine the effect of the compound identified from the screening methods of the invention on the progression of cystic fibrosis, or likelihood of the development of cystic fibrosis, in the patient. Here, a further tissue sample is taken from the patient using, for example, airway nasal brush biopsy. The location of the CFTR/CK2 polypeptides in the cells of the tissue is again determined using the kit of parts of the invention and confocal microscopy, as described above. If the sample shows an increase in localisation of CFTR and/or CK2 polypeptide to the apical membrane of a cell, in comparison to the sample taken from the patient prior to being administered the compound, then the compound can be considered as restoring CFTR and/or CK2 function.

Example 3: Further supplementary data

CK2 inhibitors

The specificity of TBB is reported in Sarno et al (2001) FEBS Lett 496, 44-48 with an IC₅₀ of 1.6 μM in vitro, in the presence of 100 μM ATP, and in vivo the concentration used is 60 μM, even higher than the inhibitory concentration reported in the previous examples. Furthermore, the IC₅O for TBB in a different in vivo assay is reported to be 20 μM (Pagano et al (2004) Biochem Biophys Res Comm 321, 1040-1044). In the Ussing chambers, the IC₅₀ for CFTR inhibition by TBB is about 12 μM.

We present data showing that other, previously-reported inhibitors of CFTR are capable of inhibiting CK2. We screened several published CFTR inhibitors using an assay for CK2 activity. Most of the inhibitors were without effect on CK2, however, two compounds commonly held to act as CFTR pore blockers; DPC and niflumic acid, were found to potently inhibit CK2 phosphorylation at the reported CFTR-inhibiting concentrations (Figures 12 and 14). DIDS, which inhibits CFTR when applied to the intracellular face, also inhibited CK2 activity. Indeed, the IC₅₀ for these compounds with respect to CK2 activity is an order of magnitude lower than the concentration normally used to 'specifically' inhibit CFTR. Thus, we suggest that these CFTR blockers act via CK2 and not directly on CFTR (Incubation with 10 microM of NAD+ was able to overcome the observed inhibitions (data not shown)). Figure 13 shows that genistein inhibits ATP binding to NBDl with an IC₅₀ of about IuM - TBB did not affect NBD l's ATP-binding activity up to ImM. Therefore, we conclude that, despite some resemblance to ATP in structure, TBB does not bind to NBDl. These data show that ATP analogues can be highly specific to the ATP-binding sites of particular proteins. We interpret this to reflect differences in the orientation of ATP in the respective binding sites of NBDl and CK2, as illustrated in figure 26.

Furthermore, genistein, the ATP analogue that binds to NBDl of CFTR₅ did not affect CK2 activity, and we find that TBB inhibits CFTR activation by genistein in a dose-dependent manner (Figure 13), despite the fact that it does not bind to the same site on NBDl.

Relating CFTR function to CK2 phosphoiylation of CFTR

We constructed mutant forms of CFTR for use in patch-clamp experiments. Two mutants, S511A and S511D were produced to mimic the non-phosphorylatable and mock-phosphorylated states, respectively. The full-length proteins were expressed in HEK/BF1K cells and their conductance characteristics assessed at both the whole cell and the single channel level (data not shown).

The data show that the S511A mutant CFTR was very slow to respond to PKA/ATP activation, compared to the wild type. In the single channel configuration, S51 IA-CFTR had a similar behaviour to that of ΔF508-CFTR (P₀=, i=). S51 ID, in contrast, was activated similar to wild type, but was insensitive to both TBB and DMAT inhibition.

Furthermore, we observe that CK2α is able to bind full length Wt CFTR, NBDl, S511D and DF508-S511D whilst it is unable to bind the ΔF508 CFTR or the S511 A CFTR mutant (Figure 15). As can be seem from Figure 16, we observe that A mutants 1 and 3 and D mutants 1, 2 and 3 seem to have no CK2 phosphorylation whilst still being recognised by Neomarkers and phosphorylated by PKA.

Also, overlay CK2α dot-blots of the mutants revealed that most likely, Al and Dl - 3 have worked when compared to wild-type / NBDl and MEK positive and negative controls respectively. Neomarkers screen of mutants showed full length protein expressed (Figure 16).

Specific CK2 assays revealed that the DF508 - S51 ID mutant was able to recover -75 - 80% of CK2 activity in the membranes of CFBE cells transfected with this mutation (Figure 17).

Also, we observe that there is an appreciable increase in membrane bound CFTR in ΔF-S51 ID transfected with respect to mock and untransfected CFBE cells and that whereas we cannot detect a CK2 signal in CFBE membranes normally, ΔF- S511D transfection results in a detectable CK2 signal in transfected CFBE membranes (Figure 18). These results suggest that ΔF-S511D either increases CFTR insertion or retention in the membranes of CFBE cells and that S511D is sufficient for localising CK2 to the membrane under these circumstances.

This data demonstrates that CK2 alpha, when overlaid onto purified CFTR, binds to wild type and S511D but not S511A full length CFTR. Further, CK2 binding abrogated by the ΔF508 mutation in CFTR is restored by the double (ΔF508+ S51 ID)-CFTR. We interpret this finding to mean that the interaction between CK2 and CFTR is not wholly dependent on F508, but might also depend on the phosphorylation state of CFTR at S511. This recovery of binding led us to test whether the CK2-CFTR interaction could affect trafficking. ΔF508 CFTR shows defective trafficking, resulting in -10% of wild type protein levels present in the membrane of CFBE cells, as shown in the manuscript. When we transfected (ΔF508+ S51 ID)-CFTR into the ΔF508 CFBE background cell line, we found near wild type levels of CFTR appearing in the membrane (Figure 18), whereas transfection of full length ΔF5O8 CFTR alone did not increase CFTR levels above background. Thus, in cells grown at 37°C, we have made the critical observation that CFTR-bound CK2 activity could be restored to the membrane with this double mutant (Figure 18). Therefore, the double mutation not only overcomes the trafficking defect of ΔF508 CFTR, but also restores the localisation of CK2.

TBB effect on PKA

Although TBB binds to the ATP -binding site of CK2, this does not mean that it is likely to bind to ATP-binding sites on other proteins. We have tested TBB for its effect on PKA and confirmed that it is ineffective up to lOOμM, as reported in Sarno, S et al. (2001) FEBS Lett 496, 44-8. Furthermore, genistein, an ATP analogue that is reported to bind to the NBDs of CFTR₅ activating Cl- transport, did not affect CK2 activity. Figure 26 shows the space-filling structure of the ATP -bound sites for NBDl and CK2. The orientation of the ATP molecule is clearly different in each, showing a different mode of binding and suggesting that ATP analogues which bind to one are unlikely to also bind to the other. These data show that ATP analogues can be highly specific to the ATP-binding sites of particular proteins.

CK2 is localised to the apical membrane

We stained wild type cells with CK2-rhodamine and CFTR-FITC separately. Gain levels were set to the optimum for each fluorophore and subsequent pictures were taken using the multi-channel setup at these levels. The data obtained show not only that CK2 is localised to the apical membrane, but also that bleed-through does not occur at these settings (Figure 27). The difference in cytosolic levels of CK2 is consistent within the technique. The fact that this is not reflected in the biochemical data suggests that epitope masking occurs in the fixed CF cells. The confocal data are an indication of the distribution of a given protein; the biochemical data reflect the absolute quantity of the protein present, as epitope masking is eliminated by SDS-PAGE. Confirmation of the importance of phosphorylation ofS511 and the dependence of CK2 binding on F508

The functional importance of CK2 phosphorylation at S511 was investigated using the patch-clamp technique and a CFTR construct containing the site-directed mutation S51 ID. This point mutation is expected both to permanently mimic the CK2-phosphorylated state and to result in CFTR function that is insensitive to CK2 inhibition. Figure 30(a) demonstrates that the S511D mutation dramatically alters channel gating, decreasing open probability three-fold, without altering single-channel current amplitude. Moreover, the sensitivity of the S51 ID-CFTR Cl^" channel to inhibition by TBB was attenuated significantly compared to wild- type CFTR (Fig. 30(a), bottom right histogram). To investigate the TBB sensitivity of the ΔF508-CFTR Cl^" channel, we employed Xenopus oocytes to overcome the temperature-dependent processing defect of ΔF508-CFTR and promote the delivery of the mutant protein to the cell surface (Denning, G. M. et al. Nature 358, 761-4 (1992)). Figure 30(b) demonstrates that TBB failed to inhibit ΔF508-CFTR Cl^" currents in Xenopus oocytes, but wild-type CFTR Cl^" currents declined as expected, confirming the functional significance of the loss of CK2 binding to ΔF508-CFTR. We conclude that (i) CK2 regulates CFTR activation, (ii) S511 is a CK2 target involved in CFTR gating and (iii) the lack of CK2 binding to ΔF508-CFTR abolishes channel inhibition by TBB.

Claims

1. A method of identifying a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 comprising exposing CFTR and/or CK2 to a test compound and assessing the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

2. A method of screening for compounds of use hi preventing or treating a secretory state or disorder and/or a cancer and/or cystic fibrosis wherein CFTR and/or CK2 is exposed to a test compound and the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 is assessed.

3. The method of claim 1 or 2 comprising exposing a cell comprising CFTR polypeptide and CK2 polypeptide and assessing the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

4. The method of claim 1 or 2 comprising exposing a non-human animal comprising CFTR polypeptide and CK2 polypeptide and assessing the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

5. The method of any one of the previous claims wherein the method further comprises the step of selecting a compound that modulates CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

6. The method of claim 5 wherein the selected compound reduces CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

7. The method of claim 5 wherein the selected compound elevates CFTR/CK2 interaction and/or CFTR phosphorylation by CK2. 8. The method of any of the previous claims wherein the method further comprises the step of assessing the specificity of the effect of the test compound on CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

9. The method of claim 8 wherein the method comprises the step of selecting a compound that specifically modulates CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

10. The method of any one of the previous claims wherein a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 is formulated into a pharmaceutically acceptable composition.

11. A method of making a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 comprising synthesising a compound identified by any one of the previous claims.

12. A method of making a pharmaceutical composition comprising the method of any one of the previous claims and the step of mixing the identified compound with a pharmaceutically acceptable carrier.

13. Use of CFTR and/or CK2 in a method of identifying a compound capable of modulating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

14. Use of CFTR and/or CK2 in a method of identifying a compound of use in preventing or treating a secretory state or disorder and/or a cancer and/or cystic fibrosis.

15. A method of assessing a patient's risk of developing cystic fibrosis or progression of cystic fibrosis comprising:

i) obtaining a sample containing protein from the patient; and, ii) determining CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in the patient's sample.

16. The method of claim 15 wherein the if sample has a reduced amount of CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 then the patient is considered to be at risk of developing cystic fibrosis.

17. A method of determining the restitution of CK2 and/or CFTR function comprising:

i) obtaining a sample containing protein from the patient; and,

ii) deterrnining CFTR and/or CK2 location in a cell.

18. The method of any one of claims 15 to 17 wherein the sample contains extracts of airway epithelial cells.

19. A polypeptide comprising all or a fragment or a variant or a fusion of a pseudo-CK2 phosphorylated CFTR polypeptide.

20. A polynucleotide encoding a polypeptide according to claim 19

21. A therapeutic entity comprising a polypeptide according to claim 19.

22. A peptidomimetic of the polypeptide according to claim 19.

23. A gene therapy vector comprising a polynucleotide according to claim 20.

24. The gene therapy vector of claim 23 wherein the vector comprises a Moloney Leukaemia Virus (MLV) based retroviral vector or a lentiviral vector or adeno-associated vector (AAV). 25. A compound identified by the method of claim 6 or 7 and/or a polypeptide according to claim 19 and/or a polynucleotide according to claim 20 and/or therapeutic entity according to claim 21 and/or a peptidomimetic according to claim 22 and/or a gene therapy vector according to claim 23 or 24 for use in medicine.

26. A pharmaceutical composition comprising a compound identified by the method of claim 6 or 7 and/or a polypeptide according to claim 19 and/or a polynucleotide according to claim 20 and/or therapeutic entity according to claim 21 and/or a peptidomimetic according to claim 22 and/or a gene therapy vector according to claim 23 or 24 and a pharmaceutically acceptable carrier.

27 Use of a compound capable of reducing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in the manufacture of a medicament for preventing or treating a secretory state or disorder and/or a cancer.

28. A method of treating a patient with or at risk of developing a secretory state or disorder and/or a cancer comprising administering to the patient an effective amount of a compound capable of reducing CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

29. The use of claim 27 or method of claim 28 wherein the compound is a compound identified by the method of claim 6.

30. The use of claim 27 or method of claim 28 wherein the compound is a peptide comprising the amino acid sequence from 500 to 518 of CFTR and having a maximum of 50 amino acids, or a fragment or a variant or fusion of said peptide, which is capable of interacting with CK2.

31. The use of claim 27 or method of claim 28 wherein the compound is a polynucleotide encoding a peptide defined in relation to claim 30. 5 >12.. The use of claim 27 or method of claim 28 wherein the compound is a therapeutic entity comprising a peptide defined in relation to claim 30.

33. The use of claim 27 or method of claim 28 wherein the compound is a peptidomimetic of a peptide defined in relation to claim 30.

34. The use of claim 27 or method of claim 28 wherein the compound is a gene therapy vector comprising a polynucleotide as defined in relation to claim 31.

35. The use of claim 27 or method of claim 28 wherein the compound is a polypeptide comprising all or a fragment or a variant of an amino acid sequence of a CFTR polypeptide and which is capable of binding to CK2.

36. The use of claim 27 or method of claim 28 wherein the compound is DPC, niflumic acid or DIDS.

37. The use or method of claim 36 for preventing or treating cancer.

38. Use of a compound capable of elevating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in the manufacture of a medicament for preventing or treating cystic fibrosis.

39. A method of treating a patient with or at risk of developing a cystic fibrosis comprising administering to the patient an effective amount of a compound capable of elevating CFTR/CK2 interaction and/or CFTR phosphorylation by CK2.

40. The use of claim 38 or method of claim 39 wherein the compound is a compound identified by the method of claim 7. 41. Use of a polypeptide as defined in claim 19 and/or a polynucleotide as defined in claim 20 and/or a therapeutic entity as defined in claim 21 and/or a peptidomimetic as defined in claim 22 and/or a gene therapy vector according to claim 23 or 24 in the manufacture of a medicament for preventing or treating cystic fibrosis.

42. A method of treating a patient with or at risk of developing cystic fibrosis comprising administering to the patient an effective amount of a polypeptide as defined in claim 19 and/or a polynucleotide as defined in claim 20 and/or a therapeutic entity as defined in claim 21 and/or a peptidomimetic as defined in claim 22 and/or a gene therapy vector according to claim 23 or 24.

43. A kit of parts useful in diagnosing cystic fibrosis comprising an agent capable of use in determining CFTR/CK2 interaction and/or CFTR phosphorylation by CK2 in a patient's sample.

44. A kit of parts of use in determining the restitution of CK2 and/or CFTR function comprising one or more antibodies capable of binding to CK2 and/or CFTR in a patient's sample.

45. A kit of parts according to Claim 43 or 44 further comprising a negative control and/or a positive control.

46 A kit of parts according to any one of Claims 43 to 44 further comprising means for separating extracts of airway epithelial cells from a sample.