Lee M Allen

Michael R G Hodskinson

Jon R Sayers
email: j.r.sayers@shef.ac.uk

Active site substitutions delineate distinct classes of eubacterial flap endonuclease

Keywords: Exonuclease IX, FEN, DNA polymerase I, Replication, metallonuclease

Flap endonucleases (FENs) play essential roles in DNA replication, pivotally in the resolution of Okazaki fragments. In eubacteria, DNA Polymerase I (PolI) contains a flap processing domain, the N-terminal 5' 3' exonuclease. We present evidence of paralogous FEN-encoding genes present in many eubacteria. Two distinct classes of these independent FEN-encoding genes exist with four groups of eubacteria being identified based on the number and type of FEN gene encoded. The respective proteins possess distinct motifs hallmarking their differentiation. Crucially, based on primary sequence and predicted secondary structural motifs, we reveal key differences at their active site. These data are supported by biochemical characterization of two family members -exonuclease IX (ExoIX) from Escherichia coli and Staphylococcus aureus flap endonuclease (SaFEN). These proteins displayed marked differences in their ability to process a range of branched and linear DNA structures. On bifurcated substrates, SaFEN exhibited similar substrate specificity to previously characterised FENs. In quantitative exonuclease assays, SaFEN maintained a comparable activity to that reported for PolI. However, ExoIX showed no observable enzymatic activity. A threaded model is presented for SaFEN, demonstrating the probable interaction of this newly identified class of flap endonuclease with divalent metal ions and a branched DNA substrate. These data provide an intriguing model for the cellular role of these FEN sub-classes and illustrate the evolutionary importance of processing aberrant DNA, which has led to their maintenance alongside DNA PolI in many eubacteria.

INTRODUCTION

Classical biochemistry conducted by Kornberg and co-workers revealed that Escherichia coli DNA Polymerase I (PolI) contained two readily separable enzymatic activities within the same polypeptide [START_REF] Setlow | Deoxyribonucleic acid polymerase: two distinct enzymes in one polypeptide. I. A proteolytic fragment containing the polymerase and 3' leads to 5' exonuclease functions[END_REF][START_REF] Setlow | Deoxyribonucleic acid polymerase: two distinct enzymes in one polypeptide. II. A proteolytic fragment containing the 5' leads to 3' exonuclease function. Restoration of intact enzyme functions from the two proteolytic fragments[END_REF]. The C-terminal 'Klenow' fragment exhibited DNA polymerase and 3' 5' 'proofreading' activity, while the N-terminal domain accounted for 5 ' 3' exonuclease activity. The 5' 3' exonuclease domain of PolI falls within of a wider class of essential metallonucleases, the flap endonuclease (FEN) family [START_REF] Robins | Structural and functional homology between mammalian DNase IV and the 5'-nuclease domain of Escherichia coli DNA polymerase I[END_REF]. FENs demonstrate both 5 ' 3' exonuclease and structure-specific endonuclease activity, playing important roles in DNA replication, repair and recombination [START_REF] Lieber | The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair[END_REF][START_REF] Ceska | Structure-specific DNA cleavage by 5' nucleases[END_REF][START_REF] Liu | Flap endonuclease 1: a central component of DNA metabolism[END_REF]. These enzymes are ubiquitous and highly conserved in all three kingdoms of life (Eubacteria, Archaea and Eukaryota), presumably due to their role in these crucial biological processes.

The importance and biological similarity of FENs has been demonstrated in several cross-species studies. In bacteriophage T4, disruption of the rnh gene (which encodes a FEN, known historically as T4 RNase H) resulted in slower, less accurate DNA replication. Furthermore, no measurable progeny were recovered from a host carrying a deletion in the PolI 5 ' 3' exonuclease domain [START_REF] Hobbs | Either bacteriophage T4 RNase H or Escherichia coli DNA polymerase I is essential for phage replication[END_REF], clearly implicating the essential requirement of an active FEN for viral replication. The lethal phenotype of rad27 (encoding another FEN homologue) in Saccharomyces cerevisiae was suppressed by the PolI 5 ' 3' exonuclease domain of E. coli [START_REF] Sun | Suppression of Saccharomyces cerevisiae rad27 null mutant phenotypes by the 5' nuclease domain of Escherichia coli DNA polymerase I[END_REF]. In higher organisms, heterozygous (Fen1 +/-) mice rapidly developed lymphoma and thymic abnormalities, while homozygous knockouts (Fen1 -/-) displayed the embryonic lethal phenotype [START_REF] Kucherlapati | Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression[END_REF]. Moreover, mice with a single point mutation (E160D) in one of the two divalent metal ion binding sites present in the FEN enzyme were reported to be predisposed to autoimmunity, chronic inflammation and cancers [START_REF] Zheng | Fen1 mutations result in autoimmunity, chronic inflammation and cancers[END_REF].

FENs recognise substrates with a DNA duplex and a single-stranded 5' overhang such as might arise during strand displacement synthesis. The principle site of hydrolytic phosphodiester backbone cleavage is one nucleotide into the DNA duplex, at the bifurcation of single and double stranded DNA [START_REF] Rossi | Reconstituted Okazaki fragment processing indicates two pathways of primer removal[END_REF]. In addition, FENs that play a role in nucleotide excision repair (e.g. XPG in humans) can cleave 'bubble' substrates that have no free 5' end at the bifurcation of single and double-stranded DNA. As exonucleases, FENs cleave free 5' termini and nicked DNA duplexes liberating mono-, di-and short poly-nucleotides [START_REF] Sayers | Properties of Overexpressed Phage T5 D15 Exonuclease[END_REF][START_REF] Lundquist | Transient generation of displaced singlestranded DNA during nick translation[END_REF]. Both endo-and exonucleolytic activities are generally accepted to play a role in Okazaki fragment processing during DNA replication [START_REF] Liu | Flap endonuclease 1: a central component of DNA metabolism[END_REF].

A number of FEN structures have been elucidated using X-ray crystallography. The structures show striking similarities, with a core -sheet adorned with a number of surrounding -helices. The FEN domain of Thermus aquaticus DNA PolI, T4RNaseH and T5FEN all reveal the presence of at least two divalent metal ion binding sites [START_REF] Kim | Crystal structure of Thermus aquaticus DNA polymerase[END_REF][15][16]. Experimental evidence based on isothermal titration calorimetry and mutagenesis studies support the presence of two metal binding sites [START_REF] Feng | Roles of divalent metal ions in flap endonuclease-substrate interactions[END_REF][START_REF] Zheng | Distinct roles of two Mg2+ binding sites in regulation of murine flap endonuclease-1 activities[END_REF]. Metal site I is required for generation of an attacking hydroxyl nucleophile during phosphodiester backbone hydrolysis [START_REF] Tock | Dynamic evidence for metal ion catalysis in the reaction mediated by a flap endonuclease[END_REF] and metal site II is required for substrate binding, particularly during exonucleolytic cleavage [START_REF] Feng | Roles of divalent metal ions in flap endonuclease-substrate interactions[END_REF]. In T5FEN a key structural motif is formed from -helices bridging above the active site and has been described as a 'helical arch ' [15] or 'helical clamp' [20,[START_REF] Hwang | The crystal structure of flap endonuclease-1 from Methanococcus jannaschii[END_REF]. This feature has been observed in a number of enzymes, and based on biochemical studies, has been proposed to play a role in substrate binding [20][START_REF] Hwang | The crystal structure of flap endonuclease-1 from Methanococcus jannaschii[END_REF][START_REF] Bhagwat | Identification of residues of T4 RNase H required for catalysis and DNA binding[END_REF][START_REF] Garforth | Mutagenesis of conserved lysine residues in bacteriophage T5 5 '-3 ' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage[END_REF]. Most recently, a 3 Å X-ray diffraction structure of T4 RNase H in complex with a forked DNA substrate was solved [24]. These data were in close agreement with models of FEN-substrate interaction previously proposed from site directed mutagenesis and substrate binding studies [START_REF] Dervan | Interactions of mutant and wild-type flap endonucleases with oligonucleotide substrates suggest an alternative model of DNA binding[END_REF][START_REF] Allawi | Modeling of flap endonuclease interactions with DNA substrate[END_REF].

An early interrogation of the incomplete E. coli genome sequence identified a putative gene (later termed xni [START_REF] Shafritz | Exonuclease IX of Escherichia coli[END_REF].), whose product shared significant homology to PolI 5' 3' exonuclease domain and other known flap endonucleases [START_REF] Lieber | The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair[END_REF][START_REF] Sayers | Computer aided identification of a potential 5'-3' exonuclease gene encoded by Escherichia coli[END_REF]. Shafritz and coworkers reported the purification and characterization of the product of the xni gene [START_REF] Shafritz | Exonuclease IX of Escherichia coli[END_REF]. Surprisingly, given the significant homology to a family of enzymes demonstrating 5' 3' exonuclease activity the authors suggested that the xni product was a 3' 5' exonuclease and designated the xni gene product exonuclease IX (ExoIX) [START_REF] Shafritz | Exonuclease IX of Escherichia coli[END_REF]. Recently we have shown that exonuclease III (a 3' 5' exonuclease) copurifies with ExoIX over a multiple-stage purification process [START_REF] Hodskinson | Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity[END_REF]. Therefore, we suggested that the 3' 5' exonuclease activity described by Shafritz et al in ExoIX was due to co-purifying exonuclease III.

Here we show that a number of prokaryotes encode a second FEN paralogue, in addition to that present as an N-terminal domain of PolI. Based on amino acid sequence analysis, these single-domain eubacterial FEN homologues can be delineated into two distinct groups. One group appears to possess binding sites for two divalent metals ions, as observed in all FENs characterized to date. The second group, exemplified by E. coli ExoIX, seems to lack a number of normally conserved amino acid residues that act as metal-binding ligands. We demonstrate that, in contrast to ExoIX which apparently lacks activities expected of a FEN when assayed in isolation, a discrete FEN homologue encoded by S. aureus has both flap endonuclease and 5' 3' exonuclease activities. We compare and contrast these two representatives of the cryptic single-domain eubacterial FEN homologues and speculate as to their biological roles.

EXPERIMENTAL

Alignments and phylogenetics

DNA PolI paralogues were identified by TBLASTN searches [START_REF] Altschul | Gapped BLAST and PSI-BLAST: a new generation of protein database search programs[END_REF] and multiple sequence alignments prepared with CLUSTALW [START_REF] Larkin | Clustal W and Clustal X version 2.0[END_REF], edited in Jalview [START_REF] Clamp | The Jalview Java alignment editor[END_REF] and annotated by hand. Identified primary motifs were analysed with Pattinprot [START_REF] Combet | NPS@: network protein sequence analysis[END_REF]. A phylogenetic tree was constructed from the alignment using Phylip algorithms [34]. These were defined using default parameters in Protdist and Neighbor-joining clustering [35], followed by radial plots, generated using the Phylemon server (http://phylemon.bioinfo.cipf.es) [START_REF] Tarraga | Phylemon: a suite of web tools for molecular evolution, phylogenetics and phylogenomics[END_REF].

Secondary and tertiary structural predictions

Structural predictions for SaFEN were generated using FUGUE (http://wwwcryst.bioc.cam.ac.uk/~fugue/prfsearch.html) [START_REF] Shi | FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structuredependent gap penalties[END_REF] and Phyre (http://www.sbg.bio.ic.ac.uk/~phyre/) [START_REF] Bennett-Lovsey | Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre[END_REF]. The quality of the derived models was checked by eye in the first instance, for the local geometry of strictly conserved residues. A model of SaFEN, docked with a branched substrate was prepared by superimposition of the Phyre Apo SaFEN model with bacteriophage T4 RNase H in complex with DNA (pdb code 2IHN), using an all atom best-fit algorithm in Deepview (http://www.expasy.org/spdbv/) [START_REF] Guex | SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling[END_REF], followed by manual rigid body docking of DNA within a 1.5 Å constraint and final remodeling of ssDNA regions to facilitate best fit. Final models were submitted to PROCHECK [START_REF] Laskowski | Main-chain bond lengths and bond angles in protein structures[END_REF], hosted on the JCSB server (http://www.jcsg.org/scripts/prod/validation/sv_final.cgi) for assessment of their

Bacterial strains and plasmids

Escherichia coli strain M72 pJONEX4ch was kindly provided by Dr. C. E. Meadows (University of Sheffield) and purified genomic DNA from Staphylococcus aureus strain 8325-4 was a generous gift from Prof. Simon Foster (University of Sheffield). Recombinant bacteriophage T5 FEN and E. coli exonuclease IX (ExoIX) were overexpressed and purified, as described previously [START_REF] Sayers | Properties of Overexpressed Phage T5 D15 Exonuclease[END_REF][START_REF] Hodskinson | Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity[END_REF].

Cloning recombinant S. aureus fen.

Polymerase chain reaction (PCR) was performed using S. aureus strain 8325-4 genomic DNA template with primers for MW1329 (forward, 5'-TAT TGA ATT CAA AGA AGG AAC GTT TAA ATG CCT-3'; reverse, 5'-TTT ATA CGG ATC CAA AAA TGG GAT GAA ATA TAT TTT CC-3'), using the Expand High Fidelity PCR System, according to the manufacturer's instructions (Roche Diagnostics GmbH). Gelpurified product was digested with BamHI and EcoRI, ligated into pJONEX4ch (a generous gift from Dr. C. Meadows, University of Sheffield) and transformed into electrocompetent E. coli M72. Positive clones were confirmed by capillary sequencing on an ABI 3730 instrument (Genetics Core Facility, University of Sheffield). The resulting expression plasmid was designated pJONEX4/fench. This plasmid encoded the S. aureus FEN-like protein as a C-terminal polyhistidine fusion linked via an enterokinase cleavage motif resulting in the addition of the sequence DDDDKHHHHHH to the C-terminus of the protein.

Preliminary inductions

Culture medium (30 ml 2xYT with 100 µg ml -1 carbenicillin [2xYT/C]) was inoculated with a 1 / 100 volume (300 µl) of an overnight culture of M72 pJONEX4/fench. The culture was grown to an A 595nm of 0.5 and induced at 42ºC for 0.5, 1, 1.5 or 2 h duration, followed by incubation at 30ºC, for up to a total of 2 h post induction (i.e. the 2 h, 42ºC culture received no further incubation). A control strain containing the empty vector was grown with continuous heat-shock or without temperatureinduction. Cells were harvested and analysed by denaturing 10 % acrylamide SDS-PAGE using either Coomassie stain or DNase substrate gel assays [START_REF] Rosenthal | Nuclease detection in SDS-polyacrylamide gel electrophoresis[END_REF].

Over-expression and purification of SaFEN

A fermenter vessel containing approx. 5 L 2xYT/C with 0.01% v/v antifoam (Sigma) was seeded with a 1 / 15 inoculum of an overnight culture of M72 pJONEX4/fench. The culture was grown to an absorbance of 0.6 (A 595 nm ) at 30ºC, followed by heat-shock induction at 42ºC for 2 h. The culture was cooled to 25ºC and incubated for a further 4.5 h, prior to harvesting. Cell pellet was recovered by centrifugation at 3,000 x g for 30 min at 4ºC, washed with 50 mM Tris•HCl, pH 8.0 containing 0.9% w/v NaCl and stored in aliquots at -80ºC. Cell pellet (7.5 g) was resuspended in 40 ml of 25 mM potassium phosphate buffer, pH 7.4, containing 20% w/v glycerol, 500 mM NaCl and 30 mM imidazole (EQ buffer). Lysozyme (200 µg ml -1 ) and sodium deoxycholate (500 µg ml -1 ) were added and the reaction incubated on ice for up to 1 h, followed by A c c e p t e d M a n u s c r i p t sonication at 20% max. amplitude in 10 s bursts. Insoluble material was removed via centrifugation at 19,000 x g for 30 min and the supernatant cleared by filtration (Whatman No. 1) to remove residual precipitate, before loading onto a 1 ml HisTrap column (GE Healthcare), equilibrated with EQ buffer, at 0.5 ml min -1 . The column was washed in excess of 30 ml EQ buffer (returning A 280 nm to baseline), followed by elution over a 15 ml 40 -500 mM imidazole gradient, collecting 0.5 ml fractions. Fractions were analysed by SDS-PAGE using Coomassie and zymogram assays for exonuclease activity [START_REF] Rosenthal | Nuclease detection in SDS-polyacrylamide gel electrophoresis[END_REF]. Fractions of greatest purity were equilibrated against a 100-fold excess of LSQ buffer (25 mM Tris•HCl, pH 8.0 containing 5% w/v glycerol, 1 mM EDTA and 5 mM DTT), adjusted to 30% w/v ammonium sulphate and incubated for 15 min at RT. Precipitated material was collected via centrifugation at 4,000 x g for 10 min and the buffer adjusted to LSQ Buffer, containing 50% w/v glycerol, for storage at -20ºC.

Nuclease assays

Structure-specific nuclease assays were performed using oligonucleotides synthesized by MWG Biotech AG (table 1) and purified by butanol precipitation [42]. Oligonucleotides were radiolabelled in a reaction (30 µl) containing 5 pmol DNA, [1x] kinase buffer (70 mM Tris•HCl, pH 7.6, 10 mM MgCl 2 , 5 mM DTT), 6 pmol [ - 32 P] ATP and 30 units polynucleotide kinase at 37ºC for 1 h, quenched by the addition of an equal volume of stop-mix (95 % deionised formamide, 10 mM EDTA and 0.05 % w/v bromophenol blue). Labelled oligonucleotides were purified using a QIAquick Nucleotide Removal Kit (Qiagen Ltd) and quantified by comparison with a serial dilution of [ -32 P] ATP using a BioRad Personal FX phosphorimager. Substrates for structure-specific assays were formed in annealling reactions (200 µl) containing 450 pM [ -32 P]-labelled DNA and 13.5 nM cold complementary oligonucleotide(s), in a buffer comprising 25 mM Tris•HCl, pH 8.0 and 500 mM KCl, incubated at 80ºC for 5 min and passively cooled to ambient temperature. Standard nuclease reactions (10 µl) were performed with 3 µl annealed labelled oligonucleotide, 5 µl of 2 X reaction buffer (50 mM Tris•HCl, pH 8.0, 200 mM KCl, 20 mM MgCl 2 and 0.6 mg ml -1 acetylated BSA), and 2 µl enzyme prepared in enzyme dilution buffer (25 mM Tris•HCl, pH 8.0, containing 10 % w/v glycerol, 1 mM EDTA, 1 mM DTT and 1 mg ml - 1 acetylated BSA). The final mixture, which contained 100 nM enzyme and 135 pM radiolabelled oligonucleotide was incubated at 37ºC for 30 min before the reaction was quenched by the addition of an equal volume of stop-mix. Reaction products were analysed by denaturing PAGE (15% 19:1 acrylamide: bisacrylamide) [42]. In addition to the structure-specific nuclease assays described above, an exonuclease assay was deployed based on that described by Fraser [43]. This assay used high molecular weight DNA as a substrate. Exposure to exonuclease releases acid soluble mononucleotides and polynucleotides from acid precipitable substrate. Release of acid soluble products was monitored by UV spectroscopy after incubation with perchloric acid and centrifugation as described [START_REF] Hodskinson | Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity[END_REF].

RESULTS AND DISCUSSION

Delineation of eubacterial FENs

We originally identified the gene now known as xni by homology searches of the sequence databases in 1994 [START_REF] Lieber | The FEN-1 family of structure-specific nucleases in eukaryotic DNA replication, recombination and repair[END_REF][START_REF] Sayers | Computer aided identification of a potential 5'-3' exonuclease gene encoded by Escherichia coli[END_REF] A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

putative active site (Supplementary figure S1). In E. coli ExoIX (EcExoIX), these are Asp10, Asp51, Glu103, Asp105 and Asp128. A key feature of the EcExoIX polypeptide sequence is the absence of certain aspartic acid residues, conserved in previously characterized FENs (Figure 1A). These conserved residues form the ligands that coordinate one of two divalent metal ions, directly observed in several FEN crystal structures [START_REF] Kim | Crystal structure of Thermus aquaticus DNA polymerase[END_REF][15][16]. EcExoIX possesses the conserved aspartates which make up the high affinity, catalytic metal binding site, known as site I [START_REF] Feng | Roles of divalent metal ions in flap endonuclease-substrate interactions[END_REF]. However, the multiple sequence alignment (Figure 1) reveals that key aspartate residues, normally involved in binding the second divalent metal ion in classical FENs have been replaced by small side chains Gly130, Ser175 and the aliphatic residue, Ile178 in EcExoIX (boxed residues in Figure 1A). These residues are unable to chelate a divalent metal ion. Indeed, when only two of the analogous mutations (D201I and D204S) were introduced into T5 FEN, the resulting mutant lost its ability to bind metal at site II [START_REF] Feng | Roles of divalent metal ions in flap endonuclease-substrate interactions[END_REF] as well as much of its potency as an exonuclease. However, the structure-specific endonuclease activity of T5FEN D201I-D204S mutant remained intact. The metal-site II substitutions form a striking motif that delineates the ExoIX subset from the wider FEN family of polypeptides (Figures 1B andS1).

Our sequence analysis revealed a further class of FEN paralogues. We observed a subset encoding short FEN-like proteins that retain all the conserved aspartic acid residues associated with both metal sites I and II. This subset encoded FEN-like proteins that were independent of the N-terminal FEN domains found in DNA PolI orthologues. Figure 1A shows exemplars of the two subsets of autonomous FEN-like proteins. The S. aureus (SaFEN) represents those containing all the conserved features of the FENs while E. coli ExoIX (EcExoIX), represents those lacking the aspartates necessary for binding metal site II. Phylogenetic analysis reveals that ExoIX and FEN fall broadly, though not exclusively in classification based on Gram type (Figure 1B). Here we analysed nineteen previously identified PolI FEN paralogues [START_REF] Fukushima | Reassessment of the in vivo functions of DNA polymerase I and RNase H in bacterial cell growth[END_REF], performing ClustalW multiple alignments (Figure S1) to generate a phylogenetic tree (Figure 1B).

In our analysis we also observed another, intriguing class of PolI FEN paralogues in the marine bacteria, Shewanella sp. and Colwellia sp. These organisms appear to fall at the divergent branch point between autonomous eubacterial FEN and ExoIX subsets (Figure 1B). They retain more conserved residues than EcExoIX (Colwellia sp. Gly150, Asp195 and N198) in place of typical FEN site II aspartate ligands. Based on site directed mutagenesis in T4 RNase H and other FENs, we suggest these gene products would be likely to exhibit diminished enzymatic activity [START_REF] Bhagwat | Identification of residues of T4 RNase H required for catalysis and DNA binding[END_REF].

We tentatively suggest that prokaryotes can be classified into four groups based on the number and type of FEN homologue that they encode: (i) a FEN only as an Nterminal domain of DNA PolI, as exemplified in Streptococcus pneumoniae (ii) Nterminal DNA PolI with a separate FEN paralogue with one divalent metal binding site e.g ExoIX in E. coli, (iii) N-terminal DNA PolI with a separate FEN paralogue with two divalent metal binding sites e.g. S. aureus and (iv) a Klenow-like PolI domain (with no attached FEN domain) and a separate FEN domain with two divalent metal ion binding sites as exists in Mycoplasma genitalium (NP_072928). Accordingly, we therefore propose a more precise nomenclature for prokaryotic FEN-like genes, to allow for this simple but important distinction. The gene name xni should be used to specify the subset containing only the site I carboxylate ligands (i.e. those genes encoding ExoIX orthologues). The gene fen should specify all other homologues, whether carried alongside polA or as the sole chromosomal copy. We support this proposition with the following biochemical analysis of the S. aureus fen gene product.

S. aureus fen encodes an active nuclease

A c c e p t e d M a n u s c r i p t

Based on the retention of a full complement of metal-binding aspartyl ligands, we reasoned that the newly identified staphylococcal enzyme should display similar structure-specific endo-and exo-nucleolytic activities to previously identified flap endonucleases. We cloned and expressed S. aureus fen, under the control of the temperature-inducible bacteriophage P L promoter in the pJONEXCH expression vector [START_REF] Sayers | A single-strand specific endonuclease activity copurifies with overexpressed T5 D15 exonuclease[END_REF]. The recombinant protein was expressed with a C-terminal polyhistidine tag. Figure 2A shows the properties of the recombinant SaFEN protein in preliminary inductions. A distinct band of the anticipated molecular mass (35 kDa) was induced upon heat-shock (0.5 -2 h) but was absent in control lysates (-/+, 0 h). Furthermore, using an in situ zymogram assay we observed a nuclease activity co-induced, under conditions of heat shock (Figure 2B, panel ii) with some evidence of proteolysis. The protein was purified to > 95% homogeneity (Figure 2B, panel i). Zymogram assays confirmed that the DNase activity shared chromatographic and physicochemical properties with the induced SaFEN protein (Figure 2B, panel ii). These data show that the novel S. aureus fen gene encodes a bona fide nuclease. The nuclease activity required divalent metal ions as expected and was not adversely affected by the C-terminal fusion (data not shown). We also obtained very similar zymogram data for an untagged version of the SaFENprotein but it proved difficult to purify without th eaffinity tag unlike the untagged T5FEN and ExoIX (data not shown).

Comparison of SaFEN with other flap endonuclease family members

Using a standard spectrophotometric assay, we quantified the exonucleolytic activity of purified SaFEN against the well-characterized control flap endonuclease T5FEN (Figure 3). This has proven to be a simple, highly reproducible and robust assay, in the characterisation of other flap endonucleases [START_REF] Sayers | Properties of Overexpressed Phage T5 D15 Exonuclease[END_REF][START_REF] Garforth | Mutagenesis of conserved lysine residues in bacteriophage T5 5 '-3 ' exonuclease suggests separate mechanisms of endo-and exonucleolytic cleavage[END_REF][START_REF] Hodskinson | Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity[END_REF]. Under similar experimental conditions (pH 8.0 at 37 C), the specific activity for T5FEN, calculated in a previous study (610 units µg -1 ) [START_REF] Hodskinson | Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity[END_REF] was similar to that reported here (420±10 units µg -1 ), The specific activity of SaFEN (90±5 units µg -1 ) was approximately fivefold lower than that of T5FEN, equivalent to a turnover rate of 106 min -1 . T5FEN is a highly processive exonuclease, which serves as a stringent control. By comparison, the reported turnover of E. coli Pol I FEN, under similar assay conditions is 37 min -1 (37ºC, pH 7.4) [START_REF] Setlow | Deoxyribonucleic acid polymerase: two distinct enzymes in one polypeptide. II. A proteolytic fragment containing the 5' leads to 3' exonuclease function. Restoration of intact enzyme functions from the two proteolytic fragments[END_REF]. Thus, the activity of SaFEN is approximately three-fold greater than E. coli PolI FEN activity under similar conditions, suggesting the activity of the novel polypeptide is biologically relevant. As reported previously ExoIX, showed no detectable activity under any assay conditions tested [START_REF] Hodskinson | Molecular interactions of Escherichia coli ExoIX and identification of its associated 3'-5' exonuclease activity[END_REF].

Structure-specific activity of SaFEN and EcExoIX

We hypothesised that the alterations in predicted active site of the two classes of divergent FENs, would lead to pronouncedly different catalytic activity. To test this we employed a substrate screen formed from partial or completely complementary oligonucleotides (Table 1). The catalytic activity of the proteins was examined for exo-and endonucleolytic cleavage under denaturing PAGE (Figure 4).

Single-stranded and fully duplexed DNA substrates were cleaved exonucleolytically by SaFEN, liberating products of the same electrophoretic mobility as those released from T5FEN, albeit to a lesser extent (Figure 4, panel A andB). The enzyme also liberated exo-product ( 4mer) from the 5' terminus of the downstream strand of a nicked DNA strand. Under the conditions tested, all substrate was degraded (Figure 4, panel C). The nicked substrate (36mer) was larger than the duplex DNA (24mer), though this difference is unlikely to account for the heightened activity on the former substrate. This is supported by the similar substrate turnover of the strand upstream of a nick, compared with purely duplex DNA (cf. Panels D and B). These data suggest SaFEN favours a free 3' terminus, upstream of the free 5' end, which perhaps facilitates loading of the enzyme onto the DNA substrate. In this assay the activity of SaFEN was apparently equivalent to T5FEN. Such activity correlates with the involvement of this class of proteins in nick translation [46]. While SaFEN did not endonucleolytically hydrolyse a 5' DNA overhang, 5' pseudo-Y (PsY) and 3' invader (Inv) bifurcations were cleaved by the staphylococcal enzyme (Panels E-G). On PsY and Inv substrates, SaFEN exhibited both endo-and exonucleolytic cleavage, the products for which corresponded to decamers (closed triangle) and tetramers (open triangle). Interestingly, a free 5' flap of a partially formed junction (panel H) was not hydrolysed by SaFEN. This structure resembled the double flap substrate used in the early characterization of human FEN [47]. In those assays, human FEN cleaved the 5' flap of double flap structures efficiently, when 3' overlaps of up to 10 nucleotides were tested. T5FEN cut the large double flap substrate at a number of sites, the most prominent product of which was a 20mer, formed from hydrolysis at the bifurcation junction. The complex cleavage pattern with this substrate is probably due to transient base pairing, causing the formation of substrates with different free 5' arms.

The length of duplex DNA also appears to play a role in cleavage efficiency. This hypothesis is supported by the cleavage properties on the dsDNA region of larger (20 bp) Holliday junction intermediates (Panels I and J). Duplex DNA regions of partially formed junctions were hydrolyzed more efficiently than shorter substrates (3' PsY and 5' Inv substrates, data not shown), with comparable activity to all duplex DNA (panel B). SaFEN activity was limited to exonucleolytic degradation of duplex DNA in Holliday junction intermediates.

In contrast to SaFEN, ExoIX did not demonstrate flap endonuclease activity when incubated with a range of substrates (Figure 4). Therefore, ExoIX (when assayed in isolation) did not possess either 5-3' exonuclease or 5' flap endonuclease activity that would be expected of a FEN enzyme. We hypothesize that the lack of observable characteristic FEN activity is due to the absence of divalent metal ion binding site II in this protein.

Modelling cofactor and substrate interactions of SaFEN

We used a structural bioinformatics approach to gain insight into the catalytic differences observed in the different FEN homologues characterised above (Figure 5). Using a coordinated approach of different structural prediction algorithms we were able to ascertain with confidence the likely structure of the newly identified staphylococcal enzyme (FUGUE z-score 43.6, where 6 represents 99% confidence [START_REF] Shi | FUGUE: sequence-structure homology recognition using environment-specific substitution tables and structuredependent gap penalties[END_REF]). A Phyre-generated model was used for analysis based on its favourable structural constraints [START_REF] Bennett-Lovsey | Exploring the extremes of sequence/structure space with ensemble fold recognition in the program Phyre[END_REF]. The model bore striking semblance to many experimentally derived FEN structures, most notably at the DNA binding helix-threeturn helix (H3TH) motif and the putative active site (panel A and B). The model reveals that both divalent metal binding sites are present in SaFEN, whereas, metal site II ligands are absent in ExoIX. We postulate this accounts for the different catalytic activities observed in our screen when the staphylococcal enzyme was compared to ExoIX. Comparison of this threaded model with other FEN family members revealed a smaller arch domain than that found in T5FEN. The first cocrystal structure of a FEN in complex with a bifurcated substrate has recently been solved [24]. We sought to use this as a model to understand the behaviour of SaFEN in its activity on a similar Ps-Y substrate assayed above (Figure 5, panel C cf. though upon interaction with a branched substrate, positioning the scissile phosphate diester close to the site I metal binding site which is known to be essential for catalysis in the FENs [START_REF] Feng | Roles of divalent metal ions in flap endonuclease-substrate interactions[END_REF][START_REF] Zheng | Distinct roles of two Mg2+ binding sites in regulation of murine flap endonuclease-1 activities[END_REF]. The more restricted arch present in SaFEN may account for the slightly different cleavage pattern it exhibits compared to the T5FEN (Figure 4).

Biological role of the cryptic FEN-like proteins

The gene now known as xni was initially designated exo by this laboratory. It was speculated at the time that the gene might encode a backup function for the FENdomain of PolI [START_REF] Sayers | Computer aided identification of a potential 5'-3' exonuclease gene encoded by Escherichia coli[END_REF]. This inference was initially undermined by the observation that Streptococcus pneumoniae polA null mutants are non-viable [START_REF] Díaz | The 5' to 3' exonuclease activity of DNA polymerase I is essential for Streptococcus pneumoniae[END_REF]. However, this organism does not encode an additional FEN-like protein. Recently, Fukushima and co-workers demonstrated that the FEN-domain encoded by either polA or xni was required for the viability of E. coli [START_REF] Fukushima | Reassessment of the in vivo functions of DNA polymerase I and RNase H in bacterial cell growth[END_REF]. Similarly, they also showed that either polA or ypcP is essential for B. subtilis. The ypcP gene encodes a two-metal-site FEN that shares 48% identity and 64% similarity to SaFEN (Figure S1). Thus, it would appear that either subclass of the single domain FEN-like proteins discussed here acts in redundancy with DNA polymerase I. The actual biochemical activity of the xniencoded proteins remains elusive, as we were unable to identify any nucleolytic activity when tested in isolation in biochemical assays, despite testing a wide range of substrates and cofactors. Interestingly, amino acid sequence alignments also suggest that the helical arch motif identified as being important in FEN/substrate interactions is truncated in EcExoIX which may be an additional factor in the lack of expected nuclease activity. Perhaps the ExoIX-type proteins need to be part of a complex of protein(s) to display nuclease activity or require some as yet unidentified cofactor. Alternatively, it could be that they process some form of unusual nucleic acid structure. However, it is clear from genetic experiments and their widespread occurrence that these small FEN-like proteins are capable of fulfilling a critical biological role. The reasons why each group of organisms has retained FEN-like proteins with differing properties is unclear and will require further investigation in to how these proteins participate in biological processes. A c c e p t e d M a n u s c r i p t Release of acid soluble nucleotides (nmol) in a standard reaction (1 ml) at pH 8.0, containing an excess of type XIV herring sperm DNA substrate, measured spectrophotometrically, adjusted to 1 µg protein to aid comparison: ExoIX ( ), SaFEN ( ), T5FEN ( ). Mean data from three independent readings were plotted, shown with standard error (error bars). Nucleotide release was determined as described elsewhere [START_REF] Sayers | Properties of Overexpressed Phage T5 D15 Exonuclease[END_REF]. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2008 as manuscript BJ20081637 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081637 A c c e p t e d M a n u s c r i p t stereo-chemical quality. MolProbity (http://molprobity.biochem.duke.edu/) was used for additional checks on the validity of ssDNA remodeling, in comparison with the original DNA template (chain CD, of pdb 2IHN [24]).

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2008 as manuscript BJ20081637 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081637

  . The xni gene encodes ExoIX, a paralogue of the N-terminal FEN domain of DNA polymerase I. The defining feature of all the eubacterial FEN family is a highly conserved cluster of carboxylate ligands at the Biochemical Journal Immediate Publication. Published on 11 Nov 2008 as manuscript BJ20081637 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081637

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2008 as manuscript BJ20081637 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081637

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2008 as manuscript BJ20081637 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081637 A c c e p t e d M a n u s c r i p t

Figure 4 ,

 4 panel F). The restricted arch of the SaFEN model could still allow DNA to thread Biochemical Journal Immediate Publication. Published on 11 Nov 2008 as manuscript BJ20081637 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081637A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Biochemical

  Journal Immediate Publication. Published on 11 Nov 2008 as manuscript BJ20081637 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081637A c c e p t e d M a n u s c r i p t Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

Figure 1 .A.

 1 Figure 1. Evaluation of eubacterial DNA Polymerase I 5' -3' exonuclease paralogues.A. ClustalW multiple sequence alignment of 5'-3'-like nucleases with their related Pol I paralogue. Both classes of eubacteria contain two putative paralogous FEN-like proteins, one associated with the Klenow fragment in the form of DNA Pol I (polA) and the other encoded autonomously. The autonomous FEN-like proteins can be divided into those which retain all the putative ligands to bind two metal ions (e.g. Staphylococcus aureus FEN, SaFEN) and those which are retain the ligands to bind a single divalent metal ion (Escherichia coli Exo IX). Perfectly conserved residues are boxed in grey. The amino acid substitutions affecting binding at the second metal site in Exo IX are shown in clear boxes. B. Phylip radial phylogenetic tree, derived from ClustalW alignment of nineteen secondary FENs from eubacteria (supplementary FigureS1). A clear differentiation of Exo IX and FEN classes. Eubacterial paralogous FENs fall into two discrete classes with predicted affinity for one (hatched area) or two (grey area) metal ions. This broadly, but not exclusively correlates with Gram positive and negative classification (bold and underlined type, respectively).

Figure 3 .

 3 Figure 3. Comparison of exonucleolytic properties of prokaryotic FEN homologues.

Figure 4 .

 4 Figure 4. Comparison of enzymes' structure-specific nucleolytic cleavage on a selection of exo-and endo-nuclease substrates.

Figure 5 .

 5 Figure 5. Homology modelling SaFEN in complex with M 2+ cofactor and a branched Pseudo-Y DNA substrate.

Table 1 . Oligonucleotides used in structure-specific nuclease assays, presented in Figure 4.

 1 Substrates were prepared as described in the Materials and Methods, in brief, cold oligonucleotides were present at a 30-fold excess (13.5 nM) when annealed to the complementary radiolabelled strand (450 pM). Radiolabelled oligonucletides are italicised and the labelled 5' terminal nucleotide indicated in bold typeface.

	Biochemical Journal Immediate Publication. Published on 11 Nov 2008 as manuscript BJ20081637

  The autonomous FEN-like proteins can be divided into those which retain all the putative ligands to bind two metal ions (e.g. Staphylococcus aureus FEN, SaFEN) and those which are retain the ligands to bind a single divalent metal ion (Escherichia coli Exo IX). Perfectly conserved residues are boxed in grey. The amino acid substitutions affecting binding at the second metal site in Exo IX are shown in clear boxes. B. Phylip radial phylogenetic tree, derived from ClustalW alignment of nineteen secondary FENs from eubacteria (supplementary FigureS1). A clear differentiation of Exo IX and FEN classes. Eubacterial paralogous FENs fall into two discrete classes with predicted affinity for one (hatched area) or two (grey area) metal ions. This broadly, but not exclusively correlates with Gram positive and negative classification (bold and underlined type, respectively).

Figure 2. SDS-PAGE (10%) analysis of recombinant S. aureus FEN. A. Heat-shock induction (0 -2 h at 42°C) of recombinant SaFEN demonstrates protein of the anticipated molecular mass (35 kDa; black arrow); i) stained with Commassie or ii) zymogram for DNase activity. B. Fractions taken during the purification of SaFEN were analysed by SDS-PAGE (10% acrylamide) and stained with i) Coomassie blue or ii) a zymogram assay of DNase activity. A replicate gel was prepared with 40 µg ml -1 Type XIV herring sperm DNA, and assayed for in situ

DNase activity, furnished with 10 mM MgCl 2 as cofactor

[START_REF] Rosenthal | Nuclease detection in SDS-polyacrylamide gel electrophoresis[END_REF]

. Abbreviations; M, Marker (BioRad precision unstained), molecular mass of standards is given in kDa; TL, total lysate; SE, soluble extract; Ch, pooled Ni-chelate eluate; AS; ammonium sulphate precipitated fraction (final 30% saturated).

Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited

© 2008 The Authors Journal compilation © 2008 Portland Press Limited

ACKNOWLEDGEMENTS

We acknowledge the BBSRC for funding to JRS (ref. no. B19466) and studentships for LMA (ref. no. 99B1B05418) and MRGH (ref. no. 02B1B08371). We thank Prof. Peter Artymiuk (University of Sheffield) for helpful advice on model building.


Dr Namir J Hassan
email: namir.j.hassan@gsk.com

Sheraz Gul

Fiona Flett

Edward Hollingsworth

Angela A Dunne

Amanda J Emmons

Jonathan P Hutchinson

Martin J Hibbs

Susan Dyos

Jeremy D Kitson

Emma Hiley

Martin Rüdiger

David G Tew

David J Powell

Mary Morse

Development of an insect cell-based assay for detection of kinase inhibition using NF-κB inducing kinase as a paradigm

Keywords: NFΚB inducing kinase, IΚB kinase-1, baculovirus expression system, cell-based assay NIK, NFκB inducing kinase, IKK, IκB kinase-1, TR-FRET, timeresolved fluorescence resonance energy transfer, FP, fluorescence polarisation, sf9, Spodoptera frugiperda, HTS, high-throughput screening

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Kinases are key regulators of many biological processes with aberrant activity of these proteins being associated with diseases such as cancer, inflammation and diabetes [START_REF] Dancey | Issues and progress with protein kinase inhibitors for cancer treatment[END_REF]. The unveiling of at least 500 distinct kinases through sequencing the human genome coupled with the recent successes of small molecule kinase inhibitors [START_REF] Druker | Inhibition of the Bcr-Abl tyrosine kinase as a therapeutic strategy for CML[END_REF][START_REF] Rubin | Molecular targeting of platelet-derived growth factor B by imatinib mesylate in a patient with metastatic dermatofibrosarcoma protuberans[END_REF] have led to an increase in drug research in this area second to G-protein coupled receptors as drug development targets [START_REF] Cohen | Protein kinases-the major drugs targets of the twenty-first century?[END_REF]. A variety of approaches have been used to develop kinase assays for inhibitor identification. These involve utilising purified protein, recombinant cell-lines or primary cells. The strategy of choice can be divided into two categories; biochemical and/or cellular. A biochemical approach to monitoring kinase activity can be further divided into i) generic and ii) antibody-based formats that detect an epitope within the phosphorylated product. In both cases suitable quantities of purified active kinase are required. This can often be challenging due to protein expression yields, degradation, aggregation and low activity protein upon purification. The majority of biochemical assays for kinases have used only the catalytic domain for high-throughput screening (HTS), thus identifying ATP-site directed inhibitors as the primary class of agents. Use of the catalytic domain can address some of the issues around yield, purity and aggregation of protein compared to full-length. However, recent studies on the Akt-family of kinases have demonstrated some of the benefits of using full-length protein by discovering a pharmacologically unique class of compounds [START_REF] Barnett | Identification and characterization of pleckstrinhomology-domain-dependent and isoenzyme-specific Akt inhibitors[END_REF].

More recently there has been increased focus on cell-based assays, where in 2006 a survey had indicated that approximately half of HTS assays were cell-based [START_REF] Fox | High-throughput screening: update on practices and success[END_REF]. This is partly due to the advances in suitable detection systems utilizing chemical or genetically encoded fluorescent probes and the development of sophisticated assay platforms such as microscope-based imaging systems or microplate cytometers [START_REF] Giepmans | The fluorescent toolbox for assessing protein location and function[END_REF]8]. Cells used in these type of assays can be primary cells or recombinant cell-lines. While primary cells offer major advantages such as physiological relevance of wildtype or disease states, they are difficult to obtain and prone to inter-individual variability. Stable cell-lines offer reliability of protein expression, and ease of continuous culture, however there is a potential for the gene product to be toxic when expressed constitutively and stable cell-lines are time-consuming to generate and clone. Transient cell-lines are frequently used, but can be costly and require large amounts of DNA that sometimes cause toxicity to cells. Modified baculoviruses termed BacMam have recently been developed to address some of these issues [START_REF] Condreay | Baculoviruses and mammalian cell-based assays for drug screening[END_REF].

The baculovirus insect cell expression system is widely used as a tool for production of recombinant proteins. The advantages of the baculovirus system over other expression systems are well documented [START_REF] Kost | Baculovirus as versatile vectors for protein expression in insect and mammalian cells[END_REF][START_REF] Berger | Baculovirus expression system for heterologous multiprotein complexes[END_REF]. Briefly, these include acceptable expression for a diverse range of proteins, the capability of insect cells for post-translational modifications such as phosphorylation and ease of culture. Insect cells have been used to configure Calcium flux assays for G-protein coupled receptor targets [START_REF] Knight | A functional assay for G-protein-coupled receptors using stably transformed insect tissue culture cell lines[END_REF], but to our knowledge there are currently no reports on the use of insect cells in a cellular assay format for any other target classes.

Herein we describe the novel utility of baculovirus-infected insect cells to monitor kinase activity using human NF-kappa B inducing kinase (NIK/MAP3K14) as a paradigm. We show that in baculovirus co-infections of insect cells, full-length NIK produced the highest phosphorylation of its downstream physiological substrate IκB kinase-1 (IKK1). These baculoviruses were then used to develop an insect cell plate based assay in a 384-well Time Resolved-Florescence Resonance Energy Transfer (TR-FRET) format that utilizes a specific antibody to the NIK mediated IKK1 phosphorylation sites. The assay was sufficiently robust to be used for screening purposes. The index that is used to measure the robustness of the assay quantitatively is the Z' value. This provides an important measure of the quality of an assay as it takes into account the signal window variability. The closer the Z' value to 1, the more robust the assay [START_REF] Zhang | A simple statistical parameter for use in evaluation and validation of high throughput screening assays[END_REF] however an acceptable Z' value for cell-based assays is greater than 0.4. The current assay yielded average Z' values greater than 0.65. Using this assay, a number of apparent NIK inhibitors were identified when screening a focussed compound set. To validate these hits a selection were further analysed and we identified an inhibitor, compound A, that was a) non-cytotoxic, b) interacted directly with NIK and c) inhibited lymphotoxin induced p52 translocation to the nucleus. This provided strong support that the insect cell system was able to identify bona fide kinase inhibitors. Thus this report has set precedence for the use of baculovirus-infected insect cells to detect kinase activity in a HTS amenable system.

MATERIALS AND METHODS

Cloning of expression constructs

Full-length and truncations of human NIK were cloned into a modified pFastBac vector containing a dual tag, FLAG-6xHis at the N-terminus followed by the restriction site NdeI. Cloning was performed by PCR amplification of NIK of interest flanked with an N-terminal TEV protease cleavage site downstream of an NdeI site. The restriction site XhoI was incorporated into the reverse PCR primers at C-terminus. Upon ligation of vector and insert the gene is in frame with the N-terminal dual tag FLAG-6xHis followed by the TEV protease cleavage site. PCR primers used for amplification of full-length or truncations of NIK are shown below (underlined codon represents the reading frame, and those in bold represent the start of NIK): The mutations KK429/430AA and T559D were generated using the Quickchange site-directed mutagenesis (SDM) system (Stratagene) using the primers detailed below: For KK429/430AA: Forward: ggcttccaatgtgccgtggctgccgtccgtctggaggtcttc Reverse: gaagacctccagacggacggcagccacggcacattggaagcc For T559D: Forward: ggcgactacatccctggagacgagacccacatggctccag Reverse: ctggagccatgtgggtctcgtctccagggatgtagtcgcc The deletion d78-84 was generated using overlap extension PCR with the primers outlined below: Forward 1: ctgaggccggtccagcggctgaatgcgagaactcgcaagaattc Reverse: gaattcttgcgagttctcgcattcagccgctggaccggcctcag Human IKK1 (5-746)-pFastBac 1 containing an N-terminal FLAG tag upstream of a TEV protease cleavage site was kindly provided by Angela Bridges (GSK, Harlow). This construct was used as template to generate the K44A kinase dead mutant using the Quickchange SDM system (Stratagene) and the primers below: Forward: CTTGATCTCAAAATAGCAATTGCCTCTTGTCGCCTAGAGCTAAG Reverse: CTTAGCTCTAGGCGACAAGAGGCAATTGCTATTTTGAGATCAAG All constructs were sequence verified.

Baculovirus generation, protein expression and analysis by western blotting

NIK (full-length and truncations) and IKK1-K44A-pFastBac constructs were used to generate P1 amplified baculoviruses using the Bac-to-Bac system (Invitrogen). NIK (full-length or truncations) and IKK1-K44A baculoviruses were mixed with Spodoptera frugiperda (sf9) insect cells at a cell density of 1x10 6 /ml and an MOI of 5-10 each. After incubating the cells for 48hrs at 27 o C while shaking at 100rpm, they were harvested by centrifugation at 805g for 20 minutes. The cell pellets were recovered and lysed using 20mM Tris-HCl pH7.5, 150mM NaCl 1% v/v triton-X100 and protease inhibitor cocktail (Roche, 1 tablet per 10ml). The lysates were placed on ice for 20 minutes then centrifuged at 17,500g for 10 minutes at 4°C. The supernatant fraction was mixed with sodium dodecyl sulphate (SDS) protein sample buffer (Invitrogen) supplemented with reducing agent (Invitrogen) and heated to 95 o C for 5 minutes. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate proteins in 4-20% Tris-Glycine gels (Invitrogen). The protein gels were then blotted onto PVDF membranes using the semi-dry western blotting apparatus (Bio-Rad). The membranes were blocked in buffer containing 5% w/v milk (Marvel) in phosphate buffered saline (PBS) and 0.1% v/v Tween-20 (Sigma). The membranes were then treated with mouse anti-FLAG (Strategene, 1:1000) and rabbit anti-phospho IKK1 ser176/180 (Cell signalling, 1:1000) diluted in blocking buffer, while shaking overnight at 4°C. The membranes were washed 5x3 minutes in blocking buffer then treated with anti-mouse and anti-rabbit antibodies (Odyssey, 1:5000 each) diluted in blocking buffer, while shaking at room temperature for 1hr. The membranes were then washed 5x3minutes in PBS/0.1% v/v Tween-20 then 2x3minutes in PBS alone. Membranes were then visualised using the Odyssey imaging system detecting in both 700 nm (red) and 800 nm (green) channels.

Development of a NIK insect cell-based assay

Once the appropriate NIK and IKK1-K44A constructs were identified using the dual detection Western methodology, we modified the assay so as to make it suitable for screening purposes allowing the identification of compounds that inhibit NIK mediated phosphorylation of IKK1-K44A. The infections of the Sf9 cells were performed in bulk solutions (typically 10-100ml). Infected cells (5µl, containing 5,000-10,000 cells) in culture media were dispensed into 384 well plates that contained 50nl of test compound in 100% v/v DMSO yielding a final concentration of 6µM for single concentration screening and 1% v/v DMSO. Plates were incubated at 27°C for 24 and 48hrs. Optimal conditions for the assay were determined by varying the number of cells per well, infection time, detergent type and concentration during the cell lysis step and antibody concentrations during the detection step. Assays were performed in black 384 well plates (Greiner 784076). The optimised lysis buffer was 20mM Tris/HCl, 150mM NaCl, 2% v/v Triton & 1 protease inhibitor tablet per 10ml buffer. Assay signal was determined with cells ranging between 5,000-10,000 per well in the 384 well plates. The antibodies used in the detection step were 25nM anti-FLAG-APC (Perkin Elmer, AD0059F), 2nM LANCE anti-rabbit Eu chelate (Perkin Elmer, AD0082) and 1nM anti-phospho IKK1/2, S176/180 in IKK sequence, rabbit monoclonal (Cell Signalling 2697). A mixture of the three antibodies was made in 50mM HEPES, 1mM CHAPS, 1mM DTT, pH 7.4. The detector used for readout was a Perkin Elmer Viewlux. The Cell Titre Glo assay kit (Promega, G7570) was used in accordance with manufacturer's instructions and replicating the same conditions for infection and lysis as the NIK/IKK1-K44A assay. The signal window for the assay was determined from assays in 384-well plate format that contained an entire column (16 wells) of dual NIK and IKK1-K44A infected Sf9 cells (high control) and another column containing cells infected with NIK KK429/430AA and IKK1-K44A (low control). The quality and robustness of the assay, represented as Z' [START_REF] Zhang | A simple statistical parameter for use in evaluation and validation of high throughput screening assays[END_REF] was calculated using equation ( 1):

) mean (mean ) S.D. 3 S.D. (3 1 Z lows highs lows highs ' - × + × - = (1)
Dose-response data were fitted to a four parameter logistic equation using non-linear regression.

NIK fluorescence polarisation binding assay

Purified NIK kinase domain (318-947) was obtained as a GST fusion from Invitrogen (PV4902). Rhodamine green labelled FP ligand was synthesised in-house (structure shown below). Structurally related kinase FP ligands have been described previously [14]. Test compounds were diluted from 10mM in DMSO in a 3-fold series of 11 points. 100nL of the dilution series were transferred to a 384-well low volume plate (Greiner 784076) using an Echo acoustic dispenser. GST-NIK and rhodamine green labelled FP ligand were added to the assay plate to final concentrations of 20nM and 5nM respectively, in a total volume of 10uL, using a Multidrop Combi. The plate was incubated at room temperature for 30 minutes after which fluorescence polarisation was measured using an Acquest plate reader (Molecular Devices). Data were converted to % displacement by normalisation to high controls (enzyme-ligand mix) and low controls (ligand only) and fitted as described above.

O O O O NH 2 NH 2 N H N H O N N N N CH 3 N H 2 N
Structure of the Rhodamine green labelled FP ligand p52 nuclear translocation assay U2OS human osteosarcoma cells were seeded into clear bottom PDL-coated 384-well plates (Greiner 781948) at 1500 cells in 45μl DMEM Ham's F-12/10% FBS (Invitrogen) per well, using a Multidrop Combi dispenser, and incubated at 37°C/5% CO2 for 24h. Test compounds were diluted from 10mM in DMSO in a 3-fold series of 11 points. 0.5 μl of the dilution series were transferred into the cell plate using a Cybiwell 384-well head pipetter. 5 μl of a 3 μg/ml solution of lymphotoxin a1/b2 (R&D Systems 678-LY) in culture media were added to each well, and the plate incubated for a further [START_REF] Regnier | Identification and characterization of an IkappaB kinase[END_REF] A c c e p t e d M a n u s c r i p t PBS, permeabilised with 0.1% Triton X 100/PBS (15 min), stained with anti-p52 antibody (Upstate 05-361) diluted 1:200 in PBS/0.5% BSA (1h), and stained with secondary goat anti-mouse Alexa488 antibody (Invitrogen A11001) (1:200) plus Hoechst 33342 (Invitrogen H-3570) (1:5000) in PBS/0.5% BSA (1h), with intermediate wash steps (PBS/0.5% BSA) between each of these steps. Plates were kept with PBS/0.5% BSA at 4°C until analysis.

Images were collected on a Perkin Elmer Opera system, using 405nm and 488nm laser excitation, and a 10x magnification objective. For image analysis by the ACapella software, nuclear and cytoplasmic areas were determined, intensities of the Alexa488 stain in these areas measured, and the 'nuclear fraction' of nuclear/(nuclear+cytoplasmic) intensities calculated for each individual cell. Typically, 150-200 cells were analysed for each well. Since not all cells within a well responded to stimulus or showed the inhibition effect, but usually around 70-80%, the most robust data were achieved by determining the 'percentage of responding cells' within each well, with 'responding cell' being defined as a cell with a 'nuclear fraction' value greater than 0.5.

RESULTS

Detection of NIK mediated IKK1 phosphorylation in co-infected insect cells

We examined the kinase activity of full-length and truncations of human NIK using the physiological downstream substrate IKK1. Reports show that IKK1 autophosphorylation occurs at the same sites as NIK mediated phosphorylation, Serine 176/180 [START_REF] Ling | NF-kappaB-inducing kinase activates IKK-alpha by phosphorylation of Ser-176[END_REF]. In order to discriminate between these events, it was necessary to use a kinase dead IKK1 mutant IKK1-K44A that renders the kinase incapable of autophosphorylation thus allowing the detection only of NIK mediated IKK1 phosphorylation [START_REF] Ling | NF-kappaB-inducing kinase activates IKK-alpha by phosphorylation of Ser-176[END_REF][START_REF] Regnier | Identification and characterization of an IkappaB kinase[END_REF].

Insect cells were co-infected with NIK and IKK1-K44A baculoviruses while shaking in flasks for 48 hours, and the resulting product detected by two colour western blotting. IKK1-K44A phosphorylation was detected in the green channel using a phospho-specific antibody recognizing phospho-Serine 176/180. Both NIK and IKK1-K44A were tagged at the N-terminus with a FLAG epitope that was also probed and visualised in the red channel (Figure 1). As shown both NIK (107kDa fulllength or smaller for truncations) and IKK1-K44A (85kDa) protein was detected. An overlay of the green and red channels indicates the detection of phosphorylated protein at that given size and was visualised in yellow. The results show the detection of IKK1-K44A phosphorylation when co-infected with full-length NIK that was not observed in cells infected with IKK1-K44A alone (Figure 1). In addition, phosphorylation was mediated by the kinase activity of NIK as a kinase dead mutant NIK KK429/430AA [START_REF] Ling | NF-kappaB-inducing kinase activates IKK-alpha by phosphorylation of Ser-176[END_REF] was unable to phosphorylate IKK1-K44A under the same conditions. A predicted constitutively active mutant of NIK T559D appeared incapable of phosphorylating IKK1-K44A. Also included was a construct containing a deletion in NIK from amino acids 78-84, associated with TRAF3 binding, and reported to enhance expression yields [START_REF] Liao | Regulation of the NF-kappaB-inducing kinase by tumor necrosis factor receptor-associated factor 3-induced degradation[END_REF]. By assessing extent of product formation (percentage of phosphorylated IKK1-K44A relative to total IKK1-K44A protein, as determined from band intensities) full-length NIK confers the greatest activity, and truncations of the protein, including those predicted to contain the kinase domain 389-658 exhibit either little or no kinase capability. These observations suggested that regions in NIK outside of the kinase domain were important for the kinase potential of the protein. Thus full-length NIK and IKK1-K44A baculoviruses were the tools selected for development of an insect cell based assay.

Development of a medium/high throughput assay to monitor NIK activity using baculovirus infected insect cells

Once the in-flask co-infection studies had allowed the triage of appropriate constructs for NIK and IKK1-K44A, the desire was to develop an assay to screen against a library of compounds. The assay format that was considered was based on the principle of Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET). This utilized commercially available antibodies which form a sandwich in the presence of phosphorylated FLAG-IKK1-K44A. A rabbit monoclonal antibody recognizing the phospho-Serine 176/180 of IKK1/2 becomes bound to the europium (Eu) labeled anti-rabbit IgG. A third antibody, the mouse monoclonal anti-FLAG-Allophycocyanin (APC) is bound to the FLAG epitope of IKK1-K44A (Figure 2). This forms a sandwich which when excited by electromagnetic radiation at 320nm results in the excitation of the Eu-label which emits light at 615nm. If the anti-FLAG-APC is in close proximity to the Eu-labelled antibody (i.e. there exists the phosphorylated FLAG-IKK1-K44A) TR-FRET takes place resulting in the excitation of the APC and subsequent emission of electromagnetic radiation at 665nm. In the absence of phosphorylated FLAG-IKK1-K44A a low signal would be obtained.

There were a number of key parameters that we addressed during the development of the insect cell assay. Some of these were necessary to understand optimal conditions required for expression of proteins in baculovirus treated insect cells when miniaturising from a typical shaking flask system to a static plate based system. Investigations were carried out to identify the optimal conditions for a) the multiplicity of infection (MOI) of baculoviruses per cell for efficient expression in a 384-well plate b) cell numbers per well c) the infection period of insect cells with NIK and IKK1-K44A baculoviruses, d) detergent type/concentration for in plate cell lysis and e) antibody concentrations to generate a robust assay that was screening compatible. The final optimised assay in 384-well plate format contained 10,000 cells/well that were infected with an MOI of 5 for each virus in a volume of 10µl for a period of 48hrs at 27°C. After this period the cells were lysed by the addition of 10µl of lysis/detection solution. This was done using Tris/HCl buffer containing Trition-X 100 at 2% v/v, 1mM DTT, 25nM anti-FLAG-APC, 2nM europium labeled anti-rabbit IgG and 2nM anti phospho-Serine 176/180 of IKK1/2. The final optimized assay yielded a Signal:Background (S:B) of ~2 which although is relatively low provided an average Z' >0.65 due to the fact that the assay produced low deviations in the values of the high and low controls (see Figure 3 for typical data quality).

Application of the NIK insect cell assay to a focussed screening campaign

Once a robust assay was configured it was applied to screen a kinase focussed compound set to identify potential NIK inhibitors. Approximately 10,000 compounds were chosen to represent the diversity of GSK kinase chemical space. This set of compounds was screened in duplicate against the NIK/IKK1-K44A assay at a concentration of 6µM compound and a range of inhibitory responses were observed in the assay at this concentration. Figure 4 shows a correlation plot for the experimental data. The number of active compounds (being classified as those giving >50% inhibition at 6µM in duplicate) was 423 and they exhibited inhibition in the assay between 50-100%. A total of 75 compounds were active on one of the two test occasions equating to a 17% false positive/negative hit rate. Of the 423 active compounds care needed to be taken to ensure their mechanism of action was due to NIK inhibition and not for example cytotoxicity. Such compounds would be of no interest as the inhibition profile is essentially an artefact. In order to increase confidence that the inhibition in the cellular assay was not due to a NIK independent process, the active compounds were investigated for their cytotoxic effects using the Promega Cell Titre Glo assay that measures intracellular ATP. To enable discrimination of the NIK dependent actives from potential cytotoxicity effects, all actives were screened in this cytotoxicity assay. In total 382 compounds were identified as exhibiting a potency in the NIK/IKK1-K44A assay pIC50 >4.5. Of these a significant number exhibited cytotoxicity (343 in total). The remaining 39 compounds exhibited activity in the NIK/IKK1-K44A assay with pIC50 >4.5 and at least a 10-fold decrease in potency in the cytotoxicity assay (Figure 5). This 10-fold window indicated that the compounds blocked the NIK/IKK1-K44A assay independent of cytotoxicity and were thus considered 'apparent NIK inhibitors'.

Use of a fluorescence polarisation assay to identify direct binding NIK inhibitors

To identify direct binding inhibitors from amongst the apparent inhibitor set of compounds a NIK fluorescence polarisation binding assay was developed using a rhodamine green labelled non-selective kinase inhibitor as an ATP binding site probe. The principle of the fluorescence polarisation assay as applied to kinases has been described elsewhere [START_REF] Eccleston | Fluorescencebased assays[END_REF]. The source of kinase was purified NIK truncate protein (318-947). The affinity of the ligand was shown to be 15nM by titration, and the interaction was competitive with ATP. The binding assay was configured with a NIK concentration slightly in excess of the dissociation constant for the ligand, and a selection of apparent NIK inhibitors were tested for their ability to compete with this interaction. Figure 6 shows a normalised displacement curve for compound A, which yielded an average pIC50 of 7.0 in duplicate determinations.

Inhibition of lymphotoxin induced p52 nuclear translocation

In order to validate compounds further, a nuclear translocation assay was configured detecting the inhibition of lymphotoxin-induced migration of p52 into the nucleus. The antibody used for immunostaining recognises both the NFkB subunit p52 and its precursor p100. p52 translocates into the nucleus upon p100 processing [START_REF] Dejardin | The Lymphotoxin-β Receptor Induces Different Patterns of Gene Expression via Two NF-κB Pathways[END_REF], which therefore can be observed as a change of staining pattern with a p100/p52 antibody. Following immunocytochemical analysis of p100/p52 localisation in various cell lines, U2OS cells were found to exhibit a reproducible response. Treated with lymphotoxin α1/β2, the majority of cells show higher intensity of p100/p52 staining in the nucleus compared with cytoplasm (Figure 7A). In the control experiment, the staining intensity is higher in the cytoplasm (Figure 7B). When cells are treated with lymphotoxin α1/β2 in the presence of compound A, the p52 nuclear translocation effect is inhibited (Figure 7C). Out of 12 selected apparent NIK inhibitors, 9 showed activity in this assay, and quantitative image analysis (as described in the Materials and Methods section) gave pIC50 values in the range of 4 to 6 (data not shown).

DISCUSSION

We have developed an insect cell based assay monitoring kinase activity that involves infection, lysis and detection within a 384-well plate. We illustrated the utility of this assay through identification of apparent NIK inhibitors following a medium throughput focussed screen. A selection of these were characterised further, and we discovered a compound that bound directly to NIK and inhibited nuclear translocation of p52 in mammalian cells. The insect cell based assay demonstrated here, provides an additional tool to be considered when a screen is desired for identifying inhibitors of a given target. The major advantages of using this system over others include achieving adequate protein expression levels, ease of use, amenability to scale-up and the flexibility to express a number of proteins by infecting with numerous baculoviruses.

When considering which assay format to adopt for screening a number of factors are taken into account, not least the question of pursuing a biochemical or cell based approach or both. In some cases, the complexity of the biological target under study can influence this decision. In this study NIK was used as a paradigm. This kinase was first cloned over a decade ago and identified as a member of the mitogenactivating protein (MAP) kinase kinase kinase (MAP3K) family, comprising 947 amino acids including a kinase domain in the region 389-658 [START_REF] Malinin | MAP3K-related kinase involved in NF-kappaB induction by TNF, CD95 and IL-1[END_REF]. In the past 10 years there have been significant advances in understanding the role of NIK in NF-κB activation. The transcription factor NFκB plays a pivotal role in immune and inflammatory responses. Nuclear translocation of NFκB is controlled by two main pathways: the classical and the alternative pathways. The classical pathway regulates innate and adaptive immune responses. In contrast, the alternative pathway appears to be activated by a more restricted set of signals principally involved in mounting a secondary adaptive immune response including those triggered by lymphotoxin β [START_REF] Xiao | NF-kappaB-inducing kinase regulates the processing of NF-kappaB2 p100[END_REF], B cell activating factor [START_REF] Claudio | BAFF-induced NEMO-independent processing of NF-kappa B2 in maturing B cells[END_REF] and CD40 ligand [START_REF] Coope | CD40 regulates the processing of NF-kappaB2 p100 to p52[END_REF]. It is now believed that NIK is an essential mediator of the alternative pathway thus representing a novel target for autoimmune diseases, such as rheumatoid arthritis. Studies have shown that NIK activates IKK1 through phosphorylation of Serine-176 leading to the processing of the NFKB precursor protein p100. Recently NIK was shown to physically associate with IKK1 and p100 functioning as a "docking" molecule [START_REF] Xiao | Induction of p100 processing by NF-kappaB-inducing kinase involves docking IkappaB kinase alpha (IKKalpha) to p100 and IKKalpha-mediated phosphorylation[END_REF]. Other NIK protein interactions have been identified including those with TRAF2 and TRAF3. The role that these interactions play in NIK activation and regulation is not fully understood. Due to the complex nature of activation and function of this target it is not surprising that to date, there are no published reports demonstrating a biochemical assay of NIK activity using its physiological downstream substrate IKK1. We have observed that part-purified NIK showed insignificant activity against purified IKK1-K44A (data not shown) using the same constructs and detection system as that described for the insect cell assay. It is tempting to speculate that the diminished activity was due to the loss of an essential component as a consequence of purification.

The western blotting data had shown that regions outside of the kinase domain of NIK (389-658) were required to achieve highest phosphorylation of IKK1 (Figure 1). Previous reports have linked some of these regions to association with IKK1 [START_REF] Lin | Molecular determinants of NF-kappaB-inducing kinase action[END_REF] and TRAF2 [START_REF] Xiao | NF-kappaB-inducing kinase regulates the processing of NF-kappaB2 p100[END_REF]. Furthermore, it has been reported that recombinant NIK spontaneously forms oligomers [START_REF] Lin | Molecular determinants of NF-kappaB-inducing kinase action[END_REF], possibly induced through its interaction with TRAF2. Reports show that in an assessment of a series of truncated NIK constructs multiple regions within NIK contribute to the oligomerisation of the kinase. Only the relatively long 200-947 construct from the series of truncations retained significant, although reduced stimulatory activity of NF-ΚB in a luciferase reporter assay. It is possible that the truncated NIK proteins described in this report fail to form suitable oligomers thus affecting catalytic function (Figure 1). Also, the lack of activity observed in purified NIK may have been caused by the disruption of oligomers. A number of kinases have been reported to form homooligomers within cells [START_REF] Noguchi | Recruitment of tumor necrosis factor receptorassociated factor family proteins to apoptosis signal-regulating kinase 1 signalosome is essential for oxidative stress-induced cell death[END_REF], thus isolating these proteins in a purified system may not be a feasible approach to assay design or representative of the mechanism in vivo.

Using the western blotting method we also investigated the potential for NIK to phosphorylate kinase dead IKK2 (K44A). Reports show that NIK preferentially phosphorylates IKK1 over IKK2 [START_REF] Ling | NF-kappaB-inducing kinase activates IKK-alpha by phosphorylation of Ser-176[END_REF]. However, we observed similar levels of 10 A c c e p t e d M a n u s c r i p t phosphorylation for both substrates (data not shown). One possible explanation is that the mechanisms involved in controlling substrate specificity for endogenous protein break down in the recombinant baculovirus expression system due to the high protein levels of kinase and substrate. This is not unique to insect cells, and extrapolation from any recombinant system to physiological systems will be limited due to high protein levels.

Cell-based assays commonly utilise recombinant systems such as stable or transiently transfected mammalian cells, or primary cells. Successful assay design using these systems relies on an enabling level of protein expression, reproducibility, and scalability. Any one of these can be limiting. The higher expression levels typically observed in a baculovirus expression system are well documented for numerous target classes [START_REF] Kost | Baculovirus as versatile vectors for protein expression in insect and mammalian cells[END_REF]. Insect cells are robust and relatively easy to use. Baculovirus generation is simple and induced protein expression usually reproducible. In a typical screening campaign a large number of microtitre (384-well) or nanotitre (1536-well) plates are used to minimize the requirements for reagents. One of the main objectives of such a screen (focussed or high throughput, comprising compound numbers in the tens of thousands to millions respectively) carried out on a target of interest is in discovering compounds that have the potential for further development in the drug-discovery process. In order to make an assay amenable for screening, a number of key requirements need to be fulfilled. These will include the cost of the assay per well. In order to achieve this with potentially expensive reagents miniaturization is an integral part of assay development. We successfully miniaturised the insect cell assay to 5μl of cells per well and 5μl of detection reagent. In addition, the large numbers of compounds involved also necessitates sizeable quantities of cellsupply to resource a screening exercise. In a typical mammalian assay system this can be labour intensive, and can suffer from cell stability issues over the course of a screening day. The insect cell system is scaleable, and robust, with a 1 litre culture providing sufficient cell numbers for up to 100,000 wells. Another important aspect for development of a screening assay is to optimize conditions to obtain a high S:B ratio. This would enable compounds that are genuine inhibitors of the assay to be identified with confidence, away from noise within the signal window. The current assay format offers a S:B ratio of 2:1 and a very good average Z' of >0.65, thus providing confidence that compounds identified in the current assay are statistically significant inhibitors.

As with other cellular assay systems it was important to incorporate further assays to determine the specificity of apparent inhibitors, and gain confidence that the inhibitory effects are due to specific inactivation of NIK rather than off target effects. Using a cell cytotoxicity assay we have discriminated between those compounds that cause cytotoxicity and those that are apparent inhibitors of NIK. In addition to cell cytotoxicity though, there are other compound activities important to avoid particularly in the insect cell assay described here. This is because compound addition is made at the point of infection thus providing multiple opportunities for compounds to inhibit the processes of infection, transcription, translation as well as the effect of interest, phosphorylation. Inhibition of one or all of these processes would produce a low assay signal and appear as NIK inhibition. Therefore in order to validate the inhibitors from the insect cell assay further a selection were assessed in a direct binding FP assay using purified NIK truncate protein. At least one compound showed binding with a pIC50 of 7.0 (compound A). Furthermore, of those apparent NIK inhibitors tested, the majority blocked the lymphotoxin induced translocation of p52 into the nucleus. We determined the intracellular localisation of p100/p52 as a measure for activation or inhibition of the endogenous NIK-p100 pathway in U2OS cells. Compound A demonstrated clear effects in this pathway assay (Figure 7). Thus compound A, an inhibitor identified from the insect cell assay (with pIC50 6.3) was a) non-cytotoxic (pIC50 < 4.5) b) showed direct binding to purified NIK protein with a pIC50 7.0 and c) illustrated activity in a NIK-dependent pathway in mammalian cells.

We also developed an insect cells assay detecting wild type IKK1 autophosphorylation (data not shown) to identify a) off target compound activities and b) selective NIK inhibitors. This used the same detection reagents as the NIK/IKK1-K44A assay thus allowing the identification of compounds that were format specific in addition to those inhibiting baculovirus infection and/or protein expression as well as IKK1 autophosphoryltion. Compound A inhibited IKK1 autophosphorylation with similar potency to NIK inhibition (pIC50 6.3) in this assay. Furthermore, inhibition of IKK1 activity against IκB alpha using compound A was also observed in vitro (data not shown). The lack of selectivity of compound A was not surprising as the compound set screened is kinase focussed and it is common to identify non-selective kinase inhibitors from this set. It is anticipated that a full-diversity screen against a complete compound library (millions of compounds) could potentially yield selective inhibitors. The downstream assays we used to validate hits from the primary screen highlights the importance of incorporating these type assays in a screening path given the potential for false positives with the insect cell (or any other cell-based) screening system. With the appropriate downstream assays in place (cytotoxicity, direct binding, format-dependent specificity and pathway assays) we demonstrate that the insect cell assay can identify compounds which are active in a NIK-dependent pathway assay in mammalian cells.

This work has set precedence for the development of a robust cellular kinase assay using insect cells expressing the kinase of interest. NIK was used to illustrate the potential of this system however this approach could be applied to other kinases, and potentially other target classes.

Figure 2 Schematic representation of TR-FRET assay for NIK

Detection of NIK mediated phosphorylation of IKK1 is measured using specific antibody interactions. In (a) cells were treated with baculoviruses and transferred from a typical shaking flask system to a static 384-well plate based system. During the next 24 and 48hrs, (b) protein (NIK and IKK1-K44A) production occurs followed by phosphorylation of IKK1-K44A, (c) the cells are lysed and the detection of the phospho-IKK1-K44A takes place using a TR-FRET system. See the Materials and Methods for more details.

Figure 3 NIK assay signal:background and pharmacology data

A) Data showing that the assay window generated is dependent on NIK catalytic activity. The assay yields a low signal when using the NIK kinase dead mutant (KK429/430AA) + IKK1-K44A and is the same when using an inhibitor (compound B), (B) typical dose-response curve for the NIK/IKK1-K44A assay with compound B. Duplicate data is shown with IC 50 values in the assay of 0.62±0.05µM, (C) Z' values for 52 plates screened as part of the kinase focussed set of compounds. The assay yielded average Z' >0.65, indicative of a very good robustness. The open circles highlight the 39 compounds that were at least 10-fold more active in the NIK/IKK1-K44A than the Cell Titre Glo cytotoxicity assay. pIC50 determined using n=2 for each assay. A range of concentrations of compound A was incubated for 30 minutes with GST-NIK (20nM) and rhodamine green labelled FP ligand (5nM). Fluorescence polarisation was recorded, and converted to % displacement by reference to high controls (enzyme-ligand mix) and low controls (ligand only). Resulting data were fitted as described in the text to yield a pIC50 for compound A of 7.0. Licenced copy. Copying is not permitted, except with prior permission and as allowed by law.

  Forward primers: NIK (a.a 1): gggaattccatatggagaacctgtacttccagggtatggccgtcatggaaatggcc NIK (a.a 48): gggaattccatatggagaacctgtacttccagggttctccagtcttctgcggaaag NIK (a.a 233): gggaattccatatggagaacctgtacttccagggtctccaatgtctcaatcacgtg NIK (aa 327): gggaattccatatggagaacctgtacttccagggtgcgcacgaaaaattcagtgtg NIK (aa 393): gggaattccatatggagaacctgtacttccagggttaccgcgaggaagttcac NIK (aa 397): gggaattccatatggagaacctgtacttccagggtgttcactgggctacccaccaattg Reverse Primers: NIK (end): ccgccgctcgagttatgggcggttttcgagctg NIK (aa903): ccgccgctcgagTTATGTTACCAAGCTGAAAGCGGCAGC NIK (aa658): ccgccgctcgagTTAGACTTGCTGCAGGGCGCGATTAAC

  -20h. Cells were fixed for 10 mins with 4% formaldehyde in 6 Biochemical Journal Immediate Publication. Published on 08 Dec 2008 as manuscript BJ20081646 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081646

Biochemical

  Journal Immediate Publication. Published on 08 Dec 2008 as manuscript BJ20081646 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081646

Figure 1

 1 Figure 1 Full-length NIK is required for maximal IKK1-K44A phosphorylation

Figure 4

 4 Figure 4 Correlation plot for n=1 vs n=2 data set for the NIK/IKK1-K44A assay

Figure 5 pIC50

 5 Figure 5 pIC50 NIK/IKK1-K44A vs pIC50 cell cytotoxicity

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 14 Journal Immediate Publication. Published on 08 Dec 2008 as manuscript BJ20081646 THIS IS NOT THE VERSION OF RECORD -see doi:10.1042/BJ20081646 A c c e p t e d M a n u s c r i p t

Figure 6

 6 Figure 6 NIK fluorescence polarisation binding assay

Figure 7

 7 Figure 7 Inhibition of lymphotoxin induced p52 nuclear translocation (A) Lymphotoxin α1/β2  (0.3 μg/ml) induces translocation of p52 into the nucleus in U2OS cells. (B) Without lymphotoxin stimulation, staining for p52/p100 is higher in the cytoplasm for the majority of cells. (C) Adding compound A (3 μM) together with lymphotoxin α1/β2 (0.3 μg/ml) prevents p52 translocation. (D) Assay window and standard deviation determined from 16 repeat wells per condition.

  Figure 1

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Licenced copy. Copying is not permitted, except with prior permission and as allowed by law. © 2008 The Authors Journal compilation © 2008 Portland Press Limited

© 2008 The Authors Journal compilation © 2008 Portland Press Limited

ACKNOWLEDGEMENTS

We would like to thank Paul Bamborough for reviewing the paper and Angela Bridges for providing IKK1 cDNA and IKK2 K44A baculovirus.